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，人们一直认为癌细胞表现出独特的生物能量需求。糖大环内酯类细胞凋亡菌素家族的特点是它们对癌基因转化细胞的选择性细胞毒性，但它们的分子机制仍不确定。我们使用凋亡肽的光亲和类似物来识别线粒体 ATP 合酶的 F₁ 亚复合物作为细胞凋亡肽 A 的靶标。细胞凋亡肽和杀虫素-ATP 合酶复合物的低温电子显微镜 （cryo-EM） 揭示了一种新的共同抑制模式，该模式通过结合界面的深度突变扫描得到证实，以揭示耐药突变，这些突变已使用 CRISPR-Cas9 证实。发现氨霉素 A 在耐受剂量下抑制体内白血病进展且毒性最小。细胞、结构、诱变和体内证据的结合定义了细胞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。