Dissecting Polypharmacology in Phenotypic Screening to Resolve Ferroptotic and Necrotic Cell-Death Mechanisms

Small molecules frequently induce heterogeneous cell-death programs, complicating the mechanistic interpretation and optimization. Here, we investigate the ferroptotic and necrotic activities of the lethal small molecule CIL56 and related analogs. Although structurally similar, these compounds induce chemically separable death phenotypes. A phenotypic suppressor screen further identified distinct sets of small molecules that selectively attenuate ferroptotic or necrotic death. Classification of suppressor compounds based on shared ligand-based target predictions suggested nonoverlapping groups of candidate protein targets linked to each death modality. Together, these results show that integrating phenotypic screening with suppressor classification and target prediction can improve the interpretability of small-molecule phenotypic screens by prioritizing candidate proteins and pathways underlying the observed biological response.