Previously Uncharacterized Vacuolar-type ATPase Binding Site Discovered from Structurally Similar Compounds with Distinct Mechanisms of Action

Using a comprehensive chemical geneticsapproach, we identified a member of the lignan natural productfamily, HTP-013, which exhibited significant cytotoxicity across various cancer cell lines. Correlation of compound activity across a panel of reporter gene assays suggested the vacuolar-type ATPase( v-ATPase) as a potential target for this compound. Additional cellular studies and a yeast haploinsufficiencyscreen strongly supported this finding. Competitive photoaffinity labelingexperiments demonstrated that the ATP6V0A2 subunit of the v-ATPasecomplex binds directly to HTP-013, and further mutagenesis library screening identified resistance-conferring mutations in ATP6V0A2. The positions of these mutations suggest the molecule binds a novel pocket within the domain of the v-ATPase complex responsiblefor proton translocation. While other mechanisms of v-ATPaseregulation have been described, such as dissociation of the complex, or inhibition by natural productsincluding BafilomycinA1 and Concanamyc...