DEVELOPMENT OF ENHANCED DIPHYLLIN ANALOGS AS BROAD-SPECTRUM ANTIVIRALS TARGETING V-ATPASE VIA COMPUTATIONAL DESIGN

Emerging viral threats such as Ebola virus (EBOV), Chikungunya virus (CHIKV), and Venezuelan equine encephalitis virus (VEEV) highlight the need for broad-spectrum antiviral strategies targeting host factors. In response, vacuolar H⁺-ATPase (V-ATPase), a proton pump essential for endosomal acidification, was explored as a universal antiviral target. Diphyllin, a natural V-ATPase inhibitor with broad antiviral activity but poor pharmacokinetic properties, served as the starting scaffold for optimization. A computational strategy was employed to identify and characterize diphyllin’s binding site on the V-ATPase V0 domain. A conserved interfacial pocket with three sub-pockets was revealed and validated through molecular modeling and structure–activity data, providing a blueprint for analog design. This model guided a series of diphyllin analogs with strategic D-ring modifications were rationally designed and synthesized to enhance antiviral potency and drug-like properties. Several D-ring–modified analogs exhibited markedly improved in vitro antiviral activity, with lead compounds 3h and 3o demonstrating potent efficacy against EBOV, CHIKV, and VEEV. These lead analogs also showed significantly improved metabolic stability and oral bioavailability, maintaining plasma drug concentrations above effective levels in vivo. Collectively, this work transformed diphyllin from a promising yet pharmacologically limited natural product into optimized analogs with robust broad-spectrum antiviral activity, validating host-directed inhibition of V-ATPase as an effective therapeutic strategy.