以铁依赖性脂质过氧化为标志的铁死亡可能是癌症治疗的致命弱点。铁死亡抑制蛋白-1 (FSP1) 作为铁死亡的第二个支柱，通过 NAD(P)H 依赖性醌还原来有效防止脂质过氧化。由于其分子机制仍不清楚，我们研究了癌症中存在的大量 FSP1 突变或通过非靶向随机诱变鉴定的突变。该突变分析阐明了 FSP1 的 FAD/NAD(P)H 结合位点和质子转移功能，这些功能在不同的 NADH 醌还原酶中在进化上是保守的。通过随机诱变筛选，我们揭示了下一代 FSP1 抑制剂的作用机制。我们的研究确定了第一个 FSP1 抑制剂 iFSP1 的结合口袋，并引入了第一个独立于物种的 FSP1 抑制剂，靶向 NAD(P)H 结合口袋。总之，我们的研究为 FSP1 的分子功能提供了新的见解，并能够合理设计针对癌细胞的 FSP1 抑制剂。© 2023。作者。

Ferroptosis, marked by iron-dependent lipid peroxidation, may present an Achilles heel for the treatment of cancers. Ferroptosis suppressor protein-1 (FSP1), as the second ferroptosis mainstay, efficiently prevents lipid peroxidation via NAD(P)H-dependent reduction of quinones. Because its molecular mechanisms have remained obscure, we studied numerous FSP1 mutations present in cancer or identified by untargeted random mutagenesis. This mutational analysis elucidates the FAD/NAD(P)H-binding site and proton-transfer function of FSP1, which emerged to be evolutionarily conserved among different NADH quinone reductases. Using random mutagenesis screens, we uncover the mechanism of action of next-generation FSP1 inhibitors. Our studies identify the binding pocket of the first FSP1 inhibitor, iFSP1, and introduce the first species-independent FSP1 inhibitor, targeting the NAD(P)H-binding pocket. Conclusively, our study provides new insights into the molecular functions of FSP1 and enables the rational design of FSP1 inhibitors targeting cancer cells.© 2023. The Author(s).