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

摘要

In previous studies (Delker et al. (2010), J. Am. Chem. Soc. 132, 5437-5442) we determined the crystal structures of neuronal nitric oxide synthase (nNOS) complexed with nNOS-selective chiral pyrrolidine inhibitors, designed to have an aminopyridine group bound over the heme where it can electrostatically interact with the conserved active site Glu residue. However, in addition to the expected binding mode with the (S, S)-cis inhibitors an unexpected “flipped” orientation was observed for the (R, R)-cis enanthiomers. In the flipped mode the aminopyridine extends out of the active site where it interacts with one heme propionate. This prompted us to design and synthesize symmetric “double headed” inhibitors with an aminopyridine at each end of a bridging ring structure (Xue, F., Delker, S. L., Li, H., Fang, J., Jamal, J., Martásek, P., Roman, L. J., L., P. T., and Silverman, R. B. Symmetric double-headed aminopyridines, a novel strategy for potent and membrane-permeable inhibitors of neuronal nitric oxide synthase, J. Med. Chem. submitted). One aminopyridine should interact with the active site Glu and the other with the heme propionate. Crystal structures of these “double headed” aminopyridine inhibitors complexed to nNOS show unexpected and significant protein and heme conformational changes induced by inhibitor binding that result in removal of the tetrahydrobiopterin (H4B) cofactor and creation of a new Zn²⁺ site. These changes are due to binding of a second inhibitor molecule that results in displacement of H4B and the placement of the inhibitor pyridine group in position to serve as a Zn²⁺ ligand together with Asp, His, and a chloride ion. Binding of the second inhibitor molecule and generation of the Zn²⁺ site does not occur in eNOS. Structural requirements for creation of the new Zn²⁺ site in nNOS were analyzed in detail. These observations open the way for the potential design of novel inhibitors selective for nNOS.