The three important mammalian isozymes of nitric oxide synthase (NOS) are neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Inhibitors of nNOS show promise as treatments for neurodegenerative diseases. Eight easily-synthesized compounds containing either one (20a,b) or two (9a-d; 15a,b) 2-amino-4-methylpyridine groups with a chiral pyrrolidine linker were designed as selective nNOS inhibitors. Inhibitor 9c is the best of these compounds, having a potency of 9.7 nM and dual selectivity of 693 and 295 against eNOS and iNOS, respectively. Crystal structures of nNOS complexed with either 9a or 9c show a double-headed binding mode, where each 2-aminopyridine head group interacts with either a nNOS active site Glu residue or a heme propionate. In addition, the pyrrolidine nitrogen of 9c contributes additional hydrogen bonds to the heme propionate, resulting in a unique binding orientation. In contrast, the lack of hydrogen bonds from the pyrrolidine of 9a to the heme propionate allows the inhibitor to adopt two different binding orientations. Both 9a and 9c bind to eNOS in a single-headed mode, which is the structural basis for the isozyme selectivity.

Department of Biochemistry The University of Texas Health Science Center San Antonio TX 78384 7760 USA

Department of Biochemistry The University of Texas Health Science Center San Antonio TX 78384 7760 USA ; Department of Pediatrics 1st Faculty of Medicine Charles University Prague Czech Republic

Department of Chemistry Department of Molecular Biosciences Chemistry of Life Processes Institute Center for Molecular Innovation and Drug Discovery Northwestern University Evanston Illinois 60208 3113 USA

Departments of Molecular Biology and Biochemistry Pharmaceutical Sciences and Chemistry University of California Irvine California 92697 3900 USA

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Combination of chiral linkers with thiophenecarboximidamide heads to improve the selectivity of inhibitors of neuronal nitric oxide synthase