TRPC6 inhibition by Z3571: A structure-based strategy to ameliorate glomerular and tubular dysfunction in chronic...

The transient receptor potential canonical 6 (TRPC6) channel represents a promising therapeutic target for chronic kidney diseases (CKDs) involving glomerular and tubular dysfunction; however, the development of highly specific inhibitors remains a challenge. A high-throughput screen of 150,000 structurally diverse compounds targeting the non-conserved C-terminal domain of TRPC6 identified promising candidates, which were validated using microscale thermophoresis and calcium imaging. Screening identified Z3571 as a top candidate targeting the non-conserved C-terminal domain of TRPC6, exhibiting strong binding affinity (K_D = 1.62 μM) and potent inhibition (IC₅₀ = 31.07 nM) with > 100-fold selectivity over TRPC3 and TRPC7. Z3571 was evaluated in both an adriamycin-induced podocyte injury model and a transforming growth factor-β1-stimulated tubular fibrosis models. Its functional efficacy was assessed in mouse models of focal segmental glomerulosclerosis (FSGS) and renal fibrosis. Z3571 demonstrated significant therapeutic effects in both in vitro and in vivo models of FSGS and renal fibrosis. Pharmacokinetic properties were analyzed via high-performance liquid chromatography. Pharmacokinetic profiling revealed favorable oral bioavailability, metabolic stability, and preferential renal accumulation. On-target effects were confirmed using Trpc6-knockout cells and mice, while binding-site-mutated cells established target specificity. Z3571 exhibited no activity in Trpc6-knockout models, confirming TRPC6 as its essential target. Additionally, site-directed mutagenesis confirmed Lys821 and His831 as critical residues mediating the interaction between Z3571 and TRPC6. These findings establish Z3571 as a highly selective and orally bioavailable TRPC6 inhibitor with robust efficacy, positioning it as a promising targeted therapy for CKDs involving glomerular and tubular dysfunction.