Abstract Fri004: AI Modeling Predicts BI 749327 disrupts Transient Receptor Potential Canonical 6 channel formation to rescue dystrophic cardiomyopathy

Introduction: Duchenne muscular dystrophy (DMD) patients suffer from progressive loss of muscle function and ambulation, but early mortality in DMD is primarily due to heart failure. DMD is caused by the loss of the structural protein dystrophin, and the subsequent membrane instability activates mechanosensitive ion channels that mediate Ca 2+ overload. TRPC6 (Transient Receptor Potential Canonical 6), a tetrameric cation channel, is a major mediator of Ca 2+ overload in DMD. We show a new specific TRPC6 inhibitor, BI 749327, dramatically improves survival and rescues heart failure in DMD mice, but the mechanism-of-action remains unknown. Hypothesis: BI 749327 attenuates Ca 2+ overload in DMD by disrupting TRPC6 channel formation. Methods: To test this hypothesis, 1) we treated DMD mice with BI 749327 (30 mg/kg) and measured isolated cardiomyocyte function. 2) Using live cell super-resolution imaging (Dragonfly SRRF), we visualized mCherry-tagged TRPC6 in vitro to assess real-time channel formation dynamics. 3) To rapidly assess BI 749327 binding and its impact on TRPC6 channel stability, we combined AI protein modeling (AlphaFold3) with traditional computational modeling (molecular docking simulation). Results: BI 749327 treatment in DMD mice confirms improvement in survival from 26 to 43 weeks (n≥56/group; p=8E-4 log rank test) and fractional shortening from 39±1.2% to 47±1.5% in vivo (n≥41/group). BI 749327 treatment in cardiomyocytes reduced peak [Ca 2+ ] IC from 0.7±0.04 to 0.5±0.02 (n≥54/group). mCherry-TRPC6 showed striking sub-second intracellular trafficking blocked with BI 749327. Molecular docking simulations showed BI 749327 binds TRPC6 at a novel TRPC6-TRPC6 interface site. In silico “site-directed mutagenesis” with AlphaFold3 showed disruption of the TRPC6 interface site impacts channel stability. Conclusions: We demonstrate the clinical potential for inhibiting TRPC6 to treat heart failure in DMD. The capacity of BI 749327 to inhibit peak (and not baseline) [Ca 2+ ] IC coupled with the incredibly rapid trafficking of TRPC6 suggests that TRPC6 channels are dynamically regulated. Our combined AI and traditional in silico modeling identified a new binding site and novel mechanism-of-action of BI 749327. Future studies will focus on in vitro validation of in silico models, determine the functional implications of rapid TRPC6 trafficking, and test an exciting new clinical version of the TRPC6 inhibitor that has completed safety clinical trials in healthy volunteers.