Dynamic tail amine interactions drive isoform selectivity for potent human neuronal nitric oxide synthase inhibitors

Abstract Achieving isoform-selective inhibition of neuronal nitric oxide synthase (nNOS) remains a significant challenge due to the high structural similarity with other NOS isoforms. Here, we report the design, synthesis, and characterization of novel nNOS inhibitors 3 and 4 , incorporating dimethylamino-substituted tail groups to exploit hnNOS-specific peripheral pocket interactions. Both compounds retained sub-20 nM potency against human nNOS with enhanced selectivity over endothelial (hn/he > 1500-fold) and inducible (hn/hi > 229-fold) isoforms. Molecular dynamics simulations and MM-GBSA calculations suggested that hnNOS selectivity arises from a dynamically formed cation-π interaction between the terminal amino group and W311(B), which is precluded in heNOS due to the steric hindrance from F105. PAMPA-BBB assays inḍdicated moderate blood-brain barrier permeability, supporting CNS applications. These findings highlight peripheral pocket interactions as key drivers of isoform selectivity and guide future nNOS inhibitor optimization for neurodegenerative diseases and melanoma.