Forward Chemical Genetic Screen for Oxygen-Dependent Cytotoxins Uncovers New Covalent Fragments that Target GPX4

The identification of growth inhibitory compounds with the ability to selectively target the cellular oxygenation state may be of therapeutic interest. Here, a phenotypic screen of a covalent fragment library revealed diverse compounds containing propiolamide warheads with selective toxicity for liver cancer cells in normoxic conditions. Target identification and validation through CETSA and direct pulldown experiments demonstrated that several compounds target glutathione peroxidase 4 (GPX4) and induce ferroptotic cell death. Although being an oxidative cell death mechanism, ferroptosis can be induced also under hypoxic conditions. Prompted by the selective toxicity discovered in the screen, we mapped the oxygen-dependence of several ferroptosis-inducing compounds across three different cell lines. These studies revealed combinations with notable reductions in sensitivity under hypoxic conditions. These observations are mechanistically interesting and may be relevant for the use of ferroptosis-inducers as anti-cancer agents.