Nitric oxide synthase (NOS) is a flavo‐hemoprotein that catalyzes the conversion of L‐arginine to L‐citrulline in the production nitric oxide (NO). Mammalian NOS exists in three isoforms: endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). Each isoform requires calmodulin (CaM) for activation as CaM binding induces a conformational change, which facilitates electron transfer within the NOS peptide via a highly dynamic process. NO overproduction is implicated in several neurodegenerative and inflammatory conditions including septic shock, epilepsy, myocarditis and Alzheimer's, thus the development of a NOS isoform‐specific inhibitor is of clinical significance. Previous research has shown that N1‐acetyl‐5‐methoxykynuramine (AMK), a metabolite of melatonin (aMT), is a CaM‐dependant inhibitor of nNOS in rat striatum1,2. Despite attenuation of nNOS activity demonstrated both in vitro and in vivo, there has yet to be a comprehensive structural analysis of AMK‐CaM‐NOS complexes. To further validate possible therapeutic applications, we conducted an investigation of AMK interactions with CaM and the CaM‐NOS binding domain for all three NOS isoforms. Our results showed CaM‐AMK binding using gel shift assays, while circular dichroism (CD) results displayed the greatest spectral differences when AMK was added to the CaM‐nNOS complex. This indicates potential isoform‐dependent variation in NOS inhibition due to differences in the CaM‐NOS binding domain. These results were substantiated by subsequent multidimensional NMR experiments, in which the largest differences in chemical shifts occurred when inhibitors were added to the CaM‐nNOS sample, primarily amongst residues found in the N‐lobe of CaM. Our investigation provides significant insight into the potential of AMK as an isoform‐specific NOS inhibitor.Support or Funding InformationThis work was funded by the NSERC of Canada.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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