In vitro studies of NOS inhibitors using purified human NOS isozymes
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Date
2017-12-18
Authors
Estavillo, Danica
Journal Title
Journal ISSN
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Publisher
University of Waterloo
Abstract
Endogenous nitric oxide (NO) is derived from L-arginine via a chemical reaction catalyzed by nitric oxide synthases (NOS). Three NOS isozymes are found in humans: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Over-activation of NOS enzymes lead to pathologies due to excessive NO production. In particular, nNOS and iNOS are implicated in serious neurodegenerative and inflammatory diseases, and are hence attractive pharmacological targets. However, off-target inhibition of eNOS results in adverse effects such as hypertension and atherosclerosis. To complicate drug design, the NOS active site sequence is highly conserved across isozymes. Most of the currently available NOS inhibitors have been designed through and assayed against non-human mammalian NOS isozymes but the recent publication of the human nNOS oxygenase crystal structures showed minute differences in structure between human NOS and their non-human counterparts. Oxyhemoglobin capture assays were performed to determine the specific activity of each NOS isoform and plot the corresponding Michaelis-Menten curves to determine their relative affinity towards the substrate L-arginine. These assays, which are performed in quadruplicates, show significant difference in the activity of human NOS and non-human NOS. Furthermore, discrepancies in the Km values of human NOS and non-human NOS were observed.
Inhibition studies using first generation active site inhibitors were performed in quadruplicates to confirm their isoform selectivity as previously determined using non-human NOS isoforms. L-arginine based inhibitors L-NMMA, L-ALA and L-NNA were shown to be non-selective over eNOS. However, the affinity ranking (in terms of Ki) of human NOS toward these L-ALA and L-NNA did not agree with the affinity rankings for non-human NOS. The Ki values of the cyclic aromatic compound 3-bromo-7-nitroindazole did not vary among isoforms for both human and non-human NOS, as previously available data. In contrast, the iNOS selective isoform 1400W showed significantly higher affinity towards iNOS over nNOS and eNOS. Given these preliminary results, the minute differences in sequence and structure between human and non-human NOS appear to have substantial effects on the selectivity of first generation inhibitors. However, it is important to note that these results need to be verified by structural studies, which can be used to pinpoint the differences in the binding of these inhibitors between human and non-human NOS.
To determine if human and non-human NOS are affected by calmodulin-binding inhibitors differently, inhibition assays were performed with trifluoperazine (TFP) and melatonin. Since both of these inhibitors bind CaM instead of NOS, it is expected that their IC50s for human and non-human NOS isoforms would be similar. Furthermore, the fact that these compounds bind CaM in a similar manner suggests that TFP and melatonin would have similar dose-inhibition curves. However, only TFP exhibited dose-dependent inhibition of NOS, while melatonin did not inhibit NOS at all. In any case, the inhibition assay results for both did not show differences between human and non-human NOS.
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Keywords
nitric oxide, nitric oxide synthase, NOS inhibitors, enzyme kinetics