Computationally and Target-Guided Discovery of Trifluoromethyl-Pyrazole Derivatives Targeting the a-c Subunit Interface of V-ATPase in Phytophthora capsici.

Vacuolar H+-ATPase (V-ATPase) is essential for vacuolar acidification, ion homeostasis, and development in various organisms, including plant pathogens. The V-ATPase a-subunit has been recently identified as the target protein of the oomycete inhibitor fluopicolide, highlighting the favorable druggability of V-ATPase. In this study, we propose a structure-based virtual screening approach to identify novel bioactive compounds targeting the a-c subunit interface of V-ATPase. Following multistep docking and molecular mechanics/generalized born surface area (MM/GBSA) analysis, compound a10 was identified as a lead and optimized. Among the analogues, the trifluoromethyl-pyrazole derivative 4j exhibited potent inhibition against Phytophthora capsici. Biochemical assays, molecular simulations, and transmission electron microscopy demonstrated that 4j inhibits V-ATPase, causing pronounced vacuolar enlargement and ultrastructural disruption. It also suppressed sporangia formation, zoospore release, and cystospore germination of P. capsici and showed notable protective efficacy. Therefore, 4j is a promising candidate for developing novel V-ATPase-targeted fungicides to control P. capsici.