Discovery of GPX4 inhibitors through FP-based high-throughput screening.

Ferroptosis is a form of non-apoptotic cell death, regulated by phospholipid hydroperoxide glutathione peroxidase 4 (GPX4), a selenoprotein with a selenocysteine residue (sec) in the active site. GPX4 is a promising target for cancer cells in therapy-resistant conditions via ferroptosis, which can reduce the level of lipid reactive oxygen species (ROS). So far, all existing GPX4 inhibitors covalently bind to GPX4 via a reactive alkyl chloride moiety or masked nitrile-oxide electrophiles with poor selectivity and pharmacokinetic properties and most were obtained by cell phenotype-based screening. Lacking of effective high-throughput screening methods for GPX4 protein limits the discovery of GPX4 inhibitors. Here, we report a fluorescence polarization (FP)-based high throughput screening (HTS) assay for GPX4-U46C-C10A-C66A in vitro , and found Metamizole sodium from our in-house compound library inhibits GPX4-U46C-C10A-C66A enzyme activity. Structure-activity relationships (SAR) demonstrated the importance of sulfonyl group on interaction between Metamizole sodium and GPX4-U46C-C10A-C66A. Our FP assay could be an effective tool for discovery of GPX4 inhibitors and Metamizole sodium was a potential inhibitor for GPX4 in vitro. [Display omitted] • Using FITC modified peptide, a FP-based HTS assay for GPX4-U46C-C10A-C66A in vitro was established. • Metamizole sodium inhibited GPX4-U46C-C10A-C66A in an uncompetitive and non-covalent reversible manner. • Using rational drug design, a series of Metamizole sodium derivatives were synthesized and evaluated. • SAR demonstrated the importance of sulfonyl group on interaction between Metamizole sodium and GPX4-U46C-C10A-C66A. • Molecular docking results showed that Metamizole sodium competitively binds to the peptide-binding pocket. [ABSTRACT FROM AUTHOR]