56. Identification of an (–)-Englerin A analogue which antagonizes (–)-Englerin A at TRPC1/4/5 channels
H. Rubaiy, T. Seitz, S. Hahn, A. Choidas, P. Habenberger, B. Klebl, K. Dinkel, P. Nussbaumer, H. Waldmann, M. Christmann, D. Beech
BACKGROUND AND PURPOSE (-)-Englerin A (EA) is a potent cytotoxic agent against renal cell carcinoma cells. It achieves its effects by activation of TRPC4/TRPC1 heteromeric channels. It is also an agonist at channels formed by the related protein, TRPC5. Here we sought an EA analogue which might enable better understanding of these EA effects. EXPERIMENTAL APPROACH Renal cell carcinoma A498 cells and HEK 293 cells overexpressing TRPC4 or TRPC5 were studied by intracellular Ca2+ measurement or whole-cell patch-clamp. The EA analogue A54 was generated by total synthesis. KEY RESULTS A54 had weak or no agonist activity at endogenous TRPC4/TRPC1 channels in A498 cells or TRPC4 or TRPC5 homomeric channels overexpressed in HEK 293 cells. A54 strongly inhibited EA-mediated activation of TRPC4/TRPC1 or TRPC5 and weakly inhibited activation of TRPC4. Studies of TRPC5 showed that A54 right-shifted the EA concentration-response curve without changing its slope, consistent with competitive antagonism. In contrast, TRPC5 activated by Gd3+ and TRPC4 activated by sphingosine-1-phosphate were not inhibited but potentiated by A54. A54 did not activate TRPC3 channels or affect activation of these channels by the agonist 1-oleoyl-2-acetylglycerol. CONCLUSIONS AND IMPLICATIONS The data suggest identification of a tool compound which could be useful for characterizing endogenous TRPC1/4/5 channels and understanding EA binding sites and their physiological relevance.