De Novo Design of Potent and Selective Neuronal Nitric Oxide Synthase (nNOS) Inhibitors by a Fragment-Based Approach
Nitric oxide, a gaseous free radical molecule (NO) behaves, as a secondary messenger in various tissues. It is responsible for different physiological functions and pathological symptoms. Mammals contain three different nitric oxide synthase (NOS) isoforms: neuronal NOS (nNOS: in the brain, in peripheral nervous system and muscle tissues), inducible NOS (iNOS: in macrophage cells), endothelial NOS (eNOS: in endothelial cells). Under certain pathological conditions and/or after certain ages excessive NO produced in brain causes tissue damage and oxidative stress. It also reacts with other free radicals to create specific molecular modifications. The excessive production of NO, especially by nNOS (in brain) is implicated in various disease states such as neurodegeneration, stroke, migraine and Parkinsons, Alzheimers, and Huntingtons diseases. The active sites of three NOS isoforms show great similarity; therefore, designing of selective nNOS inhibitors is not an easy task. The computational results carried out with all of the docking tools clearly demonstrate that the selected scaffold is a potential candidate for further modifications and optimization for designing selective and potent nNOS inhibitors. Subtle differences in the conformations of amino acid sequences (e.g. ASP597 in nNOS) of the three isoforms in the active site region were the determining factors for the selectivity and the potency of the compounds. In this study several hundred compounds were screened in silico  using the ZINCv12 lead library for prioritization of lead candidates. De novo design method was used rationally for the modifications of selected scaffold within a target-binding site in order to enhance its binding affinity and selectivity to nNOS enzyme. The potency and the selectivity of nNOS isoform were achieved by introducing 1-methyl amino group at the forth position of the imidazole moiety of the best inhibitor. The positively charged 1-methyl amino group makes three hydrogen bonds with the two-propionate groups of the heme cofactor, which does not occur in eNOS and iNOS. Removing of 1-methyl amino group from scaffold totally abolished both potency and selectivity for nNOS. Newly designed inhibitor 7  shows nNOS inhibition 23 and 17 fold better than both eNOS and iNOS, respectively.
