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

Using a comprehensive
chemical genetics approach, we identified a member of the lignan natural
product family, 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 haploinsufficiency screen strongly supported this finding.
Competitive photoaffinity labeling experiments demonstrated that the
ATP6V0A2 subunit of the v-ATPase complex 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
responsible for proton translocation. While other mechanisms of v-ATPase
regulation have been described, such as dissociation of the complex
or inhibition by natural products including bafilomycin A1 and concanamycin,
this work provides detailed insight into a distinct binding pocket
within the v-ATPase complex.