Assessing the activity of benzodioxol, phenol and aniline derivatives as radical trapping antioxidants that inhibit ferroptosis

A small molecule screen has identified a benzodioxol anilide as a ferroptosis inhibitor. To determine the structure-activity relationship (SAR) of this compound class, we have synthesized a library of 35 derivatives followed by testing their ability to inhibit cell death that was induced via RSL3, an inhibitor of the main ferroptosis regulator glutathione peroxidase 4 (GPX4), in the mouse embryonic fibroblast cell line Pfa-1. Choosing 12 representative structures, rescue experiments were performed following the tamoxifen-induced deletion of GPX4 in Pfa-1 cells to complement the pharmacological induction of ferroptosis with a genetic induction and the experiments were extended to the human epithelial lung adenocarcinoma cell line HCC827. To assess if the compounds act as radical trapping antioxidants (RTAs) that scavenge peroxy radicals which drive lipid peroxidation, a hallmark of ferroptotic cell death, we have used a fluorescence-enabled inhibited autoxidation (Fenix) assay. Mitochondria were identified as the site-of-action using an alkynylated benzodioxol probe together with a copper-catalyzed alkyne-azide cycloaddition (CuAAC)-based Alexa Fluor 488 imaging kit. Our SAR study has yielded insights into the substitution-dependent activity of benzodioxol, aniline and phenol groups as RTAs that inhibit ferroptosis which led to the design of MM-12-Urea as a new broadly applicable chemical probe. Since the investigated functional groups are common motifs in small molecule ligands or natural products, we highlight that it is worth testing their ability to act as RTAs to avoid a potentially compounding factor in their bioactivities.