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.

中文翻译：

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从具有相似作用机理的结构相似的化合物中发现以前未表征的液泡型ATPase结合位点。

使用全面的化学遗传学方法，我们确定了木脂素天然产物家族的成员HTP-013，该家族成员在各种癌细胞系中均表现出显着的细胞毒性。一系列报告基因测定中化合物活性的相关性表明，液泡型ATPase（v-ATPase）是该化合物的潜在靶标。额外的细胞研究和酵母单倍功能不足筛选强烈支持这一发现。竞争性光亲和标记实验表明，v-ATPase复合物的ATP6V0A2亚基直接与HTP-013结合，进一步的诱变文库筛选确定了ATP6V0A2中具有抗性的突变。这些突变的位置表明该分子结合了负责质子转运的v-ATPase复合物域内的一个新口袋。