A Tetrahydrobiopterin-Displacing Potent Neuronal Nitric Oxide Synthase Inhibitor with an Unprecedented Binding Mode

Nitric oxide synthase (NOS) is a pivotal enzyme that regulates various physiological processes, and the dysregulation of neuronal NOS (nNOS) is implicated in neurodegenerative diseases. In our efforts to leverage existing X-ray crystallography data to develop novel aminoquinoline-pyridine hybrid inhibitors and evaluate their inhibitory activities and interactions with NOS isoforms, we identified compounds 8 and 9 as potent human nNOS inhibitors (K _i = 38 and 22 nM, respectively). Notably, compound 8 displayed an unprecedented binding mode, displacing the essential cofactor tetrahydrobiopterin (H₄B). Furthermore, compound 9 exhibited excellent selectivity, with a 900-fold preference for human nNOS over human eNOS, making it one of the most potent and selective aminoquinoline-based nNOS inhibitors reported to date. Herein we present our inhibitor design rationale, the synthesis, and the biochemical/physical evaluation of binding along with X-ray crystallographic studies with three NOS isoforms, providing detailed insights into the observed potency and selectivity of these inhibitors.