Role of Zinc in Isoform-Selective Inhibitor Binding to Neuronal Nitric Oxide Synthase,

摘要

In previous studies [Delker, S. L., et al. (2010), J. Am. Chem. Soc. 132, 5437-5442], we determinednthe crystal structures of neuronal nitric oxide synthase (nNOS) in complex with nNOS-selective chiralnpyrrolidine inhibitors, designed to have an aminopyridine group bound over the heme where it cannelectrostatically interact with the conserved active site Glu residue. However, in addition to the expectednbindingmode with the (S,S)-cis inhibitors, an unexpected “flipped” orientation was observed for the (R,R)-cisnenantiomers. In the flipped mode, the aminopyridine extends out of the active site where it interacts with onenheme propionate. This prompted us to design and synthesize symmetric “double-headed” inhibitors with annaminopyridine at each end of a bridging ring structure [Xue, F., Delker, S. L., Li, H., Fang, J., Jamal, J.,nMartu0001 asek, P., Roman, L. J., Poulos, T. L., and Silverman, R. B. Symmetric double-headed aminopyridines, annovel strategy for potent and membrane-permeable inhibitors of neuronal nitric oxide synthase. J. Med.nChem. (submitted for publication)]. One aminopyridine should interact with the active site Glu and the othernwith the heme propionate. Crystal structures of these double-headed aminopyridine inhibitors in complexesnwith nNOS show unexpected and significant protein and heme conformational changes induced by inhibitornbinding that result in removal of the tetrahydrobiopterin (H4B) cofactor and creation of a new Zn2þ site.nThese changes are due to binding of a second inhibitor molecule that results in the displacement of H4B andnthe placement of the inhibitor pyridine group in position to serve as a Zn2þ ligand together with Asp,His, andna chloride ion. Binding of the second inhibitormolecule and generation of the Zn2þ site do not occur in eNOS.nStructural requirements for creation of the new Zn2þ site in nNOS were analyzed in detail. These observationsnopen the way for the potential design of novel inhibitors selective for nNOS.