BI-749327 inhibits the proliferation of hepatocellular carcinoma by targeting the TRPC6 channel.

[PURPOSE] Transient receptor potential canonical 6 (TRPC6), a calcium-permeable cation channel, is overexpressed in various tumors, including hepatocellular carcinoma (HCC), and its overexpression is closely associated with poor prognosis. Therefore, pharmacological targeting of TRPC6 may benefit HCC patients. BI-749327, a potent and selective small-molecule TRPC6 inhibitor, was investigated for its potential anti-HCC effects and mechanism.

[METHODS] The critical role of TRPC6 in HCC progression was demonstrated via its exogenous overexpression and siRNA-mediated knockdown. Anti-HCC effects of BI-749327 on proliferation, migration, and apoptosis were evaluated using CCK-8, colony formation, EdU, wound healing, transwell assays, flow cytometry, and Western blot (WB). CETSA, DARTS, molecular docking, dynamics, patch-clamp, Knockdown and overexpression experiments, and site-directed mutagenesis elucidated the BI-749327-TRPC6 interaction and binding sites. Intracellular Ca concentration ([Ca]) and downstream signaling were assessed via Fluo-4 AM and WB.

[RESULTS] TRPC6 overexpression promoted HCC cell proliferation, while its knockdown suppressed it. BI-749327 inhibited HCC cell proliferation, migration, and induced apoptosis. Mechanistically, BI-749327 inhibited TRPC6 activity and reduced its protein expression. CETSA and DARTS confirmed direct binding, with molecular dynamics, mutagenesis, patch-clamp identifying key residues (Tyr-612, Ser-608) in the TRPC6 VSLD. BI-749327 significantly decreased [Ca] and suppressed the PI3K/AKT/mTOR pathway.

[CONCLUSION] BI-749327 directly binds TRPC6, inhibiting its channel activity and expression, thereby reducing [Ca] and downstream Ca-related PI3K/AKT/mTOR signaling to suppress HCC progression. This study elucidates the BI-749327-TRPC6 interaction, provides a molecular basis for developing novel anti-HCC drugs, and establishes a research paradigm for other TRPC6 inhibitors.