Fig. 1.

Assay diagrams. A) GPX activity schematic; B) Schematic diagram of fluorescence-based high-throughput screening assay used to evaluate GPX activity. GPX enzymatic reaction when reducing CHP is coupled to GR with the detection of NADPH fluorescence. C) Schematic diagram of GR inhibition assay used as a counter-screen to identify small molecules that show inhibition in the primary screening assay via indirect inhibition of the coupling enzyme; D) Schematic diagram of TXNRD1 activity assay reducing selenite to selenide in an NADPH-dependent reaction, used as counter-screen to identify small molecules with broader Sec-targeting activity; E) Schematic diagram of orthogonal endpoint GPX inhibition assay. Following a reaction window, monobromobimane is added and reacts with the thiols in remaining GSH to quantify GPX activity.