CHEMBL5732703

Enzyme Assay: Rat and human nNOS, murine macrophage iNOS, and human eNOS were recombinant enzymes (expressed in E. coli and purified as reported previously in the literature). To test for NOS inhibition, the hemoglobin capture assay was used to measure nitric oxide production. The assay was performed at 37° C. in HEPES buffer (100 mM with 10% glycerol, pH 7.4) in the presence of 10 μM L-arginine. Also included were 100 μM NADPH, 0.83 mM CaCl2, approximately 320 units/mL of calmodulin, 10 μM H4B, and human oxyhemoglobin (3 μM). For iNOS, the CaCl2 and calmodulin were omitted and replaced with HEPES buffer (neither are required for activation of iNOS). The assay was performed in 96-well plates using a Synergy 4 BioTek hybrid reader. The dispensing of NOS enzyme and hemoglobin were automated, and after 30 sec (maximum delay), NO production was read by monitoring the absorbance at 401 nm (resulting from conversion of oxyhemoglobin to methemoglobin). Kinetic readouts were performed for 5 min. Each compound was assayed at least in duplicate, and six to nine concentrations (500 μM-50 nM or 100 μM-10 nM for eNOS and iNOS; 50 μM to 5 nM for rat and human nNOS) were used to construct dose-response curves. IC50 values were calculated by non-linear regression (variable slope, four parameters) using GraphPad Prism software, and Ki values were obtained using the Cheng-Prusoff equation [Ki=IC50/(1+[S]/Km)] with the following Km values: 1.3 μM (rat nNOS), 1.6 μM (human nNOS), 8.2 μM (murine macrophage iNOS), and 3.9 (human eNOS).