Fig. 2.

Validation of GPX1 qHTS primary assay. A) GPX1 assay velocity with 10 nM GPX1 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) GPX1 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 10 nM; C) effect of GR concentration on GPX1 velocity with optimized substrate and enzyme conditions, red bar denotes optimized concentration of 50 nM; D) 1536-well plate performance of optimized GR-coupled GPX1 assay, average Z′ for whole plate was 0.75; E) Prior art GPX1 inhibitor activities in optimized assay curve fit for IC50 calculation: IC₅₀ of auranofin, MSA, and mercuric(II) chloride were 4.84 μM, 1.44 μM, and 5.83 μM, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)