CHEMBL5735692

Enzyme Inhibition Assay: Enzyme inhibition was evaluated by measuring NO production with the hemoglobin capture assay, which was performed with purified NOSs in 96-well plates using a Biotek Gen5™ microplate reader. Purified NOSs used in this study, rat nNOS, human nNOS, murine macrophage iNOS, bovine eNOS, and human eNOS, are recombinant enzymes, expressed in E. coli and purified by reported procedures.37-39 For rat and human nNOS, bovine and human eNOS, the assays were performed at 37° C. in 100 mM HEPES buffer with 10% glycerol (pH 7.4) in the presence of 10/M L-arginine and tetrahydrobiopterin, 100 μM NADPH, 0.83 mM CaCl2, 320 units/mL of calmodulin, and 3 μM human oxyhemoglobin. For iNOS, the assay was performed at 37° C. in HEPES buffer in the presence of L-arginine and cofactors (NADPH, H4B, and human oxyhemoglobin). For all the assays, NO production was read by monitoring the absorbance at 401 nm, and kinetic readouts were recorded for 6 min. The inhibition constants (K,) for all NOSs were calculated from the IC50 values and Km (human nNOS: 1.6 M; rat nNOS: 1.3 μM; murine iNOS: 8.2 μM; bovine eNOS: 1.7 μM; human eNOS: 3.9 μM)40 using the Cheng-Prusoff equation: Ki=IC50/(1+[S]/Km).41