[224] T. Matziol, V. Talagayev, T. Slokan, N. Strašek Benedik, J. Holze, M. Sova, G. Wolber, G. Weindl. Discovery of Novel Isoxazole-Based Small-Molecule Toll-Like Receptor 8 Antagonists, J. Med. Chem., 68(4):4888-4907, 2025.
Links: [doi:10.1021/acs.jmedchem.4c03148] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Toll-like receptor 8 (TLR8) recognizes viral and bacterial RNA, initiating inflammatory responses that are crucial for innate immunity. Dysregulated TLR8 signaling contributes to autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis, driving chronic inflammation and tissue damage. Therefore, targeting TLR8 has gained attention as a promising therapeutic strategy. We report a novel selective TLR8 antagonist scaffold identified through computational modeling and simulation. In silico-guided rational drug design and synthesis led to potent isoxazole-based compounds that were characterized by structure–activity relationships. The most active compounds inhibited TLR8-mediated signaling in cell lines and primary cells, reduced MyD88 recruitment, suppressed NF-κB- and IRF-dependent signaling, and decreased inflammatory responses. In silico and pharmacological analyses demonstrated competitive binding to the pocket of chemical ligands within the TLR8 dimerization interface. These highly selective and potent TLR8 antagonists possess favorable physicochemical properties, representing potential clinical candidates for TLR8-targeted therapy.

[223] S. Upadhyay, V. Talagayev, S. Cho, G. Wolber, M. Gabr. Structure-based virtual screening identifies potent CD28 inhibitors that suppress T cell co-stimulation in cellular and mucosal models, Eur. J. Med. Chem., 300:118194, 2025.
Links: [doi:10.1016/j.ejmech.2025.118194] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Resistance to immune checkpoint inhibitors targeting PD-1 and CTLA-4 remains a major barrier to effective cancer immunotherapy, often arising from compensatory CD28-mediated costimulation. Here, we report the discovery and biological validation of small molecule CD28 antagonists identified through a structure-based virtual screening pipeline. Molecular dynamics and Pyrod-based water mapping revealed a cryptic lipophilic canyon on CD28 enriched in druggable features. A pharmacophore-based screen of over 7 million compounds yielded several candidates, of which compound 22VS emerged as a lead based on biophysical binding (TRIC and MST), structure–activity insights, and functional inhibition in ELISA and NanoBit assays. 22VS demonstrated potent and selective blockade of CD28–B7 interactions, with submicromolar IC50 values in cellular assays and minimal cytotoxicity. Importantly, 22VS suppressed proinflammatory cytokines (IFN-γ, IL-2, TNF-α) in human tumor–PBMC and mucosal–PBMC co-culture systems, closely mimicking the biological activity of the CD28-targeting biologic FR104. Pharmacokinetic profiling revealed favorable solubility, metabolic stability, low CYP inhibition, and excellent safety in human fibroblasts. These findings establish CD28 as a druggable immunotherapeutic checkpoint and validate 22VS as a promising lead candidate for modulating T cell responses. This small-molecule approach offers a viable pharmacological strategy to overcome resistance mechanisms associated with PD-1 and CTLA-4 blockade, with implications for autoimmune disease, transplantation, and cancer immunotherapy.

[222] N. Strašek Benedik, D. Lukić, T. Slokan, A. Dolšak, U. Švajger, S. Kranjc Brezar, V. Talagayev, G. Wolber, M. Čemažar, I. Sosič, M. Sova. Synthesis and structure-activity relationship study of novel quinazolin-4(3H)-one derivatives as Toll-like receptor 7 and 8 agonists with immunomodulatory activity, Eur. J. Med. Chem., :118360, 2025.
Links: [doi:10.1016/j.ejmech.2025.118360] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Toll-like receptors (TLRs) 7 and 8 are intracellular pattern recognition receptors that play a crucial role in the innate immune system, making them promising targets for the treatment of viral infections, autoimmune diseases and cancer. In this study, we present the synthesis and biological evaluation of quinazolin-4(3H)-one derivatives as a new class of dual TLR7/8 agonists. In a comprehensive structure- activity relationship (SAR) study, different substitutions on the quinazoline ring and modifications of the aliphatic side chain were investigated. Several compounds showed significantly improved potency compared to the original hit compound, with EC50 values in the nanomolar and low micromolar range for TLR7 and TLR8, respectively. The most potent compounds significantly increased the secretion of the proinflammatory cytokines TNF-α, IL-1β, IL-8 and interferon γ in peripheral blood mononuclear cells (PBMCs). In addition, increased secretion of TNF-α and upregulated CD86 expression in dendritic cells were also observed, indicating their immunomodulatory properties. Notably, the most potent compound 69 significantly suppressed tumor growth in vivo in the CT26 mouse tumor model after intratumoral administration. These results highlight the potential of quinazolinone-based compounds as promising candidates for further development of new immunomodulatory agents targeting TLR7 and TLR8.

[221] V. Talagayev, G. Wolber, A. Nunes-Alves. Agonists and Antagonists Show Different Unbinding Paths from the TLR8 Receptor, J. Chem. Inf. Model., 65(14):7678–7688, 2025.
Links: [doi:10.1021/acs.jcim.5c00496] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Toll-like receptors (TLRs) form the first barrier of the innate immune system. TLR8 is an important target to treat autoimmune diseases since its ligand-induced degree of activation regulates immune response and associated hyperinflammation. Molecular dynamics (MD) simulations have been used to investigate interactions of TLRs with ligands, but the mechanism of ligand unbinding remains elusive. We therefore applied τ-random acceleration molecular dynamics (τRAMD) simulations to characterize the unbinding paths of one TLR8 agonist and five TLR8 antagonists. Data analysis of the simulations led to the discovery of two possible unbinding pathways: the internal pathway, directed toward the Toll-interleukin-1 receptor (TIR) domain, and the external pathway, pointing away from the TIR domain. Remarkably, some ligands showed clear path preferences: the TLR8 agonist exited through the external unbinding pathway only, while the cationic antagonists exited through the internal pathway only. The neutral antagonists used both pathways. The mechanistic insights obtained can assist in the design of improved TLR modulators.

[220] V. Talagayev, Y. Chen, N. P. Doering, L. Obendorf, K. Denzinger, K. Puls, K. Lam, S. Liu, C. A. Wolf, T. Noonan, M. Breznik, P. Knaus, G. Wolber. OpenMMDL - Simplifying the Complex: Building, Simulating, and Analyzing Protein–Ligand Systems in OpenMM, J. Chem. Inf. Model., 65(4):1967-1978, 2025.
Links: [doi:10.1021/acs.jcim.4c02158] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Molecular dynamics (MD) simulations have become an essential tool for studying the dynamics of biological systems and exploring protein–ligand interactions. OpenMM is a modern, open-source software toolkit designed for MD simulations. Until now, it has lacked a module dedicated to building receptor–ligand systems, which is highly useful for investigating protein–ligand interactions for drug discovery. We therefore introduce OpenMMDL, an open-source toolkit that enables the preparation and simulation of protein–ligand complexes in OpenMM, along with the subsequent analysis of protein–ligand interactions. OpenMMDL consists of three main components: OpenMMDL Setup, a graphical user interface based on Python Flask to prepare protein and simulation settings, OpenMMDL Simulation to perform MD simulations with consecutive trajectory postprocessing, and finally OpenMMDL Analysis to analyze simulation results with respect to ligand binding. OpenMMDL is not only a versatile tool for analyzing protein–ligand interactions and generating ligand binding modes throughout simulations; it also tracks and clusters water molecules, particularly those exhibiting minimal displacement from their previous coordinates, providing insights into solvent dynamics. We applied OpenMMDL to study ligand–receptor interactions across diverse biological systems, including LDN-193189 and LDN-212854 with ALK2 (kinases), nifedipine and amlodipine in Cav1.1 (ion channels), LSD in 5-HT2B (G-protein coupled receptors), letrozole in CYP19A1 (cytochrome P450 oxygenases), flavin mononucleotide binding the FMN-riboswitch (RNAs), ligand C08 bound to TLR8 (toll-like receptor), and PZM21 bound to MOR (opioid receptor), highlighting distinct functionalities of OpenMMDL. OpenMMDL is publicly available at https://github.com/wolberlab/OpenMMDL.

[219] S. Liu, J. Wu, Y. Chen, C. A. Wolf, M. Bureik, J. Kirchmair, M. A. Marchisio, G. Wolber. Integrated Virtual Screening Approach Identifies New CYP19A1 Inhibitors, J. Chem. Inf. Model., 65(7):3529-3543, 2025.
Links: [doi:10.1021/acs.jcim.5c00204] [Publisher][Download citation: BibTeX | RIS]
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Abstract: The human cytochrome P450 19A1 (CYP19A1, aromatase) is a heme-containing protein catalyzing the final steps of the biosynthesis of the steroid hormone 17β-estradiol. It is a key target for the treatment of sex-hormone-related disorders due to its role in mediating the conversion of androgens to estrogens. Here, we report the development of a virtual screening workflow incorporating machine learning and structure-based modeling that has led to the discovery of new CYP19A1 inhibitors. The machine learning models were built on comprehensive CYP19A1 data sets extracted from the ChEMBL and PubChem Bioassay databases and subjected to thorough validation routines. Ten promising hits that resulted from the virtual screening campaign were selected for experimental testing in an enzymatic assay based on heterologous expression of human CYP19A1 in yeast. Among the seven structurally diverse compounds identified as new CYP19A1 inhibitors, compound 9, a novel, noncovalent inhibitor based on coumarin and imidazole substructures, stood out by its high potency, with an IC50 value of 271 ± 51 nM.

[218] O. Herasymenko, [...], M. Breznik, [...], K. Denzinger, [...], S. Liu, [...], L. Obendorf, [...], A. Pandit, [...], V. Talagayev, [...], G. Wolber, [...], M. Schapira. CACHE Challenge #2: Targeting the RNA Site of the SARS-CoV-2 Helicase Nsp13, J. Chem. Inf. Model., 65(13):6884-6898, 2025.
Links: [doi:10.1021/acs.jcim.5c00535] [Publisher][Download citation: BibTeX | RIS]
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Abstract: AbstractA critical assessment of computational hit-finding experiments (CACHE) challenge was conducted to predict ligands for the SARS-CoV-2 Nsp13 helicase RNA binding site, a highly conserved COVID-19 target. Twenty-three participating teams comprised of computational chemists and data scientists used protein structure and data from fragment-screening paired with advanced computational and machine learning methods to each predict up to 100 inhibitory ligands. Across all teams, 1957 compounds were predicted and were subsequently procured from commercial catalogs for biophysical assays. Of these compounds, 0.7% were confirmed to bind to Nsp13 in a surface plasmon resonance assay. The six best-performing computational workflows used fragment growing, active learning, or conventional virtual screening with and without complementary deep-learning scoring functions. Follow-up functional assays resulted in identification of two compound scaffolds that bound Nsp13 with a Kd below 10 μM and inhibited in vitro helicase activity. Overall, CACHE #2 participants were successful in identifying hit compound scaffolds targeting Nsp13, a central component of the coronavirus replication-transcription complex. Computational design strategies recurrently successful across the first two CACHE challenges include linking or growing docked or crystallized fragments and docking small and diverse libraries to train ultrafast machine-learning models. The CACHE #2 competition reveals how crowd-sourcing ligand prediction efforts using a distinct array of approaches followed with critical biophysical assays can result in novel lead compounds to advance drug discovery efforts.

[217] N. P. Doering, M. Taterra, M. Bermúdez, G. Wolber. MDPath: Unraveling Allosteric Communication Paths of Drug Targets through Molecular Dynamics Simulations, J. Chem. Inf. Model., 65(20):11123-11135, 2025.
Links: [doi:10.1021/acs.jcim.5c01590] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Understanding allosteric communication in proteins remains a critical challenge for structure-based, rational drug design. We present MDPath, a Python toolkit for analyzing allosteric communication paths in molecular dynamics simulations using NMI-based analysis. We demonstrate MDPath’s ability to identify both established and novel GPCR allosteric mechanisms using the β2-adrenoceptor, adenosine A2A receptor, and μ-opioid receptor as model systems. The toolkit reveals ligand-specific allosteric effects in β2-adrenoceptor and MOR, illustrating how protein–ligand interactions drive conformational changes. Analysis of ABL1 kinase in complex with allosteric and orthosteric inhibitors demonstrates the broader applicability of the approach. Ultimately, MDPath provides an open-source framework for mapping allosteric communication within proteins, advancing structure-based drug design (https://github.com/wolberlab/mdpath).

[216] L. L. Sallandt, C. A. Wolf, S. Schuster, H. Enke, D. Enke, G. Wolber, T. H. J. Niedermeyer. Derivatization of Microcystins Can Increase Target Inhibition while Reducing Cellular Uptake, J. Nat. Prod., 88(1):3-14, 2025.
Links: [doi:10.1021/acs.jnatprod.4c00688] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Microcystins, a large family of nonribosomal cyclic heptapeptides known for their hepatotoxicity, are among the best-studied cyanobacterial toxins. Recently, they have been discussed as leads for the development of anticancer drug substances. Their main mode-of-action is inhibition of the eukaryotic serine/threonine protein phosphatases 1 and 2A. Unlike many cytotoxins that can cross cell membranes by passive diffusion, microcystins depend on active uptake via organic anion transporting polypeptides 1B1 or 1B3. Both phosphatase inhibition and transportability strongly depend on the structure of the individual microcystin. Here, we present how chemical modification of positions 2 and 4 of the microcystin core structure can alter these two properties. Aiming to reduce transportability and increase phosphatase inhibition, we used pharmacophore modeling to investigate the phosphatase inhibition potential of microcystins derivatized with small molecules containing a variety of functional groups. The respective derivatives were synthesized using click chemistry. We discovered that some derivatized microcystins can address a yet undescribed subpocket of the protein phosphatase 1. The derivatized microcystins were tested for phosphatase 1 inhibition and cytotoxicity on transporter-expressing cell lines, revealing that target inhibition and transportability of microcystins can independently be influenced by the physicochemical properties, especially of the residue located in position 2 of the microcystin. Derivatization with small acids or amino acids resulted in microcystins with a favorable ratio of inhibition to transportability, making these derivatives potentially suitable for drug development.

[215] H. Nada, G. Wolber, M. T. Gabr. Targeting CAPON to modulate the CAPON–NOS Axis: a computational approach, Comput. Struct. Biotechnol. J., 27:4813-4824, 2025.
Links: [doi:10.1016/j.csbj.2025.11.001] [Download citation: BibTeX | RIS]
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Abstract: The carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) serves as a critical regulatory protein controlling nitric oxide (NO) signaling across multiple physiological and pathological processes which encompass neurological, cardiac and metabolic functions. These diverse physiological roles of CAPON marks it as a key therapeutic target for conditions associated with its dysregulation. Despite this therapeutic potential there are no specific CAPON or nNOS/CAPON modulators which have been developed to date, highlighting a significant gap in targeted drug discovery. Herein, we report the first strategy specifically focused on disrupting the nNOS/CAPON protein-protein interface. Through screening of a chemical library composed of 4.6 million compounds and 13 molecular dynamics simulations, nine potential hit compounds were identified. This work represents a foundational step toward developing targeted therapies for CAPON-mediated disorders. Beyond identifying these promising hits, our approach introduces three python-based drug discovery tools: (i) a Python-based toolset for NMR structural analysis, clustering and visualization, (ii) accelerated ligand preparation toolkit, (iii) Automated hit prioritization pipeline based on multi-method consensus scoring approach that takes in account docking scores and MMGBSA. Collectively, these tools form an accelerated drug discovery pipeline that automates most of the virtual screening process and offers a scalable computational framework to support future drug discovery targeting protein–protein interactions.

[214] T. Matziol, V. Talagayev, G. Weindl, G. Wolber. Structure-guided approaches to modulate endosomal toll-like receptors TLR7, TLR8 and TLR9: advances, challenges and therapeutic promise, Drug Discov. Today, 30(11):104495, 2025.
Links: [doi:https://doi.org/10.1016/j.drudis.2025.104495] [Publisher][Download citation: BibTeX | RIS]
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Abstract: TLR7, TLR8 and TLR9 are endosomal immune receptors central to antiviral defense, autoimmunity and cancer immunotherapy. Recent structural insights have revealed distinct ligand-binding mechanisms and conformational dynamics, enabling the design of selective small-molecule agonists and antagonists. This review summarizes key advances in TLR7/8/9 biology, pharmacology and structure-guided drug discovery. We highlight clinical progress, delivery strategies and translational challenges including species-specific differences and immune-related toxicities. Novel approaches such as nanoparticle systems and endogenous RNA mimetics promise targeted modulation of TLR activity. Together, these developments emphasize the therapeutic potential of precision TLR modulation in immunologically complex diseases.

[213] E. Koçak Aslan, A. Karagüzel, K. Lam, C. Dengiz, S. Huang, R. J. Burk, R. Salehi, S. J. Sajeevan J, G. W. Zamponi, G. Wolber, D. W. Armstrong, M. G. Gunduz. Synthesis, molecular modeling, DFT studies, and enantioseparation of tetrahydro-4H-chromene derivatives with calcium channel blocking activity, J. Mol. Struct., 1330:141457, 2025.
Links: [doi:10.1016/j.molstruc.2025.141457] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Calcium channel blockers are considered effective therapeutics in cardiovascular and neurological disorders. Herein, we replaced the nitrogen atom of 1,4-dihydropyridines (DHPs), the most popular group of calcium channel blockers, with oxygen to yield 4H-pyrans. The target compounds (EA1-EA3) were obtained by the reaction of 4,4-dimethyl-1,3-cyclohexanedione, substituted benzaldehyde, malononitrile, and excess ammonium acetate in ethanol in which ammonium acetate functioned as a catalyst rather than a reactant resulting in the formation of tetrahydrochromenes over hexahydroquinolines. After confirming their proposed chemical structures, the title compounds were tested on two types of calcium channels, L-type (Cav1.2) and T-type (Cav3.2), to determine whether physiological activity was still maintained after this bioisosteric substitution. According to the data obtained from patch-clamp experiments, EA1 and EA3 carrying lipophilic halogens on C-4 phenyl ring were found to be more effective inhibitors of both calcium channels compared to EA2 with a nitro group at the same position. Molecular docking studies and molecular dynamics simulations were conducted on EA1 in Cav1.2 to investigate the binding mode of tetrahydro-4H-chromenes in the DHP binding site. EA1 achieves a binding pose similar to other DHPs in L-type channels. Density functional theory (DFT) vibrational frequency calculations were carried out for all synthesized compounds to assist in assigning the bands observed in the experimental IR spectra. Additionally, electrostatic potential (ESP) map analysis was utilized to visualize the charge distributions within the molecules. Frontier molecular orbital visualizations were employed to examine the reactivity of the compounds, along with other chemical parameters such as the HOMO-LUMO gap, electronegativity, global chemical hardness, and global softness. Finally, enantioseparation of EA1-EA3 was carried out on chiral stationary phases in three different chromatographic modes.

[212] B. Kaur, K. Denzinger, L. Zhang, N. García-Vázquez, G. Wolber, M. Gabr. Virtual Screening-Guided Discovery of Small-Molecule CHI3L1 Inhibitors with Functional Activity in Glioblastoma Spheroids, ACS Med. Chem. Lett., , 2025.
Links: [doi:10.1021/acsmedchemlett.5c00550] [Publisher][Download citation: BibTeX | RIS]

[211] H. Karami, S. Soltani, G. Wolber, S. Sadigh-Eteghad, R. Nikbakht, H. Farrokhi, F. Narimani, R. Teimuri-Mofrad, M. Rashidi. Anti-Alzheimer effects of the newly synthesized cationic compounds as multi-target dual hAChE/hBuChE inhibitor: An in silico, in vitro, and in vivo approach, Bioimpacts, 15(1):24196-24196, 2025.
Links: [doi:10.34172/bi.24196] [Publisher][Download citation: BibTeX | RIS]
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Abstract: <em><strong>Introduction:</strong></em> Multi-target anti-Alzheimer's disease (AD) compounds are promising leads for the development of AD modifying agents. Ionic compounds containing quaternary ammonium moiety were synthesized, and their multi-targeted anti-AD effects were examined. <em><strong> Methods:</strong></em> Imidazole derivatives containing a quaternary ammonium moiety were synthesized and evaluated for their potential anti-Alzheimer properties using computational (<em>in silico</em>), cellular (<em>in vitro</em>), and animal (<em>in vivo</em>) models. The inhibition kinetics of both human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBuChE) were assessed. Neuroprotective effects in amyloid-beta (Aβ)-exposed PC12 cells were also examined. Furthermore, the compounds' impact on Aβ-induced memory impairment in Wistar rats was evaluated, with a detailed analysis of the underlying mechanisms. <em><strong>Results: </strong></em>Compound 5g demonstrated acceptable cytotoxicity against human cells. This compound exhibited non-competitive dual inhibition of both hAChE and hBuChE. Additionally, compound 5g mitigated the morphological changes induced by amyloid-beta (Aβ) in PC12 cells and decreased cell mortality. It exhibited anti-oxidative stress properties, evident by reduction in reactive oxygen species (ROS) production, and inhibition of lipid peroxidation. The compound also down regulated the expression of pro-inflammatory genes IL-1β and TNF-α. In vitro studies validated compound 5g's ability to inhibit lactate dehydrogenase (LDH), attenuate neuroinflammation, and prevent the autophagy-apoptosis cascade. When administered to rats with Aβ-induced memory dysfunction, compound 5g enhanced cognitive function and improved spatial memory. In the hippocampi of treated rats, there was a noted downregulation of TNF-α and NF-kB. Furthermore, compound 5g counteracted the elevated activity of AChE. Molecular modeling validated the binding of compound 5g to both steric and catalytic sites of cholinesterase enzymes. <em><strong>Conclusion:</strong></em> The novel quaternary ammonium derivative, compound 5g, demonstrated multi-target anti-AD properties, as evidenced by i<em>n silico, in vitro </em>and <em>in vivo</em> studies. Behavioral assessments and molecular analyses further confirmed its therapeutic efficacy in amyloid-beta (Aβ)-challenged rats.

[210] M. G. Gündüz, C. Dengiz, K. Denzinger, S. Huang, J. T. Lee, J. W. Nafie, D. W. Armstrong, G. Wolber, G. W. Zamponi. Biginelli dihydropyrimidines and their acetylated derivatives as L-/T-type calcium channel blockers: Synthesis, enantioseparation, and molecular modeling studies, Arch. Pharm., 358(3):e2400584, 2025.
Links: [doi:10.1002/ardp.202400584] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Abstract Biginelli dihydropyrimidines (DHPMs) are considered superior over 1,4-dihydropyridines (DHPs) in terms of both light and metabolic stabilities. Nevertheless, DHPs dominate the market as the most prescribed calcium channel blockers with strong therapeutic potential in managing cardiovascular ailments. To overcome the restrictions that complicate the formulation and postadministration of DHPs, employing bioisosteric replacement by exchanging the DHP ring with DHPM appears as a logical approach for the improved formulations of new calcium channel blockers. In this study, we obtained DHPM derivatives via Biginelli synthesis and acetylated their N-3 position by heating them in acetic anhydride (GD1–GD12). We also incorporated the DHPM scaffold into a condensed ring system (GD13 and GD14). These DHPMs were evaluated for their ability to block both L- (Cav1.2) and T- (Cav3.2) type calcium channels. Compounds carrying acetyl moiety on the N-3 position of the DHPM scaffold appeared to be more effective inhibitors of both channels. Retesting GD4 enantiomers, separated using high-performance liquid chromatography (HPLC) on a chiral stationary phase, revealed that the (R)-isomer predominantly contributes to the outstanding inhibitory activity of GD4 on calcium channels. Molecular modeling studies, including docking, molecular dynamics simulations, and dynophore analysis, provided insights into the binding mechanism of DHPMs to Cav1.2 and Cav3.2, for the first time.

[209] D. Derwand, O. Zierau, C. A. Wolf, G. Wolber, A. M. Keiler. Effects of the dietary supplement 5α-hydroxy-laxogenin in the orchiectomized rat model, Drug Test. Anal., 17(9):1743-1749, 2025.
Links: [doi:10.1002/dta.3881] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Dietary supplements used by recreational and elite athletes for performance enhancement might contain undeclared, unlawfully added ingredients. One of those ingredients is 5α-hydroxy-laxogenin, which is sold in dietary supplements marketed as a natural compound with anabolic effects. It has been shown that 5α-hydroxy-laxogenin is not naturally occurring, but rather of synthetic origin. Previously, we observed that 5α-hydroxy-laxogenin can bind to and activate the androgen receptor in a cell-based bioassay. To investigate its androgenic potential in vivo, we treated orchiectomized rats with three different dosages of 5α-hydroxy-laxogenin for 2 weeks. Effects were neither observed on the wet weights of the androgen target tissues prostate, seminal vesicle or penis nor on the wet weights of the anabolic target tissue musculus levator ani or on skeletal hindlimb muscles. Au contraire, significantly higher atrophy was seen for some of the target tissues in the animals treated with the highest 5α-hydroxy-laxogenin dosage (36 mg/kg bw). While in silico docking supports the androgen receptor binding previously observed in vitro, we observed neither androgenic nor anabolic effects of 5α-hydroxy-laxogenin in vivo in castrated male rats.

[208] N. S. Benedik, V. Talagayev, T. Matziol, A. Dolsak, I. Sosic, G. Weindl, G. Wolber, M. Sova. Optimization of 6-(trifluoromethyl)pyrimidine derivatives as TLR8 antagonists, Acta. Pharm., 75(2):159-183, 2025.
Links: [doi:10.2478/acph-2025-0011] [Download citation: BibTeX | RIS]
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Abstract: Toll-like receptors (TLRs) are essential for the innate immune system as they recognize pathogen-associated molecular patterns and trigger immune responses. Overactivation of TLR8 by endogenous nucleic acids is associated with the development of autoimmune diseases and promotes inflammatory responses. This study presents the design, synthesis, and evaluation of a series of TLR8 antagonists based on the optimization of previously reported 6-(trifluoromethyl)pyrimidin-2-amines, with targeted modifications to further explore structure-activity relationships (SAR) and increase potency. A two-step synthesis involving nucleophilic aromatic substitution and Suzuki coupling was used to prepare two series of new compounds. The biological evaluation revealed that compounds 14 and 26 exhibited promising TLR8 antagonistic activity with IC (50) values of 6.5 and 8.7 mumol L(-) (1), respectively. Compound 14 showed reduced cell viability at higher concentrations, while compound 26 showed no cytotoxic effects, making it a promising candidate for further investigation.

[207] K. Puls, A. Olivé-Marti, S. Hongnak, D. Lamp, M. Spetea, G. Wolber. Discovery of Novel, Selective, and Nonbasic Agonists for the Kappa-Opioid Receptor Determined by Salvinorin A-Based Virtual Screening, J. Med. Chem., 67(16):13788-13801, 2024.
Links: [doi:10.1021/acs.jmedchem.4c00590] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Modulating the kappa-opioid receptor (KOR) is a promising strategy for treating various human diseases. KOR agonists show potential for treating pain, pruritus, and epilepsy, while KOR antagonists show potential for treating depression, anxiety, and addiction. The diterpenoid Salvinorin A (SalA), a secondary metabolite of Salvia divinorum, is a potent and selective KOR agonist. Unlike typical opioids, SalA lacks a basic nitrogen, which encouraged us to search for nonbasic KOR ligands. Through structure-based virtual screening using 3D pharmacophore models based on the binding mode of SalA, we identified novel, nonbasic, potent, and selective KOR agonists. In vitro studies confirmed two virtual hits, SalA-VS-07 and SalA-VS-08, as highly selective for the KOR and showing G protein-biased KOR agonist activity. Both KOR ligands share a novel spiro-moiety and a nonbasic scaffold. Our findings provide novel starting points for developing therapeutics aimed at treating pain and other conditions in which KOR is a central player.

[206] F. Li, [...], M. Breznik, [...], K. Denzinger, [...], S. Liu, [...], T. Noonan, L. Obendorf, S. Pach, A. Pandit, [...], K. Puls, [...], V. Talagayev, [...], G. Wolber, C. A. Wolf, [...], M. Schapira. CACHE Challenge #1: Targeting the WDR Domain of LRRK2, A Parkinson’s Disease Associated Protein, J. Chem. Inf. Model., 64(22):8521-8536, 2024.
Links: [doi:10.1021/acs.jcim.4c01267] [Publisher][Download citation: BibTeX | RIS]
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Abstract: The CACHE challenges are a series of prospective benchmarking exercises to evaluate progress in the field of computational hit-finding. Here we report the results of the inaugural CACHE challenge in which 23 computational teams each selected up to 100 commercially available compounds that they predicted would bind to the WDR domain of the Parkinson’s disease target LRRK2, a domain with no known ligand and only an apo structure in the PDB. The lack of known binding data and presumably low druggability of the target is a challenge to computational hit finding methods. Of the 1955 molecules predicted by participants in Round 1 of the challenge, 73 were found to bind to LRRK2 in an SPR assay with a KD lower than 150 μM. These 73 molecules were advanced to the Round 2 hit expansion phase, where computational teams each selected up to 50 analogs. Binding was observed in two orthogonal assays for seven chemically diverse series, with affinities ranging from 18 to 140 μM. The seven successful computational workflows varied in their screening strategies and techniques. Three used molecular dynamics to produce a conformational ensemble of the targeted site, three included a fragment docking step, three implemented a generative design strategy and five used one or more deep learning steps. CACHE #1 reflects a highly exploratory phase in computational drug design where participants adopted strikingly diverging screening strategies. Machine learning-accelerated methods achieved similar results to brute force (e.g., exhaustive) docking. First-in-class, experimentally confirmed compounds were rare and weakly potent, indicating that recent advances are not sufficient to effectively address challenging targets.

[205] E. Koçak Aslan, K. Lam, C. Dengiz, K. Denzinger, I. Y. Dicle Erdamar, S. Huang, G. W. Zamponi, G. Wolber, M. G. Gunduz. Synthesis, molecular modeling, DFT studies, and EPR analysis of 1,4-dihydropyridines as potential calcium channel blockers, J. Mol. Struct., 1307:137983, 2024.
Links: [doi:10.1016/j.molstruc.2024.137983] [Publisher][Download citation: BibTeX | RIS]
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Abstract: 1,4-Dihydropyridines (DHPs) are widely recognized as a highly effective class of L-type calcium channel blockers that offer significant therapeutic potential in managing cardiovascular conditions. Furthermore, their ability to target other types of calcium channels makes DHPs attractive candidates for therapeutic applications in neurological and psychiatric disorders. Close examination of the chemical structures of approved DHP-based antihypertensive drugs with a history of over forty years in the market reveals that the C-4 position is the least altered part of this privileged ring system. In the present study, we focused on this position and synthesized two novel compounds (DB1 and DB2) by carrying out chemical modifications on suitable positions of the main scaffold of DA1 (isobutyl 4-(benzo[d][1,3]dioxol-5-yl)-2,6,6-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate) that was previously identified as a DHP-based effective and selective inhibitor of T-type (Cav3.2) over L-type (Cav1.2) calcium channel. Based on whole-cell patch-clamp analysis on Cav1.2 and Cav3.2, DB1 with bromine group on the benzodioxole ring appeared to be a more effective and selective inhibitor of Cav3.2 compared to DB2 with nitro at the same locus. Molecular docking and molecular dynamics (MD) simulations were performed to investigate the binding mode of both DB1 isomers to Cav3.2. Furthermore, density functional theory (DFT) methods were employed to obtain information regarding the stability of the molecules by computing various parameters, such as electric dipole moment, band gap, electronegativity, and global chemical hardness-softness, related to their charge-transfer characteristics. According to DFT studies, DB1 also appeared to be chemically more stable than DB2. Finally, ionizing radiation-induced free radicals of gamma-irradiated DB1 and DB2 in powder form were examined utilizing the electron paramagnetic resonance (EPR) technique and the obtained data demonstrated that radiation sterilization is suitable for the dosage forms including DB1 and DB2.

[204] N. Fuchs, L. Calvo-Barreiro, V. Talagayev, S. Pach, G. Wolber, M. T. Gabr. From Virtual Screens to Cellular Target Engagement: New Small Molecule Ligands for the Immune Checkpoint LAG-3, ACS Med. Chem. Lett., 15(11):1884-1890, 2024.
Links: [doi:10.1021/acsmedchemlett.4c00350] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Herein, we performed a virtual screening study to discover new scaffolds for small molecule-based ligands of the immune checkpoint lymphocyte-activation gene 3 (LAG-3). Molecular dynamics (MD) simulations using the LAG-3 structure revealed two putative binding sites for small molecules: the antibody interface and the lipophilic canyon. A 3D pharmacophore screening resulted in the identification of potential ligands for these binding sites and afforded a library of 25 compounds. We then evaluated the screening hits for LAG-3 binding via microscale thermophoresis (MST) and surface plasmon resonance (SPR). Our biophysical screening identified two binders with KD values in the low micromolar range, compounds 3 (antibody interface) and 25 (lipophilic canyon). Furthermore, we investigated the ability of LAG-3 hits to engage LAG-3 on a cellular level using a cellular thermal shift assay (CETSA). In summary, compound 3 shows potential as a lead but is not yet a development candidate.

[203] K. E. Elagib, S. Liu, V. Burguener, R. Sahu, D. Kotay, G. Wolber, C. Watts, A. Goldfarb. Molecular Delineation and Targeting of a Megakaryocyte Ontogenic Switch for Therapeutic Applications, Blood, 144:1172, 2024.
Links: [doi:https://doi.org/10.1182/blood-2024-203846] [Publisher][Download citation: BibTeX | RIS]
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Abstract: In contrast to adult-type megakaryocytes (Mk), fetal-type Mk display increased progenitor proliferation, decreased morphogenesis, and decreased platelet release. Mk morphogenesis comprises a coordinated sequence of events that primes the cells for intravascular platelet release: endomitosis, polyploidization, cellular enlargement, and polarized process extension. Impairment in this program occurs most prominently during fetal development but persists in normal infants for up to a year and strongly manifests in iPSC-derived megakaryocytes. These ontogenically controlled features ensure functional adaptation to host needs. Thus, overall development of the fetus is best supported by rapid Mk expansion and restrained production of platelets. However, fetal-type megakaryopoiesis lacks the capacity for stress thrombopoiesis, leading to clinical problems in situations requiring rapidly augmented platelet production, and is also uniquely susceptible to neoplastic transformation. Pharmacologic interventions to induce adult-type megakaryopoiesis, i.e. ontogenic switching, do not exist but would greatly benefit the following clinical problems: 1) neonatal thrombocytopenia, 2) platelet recovery post umbilical cord blood (UCB) transplant, 3) Down syndrome-associated megakaryocyte (Mk) neoplasms, and 4) large scale ex vivo platelet production. Our lab over the past 15 years has characterized several of the molecular determinants of Mk ontogeny. We recently found that Dyrk kinase signaling, by repressing Mkl1 co-activator function, maintains the fetal program in human neonatal Mk (Elagib et al. J. Clin. Invest., 2022). While Dyrk inhibition induced ontogenic switching in vitro, in vivo application was precluded by broad toxicity. In pursuing a downstream target of Dyrk signaling, we have discovered a novel ontogenic control factor, Kifc3, a minus-end-directed kinesin involved in delivery of proteins to the centrosome. Therapeutically attractive features of this protein include a unique, structurally characterized ATP-binding pocket and absence of abnormalities in knockout mice. Our functional analysis of Kifc3 in human progenitors has revealed: 1) 2-fold lower levels of protein in adult vs neonatal cells (P < 0.005); 2) induction of adult morphogenesis in neonatal cells subjected to knockdown (kd), with 2.5-fold enhanced polyploidization (P < 0.005); 3) 3-fold enhancement in functional platelet production by neonatal cells subjected to kd (P < 0.005). In silico modelling using motor domain structures of Kifc3 and of Kifc1 bound to an inhibitor has enabled identification of candidate Kifc3 small molecule inhibitors that elicit the same phenotype as Kifc3 kd, including 1.7-fold enhancement in platelet production by neonatal Mk (P < 0.05). Our recent studies have also identified a key target of Kifc3 in megakaryocyte ontogeny, Cep192. Cep192 is known to be a scaffold for aurora (Aurk) and polo-like (Plk) kinases, recruiting them to centrosomes to coordinate mitotic maturation. A recent publication shows that Cep192 also recruits these kinases to cortical actin to promote cytoskeletal remodeling and process extension (Luo et al., Nat. Commun., 2022). Elevated levels of Cep192 are known to sequester these kinases away from mitotic scaffolds that coordinate cytokinesis. Comparison of neonatal and adult Mk by immunofluorescence showed marked differences in Cep192: focal-centriolar in > 80% neonatal Mk and broadly dispersed in 60% adult Mk (P < 0.01); by immunoblot, adult Mk expressed 3-fold higher levels (P < 0.01). Knocking down or inhibiting Kifc3 in neonatal Mk caused Cep192 to increase 2-fold in amount (P < 0.01)) and disperse from centrosomes in 60% of cells (P < 0.05)). Knocking down Cep192 in adult Mk blocked morphogenesis (2-fold decreased polyploidization, P < 0.005), and a CRISPR hypomorph of Cep192 in mice caused a 17% decrease in platelet counts (P = 0.015) with no effect on red cells or white cells. Overall, our findings provide a novel, therapeutically relevant, mechanistic pathway in which Kifc3 levels dictate Mk ontogeny via Cep192. We propose a model in which Kifc3-Cep192 signaling acts on cytoskeletal kinases (Aurk and Plk) to coordinate loss of cytokinesis with process extension. Clinically, these findings provide proof of principle for therapeutic induction of Mk ontogenic switching to enable stress thrombopoiesis in settings of infantile hematopoiesis.

[202] U. B. A. Aziz, A. Saoud, M. Bermudez, M. Mieth, A. Atef, T. Rudolf, C. Arkona, T. Trenkner, C. Böttcher, K. Ludwig, A. Hoelzemer, A. C. Hocke, G. Wolber, J. Rademann. Targeted small molecule inhibitors blocking the cytolytic effects of pneumolysin and homologous toxins, Nat. Commun., 15(1):3537, 2024.
Links: [doi:10.1038/s41467-024-47741-3] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.

[201] R. A. Ashraf, S. Liu, C. A. Wolf, G. Wolber, M. Bureik. Identification of new substrates and inhibitors of human CYP2A7, Molecules, 29(10):2191, 2024.
Links: [doi:10.3390/molecules29102191] [Download citation: BibTeX | RIS]
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Abstract: CYP2A7 is one of the most understudied human cytochrome P450 enzymes and its contributions to either drug metabolism or endogenous biosynthesis pathways are not understood, as its only known enzymatic activities are the conversions of two proluciferin probe substrates. In addition, the CYP2A7 gene contains four single-nucleotide polymorphisms (SNPs) that cause missense mutations and have minor allele frequencies (MAFs) above 0.5. This means that the resulting amino acid changes occur in the majority of humans. In a previous study, we employed the reference standard sequence (called CYP2A7*1 in P450 nomenclature). For the present study, we created another CYP2A7 sequence that contains all four amino acid changes (Cys311, Glu169, Gly479, and Arg274) and labeled it CYP2A7-WT. Thus, it was the aim of this study to identify new substrates and inhibitors of CYP2A7 and to compare the properties of CYP2A7-WT with CYP2A7*1. We found several new proluciferin probe substrates for both enzyme variants (we also performed in silico studies to understand the activity difference between CYP2A7-WT and CYP2A7*1 on specific substrates), and we show that while they do not act on the standard CYP2A6 substrates nicotine, coumarin, or 7-ethoxycoumarin, both can hydroxylate diclofenac (as can CYP2A6). Moreover, we found ketoconazole, 1-benzylimidazole, and letrozole to be CYP2A7 inhibitors.

[200] P. P. Adhikary, T. Idowu, Z. Tan, C. Hoang, S. Shanta, M. Dumbani, L. Mappalakayil, B. Awasthi, M. Bermudez, J. Weiner, D. Beule, G. Wolber, B. D. G. Page, S. Hedtrich. Disrupting TSLP–TSLP receptor interactions via putative small molecule inhibitors yields a novel and efficient treatment option for atopic diseases, EMBO Mol. Med., :1-27-27, 2024.
Links: [doi:10.1038/s44321-024-00085-3] [Publisher][Download citation: BibTeX | RIS]
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Abstract: AbstractThymic stromal lymphopoietin (TSLP) is a key player in atopic diseases, which has sparked great interest in therapeutically targeting TSLP. Yet, no small-molecule TSLP inhibitors exist due to the challenges of disrupting the protein?protein interaction between TSLP and its receptor. Here, we report the development of small-molecule TSLP receptor inhibitors using virtual screening and docking of >1,000,000 compounds followed by iterative chemical synthesis. BP79 emerged as our lead compound that effectively abrogates TSLP-triggered cytokines at low micromolar concentrations. For in-depth analysis, we developed a human atopic disease drug discovery platform using multi-organ chips. Here, topical application of BP79 onto atopic skin models that were co-cultivated with lung models and Th2 cells effectively suppressed immune cell infiltration and IL-13, IL-4, TSLP, and periostin secretion, while upregulating skin barrier proteins. RNA-Seq analysis corroborate these findings and indicate protective downstream effects on the lungs. To the best of our knowledge, this represents the first report of a potent putative small molecule TSLPR inhibitor which has the potential to expand the therapeutic and preventive options in atopic diseases.

[199] Y. Jia, B. Schroeder, Y. Pfeifer, C. Fröhlich, L. Deng, C. Arkona, B. Kuropka, J. Sticht, K. Ataka, S. Bergemann, G. Wolber, C. Nitsche, M. Mielke, H. S. Leiros, G. Werner, J. Rademann. Kinetics, Thermodynamics, and Structural Effects of Quinoline-2-Carboxylates, Zinc-Binding Inhibitors of New Delhi Metallo-β-lactamase-1 Re-sensitizing Multidrug-Resistant Bacteria for Carbapenems, J. Med. Chem., 66(17):11761-11791, 2023.
Links: [doi:10.1021/acs.jmedchem.3c00171] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Carbapenem resistance mediated by metallo-β-lactamases (MBL) such as New Delhi metallo-β-lactamase-1 (NDM-1) has become a major factor threatening the efficacy of essential β-lactam antibiotics. Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy- and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors of NDM-1 with highly improved inhibitory activity and binding affinity. The most active compounds formed reversibly inactive ternary protein-inhibitor complexes with two zinc ions as proven by native protein mass spectrometry and bio-layer interferometry. Modification of the NDM-1 structure with remarkable entropic gain was shown by isothermal titration calorimetry and NMR spectroscopy of isotopically labeled protein. The best compounds were potent inhibitors of NDM-1 and other representative MBL with no or little inhibition of human zinc-binding enzymes. These inhibitors significantly reduced the minimum inhibitory concentrations (MIC) of meropenem for multidrug-resistant bacteria recombinantly expressing blaNDM-1 as well as for several multidrug-resistant clinical strains at concentrations non-toxic to human cells.

[198] S. A. Abdel-Rahman, V. Talagayev, S. Pach, G. Wolber, M. T. Gabr. Discovery of Small-Molecule TIM-3 Inhibitors for Acute Myeloid Leukemia Using Pharmacophore-Based Virtual Screening, J. Med. Chem., 66(16):11464-11475, 2023.
Links: [doi:10.1021/acs.jmedchem.3c00960] [Publisher][Download citation: BibTeX | RIS]
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Abstract: T-cell immunoglobulin and mucin domain 3 (TIM-3) is a negative immune checkpoint that represents a promising target for cancer immunotherapy. Although encouraging results have been observed for TIM-3 inhibition in the context of acute myeloid leukemia (AML), targeting TIM-3 is currently restricted to monoclonal antibodies (mAbs). To fill this gap, we implemented a pharmacophore-based screening approach to identify small-molecule TIM-3 inhibitors. Our approach resulted in the identification of hit compounds with TIM-3 binding affinity. Subsequently, we used the structure–activity relationship (SAR) by a catalog approach to identify compound A-41 with submicromolar TIM-3 binding affinity. Remarkably, A-41 demonstrated the ability to block TIM-3 interactions with key ligands and inhibited the immunosuppressive function of TIM-3 using an in vitro coculture assay. This work will pave the way for future drug discovery efforts aiming at the development of small-molecule inhibitors TIM-3 for AML.

[197] F. Ricci, K. Schira, L. Khettabi, L. Lombardo, S. Mirabile, R. Gitto, M. Soler-Lopez, J. Scheuermann, G. Wolber, L. De Luca. Computational methods to analyze and predict the binding mode of inhibitors targeting both human and mushroom tyrosinase, Eur. J. Med. Chem., 260:115771, 2023.
Links: [doi:10.1016/j.ejmech.2023.115771] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Tyrosinase, a copper-containing enzyme critical in melanin biosynthesis, is a key drug target for hyperpigmentation and melanoma in humans. Testing the inhibitory effects of compounds using tyrosinase from Agaricus bisporus (AbTYR) has been a common practice to identify potential therapeutics from synthetic and natural sources. However, structural diversity among human tyrosinase (hTYR) and AbTYR presents a challenge in developing drugs that are therapeutically effective. In this study, we combined retrospective and computational analyses with experimental data to provide insights into the development of new inhibitors targeting both hTYR and AbTYR. We observed contrasting effects of Thiamidol™ and our 4-(4-hydroxyphenyl)piperazin-1-yl-derivative (6) on both enzymes; based on this finding, we aimed to investigate their binding modes in hTYR and AbTYR to identify residues that significantly improve affinity. All the information led to the discovery of compound [4-(4-hydroxyphenyl)piperazin-1-yl](2-methoxyphenyl)methanone (MehT-3, 7), which showed comparable activity on AbTYR (IC50 = 3.52 μM) and hTYR (IC50 = 5.4 μM). Based on these achievements we propose the exploitation of our computational results to provide relevant structural information for the development of newer dual-targeting molecules, which could be preliminarily tested on AbTYR as a rapid and inexpensive screening procedure before being tested on hTYR.

[196] R. Maccari, G. Wolber, M. Genovese, G. Sardelli, V. Talagayev, F. Balestri, S. Luti, A. Santi, R. Moschini, A. Del Corso, P. Paoli, R. Ottana. Designed multiple ligands for the treatment of type 2 diabetes mellitus and its complications: Discovery of (5-arylidene-4-oxo-2-thioxothiazolidin-3-yl)alkanoic acids active as novel dual-targeted PTP1B/AKR1B1 inhibitors, Eur. J. Med. Chem., 252:115270, 2023.
Links: [doi:10.1016/j.ejmech.2023.115270] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Type 2 diabetes mellitus (T2DM) is a serious chronic disease with an alarmingly growing worldwide prevalence. Current treatment of T2DM mainly relies on drug combinations in order to control blood glucose levels and consequently prevent the onset of hyperglycaemia-related complications. The development of multiple-targeted drugs recently emerged as an attractive alternative to drug combinations for the treatment of complex diseases with multifactorial pathogenesis, such as T2DM. Protein tyrosine phosphatase 1B (PTP1B) and aldose reductase (AKR1B1) are two enzymes crucially involved in the development of T2DM and its chronic complications and, therefore, dual inhibitors targeted to both these enzymes could provide novel agents for the treatment of this complex pathological condition. In continuing our search for dual-targeted PTP1B/AKR1B1 inhibitors, we designed new (5-arylidene-4-oxo-2-thioxothiazolidin-3-yl)alkanoic acids. Among them, 3-(4-phenylbutoxy)benzylidene derivatives 6f and 7f, endowed with interesting inhibitory activity against both targets, proved to control specific cellular pathways implicated in the development of T2DM and related complications.

[195] F. Wunsch, T. N. Nguyen, G. Wolber, M. Bermudez. Structural determinants of sphingosine-1-phosphate receptor selectivity, Arch. Pharm., :e2300387, 2023.
Links: [doi:10.1002/ardp.202300387] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Fingolimod, the prodrug of fingolimod-1-phosphate (F1P), was the first sphingosine-1-phosphate receptor (S1PR) modulator approved for multiple sclerosis. F1P unselectively targets all five S1PR subtypes. While agonism (functional antagonism via receptor internalization) at S1PR(1) leads to the desired immune modulatory effects, agonism at S1PR(3) is associated with cardiac adverse effects. This motivated the development of S1PR(3) -sparing compounds and led to a second generation of S1PR(1,5) -selective ligands like siponimod and ozanimod. Our method combines molecular dynamics simulations and three-dimensional pharmacophores (dynophores) and enables the elucidation of S1PR subtype-specific binding site characteristics, visualizing also subtle differences in receptor-ligand interactions. F1P and the endogenous ligand sphingosine-1-phosphate bind to the orthosteric pocket of all S1PRs, but show different binding mode dynamics, uncovering potential starting points for the development of subtype-specific ligands. Our study contributes to the mechanistic understanding of the selectivity profile of approved drugs like ozanimod and siponimod and pharmaceutical tool compounds like CYM5541.

[194] R. Wamser, S. Pach, C. Arkona, M. Baumgardt, U. B. A. Aziz, A. C. Hocke, G. Wolber, J. Rademann. A Critical Study on Acylating and Covalent Reversible Fragment Inhibitors of SARS-CoV-2 Main Protease Targeting the S1 Site with Pyridine, ChemMedChem, 18:e202200635, 2023.
Links: [doi:10.1002/cmdc.202200635] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Abstract SARS coronavirus main proteases (3CL proteases) have been validated as pharmacological targets for the treatment of coronavirus infections. Current inhibitors of SARS main protease, including the clinically admitted drug nirmatrelvir are peptidomimetics with the downsides of this class of drugs including limited oral bioavailability, cellular permeability, and rapid metabolic degradation. Here, we investigate covalent fragment inhibitors of SARS Mpro as potential alternatives to peptidomimetic inhibitors in use today. Starting from inhibitors acylating the enzyme's active site, a set of reactive fragments was synthesized, and the inhibitory potency was correlated with the chemical stability of the inhibitors and the kinetic stability of the covalent enzyme-inhibitor complex. We found that all tested acylating carboxylates, several of them published prominently, were hydrolyzed in assay buffer and the inhibitory acyl-enzyme complexes were rapidly degraded leading to the irreversible inactivation of these drugs. Acylating carbonates were found to be more stable than acylating carboxylates, however, were inactive in infected cells. Finally, reversibly covalent fragments were investigated as chemically stable SARS CoV-2 inhibitors. Best was a pyridine-aldehyde fragment with an IC50 of 1.8??M at a molecular weight of 211?g/mol, showing that pyridine fragments indeed are able to block the active site of SARS-CoV-2 main protease.

[193] Y. Shi, C. A. Wolf, R. Lotfy, S. S. Sharma, A. F. Tesfa, G. Wolber, M. Bureik, B. R. Clark. Deciphering the biotransformation mechanism of dialkylresorcinols by CYP4F11, Bioorg. Chem., 131:106330, 2023.
Links: [doi:10.1016/j.bioorg.2022.106330] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Cytochrome P450 enzymes (CYPs) are one of the most important classes of oxidative enzymes in the human body, carrying out metabolism of various exogenous and endogenous substrates. In order to expand the knowledge of these enzymes’ specificity and to obtain new natural product derivatives, CYP4F11, a cytochrome P450 monooxygenase, was used in the biotransformation of dialkylresorcinols 1 and 2, a pair of antibiotic microbial natural products. This investigation resulted in four biotransformation products including two oxidative products: a hydroxylated derivative (3) and a carboxylic acid derivative (4). In addition, acetylated (5) and esterified products (6) were isolated, formed by further metabolism by endogenous yeast enzymes. Oxidative transformations were highly regioselective, and took place exclusively at the ω-position of the C-5 alkyl chain. Homology modeling studies revealed that optimal hydrogen bonding between 2 and the enzyme can only be established with the C-5 alkyl chain pointing towards the heme. The closely-related CYP4F12 was not capable of oxidizing the dialkylresorcinol 2. Modeling experiments rationalize these differences by the different shapes of the binding pockets with respect to the non-oxidized alkyl chain. Antimicrobial testing indicated that the presence of polar groups on the side-chains reduces the antibiotic activity of the dialkylresorcinols.

[192] Y. Shi, J. Li, C. A. Wolf, S. Liu, S. S. Sharma, G. Wolber, M. Bureik, B. R. Clark. Expected and Unexpected Products from the Biochemical Oxidation of Bacterial Alkylquinolones with CYP4F11, J. Nat. Prod., , 2023.
Links: [doi:10.1021/acs.jnatprod.3c00689] [Publisher][Download citation: BibTeX | RIS]
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Abstract: 2-Alkylquinolones are a class of microbial natural products primarily produced in the Pseudomonas and Burkholderia genera that play a key role in modulating quorum sensing. Bacterial alkylquinolones were synthesized and then subjected to oxidative biotransformation using human cytochrome P450 enzyme CYP4F11, heterologously expressed in the fission yeast Schizosaccharomyces pombe. This yielded a range of hydroxylated and carboxylic acid derivatives which had undergone ω-oxidation of the 2-alkyl chain, the structures of which were determined by analysis of NMR and MS data. Oxidation efficiency depended on chain length, with a chain length of eight or nine carbon atoms proving optimal for high yields. Homology modeling suggested that Glu233 was relevant for binding, due to the formation of a hydrogen bond from the quinolone nitrogen to Glu233, and in this position only the longer alkyl chains could come close enough to the heme moiety for effective oxidation. In addition to the direct oxidation products, a number of esters were also isolated, which was attributed to the action of endogenous yeast enzymes on the newly formed ω-hydroxy-alkylquinolones. ω-Oxidation of the alkyl chain significantly reduced the antimicrobial and antibiofilm activity of the quinolones.

[191] K. Puls, G. Wolber. Solving an Old Puzzle: Elucidation and Evaluation of the Binding Mode of Salvinorin A at the Kappa Opioid Receptor, Molecules, 28(2):718, 2023.
Links: [doi:10.3390/molecules28020718] [Download citation: BibTeX | RIS]
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Abstract: The natural product Salvinorin A (SalA) was the first nitrogen-lacking agonist discovered for the opioid receptors and exhibits high selectivity for the kappa opioid receptor (KOR) turning SalA into a promising analgesic to overcome the current opioid crisis. Since SalA&rsquo;s suffers from poor pharmacokinetic properties, particularly the absence of gastrointestinal bioavailability, fast metabolic inactivation, and subsequent short duration of action, the rational design of new tailored analogs with improved clinical usability is highly desired. Despite being known for decades, the binding mode of SalA within the KOR remains elusive as several conflicting binding modes of SalA were proposed hindering the rational design of new analgesics. In this study, we rationally determined the binding mode of SalA to the active state KOR by in silico experiments (docking, molecular dynamics simulations, dynophores) in the context of all available mutagenesis studies and structure-activity relationship (SAR) data. To the best of our knowledge, this is the first comprehensive evaluation of SalA&rsquo;s binding mode since the determination of the active state KOR crystal structure. SalA binds above the morphinan binding site with its furan pointing toward the intracellular core while the C2-acetoxy group is oriented toward the extracellular loop 2 (ECL2). SalA is solely stabilized within the binding pocket by hydrogen bonds (C210ECL2, Y3127.35, Y3137.36) and hydrophobic contacts (V1182.63, I1393.33, I2946.55, I3167.39). With the disruption of this interaction pattern or the establishment of additional interactions within the binding site, we were able to rationalize the experimental data for selected analogs. We surmise the C2-substituent interactions as important for SalA and its analogs to be experimentally active, albeit with moderate frequency within MD simulations of SalA. We further identified the non-conserved residues 2.63, 7.35, and 7.36 responsible for the KOR subtype selectivity of SalA. We are confident that the elucidation of the SalA binding mode will promote the understanding of KOR activation and facilitate the development of novel analgesics that are urgently needed.

[190] V. Kremling, B. Loll, S. Pach, I. Dahmani, C. Weise, G. Wolber, S. Chiantia, M. C. Wahl, N. Osterrieder, W. Azab. Crystal structures of glycoprotein D of equine alphaherpesviruses reveal potential binding sites to the entry receptor MHC-I, Front. Microbiol., 14:1197120, 2023.
Links: [doi:10.3389/fmicb.2023.1197120] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Cell entry of most alphaherpesviruses is mediated by the binding of glycoprotein D (gD) to different cell surface receptors. Equine herpesvirus type 1 (EHV-1) and EHV-4 gDs interact with equine major histocompatibility complex I (MHC-I) to initiate entry into equine cells. We have characterized the gD-MHC-I interaction by solving the crystal structures of EHV-1 and EHV-4 gDs (gD1, gD4), performing protein-protein docking simulations, surface plasmon resonance (SPR) analysis, and biological assays. The structures of gD1 and gD4 revealed the existence of a common V-set immunoglobulin-like (IgV-like) core comparable to those of other gD homologs. Molecular modeling yielded plausible binding hypotheses and identified key residues (F213 and D261) that are important for virus binding. Altering the key residues resulted in impaired virus growth in cells, which highlights the important role of these residues in the gD-MHC-I interaction. Taken together, our results add to our understanding of the initial herpesvirus-cell interactions and will contribute to the targeted design of antiviral drugs and vaccine development.

[189] L. C. Harps, A. L. Jendretzki, C. A. Wolf, U. Girreser, G. Wolber, M. K. Parr. Development of an HPLC-MS/MS Method for Chiral Separation and Quantitation of (R)- and (S)-Salbutamol and Their Sulfoconjugated Metabolites in Urine to Investigate Stereoselective Sulfonation, Molecules, 28(20), 2023.
Links: [doi:10.3390/molecules28207206] [Download citation: BibTeX | RIS]
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Abstract: The aim of this study was to develop and optimize a chiral HPLC-MS/MS method for quantitative analysis of (R)-/(S)-salbutamol and (R)-/(S)-salbutamol-4&prime;-O-sulfate in human urine to allow for bioanalytical quantitation of the targeted analytes and investigations of stereoselectivity in the sulfonation pathway of human phase &#8545; metabolism. For analytical method development, a systematic screening of columns and mobile phases to develop a separation via enantiomerically selective high performance liquid chromatography was performed. Electrospray ionization settings were optimized via multiple-step screening and a full factorial design-of-experiment. Both approaches were performed matrix-assisted and the predicted values were compared. The full factorial design was superior in terms of prediction power and knowledge generation. Performing a longitudinal excretion study in one healthy volunteer allowed for the calculation of excretion rates for all four targeted analytes. For this proof-of-concept, either racemic salbutamol or enantiopure levosalbutamol was administered perorally or via inhalation, respectively. A strong preference for sulfonation of (R)-salbutamol for inhalation and peroral application was found in in vivo experiments. In previous studies phenol sulfotransferase 1A3 was described to be mainly responsible for salbutamol sulfonation in humans. Thus, in vitro and in silico investigations of the stereoselectivity of sulfotransferase 1A3 complemented the study and confirmed these findings.

[188] L. Calvo-Barreiro, V. Talagayev, S. Pach, S. A. Abdel-Rahman, G. Wolber, M. T. Gabr. Discovery of ICOS-Targeted Small Molecules Using Pharmacophore-Based Screening, ChemMedChem, n/a(n/a):e202300305, 2023.
Links: [doi:https://doi.org/10.1002/cmdc.202300305] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Abstract There are currently no small molecules clinically approved as immune checkpoint modulators. Besides possessing oral bioavailability, cell-penetrating capabilities and enhanced tumor penetration compared to monoclonal antibodies (mAbs), small molecules are amenable to pharmacokinetic optimization, which allows adopting flexible dosage regimens that may avoid immune-related adverse events associated with mAbs. The interaction of inducible co-stimulator (ICOS) with its ligand (ICOS-L) plays key roles in T-cell differentiation and activation of T-cell to B-cell functions. This study represents the development and validation of a virtual screening strategy to identify small molecules that bind a novel druggable binding pocket in human ICOS. We used a lipophilic canyon in the apo-structure of ICOS and the ICOS/ICOS-L interface individually as templates for molecular dynamics simulation to generate 3D pharmacophores subsequently used for virtual screening campaigns. Our strategy was successful finding a first-in-class small molecule ICOS binder (5P, KD value=108.08±26.76??M) and validating biophysical screening platforms for ICOS-targeted small molecules. We anticipate that future structural optimization of 5P will result in the discovery of high affinity chemical ligands for ICOS.

[187] D. Akman, K. Denzinger, S. Huang, J. T. Lee, J. W. Nafie, G. Wolber, G. W. Zamponi, D. W. Armstrong, M. G. Gunduz. Focusing on C-4 position of Hantzsch 1,4-dihydropyridines: Molecular modifications, enantioseparation, and binding mechanism to L- and T-type calcium channels, Eur. J. Med. Chem., 244:114787, 2022.
Links: [doi:10.1016/j.ejmech.2022.114787] [Publisher][Download citation: BibTeX | RIS]
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Abstract: 1,4-Dihydropyridines (DHPs) represent the blockbuster class of L-type calcium channel blockers that have tremendous therapeutic value against cardiovascular conditions. Due to their abilities to additionally target other subtypes of calcium channels, DHPs are also considered promising molecules for the treatment of neurological and psychiatric disorders. Having been in the market for more than forty years, DHP is one of the most modified scaffolds for the development of novel molecules acting on calcium channels. Taking the chemical structures of approved DHPs into account, it is noteworthy that C-4 position is the least modified part of the ring system. Therefore, in the present study, we focused on this location and carried out various molecular modifications to obtain twelve potential calcium channel blockers with a DHP-based hexahydroquinoline scaffold (DA1-DA12). The whole-cell patch clamp technique applied to analyze the blocking ability of the synthesized compounds on both L- (Cav1.2) and T- (Cav3.2) type calcium channels revealed five blockers with different selectivity profiles. Introducing naphthyl moiety onto the C-4 position of the main scaffold led to the identification of a selective blocker of Cav1.2 (DA8). The benzodioxole-substituted derivative (DA1) was the most potent and selective Cav3.2 inhibitor, therefore, its enantiomers were separated using HPLC on a chiral stationary phase. Retesting single isomers on Cav3.2 revealed that S-enantiomer was mainly responsible for the block. Finally, DA compounds were docked into two generated homology models of L- and T-type calcium channels. Molecular dynamics (MD) simulations and 3D pharmacophore modeling provided further insights into the detailed binding mechanism of DHPs to Cav1.2 as well as to Cav3.2.

[186] J. Zhao, X. Zhang, Y. Wang, H. Huang, S. Sharma, S. S. Sharma, C. A. Wolf, S. Liu, G. Wolber, E. J. Sorensen, M. Bureik. Exploring the chemical space of Proluciferins as probe substrates for human Cytochrome P450 enzymes, Appl. Biochem. Biotechnol., 195:1042-1058, 2022.
Links: [doi:10.1007/s12010-022-04184-0] [Publisher][Download citation: BibTeX | RIS]
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Abstract: We report the synthesis of 21 new proluciferin compounds that bear a small aliphatic ether group connected to the 6’ hydroxy function of firefly luciferin and either contain an acid or methyl ester function at the dihydrothiazole ring. Each of these compounds was found to be a substrate for some members of the human CYP1 and CYP3 families; a total of 92 new enzyme–substrate pairs were identified. In a screen of the whole human P450 complement (CYPome) with three selected proluciferin acid substrates, another 13 enzyme–substrate pairs were detected, which involve enzymes belonging to the CYP2, CYP4, CYP7, CYP21, and CYP27 families. All in all, we identified new probe substrates for members of seven out of 18 human CYP families.

[185] J. Zhao, D. Machalz, S. Liu, C. A. Wolf, G. Wolber, M. K. Parr, M. Bureik. Metabolism of the antipsychotic drug olanzapine by CYP3A43, Xenobiotica, :1-29, 2022.
Links: [doi:10.1080/00498254.2022.2078751] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Abstract1. Olanzapine is an atypical antipsychotic primarily used to treat schizophrenia and bipolar disorder. An intronic single nucleotide polymorphism (SNP) that highly significantly predicts increased olanzapine clearance (rs472660) was previously identified in the CYP3A43 gene, which encodes a cytochrome P450 enzyme. But until now there was no experimental evidence for the metabolism of olanzapine by the CYP3A43 enzyme.2. In the present study we provide this evidence, together with a thorough analysis of olanzapine metabolism by all human CYP3A enzymes. We also rationalize our findings by molecular docking experiments. Moreover, we describe the activities of several CYP3A43 mutants and present the first enzymatic activity data for the CYP3A43.3 variant; with respect to prostate cancer, this polymorphic variant is associated with both increased risk and increased mortality. The catalytic properties of the wild type enzyme and the tumor mutant were analyzed by molecular dynamics simulations, which fit very well with the observed experimental results.3. Our finding suggests that the SNP rs472660 likely causes an increased CYP3A43 expression level and demonstrate that, depending on the substrate under study, the tumor mutant CYP3A43.3 can have increased activity in comparison to the wild type enzyme CYP3A43.1.

[184] J. Zhao, S. Liu, C. A. Wolf, G. Wolber, M. K. Parr, M. Bureik. Changes in Alprazolam metabolism by CYP3A43 mutants, Biomedicines, 10(12):3022, 2022.
Links: [doi:10.3390/biomedicines10123022] [Publisher][Download citation: BibTeX | RIS]

[183] F. Yang, S. Liu, G. Wolber, M. Bureik, M. K. Parr. Complete reaction phenotyping of Propranolol and 4-Hydroxypropranolol with the 19 enzymes of the human UGT1 and UGT2 families, Int. J. Mol. Sci., 23(13):7476, 2022.
Links: [doi:10.3390/ijms23137476] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Propranolol is a competitive non-selective beta-receptor antagonist that is available on the market as a racemic mixture. In the present study, glucuronidation of propranolol and its equipotent phase I metabolite 4-hydroxypropranolol by all 19 members of the human UGT1 and UGT2 families was monitored. UGT1A7, UGT1A9, UGT1A10 and UGT2A1 were found to glucuronidate propranolol, with UGT1A7, UGT1A9 and UGT2A1 mainly acting on (S)-propranolol, while UGT1A10 displays the opposite stereoselectivity. UGT1A7, UGT1A9 and UGT2A1 were also found to glucuronidate 4-hydroxypropranolol. In contrast to propranolol, 4-hydroxypropranolol was found to be glucuronidated by UGT1A8 but not by UGT1A10. Additional biotransformations with 4-methoxypropanolol demonstrated different regioselectivities of these UGTs with respect to the aliphatic and aromatic hydroxy groups of the substrate. Modeling and molecular docking studies were performed to explain the stereoselective glucuronidation of the substrates under study.

[182] D. Thieme, P. Anielski, S. Rzeppa, C. A. Wolf, G. Wolber, A. M. Keiler. Detection of 18-methyl steroids: Case report on a forensic urine sample and corresponding dietary supplements, Drug Test. Anal., 14(11-12):1864-1870, 2022.
Links: [doi:10.1002/dta.3389] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Abstract The detection of a putative 18-methyl-19-nortestosterone metabolite in a forensic bodybuilder's urine sample collected as part of a criminal proceeding has triggered a follow-up investigation. Four different dietary supplements in the possession of the suspect were examined with regard to possible precursor steroids. This led to the detection of the declared ingredient methoxydienone, which was confirmed by both, GC–MSMS and LC-HRMSMS. As neither 18-methyl-testosterone, nor 18-methyl-19-nortestosterone were detectable in the supplements, the possibility that the metabolite originates from methoxydienone was investigated. For this purpose, the metabolic fate of methoxydienone was studied in vitro using human HepG2 cells and in vivo by a single oral administration. While the 18-methyl-19-nortestosterone metabolite was not generated by HepG2 cells incubated with methoxydienone, it was observed in the urine samples collected at 2, 6, 10 and 24 h after methoxydienone administration. Moreover, the potential binding of methoxydienone as ligand to the human androgen receptor was modelled in silico in comparison with 18-methylnandrolone, for which androgen receptor activation had been shown in an in vitro approach before. In conclusion, we could ascribe the presence of the 18-methyl-19-nortestosterone metabolite in a forensic urine sample to originate from methoxydienone present in dietary supplements. Methoxydienone was observed to slowly degrade by demethylation of the methoxy substituent in liquid solutions. While no compound-specific intermediates were identified that allowed differentiation from other 18-methyl steroids, the 18-methyl-19-nortestosterone metabolite proved to be a suitable marker for reliable detection in doping analysis.

[181] D. Stepanov, D. Buchmann, N. Schultze, G. Wolber, K. Schaufler, S. Guenther, V. Belik. A combined bayesian and similarity-based approach for predicting E. coli biofilm inhibition by phenolic natural compounds, J. Nat. Prod., , 2022.
Links: [doi:10.1021/acs.jnatprod.2c00005] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Screening for biofilm inhibition by purified natural compounds is difficult due to compounds’ chemical diversity and limited commercial availability, combined with time- and cost-intensiveness of the laboratory process. In silico prediction of chemical and biological properties of molecules is a widely used technique when experimental data availability is of concern. At the same time, the performance of predictive models directly depends on the amount and quality of experimental data. Driven by the interest in developing a model for prediction of the antibiofilm effect of phenolic natural compounds such as flavonoids, we performed experimental assessment of antibiofilm activity of 320 compounds from this subset of chemicals. The assay was performed once on two Escherichia coli strains on agar in 24-well microtiter plates. The inhibition was assessed visually by detecting morphological changes in macrocolonies. Using the data obtained, we subsequently trained a Bayesian logistic regression model for prediction of biofilm inhibition, which was combined with a similarity-based method in order to increase the overall sensitivity (at the cost of accuracy). The quality of the predictions was subsequently validated by experimental assessment in three independent experiments with two resistant E. coli strains of 23 compounds absent in the initial data set. The validation demonstrated that the model may successfully predict the targeted effect as compared to the baseline accuracy. Using a randomly selected database of commercially available natural phenolics, we obtained approximately 6.0% of active compounds, whereas using our prediction-based substance selection, the percentage of phenolics found to be active increased to 34.8%.

[180] K. Puls, H. Schmidhammer, G. Wolber, M. Spetea. Mechanistic characterization of the pharmacological profile of hs-731, a peripherally acting opioid analgesic, at the µ-, δ, κ-opioid and nociceptin receptors, Molecules, 27(3):919, 2022.
Links: [doi:10.3390/molecules27030919] [Download citation: BibTeX | RIS]
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Abstract: Accumulated preclinical and clinical data show that peripheral restricted opioids provide pain relief with reduced side effects. The peripherally acting opioid analgesic HS-731 is a potent dual &mu;-/&delta;-opioid receptor (MOR/DOR) full agonist, and a weak, partial agonist at the &kappa;-opioid receptor (KOR). However, its binding mode at the opioid receptors remains elusive. Here, we present a comprehensive in silico evaluation of HS-731 binding at all opioid receptors. We provide insights into dynamic interaction patterns explaining the different binding and activity of HS-731 on the opioid receptors. For this purpose, we conducted docking, performed molecular dynamics (MD) simulations and generated dynamic pharmacophores (dynophores). Our results highlight two residues important for HS-731 recognition at the classical opioid receptors (MOR, DOR and KOR), particular the conserved residue 5.39 (K) and the non-conserved residue 6.58 (MOR: K, DOR: W and KOR: E). Furthermore, we assume a salt bridge between the transmembrane helices (TM) 5 and 6 via K2275.39 and E2976.58 to be responsible for the partial agonism of HS-731 at the KOR. Additionally, we experimentally demonstrated the absence of affinity of HS-731 to the nociceptin/orphanin FQ peptide (NOP) receptor. We consider the morphinan phenol Y1303.33 responsible for this affinity lack. Y1303.33 points deep into the NOP receptor binding pocket preventing HS-731 binding to the orthosteric binding pocket. These findings provide significant structural insights into HS-731 interaction pattern with the opioid receptors that are important for understanding the pharmacology of this peripheral opioid analgesic.

[179] K. Puls, A. Olivé-Marti, S. Pach, B. Pinter, F. Erli, G. Wolber, M. Spetea. In vitro, in vivo and In silico characterization of a novel kappa-opioid receptor antagonist, Pharmaceuticals, 15(6):680, 2022.
Links: [doi:10.3390/ph15060680] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Kappa-opioid receptor (KOR) antagonists are promising innovative therapeutics for the treatment of the central nervous system (CNS) disorders. The new scaffold opioid ligand, Compound A, was originally found as a mu-opioid receptor (MOR) antagonist but its binding/selectivity and activation profile at the KOR and delta-opioid receptor (DOR) remain elusive. In this study, we present an in vitro, in vivo and in silico characterization of Compound A by revealing this ligand as a KOR antagonist in vitro and in vivo. In the radioligand competitive binding assay, Compound A bound at the human KOR, albeit with moderate affinity, but with increased affinity than to the human MOR and without specific binding at the human DOR, thus displaying a preferential KOR selectivity profile. Following subcutaneous administration in mice, Compound A effectively reverse the antinociceptive effects of the prototypical KOR agonist, U50,488. In silico investigations were carried out to assess the structural determinants responsible for opioid receptor subtype selectivity of Compound A. Molecular docking, molecular dynamics simulations and dynamic pharmacophore (dynophore) generation revealed differences in the stabilization of the chlorophenyl moiety of Compound A within the opioid receptor binding pockets, rationalizing the experimentally determined binding affinity values. This new chemotype bears the potential for favorable ADMET properties and holds promise for chemical optimization toward the development of potential therapeutics. View Full-Text

[178] T. Noonan, K. Denzinger, V. Talagayev, Y. Chen, K. Puls, C. A. Wolf, S. Liu, T. N. Nguyen, G. Wolber. Mind the gap - deciphering GPCR pharmacology using 3D pharmacophores and artificial intelligence, Pharmaceuticals, 15(11):1304, 2022.
Links: [doi:10.3390/ph15111304] [Publisher][Download citation: BibTeX | RIS]
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Abstract: G protein-coupled receptors (GPCRs) are amongst the most pharmaceutically relevant and well-studied protein targets, yet unanswered questions in the field leave significant gaps in our understanding of their nuanced structure and function. Three-dimensional pharmacophore models are powerful computational tools in in silico drug discovery, presenting myriad opportunities for the integration of GPCR structural biology and cheminformatics. This review highlights success stories in the application of 3D pharmacophore modeling to de novo drug design, the discovery of biased and allosteric ligands, scaffold hopping, QSAR analysis, hit-to-lead optimization, GPCR de-orphanization, mechanistic understanding of GPCR pharmacology and the elucidation of ligand–receptor interactions. Furthermore, advances in the incorporation of dynamics and machine learning are highlighted. The review will analyze challenges in the field of GPCR drug discovery, detailing how 3D pharmacophore modeling can be used to address them. Finally, we will present opportunities afforded by 3D pharmacophore modeling in the advancement of our understanding and targeting of GPCRs

[177] Y. Lu, X. Liu, R. Lotfy, S. Liu, A. F. Tesfa, G. Wolber, M. Bureik, B. R. Clark. Experimental and computational studies on the biotransformation of pseudopyronines with human Cytochrome P450 CYP4F2, J. Nat. Prod., , 2022.
Links: [doi:10.1021/acs.jnatprod.2c00616] [Publisher][Download citation: BibTeX | RIS]
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Abstract: The secondary metabolite pseudopyronine B, isolated from Pseudomonas mosselii P33, was biotransformed by human P450 enzymes, heterologously expressed in the fission yeast Schizosaccharomyces pombe. Small-scale studies confirmed that both CYP4F2 and CYP4F3A were capable of oxidizing the substrate, with the former achieving a higher yield. In larger-scale studies using CYP4F2, three new oxidation products were obtained, the structures of which were elucidated by UV–vis, 1D and 2D NMR, and HR-MS spectroscopy. These corresponded to hydroxylated, carboxylated, and ester derivatives (1–3) of pseudopyronine B, all of which had been oxidized exclusively at the ω-position of the C-6 alkyl chain. In silico homology modeling experiments highlighted key interactions between oxygen atoms of the pyrone ring and two serine residues and a histidine residue of CYP4F2, which hold the substrate in a suitable orientation for oxidation at the terminus of the C-6 alkyl chain. Additional modeling studies with all three pseudopyronines revealed that the seven-carbon alkyl chain of pseudopyronine B was the perfect length for oxidation, with the terminal carbon lying close to the heme iron. The antibacterial activity of the substrates and three oxidation products was also assessed, revealing that oxidation at the ω-position removes all antimicrobial activity. This study both increases the range of known substrates for human CYF4F2 and CYP4F3A enzymes and demonstrates their utility in producing additional natural product derivatives.

[176] S. El Deeb, A. E. Ibrahim, A. Al-Harrasi, G. Wolber, R. Gust. Validated capillary zone electrophoresis method for impurity profiling and determination of niII(3-ome-salophene), Separations, 9(2):25, 2022.
Links: [doi:10.3390/separations9020025] [Download citation: BibTeX | RIS]
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Abstract: A capillary zone electrophoresis method was developed for the determination of NiII(3-OMe-salophene), a substance with anticancer activity in vitro. A fused silica capillary (56 cm × 100 µm) was used for this purpose. The method was optimized in terms of parameters affecting the electrophoretic conditions in order to optimize separation efficiency and total time of migration. The analysis was best performed using an operating buffer of 50 mM borate, adjusted to pH 9.3, mixed with acetonitrile (50%, v/v) as organic modifier. Injections were performed hydrodynamically by applying a pressure of 50 mbar for 8 s, and a 30 kV separation voltage was selected at 25 ◦C. Detection was carried out at 250 nm using diode array detector (DAD). The method allowed the separation of NiII(3-OMe-salophene) from four other structurally related impurities in a total migration time (tm) of 8 min. Peak identification was achieved using the standard reference of individual impurities. The purity of the migrated NiII(3-OMe-salophene) was confirmed by Ultra-violet (UV) scan overlay depending on DAD. The linear ranges for the determination of NiII(3-OMe-salophene) was 400–20,000 ng mL−1 with limit of detection (LOD) of 120 ng mL−1. Acceptable intra-day and inter-day precisions were achieved (%relative standard deviation (RSD) results were less than 0.76% and 0.30%, respectively). The proposed method was assessed for greenness and compared to reported methodologies to prove superiority.

[175] D. Machalz, H. Li, W. Du, S. Sharma, S. Liu, M. Bureik, G. Wolber. Discovery of a novel potent cytochrome P450 CYP4Z1 inhibitor, Eur. J. Med. Chem., 215:113255, 2021.
Links: [doi:10.1016/j.ejmech.2021.113255] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Human cytochrome P450 enzyme CYP4Z1 represents a promising target for the treatment of a multitude of malignancies including breast cancer. The most active known non-covalent inhibitor (1-benzylimidazole) only shows low micromolar affinity to CYP4Z1. We report a new, highly active inhibitor for CYP4Z1 showing confirmed binding in an enzymatic assay and an IC50 value of 63 ± 19 nM in stably transfected MCF-7 cells overexpressing CYP4Z1. The new inhibitor was identified by a systematically developed virtual screening protocol. Binding was rationalized using a carefully elaborated 3D pharmacophore hypothesis and thoroughly characterized using extensive molecular dynamics simulations and dynamic 3D pharmacophore (dynophore) analyses. This novel inhibitor represents a valuable pharmacological tool to accelerate characterization of the still understudied CYP4Z1 and might pave the way for a new treatment strategy in CYP4Z1-associated malignancies. The presented in silico model for predicting CYP4Z1 interaction provides novel mechanistic insights and revealed that the drug ozagrel interacts with CYP4Z1.

[174] A. Dolšak, D. Šribar, A. Scheffler, M. Grabowski, U. Švajger, S. Gobec, J. Holze, G. Weindl, G. Wolber, M. Sova. Further hit optimization of 6-(trifluoromethyl)pyrimidin-2-amine based TLR8 modulators: Synthesis, biological evaluation and structure–activity relationships, Eur. J. Med. Chem., 225:113809, 2021.
Links: [doi:10.1016/j.ejmech.2021.113809] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Toll-like receptor 8 (TLR8) is an endosomal TLR that has an important role in the innate human immune system, which is involved in numerous pathological conditions. Excessive activation of TLR8 can lead to inflammatory and autoimmune diseases, which highlights the need for development of TLR8 modulators. However, only a few small-molecule modulators that selectively target TLR8 have been developed. Here, we report the synthesis and systematic investigation of the structure–activity relationships of a series of novel TLR8 negative modulators based on previously reported 6-(trifluoromethyl)pyrimidin-2-amine derivatives. Four compounds showed low-micromolar concentration-dependent inhibition of TLR8-mediated signaling in HEK293 cells. These data confirm that the 6-trifluoromethyl group and two other substituents on positions 2 and 4 are important structural elements of pyrimidine-based TLR8 modulators. Substitution of the main scaffold at position 2 with a methylsulfonyl group or para hydroxy/hydroxymethyl substituted benzylamine is essential for potent negative modulation of TLR8. Our best-in-class TLR8-selective modulator 53 with IC50 value of 6.2 μM represents a promising small-molecule chemical probe for further optimization to a lead compound with potent immunomodulatory properties.

[173] C. Tauber, R. Wamser, C. Arkona, M. Tuegend, U. B. Abdul Aziz, S. Pach, R. Schulz, D. Jochmans, G. Wolber, J. Neyts, J. Rademann. Chemical evolution of antivirals against enterovirus D68 through protein-templated Knoevenagel reactions, Angew. Chem. Int. Ed., 60(24):13294-13301, 2021.
Links: [doi:10.1002/anie.202102074] [Publisher][Download citation: BibTeX | RIS]
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Abstract: The generation of bioactive molecules from inactive precursors is a crucial step in the chemical evolution of life, however, mechanistic insights into this aspect of abiogenesis are scarce. Here, we investigate the protein-catalyzed formation of antivirals by the 3C-protease of enterovirus D68. The enzyme induces aldol condensations yielding inhibitors with antiviral activity in cells. Kinetic and thermodynamic analyses reveal that the bioactivity emerges from a dynamic reaction system including inhibitor formation, alkylation of the protein target by the inhibitors, and competitive addition of non-protein nucleophiles to the inhibitors. The most active antivirals are slowly reversible inhibitors with elongated target residence times. The study reveals first examples for the chemical evolution of bio-actives through protein-catalyzed, non-enzymatic CC-couplings. The discovered mechanism works under physiological conditions and might constitute a native process of drug development.

[172] Szymon Pach, Trung Ngoc Nguyen, Jakob Trimpert, Dusan Kunec, Nikolaus Osterrieder, Gerhard Wolber. ACE2 variants indicate potential SARS-CoV-2-Susceptibility in animals: An extensive molecular dynamics study, Mol. Inform., 40(9):2100031, 2021.
Links: [doi:10.1002/minf.202100031] [Publisher][Download citation: BibTeX | RIS]

[171] S. Sharma, S. Liu, P. Durairaj, D. Machalz, G. Wolber, M. Bureik. A convenient test system for the identification of CYP4V2 inhibitors, Mol. Vis., 27:601-607, 2021. [Publisher][Download citation: BibTeX | RIS]
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Abstract: Purpose: Polymorphisms in the gene that codes for the human cytochrome P450 enzyme CYP4V2 are a cause of Bietti crystalline dystrophy (BCD). Therefore, inhibition of CYP4V2 activity may well be a cause of visual disability. However, monitoring the fatty acid hydroxylation reactions catalyzed by this enzyme is tedious and not well suited for inhibitor screening. Methods: We investigated the use of proluciferin compounds as probe substrates for efficient and convenient determination of CYP4V2 activity. Results: Ten proluciferins were tested for conversion by CYP4V2, and eight were found to be substrates of this enzyme. One point inhibitor assays were performed using luciferin 6' 3-furfuryl ether methyl ester (luciferin-3FEME) as the probe substrate and 12 test compounds. As expected, HET0016 had by far the strongest effect, while two other compounds (including osilodrostat) also displayed statistically significant inhibitory potency. The half maximal inhibitory concentration (IC50) for HET0016 was determined to be 179 nM. A recently identified potent inhibitor of human CYP4Z1 was found not to inhibit CYP4V2. To explore the selectivity of this compound between CYP4Z1 and CYP4V2, we developed a homology model of CYP4V2 and conducted docking experiments. Conclusions: We provide the first protocol for a robust and convenient CYP4V2 inhibitor assay that does not depend on fatty acid analysis but can be simply monitored with luminescence. Moreover, we demonstrate additional evidence for the concern that compounds with CYP-inhibitory properties may inhibit CYP4V2 activity and thus, possibly cause visual disability.

[170] R. Ottanà, P. Paoli, M. Cappiello, T. N. Nguyen, I. Adornato, A. Del Corso, M. Genovese, I. Nesi, R. Moschini, A. Naß, G. Wolber, R. Maccari. In search for multi-target ligands as potential agents for diabetes mellitus and its complications—a structure-activity relationship study on inhibitors of aldose reductase and Protein Tyrosine Phosphatase 1B, Molecules, 26(2):330, 2021.
Links: [doi:10.3390/molecules26020330] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Diabetes mellitus (DM) is a complex disease which currently affects more than 460 million people and is one of the leading cause of death worldwide. Its development implies numerous metabolic dysfunctions and the onset of hyperglycaemia-induced chronic complications. Multiple ligands can be rationally designed for the treatment of multifactorial diseases, such as DM, with the precise aim of simultaneously controlling multiple pathogenic mechanisms related to the disease and providing a more effective and safer therapeutic treatment compared to combinations of selective drugs. Starting from our previous findings that highlighted the possibility to target both aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two enzymes strictly implicated in the development of DM and its complications, we synthesised 3-(5-arylidene-4-oxothiazolidin-3-yl)propanoic acids and analogous 2-butenoic acid derivatives, with the aim of balancing the effectiveness of dual AR/PTP1B inhibitors which we had identified as designed multiple ligands (DMLs). Out of the tested compounds, 4f exhibited well-balanced AR/PTP1B inhibitory effects at low micromolar concentrations, along with interesting insulin-sensitizing activity in murine C2C12 cell cultures. The SARs here highlighted along with their rationalization by in silico docking experiments into both target enzymes provide further insights into this class of inhibitors for their development as potential DML antidiabetic candidates.

[169] T. Meşeli, S. D. Doğan, M. G. Gündüz, Z. Kökbudak, S. Skaro Bogojevic, T. Noonan, S. Vojnovic, G. Wolber, J. Nikodinovic-Runic. Design, synthesis, antibacterial activity evaluation and molecular modeling studies of new sulfonamides containing a sulfathiazole moiety, New J. Chem., 45(18):8166-8177, 2021.
Links: [doi:10.1039/D1NJ00150G] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Sulfonamides represent the oldest synthetic antibacterial agents; however, their central position in controlling bacterial diseases has been seriously damaged by the development of widespread resistance. Herein, we revisited sulfathiazole, a commercial member of antibacterial sulfa drugs, intending to overcome sulfonamide resistance and identify new drug candidates through molecular modifications. We synthesized twelve sulfonamides (SA1–SA12) by replacing the amino group on the phenyl ring with various substituents and introducing a thiophene ring on the core scaffold of sulfathiazole. The obtained compounds and additionally two commercial sulfonamides, sulfathiazole and sulfadiazine, were extensively screened for their antimicrobial activities. The results indicated that new sulfonamides, unlike traditional ones, were selectively effective against various Staphylococcus aureus strains. Introducing a bulky lipophilic substituent at the para position of the phenyl ring significantly increased the antibacterial activities of the compounds against Staphylococcus aureus. The compounds demonstrating favourable selectivity indices were further evaluated for their membrane potential perturbation and DNA interaction properties. The obtained data showed that these are not supporting mechanisms for the antibacterial activities of the modified sulfathiazole derivatives. In order to rationalize the activity of the three most active compounds, SA7, SA11 and SA12, against S. aureus ATCC 25923, their binding hypotheses within the catalytic site of Staphylococcus aureus dihydropteroate synthase, the validated target enzyme of sulfonamides, were generated via molecular docking and further dissected using molecular dynamics simulations and dynamic 3D pharmacophores (dynophores).

[168] D. Machalz, S. Pach, M. Bermudez, M. Bureik, G. Wolber. Structural insights into understudied human cytochrome P450 enzymes, Drug Discov. Today, 26(10):2456-2464, 2021.
Links: [doi:10.1016/j.drudis.2021.06.006] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Human cytochrome P450 (CYP) enzymes are widely known for their pivotal role in the metabolism of drugs and other xenobiotics as well as of endogenous chemicals. In addition, CYPs are involved in numerous pathophysiological pathways and, hence, are therapeutically relevant. Remarkably, a portion of promising CYP targets is still understudied and, as a consequence, untargeted, despite their huge therapeutic potential. An increasing number of X-ray and cryo-electron microscopy (EM) structures for CYPs have recently provided new insights into the structural basis of CYP function and potential ligand binding. This structural knowledge of CYP functionality is essential for both understanding metabolism and exploiting understudied CYPs as drug targets. In this review, we summarize and highlight structural knowledge about this enzyme class, with a focus on understudied CYPs and resulting opportunities for structure-based drug design. Teaser: This review summarizes recent structural insights into understudied cytochrome P450 enzymes. We highlight the impact of molecular modeling for mechanistically explaining pathophysiological effects establishing understudied CYPs as promising drug targets.

[167] S. Loke, A. Stoll, D. Machalz, F. Botrè, G. Wolber, M. Bureik, M. K. Parr. Corticosteroid biosynthesis revisited: No direct hydroxylation of Pregnenolone by Steroid 21-Hydroxylase, Front. Endocrinol., 12(629), 2021.
Links: [doi:10.3389/fendo.2021.633785] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism.

[166] J. Y. Liu, D. Machalz, G. Wolber, E. J. Sorensen, M. Bureik. New proluciferin substrates for human CYP4 family enzymes, Appl. Biochem. Biotechnol., 193(1):218-237, 2021.
Links: [doi:10.1007/s12010-020-03388-6] [Publisher][Download citation: BibTeX | RIS]
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Abstract: We report the synthesis of seven new proluciferins for convenient activity determination of enzymes belonging to the cytochrome P450 (CYP) 4 family. Biotransformation of these probe substrates was monitored using each of the twelve human CYP4 family members, and eight were found to act at least on one of them. For all substrates, activity of CYP4Z1 was always highest, while that of CYP4F8 was always second highest. Site of metabolism (SOM) predictions involving SMARTCyp and docking experiments helped to rationalize the observed activity trends linked to substrate accessibility and reactivity. We further report the first homology model of CYP4F8 including suggested substrate recognition residues in a catalytically competent conformation accessed by replica exchange solute tempering (REST) simulations.

[165] M. Janežič, K. Valjavec, K. B. Loboda, B. Herlah, I. Ogris, M. Kozorog, M. Podobnik, S. G. Grdadolnik, G. Wolber, A. Perdih. Dynophore-based approach in virtual screening: a case of human DNA Topoisomerase IIα, Int. J. Mol. Sci., 22(24):13474, 2021.
Links: [doi:10.3390/ijms222413474] [Publisher][Download citation: BibTeX | RIS]
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Abstract: In this study, we utilized human DNA topoisomerase IIα as a model target to outline a dynophore-based approach to catalytic inhibitor design. Based on MD simulations of a known catalytic inhibitor and the native ATP ligand analog, AMP-PNP, we derived a joint dynophore model that supplements the static structure-based-pharmacophore information with a dynamic component. Subsequently, derived pharmacophore models were employed in a virtual screening campaign of a library of natural compounds. Experimental evaluation identified flavonoid compounds with promising topoisomerase IIα catalytic inhibition and binding studies confirmed interaction with the ATPase domain. We constructed a binding model through docking and extensively investigated it with molecular dynamics MD simulations, essential dynamics, and MM-GBSA free energy calculations, thus reconnecting the new results to the initial dynophore-based screening model. We not only demonstrate a new design strategy that incorporates a dynamic component of molecular recognition, but also highlight new derivates in the established flavonoid class of topoisomerase II inhibitors.

[164] M. Dumitrascuta, M. Bermudez, O. Trovato, J. De Neve, S. Ballet, G. Wolber, M. Spetea. Antinociceptive efficacy of the μ-opioid/nociceptin peptide-based hybrid KGNOP1 in inflammatory pain without rewarding effects in mice: an experimental assessment and molecular docking, Molecules, 26(11):3267, 2021.
Links: [doi:10.3390/molecules26113267] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Opioids are the most effective analgesics, with most clinically available opioids being agonists to the µ-opioid receptor (MOR). The MOR is also responsible for their unwanted effects, including reward and opioid misuse leading to the current public health crisis. The imperative need for safer, non-addictive pain therapies drives the search for novel leads and new treatment strategies. In this study, the recently discovered MOR/nociceptin (NOP) receptor peptide hybrid KGNOP1 (H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) was evaluated following subcutaneous administration in mouse models of acute (formalin test) and chronic inflammatory pain (Complete Freund’s adjuvant-induced paw hyperalgesia), liabilities of spontaneous locomotion, conditioned place preference, and the withdrawal syndrome. KGNOP1 demonstrated dose-dependent antinociceptive effects in the formalin test, and efficacy in attenuating thermal hyperalgesia with prolonged duration of action. Antinociceptive effects of KGNOP1 were reversed by naltrexone and SB-612111, indicating the involvement of both MOR and NOP receptor agonism. In comparison with morphine, KGNOP1 was more potent and effective in mouse models of inflammatory pain. Unlike morphine, KGNOP1 displayed reduced detrimental liabilities, as no locomotor impairment nor rewarding and withdrawal effects were observed. Docking of KGNOP1 to the MOR and NOP receptors and subsequent 3D interaction pattern analyses provided valuable insights into its binding mode. The mixed MOR/NOP receptor peptide KGNOP1 holds promise in the effort to develop new analgesics for the treatment of various pain states with fewer MOR-mediated side effects, particularly abuse and dependence liabilities.

[163] D. Volpato, M. Kauk, R. Messerer, M. Bermudez, G. Wolber, A. Bock, C. Hoffmann, U. Holzgrabe. The role of orthosteric building blocks of bitopic ligands for muscarinic M1 receptors, ACS Omega, 5(49):31706-31715, 2020.
Links: [doi:10.1021/acsomega.0c04220] [Publisher][Download citation: BibTeX | RIS]
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Abstract: The muscarinic M1 acetylcholine receptor is an important drug target for the treatment of various neurological disorders. Designing M1 receptor-selective drugs has proven challenging, mainly due to the high conservation of the acetylcholine binding site among muscarinic receptor subtypes. Therefore, less conserved and topographically distinct allosteric binding sites have been explored to increase M1 receptor selectivity. In this line, bitopic ligands, which target orthosteric and allosteric binding sites simultaneously, may provide a promising strategy. Here, we explore the allosteric, M1-selective BQCAd scaffold derived from BQCA as a starting point for the design, synthesis, and pharmacological evaluation of a series of novel bitopic ligands in which the orthosteric moieties and linker lengths are systematically varied. Since β-arrestin recruitment seems to be favorable to therapeutic implication, all the compounds were investigated by G protein and β-arrestin assays. Some bitopic ligands are partial to full agonists for G protein activation, some activate β-arrestin recruitment, and the degree of β-arrestin recruitment varies according to the respective modification. The allosteric BQCAd scaffold controls the positioning of the orthosteric ammonium group of all ligands, suggesting that this interaction is essential for stimulating G protein activation. However, β-arrestin recruitment is not affected. The novel set of bitopic ligands may constitute a toolbox to study the requirements of β-arrestin recruitment during ligand design for therapeutic usage.

[162] Y. Sun, D. Machalz, G. Wolber, M. K. Parr, M. Bureik. Functional expression of all human sulfotransferases in fission yeast, assay development, and structural models for isoforms SULT4A1 and SULT6B1, Biomolecules, 10(11):1517, 2020.
Links: [doi:10.3390/biom10111517] [Publisher][Download citation: BibTeX | RIS]

[161] D. Stepanov, S. Canipa, G. Wolber. HuskinDB, a database for skin permeation of xenobiotics, Sci. Data, 7(1):426, 2020.
Links: [doi:10.1038/s41597-020-00764-z] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Skin permeation is an essential biological property of small organic compounds our body is exposed to, such as drugs in topic formulations, cosmetics, and environmental toxins. Despite the limited availability of experimental data, there is a lack of systematic analysis and structure. We present a novel resource on skin permeation data that collects all measurements available in the literature and systematically structures experimental conditions. Besides the skin permeation value kp, it includes experimental protocols such as skin source site, skin layer used, preparation technique, storage conditions, as well as test conditions such as temperature, pH as well as the type of donor and acceptor solution. It is important to include these parameters in the assessment of the skin permeation data. In addition, we provide an analysis of physicochemical properties and chemical space coverage, laying the basis for applicability domain determination of insights drawn from the collected data points. The database is freely accessible under https://huskindb.drug-design.deor https://doi.org/10.7303/syn21998881.

[160] D. Schaller, G. Wolber. Pyrod enables rational homology model-based virtual screening against MCHR1, Mol. Inform., 39(6):e2000020, 2020.
Links: [doi:10.1002/minf.202000020] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Several encouraging pre-clinical results highlight the melanin-concentrating hormone receptor 1 (MCHR1) as promising target for anti-obesity drug development. Currently however, experimentally resolved structures of MCHR1 are not available, which complicates rational drug design campaigns. In this study, we aimed at developing accurate, homologymodel-based 3D pharmacophores against MCHR1. We show that traditional approaches involving docking of known active small molecules are hindered by the flexibility of binding pocket residues. Instead, we derived three-dimensional pharmacophores from molecular dynamics simulations by employing our novel open-source software PyRod. In a retrospective evaluation, the generated 3D pharmacophores were highly predictive returning up to 35 % of active molecules and showing an early enrichment (EF1) of up to 27.6. Furthermore, PyRod pharmacophores demonstrate higher sensitivity than ligand-based pharmacophores and deliver structural insights, which are key to rational lead optimization.

[159] D. Schaller, D. Sribar, T. Noonan, L. H. Deng, T. N. Nguyen, S. Pach, D. Machalz, M. Bermudez, G. Wolber. Next generation 3D pharmacophore modeling, WIREs Comput Mol Sci., 10(4):e1468, 2020.
Links: [doi:10.1002/wcms.1468] [Publisher][Download citation: BibTeX | RIS]
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Abstract: 3D pharmacophore models are three-dimensional ensembles of chemically defined interactions of a ligand in its bioactive conformation. They represent an elegant way to decipher chemically encoded ligand information and have therefore become a valuable tool in drug design. In this review, we provide an overview on the basic concept of this method and summarize key studies for applying 3D pharmacophore models in virtual screening and mechanistic studies for protein functionality. Moreover, we discuss recent developments in the field. The combination of 3D pharmacophore models with molecular dynamics simulations could be a quantum leap forward since these approaches consider macromolecule-ligand interactions as dynamic and therefore show a physiologically relevant interaction pattern. Other trends include the efficient usage of 3D pharmacophore information in machine learning and artificial intelligence applications or freely accessible web servers for 3D pharmacophore modeling. The recent developments show that 3D pharmacophore modeling is a vibrant field with various applications in drug discovery and beyond. This article is categorized under: Computer and Information Science > Chemoinformatics Computer and Information Science > Computer Algorithms and Programming Molecular and Statistical Mechanics > Molecular Interactions

[158] S. Pach, T. M. Sarter, R. Yousef, D. Schaller, S. Bergemann, C. Arkona, J. Rademann, C. Nitsche, G. Wolber. Catching a moving target: comparative modeling of flaviviral NS2B-NS3 reveals small molecule Zika protease inhibitors, ACS Med. Chem. Lett., 11(4):514-520, 2020.
Links: [doi:10.1021/acsmedchemlett.9b00629] [Publisher][Download citation: BibTeX | RIS]
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Abstract: The pivotal role of viral proteases in virus replication has already been successfully exploited in several antiviral drug design campaigns. However, no efficient antivirals are currently available against flaviviral infections. In this study, we present lead-like small molecule inhibitors of the Zika Virus (ZIKV) NS2B-NS3 protease. Since only few nonpeptide competitive ligands are known, we take advantage of the high structural similarity with the West Nile Virus (WNV) NS2B-NS3 protease. A comparative modeling approach involving our in-house software PyRod was employed to systematically analyze the binding sites and develop molecular dynamics-based 3D pharmacophores for virtual screening. The identified compounds were biochemically characterized revealing low micromolar affinity for both ZIKV and WNV proteases. Their lead-like properties together with rationalized binding modes represent valuable starting points for future lead optimization. Since the NS2B-NS3 protease is highly conserved among flaviviruses, these compounds may also drive the development of pan-flaviviral antiviral drugs.

[157] K. B. Loboda, K. Valjavec, M. Štampar, G. Wolber, B. Žegura, M. Filipič, M. S. Dolenc, A. Perdih. Design and synthesis of 3,5-substituted 1,2,4-oxadiazoles as catalytic inhibitors of human DNA topoisomerase IIα, Bioorg. Chem., 99:103828, 2020.
Links: [doi:10.1016/j.bioorg.2020.103828] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Cancer constitutes a group of diseases linked to abnormal cell growth that can potentially spread to other parts of the body and is one of the most common causes of death. The molecular motors - DNA topoisomerases - that enable topological changes of the DNA molecule are one of the most established targets of cancer therapies. Due to known limitations of established topo II poisons such as cardiotoxicity, induction of secondary malignancies and recognized cancer cell resistance, an emerging group of catalytic topo II inhibitors attempts to circumvent these challenges. Currently, this approach comprises several subgroups of mechanistically diverse inhibitors, one of which are compounds that act by binding to their ATPase domain. In this study we have designed, synthesized and characterized a new series of 3,5-substituted 1,2,4-oxadiazoles that act as catalytic inhibitors of human topo IIα. The introduction of the substituted rigid substitutions on the oxadiazole backbone was intended to enhance the interactions with the ATP binding site. In the inhibition assays selected compounds revealed a new class of catalytic inhibitors targeting this molecular motor and showed binding to the isolated topo IIα ATPase domain. The predicted inhibitor binding geometries were evaluated in molecular dynamics simulations and subsequently dynophore models were derived, which provided a deeper insight into molecular recognition with its macromolecular target. Selected compounds also displayed in vitro cytotoxicity on the investigated MCF-7 cancer cell line and did not induce double-strand breaks (DSB), thus displaying a mechanism of action diverse from the topo II poisons also on the cellular level. The substituted oxadiazoles thus comprise a chemical class of interesting compounds that are synthetically fully amenable for further optimization to anticancer drugs.

[156] J. Holze, M. Bermudez, E. M. Pfeil, M. Kauk, T. Bödefeld, M. Irmen, C. Matera, C. Dallanoce, M. De Amici, U. Holzgrabe, G. M. König, C. Tränkle, G. Wolber, R. Schrage, K. Mohr, C. Hoffmann, E. Kostenis, A. Bock. Ligand-specific allosteric coupling controls G-Protein-Coupled receptor signaling, ACS Pharmacol. Transl. Sci., 3(5):859-867, 2020.
Links: [doi:10.1021/acsptsci.0c00069] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Allosteric coupling describes a reciprocal process whereby G-protein-coupled receptors (GPCRs) relay ligand-induced conformational changes from the extracellular binding pocket to the intracellular signaling surface. Therefore, GPCR activation is sensitive to both the type of extracellular ligand and intracellular signaling protein. We hypothesized that ligand-specific allosteric coupling may result in preferential (i.e., biased) engagement of downstream effectors. However, the structural basis underlying ligand-dependent control of this essential allosteric mechanism is poorly understood. Here, we show that two sets of extended muscarinic acetylcholine receptor M1 agonists, which only differ in linker length, progressively constrain receptor signaling. We demonstrate that stepwise shortening of their chemical linker gradually hampers binding pocket closure, resulting in divergent coupling to distinct G-protein families. Our data provide an experimental strategy for the design of ligands with selective G-protein recognition and reveal a potentially general mechanism of ligand-specific allosteric coupling.

[155] M. Grabowski, M. S. Murgueitio, M. Bermudez, G. Wolber, G. Weindl. The novel small-molecule antagonist MMG-11 preferentially inhibits TLR2/1 signaling, Biochem. Pharmacol., 171:113687, 2020.
Links: [doi:10.1016/j.bcp.2019.113687] [Download citation: BibTeX | RIS]
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Abstract: Toll-like receptor 2 (TLR2) forms heterodimers with either TLR1 or TLR6 to induce protective early inflammatory responses to pathogen- and damage-associated molecular patterns. However, excessive activation is associated with inflammatory and metabolic diseases. Several TLR2 antagonists have been described but pharmacological characterization is still at an early stage. Previously, we identified the potent and selective TLR2 antagonist MMG-11 by computational modelling and experimental validation. Here, we characterized the TLR2 antagonists MMG-11 and CU-CPT22 as well as the TIR-domain binding TLR2 antagonist C29 in TLR-overexpressing promoter cells as well as human and mouse macrophages. In line with our recent studies, MMG-11 abrogated pro-inflammatory cytokine secretion and NF-κB activation induced by different bacterial TLR2 agonists. MMG-11 preferentially inhibited TLR2/1 signaling in promoter cells stably expressing TLR2 heterodimers and mouse macrophages. Furthermore, the TLR2 antagonist blocked ligand-induced interaction of TLR2 with MyD88 and reduced MAP kinase and NF-κB activation. MMG-11 and CU-CPT22 but not C29 displaced Pam3CSK4 in an indirect binding assay confirming the competitive mode of action of MMG-11 and CU-CPT22. Isobologram analysis revealed additive and synergistic effects when the non-competitive antagonist C29 was combined with the competitive antagonist MMG-11 or CU-CPT22, respectively. In conclusion, we provide evidence that MMG-11 acts as a competitive antagonist with a predominance for the TLR2/1 heterodimer in human and mouse cells. Our results also indicate that MMG-11 is a model compound for studying TLR2 signaling.

[154] M. Grabowski, M. Bermudez, T. Rudolf, D. Sribar, P. Varga, M. S. Murgueitio, G. Wolber, J. Rademann, G. Weindl. Identification and validation of a novel dual small-molecule TLR2/8 antagonist, Biochem. Pharmacol., 177:113957, 2020.
Links: [doi:10.1016/j.bcp.2020.113957] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Toll-like receptor 2 (TLR2) and TLR8 are involved in the recognition of bacterial and viral components and are linked not only to protective antimicrobial immunity but also to inflammatory diseases. Recently, increasing attention has been paid to the receptor crosstalk between TLR2 and TLR8 to fine-tune innate immune responses. In this study, we report a novel dual TLR2/TLR8 antagonist, compound 24 that was developed by a modeling-guided synthesis approach. The modulator was optimized from the previously reported 1,3-benzothiazole derivative, compound 8. Compound 24 was pharmacologically characterized for the ability to inhibit TLR2- and TLR8-mediated responses in TLR-overexpressing reporter cells and THP-1 macrophages. The modulator showed high efficacy with IC50, values in the low micromolar range for both TLR5, selectivity towards other TLR5 and low cytotoxicity. At TLR2, a slight predominance for the TLR2/1 heterodimer was found in reporter cells selectively expressing TLR2/1 or TLR2/6 heterodimers. Concentration ratio analysis in the presence of Pam(3)CSK(4) or Pam(2)CSK(4) indicated non-competitive antagonist behavior at hTLR2. In computational docking studies, a plausible alternative binding mode of compound 24 was predicted for both TLR2 and TLR8. Our results provide evidence that it is feasible to simultaneously and selectively target endosomal- and surface-located TLR5. We identified a small-molecule dual TLR2/8 antagonist that may serve as a valuable pharmacological tool to decipher the role of TLR2/8 co-signaling in inflammation.

[153] M. T. Gabr, D. Machalz, S. Pach, G. Wolber. A benzoxazole derivative as an inhibitor of anaerobic choline metabolism by human gut microbiota, RSC Med. Chem., 11(12):1402-1412, 2020.
Links: [doi:10.1039/D0MD00218F] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Metabolic pathways mediated by human gut bacteria have emerged as potential therapeutic targets because of their association with the pathophysiology of various human diseases. The anaerobic transformation of choline into trimethylamine (TMA) by gut microbiota is directly linked to type 2 diabetes, fatty liver disease, and cardiovascular diseases. Structural analogs of choline have been developed as competitive inhibitors of choline TMA-lyase (CutC), a key enzyme for the conversion of choline to TMA. However, weak to moderate CutC inhibitory profiles of the choline analogs limit their further advancement into clinical translation. In this study, we introduce a glycomimetic-based approach for the identification of CutC inhibitors with intestinal metabolic stability. Our workflow started with screening of a small library of glycomimetics for metabolic stability in the presence of human intestinal S9 fraction. Further screening using an in vitro CutC inhibitory assay identified a benzoxazole ligand (BO-I) as a CutC inhibitor with an IC50 value of 2.4 ± 0.3 μM. Kinetic analysis revealed that BO-I functions as a non-competitive inhibitor of CutC. Interestingly, BO-I reduced the production of TMA in whole cell assays of multiple bacterial strains as well as in complex biological environments. Therefore, structural optimization of BO-I holds promise for the development of efficient gut microbiota-targeted small molecules.

[152] M. Dumitrascuta, M. Bermudez, T. Ben Haddou, E. Guerrieri, L. Schlafer, A. Ritsch, S. Hosztafi, A. Lantero, C. Kreutz, D. Massotte, H. Schmidhammer, G. Wolber, M. Spetea. N-phenethyl substitution in 14-methoxy-n-methylmorphinan-6-ones turns selective mu opioid receptor ligands into dual µ/δ opioid receptor agonists, Sci Rep-Uk, 10(1):5653, 2020.
Links: [doi:10.1038/s41598-020-62530-w] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Morphine and structurally-derived compounds are mu opioid receptor (mu OR) agonists, and the most effective analgesic drugs. However, their usefulness is limited by serious side effects, including dependence and abuse potential. The N-substituent in morphinans plays an important role in opioid activities in vitro and in vivo. This study presents the synthesis and pharmacological evaluation of new N-phenethyl substituted 14-O-methylmorphinan-6-ones. Whereas substitution of the N-methyl substituent in morphine (1) and oxymorphone (2) by an N-phenethyl group enhances binding affinity, selectivity and agonist potency at the mu OR of 1a and 2a, the N-phenethyl substitution in 14-methoxy-N-methylmorphinan-6-ones (3 and 4) converts selective mu OR ligands into dual mu/delta OR agonists (3a and 4a). Contrary to N-methylmorphinans 1-4, the N-phenethyl substituted morphinans 1a-4a produce effective and potent antinociception without motor impairment in mice. Using docking and molecular dynamics simulations with the mu OR, we establish that N-methylmorphinans 1-4 and their N-phenethyl counterparts 1a-4a share several essential receptor-ligand interactions, but also interaction pattern differences related to specific structural features, thus providing a structural basis for their pharmacological profiles. The emerged structure-activity relationships in this class of morphinans provide important information for tuning in vitro and in vivo opioid activities towards discovery of effective and safer analgesics.

[151] M. Dumitrascuta, M. Bermudez, S. Ballet, G. Wolber, M. Spetea. Mechanistic understanding of peptide analogues, dalda, [Dmt1]DALDA, and KGOP01, binding to the µ opioid receptor, Molecules, 25(9):2087, 2020.
Links: [doi:10.3390/molecules25092087] [Publisher][Download citation: BibTeX | RIS]
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Abstract: The mu opioid receptor (MOR) is the primary target for analgesia of endogenous opioid peptides, alkaloids, synthetic small molecules with diverse scaffolds, and peptidomimetics. Peptide-based opioids are viewed as potential analgesics with reduced side effects and have received constant scientific interest over the years. This study focuses on three potent peptide and peptidomimetic MOR agonists, DALDA, [Dmt(1)]DALDA, and KGOP01, and the prototypical peptide MOR agonist DAMGO. We present the first molecular modeling study and structure-activity relationships aided by in vitro assays and molecular docking of the opioid peptide analogues, in order to gain insight into their mode of binding to the MOR. In vitro binding and functional assays revealed the same rank order with KGOP01 > [Dmt(1)]DALDA > DAMGO > DALDA for both binding and MOR activation. Using molecular docking at the MOR and three-dimensional interaction pattern analysis, we have rationalized the experimental outcomes and highlighted key amino acid residues responsible for agonist binding to the MOR. The Dmt (2 ',6 '-dimethyl-L-Tyr) moiety of [Dmt(1)]DALDA and KGOP01 was found to represent the driving force for their high potency and agonist activity at the MOR. These findings contribute to a deeper understanding of MOR function and flexible peptide ligand-MOR interactions, that are of significant relevance for the future design of opioid peptide-based analgesics.

[150] W. Du, D. Machalz, Q. Yan, E. J. Sorensen, G. Wolber, M. Bureik. Importance of asparagine-381 and arginine-487 for substrate recognition in CYP4Z1, Biochem. Pharmacol., 174:113850, 2020.
Links: [doi:10.1016/j.bcp.2020.113850] [Publisher][Download citation: BibTeX | RIS]
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Abstract: The human cytochrome P450 enzyme CYP4Z1 remains an understudied enzyme despite its association with poor prognosis and overexpression in breast cancer. Hence, CYP4Z1 has previously been suggested as an anti-breast cancer target. In the present study we employed extended mutation analysis to increase our understanding of the substrate binding mode of this enzyme. In a combined in vitro and in silico approach we show for the first time that residue Arg487 plays an important role in substrate recognition and binding of CYP4Z1. Using a large array of recombinant CYP4Z1 mutants we show that, apart from Asn381, all other postulated binding residues only play an auxiliary role in substrate recognition and binding. Different substrate interaction motifs were identified via dynamic pharmacophores (dynophores) and their impact on catalytically competent substrate binding was classified. These new insights on the substrate recognition and binding mode represent an important step towards the rational design of CYP4Z1 prodrugs and guide further investigations into the so far poorly understood physiological role of CYP4Z1.

[149] K. Denzinger, T. N. Nguyen, T. Noonan, G. Wolber, M. Bermudez. Biased ligands differentially shape the conformation of the extracellular loop region in 5-HT2B receptors, International Journal of Molecular Sciences, 21(24):9728, 2020.
Links: [doi:10.3390/ijms21249728] [Publisher][Download citation: BibTeX | RIS]
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Abstract: G protein-coupled receptors are linked to various intracellular transducers, each pathway associated with different physiological effects. Biased ligands, capable of activating one pathway over another, are gaining attention for their therapeutic potential, as they could selectively activate beneficial pathways whilst avoiding those responsible for adverse effects. We performed molecular dynamics simulations with known beta-arrestin-biased ligands like lysergic acid diethylamide and ergotamine in complex with the 5-HT2B receptor and discovered that the extent of ligand bias is directly connected with the degree of closure of the extracellular loop region. Given a loose allosteric coupling of extracellular and intracellular receptor regions, we delineate a concept for biased signaling at serotonin receptors, by which conformational interference with binding pocket closure restricts the signaling repertoire of the receptor. Molecular docking studies of biased ligands gathered from the BiasDB demonstrate that larger ligands only show plausible docking poses in the ergotamine-bound structure, highlighting the conformational constraints associated with bias. This emphasizes the importance of selecting the appropriate receptor conformation on which to base virtual screening workflows in structure-based drug design of biased ligands. As this mechanism of ligand bias has also been observed for muscarinic receptors, our studies provide a general mechanism of signaling bias transferable between aminergic receptors.

[148] M. Bermudez, M. Grabowski, M. S. Murgueitio, M. Tiemann, P. Varga, T. Rudolf, G. Wolber, G. Weindl, J. Rademann. Biological characterization, mechanistic investigation and structure-activity relationships of chemically stable TLR2 antagonists, ChemMedChem, 15(14):1364-1371, 2020.
Links: [doi:10.1002/cmdc.202000060] [Publisher][Download citation: BibTeX | RIS]
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Abstract: Toll-like receptors (TLRs) build the first barrier in the innate immune response and therefore represent promising targets for the modulation of inflammatory processes. Recently, the pyrogallol-containing TLR2 antagonists CU-CPT22 and MMG-11 were reported; however, their 1,2,3-triphenol motif renders them highly susceptible to oxidation and excludes them from use in extended experiments under aerobic conditions. Therefore, we have developed a set of novel TLR2 antagonists (1-9) based on the systematic variation of substructures, linker elements, and the hydrogen-bonding pattern of the pyrogallol precursors by using chemically robust building blocks. The novel series of chemically stable and synthetically accessible TLR2 antagonists (1-9) was pharmacologically characterized, and the potential binding modes of the active compounds were evaluated structurally. Our results provide new insights into structure-activity relationships and allow rationalization of structural binding characteristics. Moreover, they support the hypothesis that this class of TLR ligands bind solely to TLR2 and do not directly interact with TLR1 or TLR6 of the functional heterodimer. The most active compound from this series (6), is chemically stable, nontoxic, TLR2-selective, and shows a similar activity with regard to the pyrogallol starting points, thus indicating the variability of the hydrogen bonding pattern.

[147] L. Agnetta, M. Bermudez, F. Riefolo, C. Matera, E. Claro, R. Messerer, T. Littmann, G. Wolber, U. Holzgrabe, M. Decker. Fluorination of photoswitchable muscarinic agonists tunes receptor pharmacology and photochromic properties, J. Med. Chem., 62(6):3009-3020, 2019.
Links: doi:10.1021/acs.jmedchem.8b01822 | Publisher | Download citation: BibTeX RIS

[146] D. Sribar, M. Grabowski, M. S. Murgueitio, M. Bermudez, G. Weindl, G. Wolber. Identification and characterization of a novel chemotype for human TLR8 inhibitors, Eur. J. Med. Chem., 179:744-752, 2019.
Links: doi:10.1016/j.ejmech.2019.06.084 | Download citation: BibTeX RIS
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Abstract: The endosomal Toll-like receptor 8 (TLR8) recognizes single-stranded RNA and initiates early inflammatory responses. Despite the importance of endosomal TLRs for human host defense against microbial pathogens, extensive activation may contribute to autoimmune and inflammatory diseases. In contrast to the recent progress made in the development of modulators of plasma membrane-bound TLRs, little is known about endosomal TLR modulation and very few TLR8 inhibitors have been reported. In this study, we discovered and validated novel small-molecule TLR8 inhibitors. Fourteen potential TLR8 modulators were experimentally validated in HEK293T cells stably overexpressing human TLR8 and THP-1 macrophages. Five compounds inhibited TLR8-mediated signaling, representing a hit rate of 36%. The three most potent compounds neither cause cellular toxicity nor inhibition of TLR signaling induced by other receptor subtypes. Conclusively, we experimentally confirm novel and selective, pyrimidine-based TLR8 inhibitors with low cytotoxicity that are relevant candidates for lead optimization and further mechanistic studies.

[145] D. Schaller, S. Pach, G. Wolber. PyRod: Tracing water molecules in molecular dynamics simulations, J. Chem. Inf. Model., 59(6):2818-2829, 2019.
Links: doi:10.1021/acs.jcim.9b00281 | Publisher | Download citation: BibTeX RIS
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Abstract: Ligands entering a protein binding pocket essentially compete with water molecules for binding to the protein. Hence, the location and thermodynamic properties of water molecules in protein structures have gained increased attention in the drug design community. Including corresponding data into 3D pharmacophore modeling is essential for efficient high throughput virtual screening. Here, we present PyRod, a free and open-source Python software that allows for visualization of pharmacophoric binding pocket characteristics, identification of hot spots for ligand binding, and subsequent generation of pharmacophore features for virtual screening. The implemented routines analyze the protein environment of water molecules in molecular dynamics (MD) simulations and can differentiate between hydrogen bonded waters as well as waters in a protein environment of hydrophobic, charged, or aromatic atom groups. The gathered information is further processed to generate dynamic molecular interaction fields (dMIFs) for visualization and pharmacophoric features for virtual screening. The described software was applied to 5 therapeutically relevant drug targets, and generated pharmacophores were evaluated using DUD-E benchmarking sets. The best performing pharmacophore was found for the HIV1 protease with an early enrichment factor of 54.6. PyRod adds a new perspective to structure-based screening campaigns by providing easy-to-interpret dMIFs and purely protein-based pharmacophores that are solely based on tracing water molecules in MD simulations. Since structural information about cocrystallized ligands is not needed, screening campaigns can be followed, for which less or no ligand information is available. PyRod is freely available at https://github.com/schallerdavid/pyrod .

[144] O. Zierau, A. Kolodziejczyk, G. Vollmer, D. Machalz, G. Wolber, D. Thieme, A. M. Keiler. Comparison of the three SARMs RAD-140, GLPG0492 and GSK-2881078 in two different in vitro bioassays, and in an in silico androgen receptor binding assay, J. Steroid Biochem. Mol. Biol., 189:81-86, 2019.
Links: doi:10.1016/j.jsbmb.2019.02.014 | Download citation: BibTeX RIS
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Abstract: Selective androgen receptor modulators comprise compounds that bind as ligands to the androgen receptor and possess tissue-selective activities. Ideally, they show agonistic properties in anabolic target tissues, while inducing antagonistic or only weak agonistic effects in reproductive organs. Due to their myoanabolic effects, selective androgen receptor modulators are included in the list of prohibited substances and methods of the World Anti-Doping Agency. In the current investigation, the androgenic potential of RAD-140, GSK-2881078 and GLPG0492 was comparably investigated in two different in vitro bioassays. In the yeast androgen screen, the androgenic effects were lower than in the reporter gene assay in prostate carcinoma cells (e.g. for GSK-2881078, the EC50 values were 4.44 × 10−6M in the yeast screen and 3.99 × 10-9M in the prostate cells respectively). For future investigations, it is of importance whether the yeast androgen screen, which has been proven to detect androgenic compounds in urine, can detect an abuse of the selective androgen receptor modulators. Molecular modeling of the binding to the androgen receptor ligand binding domain suggests slight differences in the binding modes of RAD-140, GSK-2881078 and GLPG0492. In conclusion, androgenic activity of the three non-steroidal compounds in the two different in vitro test systems confirmed the results of the in silico modeling of the androgen receptor binding.

[143] N. K. Wenke, J. Kreye, E. Andrzejak, A. van Casteren, J. Leubner, M. S. Murgueitio, S. M. Reincke, C. Secker, L. Schmidl, C. Geis, F. Ackermann, M. Nikolaus, C. C. Garner, H. Wardemann, G. Wolber, H. Pruess. NMDA receptor dysfunction via unmutated human antibodies against the NR1 subunit, Ann. Neurol., 85(5):771-776, 2019.
Links: doi:10.1002/ana.25460 | Publisher | Download citation: BibTeX RIS
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Abstract: Anti‐N‐methyl‐D‐aspartate‐receptor (NMDAR) encephalitis is the most common autoimmune encephalitis related to autoantibody‐mediated synaptic dysfunction. Cerebrospinal fluid‐derived human monoclonal NR1 autoantibodies showed low numbers of somatic hypermutations or were unmutated. These unexpected germline‐configured antibodies showed weaker binding to the NMDAR than matured antibodies from the same patient. In primary hippocampal neurons, germline NR1 autoantibodies strongly and specifically reduced total and synaptic NMDAR currents in a dose‐ and time‐dependent manner. The findings suggest that functional NMDAR antibodies are part of the human naïve B cell repertoire. Given their effects on synaptic function, they might contribute to a broad spectrum of neuropsychiatric symptoms.

[142] A. Stoll, S. Loke, J. F. Joseph, D. Machalz, X. de la Torre, F. Botrè, G. Wolber, M. Bureik, M. K. Parr. Fine-mapping of the substrate specificity of human steroid 21-hydroxylase (CYP21A2), J. Steroid Biochem. Mol. Biol., 194:105446, 2019.
Links: doi:10.1016/j.jsbmb.2019.105446 | Download citation: BibTeX RIS
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Abstract: Cytochrome P450 enzymes (CYPs) are capable of catalyzing regio- and stereo-specific oxy functionalization reactions which otherwise are major challenges in organic chemistry. In order to make the best possible use of these biocatalysts it is imperative to understand their specificities. Human CYP21A2 (steroid 21-hydroxylase) acts on the side-chain attached to C-17 in ring D of a steroid substrate, but the configuration of ring A also plays a prominent role in substrate cognition. Here, we comprehensively investigated this relationship using sixteen 17,17-dimethyl-18-nor-13-ene steroids with different arrangements of hydroxy-, oxo-, fluoro- and chloro-groups and in the presence or absence of double bonds (Δ1 and/or Δ4) and heteroatoms in ring A. The results show that presence of a 3-oxo group is a strict requirement for a CYP21A2 substrate, while the other configurations tested were all tolerated. This was also confirmed by control experiments using endogenous steroids. While progesterone and 17-hydroxyprogesterone were hydroxylated at C-21, (17-hydroxy-) pregnenolone did not react. Molecular docking experiments indicate that the interaction of the carbonyl group at C-3 to the side-chain Arg234 of the enzyme is indispensable.

[141] S. Schramm, L. Agnetta, M. Bermudez, H. Gerwe, M. Irmen, J. Holze, T. Littmann, G. Wolber, C. Trankle, M. Decker. Novel BQCA- and TBPB-derived M1 receptor hybrid ligands: Orthosteric carbachol differentially regulates partial agonism, ChemMedChem, 14(14):1349-1358, 2019.
Links: doi:10.1002/cmdc.201900283 | Publisher | Download citation: BibTeX RIS
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Abstract: Recently, investigations of the complex mechanisms of allostery have led to a deeper understanding of G protein-coupled receptor (GPCR) activation and signaling processes. In this context, muscarinic acetylcholine receptors (mAChRs) are highly relevant due to their exemplary role in the study of allosteric modulation. In this work, we compare and discuss two sets of putatively dualsteric ligands, which were designed to connect carbachol to different types of allosteric ligands. We chose derivatives of TBPB [1-(1'-(2-tolyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one] as M1 -selective putative bitopic ligands, and derivatives of benzyl quinolone carboxylic acid (BQCA) as an M1 positive allosteric modulator, varying the distance between the allosteric and orthosteric building blocks. Luciferase protein complementation assays demonstrated that linker length must be carefully chosen to yield either agonist or antagonist behavior. These findings may help to design biased signaling and/or different extents of efficacy.

[140] D. Schaller, S. Hagenow, H. Stark, G. Wolber. Ligand-guided homology modeling drives identification of novel histamine H3 receptor ligands, PLoS One, 14(6):e0218820, 2019.
Links: doi:10.1371/journal.pone.0218820 | Publisher | Download citation: BibTeX RIS
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Abstract: In this study, we report a ligand-guided homology modeling approach allowing the analysis of relevant binding site residue conformations and the identification of two novel histamine H3 receptor ligands with binding affinity in the nanomolar range. The newly developed method is based on exploiting an essential charge interaction characteristic for aminergic G-protein coupled receptors for ranking 3D receptor models appropriate for the discovery of novel compounds through virtual screening.

[139] R. Ottanà, P. Paoli, G. Lori, I. Adornato, S. Previti, A. Naß, G. Wolber, R. Maccari. Design and evaluation of non-carboxylate 5-arylidene-2-thioxo-4-imidazolidinones as novel non-competitive inhibitors of protein tyrosine phosphatase 1B, Bioorg. Chem., 92:103211, 2019.
Links: doi:10.1016/j.bioorg.2019.103211 | Publisher | Download citation: BibTeX RIS
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Abstract: Protein tyrosine phosphatase 1B (PTP1B) acts as a negative regulator of insulin and leptin signalling and is crucially involved in the development of type 2 diabetes mellitus, obesity, cancer and neurodegenerative diseases. Pursuing our efforts to identify PTP1B inhibitors endowed with drug-like properties, we designed and evaluated 3-aryl-5-arylidene-2-thioxo-4-imidazolidinones (7) as a novel class of non-carboxylate PTP1B inhibitors. In agreement with our design, kinetic studies demonstrated that selected compounds 7 act as reversible, non-competitive inhibitors of the target enzyme at low micromolar concentrations. Accordingly, molecular docking experiments suggested that these inhibitors can fit an allosteric site of PTP1B that we previously individuated. Moreover, cellular assays demonstrated that compound 7e acts as a potent insulin-sensitizing agent in human liver HepG2 cells. Taken together, our results showed that these non-competitive PTP1B inhibitors can be considered promising lead compounds aimed to enhance druggability of the target enzyme and identify novel antidiabetic drugs.

[138] M. J. Ojeda-Montes, A. Casanova-Marti, A. Gimeno, S. Tomas-Hernandez, A. Cereto-Massague, G. Wolber, R. Beltran-Debon, C. Valls, M. Mulero, M. Pinent, G. Pujadas, S. Garcia-Vallve. Mining large databases to find new leads with low similarity to known actives: application to find new DPP-IV inhibitors, Future Med. Chem., 11(12):1387-1401, 2019.
Links: doi:10.4155/fmc-2018-0597 | Publisher | Download citation: BibTeX RIS
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Abstract: Aim: Fragment-based drug design or bioisosteric replacement is used to find new actives with low (or no) similarity to existing ones but requires the synthesis of nonexisting compounds to prove their predicted bioactivity. Protein-ligand docking or pharmacophore screening are alternatives but they can become computationally expensive when applied to very large databases such as ZINC. Therefore, fast strategies are necessary to find new leads in such databases. Materials & methods: We designed a computational strategy to find lead molecules with very low (or no) similarity to existing actives and applied it to DPP-IV. Results: The bioactivity assays confirm that this strategy finds new leads for DPP-IV inhibitors. Conclusion: This computational strategy reduces the time of finding new lead molecules.

[137] A. Nass, D. Schaller, G. Wolber. Assessment of flexible shape complementarity: New opportunities to explain and induce selectivity in ligands of protein tyrosine phosphatase 1B, Mol. Inform., 38(5):1800141, 2019.
Links: doi:10.1002/minf.201800141 | Publisher | Download citation: BibTeX RIS
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Abstract: For drug design projects it is essential to rationally induce and explain selectivity. In this context shape complementarity as well as protein and ligand flexibility represent important factors. Currently available tools for the analysis of protein-ligand interactions focus mainly on electrostatic complementarity and/or static structures. Here we address the shortcomings of available methods by presenting two new tools: The first one can be used to assess steric complementarity in flexible protein-ligand complexes in order to explain selectivity of known ligands. It further allows to determine ligand atoms with especially good or bad shape-fit which can be of use in lead optimization projects. The second tool was designed to detect differences in protein flexibility in similar proteins along with their exploitation for virtual screening. Both tools yield interesting results when applied to data of protein tyrosine phosphatase 1B (PTP1B): The case of PTP1B has proven especially difficult in terms of selectivity, due to a closely related phosphatase connected to severe undesired effects. With our tool for steric complementarity assessment we were able to explain previously undisclosed causes of moderate selectivity of selected PTP1B ligands. The second tool allowed us to find differences of flexibility in the two highly similar proteins and give directions for exploitation in virtual screening.

[136] P. Durairaj, L. Fan, D. Machalz, G. Wolber, M. Bureik. Functional characterization and mechanistic modeling of the human cytochrome P450 enzyme CYP4A22, FEBS Lett., 593(16):2214-2225, 2019.
Links: doi:10.1002/1873-3468.13489 | Publisher | Download citation: BibTeX RIS
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Abstract: The human cytochrome P450 (CYP) enzyme CYP4A22 is an orphan CYP with unknown function. Here, through functional expression in fission yeast, we show that CYP4A22 catalyzes fatty acid hydroxylation as well as aliphatic or aromatic hydroxylations of luciferin-based probe substrates. Mechanistic molecular modeling of CYP4A22 suggests that its ω-hydroxylation activity is hampered by a more spacious active site compared to CYP4B1. Substrate recognition via side-chains R96 and R233 is indicated by dynamic three-dimensional pharmacophores (dynophores) derived from molecular dynamics simulations. CYP4A22 activity is inhibited by three unspecific CYP inhibitors. A comparison of CYP4A22*1 (the reference standard sequence) with CYP4A22-WT (the most common allele) revealed that for the four substrates tested the WT-enzyme always had lower activity.

[135] M. Bermudez, T. N. Nguyen, C. Omieczynski, G. Wolber. Strategies for the discovery of biased GPCR ligands, Drug Discov. Today, 24(4):1031-1037, 2019.
Links: doi:10.1016/j.drudis.2019.02.010 | Download citation: BibTeX RIS
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Abstract: G-protein-coupled receptors (GPCRs) represent important drug targets with complex pharmacological characteristics. Biased signaling represents one important dimension, describing ligand-dependent shifts of naturally imprinted signaling profiles. Because biased GPCR modulators provide potential therapeutic benefits including higher efficiencies and reduced adverse effects, the identification of such ligands as drug candidates is highly desirable. This review aims to provide an overview of the challenges and strategies in the discovery of biased ligands. We show different approaches for biased ligand discovery in the example of G-protein-biased opioid analgesics and discuss possibilities to design biased ligands by targeting extracellular receptor regions.

[134] H. Aygun Cevher, D. Schaller, M. A. Gandini, O. Kaplan, E. Gambeta, F. X. Zhang, M. Celebier, M. N. Tahir, G. W. Zamponi, G. Wolber, M. G. Gunduz. Discovery of Michael acceptor containing 1,4-dihydropyridines as first covalent inhibitors of L-/T-type calcium channels, Bioorg. Chem., 91:103187, 2019.
Links: doi:10.1016/j.bioorg.2019.103187 | Publisher | Download citation: BibTeX RIS
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Abstract: 1,4-Dihydropyridines (DHPs) are an important class of blockers targeting different calcium channel subtypes and have great therapeutic value against cardiovascular and neurophysiologic conditions. Here, we present the design of DHP-based hexahydroquinoline derivatives as either selective or covalent inhibitors of calcium channels. These compounds were synthesized via a modified Hantzsch reaction under microwave irradiation and characterized by IR, (1)H NMR, (13)C NMR and mass spectra. Additionally, the proposed structure of HM12 was resolved by single crystal X-ray analysis. The abilities of the target compounds to block both L- and T-type calcium channels were evaluated by utilizing the whole-cell patch clamp technique. Our results identified covalent inhibitors of calcium channels for the first time, which could be achieved by introducing a Michael acceptor group into the ester side chain of the compounds. The proposed covalent binding between the compounds and the cysteine amino acid (Cys1492) within the DHP binding pocket of L-type calcium channel was supported by docking and pharmacophore analysis as well as a glutathione reactivity assay.

[133] R. Schulz, A. Atef, D. Becker, F. Gottschalk, C. Tauber, S. Wagner, C. Arkona, A. A. Abdel-Hafez, H. H. Farag, J. Rademann, G. Wolber. Phenylthiomethyl ketone-based fragments show selective and irreversible inhibition of enteroviral 3C proteases, J. Med. Chem., 61(3):1218-1230, 2018.
Links: doi:10.1021/acs.jmedchem.7b01440 | Publisher | Download citation: BibTeX RIS
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Abstract: Lead structure discovery mainly focuses on the identification of noncovalently binding ligands. Covalent linkage, however, is an essential binding mechanism for a multitude of successfully marketed drugs although discovered by serendipity in most cases. We present a concept for the design of fragments covalently binding to proteases. Covalent linkage enables fragment binding unrelated to affinity to shallow protein binding sites and at the same time allows differentiated targeted hit verification and binding location verification through mass spectrometry. We describe a systematic and rational computational approach for the identification of covalently binding fragments from compound collections inhibiting enteroviral 3C protease, a target with high therapeutic potential. By implementing reactive groups potentially forming covalent bonds as chemical feature in our 3D pharmacophore methodology, covalent binders were discovered by high-throughput virtual screening. We present careful experimental validation of the virtual hits using enzymatic assays and mass spectrometry unraveling a novel, previously unknown irreversible inhibition of the 3C protease by phenylthiomethyl ketone-based fragments. Subsequent synthetic optimization through fragment growing and reactivity analysis against catalytic and non-catalytic cysteines revealed specific irreversible 3C protease inhibition.

[132] D. Schaller, M. G. Gündüz, F. X. Zhang, G. W. Zamponi, G. Wolber. Binding mechanism investigations guiding the synthesis of novel condensed 1,4-dihydropyridine derivatives with L-/T-type calcium channel blocking activity, Eur. J. Med. Chem., 155:1-12, 2018.
Links: doi:10.1016/j.ejmech.2018.05.032 | Download citation: BibTeX RIS
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Abstract: Nifedipine and isradipine are prominent examples of calcium channel blockers with a 1,4-dihydropyridine (DHP) scaffold. Although successfully used in clinics since decades for the treatment of hypertension, the binding mechanism to their target, the L-type voltage-gated calcium channel Cav1.2, is still incompletely understood. Recently, novel DHP derivatives with a condensed ring system have been discovered that show distinct selectivity profiles to different calcium channel subtypes. This property renders this DHP class as a promising tool to achieve selectivity towards distinct calcium channel subtypes. In this study, we identified a common binding mode for prominent DHPs nifedipine and isradipine using docking and pharmacophore analysis that is also able to explain the structure-activity relationship of a small subseries of DHP derivatives with a condensed ring system. These findings were used to guide the synthesis of twenty-two novel DHPs. An extensive characterization using 1H NMR, 13C NMR, mass spectra and elemental analysis was followed by whole cell patch clamp assays for analyzing activity at Cav1.2 and Cav3.2. Two compounds were identified with significant activity against Cav1.2. Additionally, we identified four compounds active against Cav3.2 of which three were selective over Cav1.2. Novel binding modes were analyzed using docking and pharmacophore analysis as well as molecular dynamics simulations.

[131] A. Tkachenko, M. Bermudez, S. Irmer-Stooff, D. Genkinger, F. Henkler-Stephani, G. Wolber, A. Luch. Nuclear transport of the human aryl hydrocarbon receptor and subsequent gene induction relies on its residue histidine 291, Arch. Toxicol., 92(3):1151-1160, 2018.
Links: doi:10.1007/s00204-017-2129-0 | Publisher | Download citation: BibTeX RIS
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Abstract: The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor involved in the metabolism of physiological substances and xenobiotics, representing an interesting target in both toxicology and pharmacology. In this study, we investigated the ligand-dependent conjunction of nuclear import of the human AHR in living cells and target gene induction. Our findings strengthen the theory that the AHR triggers a precisely defined and rapid reaction upon binding to endogenous ligands, while the xenobiotic β-naphthoflavone only induces rather unspecific and slow effects. To better illuminate the ligand-mediated responses of the human AHR, we applied site-directed mutagenesis and identified histidine 291 as key residue for AHR functionality, essential for both nuclear import and target gene induction. Contrary, replacing histidine at position 291 by alanine did not affect nucleo-cytoplasmic shuttling, showing that permanent endogenous import and ligand-induced import of the AHR into the nucleus are two independent and differently regulated processes. Combining these observations with our structural investigations using a homology model of the AHR-PAS B domain, we suggest a dual role of histidine 291: (1) a major role for shaping the ligand binding site including direct interactions with ligands and, (2) an essential role for the conformational dynamics of a PAS B loop, which most likely influences the association of the AHR with the AHR nuclear translocator through interference with their protein–protein interface.

[130] R. Maccari, A. Del Corso, P. Paoli, I. Adornato, G. Lori, F. Balestri, M. Cappiello, A. Naß, G. Wolber, R. Ottana. An investigation on 4-thiazolidinone derivatives as dual inhibitors of aldose reductase and protein tyrosine phosphatase 1B, in the search for potential agents for the treatment of type 2 diabetes mellitus and its complications, Bioorg. Med. Chem. Lett., 28(23-24):3712-3720, 2018.
Links: doi:10.1016/j.bmcl.2018.10.024 | Download citation: BibTeX RIS
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Abstract: Designed multiple ligands (DMLs), developed to modulate simultaneously a number of selected targets involved in etiopathogenetic mechanisms of a multifactorial disease, such as diabetes mellitus (DM), are considered a promising alternative to combinations of drugs, when monotherapy results to be unsatisfactory. In this work, compounds 1–17 were synthesized and in vitro evaluated as DMLs directed to aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two key enzymes involved in different events which are critical for the onset and progression of type 2 DM and related pathologies. Out of the tested 4-thiazolidinone derivatives, compounds 12 and 16, which exhibited potent AR inhibitory effects along with interesting inhibition of PTP1B, can be assumed as lead compounds to further optimize and balance the dual inhibitory profile. Moreover, several structural portions were identified as features that could be useful to achieve simultaneous inhibition of both human AR and PTP1B through binding to non-catalytic regions of both target enzymes.

[129] J. Liu, L. Chen, J. F. Joseph, A. Naß, A. Stoll, X. de la Torre, F. Botrè, G. Wolber, M. K. Parr, M. Bureik. Combined chemical and biotechnological production of 20βOH-NorDHCMT, a long-term metabolite of Oral-Turinabol (DHCMT), J. Inorg. Biochem., 183:165-171, 2018.
Links: doi:10.1016/j.jinorgbio.2018.02.020 | Download citation: BibTeX RIS
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Abstract: Anabolic androgenic steroids (AAS) are misused very frequently in sport competitions as performance enhancing agents. One of the doping compounds that has been detected with increased frequency in the last few years is dehydrochloromethyltestosterone (DHCMT, 4-chloro-17β-hydroxy-17α-methylandrosta-1,4-dien-3-one; brand name Oral Turinabol). The long-term DHCMT metabolite 20βOH-NorDHCMT (4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one) was reported earlier to be detectable in urine samples for more than 22 days after DHCMT administration; however, purified reference material was not available so far. In this study we demonstrate a successful combination of Wagner-Meerwein rearrangement of DHCMT to NorDHCMT (4-chloro-17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one) and subsequent whole-cell biotransformation with a recombinant fission yeast strain expressing the human cytochrome P450 enzyme (CYP or P450) CYP21A2 for the synthesis of mg amounts of this metabolite. It was then used as reference for the analysis of a post administration urine of DHCMT. The availability of this reference compound will provide an incontestable proof for DHCMT abuse.

[128] A. M. Keiler, O. Zierau, S. Wolf, P. Diel, W. Schänzer, G. Vollmer, D. Machalz, G. Wolber, M. K. Parr. Androgen- and estrogen-receptor mediated activities of 4-hydroxytestosterone, 4-hydroxyandrostenedione and their human metabolites in yeast based assays, Toxicol. Lett., 292:39-45, 2018.
Links: doi:10.1016/j.toxlet.2018.04.026 | Publisher | Download citation: BibTeX RIS
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Abstract: 4-Hydroxyandrost-4-ene-3,17-dione, also named formestane, is an irreversible aromatase inhibitor and therapeutically used as anti-breast cancer medication in post-menopausal women. Currently, no therapeutical indication led to approval of its 17-hydroxylated analog 4-hydroxytestosterone, an anabolic steroid. However, it is currently investigated in a clinical trial for breast cancer. In context with sports doping, aromatase inhibitors are administered to reduce estrogenic side effects of misused anabolic substances or their metabolites. Therefore, both substances are prohibited in sports by the World Anti-Doping Agency (WADA). Analysis of urinary phase I and phase II metabolites showed similar results for both compounds. In the current investigation, 4-hydroxyandrost-4-ene-3,17-dione, 4-hydroxytestosterone and seven of their described urinary metabolites as well as 2α-hydroxyandrostenedione were tested in the yeast androgen screen and the yeast estrogen screen. Androgenic effects were observed for all tested substances, except for one, which showed anti-androgenic properties. With regard to the yeast estrogen screen, estrogenic effects were observed for only two metabolites at rather high concentrations, while six out of the ten substances tested showed anti-estrogenic properties. In terms of the strong androgenic effect observed for 4-hydroxytestosterone (10−8 M), 4-hydroxyandrost-4-ene-3,17-dione (10−8 M) and two more urinary metabolites, the yeast androgen assay may also be used to trace abuse in urine samples.

[127] M. Grabowski, M. S. Murgueitio, M. Bermudez, J. Rademann, G. Wolber, G. Weindl. Identification of a pyrogallol derivative as a potent and selective human TLR2 antagonist by structure-based virtual screening, Biochem. Pharmacol., 154:148-160, 2018.
Links: doi:10.1016/j.bcp.2018.04.018 | Download citation: BibTeX RIS
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Abstract: Toll-like receptor 2 (TLR2) induces early inflammatory responses to pathogen and damage-associated molecular patterns trough heterodimerization with either TLR1 or TLR6. Since overstimulation of TLR2 signaling is linked to several inflammatory and metabolic diseases, TLR2 antagonists may provide therapeutic benefits for the control of inflammatory conditions. We present virtual screening for the identification of novel TLR2 modulators, which combines analyses of known ligand sets with structure-based approaches. The 13 identified compounds were pharmacologically characterized in HEK293-hTLR2 cells, THP-1 macrophages and peripheral blood mononuclear cells for their ability to inhibit TLR2-mediated responses. Four out of 13 selected compounds show concentration-dependent activity, representing a hit rate of 31%. The most active compound is the pyrogallol derivative MMG-11 that inhibits both TLR2/1 and TLR2/6 signaling and shows a higher potency than the previously discovered CU-CPT22. Concentration ratio analysis identified both compounds as competitive antagonists of Pam3CSK4- and Pam2CSK4-induced responses. Schild plot analysis yielded apparent pA2 values of 5.73 and 6.15 (TLR2/1), and 5.80 and 6.65 (TLR2/6) for CU-CPT22 and MMG-11, respectively. MMG-11 neither shows cellular toxicity nor interference with signaling induced by other TLR agonists, IL-1β or TNF. Taken together, we demonstrate that MMG-11 is a potent and selective TLR2 antagonist with low cytotoxicity rendering it a promising pharmacological tool for the investigation of TLR signaling and a suitable lead structure for further chemical optimization.

[126] Fan Yang, David Machalz, Sisi Wang, Zhengyi Li, Gerhard Wolber, Matthias Bureik. A common polymorphic variant of UGT1A5 displays increased activity due to optimized cofactor binding, FEBS Lett., 592(11):1837-1846, 2018.
Links: doi:10.1002/1873-3468.13072 | Publisher | Download citation: BibTeX RIS
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Abstract: Uridine diphosphate‐glucuronosyltransferases (UGTs) are the most important phase II enzymes in human drug metabolism. Using permeabilized recombinant fission yeast cells (enzyme bags), we demonstrate that UGT1A5 can catalyze an N‐glucuronidation reaction. We characterized two new polymorphic UGT1A5 variants: a common ninefold mutant (UGT1A5*8) with double‐fold activity and a much rarer sixfold mutant (UGT1A5*9), which has the same activity as the wild‐type. Molecular modeling studies indicate that the minor effects of all mutations, except for Gly259Arg, are due to their distance to the substrate binding site. Extensive molecular dynamics simulations revealed that the Gly259Arg mutation stabilizes helix Q through a newly formed hydrogen bonding network, which places the cofactor in a much more favorable geometry in UGT1A5*8 as compared to the wild‐type.

[125] R. Ottana, P. Paoli, A. Naß, G. Lori, V. Cardile, I. Adornato, A. Rotondo, A. C. Eleonora Graziano, G. Wolber, R. Maccari. Discovery of 4-[(5-arylidene-4-oxothiazolidin-3-yl)methyl]benzoic acid derivatives active as novel potent allosteric inhibitors of protein tyrosine phosphatase 1B: In silico studies and in vitro evaluation as insulinomimetic and anti-inflammatory agents, Eur. J. Med. Chem., 127:840-858, 2017.
Links: doi:10.1016/j.ejmech.2016.10.063 | Publisher | Download citation: BibTeX RIS
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Abstract: New 4-{[5-arylidene-2-(4-fluorophenylimino)-4-oxothiazolidin-3-yl]methyl}benzoic acids (5) and 2-thioxo-4-thiazolidinone analogues (6) were synthesised as a part of a continuing search for new inhibitors of protein tyrosine phosphatase 1B (PTP1B), an enzyme which is implicated in metabolic disorders and inflammatory signaling. Most of the tested compounds were shown to be potent PTP1B inhibitors. Moreover, their inhibition mechanism was markedly influenced by the substituents in the positions 2 and 5, as kinetic studies indicated. Docking experiments suggested that certain derivatives 5 and 6 may efficiently fit into an allosteric site positioned between the β-sheet including Leu71 and Lys73 and a lipophilic pocket closed by the loop consisting of Pro210 to Leu 204. In cellular assays, several of these new 4-thiazolidinone derivatives showed insulinomimetic and anti-inflammatory properties. Out of them, compound 5b exhibited the most promising profile, being able to promote the activation of both insulin receptor and downstream Akt protein as well as to increase 2-deoxyglucose cellular uptake. Interestingly, compound 5b was also able to interrupt critical events in inflammatory signalling.

[124] Q. Yan, D. Machalz, A. Zöllner, E. J. Sorensen, G. Wolber, M. Bureik. Efficient substrate screening and inhibitor testing of human CYP4Z1 using permeabilized recombinant fission yeast, Biochem. Pharmacol., 146:174-187, 2017.
Links: doi:10.1016/j.bcp.2017.09.011 | Publisher | Download citation: BibTeX RIS
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Abstract: We have established a protocol for the preparation of permeabilized fission yeast cells (enzyme bags) that recombinantly express human cytochrome P450 enzymes (CYPs). A direct comparison of CYP3A4 activity gave an eightfold higher space-time yield for enzyme bag-catalyzed biotransformation as compared to whole-cell biotransformation, even though the total number of cells employed was lower by a factor of 150. Biotransformation of the luminogenic substrate Luciferin-H using CYP2C9-containing enzyme bags proceeded efficiently and stably for 24h. CYP4Z1 is of interest because it is strongly overexpressed both in breast cancer cells and in breast cancer metastases; however, current knowledge about its catalytic properties is very limited. Screening of CYP4Z1-containing enzyme bags with 15 luminogenic substrates enabled us to identify two new hydroxylations and eleven ether cleavage reactions that are catalyzed by CYP4Z1. By far the best substrate found in this study was Luciferin benzyl ether (Luciferin-BE). On the basis of the recently published crystal structure of CYP4B1 we created a new homology model of CYP4Z1 and performed molecular docking experiments, which indicate that all active substrates show a highly similar binding geometry compared to the endogenous substrates. The model predicts that Ser113, Ser222, Asn381, and Ser383 are key hydrogen bonding residues. We also identified five new inhibitors of CYP4Z1: miconazole, econazole, aminobenzotriazole, tolazoline, and 1-benzylimidazole respectively, with the last compound being the most potent giving an IC50 value of 180nM in our test system.

[123] D. Schaller, S. Hagenow, G. Alpert, A. Naß, R. Schulz, M. Bermudez, H. Stark, G. Wolber. Systematic data mining reveals synergistic H3R/MCHR1 ligands, ACS Med. Chem. Lett., 8(6):648-653, 2017.
Links: doi:10.1021/acsmedchemlett.7b00118 | Publisher | Download citation: BibTeX RIS
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Abstract: In this study, we report a ligand-centric data mining approach that guided the identification of suitable target profiles for treating obesity. The newly developed method is based on identifying target pairs for synergistic positive effects and also encompasses the exclusion of compounds showing a detrimental effect on obesity treatment (off-targets). Ligands with known activity against obesity-relevant targets were compared using fingerprint representations. Similar compounds with activities to different targets were evaluated for the mechanism of action since activation or deactivation of drug targets determines the pharmacological effect. In vitro validation of the modeling results revealed that three known modulators of melanin-concentrating hormone receptor 1 (MCHR1) show a previously unknown submicromolar affinity to the histamine H3 receptor (H3R). This synergistic activity may present a novel therapeutic option against obesity.

[122] F. Sanz, [...], G. Wolber, I. Zamora. Legacy data sharing to improve drug safety assessment: the eTOX project, Nature Reviews Drug Discovery, 16:811, 2017.
Links: doi:10.1038/nrd.2017.177 | Publisher | Download citation: BibTeX RIS

[121] M. S. Murgueitio, C. Rakers, A. Frank, G. Wolber. Balancing Inflammation: Computational Design of Small-Molecule Toll-like Receptor Modulators, Trends Pharmacol. Sci., 38(2):155-168, 2017.
Links: doi:10.1016/j.tips.2016.10.007 | Publisher | Download citation: BibTeX RIS
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Abstract: As essential proteins of the innate immune system, Toll-like receptors (TLRs) are involved in a plethora of physiological pathologies and their modulation is an ongoing quest in the field of drug discovery. Although TLRs recognize an unusually broad range of different molecular patterns, only a few small-molecule TLR modulators have been reported to date. Recent advances in crystallography and in silico techniques provide promising opportunities for TLR investigations and drug design. Here, three application areas for computational approaches are considered: (i) exploration of TLR structure and activation; (ii) understanding TLR modulation; and (iii) TLR drug discovery. By providing an overview on state-of-the-art computational methods, we highlight the value of molecular modeling in mechanistically understanding TLR function and guiding drug design.

[120] M. S. Murgueitio, S. Ebner, P. Hörtnagl, C. Rakers, R. Bruckner, P. Henneke, G. Wolber, S. Santos-Sierra. Enhanced immunostimulatory activity of in silico discovered agonists of Toll-like receptor 2 (TLR2), Biochimica et Biophysica Acta (BBA), General Subjects, 1861(11):2680-2689, 2017.
Links: doi:10.1016/j.bbagen.2017.07.011 | Publisher | Download citation: BibTeX RIS
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Abstract: Emergent therapies in anticancer vaccination use Toll-like receptors (TLRs) agonists as dendritic cell (DC) vaccine adjuvants. DCs from the patient are isolated, stimulated with TLR agonists and tumor antigens ex vivo and then infused back into the patient. Although some TLR ligands have been tested in clinical trials, novel TLR agonists with improved immunomodulatory properties are essential to optimize treatment success. We report on the discovery of small-molecule TLR2 agonists, with favorable properties as synthetic adjuvants. We performed a shape- and featured-based similarity virtual screening against a commercially available compound library. The selected virtual hits were experimentally tested in TLR2-reporter cells and their activity in phagocytes and DCs was characterized. A binding model of the compounds to TLR2 (docking studies) was proposed. Through a virtual screening approach against a library of three million compounds four virtual hits (AG1, AG2, AG3, AG4) were found to synergistically augment the NF-kB activation induced by the lipopeptide ligand Pam3CSK4 in luciferase reporter assays using HEK293-TLR2 cells. Biacore experiments indicated that AG1–AG4 are ago-allosteric modulators of TLR2 and AG2 bound TLR2 with high affinity (KD 0.8μM). The compounds induced TNF-α production in human peripheral blood mononuclear cells (PBMCs) and they activated DCs as indicated by IL-12 production and upregulation of CD83/CD86. Following a combined in silico/in vitro approach we have discovered TLR2-agonists (AG1–AG4) that activate human and mouse immune cells. We introduce four novel TLR2 ago-allosteric modulators that stimulate myeloid cell activity and constitute promising candidates as synthetic adjuvants.

[119] J. Mortier, J. R. C. Prévost, D. Sydow, S. Teuchert, C. Omieczynski, M. Bermudez, R. Frédérick, G. Wolber. Arginase structure and inhibition: Catalytic site plasticity reveals new modulation possibilities, Scientific Reports, 7(1):13616, 2017.
Links: doi:10.1038/s41598-017-13366-4 | Publisher | Download citation: BibTeX RIS
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Abstract: Metalloenzyme arginase is a therapeutically relevant target associated with tumor growth. To fight cancer immunosuppression, arginase activity can be modulated by small chemical inhibitors binding to its catalytic center. To better understand molecular mechanisms of arginase inhibition, a careful computer-aided mechanistic structural investigation of this enzyme was conducted. Using molecular dynamics (MD) simulations in the microsecond range, key regions of the protein active site were identified and their flexibility was evaluated and compared. A cavity opening phenomenon was observed, involving three loops directly interacting with all known ligands, while metal coordinating regions remained motionless. A novel dynamic 3D pharmacophore analysis method termed dynophores has been developed that allows for the construction of a single 3D-model comprising all ligand-enzyme interactions occurring throughout a complete MD trajectory. This new technique for the in silico study of intermolecular interactions allows for loop flexibility analysis coupled with movements and conformational changes of bound ligands. Presented MD studies highlight the plasticity of the size of the arginase active site, leading to the hypothesis that larger ligands can enter the cavity of arginase. Experimental testing of a targeted fragment library substituted by different aliphatic groups validates this hypothesis, paving the way for the design of arginase inhibitors with novel binding patterns.

[118] Marcel Bermudez, Andreas Bock, Fabian Krebs, Ulrike Holzgrabe, Klaus Mohr, Martin J. Lohse, Gerhard Wolber. Ligand-specific restriction of extracellular conformational dynamics constrains signaling of the M2 muscarinic receptor, ACS Chem. Biol., 12(7):1743-1748, 2017.
Links: doi:10.1021/acschembio.7b00275 | Publisher | Download citation: BibTeX RIS
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Abstract: G protein-coupled receptors transmit extracellular signals across cell membranes via different G protein classes and β-arrestins. Some pathways may be therapeutically beneficial, whereas others may be detrimental under certain pathophysiological conditions. For many GPCRs, biased agonists are available, which preferentially signal through one pathway or a subset of pathways, and harnessing biased agonism could be a potential novel therapeutic strategy. However, the incomplete mechanistic understanding of biased agonism hampers rational design of biased ligands. Using the muscarinic M2 receptor as a model system, we have analyzed the relationship between ligand-dependent conformational changes as revealed in all-atom MD simulations and the activation of specific G proteins. We find that the extent of closure of the extracellular, allosteric binding site interferes with the activation of certain G proteins. Our data allow the rational design of Gi-biased agonists at the M2 receptor and delineate a simple principle which may be translated to other GPRCs.

[117] R. Maccari, R. Ettari, I. Adornato, A. Naß, G. Wolber, A. Bitto, F. Mannino, F. Aliquo, G. Bruno, F. Nicolo, S. Previti, S. Grasso, M. Zappala, R. Ottana. Identification of 2-thioxoimidazolidin-4-one derivatives as novel noncovalent proteasome and immunoproteasome inhibitors, Bioorg. Med. Chem. Lett., 28(3):278-283, 2017.
Links: doi:10.1016/j.bmcl.2017.12.053 | Download citation: BibTeX RIS
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Abstract: This paper describes the design, synthesis, and biological evaluation of 2-thioxoimidazolidin-4-one derivatives as inhibitors of proteasome and immunoproteasome, potential targets for the treatment of hematological malignancies. In particular, we focused our efforts on the design of noncovalent inhibitors, which might be a promising therapeutic option potentially devoid of drawbacks and side-effects related to irreversible inhibition. Among all the synthesized compounds, we identified a panel of active inhibitors with Ki values towards one or two chymotrypsin-like activities of proteasome (β5c) and immunoproteasome (β5i and β1i subunits) in the low micromolar range. Docking studies suggested a unique binding mode of the molecules in the catalytic site of immunoproteasome proteolytic subunits.

[116] H. A. Abuelizz, R. Al-Salahi, J. Al-Asri, J. Mortier, M. Marzouk, E. Ezzeldin, A. A. Ali, M. G. Khalil, G. Wolber, H. A. Ghabbour, A. A. Almehizia, G. A. Abdel Jaleel. Synthesis, crystallographic characterization, molecular docking and biological activity of isoquinoline derivatives, Chem. Cent. J., 11(1):103, 2017.
Links: doi:10.1186/s13065-017-0321-1 | Publisher | Download citation: BibTeX RIS
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Abstract: The main objective of this work was to synthesize novel compounds with a benzo[de][1,2,4]triazolo[5,1-a]isoquinoline scaffold by employing (dioxo-benzo[de]isoquinolin-2-yl) thiourea as a building block. Molecular docking was conducted in the COX-2 active site to predict the plausible binding mode and rationalize the structure–activity relationship of the synthesized compounds. The structures of the synthesized compounds were confirmed by HREI-MS, and NMR spectra along with X-ray diffraction were collected for products 1 and 5. Thereafter, anti-inflammatory effect of molecules 1–20 was evaluated in vivo using carrageenan-induced paw edema method, revealing significant inhibition potency in albino rats with an activity comparable to that of the standard drugs indomethacin. Compounds 8, 9, 15 and 16 showed the highest anti-inflammatory activity. However, thermal sensitivity-hot plat test, a radiological examination and motor coordination assessment were performed to test the activity against rheumatoid arthritis. The obtained results indicate promising anti-arthritic activity for compounds 9 and 15 as significant reduction of the serum level of interleukin-1β [IL-1β], cyclooxygenase-2 [COX-2] and prostaglandin E2 [PGE2] was observed in CFA rats.

[115] V. Obermoser, M. E. Urban, M. S. Murgueitio, G. Wolber, U. Kintscher, R. Gust. New telmisartan-derived PPARγ agonists: Impact of the 3D-binding mode on the pharmacological profile, Eur. J. Med. Chem., 124:138-152, 2016.
Links: doi:10.1016/j.ejmech.2016.08.027 | Download citation: BibTeX RIS
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Abstract: In previous studies, the 4′-((2-propyl-1H-benzo[d]imidazol-1-yl)methyl)-[1,1′-biphenyl]-2-carboxylic acid was identified as pharmacophoric core for PPARγ activation. In this structure-activity relationship study the C2-alkyl chain was elongated and the 2-COOH group was changed to a carbamide/nitrile or shifted to the 3- or 4-position. Furthermore, the benzo[d]imidazole was exchanged by 2,3-dihydrobenzo[d]thiazole or 1H-indole. C2-propyl derivatives showed the profile of partial agonists, while elongation of the C2-chain to that of an n-heptyl group or a 4-COOH shift changed the pharmacological profile to that of a potent full agonist. This finding can be explained by binding to the LBD in different ligand conformations. Two anchoring points (Tyr473 and Arg288) exist in the LBD, which have to be contacted to achieve receptor activation. In a crystal violet chemosensitivity assay using COS-7 cells and LNCaP cells expressing PPARγ only the carboxamide derivatives influenced the cell growth, independently on the presence of the PPARγ. Therefore, receptor mediated cytotoxicity can be excluded.

[114] C. Rakers, F. Schumacher, W. Meinl, H. Glatt, B. Kleuser, G. Wolber. In silico prediction of human sulfotransferase 1E1 activity guided by pharmacophores from molecular dynamics simulations, Journal of Biological Chemistry, 291(1):58-71, 2016.
Links: doi:10.1074/jbc.M115.685610 | Publisher | Download citation: BibTeX RIS
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Abstract: Acting during phase II metabolism, sulfotransferases (SULTs) serve detoxification by transforming a broad spectrum of compounds from pharmaceutical, nutritional, or environmental sources into more easily excretable metabolites. However, SULT activity has also been shown to promote formation of reactive metabolites that may have genotoxic effects. SULT subtype 1E1 (SULT1E1) was identified as a key player in estrogen homeostasis, which is involved in many physiological processes and the pathogenesis of breast and endometrial cancer. The development of an in silico prediction model for SULT1E1 ligands would therefore support the development of metabolically inert drugs and help to assess health risks related to hormonal imbalances. Here, we report on a novel approach to develop a model that enables prediction of substrates and inhibitors of SULT1E1. Molecular dynamics simulations were performed to investigate enzyme flexibility and sample protein conformations. Pharmacophores were developed that served as a cornerstone of the model and machine learning techniques were applied for prediction refinement. The prediction model was used to screen the DrugBank (a database of experimental and approved drugs): 28 % of the predicted hits were reported in literature as ligands of SULT1E1. From the remaining hits, a selection of nine molecules was subjected to biochemical assay validation and experimental results were in accordance with the in silico prediction of SULT1E1 inhibitors and substrates, thus affirming our prediction hypotheses.

[113] S. Köhling, G. Künze, K. Lemmnitzer, M. Bermudez, G. Wolber, J. Schiller, D. Huster, J. Rademann. Chemoenzymatic synthesis of nonasulfated tetrahyaluronan with a paramagnetic tag for studying its complex with Interleukin-10, Chem. Eur. J., 22(16):5563-5574, 2016.
Links: doi:10.1002/chem.201504459 | Publisher | Download citation: BibTeX RIS
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Abstract: Implants and artificial biomaterials containing sulfated hyaluronans have been shown to improve the healing of injured skin and bones. It is hypothesized that these effects are mediated by the binding of sulfated glycosaminoglycans (GAGs) to growth factors and cytokines, resulting in the sequestering of proteins to the wound healing site and in modulated protein activity. Given that no direct synthetic access to sulfated oligohyaluronans has been available, little is known about their protein binding and the structure of the resulting protein complexes. Here, the chemoenzymatic preparation of oligohyaluronans on the gram scale is described. Oligohyaluronans are converted into anomeric azides at the reducing end, enabling the attachment of analytical labels through an anomeric ligation reaction. A nonasulfated tetrahyaluronan–ethylenediaminetetraacetic acid derivative has been produced and used as a paramagnetic tag for the elucidation of the complex of this ligand with interleukin-10 using paramagnetic relaxation enhancement NMR analysis. The metal ion position is resolved with 1.0 Å, enabling a refined structural model of the complex.

[112] J. R. Homoki, A. Nemes, E. Fazekas, G. Gyémánt, P. Balogh, F. Gál, J. Al-Asri, J. Mortier, G. Wolber, L. Babinszky, J. Remenyik. Anthocyanin composition, antioxidant efficiency, and α-amylase inhibitor activity of different Hungarian sour cherry varieties (Prunus cerasus L.), Food Chemistry, 194:222-229, 2016.
Links: doi:10.1016/j.foodchem.2015.07.130 | Publisher | Download citation: BibTeX RIS
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Abstract: Five Hungarian sour cherry cultivars were studied to determine their anthocyanin contents and their possible inhibitory properties. The water and methanol soluble antioxidant capacities were separately assessed by photoluminescence showing values ranged from 3.4 μg mg−1 to 15.4 μg mg−1, respectively. The “VN1” variety (selected from “Csengődi csokros”) showed the highest antioxidant capacity. The anthocyanin content, measured by pH differential method or isolated by solid phase extraction, was the highest also in “VN1”. Correlation was found between the anthocyanin content and the high antioxidant capacity. The main anthocyanin components were cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside. The presence of malvidin-3,5-O-diglycoside was verified by MALDI-TOF MS. Sour cherry extracts and selected anthocyanins inhibited the human salivary alpha-amylase catalyzed hydrolysis competitively. The lowest IC50 value, 55 μg mL−1 or 80 μM, was measured for malvidin-3,5-O-diglycoside, for which possible binding modes within the alpha-amylase active site could be investigated in silico using molecular docking and molecular dynamics.

[111] E. Guerrieri, M. Bermudez, G. Wolber, I. P. Berzetei-Gurske, H. Schmidhammer, M. Spetea. Structural determinants of diphenethylamines for interaction with the κ opioid receptor: Synthesis, pharmacology and molecular modeling studies, Bioorg. Med. Chem. Lett., 26(19):4769-4774, 2016.
Links: doi:10.1016/j.bmcl.2016.08.031 | Download citation: BibTeX RIS
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Abstract: The κ opioid (KOP) receptor crystal structure in an inactive state offers nowadays a valuable platform for inquiry into receptor function. We describe the synthesis, pharmacological evaluation and docking calculations of KOP receptor ligands from the class of diphenethylamines using an active-like structure of the KOP receptor attained by molecular dynamics simulations. The structure–activity relationships derived from computational studies was in accordance with pharmacological activities of targeted diphenethylamines at the KOP receptor established by competition binding and G protein activation in vitro assays. Our analysis identified that agonist binding results in breaking of the Arg156-Thr273 hydrogen bond, which stabilizes the inactive receptor conformation, and a crucial hydrogen bond with His291 is formed. Compounds with a phenolic 4-hydroxy group do not form the hydrogen bond with His291, an important residue for KOP affinity and agonist activity. The size of the N-substituent hosted by the hydrophobic pocket formed by Val108, Ile316 and Tyr320 considerably influences binding and selectivity, with the n-alkyl size limit being five carbon atoms, while bulky substituents turn KOP agonists in antagonists. Thus, combination of experimental and molecular modeling strategies provides an initial framework for understanding the structural features of diphenethylamines that are essential to promote binding affinity and selectivity for the KOP receptor, and may be involved in transduction of the ligand binding event into molecular changes, ultimately leading to receptor activation.

[110] S. Bock, M. S. Murgueitio, G. Wolber, G. Weindl. Acute myeloid leukaemia-derived Langerhans-like cells enhance Th1 polarization upon TLR2 engagement, Pharmacological Research, 105:44-53, 2016.
Links: doi:10.1016/j.phrs.2016.01.016 | Publisher | Download citation: BibTeX RIS
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Abstract: Langerhans cells (LCs) represent a highly specialized subset of epidermal dendritic cells (DCs), yet not fully understood in their function of balancing skin immunity. Here, we investigated in vitro generated Langerhans-like cells obtained from the human acute myeloid leukaemia cell line MUTZ-3 (MUTZ-LCs) to study TLR- and cytokine-dependent activation of epidermal DCs. MUTZ-LCs revealed high TLR2 expression and responded robustly to TLR2 engagement, confirmed by increased CD83, CD86, PD-L1 and IDO expression, upregulated IL-6, IL-12p40 and IL-23p19 mRNA levels IL-8 release. TLR2 activation reduced CCR6 and elevated CCR7 mRNA expression and induced migration of MUTZ-LCs towards CCL21. Similar results were obtained by stimulation with pro-inflammatory cytokines TNF-α and IL-1β whereas ligands of TLR3 and TLR4 failed to induce a fully mature phenotype. Despite limited cytokine gene expression and production for TLR2-activated MUTZ-LCs, co-culture with naive CD4+ T cells led to significantly increased IFN-γ and IL-22 levels indicating Th1 differentiation independent of IL-12. TLR2-mediated effects were blocked by the putative TLR2/1 antagonist CU-CPT22, however, no selectivity for either TLR2/1 or TLR2/6 was observed. Computer-aided docking studies confirmed non-selective binding of the TLR2 antagonist. Taken together, our results indicate a critical role for TLR2 signalling in MUTZ-LCs considering the leukemic origin of the generated Langerhans-like cells.

[109] A. Bock, M. Bermudez, F. Krebs, C. Matera, B. Chirinda, D. Sydow, C. Dallanoce, U. Holzgrabe, M. De Amici, M. J. Lohse, G. Wolber, K. Mohr. Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-Coupled Receptor, Journal of Biological Chemistry, 291(31):16375-16389, 2016.
Links: doi:10.1074/jbc.M116.735431 | Publisher | Download citation: BibTeX RIS
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Abstract: G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site.

[108] Bilal Nizami, Dominique Sydow, Gerhard Wolber, Bahareh Honarparvar. Molecular insight on the binding of NNRTI to K103N mutated HIV-1 RT: molecular dynamics simulations and dynamic pharmacophore analysis, Mol. BioSyst., 12:3385-3395, 2016.
Links: doi:10.1039/C6MB00428H | Publisher | Download citation: BibTeX RIS

[107] M. Bermudez, J. Mortier, C. Rakers, D. Sydow, G. Wolber. More than a look into a crystal ball: protein structure elucidation guided by molecular dynamics simulations, Drug Discov. Today, 21(11):1799-1805, 2016.
Links: doi:10.1016/j.drudis.2016.07.001 | Download citation: BibTeX RIS
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Abstract: The ‘form follows function’ principle implies that a structural determination of protein structures is indispensable to understand proteins in their biological roles. However, experimental methods still show shortcomings in the description of the dynamic properties of proteins. Therefore, molecular dynamics (MD) simulations represent an essential tool for structural biology to investigate proteins as flexible and dynamic entities. Here, we will give an overview on the impact of MD simulations on structural investigations, including studies that aim at a prediction of protein-folding pathways, protein-assembly processes and the sampling of conformational space by computational means.

[106] D. Becker, Z. Kaczmarska, C. Arkona, R. Schulz, C. Tauber, G. Wolber, R. Hilgenfeld, M. Coll, J. Rademann. Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments, Nat. Commun., 7:12761, 2016.
Links: doi:10.1038/ncomms12761 | Publisher | Download citation: BibTeX RIS

[105] J. Al-Asri, G. Gyémánt, E. Fazekas, G. Lehoczki, M. F. Melzig, G. Wolber, J. Mortier. α-Amylase Modulation: Discovery of Inhibitors Using a Multi-Pharmacophore Approach for Virtual Screening, ChemMedChem, 11(21):2372-2377, 2016.
Links: doi:10.1002/cmdc.201600427 | Publisher | Download citation: BibTeX RIS
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Abstract: Better control of postprandial hyperglycemia can be achieved by delaying the absorption of glucose resulting from carbohydrate digestion. Because α-amylase initiates the hydrolysis of polysaccharides, the design of α-amylase inhibitors can lead to the development of new treatments for metabolic disorders such as type II diabetes and obesity. In this study, a rational computer-aided approach was developed to identify novel α-amylase inhibitors. Three-dimensional pharmacophores were developed based on the binding mode analysis of six different families of compounds that bind to this enzyme. In a stepwise virtual screening workflow, seven molecules were selected from a library of 1.4 million. Five out of seven biologically tested compounds showed α-amylase inhibition, and the two most potent compounds inhibited α-amylase with IC50 values of 17 and 27 μm. The scaffold benzylideneacetohydrazide was shared by four of the discovered inhibitors, emerging as a novel drug-like non-carbohydrate fragment and constituting a promising lead scaffold for α-amylase inhibition.

[104] C. Meinguet, C. Bruyère, R. Frédérick, V. Mathieu, C. Vancraeynest, L. Pochet, J. Laloy, J. Mortier, G. Wolber, R. Kiss, B. Masereel, J. Wouters. 3D-QSAR, design, synthesis and characterization of trisubstituted harmine derivatives with in vitro antiproliferative properties, Eur. J. Med. Chem., 94:45-55, 2015.
Links: doi:10.1016/j.ejmech.2015.02.044 | Publisher | Download citation: BibTeX RIS
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Abstract: Apolar trisubstituted derivatives of harmine show high antiproliferative activity on diverse cancer cell lines. However, these molecules present a poor solubility making these compounds poorly bioavailable. Here, new compounds were synthesized in order to improve solubility while retaining antiproliferative activity. First, polar substituents have shown a higher solubility but a loss of antiproliferative activity. Second, a Comparative Molecular Field Analysis (CoMFA) model was developed, guiding the design and synthesis of eight new compounds. Characterization has underlined the in vitro antiproliferative character of these compounds on five cancerous cell lines, combining with a high solubility at physiological pH, making these molecules druggable. Moreover, targeting glioma treatment, human intestinal absorption and blood brain penetration have been calculated, showing high absorption and penetration properties.

[103] J. Mortier, E. K. Nyakatura, O. Reimann, S. Huhmann, J. O. Daldrop, C. Baldauf, G. Wolber, M. S. Miettinen, B. Koksch. Coiled-coils in phage display screening: Insight into exceptional selectivity provided by molecular dynamics, J. Chem. Inf. Model., 55(3):495-500, 2015.
Links: doi:10.1021/ci500689c | Publisher | Download citation: BibTeX RIS
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Abstract: Involved in numerous key biological functions, protein helix-helix interactions follow a well-defined intermolecular recognition pattern. The characteristic structure of the alpha-helical coiled-coil allows for the specific randomization of clearly defined interaction partners within heteromeric systems. In this work, a rationally designed heterodimeric coiled-coil was used to investigate potential factors influencing the sequence selectivity in interhelical interactions.

[102] A. Perdih, M. Hrast, K. Pureber, H. Barreteau, S. Grdadolnik, D. Kocjan, S. Gobec, T. Solmajer, G. Wolber. Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization, J. Comput. Aided Mol. Des., :1-20, 2015.
Links: doi:10.1007/s10822-015-9843-6 | Publisher | Download citation: BibTeX RIS
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Abstract: Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

[101] Stefanie Grosskopf, Chris Eckert, Christoph Arkona, Silke Radetzki, Kerstin Böhm, Udo Heinemann, Gerhard Wolber, Jens-Peter von Kries, Walter Birchmeier, Jörg Rademann. Selective inhibitors of the protein tyrosine phosphatase SHP2 block cellular motility and growth of cancer cells in-vitro and in-vivo, ChemMedChem, 10(5):815-826, 2015.
Links: doi:10.1002/cmdc.201500015 | Publisher | Download citation: BibTeX RIS
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Abstract: Selective inhibitors of the protein tyrosine phosphatase SHP2 (PTPN11), an enzyme, deregulated in numerous human tumors, were generated by combination of chemical synthesis with structure-based rational design. 70 pyridazolon-4-ylidene-hydrazinyl-benzene sulfonates were prepared and evaluated in enzyme assays. Binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful molecule 25 (GS-493) inhibited SHP2 with an IC50 value of 71 ± 15 nM in the enzyme assay and was 29- and 45-fold more active than against related SHP1 and PTP1B. In cell culture experiments 25 blocked HGF-stimulated epithelial-mesenchymal transition of human pancreatic tumor cells HPAF as indicated by the reduction of minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation of the non-small cell lung cancer cell line LXFA 526L in soft agar. Finally, it inhibited tumor growth in a murine xenograft model. Thus, the novel specific molecule strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells.

[100] J. Mortier, C. Rakers, M. Bermudez, M. S. Murgueitio, S. Riniker, G. Wolber. The impact of molecular dynamics on drug design: applications for the characterization of ligand-macromolecule complexes, Drug Discov. Today, 20(6):686-702, 2015.
Links: doi:10.1016/j.drudis.2015.01.003 | Publisher | Download citation: BibTeX RIS
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Abstract: Among all tools available to design new drugs, molecular dynamics (MD) simulations have become an essential technique. Initially developed to investigate molecular models with a limited number of atoms, computers now enable investigations of large macromolecular systems with a simulation time reaching the microsecond range. The reviewed articles cover four years of research to give an overview on the actual impact of MD on the current medicinal chemistry landscape with a particular emphasis on studies of ligand-protein interactions. With a special focus on studies combining computational approaches with data gained from other techniques, this review shows how deeply embedded MD simulations are in drug design strategies and articulates what the future of this technique could be.

[99] Maria Kristina Parr, Francesco Botrè, Alexandra Naß, Jonas Hengevoss, Patrick Diel, Gerhard Wolber. Ecdysteroids: A novel class of anabolic agents?, Biology of Sport, 32(2):169-173, 2015.
Links: doi:10.5604/20831862.1144420 | Publisher | Download citation: BibTeX RIS
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Abstract: Increasing numbers of dietary supplements with ecdysteroids are marketed as "natural anabolic agents". Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 uM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 uM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERβ mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class "S1.2 Other Anabolic Agents" of the list of prohibited substances of the World Anti-Doping Agency.

[98] R. B. El-Houri, D. E. Kotowska, K. B. Christensen, S. Bhattacharya, N. Oksbjerg, G. Wolber, K. Kristiansen, L. P. Christensen. Polyacetylenes from carrots (Daucus carota) improve glucose uptake in vitro in adipocytes and myotubes, Food Funct., 6(7):2135-44, 2015.
Links: doi:10.1039/C5FO00223K | Publisher | Download citation: BibTeX RIS
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Abstract: A dichloromethane (DCM) extract of carrot roots was found to stimulate insulin-dependent glucose uptake (GU) in adipocytes in a dose dependent manner. Bioassay-guided fractionation of the DCM extract resulted in the isolation of the polyacetylenes falcarinol and falcarindiol. Both polyacetylenes were able to significantly stimulate basal and/or insulin-dependent GU in 3T3-L1 adipocytes and porcine myotube cell cultures in a dose-dependent manner. Falcarindiol increased peroxisome proliferator-activated receptor (PPAR)γ-mediated transactivation significantly at concentrations of 3, 10 and 30 μM, while PPARγ-mediated transactivation by falcarinol was only observed at 10 μM. Docking studies accordingly indicated that falcarindiol binds to the ligand binding domain of PPARγ with higher affinity than falcarinol and that both polyacetylenes exhibit characteristics of PPARγ partial agonists. Falcarinol was shown to inhibit adipocyte differentiation as evident by gene expression studies and Oil Red O staining, whereas falcarindiol did not inhibit adipocyte differentiation, which indicates that these polyacetylenes have distinct modes of action. The results of the present study suggest that falcarinol and falcarindiol may represent scaffolds for novel partial PPARγ agonists with possible antidiabetic properties.

[97] R. B. El-Houri, J. Mortier, M. S. Murgueitio, G. Wolber, L. P. Christensen. Identification of PPARγ Agonists from Natural Sources Using Different In Silico Approaches, Planta Med., 81(06):488-494, 2015.
Links: doi:10.1055/s-0034-1383119 | Publisher | Download citation: BibTeX RIS
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Abstract: Peroxisome proliferator-activated receptor γ plays an important role in lipid and glucose homeostasis and is the target of many drug discovery investigations because of its role in diseases such as type 2 diabetes. Activation of peroxisome proliferator-activated receptor γ by agonists leads to a conformational change in the ligand-binding domain altering the transcription of several target genes involved in glucose and lipid metabolism, resulting in, for example, facilitation of glucose and lipid uptake and amelioration of insulin resistance, and other effects that are important in the treatment of type 2 diabetes. Peroxisome proliferator-activated receptor γ partial agonists are compounds with diminished agonist efficacy compared to full agonists; however, they maintain the antidiabetic effect of full agonists but do not induce the same magnitude of side effects. This mini-review gives a short introduction to in silico screening methods and recent research advances using computational approaches to identify peroxisome proliferator-activated receptor γ agonists, especially partial agonists, from natural sources and how these ligands bind to the peroxisome proliferator-activated receptor γ in order to better understand their biological effects.

[96] Christin Rakers, Marcel Bermudez, Bettina G. Keller, Jérémie Mortier, Gerhard Wolber. Computational close up on protein-protein interactions: how to unravel the invisible using molecular dynamics simulations?, Wiley Interdisciplinary Reviews: Computational Molecular Science, 5(5):345-359, 2015.
Links: doi:10.1002/wcms.1222 | Publisher | Download citation: BibTeX RIS
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Abstract: As an essential part of many biological processes, protein–protein interactions (PPIs) offer exciting and promising opportunities for drug discovery by extension of the druggable target space. Over the last decade, studies on protein networks have significantly increased the number of identified PPIs. However, despite steadily growing data on PPIs, detailed understanding of the interaction surfaces and their dynamics remains limited. Furthermore, the development of small‐molecule inhibitors of PPIs faces technological challenges, leaving the question about the ‘druggability’ of PPIs open. Molecular dynamics (MD) simulations may facilitate the prediction of druggable binding sites on protein–protein interfaces by detecting binding hot spots and transient pockets. MD allows for a detailed analysis of structural and functional aspects of PPIs and thus provides valuable insights into PPI mechanisms and supports the design of PPI modulators. We provide an overview on the main areas of MD applications to PPIs including structural investigations and the design of PPI disruptors. Emphasizing the beneficial synergies between computational and experimental techniques, MD techniques are also frequently applied to low‐resolution structural data and have been used to elucidate structure and movements of complex macromolecular structures relevant for biological processes.

[95] M. Bermudez, G. Wolber. Structure vs. function - the impact of computational methods on the discovery of specific GPCR-ligands, Bioorg. Med. Chem., 23(14):3907-3912, 2015.
Links: doi:10.1016/j.bmc.2015.03.026 | Publisher | Download citation: BibTeX RIS
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Abstract: Over the past decades, computational methods have become invaluable for drug design campaigns but also as auxiliary tool for structural biology. The combination of experimental and in silico methods in the field of G protein coupled receptors (GPCRs) is indispensable. Despite recent groundbreaking achievements in GPCR crystallography, structural information for the vast majority of this physiologically important protein class is only accessible through homology models. Since the understanding of the conformational changes resulting in multiple activation pathways is incomplete, the design of specific GPCR modulating drugs remains a major challenge. However, due to the highly interdisciplinary requirements for the investigation of receptor function and the necessity of joining scientist from different fields, computational approaches gain importance in rationalizing and illustrating certain specific effects. In silico methods, such as molecular dynamics (MD) simulations, pharmacophore modeling or docking, proved to be suitable to complement experimental approaches. In this review, we highlight recent examples of in silico studies that were successfully applied in the field of GPCR research. Those approaches follow two main goals: Firstly, structural investigations that help to understand the receptor function and the characterization of ligand binding and secondly the identification of novel GPCR modulators as potential drugs.

[94] M. Bermudez, C. Rakers, G. Wolber. Structural characteristics of the allosteric binding site represent a key to subtype selective modulators of muscarinic acetylcholine receptors, Mol. Inform., 34(8):526-530, 2015.
Links: doi:10.1002/minf.201500025 | Publisher | Download citation: BibTeX RIS
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Abstract: The high conservation of the orthosteric acetylcholine binding site of muscarinic receptors (MAChR) represents a considerable challenge in terms of designing subtype selective drugs. A promising approach to gain subtype selectivity is to include allosteric or dualsteric targeting that aims to address more specific extracellular binding sites. Despite recent advances in crystallography of G protein coupled receptors (GPCRs), structural information for all 5 MAChR subtypes is not yet available. Here we report structural models of the active and the inactive receptor state of all subtypes derived by homology modelling in combination with MD simulations. The comparison of the allosteric binding site unveils the characteristics for each subtype on a structural level and indicates anchor points for rational design of selective drugs. Additionally, homology models offer the possibility for a rational explanation of dualsteric subtype selectivity, as we show for the M2 over M5 selectivity of the dualsteric ligands Atr-6-naph and Iper-6-phth.

[93] J. Al-Asri, E. Fazekas, G. Lehoczki, A. Perdih, C. Görick, M. F. Melzig, G. Gyémánt, G. Wolber, J. Mortier. From carbohydrates to drug-like fragments: Rational development of novel α-amylase inhibitors, Bioorg. Med. Chem., 23(20):6725-6732, 2015.
Links: doi:10.1016/j.bmc.2015.09.007 | Publisher | Download citation: BibTeX RIS
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Abstract: Starch catabolism leading to high glucose level in blood is highly problematic in chronic metabolic diseases, such as type II diabetes and obesity. α-Amylase catalyzes the hydrolysis of starch, increasing blood sugar concentration. Its inhibition represents a promising therapeutic approach to control hyperglycaemia. However, only few drug-like molecule inhibitors without sugar moieties have been discovered so far, and little information on the enzymatic mechanism is available. This work aims at the discovery of novel small α-amylase binders using a systematic in silico methodology. 3D-pharmacophore-based high throughput virtual screening of small compounds libraries was performed to identify compounds with high α-amylase affinity. Twenty-seven compounds were selected and biologically tested, revealing IC50 values in the micromolar range and ligand efficiency higher than the one of the bound form of acarbose, which is used as a reference for α-amylase inhibition.

[92] J. Schmitz, D. van der Mey, M. Bermudez, J. Klöckner, R. Schrage, E. Kostenis, C. Tränkle, G. Wolber, K. Mohr, U. Holzgrabe. Dualsteric muscarinic antagonists - orthosteric binding pose controls allosteric subtype selectivity, J. Med. Chem., 57(15):6739-6750, 2014.
Links: doi:10.1021/jm500790x | Publisher | Download citation: BibTeX RIS
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Abstract: Bivalent ligands of G protein-coupled receptors have been shown to simultaneously either bind to two adjacent receptors or to bridge different parts of one receptor protein. Recently, we found that bivalent agonists of muscarinic receptors can simultaneously occupy both the orthosteric transmitter binding site and the allosteric vestibule of the receptor protein. Such dualsteric agonists display a certain extent of subtype selectivity, generate pathway-specific signaling, and in addition may allow for designed partial agonism. Here, we want to extend the concept to bivalent antagonism. Using the phthal- and naphthalimide moieties, which bind to the allosteric, extracellular site, and atropine or scopolamine as orthosteric building blocks, both connected by a hexamethonium linker, we were able to prove a bitopic binding mode of antagonist hybrids for the first time. This is demonstrated by structure–activity relationships, site-directed mutagenesis, molecular docking studies, and molecular dynamics simulations. Findings revealed that a difference in spatial orientation of the orthosteric tropane moiety translates into a divergent M2/M5 subtype selectivity of the corresponding bitopic hybrids.

[91] R. Ottana, R. Maccari, J. Mortier, A. Caselli, S. Amuso, G. Camici, A. Rotondo, G. Wolber, P. Paoli. Synthesis, biological activity and structure-activity relationships of new benzoic acid-based protein tyrosine phosphatase inhibitors endowed with insulinomimetic effects in mouse C2C12 skeletal muscle cells, Eur. J. Med. Chem., 71:112-127, 2014.
Links: doi:10.1016/J.Ejmech.2013.11.001 | Publisher | Download citation: BibTeX RIS
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Abstract: Insulin resistance is a complex altered metabolic condition characterized by impaired insulin signaling and implicated in the pathogenesis of serious human diseases, such as diabetes, obesity, neurodegenerative pathologies. In pursuing our aim to identify new agents able to improve cellular insulin sensitivity, we have synthesized new 4-[(5-arylidene-4-oxo-2-phenylimino/oxothiazolidin-3-yl)methyl] benzoic acids (5, 8) and evaluated their inhibitory activity towards human protein tyrosine phosphatases PTP1B, LMW-PTP and TCPTP, enzymes which are involved in the development of insulin resistance. Compounds 5 and 8 showed from moderate to significant selectivity toward PTP1B over both the highly homologous TCPTP and the two isoforms of human LMW-PTP. In addition, most of the tested compounds selectively inhibited LMW-PTP IF1 over the isoform IF2. Docking studies into the active sites of PTP1B and LMW-PTP aided the rationalization of the observed PTP inhibitory profile. Moreover, most tested compounds were capable to induce the insulin metabolic pathway in mouse C2C12 skeletal muscle cells by remarkably stimulating both IR beta phosphorylation and 2-deoxyglucose cellular uptake. (C) 2013 Elsevier Masson SAS. All rights reserved.

[90] S. A. Galal, S. H. M. Khairat, F. A. F. Ragab, A. S. Abdelsamie, M. M. Ali, S. M. Soliman, J. Mortier, G. Wolber, H. I. El Diwani. Design, synthesis and molecular docking study of novel quinoxalin-2(1H)-ones as anti-tumor active agents with inhibition of tyrosine kinase receptor and studying their cyclooxygenase-2 activity, Eur. J. Med. Chem., 86(0):122-132, 2014.
Links: doi:10.1016/j.ejmech.2014.08.048 | Publisher | Download citation: BibTeX RIS
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Abstract: On continuation to our work, new quinoxalin-2(1H)-ones were synthesized to study their cytotoxic effect against HepG-2 and MCF-7 with their effect on the human tyrosine kinase (TRK). Compounds 12, 18, 15, 13, 11a, 20 and 16, respectively, were found to be more potent than cisplatin against HepG2 and selective to TRK. Also, compounds 12, 18, 20, 13, 14, and 22, respectively, exhibited decidedly activity against MCF-7 and selectivity against human TRK compared to cisplatin. A molecular docking study was also performed to gain comprehensive understanding into plausible binding modes and to conclude the structure activity relationships of the synthesized compounds. Moreover, anti-inflammatory activity was studied. Compounds 12, 15, 18 and 22 were found to be potent and selective against COX-2.

[89] A. Perdih, M. Hrast, H. Barreteau, S. Gobec, G. Wolber, T. Solmajer. Inhibitor Design Strategy Based on an Enzyme Structural Flexibility: A Case of Bacterial MurD Ligase, J. Chem. Inf. Model., 54(5):1451-1466, 2014.
Links: doi:10.1021/Ci500104m | Publisher | Download citation: BibTeX RIS
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Abstract: Increasing bacterial resistance to available antibiotics stimulated the discovery of novel efficacious antibacterial agents. The biosynthesis of the bacterial peptidoglycan, where the MurD enzyme is involved in the intracellular phase of the UDP-MurNAc-pentapeptide formation, represents a collection of highly selective targets for novel antibacterial drug design. In our previous computational studies, the C-terminal domain motion of the MurD ligase was investigated using Targeted Molecular Dynamic (TMD) simulation and the Off-Path Simulation (OPS) technique. In this study, we present a drug design strategy using multiple protein structures for the identification of novel MurD ligase inhibitors. Our main focus was the ATP-binding site of the MurD enzyme. In the first stage, three MurD protein conformations were selected based on the obtained OPS/TMD data as the initial criterion. Subsequently, a two-stage virtual screening approach was utilized combining derived structure-based pharmacophores with molecular docking calculations. Selected compounds were then assayed in the established enzyme binding assays, and compound 3 from the aminothiazole class was discovered to act as a dual MurC/MurD inhibitor in the micomolar range. A steady-state kinetic study was performed on the MurD enzyme to provide further information about the mechanistic aspects of its inhibition. In the final stage, all used conformations of the MurD enzyme with compound 3 were simulated in classical molecular dynamics (MD) simulations providing atomistic insights of the experimental results. Overall, the study depicts several challenges that need to be addressed when trying to hit a flexible moving target such as the presently studied bacterial MurD enzyme and show the possibilities of how computational tools can be proficiently used at all stages of the drug discovery process.

[88] A. Temirak, Y. M. Shaker, F. A. F. Ragab, M. M. Ali, S. M. Soliman, J. Mortier, G. Wolber, H. I. Ali, H. I. El Diwani. Synthesis, biological evaluation, and docking studies of new 2-furylbenzimidazoles as anti- angiogenic agents: Part II, Arch. Pharm., 347(4):291-304, 2014.
Links: doi:10.1002/Ardp.201300356 | Publisher | Download citation: BibTeX RIS
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Abstract: The 2-(5-methyl-2-furyl)-1H-benzimidazole moiety has shown promising activity against vascular endothelial growth factor (VEGF)-induced angiogenesis. In part I of this study, we have synthesized new analogs and tested their anti-angiogenic potentials. Here, we continue our previous study with different new analogs. Some compounds show promising cytotoxic activity against the human breast cancer cell line MCF-7, with IC50 in the range of 7.80-13.90 mu g/mL, and exhibited remarkable in vitro inhibition against VEGF in the MCF-7 cancer cell line, with 95-98% of inhibition in comparison to tamoxifen as reference (IC50: 8.00 mu g/mL, % of inhibition=98%). Additionally, a molecular docking study was carried out to gain insight into plausible binding modes and to understand the structure-activity relationships of the synthesized compounds.

[87] M. A. Seiter, S. Salcher, M. Rupp, J. Hagenbuchner, U. Kiechl-Kohlendorfer, J. Mortier, G. Wolber, J. M. Rollinger, P. Obexer, M. J. Ausserlechner. Discovery of Sanggenon G as a natural cell-permeable small-molecular weight inhibitor of X-linked inhibitor of apoptosis protein (XIAP), FEBS Open Bio, 4(0):659-671, 2014.
Links: doi:10.1016/j.fob.2014.07.001 | Publisher | Download citation: BibTeX RIS
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Abstract: Defects in the regulation of apoptosis are one main cause of cancer development and may result from overexpression of anti-apoptotic proteins such as the X-linked inhibitor of apoptosis protein (XIAP). XIAP is frequently overexpressed in human leukemia and prostate and breast tumors. Inhibition of apoptosis by XIAP is mainly coordinated through direct binding to the initiator caspase-9 via its baculovirus-IAP-repeat-3 (BIR3) domain. XIAP inhibits caspases directly making it to an attractive target for anti-cancer therapy. In the search for novel, non-peptidic XIAP inhibitors in this study we focused on the chemical constituents of sāng bái pí (mulberry root bark). Most promising candidates of this plant were tested biochemically in vitro by a fluorescence polarization (FP) assay and in vivo via protein fragment complementation analysis (PCA). We identified the Diels Alder adduct Sanggenon G (SG1) as a novel, small-molecular weight inhibitor of XIAP. As shown by FP and PCA analyses, SG1 binds specifically to the BIR3 domain of XIAP with a binding affinity of 34.26 μM. Treatment of the transgenic leukemia cell line Molt3/XIAP with SG1 enhances caspase-8, -3 and -9 cleavage, displaces caspase-9 from XIAP as determined by immunoprecipitation experiments and sensitizes these cells to etoposide-induced apoptosis. SG1 not only sensitizes the XIAP-overexpressing leukemia cell line Molt3/XIAP to etoposide treatment but also different neuroblastoma cell lines endogenously expressing high XIAP levels. Taken together, Sanggenon G (SG1) is a novel, natural, non-peptidic, small-molecular inhibitor of XIAP that can serve as a starting point to develop a new class of improved XIAP inhibitors.

[86] C. Rakers, S. Schwerdtfeger, J. Mortier, S. Duwe, T. Wolff, G. Wolber, M. F. Melzig. Inhibitory potency of flavonoid derivatives on influenza virus neuraminidase, Bioorg. Med. Chem. Lett., 24(17):4312-4317, 2014.
Links: doi:10.1016/j.bmcl.2014.07.010 | Publisher | Download citation: BibTeX RIS
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Abstract: The constant risk of emerging new influenza virus strains that are resistant to established inhibitors like oseltamivir leaves influenza neuraminidase (NA) a prominent target for drug design. The inhibitory activity of several flavonoid derivatives was experimentally tested in comparison to oseltamivir for the NA expressed by the seasonal influenza virus strains A/California/7/09 (A(H1N1)pdm09), A/Perth/16/09 (A(H3N2)), and B/Brisbane/60/08. IC50 values of polyphenols confirmed moderate inhibition in the μM range. Structurally, the amount and site of glycosylation of tested flavonoids have no significant influence on their inhibitory potency. In a pharmacophore-based docking approach the structure–activity relationship was evaluated. Molecular dynamics simulations revealed highly flexible parts of the enzyme and the contribution of salt bridges to the structural stability of NA. The findings of this study elucidate the impact of flavonoids on viral neuraminidase activity and the analysis of their modes of action provide valuable information about the mechanism of NA inhibition.

[85] A. Perdih, M. Hrast, H. Barreteau, S. Gobec, G. Wolber, T. Solmajer. Benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives as multiple inhibitors of bacterial Mur ligases (MurC-MurF), Bioorg. Med. Chem., 22(15):4124-4134, 2014.
Links: doi:10.1016/j.bmc.2014.05.058 | Publisher | Download citation: BibTeX RIS
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Abstract: Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of the bacterial Mur ligase family—MurC, MurD, MurE and MurF—are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Park’s nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC–MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC–MurF enzymes in biochemical inhibition assays and molecules 10–14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC–MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d-Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents.

[84] E. K. Nyakatura, J. Mortier, V. S. Radtke, S. Wieczorek, R. Rezaei Araghi, C. Baldauf, G. Wolber, B. Koksch. β- and γ-amino acids at α-helical interfaces: Toward the formation of highly stable foldameric coiled coils, ACS Med. Chem. Lett., 5(12):1300-1303, 2014.
Links: doi:10.1021/ml500361c | Publisher | Download citation: BibTeX RIS
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Abstract: Since peptides are vital for cellular and pathogenic processes, much effort has been put into the design of unnatural oligomers that mimic natural peptide structures, also referred to as foldamers. However, to enable the specific application of foldamers, a thorough characterization of their interaction profiles in native protein environments is required. We report here the application of phage display for the identification of suitable helical environments for a sequence comprising an alternating set of ?- and ?-amino acids. In vitro selected sequences show that an increase in the hydrophobic surface area at the helical interface as well as the incorporation of a polar H-bond donor functionality can significantly improve interhelical interactions involving backbone-extended amino acids. Thus, our data provide insight into the principles of the rational design of foldameric inhibitors for protein?protein interactions.

[83] E. K. Nyakatura, R. R. Araghi, J. Mortier, S. Wieczorek, C. Baldauf, G. Wolber, B. Koksch. An unusual interstrand h-bond stabilizes the heteroassembly of helical alpha beta gamma-chimeras with natural peptides, ACS Chem. Biol., 9(3):613-616, 2014.
Links: doi:10.1021/Cb4007979 | Publisher | Download citation: BibTeX RIS
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Abstract: The substitution of alpha-amino acids by homologated amino acids has a strong impact on the overall structure and topology of peptides, usually leading to a loss in thermal stability. Here, we report on the identification of an ideal core packing between an alpha-helical peptide and an alpha beta gamma-chimera via phage display. Selected peptides assemble with the chimeric sequence with thermal stabilities that are comparable to that of the parent bundle consisting purely of alpha-amino acids. With the help of MD simulations and mutational analysis this stability could be explained by the formation of an interhelical H-bond between the selected cysteine and a backbone carbonyl of the beta/gamma-segment. Gained results can be directly applied in the design of biologically relevant peptides containing beta- and gamma-amino acids.

[82] M. S. Murgueitio, P. Henneke, H. Glossmann, S. Santos-Sierra, G. Wolber. Prospective virtual screening in a sparse data scenario: design of small-molecule TLR2 antagonists, ChemMedChem, 9(4):813-822, 2014.
Links: doi:10.1002/Cmdc.201300445 | Publisher | Download citation: BibTeX RIS
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Abstract: Toll-like receptors (TLRs) are critical signaling molecules with roles in various severe clinical conditions such as sepsis and rheumatoid arthritis, and have therefore been advocated as promising drug targets for the treatment of these diseases. The aim of this study was to discover small-molecule antagonists of TLR2 by computer-aided drug design. This goal poses several challenges due to the lack of available data on TLR2 modulators. To overcome these hurdles we developed a combined structure- and ligand-based virtual screening approach. First, we calculated molecular interaction fields of the TLR2 binding site to derive a structure-based 3D pharmacophore, which was then used for virtual screening. We then performed a two-step shape- and feature-based similarity search using known TLR2 ligands as query structures. A selection of virtual screening hits was biologically tested in a cell-based assay for TLR2 signaling inhibition, leading to the identification of several compounds with antagonistic activity (IC50 values) in the low-micromolar range.

[81] D. Kotowska, R. B. El-Houri, K. Borkowski, R. K. Petersen, X. C. Fretté, G. Wolber, K. Grevsen, K. B. Christensen, L. P. Christensen, K. Kristiansen. Isomeric C12-alkamides from the roots of Echinacea Purpurea improve basal and insulin-dependent glucose uptake in 3T3-L1 adipocytes, Planta Med., 80(18):1712-1720, 2014.
Links: doi:10.1055/s-0034-1383252 | Publisher | Download citation: BibTeX RIS

[80] V. Asante, J. Mortier, G. Wolber, B. Koksch. Impact of fluorination on proteolytic stability of peptides: a case study with α-chymotrypsin and pepsin, Amino Acids, 46(12):2733-2744, 2014.
Links: doi:10.1007/s00726-014-1819-7 | Download citation: BibTeX RIS
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Abstract: Protease stability is a key consideration in the development of peptide-based drugs. A major approach to increase the bioavailability of pharmacologically active peptides is the incorporation of non-natural amino acids. Due to the unique properties of fluorine, fluorinated organic molecules have proven useful in the development of therapeutically active small molecules as well as in materials and crop science. This study presents data on the ability of fluorinated amino acids to influence proteolytic stability when present in peptide sequences that are based on ideal protease substrates. Different model peptides containing fluorinated amino acids or ethylglycine in the P2, P1′or P2′ positions were designed according to the specificities of the serine protease, α-chymotrypsin (EC 3.4.21.1) or the aspartic protease, pepsin (EC 3.4.23.1). The proteolytic stability of the peptides toward these enzymes was determined by an analytical RP-HPLC assay with fluorescence detection and compared to a control sequence. Molecular modeling was used to support the interpretation of the structure–activity relationship based on the analysis of potential ligand-enzyme interactions. Surprisingly, an increase in proteolytic stability was observed only in a few cases. Thus, this systematic study shows that the proteolytic stability of fluorinated peptides is not predictable, but rather is a very complex phenomenon that depends on the particular enzyme, the position of the substitution relative to the cleavage site and the fluorine content of the side chain.

[79] S. A. Galar, A. S. Abdelsamie, S. M. Soliman, J. Mortier, G. Wolber, M. M. Ali, H. Tokuda, N. Suzuki, A. Lida, R. A. Ramadan, H. I. El Diwani. Design, synthesis and structure-activity relationship of novel quinoxaline derivatives as cancer chemopreventive agent by inhibition of tyrosine kinase receptor, Eur. J. Med. Chem., 69:115-124, 2013.
Links: doi:10.1016/J.Ejmech.2013.07.049 | Publisher | Download citation: BibTeX RIS
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Abstract: The cancer chemopreventive activity of quinoxaline derivatives 1-20 has been evaluated by studying the inhibitory effect on Epstein-Barr virus early antigen (EBV-EA) activation. The quinoxaline derivatives 1-20 showed inhibitory effect on EBV-EA activation without cytotoxicity on Raji cells. All compounds exhibited dose dependent inhibitory activities, most of them showed significant activity at 1000 mol ratio/12-O-tetradecanoylphorbol-13-acetate (TPA). Compounds 7 and 9 exhibited stronger inhibitory effects on the EBV-EA activation than that of the representative control, oleanolic acid, at the highest measured concentration. In addition, compounds 7-10 showed potent and selective inhibition of human tyrosine kinase (TRK) in liver cancer HepG2 and breast cancer MCF-7 cell lines similar to the positive control, doxorubicin. (C) 2013 Published by Elsevier Masson SAS.

[78] A. Perdih, G. Wolber, T. Solmajer. Molecular dynamics simulation and linear interaction energy study of d-Glu-based inhibitors of the MurD ligase, J. Comput. Aided Mol. Des., 27(8):723-738, 2013.
Links: doi:10.1007/S10822-013-9673-3 | Publisher | Download citation: BibTeX RIS
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Abstract: The biosynthetic pathway of the bacterial peptidoglycan, where MurD is an enzyme involved at the intracellular stage of its construction, represents a collection of highly selective macromolecular targets for novel antibacterial drug design. In this study as part of our investigation of the MurD bacterial target two recently discovered classes of the MurD ligase inhibitors were investigated resulting from the lead optimization phases of the N-sulfonamide d-Glu MurD inhibitors. Molecular dynamics simulations, based on novel structural data, in conjunction with the linear interaction energy (LIE) method suggested the transferability of our previously obtained LIE coefficients to further d-Glu based classes of MurD inhibitors. Analysis of the observed dynamical behavior of these compounds in the MurD active site was supported by static drug design techniques. These results complement the current knowledge of the MurD inhibitory mechanism and provide valuable support for the d-Glu paradigm of the inhibitor design.

[77] M. Spetea, M. F. Asim, G. Wolber, H. Schmidhammer. The µ opioid receptor and ligands acting at the µ opioid receptor as therapeutics and potential therapeutics, Curr. Pharm. Des., 19(42):7415-7434, 2013.
Links: doi:10.2174/13816128113199990362 | Publisher | Download citation: BibTeX RIS
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Abstract: Although the mu opioid receptor (MOR) was pharmacologically and biochemically identified in binding studies forty years ago, its structure, function, and true complexity only have emerged after its cloning in 1993. Continuous efforts from many laboratories have greatly advanced our understanding of MORs, ranging from their anatomic distribution to cellular and molecular mechanisms, and from cell lines to in vivo systems. The MOR is recognized as the main target for effective pain relief, but its involvement in many other physiological functions has also been recognized. This review provides a synopsis on the history of research on MORs and ligands acting at the MOR with the focus on their clinical and potential use as therapeutic drugs, or as valuable research tools. Since the elucidation of the chemical structure of morphine and the characterization of endogenous opioid peptides, research has stimulated the development of new generations of MOR ligands with distinct pharmacological profiles (agonist, antagonist, mixed agonist/antagonist and partial agonist) or site of action (central/peripheral). Discovery of therapeutically useful morphine-like drugs and innovative drugs with new scaffolds, with several outstanding representatives, is discussed. Extensive efforts on modifications of endogenous peptides to attain stable and MOR selective analogs are overviewed with stimulating results for the development of peptide-based pharmaceuticals. With pharmacophore modeling as an important tool in drug discovery, application of modern computational methodologies for the development of morphinans as new MOR ligands is described. Moreover, the crystal structure of the MOR available today will enable the application of structure-based approaches to design better drugs for the management of pain, addiction and other human diseases, where MORs play a key role.

[76] M. Spetea, M. F. Asim, S. Noha, G. Wolber, H. Schmidhammer. Current κ opioid receptor ligands and discovery of a new molecular scaffold as a κ opioid receptor antagonist using pharmacophore-based virtual screening, Curr. Pharm. Des., 19(42):7362-7372, 2013.
Links: doi:10.2174/138161281942140105162601 | Publisher | Download citation: BibTeX RIS
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Abstract: The kappa opioid receptor (KOR) plays a significant role in many physiological functions, including pain relief, stress, depression, drug abuse, anxiety and psychotic behaviors. KORs are widely distributed in the central and peripheral nervous systems, and are specifically activated by endogenous opioids derived from prodynorphin. They are members of the G protein-coupled receptor superfamily, and the crystal structure of the human KOR was recently elucidated. KORs were initially studied for their involvement in mediation of pain as stimulation of KOR produces analgesia and minimizes abuse liability and other side effects. Nowadays, the KOR is rapidly emerging as an important target for the treatment of a variety of other human disorders. Specifically, the KOR system has become increasingly implicated as a modulator of stress-related and addictive behaviors. Several selective KOR partial agonists and antagonists have been developed as potential antidepressants, anxiolytic and anti-addiction medications. Although many KOR ligands have not demonstrated desirable pharmacological properties, some have been shown to be viable drug candidates. Herein, we describe chemical and pharmacological developments on KOR ligands, advantages and challenges, and potential therapeutic applications of ligands for KORs. In the second part, recent advances in the KOR drug design utilizing computational approaches are presented, with focus on the discovery of a new naturally derived scaffold, sewarine, as a novel class of selective KOR ligands with antagonist properties, using a pharmacophore-based virtual screening strategy.

[75] M. Levay, K. A. Krobert, K. Wittig, N. Voigt, M. Bermudez, G. Wolber, D. Dobrev, F. O. Levy, T. Wieland. NSC23766, a widely used inhibitor of rac1 activation, additionally acts as a competitive antagonist at muscarinic acetylcholine receptors, J. Pharmacol. Exp. Ther., 347(1):69-79, 2013.
Links: doi:10.1124/Jpet.113.207266 | Publisher | Download citation: BibTeX RIS
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Abstract: Small molecules interfering with Rac1 activation are considered as potential drugs and are already studied in animal models. A widely used inhibitor without reported attenuation of RhoA activity is NSC23766 [(N-6-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride]. We found that NSC23766 inhibits the M-2 muscarinic acetylcholine receptor (M-2 mAChR)-induced Rac1 activation in neonatal rat cardiac myocytes. Surprisingly, NSC27366 concomitantly suppressed the carbachol-induced RhoA activation and a M-2 mAChR-induced inotropic response in isolated neonatal rat hearts requiring the activation of Rho-dependent kinases. We therefore aimed to identify the mechanisms by which NSC23766 interferes with the differentially mediated, M-2 mAChR-induced responses. Interestingly, NSC23766 caused a rightward shift of the carbachol concentration response curve for the positive inotropic response without modifying carbachol efficacy. To analyze the specificity of NSC23766, we compared the carbachol and the similarly G(i)beta gamma-mediated, adenosine-induced activation of G(i) protein-regulated potassium channel (GIRK) channels in human atrial myocytes. Application of NSC23766 blocked the carbachol-induced K+ current but had no effect on the adenosine-induced GIRK current. Similarly, an adenosine A(1) receptor-induced positive inotropic response in neonatal rat hearts was not attenuated by NSC23766. To investigate its specificity toward the different mAChR types, we studied the carbachol-induced elevation of intracellular Ca2+ concentrations in human embryonic kidney 293 (HEK-293) cells expressing M-1, M-2, or M-3 mAChRs. NSC23766 caused a concentration-dependent rightward shift of the carbachol concentration response curves at all mAChRs. Thus, NSC23766 is not only an inhibitor of Rac1 activation, but it is within the same concentration range a competitive antagonist at mAChRs. Molecular docking analysis at M-2 and M-3 mAChR crystal structures confirmed this interpretation.

[74] J. Leschner, G. Wennerberg, J. Feierler, M. Bermudez, B. Welte, I. Kalatskaya, G. Wolber, A. Faussner. Interruption of the ionic lock in the Bradykinin B-2 receptor results in constitutive internalization and turns several antagonists into strong agonists, J. Pharmacol. Exp. Ther., 344(1):85-95, 2013.
Links: doi:10.1124/Jpet.112.199190 | Publisher | Download citation: BibTeX RIS
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Abstract: The DRY motif with the highly conserved R3.50 is a hallmark of family A G protein-coupled receptors (GPCRs). The crystal structure of rhodopsin revealed a salt bridge between R135(3.50) and another conserved residue, E247(6.30), in helix 6. This ionic lock was shown to maintain rhodopsin in its inactive state. Thus far, little information is available on how interruption of this ionic bond affects signaling properties of nonrhodopsin GPCRs, because the focus has been on mutations of R3.50, although this residue is indispensable for G protein activation. To investigate the importance of an ionic lock for overall receptor activity in a nonrhodopsin GPCR, we mutated R128(3.50) and E238(6.30) in the bradykinin (BK) B-2 receptor (B2R) and stably expressed the constructs in HEK293 cells. As expected, mutation of R3.50 resulted in lack of G protein activation. In addition, this mutation led to considerable constitutive receptor internalization. Mutation of E6.30 (mutants E6.30A and E6.30R) also caused strong constitutive internalization. Most intriguingly, however, although the two E6.30 mutants displayed no increased basal phosphatidylinositol hydrolysis, they gave a response to three different B2R antagonists that was almost comparable to that obtained with BK. In contrast, swapping of R3.50 and E6.30, thus allowing the formation of an inverse ionic bond, resulted in rescue of the wild type phenotype. These findings demonstrate for the first time, to our knowledge, that interruption of the ionic lock in a family A GPCR can have distinctly different effects on receptor internalization and G protein stimulation, shedding new light on its role in the activation process.

[73] M. Enthammer, E. S. Papadakis, M. S. Gachet, M. Deutsch, S. Schwaiger, K. Koziel, M. I. Ashraf, S. Khalid, G. Wolber, G. Packham, R. I. Cutress, H. Stuppner, J. Troppmair. Isolation of a novel Thioflavin S-derived compound that inhibits BAG-1-mediated protein interactions and targets BRAF inhibitor-resistant cell lines, Mol. Cancer Ther., 12(11):2400-2414, 2013.
Links: doi:10.1158/1535-7163.Mct-13-0142 | Publisher | Download citation: BibTeX RIS
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Abstract: Protein-protein interactions mediated through the C-terminal Bcl-2-associated athanogene (BAG) domain of BAG-1 are critical for cell survival and proliferation. Thioflavin S (NSC71948)-a mixture of compounds resulting from the methylation and sulfonation of primulin base-has been shown to dose-dependently inhibit the interaction between BAG-1 and Hsc70 in vitro. In human breast cancer cell lines, with high BAG-1 expression levels, Thioflavin S reduces the binding of BAG-1 to Hsc70, Hsp70, or CRAF and decreases proliferation and viability. Here, we report the development of a protocol for the purification and isolation of biologically active constituents of Thioflavin S and the characterization of the novel compound Thio-2. Thio-2 blocked the growth of several transformed cell lines, but had much weaker effects on untransformed cells. Thio-2 also inhibited the proliferation of melanoma cell lines that had become resistant to treatment with PLX4032, an inhibitor of mutant BRAF. In transformed cells, Thio-2 interfered with intracellular signaling at the level of RAF, but had no effect on the activation of AKT. Thio-2 decreased binding of BAG-1 to Hsc70 and to a lesser extent BRAF in vitro and in vivo, suggesting a possible mechanism of action. Given that tumors frequently develop resistance to kinase inhibitors during treatment, Thio-2 and related compounds may offer promising alternative strategies to currently available therapies. (C) 2013 AACR.

[72] K. N. de Oliveira, V. Andermark, S. von Grafenstein, L. A. Onambele, G. Dahl, R. Rubbiani, G. Wolber, C. Gabbiani, L. Messori, A. Prokop, I. Ott. Butyltin(IV) benzoates: Inhibition of thioredoxin reductase, tumor cell growth inhibition, and interactions with proteins, ChemMedChem, 8(2):256-264, 2013.
Links: doi:10.1002/Cmdc.201200505 | Publisher | Download citation: BibTeX RIS
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Abstract: Thioredoxin reductase (TrxR) is overexpressed in cancer cells and is therefore a putative cancer target. Inhibition of this enzyme is considered an important strategy for the development of new chemotherapeutic agents with a specific mechanism of action. Organotin compounds have been described as experimental antitumor agents, yet their mechanism of action remains largely unknown. Based on the outcome of a virtual screening study, various di- and tri-n-butyltin(IV) carboxylates were synthesized, and their biological properties were evaluated. All synthesized compounds were able to inhibit TrxR selectively within the micromolar range and showed potent antitumor activity against HT-29 and MCF-7 cancer cell lines. Moreover, tin(IV) organometallics were found to strongly induce apoptosis in the BJAB lymphoma cell line. Mass spectrometry and atomic absorption spectroscopy experiments revealed metal binding to proteins, and efficient cellular uptake was observed using a di-n-butyltin(IV) complex as an example.

[71] C. D. Cadicamo, J. Mortier, G. Wolber, M. Hell, I. E. Heinrich, D. Michel, L. Semlin, U. Berger, H. C. Korting, H. D. Holtje, B. Koksch, C. Borelli. Design, synthesis, inhibition studies, and molecular modeling of pepstatin analogues addressing different secreted aspartic proteinases of Candida albicans, Biochem. Pharmacol., 85(7):881-887, 2013.
Links: doi:10.1016/J.Bcp.2012.12.008 | Publisher | Download citation: BibTeX RIS
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Abstract: The family of secreted aspartic proteinases is known as an important virulence factor of yeast infections by Candida albicans in particular, which is the most common fungal pathogen for humans with respect to systemic disease. Due to the continuing increase of drug resistant strains, these proteinases are currently considered as promising drug target candidates. Based on the known Sap2-substrate specificity data and X-ray analyses of Sap/inhibitor complexes, three libraries of inhibitors were designed and synthesized by modifying the structure of pepstatin A, a common non-selective aspartic proteinase inhibitor, at the P3, P2, or P2' position. These novel inhibitors showed high inhibitory potencies for the isoenzymes Sap1, Sap3, Sap5 and Sap6. Then, the affinity and selectivity of the peptide ligands were investigated by molecular modeling, highlighting new key structural information for the design of potent and selective anti-virulence agents targeting Candida albicans. (c) 2012 Elsevier Inc. All rights reserved.

[70] A. Schafer, A. Wellner, M. Strauss, A. Schafer, G. Wolber, R. Gust. Influence of chlorine or fluorine substitution on the estrogenic properties of 1-alkyl-2,3,5-tris(4-hydroxyphenyl)-1h-pyrroles, J. Med. Chem., 55(22):9607-9618, 2012. Publisher | Download citation: BibTeX RIS
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Abstract: In continuation of Our previous work, several 1- alkyl-2,3,5-tris(4-hydroxyphenyl)aryl-1H-pyrroles with chlor-Me or fluorine substituents in the aryl residues were synthesized and tested for estrogen receptor (ER) binding at isolated ER alpha/ER beta. receptors (HAP assay), and. in trans activation assays using ER alpha-positive MCF-7/2a as well as U2-OS/ER alpha and U2-OS/ER beta cells. In the competition experiment at ER alpha the compounds displayed very high relative binding-affinities of up to 37% (determined for 8m) but with restricted subtype selectivity (e.g., ER alpha/ER beta (8ma) = 9). The highest estrogenic potency in ER alpha-positive MCP-7/2a cells was determined for 2,3,5-tris(2-fluoro-4-hydroxyphenyl)-1-prolayl-1H-pyriole 8m (EC50 = 23 nM), while in U2-OS/ER alpha cells 2-(2-fluoro-4-hydroxyphenyl)-3,5-bis(4-hydroxyphenyl)-1H propyl-1H pyrrole 8b (EC50 = 0.12 nM) was the Most potent agonist, only 30 fold less active than estradiol (E2, EC50 = 0.004 nM). In U2-OS/ER beta cells for all pyrroles no transactivation could be observed, which indicates that they are selective ER alpha agonists in Cellular systems.

[69] R. Ottana, R. Maccari, S. Amuso, G. Wolber, D. Schuster, S. Herdlinger, G. Manao, G. Camici, P. Paoli. New 4-[(5-arylidene-2-arylimino-4-oxo-3-thiazolidinyl)methyl]benzoic acids active as protein tyrosine phosphatase inhibitors endowed with insulinomimetic effect on mouse C2C12 skeletal muscle cells, Eur. J. Med. Chem., 50:332-343, 2012.
Links: doi:10.1016/J.Ejmech.2012.02.012 | Publisher | Download citation: BibTeX RIS
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Abstract: In pursuing our research targeting the identification of potent inhibitors of PTP1B and LMW-PTP, we have identified new 4-[(5-arylidene-2-arylimino-4-oxo-3-thiazolidinyl)methyl]benzoic acids endowed with interesting in vitro inhibitory profiles. Most compounds proved to be inhibitors of PTP1B and LMW-PTP isoform IF1. The tested inhibitors also showed selectivity towards PTP1B over the closely related TC-PTP. These compounds were found to activate the insulin-mediated signalling on mouse C2C12 skeletal muscle cells by increasing the phosphorylation levels of the insulin receptor and promoting cellular 2-deoxyglucose uptake. Interestingly, 4-{[5-(4-benzyloxybenzylidene)-2-(4-trifluoromethylphenylmino)-4-oxo-3-thiazolidinyl]methyl}benzoic acid (7d), the best in vitro inhibitor of PTP1B and the isoform IF1 of LMW-PTP, provided the highest activation level of the insulin receptor and was found to be endowed with an excellent insulinomimetic effect on the selected cells. This compound therefore represents an interesting lead compound for developing novel PTP1B and LMW-PTP inhibitors which could be achieved by improving both its pharmacological profile and its potentiating effects on insulin signalling. (C) 2012 Elsevier Masson SAS. All rights reserved.

[68] S. Distinto, M. Yanez, S. Alcaro, M. C. Cardia, M. Gaspari, M. L. Sanna, R. Meleddu, F. Ortuso, J. Kirchmair, P. Markt, A. Bolasco, G. Wolber, D. Secci, E. Maccioni. Synthesis and biological assessment of novel 2-thiazolylhydrazones and computational analysis of their recognition by monoamine oxidase B, Eur. J. Med. Chem., 48:284-295, 2012.
Links: doi:10.1016/J.Ejmech.2011.12.027 | Publisher | Download citation: BibTeX RIS
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Abstract: Monoamine oxidase B (MAO-B) is a promising target for the treatment of neurodegenerative disorders. We report the synthesis and the biological evaluation of halogenated derivatives of 1-aryliden-2-(4-phenylthiazol-2-yl)hydrazines. The fluorinated series shows interesting activity and great selectivity toward the human recombinant MAO-B isoform expressed in baculovirus infected BTI insect cells. The multiple crystal structures alignment of the enzyme highlighted pronounced induced fit (IF) adaptations with respect to bound ligands. Therefore, IF docking (IFD) experiments and molecular dynamic (MD) simulations were carried out to reveal the putative binding mode and to explain the experimentally observed differences in the activity of 1-(aryliden-2-(4-(4-chlorophenyl)thiazol-2-yl)hydrazines. The importance of water molecules within the binding site was also investigated. These are known to play an important role in the binding site cavity and to mediate protein ligand interactions. Detailed analyses of the trajectories provide insights on the chemical features required for the activity of this scaffold. In particular it was highlighted the importance of fluorine atom interacting with the water close to the cofactor and the influence of steric bulkiness of substituents in the arylidene moiety. Free energy perturbation (FEP) analysis confirmed experimental data. The information we deduced will help to develop novel high-affinity MAO-B inhibitors. (C) 2011 Elsevier Masson SAS. All rights reserved.

[67] S. Distinto, F. Esposito, J. Kirchmair, M. C. Cardia, M. Gaspari, E. Maccioni, S. Alcaro, P. Markt, G. Wolber, L. Zinzula, E. Tramontano. Identification of HIV-1 reverse transcriptase dual inhibitors by a combined shape-, 2D-fingerprint- and pharmacophore-based virtual screening approach, Eur. J. Med. Chem., 50:216-229, 2012.
Links: doi:10.1016/J.Ejmech.2012.01.056 | Publisher | Download citation: BibTeX RIS
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Abstract: We report the first application of ligand-based virtual screening (VS) methods for discovering new compounds able to inhibit both human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT)-associated functions, DNA polymerase and ribonuclease H (RNase H) activities. The overall VS campaign consisted of two consecutive screening processes. In the first, the VS platform Rapid Overlay of Chemical Structures (ROCS) was used to perform in silico shape-based similarity screening on the NCI compounds database in which a hydrazone derivative, previously shown to inhibit the HIV-1 RI, was chosen. As a result, 34 hit molecules were selected and assayed on both RI-associated functions. In the second, the 4 most potent RI inhibitors identified were selected as queries for parallel VS performed by combining shape-based, 2D-fingerprint and 3D-pharmacophore VS methods. Overall, a set of molecules characterized by new different scaffolds were identified as novel inhibitors of both HIV-1 RI-associated activities in the low micromolar range. (C) 2012 Elsevier Masson SAS. All rights reserved.

[66] S. von Grafenstein, J. Mihaly-Bison, G. Wolber, V. N. Bochkov, K. R. Liedl, D. Schuster. Identification of Novel Liver X Receptor Activators by Structure-Based Modeling, J. Chem. Inf. Model., 52(5):1391-1400, 2012.
Links: doi:10.1021/Ci300096c | Publisher | Download citation: BibTeX RIS
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Abstract: Liver X receptors (LXRs) are members of the nuclear receptor family. Activators of LXRs are of high pharmacological interest as LXRs regulate cholesterol, fatty acid, and carbohydrate metabolism as well as inflammatory processes. On the basis of different X-ray crystal structures, we established a virtual screening workflow for the identification of novel LXR modulators. A two-step screening concept to identify active compounds included 3D-pharmacophore filters and rescoring by shape alignment. Eighteen virtual hits were tested in vitro applying a reporter gene assay, where concentration-dependent activity was proven for four novel lead structures. The most active compound 10, a 1,4-naphthochinone, has an estimated EC50 of around 5 mu M.

[65] S. Santos-Sierra, J. Kirchmair, A. M. Perna, D. Reiss, K. Kemter, W. Roschinger, H. Glossmann, S. W. Gersting, A. C. Muntau, G. Wolber, F. B. Lagler. Novel pharmacological chaperones that correct phenylketonuria in mice, Hum. Mol. Genet., 21(8):1877-1887, 2012.
Links: doi:10.1093/Hmg/Dds001 | Publisher | Download citation: BibTeX RIS
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Abstract: Phenylketonuria (PKU) is caused by inherited phenylalanine-hydroxylase (PAH) deficiency and, in many genotypes, it is associated with protein misfolding. The natural cofactor of PAH, tetrahydrobiopterin (BH4), can act as a pharmacological chaperone (PC) that rescues enzyme function. However, BH4 shows limited efficacy in some PKU genotypes and its chemical synthesis is very costly. Taking an integrated drug discovery approach which has not been applied to this target before, we identified alternative PCs for the treatment of PKU. Shape-focused virtual screening of the National Cancer Institutes chemical library identified 84 candidate molecules with potential to bind to the active site of PAH. An in vitro evaluation of these yielded six compounds that restored the enzymatic activity of the unstable PAHV106A variant and increased its stability in cell-based assays against proteolytic degradation. During a 3-day treatment study, two compounds (benzylhydantoin and 6-amino-5-(benzylamino)-uracil) substantially improved the in vivo Phe oxidation and blood Phe concentrations of PKU mice (Pah(enu1)). Notably, benzylhydantoin was twice as effective as tetrahydrobiopterin. In conclusion, we identified two PCs with high in vivo efficacy that may be further developed into a more effective drug treatment of PKU.

[64] S. M. Noha, B. Jazzar, S. Kuehnl, J. M. Rollinger, H. Stuppner, A. M. Schaible, O. Werz, G. Wolber, D. Schuster. Pharmacophore-based discovery of a novel cytosolic phospholipase A(2)alpha inhibitor, Bioorg Med Chem Lett, 22(2):1202-1207, 2012.
Links: doi:10.1016/J.Bmcl.2011.11.093 | Publisher | Download citation: BibTeX RIS
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Abstract: The release of arachidonic acid, a precursor in the production of prostaglandins and leukotrienes, is achieved by activity of the cytosolic phospholipase A(2)alpha (cPLA(2)alpha). Signaling mediated by this class of bioactive lipids, which are collectively referred to as eicosanoids, has numerous effects in physiological and pathological processes. Herein, we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual screening of the National Cancer Institute (NCI) database, leading to the identification of 4-(hexadecyloxy)-3-(2-(hydroxyimino)-3-oxobutanamido)benzoic acid (NSC 119957) as cPLA(2)alpha inhibitor in cell-free and cell-based in vitro assays. (C) 2011 Elsevier Ltd. All rights reserved.

[63] M. S. Murgueitio, M. Bermudez, J. Mortier, G. Wolber. In silico virtual screening approaches for anti-viral drug discovery, Drug Discov. Today Technol., 9(3):e219-25, 2012.
Links: doi:10.1016/j.ddtec.2012.07.009 | Publisher | Download citation: BibTeX RIS
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Abstract: Despite the considerable advances in medical and pharmaceutical research during the past years, diseases caused by viruses have remained a major burden to public health. Virtual in silico screening has repeatedly proven to be useful to meet the special challenges of antiviral drug discovery. Large virtual compound libraries are filtered by different computational screening methods such as docking, ligand-based similarity searches or pharmacophore-based screening, reducing the number of candidate molecules to a smaller set of promising candidates that are then tested biologically. This rational approach makes the drug discovery process more goal-oriented and saves resources in terms of time and money. In this review we discuss how different virtual screening techniques can be applied to antiviral drug discovery, present recent success stories in this field and finally address the main differences between the methods.:

[62] J. Mortier, C. Rakers, R. Frederick, G. Wolber. Computational tools for in silico fragment-based drug design, Curr. Top. Med. Chem., 12(17):1935-1943, 2012. Download citation: BibTeX RIS
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Abstract: Fragment-based strategy in drug design involves the initial discovery of low-molecular mass molecules. Owing to their small-size, fragments are molecular tools to probe specific sub-pockets within a protein active site. Once their interaction within the enzyme cavity is clearly understood and experimentally validated, they represent a unique opportunity to design potent and efficient larger compounds. Computer-aided methods can essentially support the identification of suitable fragments. In this review, available tools for computational drug design are discussed in the frame of fragment-based approaches. We analyze and review (i) available commercial fragment libraries with respect to their properties and size, (ii) computational methods for the construction of such a library, (iii) the different strategies and software packages for the selection of the fragments with predicted affinity to a given target, and (iv) tools for the in silico linkage of fragments into an actual high-affinity lead structure candidate.

[61] L. Guasch, E. Sala, A. Castell-Auvi, L. Cedo, K. R. Liedl, G. Wolber, M. Muehlbacher, M. Mulero, M. Pinent, A. Ardevol, C. Valls, G. Pujadas, S. Garcia-Vallve. Identification of PPARγ partial agonists of natural origin (I): Development of a virtual screening procedure and in vitro validation, PLoS One, 7(11):e50816, 2012.
Links: doi:10.1371/journal.pone.0050816 | Publisher | Download citation: BibTeX RIS
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Abstract: Background: Although there are successful examples of the discovery of new PPAR gamma agonists, it has recently been of great interest to identify new PPAR gamma partial agonists that do not present the adverse side effects caused by PPAR gamma full agonists. Consequently, the goal of this work was to design, apply and validate a virtual screening workflow to identify novel PPAR gamma partial agonists among natural products. Methodology/Principal Findings: We have developed a virtual screening procedure based on structure-based pharmacophore construction, protein-ligand docking and electrostatic/shape similarity to discover novel scaffolds of PPAR gamma partial agonists. From an initial set of 89,165 natural products and natural product derivatives, 135 compounds were identified as potential PPAR gamma partial agonists with good ADME properties. Ten compounds that represent ten new chemical scaffolds for PPAR gamma partial agonists were selected for in vitro biological testing, but two of them were not assayed due to solubility problems. Five out of the remaining eight compounds were confirmed as PPAR gamma partial agonists: they bind to PPAR gamma do not or only moderately stimulate the transactivation activity of PPAR gamma do not induce adipogenesis of preadipocyte cells and stimulate the insulin-induced glucose uptake of adipocytes. Conclusions/Significance: We have demonstrated that our virtual screening protocol was successful in identifying novel scaffolds for PPAR gamma partial agonists.

[60] A. Faussner, S. Schussler, J. Feierler, M. Bermudez, J. Pfeifer, K. Schnatbaum, T. Tradler, M. Jochum, G. Wolber, C. Gibson. Binding characteristics of [3H]-JSM10292: A new cell membrane-permeant non-peptide bradykinin B2 receptor antagonist, Br. J. Pharmacol., 167(4):839-853, 2012.
Links: doi:10.1111/J.1476-5381.2012.02054.X | Publisher | Download citation: BibTeX RIS
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Abstract: BACKGROUND AND PURPOSE A 3H-labelled derivative of the novel small-molecule bradykinin (BK) B2 receptor antagonist JSM10292 was used to directly study its binding properties to human and animal B2 receptors in intact cells and to closely define its binding site. EXPERIMENTAL APPROACH Equilibrium binding, dissociation and competition studies with various B2 receptor ligands and [3H]-JSM10292 were performed at 4 degrees C and 37 degrees C. The experiments were carried out using HEK293 cells stably (over)expressing wild-type and mutant B2 receptors of human and animal origin. KEY RESULTS [3H]-JSM10292 bound to B2 receptors at 4 degrees C and at 37 degrees C with the same high affinity. Its dissociation strongly depended on the temperature and increased when unlabelled B2 receptor agonists or antagonists were added. [3H]-JSM10292 is cell membrane-permeant and thus also bound to intracellular, active B2 receptors, as indicated by the different nonspecific binding in the presence of unlabelled JSM10292 or of membrane-impermeant BK. Equilibrium binding curves with [3H]-JSM10292 and competition experiments with unlabelled JSM10292 and [3H]-BK showed a different affinity profile for the wild-type B2 receptor in different species (man, cynomolgus, rabbit, mouse, rat, dog, pig, guinea pig). Characterization of B2 receptor mutants and species orthologues combined with homology modelling, using the CXCR4 as template, suggests that the binding site of JSM10292 is different from that of BK but overlaps with that of MEN16132, another small non-peptide B2 receptor ligand. CONCLUSIONS AND IMPLICATIONS [3H]-JSM10292 is a novel, cell membrane-permeant, high-affinity B2 receptor antagonist that allows direct in detail studies of active, surface and intracellularly located wild-type and mutant B2 receptors.

[59] B. Waltenberger, K. Wiechmann, J. Bauer, P. Markt, S. M. Noha, G. Wolber, J. M. Rollinger, O. Werz, D. Schuster, H. Stuppner. Pharmacophore modeling and virtual screening for novel acidic inhibitors of microsomal prostaglandin E-2 synthase-1 (mPGES-1), J. Med. Chem., 54(9):3163-3174, 2011.
Links: doi:10.1021/Jm101309g | Publisher | Download citation: BibTeX RIS
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Abstract: Microsomal prostaglandin E-2 synthase-1 (mPGES-1) catalyzes prostaglandin E-2 formation and is considered as a potential anti-inflammatory pharmacological target. To identify novel chemical scaffolds active on this enzyme, two pharmacophore models for acidic mPGES-1 inhibitors were developed and theoretically validated using information on mPGES-1 inhibitors from literature. The models were used to screen chemical databases supplied from the National Cancer Institute (NCI) and the Specs. Out of 29 compounds selected for biological evaluation, nine chemically diverse compounds caused concentration-dependent inhibition of mPGES-1 activity in a cell-free assay with IC50 values between 0.4 and 7.9 mu M, respectively. Further pharmacological characterization revealed that also 5-lipoxygenase (5-LO) was inhibited by most of these active compounds in cell-free and cell-based assays with IC50 values in the low micromolar range. Together, nine novel chemical scaffolds inhibiting mPGES-1 are presented that may possess anti-inflammatory properties based on the interference with eicosanoid biosynthesis.

[58] D. Schuster, P. Markt, U. Grienke, J. Mihaly-Bison, M. Binder, S. M. Noha, J. M. Rollinger, H. Stuppner, V. N. Bochkov, G. Wolber. Pharmacophore-based discovery of FXR agonists. Part I: Model development and experimental validation, Bioorgan Med Chem, 19(23):7168-7180, 2011.
Links: doi:10.1016/J.Bmc.2011.09.056 | Publisher | Download citation: BibTeX RIS
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Abstract: The farnesoid X receptor (FXR) is involved in glucose and lipid metabolism regulation, which makes it an attractive target for the metabolic syndrome, dyslipidemia, atherosclerosis, and type 2 diabetes. In order to find novel FXR agonists, a structure-based pharmacophore model collection was developed and theoretically evaluated against virtual databases including the ChEMBL database. The most suitable models were used to screen the National Cancer Institute (NCI) database. Biological evaluation of virtual hits led to the discovery of a novel FXR agonist with a piperazine scaffold (compound 19) that shows comparable activity as the endogenous FXR agonist chenodeoxycholic acid (CDCA, compound 2). (C) 2011 Elsevier Ltd. All rights reserved.

[57] P. H. Pfisterer, C. X. Shen, Z. Nikolovska-Coleska, L. Schyschka, D. Schuster, A. Rudy, G. Wolber, A. M. Vollmar, J. M. Rollinger, H. Stuppner. In silico discovery of acylated flavonol monorhamnosides from Eriobotrya japonica as natural, small-molecular weight inhibitors of XIAP BIR3, Bioorgan Med Chem, 19(2):1002-1009, 2011.
Links: doi:10.1016/J.Bmc.2010.10.046 | Publisher | Download citation: BibTeX RIS
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Abstract: Targeting the baculoviral inhibitor of apoptosis proteins repeat (BIR) 3 of X-linked inhibitor of apoptosis proteins (XIAP) represents an innovative strategy for the design of chemosensitizers. Acylated flavonol monorhamnosides (AFMR) from Eriobotrya japonica Lindl. (Rosaceae) were virtually predicted as ligands of the XIAP BIR3 domain by using a previously generated pharmacophore model. From the methanol leaf extract of E. japonica an enriched mixture of AFMR was obtained showing chemosensitizing potential in combination with etoposide in XIAP-overexpressing Jurkat cells. The HPLC-SPE-NMR hyphenated technique facilitated the structure elucidation of three known and two new natural AFMR. The main constituent and virtual hit, kaempferol-3-O-alpha-L-(2 '',4 ''-di-E-p-coumaroyl)-rhamnoside (3) was isolated from the enriched fraction. Applying a fluorescence polarization based binding assay, 3 was identified as XIAP BIR3 ligand with a dose-dependent affinity (IC(50) 10.4 mu M). Further, 3 induced apoptosis in XIAP-overexpressing Jurkat cells and activated caspase-9 in combination with etoposide. Docking experiments revealed a major impact of the coumaric acid and sugar moieties of 3 on XIAP BIR3 binding, which was experimentally confirmed. To conclude, this study elucidates 3 as natural, small-molecular weight XIAP BIR3 inhibitor using a combination of in silico and HPLC-SPE-NMR hyphenated techniques. (C) 2010 Published by Elsevier Ltd.

[56] U. Grienke, J. Mihaly-Bison, D. Schuster, T. Afonyushkin, M. Binder, S. H. Guan, C. R. Cheng, G. Wolber, H. Stuppner, D. A. Guo, V. N. Bochkov, J. M. Rollinger. Pharmacophore-based discovery of FXR-agonists. Part II: Identification of bioactive triterpenes from Ganoderma lucidum, Bioorgan Med Chem, 19(22):6779-6791, 2011.
Links: doi:10.1016/J.Bmc.2011.09.039 | Publisher | Download citation: BibTeX RIS
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Abstract: The farnesoid X receptor (FXR) belonging to the metabolic subfamily of nuclear receptors is a ligand-induced transcriptional activator. Its central function is the physiological maintenance of bile acid homeostasis including the regulation of glucose and lipid metabolism. Accessible structural information about its ligand-binding domain renders FXR an attractive target for in silico approaches. Integrated to natural product research these computational tools assist to find novel bioactive compounds showing beneficial effects in prevention and treatment of, for example, the metabolic syndrome, dyslipidemia, atherosclerosis, and type 2 diabetes. Virtual screening experiments of our in-house Chinese Herbal Medicine database with structure-based pharmacophore models, previously generated and validated, revealed mainly lanostane-type triterpenes of the TCM fungus Ganoderma lucidum Karst. as putative FXR ligands. To verify the prediction of the in silico approach, two Ganoderma fruit body extracts and compounds isolated thereof were pharmacologically investigated. Pronounced FXR-inducing effects were observed for the extracts at a concentration of 100 mu g/mL. Intriguingly, five lanostanes out of 25 secondary metabolites from G. lucidum, that is, ergosterol peroxide (2), lucidumol A (11), ganoderic acid TR (12), ganodermanontriol (13), and ganoderiol F (14), dose-dependently induced FXR in the low micromolar range in a reporter gene assay. To rationalize the binding interactions, additional pharmacophore profiling and molecular docking studies were performed, which allowed establishing a first structure-activity relationship of the investigated triterpenes. (C) 2011 Elsevier Ltd. All rights reserved.

[55] B. Waltenberger, D. Schuster, S. Paramapojn, W. Gritsanapan, G. Wolber, J. M. Rollinger, H. Stuppner. Predicting cyclooxygenase inhibition by three-dimensional pharmacophoric profiling. Part II: Identification of enzyme inhibitors from Prasaplai, a Thai traditional medicine, Phytomedicine, 18(2-3):119-133, 2011.
Links: doi:10.1016/J.Phymed.2010.08.002 | Publisher | Download citation: BibTeX RIS
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Abstract: Prasaplai is a medicinal plant mixture that is used in Thailand to treat primary dysmenorrhea, which is characterized by painful uterine contractility caused by a significant increase of prostaglandin release. Cyclooxygenase (COX) represents a key enzyme in the formation of prostaglandins. Former studies revealed that extracts of Prasaplai inhibit COX-1 and COX-2. In this study, a comprehensive literature survey for known constituents of Prasaplai was performed. A multiconformational 3D database was created comprising 683 molecules. Virtual parallel screening using six validated pharmacophore models for COX inhibitors was performed resulting in a hit list of 166 compounds. 46 Prasaplai components with already determined COX activity were used for the external validation of this set of COX pharmacophore models. 57% of these components were classified correctly by the pharmacophore models. These findings confirm that the virtual approach provides a helpful tool (i) to unravel which molecular compounds might be responsible for the COX-inhibitory activity of Prasaplai and (ii) for the fast identification of novel COX inhibitors. (C) 2010 Elsevier GmbH. All rights reserved.

[54] D. Schuster, D. Kowalik, J. Kirchmair, C. Laggner, P. Markt, C. Aebischer-Gumy, F. Strohle, G. Moller, G. Wolber, T. Wilckens, T. Langer, A. Odermatt, J. Adamski. Identification of chemically diverse, novel inhibitors of 17 beta-hydroxysteroid dehydrogenase type 3 and 5 by pharmacophore-based virtual screening, J. Steroid Biochem., 125(1-2):148-161, 2011.
Links: doi:10.1016/J.Jsbmb.2011.01.016 | Publisher | Download citation: BibTeX RIS
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Abstract: 17 beta-Hydroxysteroid dehydrogenase type 3 and 5 (17 beta-HSD3 and 17 beta-HSD5) catalyze testosterone biosynthesis and thereby constitute therapeutic targets for androgen-related diseases or endocrine-disrupting chemicals. As a fast and efficient tool to identify potential ligands for 17 beta HSD3/5, ligand-and structure-based pharmacophore models for both enzymes were developed. The models were evaluated first by in silico screening of commercial compound databases and further experimentally validated by enzymatic efficacy tests of selected virtual hits. Among the 35 tested compounds, 11 novel inhibitors with distinct chemical scaffolds, e.g. sulfonamides and triazoles, and with different selectivity properties were discovered. Thereby, we provide several potential starting points for further 17 beta-HSD3 and 17 beta-HSD5 inhibitor development. Article from the Special issue on Targeted Inhibitors. (C) 2011 Elsevier Ltd. All rights reserved.

[53] A. Schafer, A. Wellner, M. Strauss, G. Wolber, R. Gust. Development of 2,3,5-triaryl-1h-pyrroles as Estrogen receptor α selective ligands, ChemMedChem, 6(11):2055-2062, 2011.
Links: doi:10.1002/Cmdc.201100283 | Publisher | Download citation: BibTeX RIS
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Abstract: 1-Alkyl-2,3,5-triaryl-1H-pyrroles (for which alkyl=methyl, ethyl, n-propyl, or 2-methylpropyl) were tested for stability, estrogen receptor (ER) binding, and inhibition of tumor cell growth. These pyrroles (type B) showed higher stability in aqueous solution than their 1,2,4-triaryl-1H-pyrrole congeners (type A pyrroles), exclusive ER alpha binding (no ER beta interaction), and a hormonal profile of partial agonists at ER alpha. The most potent compound, 1-(2-methylpropyl)-2,3,5-tris(4-hydroxyphenyl)-1H-pyrrole(5d), was less active than the lead structure 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) in MCF-7 cells stably transfected with the plasmid ERE(wtc)luc (MCF-7/2a), but more potent in U2-OS/alpha cells. Furthermore, 5d showed weak antiestrogenic properties (IC(50)= 310 nM). An additional propyl chain at C4 decreased the stability and pharmacological effects.

[52] S. M. Noha, A. G. Atanasov, D. Schuster, P. Markt, N. Fakhrudin, E. H. Heiss, O. Schrammel, J. M. Rollinger, H. Stuppner, V. M. Dirsch, G. Wolber. Discovery of a novel IKK-β inhibitor by ligand-based virtual screening techniques, Bioorg Med Chem Lett, 21(1):577-583, 2011.
Links: doi:10.1016/J.Bmcl.2010.10.051 | Publisher | Download citation: BibTeX RIS
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Abstract: Various inflammatory stimuli that activate the nuclear factor kappa B (NF-kappa B) signaling pathway converge on a serine/threonine kinase that displays a key role in the activation of NF-kappa B: the I kappa beta Bkinase beta (IKK-beta). Therefore, IKK-beta is considered an interesting target for combating inflammation and cancer. In our study, we developed a ligand-based pharmacophore model for IKK-beta inhibitors. This model was employed to virtually screen commercial databases, giving a focused hit list of candidates. Subsequently, we scored by molecular shape to rank and further prioritized virtual hits by three-dimensional shape-based alignment. One out of ten acquired and biologically tested compounds showed inhibitory activity in the low micromolar range on IKK-beta enzymatic activity in vitro and on NF-kappa B transactivation in intact cells. Compound 8 (2-(1-adamantyl) ethyl 4-[(2,5-dihydroxyphenyl) methylamino] benzoate) represents a novel chemical class of IKK-beta inhibitors and shows that the presented model is a valid approach for identification and development of new IKK-beta ligands. (C) 2010 Elsevier Ltd. All rights reserved.

[51] D. V. Kratschmar, A. Vuorinen, T. Da Cunha, G. Wolber, D. Classen-Houben, O. Doblhoffe, D. Schuster, A. Odermatt. Characterization of activity and binding mode of glycyrrhetinic acid derivatives inhibiting 11 beta-hydroxysteroid dehydrogenase type 2, J. Steroid Biochem., 125(1-2):129-142, 2011.
Links: doi:10.1016/J.Jsbmb.2010.12.019 | Publisher | Download citation: BibTeX RIS
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Abstract: Modulation of intracellular glucocorticoid availability is considered as a promising strategy to treat glucocorticoid-dependent diseases. 18 beta-Glycyrrhetinic acid (GA), the biologically active triterpenoid metabolite of glycyrrhizin, which is contained in the roots and rhizomes of licorice (Glycyrrhiza spp.), represents a well-known but non-selective inhibitor of 11 beta-hydroxysteroid dehydrogenases (11 beta-HSDs). However, to assess the physiological functions of the respective enzymes and for potential therapeutic applications selective inhibitors are needed. In the present study, we applied bioassays and 3D-structure modeling to characterize nine 11 beta-HSD1 and fifteen 11 beta-HSD2 inhibiting GA derivatives. Comparison of the GA derivatives in assays using cell lysates revealed that modifications at the 3-hydroxyl and/or the carboxyl led to highly selective and potent 11 beta-HSD2 inhibitors. The data generated significantly extends our knowledge on structure-activity relationship of GA derivatives as 11 beta-HSD inhibitors. Using recombinant enzymes we found also potent inhibition of mouse 11 beta-HSD2, despite significant species-specific differences. The selected GA derivatives potently inhibited 11 beta-HSD2 in intact SW-620 colon cancer cells, although the rank order of inhibitory potential differed from that obtained in cell lysates. The biological activity of compounds was further demonstrated in glucocorticoid receptor (GR) transactivation assays in cells coexpressing GR and 11 beta-HSD1 or 11 beta-HSD2. 3D-structure modeling provides an explanation for the differences in the selectivity and activity of the GA derivatives investigated. The most potent and selective 11 beta-HSD2 inhibitors should prove useful as mechanistic tools for further anti-inflammatory and anti-cancer in vitro and in vivo studies. Article from the Special issue on Targeted Inhibitors. (C) 2011 Elsevier Ltd. All rights reserved.

[50] J. Kirchmair, S. Distinto, K. R. Liedl, P. Markt, J. M. Rollinger, D. Schuster, G. M. Spitzer, G. Wolber. Development of anti-viral agents using molecular modeling and virtual screening techniques, Infect. Disord. Drug Targets, 11(1):64-93, 2011.
Links: doi:10.2174/187152611794407782 | Publisher | Download citation: BibTeX RIS

[49] R. Rubbiani, I. Kitanovic, H. Alborzinia, S. Can, A. Kitanovic, L. A. Onambele, M. Stefanopoulou, Y. Geldmacher, W. S. Sheldrick, G. Wolber, A. Prokop, S. Wolfl, I. Ottt. Benzimidazol-2-ylidene gold(I) complexes are thioredoxin reductase inhibitors with multiple antitumor properties, J. Med. Chem., 53(24):8608-8618, 2010.
Links: doi:10.1021/Jm100801e | Publisher | Download citation: BibTeX RIS
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Abstract: Gold(I) complexes such as auranofin have been used for decades to treat symptoms of rheumatoid arthritis and have also demonstrated a considerable potential as new anticancer drugs. The enzyme thioredoxin reductase (TrxR) is considered as the most relevant molecular target for these species. The here investigated gold(I) complexes with benzimidazole derived N-heterocyclic carbene (NHC) ligands 1a-4a represent a promising class of gold coordination compounds with a good stability against the thiol glutathione. TrxR was selectively inhibited by 1a-4a in comparison to the closely related enzyme glutathione reductase, and all complexes triggered significant antiproliferative effects in cultured tumor cells. More detailed studies on a selected complex (2a) revealed a distinct pharmacodynamic profile including the high increase of reactive oxygen species formation, apoptosis induction, strong effects on cellular metabolism (related to cell surface properties, respiration, and glycolysis), inhibition of mitochondrial respiration and activity against resistant cell lines.

[48] U. Grienke, M. Schmidtke, J. Kirchmair, K. Pfarr, P. Wutzler, R. Durrwald, G. Wolber, K. R. Liedl, H. Stuppner, J. M. Rollinger. Antiviral potential and molecular insight into neuraminidase inhibiting diarylheptanoids from Alpinia Katsumadai, J. Med. Chem., 53(2):778-786, 2010.
Links: doi:10.1021/Jm901440f | Publisher | Download citation: BibTeX RIS
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Abstract: At present, neuraminidase (NA) inhibitors are the mainstay of pharmacological strategies to fight against global pandemic influenza. In the search for new antiviral drug leads from nature, the seed extract of Alpinia katsumadai has been phytochemically investigated. Among the six isolated constituents, four diarylheptanoids showed in vitro NA inhibitory activities in low micromolar ranges against human influenza virus A/PR/8/34 of subtype H(1)N(1). The most promising constituent, katsumadain A (4; IC(50) = 1.05 +/- 0.42 mu M), also inhibited the NA of Four H(1)N(1) swine influenza viruses, with IC(50) values between 0.9 and 1.64 mu M, and showed antiviral effects in plaque reduction assays. Considering the flexible loop regions of NA, extensive molecular dynamics (MD) simulations were performed to study the putative binding mechanism of the T-shaped diarylheptanoid 4. Docking results showed well-established interactions between the protein and the core of this novel NA-inhibiting natural scaffold, excellent surface complementarity to the simulated binding pocket, and concordance with experimentally derived SAR data.

[47] G. M. Spitzer, M. Heiss, M. Mangold, P. Mark, J. Kirchmair, G. Wolber, K. R. Liedl. One concept, three implementations of 3D pharmacophore-based virtual screening: Distinct coverage of chemical search space, J. Chem. Inf. Model., 50(7):1241-1247, 2010.
Links: doi:10.1021/Ci100136b | Publisher | Download citation: BibTeX RIS
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Abstract: Feature-based pharmacophore modeling is a well-established concept to support early stage drug discovery, where large virtual databases are filtered for potential drug candidates. The concept is implemented in popular molecular modeling software, including Catalyst, Phase, and MOE. With these software tools we performed a comparative virtual screening campaign on HSP90 and FXIa, taken from the 'maximum unbiased validation' data set. Despite the straightforward concept that pharmacophores are based on, we observed an unexpectedly high degree of variation among the hit lists obtained. By harmonizing the pharmacophore feature definitions of the investigated approaches, the exclusion volume sphere settings, and the screening parameters, we have derived a rationale for the observed differences, providing insight on the strengths and weaknesses of these algorithms. Application of more than one of these software tools in parallel will result in a widened coverage of chemical space. This is not only rooted in the dissimilarity of feature definitions but also in different algorithmic search strategies.

[46] D. Schuster, M. Spetea, M. Music, S. Rief, M. Fink, J. Kirchmair, J. Schutz, G. Wolber, T. Langer, H. Stuppner, H. Schmidhammer, J. M. Rollinger. Morphinans and isoquinolines: Acetylcholinesterase inhibition, pharmacophore modeling, and interaction with opioid receptors, Bioorgan Med Chem, 18(14):5071-5080, 2010.
Links: doi:10.1016/J.Bmc.2010.05.071 | Publisher | Download citation: BibTeX RIS
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Abstract: Following indications from pharmacophore-based virtual screening of natural product databases, morphinan and isoquinoline compounds were tested in vitro for acetylcholinesterase (AChE) inhibition. After the first screen, active and inactive compounds were used to build a ligand-based pharmacophore model in order to prioritize compounds for biological testing. Among the virtual hits tested, the enrichment of actives was significantly higher than in a random selection of test compounds. The most active compounds were biochemically tested for their activity on mu, delta, and kappa opioid receptors. (C) 2010 Elsevier Ltd. All rights reserved.

[45] J. M. Rollinger, D. V. Kratschmar, D. Schuster, P. H. Pfisterer, C. Gumy, E. M. Aubry, S. Brandstotter, H. Stuppner, G. Wolber, A. Odermatt. 11β-Hydroxysteroid dehydrogenase 1 inhibiting constituents from Eriobotrya Japonica revealed by bioactivity-guided isolation and computational approaches, Bioorgan Med Chem, 18(4):1507-1515, 2010.
Links: doi:10.1016/J.Bmc.2010.01.010 | Publisher | Download citation: BibTeX RIS
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Abstract: The inhibition of 11 beta-hydroxysteroid dehydrogenase 1 (11 beta-HSD1), which catalyzes the conversion of inactive 11-ketoglucocorticoids to active 11 beta-hydroxyglucocorticoids, emerged as promising strategy to treat symptoms of the metabolic syndrome, including obesity and type 2 diabetes. In this study the leaves of the anti-diabetic medicinal plant loquat (Eriobotrya japonica) were phytochemically investigated following hints from a pharmacophore-based virtual screening and a bioactivity-guided approach. Determination of the 11 beta-HSD1 and 11 beta-HSD2 inhibitory activities in cell lysates revealed triterpenes from the ursane type as selective, low micro-molar inhibitors of 11 beta-HSD1, that is, corosolic acid (1), 3-epicorosolic acid methyl ester (4), 2-alpha hydroxy-3-oxo urs-12-en-28-oic acid (6), tormentic acid methyl ester (8), and ursolic acid (9). Importantly, a mixture of loquat constituents with moderate activities displayed a pronounced additive effect. By means of molecular modeling studies and the identification of the 11 beta-HSD1-inhibiting 11-keto-ursolic acid (17) and 3-acetyl-11-keto-ursolic acid (18) a structure -activity relationship was deduced for this group of pentacyclic triterpenes. The mechanism of action elucidated in the present work together with the previously determined pharmacological activities provides these natural products with an astonishing multi-targeted anti-diabetic profile. (C) 2010 Elsevier Ltd. All rights reserved.

[44] M. Goebel, G. Wolber, P. Markt, B. Staels, T. Unger, U. Kintscher, R. Gust. Characterization of new PPARγ agonists: Benzimidazole derivatives-importance of positions 5 and 6, and computational studies on the binding mode, Bioorgan Med Chem, 18(16):5885-5895, 2010.
Links: doi:10.1016/J.Bmc.2010.06.102 | Publisher | Download citation: BibTeX RIS
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Abstract: In this and previous studies we investigated the importance of partial structures of Telmisartan on PPAR gamma activation. The biphenyl-4-ylmethyl moiety at N1 and residues at C2 of the central benzimidazole were identified to be essential for receptor activation and potency of receptor binding. Now we focused our attention on positions 5 and 6 of the central benzimidazole and introduced bromine (3b-5/6, 3c), phenylcarbonyl (3d-5/6), hydroxy(phenyl) methyl (3g-5/6), hydroxymethyl (3h-5/6) and formyl (3i) groups. The selection of these moieties was inspired by the structure of Losartan and its metabolite EXP3179. In order to increase the hydrophobicity of the central part of the molecule, the benzimidazole was exchanged by a naphtho[2,3-d] imidazole (5). The compounds 3a-3i and 5 were tested in a differentiation assay using 3T3-L1 preadipocytes and a luciferase assay using COS-7 cells, transiently transfected with pGal4-hPPAR gamma DEF, pGal5-TK-pGL3 and pRL-CMV, as established models for the assessment of cellular PPAR gamma activation. An enhanced effect on PPAR gamma activation could be observed if lipophilic moieties are introduced in these positions. 4 '-[(2-Propyl-1H-naphtho[2,3-d] imidazol-1-yl) methyl] biphenyl-2-carboxylic acid (5) was identified as the most potent compound with an EC(50) of 0.26 mu M and the profile of a full agonist. Together with compounds of the former structure-activity relationship study (position 2-substituted benzimidazole derivatives 4a-4j), the binding mode of Telmisartan and its derivatives have been analyzed in 3D pharmacophore-driven docking experiments. (C) 2010 Elsevier Ltd. All rights reserved.

[43] G. Wolber. 3D pharmacophore elucidation and virtual screening, Drug Discov. Today Technol., 7(4):e203-e204, 2010.
Links: doi:10.1016/j.ddtec.2010.12.004 | Publisher | Download citation: BibTeX RIS

[42] T. Seidel, G. Ibis, F. Bendix, G. Wolber. Strategies for 3D pharmacophore-based virtual screening, Drug Discov. Today Technol., 7(4):e221-e228, 2010.
Links: doi:10.1016/j.ddtec.2010.11.004 | Publisher | Download citation: BibTeX RIS
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Abstract: 3D pharmacophore-based techniques have become one of the most important approaches for the fast and accurate virtual screening of databases with millions of compounds. The success of 3D pharmacophores is largely based on their intuitive interpretation and creation, but the virtual screening with such three-dimensional geometric models still poses a considerable algorithmic and conceptual challenge. Most current implementations favor fast screening speed at the detriment of accuracy. This review describes the general strategies and algorithms employed for 3D pharmacophore searching by some current pharmacophore modeling platforms and will highlight their differences.

[41] A. M. Scutaru, M. Wenzel, H. Scheffler, G. Wolber, R. Gust. Optimization of the N-lost drugs Melphalan and Bendamustine: Synthesis and cytotoxicity of a new set of dendrimer-drug conjugates as tumor therapeutic agents, Bioconjug. Chem., 21(10):1728-1743, 2010.
Links: doi:10.1021/Bc900453f | Publisher | Download citation: BibTeX RIS
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Abstract: Bendamustine and melphalan are very promising alkylating drugs with applicability in the treatment of various tumoral diseases, e.g., chronic lymphocytic leukemia (CLL) or breast cancer. However, numerous adverse effects limited their use. Therefore, 1,3,5-tris(3-aminopropyl)benzene (G0) and its G1 analogue 3,5-bis(3-aminopropyl)-N-(3-{3,5-bis[3-{3,5-bis(3-aminopropyl)benzoylamino}propyl]phenyl}propyl)benzamide were selected to design cytostatic drug-dendrimer conjugates to achieve tumor cell accumulation by endocytosis as already demonstrated for platinum complexes. The dendrimers act as carriers and an N-(2-hydroxyethyl)maleimide spacer between drug and carrier should guarantee a selective release of the cytostatics in the tumor cells. The resulting cytotoxicity was determined in vitro using the human MCF-7 and MDA-MB-231 breast cancer cell lines. It was demonstrated that melphalan caused cytotoxic effects depending on its free amino group (Boc protection strongly decreased the activity) but independent of a derivation of the carboxylic group (dendrimers and spacer binding). Esterification of bendamustine with the N-(2-hydroxyethyl)maleimide spacer strongly increased the hydrolytic stability of the N-lost moiety, so antiproliferative effects were yet observed in vitro.

[40] D. Schuster, G. Wolber. Identification of bioactive natural products by pharmacophore-based virtual screening, Curr. Pharm. Des., 16(15):1666-1681, 2010.
Links: doi:10.2174/138161210791164072 | Publisher | Download citation: BibTeX RIS
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Abstract: Natural products have been exposed to a long selection process to interact with biological targets and are therefore a valuable source for ideas for novel chemical entities in drug development. However, the process to determine activities of natural products is mainly based on serendipity, and can thus become time- and cost-intensive. In this review we present strategies on how modern in-silico molecular modeling techniques can be used to make this process more efficient and discuss how to discover and optimize drug candidates inspired by nature. Focusing on 3D pharmacophore modeling techniques, we provide an overview of virtual screening and modeling methods, review available in silico databases as sources for chemical structures of natural products, discuss techniques for biological activity profiling, and summarize recent success stories for the combination of in-silico approaches and pharmacognosy.

[39] D. Schuster, B. Waltenberger, J. Kirchmair, S. Distinto, P. Markt, H. Stuppner, J. M. Rollinger, G. Wolber. Predicting Cyclooxygenase inhibition by three-dimensional pharmacophoric profiling. Part I: model generation, validation, and applicability in ethnopharmacology, Mol. Inform., 29(1-2):75-86, 2010.
Links: doi:10.1002/Minf.200900071 | Publisher | Download citation: BibTeX RIS
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Abstract: 3D pharmacophore modeling has evolved as an established and state-of-the-art method for performing in-silico predictions of biological activity. Using one single model is limited to single binding modes, while the combination of several models bears a broader application scope. We demonstrate the generation of a complete and predictive 3D model set for cyclooxygenase 1 and 2 inhibitors, along with a selection and validation protocol optimized for parallel virtual screening. This model set was applied to explain the cyclooxygenase activity of an ethnopharmacologically known mixture of natural products, the Thai traditional medicine "Prasaplai". Results show that rationalizing natural product activity by modern in-silico approaches is promising and can be tremendously useful in the identification of the mechanisms of action for known biological effects of complex herbal remedies.

[38] D. Schuster, L. Kern, D. P. Hristozov, L. Terfloth, B. Bienfait, C. Laggner, J. Kirchmair, U. Grienke, G. Wolber, T. Langer, H. Stuppner, J. Gasteiger, J. M. Rollinger. Applications of integrated data mining methods to explore natural product space for Acetylcholinesterase inhibitors, Comb. Chem. High Throughput Screen., 13(1):54-66, 2010.
Links: doi:10.2174/138620710790218212 | Download citation: BibTeX RIS
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Abstract: Nature, especially the plant kingdom, is a rich source for novel bioactive compounds that can be used as lead compounds for drug development. In order to exploit this resource, the two neural network-based virtual screening techniques novelty detection with self-organizing maps (SOMs) and counterpropagation neural network were evaluated as tools for efficient lead structure discovery. As application scenario, significant descriptors for acetylcholinesterase (AChE) inhibitors were determined and used for model building, theoretical model validation, and virtual screening. Top-ranked virtual hits from both approaches were docked into the AChE binding site to approve the initial hits. Finally, in vitro testing of selected compounds led to the identification of forsythoside A and (+)-sesamolin as novel AChE inhibitors.

[37] P. H. Pfisterer, G. Wolber, T. Efferth, J. M. Rollinger, H. Stuppner. Natural products in structure-assisted design of molecular cancer therapeutics, Curr. Pharm. Des., 16(15):1718-1741, 2010.
Links: doi:10.2174/138161210791164027 | Publisher | Download citation: BibTeX RIS
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Abstract: Since the late 1990's, novel insights into molecular biology and carcinogenesis enabled the rational design of mechanismbased anticancer therapeutics. The large number of natural product (NP)-derived drugs currently under clinical evaluation and the recent approval of temsirolimus (Torisel (R)) as a first mTOR protein kinase inhibitor indicate that NPs have to be considered not only as a seminal source of cytotoxic, but also as a source of molecularly targeted agents. Whereas molecular modeling is well established as an important and successful method to discover and rationalize bioactivities in medicinal chemistry research, its application has proven to be also a powerful tool in the field of NPs. This review highlights the impact of computer-assisted approaches on NPs as molecularly targeted anticancer drugs. Examples of applications are provided focusing on innovative targets such as protein kinases, tumour vasculature, epigenetic modulators, heat shock protein (Hsp) 90, and direct apoptosis enhancers.

[36] L. G. Nashev, D. Schuster, C. Laggner, S. Sodha, T. Langer, G. Wolber, A. Odermatt. The UV-filter benzophenone-1 inhibits 17 beta-hydroxysteroid dehydrogenase type 3: Virtual screening as a strategy to identify potential endocrine disrupting chemicals, Biochem. Pharmacol., 79(8):1189-1199, 2010.
Links: doi:10.1016/J.Bcp.2009.12.005 | Publisher | Download citation: BibTeX RIS
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Abstract: The prevalence of male reproductive disorders and testicular cancer is steadily increasing. Because the exposure to chemicals disrupting natural hormone action has been associated with these diseases, it is important to identify endocrine disrupting chemicals (EDCs) and their targets of action. Here, a 3D-structural database that can be applied for virtual screening approaches to facilitate the identification of EDCs was constructed. The database was screened using pharmacophores of 17 beta-hydroxysteroid dehydrogenase type 3 (17 beta-HSD3), which catalyzes the last step of testosterone synthesis in testicular Leydig cells and plays an essential role during male sexual development. Among other chemicals, benzophenone (BP) UV-filters were predicted as potential 17 beta-HSD3 inhibitors. Biological analyses revealed (2,4-dihydroxyphenyl)-phenylmethanone (also known as benzophenone-1, BP-1) as an inhibitor of human 17 beta-HSD3 (IC50 1.05 mu M). BP-1 also efficiently blocked conversion of androstenedione to testosterone by mouse and rat 17 beta-HSD3 in whole-organ enzyme assays. Moreover, BP-I antagonized the testosterone-dependent activation of androgen receptors (IC50 5.7 mu M), suggesting synergistic anti-androgenic effects of BP-1 by preventing testosterone formation and blocking receptor activation. In addition, analyses of several commonly used UV-filters on estrogen- and androgen-metabolizing 17 beta-HSD enzymes revealed 3-benzylidene camphor (3-BC) and 4-methylbenzylidene camphor (4-MBC) as low micromolar 17 beta-HSD2 inhibitors. In conclusion, screening of virtual chemical structure libraries can facilitate the identification of compounds interfering with hormone action. The potential disruption of 17 beta-HSD enzyme function by the UV-filters BP-1, 3-BC and 4-MBC requires further investigation and should be considered for safety assessment of these chemicals. (C) 2009 Elsevier Inc. All rights reserved.

[35] N. Fakhrudin, A. Ladurner, A. G. Atanasov, E. H. Heiss, L. Baumgartner, P. Markt, D. Schuster, E. P. Ellmerer, G. Wolber, J. M. Rollinger, H. Stuppner, V. M. Dirsch. Computer-aided discovery, validation, and mechanistic characterization of novel neolignan activators of peroxisome proliferator-activated receptor γ, Mol. Pharmacol., 77(4):559-566, 2010.
Links: doi:10.1124/mol.109.062141 | Publisher | Download citation: BibTeX RIS
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Abstract: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are used for the treatment of type 2 diabetes and metabolic syndrome. However, the currently used PPARγ agonists display serious side effects, which has led to a great interest in the discovery of novel ligands with favorable properties. The aim of our study was to identify new PPARγ agonists by a PPARγ pharmacophore–based virtual screening of 3D natural product libraries. This in silico approach led to the identification of several neolignans predicted to bind the receptor ligand binding domain (LBD). To confirm this prediction, the neolignans dieugenol, tetrahydrodieugenol, and magnolol were isolated from the respective natural source or synthesized and subsequently tested for PPARγ receptor binding. The neolignans bound to the PPARγ LBD with EC50 values in the nanomolar range, exhibiting a binding pattern highly similar to the clinically used agonist pioglitazone. In intact cells, dieugenol and tetrahydrodieugenol selectively activated human PPARγ-mediated, but not human PPARα- or -β/δ-mediated luciferase reporter expression, with a pattern suggesting partial PPARγ agonism. The coactivator recruitment study also demonstrated partial agonism of the tested neolignans. Dieugenol, tetrahydrodieugenol, and magnolol but not the structurally related eugenol induced 3T3-L1 preadipocyte differentiation, confirming effectiveness in a cell model with endogenous PPARγ expression. In conclusion, we identified neolignans as novel ligands for PPARγ, which exhibited interesting activation profiles, recommending them as potential pharmaceutical leads or dietary supplements.

[34] P. Markt, C. Feldmann, J. M. Rollinger, S. Raduner, D. Schuster, J. Kirchmair, S. Distinto, G. M. Spitzer, G. Wolber, C. Laggner, K. H. Altmann, T. Langer, J. Gertsch. Discovery of novel CB2 receptor ligands by a pharmacophore-based virtual screening workflow, J. Med. Chem., 52(2):369-378, 2009.
Links: doi:10.1021/Jm801044g | Publisher | Download citation: BibTeX RIS
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Abstract: Cannabinoid receptor 2 (CB2 receptor) ligands are potential candidates for the therapy of chronic pain, inflammatory disorders, atherosclerosis, and osteoporosis. We describe the development of pharmacophore models for CB2 receptor ligands, as well as a pharmacophore-based virtual screening workflow, which resulted in 14 hits for experimental follow-up. Seven compounds were identified with K-i values below 25 mu M. The CB2 receptor-selective pyridine tetrahydrocannabinol analogue 8 (K-i = 1.78 mu M) was identified as a CB2 partial agonist. Acetamides 12 (K-i = 1.35 mu M) and 18 (K-i = 2.1 mu M) represent new scaffolds for CB2 receptor-selective antagonists and inverse agonists, respectively. Overall, our pharmacophore-based workflow yielded three novel scaffolds for the chemical development of CB2 receptor ligands.

[33] P. Tiikkainen, P. Markt, G. Wolber, J. Kirchmair, S. Distinto, A. Poso, O. Kallioniemi. Critical comparison of virtual screening methods against the MUV data set, J. Chem. Inf. Model., 49(10):2168-2178, 2009.
Links: doi:10.1021/Ci900249b | Publisher | Download citation: BibTeX RIS
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Abstract: In the current work, we measure the performance of seven ligand-based virtual screening tools - five similarity search methods and two pharmacophore elucidators - against the MUV data set. For the similarity search tools, single active molecules as well as active compound sets clustered in terms of their chemical diversity were used as templates., Their score was calculated against all inactive and active compounds in their target class. Subsequently, the scores were used to calculate different performance metrics in eluding enrichment factors and AUC values. We also studied the effect of data fusion on the results. To measure the performance of the pharmacophore tools, a set of active molecules was picked either random- or chemical diversity-based from each target class to build a pharmacophore model which was then used to screen the remaining compounds in the set. Our results indicate that template sets selected by their chemical diversity are the best choice for similarity search tools, whereas the optimal training sets for pharmacophore elucidators are based on random selection underscoring that pharmacophore modeling cannot be easily automated. We also suggest a number of improvements for future benchmark sets and discuss activity cliffs as a potential problem in ligand-based virtual screening.

[32] J. Kirchmair, S. Distinto, P. Markt, D. Schuster, G. M. Spitzer, K. R. Liedl, G. Wolber. How to optimize shape-based virtual screening: Choosing the right query and including chemical information, J. Chem. Inf. Model., 49(3):678-692, 2009.
Links: doi:10.1021/Ci8004226 | Publisher | Download citation: BibTeX RIS
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Abstract: Shape-based molecular similarity approaches have been established as important and popular virtual screening techniques. Recent applications have shown successful screening campaigns using different parameters and query selection. It is common sense that pure volume overlap scoring (or "shape-based screening") under-represents chemical or pharmacophoric information of a molecule. Using the "Directory of Useful Decoys" (DUD) as a benchmark set, we systematically evaluate how (i) the choice of query conformations, (ii) the selection of the active compound to be used as a query structure, and (iii) the inclusion of chemical information (i.e., the pharmacophoric properties of the query molecule) affect screening performance. Varying these parameters bears remarkable potential for improvements and delivers the best screening performance reported using these tools so far. From these insights, guidelines on how to reach optimum performance during virtual screening are developed.

[31] J. M. Rollinger, D. Schuster, B. Danzl, S. Schwalger, P. Markt, M. Schmidtke, J. Gertsch, S. Raduner, G. Wolber, T. Langer, H. Stuppner. In silico target fishing for rationalized ligand discovery exemplified on constituents of Ruta Graveolens, Planta Med., 75(3):195-204, 2009.
Links: doi:10.1055/S-0028-1088397 | Publisher | Download citation: BibTeX RIS
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Abstract: The identification of targets whose interaction is likely to result in the successful treatment of a disease is of growing interest for natural product scientists. In the Current Study we performed an exemplary application of a virtual parallel screening approach to identify potential targets for 16 secondary metabolites isolated and identified from the aerial parts of the medicinal plant Ruta graveolens L. Low energy conformers of the isolated constituents were simultaneously screened against a set of 2208 pharmacophore models generated in-house for the in silico prediction of putative biological targets, i.e., target fishing, Based on the predicted ligand-target interactions, we focused on three biological targets, namely acetylcholinesterase (AChE), the human rhinovirus (HRV) coat protein and the cannabinoid receptor type-2 (CB(2)). For a critical evaluation of the applied parallel screening approach, virtual hits and non-hits were assayed on the respective targets. For AChE the highest scoring virtual hit, arborinine, showed the best inhibitory in vitro activity on AChE (IC(50) 34.7 mu M). Determination of the anti-HRV-2 effect revealed 6,7,8-trimethoxycoumarin and arborinine to be the most active antiviral constituents with IC(50) values of 11.98 mu M and 3.19 mu M, respectively. Of these, arborinine was predicted virtually. Of all the molecules subjected to parallel screening, one virtual CB(2) ligand was obtained, i.e., rutamarin. Interestingly, in experimental Studies only this compound showed a selective activity to the CB(2) receptor (Ki of 7.4 mu M) by using a radioligand displacement assay. The applied parallel screening paradigm with constituents of R. graveolens on three different proteins has shown promise as an in silico tool for rational target fishing and pharmacological profiling of extracts and single chemical entities in natural product research.

[30] A. Perdih, A. Kovac, G. Wolber, D. Blanot, S. Gobec, T. Solmajer. Discovery of novel benzene 1,3-dicarboxylic acid inhibitors of bacterial MurD and MurE ligases by structure-based virtual screening approach, Bioorg Med Chem Lett, 19(10):2668-2673, 2009.
Links: doi:10.1016/J.Bmcl.2009.03.141 | Publisher | Download citation: BibTeX RIS
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Abstract: The peptidoglycan biosynthetic pathway provides an array of potential targets for antibacterial drug design, attractive especially with respect to selective toxicity. Within this pathway, the members of the Mur ligase family are considered as promising emerging targets for novel antibacterial drug design. Based on the available MurD crystal structures co-crystallised with N-sulfonyl glutamic acid inhibitors, a virtual screening campaign was performed, combining three-dimensional structure-based pharmacophores and molecular docking calculations. A novel class of glutamic acid surrogates-benzene 1,3-dicarboxylic acid derivatives-were identified and compounds 14 and 16 found to possess dual MurD and MurE inhibitory activity. (C) 2009 Elsevier Ltd. All rights reserved.

[29] D. Classen-Houben, D. Schuster, T. Da Cunha, A. Odermatt, G. Wolber, U. Jordis, B. Kueenburg. Selective inhibition of 11β-hydroxysteroid dehydrogenase 1 by 18 alpha-glycyrrhetinic acid but not 18 beta-glycyrrhetinic acid, J. Steroid Biochem., 113(3-5):248-252, 2009.
Links: doi:10.1016/J.Jsbmb.2009.01.009 | Publisher | Download citation: BibTeX RIS
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Abstract: Elevated cortisol concentrations have been associated with metabolic diseases such as diabetes type 2 and obesity. 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) type 1, catalyzing the conversion of inactive 11-ketoglucocorticoids into their active Ilp-hydroxy forms, plays an important role in the regulation of cortisol levels within specific tissues. The selective inhibition of 11 beta-HSD1 is currently considered as promising therapeutic strategy for the treatment of metabolic diseases. In recent years, natural compound-derived drug design has gained considerable interest. 18 beta-glycyrrhetinic acid (GA), a metabolite of the natural product glycyrrhizin, is not selective and inhibits both 11 beta-HSD1 and 11 beta-HSD2. Here, we compare the biological activity of 18 beta-GA and its diastereomer 18 alpha-GA against the two enzymes in lysates of transfected HEK-293 cells and show that 18 alpha-GA selectively inhibits 11 beta-HSD1 but not 11 beta-HSD2. This is in contrast to 18 beta-GA, which preferentially inhibits 11 beta-HSD2. Using a pharmacophore model based on the crystal structure of the GA-derivative carbenoxolone in complex with human 11 beta-HSD1, we provide an explanation for the differences in the activities of 18 alpha-GA and 18 beta-GA. This model will be used to design novel selective derivatives of GA. (C) 2009 Elsevier Ltd. All rights reserved.

[28] D. Schuster, L. G. Nashev, J. Kirchmair, C. Laggner, G. Wolber, T. Langer, A. Odermatt. Discovery of nonsteroidal 17β-hydroxysteroid dehydrogenase 1 inhibitors by pharmacophore-based screening of virtual compound libraries, J. Med. Chem., 51(14):4188-4199, 2008.
Links: doi:10.1021/Jm800054h | Publisher | Download citation: BibTeX RIS
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Abstract: 17 beta-Hydroxysteroid dehydrogenase type 1 (17 beta-HSD1) plays a pivotal role in the local synthesis of the most potent estrogen estradiol. Its expression is a prognostic marker for the outcome of patients with breast cancer and inhibition of 17 beta-HSD I is currently under consideration for breast cancer prevention and treatment. We aimed to identify nonsteroidal 17 beta-HSD1 inhibitor scaffolds by virtual screening with pharmacophore models built from crystal structures containing steroidal compounds. The most promising model was validated by comparing predicted and experimentally determined inhibitory activities of several flavonoids. Subsequently, a virtual library of nonsteroidal compounds was screened against the 3D pharmacophore. Analysis of 14 selected compounds yielded four that inhibited the activity of human 17 beta-HSD1 (IC(50) below 50 mu M). Specificity assessment of identified 17 beta-HSD1 inhibitors emphasized the importance of including related short-chain dehydrogenase/reductase (SDR) members to analyze off-target effects. Compound 29 displayed at least 10-fold selectivity over the related SDR enzymes tested.

[27] P. Markt, R. K. Petersen, E. N. Flindt, K. Krjstiansen, J. Kirchmair, G. Spitzer, S. Distinto, D. Schuster, G. Wolber, C. Laggner, T. Langer. Discovery of novel PPAR ligands by a virtual screening approach based on pharmacophore modeling, 3D shape, and electrostatic similarity screening, J. Med. Chem., 51(20):6303-6317, 2008.
Links: doi:10.1021/Jm800128k | Publisher | Download citation: BibTeX RIS
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Abstract: Peroxisome proliferator-activated receptors (PPARs) are important targets for drugs used in the treatment of atherosclerosis, dyslipidaemia, obesity, type 2 diabetes, and other diseases caused by abnormal regulation of the glucose and lipid metabolism. We applied a virtual screening workflow based on a combination of pharmacophore modeling with 3D shape and electrostatic similarity screening techniques to discover novel scaffolds for PPAR ligands. From the resulting 10 virtual screening hits, five tested positive in human PPAR ligand-binding domain (hPPAR-LBD) transactivation assays and showed affinities for PPAR in a competitive binding assay. Compounds 5, 7, and 8 were identified as PPAR-alpha agonists, whereas compounds 2 and 9 showed agonistic activity for hPPAR-gamma. Moreover, compound 9 was identified as a PPAR-delta antagonist. These results demonstrate that our virtual screening protocol is able to enrich novel scaffolds for PPAR ligands that could be useful for drug development in the area of atherosclerosis, dyslipidaemia, and type 2 diabetes.

[26] J. Kirchmair, P. Markt, S. Distinto, D. Schuster, G. M. Spitzer, K. R. Liedl, T. Langer, G. Wolber. The Protein Data Bank (PDB), its related services and software tools as key components for in silico guided drug discovery, J. Med. Chem., 51(22):7021-7040, 2008.
Links: doi:10.1021/Jm8005977 | Publisher | Download citation: BibTeX RIS

[25] P. Markt, C. McGoohan, B. Walker, J. Kirchmair, C. L. Feldmann, G. De Martino, G. Spitzer, S. Distinto, D. Schuster, G. Wolber, C. Laggner, T. Langer. Discovery of novel cathepsin S inhibitors by pharmacophore-based virtual high-throughput screening, J. Chem. Inf. Model., 48(8):1693-1705, 2008.
Links: doi:10.1021/Ci800101j | Publisher | Download citation: BibTeX RIS
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Abstract: The cysteine protease cathepsin S (CatS) is involved in the pathogenesis of autoimmune disorders, atherosclerosis, and obesity. Therefore, it represents a promising pharmacological target for drug development. We generated ligand-based and structure-based pharmacophore models for noncovalent and covalent CatS inhibitors to perform virtual high-throughput screening of chemical databases in order to discover novel scaffolds for CatS inhibitors. An in vitro evaluation of the resulting 15 structures revealed seven CatS inhibitors with kinetic constants in the low micromolar range. These compounds can be subjected to further chemical modifications to obtain drugs for the treatment of autoimmune disorders and atherosclerosis.

[24] J. Kirchmair, P. Markt, S. Distinto, G. Wolber, T. Langer. Evaluation of the performance of 3D virtual screening protocols: RMSD comparisons, enrichment assessments, and decoy selection - What can we learn from earlier mistakes?, J. Comput. Aided Mol. Des., 22(3-4):213-228, 2008.
Links: doi:10.1007/S10822-007-9163-6 | Publisher | Download citation: BibTeX RIS
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Abstract: Within the last few years a considerable amount of evaluative studies has been published that investigate the performance of 3D virtual screening approaches. Thereby, in particular assessments of protein-ligand docking are facing remarkable interest in the scientific community. However, comparing virtual screening approaches is a non-trivial task. Several publications, especially in the field of molecular docking, suffer from shortcomings that are likely to affect the significance of the results considerably. These quality issues often arise from poor study design, biasing, by using improper or inexpressive enrichment descriptors, and from errors in interpretation of the data output. In this review we analyze recent literature evaluating 3D virtual screening methods, with focus on molecular docking. We highlight problematic issues and provide guidelines on how to improve the quality of computational studies. Since 3D virtual screening protocols are in general assessed by their ability to discriminate between active and inactive compounds, we summarize the impact of the composition and preparation of test sets on the outcome of evaluations. Moreover, we investigate the significance of both classic enrichment parameters and advanced descriptors for the performance of 3D virtual screening methods. Furthermore, we review the significance and suitability of RMSD as a measure for the accuracy of protein-ligand docking algorithms and of conformational space sub sampling algorithms.

[23] G. Wolber, T. Seidel, F. Bendix, T. Langer. Molecule-pharmacophore superpositioning and pattern matching in computational drug design, Drug Discov. Today, 13(1–2):23-29, 2008.
Links: doi:10.1016/j.drudis.2007.09.007 | Download citation: BibTeX RIS
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Abstract: Three-dimensional (3D) pharmacophore modeling is a technique for describing the interaction of a small molecule ligand with a macromolecular target. Since chemical features in a pharmacophore model are well known and highly transparent for medicinal chemists, these models are intuitively understandable and have been increasingly successful in computational drug discovery in the past few years. The performance and applicability of pharmacophore modeling depends on two main factors: the definition and placement of pharmacophoric features and the alignment techniques used to overlay 3D pharmacophore models and small molecules. An overview of key technologies and latest developments in the area of 3D pharmacophores is given and provides insight into different approaches as implemented by the 3D pharmacophore modeling packages like Catalyst, MOE, Phase and LigandScout.

[22] J. Kirchmair, S. Distinto, D. Schuster, G. Spitzer, T. Langer, G. Wolber. Enhancing drug discovery through in silico screening: Strategies to increase true positives retrieval rates, Curr. Med. Chem., 15(20):2040-2053, 2008.
Links: doi:10.2174/092986708785132843 | Publisher | Download citation: BibTeX RIS
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Abstract: Computational chemistry software for lead discovery has become well established in pharmaceutical industry and has found its way to the desktop computers of medicinal chemists for different purposes, providing insight on the mode of action and binding properties, and creating new ideas for lead structure refinement. In this review we investigate the performance and reliability of recent state-of-the-art data modeling techniques, as well as ligand-based and structure-based modeling approaches for 3D virtual screening. We discuss and summarize recently published success stories and lately developed techniques. Parallel screening is one of these emerging approaches allowing for efficient activity in silico profiling of several compounds against different targets or anti-targets simultaneously. This is of special interest to medicinal chemists, as the approach allows revealing unknown binding modes ('target-fishing') as well as integrated ADME profiling or - more generally - the prediction of off-target effects.

[21] J. Kirchmair, S. Ristic, K. Eder, P. Markt, G. Wolber, C. Laggner, T. Langer. Fast and efficient in silico 3D screening: Toward maximum computational efficiency of pharmacophore-based and shape-based approaches, J. Chem. Inf. Model., 47(6):2182-2196, 2007.
Links: doi:10.1021/Ci700024q | Publisher | Download citation: BibTeX RIS
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Abstract: In continuation of our recent studies on the quality of conformational models generated with CATALYST and OMEGA we present a large-scale survey focusing on the impact of conformational model quality and several screening parameters on pharmacophore-based and shape-based virtual high throughput screening (vHTS). Therefore, we collected known active compounds of CDK2, p38 MAPK, PPAR-gamma, and factor Xa and built a set of druglike decoys using ilib:diverse. Subsequently, we generated 3D structures using CORINA and also calculated conformational models for all compounds using CAESAR, CATALYST FAST, and OMEGA. A widespread set of 103 structure-based pharmacophore models was developed with LigandScout for virtual screening with CATALYST. The performance of both database search modes (FAST and BEST flexible database search) as well as the fit value calculation procedures (FAST and BEST fit) available in CATALYST were analyzed in terms of their ability to discriminate between active and inactive compounds and in terms of efficiency. Moreover, these results are put in direct comparison to the performance of the shape-based virtual screening platform ROCS. Our results prove that high enrichment rates are not necessarily in conflict with efficient vHTS settings: In most of the experiments, we obtained the highest yield of actives in the hit list when parameter sets for the fastest search algorithm were used.

[20] J. Kirchmair, G. Wolber, C. Laggner, T. Langer. Comparative performance assessment of the conformational model generators omega and catalyst: A large-scale survey on the retrieval of protein-bound ligand conformations, J. Chem. Inf. Model., 46(4):1848-1861, 2006.
Links: doi:10.1021/Ci060084g | Publisher | Download citation: BibTeX RIS
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Abstract: In continuation of our studies to evaluate the ability of various conformer generators to produce bioactive conformations, we present the extension of our work on the analysis of Catalyst's conformational subsampling algorithm in a comparative evaluation with OpenEye's currently updated tool Omega 2.0. Our study is based on an enhanced test set of 778 drug molecules and pharmacologically relevant compounds extracted from the Protein Data Bank (PDB). We elaborated protocols for two common conformer generation use cases and applied them to both programs: (i) high-throughput settings for processing large databases and (ii) high-quality settings for binding site exploration or lead structure refinement. While Catalyst is faster in the first case, Omega 2.0 better reproduces the bound ligand conformations from the PDB in less time for the latter case.

[19] G. Wolber, A. A. Dornhofer, T. Langer. Efficient overlay of small organic molecules using 3D pharmacophores, J. Comput. Aided Mol. Des., 20(12):773-788, 2006.
Links: doi:10.1007/S10822-006-9078-7 | Publisher | Download citation: BibTeX RIS
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Abstract: Aligning and overlaying two or more bio-active molecules is one of the key tasks in computational drug discovery and bio-activity prediction. Especially chemical-functional molecule characteristics from the view point of a macromolecular target represented as a 3D pharmacophore are the most interesting similarity measure when describing and analyzing macromolecule-ligand interaction. In this study, a novel approach for aligning rigid three-dimensional molecules according to their chemical-functional pharmacophoric features is presented and compared to the overlay of experimentally determined poses in a comparable macromolecule coordinate frame. The presented approach identifies optimal chemical feature pairs using distance and density characteristics obtained by correlating pharmacophoric geometries and thus proves to be faster than existing combinatorial alignment methods and creates more reasonable alignments than pure atom-based methods. Examples will be provided to demonstrate the feasibility, speed and intuitiveness of this method.

[18] T. M. Steindl, D. Schuster, G. Wolber, C. Laggner, T. Langer. High-throughput structure-based pharmacophore modelling as a basis for successful parallel virtual screening, J. Comput. Aided Mol. Des., 20(12):703-715, 2006.
Links: doi:10.1007/S10822-006-9066-Y | Publisher | Download citation: BibTeX RIS
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Abstract: In order to assess bioactivity profiles for small organic molecules we propose to use parallel pharmacophore-based virtual screening. Our aim is to provide a fast, reliable and scalable system that allows for rapid in silico activity profile prediction of virtual molecules. In this proof of principle study, carried out with the new structure-based pharmacophore modelling tool LigandScout and the high-performance database mining platform Catalyst, we present a model work for the application of parallel pharmacophore-based virtual screening on a set of 50 structure-based pharmacophore models built for various viral targets and 100 antiviral compounds. The latter were screened against all pharmacophore models in order to determine if their known biological targets could be correctly predicted via an enrichment of corresponding pharmacophores matching these ligands. The results demonstrate that the desired enrichment, i.e. a successful activity profiling, was achieved for approximately 90% of all input molecules. Additionally, we discuss descriptors for output validation, as well as various aspects influencing the analysis of the obtained activity profiles, and the effect of the searching mode utilized for screening. The results of the study presented here clearly indicate that pharmacophore-based parallel screening comprises a reliable in silico method to predict the potential biological activities of a compound or a compound library by screening it against a series of pharmacophore queries.

[17] G. Wolber, T. Langer. LigandScout: 3D pharmacophores derived from protein-bound Ligands and their use as virtual screening filters, J. Chem. Inf. Model., 45(1):160-169, 2005.
Links: doi:10.1021/Ci049885e | Publisher | Download citation: BibTeX RIS
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Abstract: From the historically grown archive of protein-ligand complexes in the Protein Data Bank small organic ligands are extracted and interpreted in terms of their chemical characteristics and features. Subsequently, pharmacophores representing ligand-receptor interaction are derived from each of these small molecules and its surrounding amino acids. Based on a defined set of only six types of chemical features and volume constraints, three-dimensional pharmacophore models are constructed, which are sufficiently selective to identify the described binding mode and are thus a useful tool for in-silico screening of large compound databases. The algorithms for ligand extraction and interpretation as well as the pharmacophore creation technique from the automatically interpreted data are presented and applied to a rhinovirus capsid complex as application example.

[16] J. Kirchmair, C. Laggner, G. Wolber, T. Langer. Comparative analysis of protein-bound ligand conformations with respect to catalyst's conformational space subsampling algorithms, J. Chem. Inf. Model., 45(2):422-430, 2005.
Links: doi:10.1021/Ci0497531 | Publisher | Download citation: BibTeX RIS
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Abstract: We examined the quality of Catalyst's conformational model generation algorithm via a large scale study based on the crystal structures of a sample of 510 pharmaceutically relevant protein-ligand complexes extracted from the Protein Data Bank (PDB). Our results show that the tested algorithms implemented within Catalyst are able to produce high quality conformers, which in most of the cases are well suited for in silico drug research. Catalyst-specific settings were analyzed, such as the method used for the conformational model generation (FAST vs BEST) and the maximum number of generated conformers. By setting these options for higher fitting quality, the average RMS values describing the similarity of experimental and simulated conformers were improved from an RMS of 1.06 with max. 50 FAST generated conformers to an RMS of 0.93 with max. 255 BEST generated conformers, which represents an improvement by 12%. Each method provides best fitting conformers with an RMS value < 1.50 in more than 80% of all cases. We analyzed the computing time/quality ratio of various conformational model generation settings and examined ligands in high energy conformations. Furthermore, properties of the same ligands in various proteins were investigated, and the fitting qualities of experimental conformations from the PDB and the Cambridge Structural Database (CSD) were compared. One of the most important conclusions of former studies, the fact that bioactive conformers often have energy high above that of Global minima, was confirmed.

[15] T. Langer, G. Wolber. Virtual combinatorial chemistry and in silico screening: Efficient tools for lead structure discovery?, Pure Appl. Chem., 76(5):991-996, 2004.
Links: doi:10.1351/Pac200476050991 | Publisher | Download citation: BibTeX RIS
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Abstract: In this article, an overview of the most common ligand-based in silico screening techniques is given together with an example on the recent successful application of combined use of pharmacophore modeling, database mining, and biological assays. Additionally, a new approach for structure-based high-throughput pharmacophore model generation is presented. The LigandScout program contains an automated method for creating pharmacophore models from experimentally determined structure data, e.g., publicly available from the Brookhaven Protein Databank (PDB). In a first step, known algorithms were implemented and improved to extract small-molecule ligands from the PDB including assignment of hybridization states and bond orders. Second, from the interactions of the interpreted ligands with relevant surrounding amino acids, pharmacophore models reflecting functional interactions like H-bonds or ionic transfer interactions were created. These models can be used for screening molecular databases for similar modes of actions on the one hand, or for screening one single compound for potential side-effects (reversed screening) on the other hand. The implementation was done using the ilib framework, which also formed the basis of the software tool Comb(i)Gen, a fragment-based virtual combinatorial library generation program enabling the user to obtain in silico compound collections with high drug-likeness.

[14] T. Langer, G. Wolber. Pharmacophore definition and 3D searches, Drug Discov. Today Technol., 1(3):203-207, 2004.
Links: doi:10.1016/j.ddtec.2004.11.015 | Publisher | Download citation: BibTeX RIS
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Abstract: The most common pharmacophore building concepts based on either 3D structure of the target or ligand information are discussed together with the application of such models as queries for 3D database search. An overview of the key techniques available on the market is given and differences with respect to algorithms used and performance obtained are highlighted. Pharmacophore modelling and 3D database search are shown to be successful tools for enriching screening experiments aimed at the discovery of novel bio-active compounds. Section Editor: Hugo Kubiniyi – University of Heidelberg, Germany Pharmacophore models are hypotheses on the 3D arrangement of structural properties, such as hydrogen bond donar and acceptor properties, hydrophobic groups and aromatic rings of compounds that bind to a biological target. In the presence of the 3D structure of this target of by comparison with inactive analogs, further geometric and/or steric constraints can be defined. The article describes and evaluates strategies and commercial software for pharmacophore definition, starting from the 3D structures of ligand-protein complexes or from ligands alone. Once a pharmacophore model is established, 3D searches in large databases can be performed, leading to a significant enrichment of active analogs.

[13] D. Schaller, S. Pach, M. Bermudez, G. Wolber. Exploiting water dynamics for pharmacophore screening, In: Protein-Ligand Interactions and Drug Design, Flavio Ballante, editor, Springer, ISBN: 1064-3745, pp. 227-238, 2021.
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Abstract: Three-dimensional pharmacophore models have been proven extremely valuable in exploring novel chemical space through virtual screening. However, traditional pharmacophore-based approaches need ligand information and rely on static snapshots of highly dynamic systems. In this chapter, we describe PyRod, a novel tool to generate three-dimensional pharmacophore models based on water traces of a molecular dynamics simulation of an apo-protein.The protocol described herein was successfully applied for the discovery of novel drug-like inhibitors of West Nile virus NS2B-NS3 protease. By using this recent example, we highlight the key steps of the generation and validation of PyRod-derived pharmacophore models and their application for virtual screening.

[12] M. Dumitrascuta, M. Bermudez, S. Ballet, G. Wolber, M. Spetea. Mechanistic understanding of peptide analogues, dalda, [Dmt1]DALDA, and KGOP01, binding to the µ opioid receptor, In: Opioids and their receptors - Present and emerging concepts in opioid drug discovery, M. Spetea, H. Schmidhammer, editors, MDPI, Basel, Switzerland, ISBN: 978-3-0365-0046-1, pp. 99-110, 2020.
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Abstract: The mu opioid receptor (MOR) is the primary target for analgesia of endogenous opioid peptides, alkaloids, synthetic small molecules with diverse scaffolds, and peptidomimetics. Peptide-based opioids are viewed as potential analgesics with reduced side effects and have received constant scientific interest over the years. This study focuses on three potent peptide and peptidomimetic MOR agonists, DALDA, [Dmt(1)]DALDA, and KGOP01, and the prototypical peptide MOR agonist DAMGO. We present the first molecular modeling study and structure-activity relationships aided by in vitro assays and molecular docking of the opioid peptide analogues, in order to gain insight into their mode of binding to the MOR. In vitro binding and functional assays revealed the same rank order with KGOP01 > [Dmt(1)]DALDA > DAMGO > DALDA for both binding and MOR activation. Using molecular docking at the MOR and three-dimensional interaction pattern analysis, we have rationalized the experimental outcomes and highlighted key amino acid residues responsible for agonist binding to the MOR. The Dmt (2 ',6 '-dimethyl-L-Tyr) moiety of [Dmt(1)]DALDA and KGOP01 was found to represent the driving force for their high potency and agonist activity at the MOR. These findings contribute to a deeper understanding of MOR function and flexible peptide ligand-MOR interactions, that are of significant relevance for the future design of opioid peptide-based analgesics.

[11] Thomas Seidel, Gerhard Wolber, Manuela S. Murgueitio. Pharmacophore perception and applications, In: Applied Chemoinformatics: Achievements and Future Opportunities, Johann Gasteiger, Thomas Engel, editors, pp. 259-282, 2018. [Download citation: BibTeX | RIS]

[10] G. Wolber, W. Sippl. Pharmacophore identification and pseudo-receptor modelling, In: The Practice of Medicinal Chemistry (4th edition), C. G. Wermuth, D. Rognan, editors, Elsevier Ltd, Philadelphia, PA, USA, pp. 489-507, 2015.
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[9] G. Wolber, J. M. Rollinger. Virtual screening and target fishing for natural products using 3D pharmacophores, In: Computational Chemogenomics, E. Jacoby, editor, Pan Stanford Publishing Pte Ltd, Singapore, Malaysia, ISBN: 978-981-4411-39-4, pp. 117-139, 2013.
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[8] J. M. Rollinger, G. Wolber. Computational approaches for the discovery of natural lead structures, In: Bioactive Compounds from Natural Sources, 2nd edition, C. Tringali, editor, CRC Press, London, United Kingdom, ISBN: 1-4398-2229-8, pp. 97-132, 2011.
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[7] C. Laggner, G. Wolber, J. Kirchmair, Schuster D, Langer T. Pharmacophore-based virtual screening in drug discovery, In: Chemoinformatics: An approach to virtual screening, A. Varnek, A. Tropsha, editors, Royal Society of Chemistry, Cambridge, United Kingdom, ISBN: 978-0-85404-144-2, pp. 76-119, 2008.
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[6] G. Wolber, M. Biely, T. Langer. De novo drug design using randomized virtual chemistry and property filtering: a heuristic approach, In: QSAR and Molecular Modelling in Rational Design of Bioactive Molecules, E. Aki Sener, I. Yalcin, editors, Computer Aided Drug Design & Development Society, Ankara, Turkey, ISBN: 975-00782-0-9, 2006. [Download citation: BibTeX | RIS]

[5] G. Wolber, R. Kosara. Pharmacophores from macromolecular complexes with LigandScout, In: Pharmacophores and pharmacophore searches, T. Langer, R. Hofmann, editors, Wiley-VCH, Weinheim, Germany, ISBN: 3-527-31250-1, pp. 131-148, 2006.
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[4] T. Langer, G. Wolber. Extracting pharmacophores from bio-active molecules, In: Virtual ADMET assessment in target Selection and maturation, B. Testa, L. Turski, editors, IOS Press, Amsterdam, Netherlands, ISBN: 978-1-58603-703-1, pp. 133-150, 2006. [Download citation: BibTeX | RIS]

[3] A. A. Dornhofer, M. Biely, G. Wolber, T. Langer. A novel 2D depiction method using breadth-first ordering and an adapted 2D force field, In: QSAR and Molecular Modelling in Rational Design of Bioactive Molecules, E. Aki Sener, I. Yalcin, editors, Computer Aided Drug Design & Development Society, Ankara, Turkey, ISBN: 975-00782-0-9, pp. 421ff., 2006. [Download citation: BibTeX | RIS]

[2] G. Wolber. Structure-based Pharmacophores From Protein-Bound Ligands (German: Strukturbasierte Pharmacophore aus proteingebundenen Liganden), In: Lecture notes in informatics (LNI) D-5, D. Wagner, editor, Bonner Koellen Verlag, Bonn-Buschdorf, Germany, ISBN: 3-88579-409-8, pp. 209-218, 2004. [Download citation: BibTeX | RIS]

[1] T. Langer, G. Wolber. CombiGen: A novel software package for the rapid generation of virtual combinatorial libraries, In: Rational Approaches to Drug Design, H. Höltje, W. Sippl, editors, Prous Science, Barcelona, Spain, ISBN: 84-8124-176-8, pp. 390-399, 2001. [Download citation: BibTeX | RIS]