Analysis of compound intrinsic reactivity against TNB 2-in a colorimetric assay Thiol reactivity assays were carried out based on a previously reported assay by Resnick et al. All assays were performed in buffer containing 20 mM sodium phosphates and 150 mM NaCl at pH 7.4. All measurements were taken at 37 ºC. 50 µM DTNB and 200 µM TCEP were incubated in sodium phosphate buffer for 5 min at 37 ºC to obtain TNB 2-. Separately, 200 µM TCEP was incubated without DTNB in sodium phosphate buffer for 5 min at 37 ºC. For each compound, TNB 2-solution (73.5 µL) was treated with 200 µM compound (1.5 µL of 10 mM stock in DMSO) in triplicate in a 384-well plate. Additionally, TCEP solution without TNB 2-was treated with 200µM compound in triplicate to control for background absorbance. A DMSO group was included to serve as a negative control. UV absorbance measurements were acquired every 7.5 min for 7 hours at 412 nm on a microplate reader. For each compound, the background absorbance without TNB 2-was subtracted from the absorbance with TNB 2-. The initial inhibitor concentration, [I]0, and TNB 2- concentration, [TNB 2-]0, were 200 µM and 100 µM, respectively. The remaining concentrations [I] and [TNB 2- ] were determined as a function of time from absorbance data using absorbance of the DMSO group as a reference for 100 µM TNB 2-. Subtracted reaction progress curves were directly fit to the second-order rate equation below, using GraphPad Prism, to extract a second-order rate constant. [TNB