Fig. 3.

Validation of GPX4 qHTS primary assay. A) GPX4 assay velocity with 200 nM GPX4 and 0.5 mM CHP with varying GSH concentrations, red denotes optimized 2:1 ratio of 1 mM GSH and 0.5 mM CHP (8:1, 4 mM GSH; 4:1, 2 mM GSH; 2:1, 1 mM GSH; 1:1, 0.5 mM GSH; 1:2, 0.25 mM GSH; 1:4, 0.125 mM GSH); B) GPX4 dilution and time course of GR-coupled activity assay with 100 nM GR, 1 mM GSH, 0.5 mM CHP, and 0.5 mM NADPH, red denotes optimized concentration of 200 nM; C) effect of GR concentration on GPX4 velocity with optimized substrate and enzyme conditions, red bar denotes optimized concentration of 50 nM; D) 1536-well plate performance of optimized GR-coupled GPX4 assay, average Z′ for whole plate was 0.75; E) Prior art GPX4 inhibitor activities in optimized assay curve fit for IC50 calculation: IC₅₀ of MSA, and mercuric(II) chloride were 57.74 μM, and 7.90 μM, respectively. None of the Class II FINs showed inhibitory activity, as reported elsewhere [42]. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)