Cancer cells have long been recognized to exhibit unique bioenergetic requirements. The apoptolidin family of glycomacrolides are distinguished by their selective cytotoxicity towards oncogene-transformed cells, yet their molecular mechanism remains uncertain. We used photoaffinity analogs of the apoptolidins to identify the F₁ subcomplex of mitochondrial ATP synthase as the target of apoptolidin A. Cryogenic electron microscopy (cryo-EM) of apoptolidin and ammocidin–ATP synthase complexes revealed a novel shared mode of inhibition that was confirmed by deep mutational scanning of the binding interface to reveal resistance mutations which were confirmed using CRISPR–Cas9. Ammocidin A was found to suppress leukemia progression in vivo at doses that were tolerated with minimal toxicity. The combination of cellular, structural, mutagenesis, and in vivo evidence defines the mechanism of action of apoptolidin family glycomacrolides and establishes a path to address oxidative phosphorylation-dependent cancers.

长期以来，癌细胞一直被认为表现出独特的生物能量需求。糖大环内酯的凋亡素家族以其对癌基因转化细胞的选择性细胞毒性而著称，但其分子机制仍不确定。我们使用细胞凋亡素的光亲和类似物来识别 F ₁线粒体 ATP 合成酶的亚复合体作为凋亡素 A 的靶标。凋亡素和氨化酶-ATP 合成酶复合物的低温电子显微镜 (cryo-EM) 揭示了一种新的共享抑制模式，该模式通过结合界面的深度突变扫描证实，以揭示抗性突变使用 CRISPR–Cas9 进行了确认。发现 Ammocidin A 在体内以最小毒性耐受的剂量抑制白血病进展。细胞、结构、诱变和体内证据的结合定义了凋亡素家族糖大环内酯的作用机制，并建立了解决氧化磷酸化依赖性癌症的途径。