Integrated chemical-genetic screens unveil FSP1 mechanisms and ferroptosis vulnerabilities

Ferroptosis, a cell death modality marked by iron-dependent lipid peroxidation, may present an Achilles heel for the treatment of difficult-to-treat cancers. Recently, ferroptosis suppressor protein-1 (FSP1) was identified as the second ferroptosis mainstay, efficiently preventing lipid peroxidation via NAD(P)H-dependent reduction of quinones. Since its molecular mechanisms have remained obscure, we conducted targeted and untargeted chemical-genetic screens by studying somatic mutations occurring in cancer, by performing random mutagenesis on FSP1 and elucidating the mechanism-of-action of next generation FSP1 inhibitors. Our studies identify the NAD(P)H binding site and proton transfer pathway of FSP1, which emerge to be evolutionary conserved among different NADH quinone reductases. We further uncover the binding pocket of the FSP1 inhibitor iFSP1 and introduce the first species-independent FSP1 inhibitor, targeting the NAD(P)H binding pocket. Conclusively, our study provides intriguing insights into the molecular functions of FSP1 and enables the rationale design of FSP1 inhibitors targeting certain cancer states.