Structural Biology of Neuronal Nitric Oxide Synthase

说明书(申请人提供)：一氧化氮(NO)是一种由L精氨酸和分子氧通过三种结构不同的一氧化氮合酶(NOS)合成的气体信使：神经型(NNOS)、内皮(ENOS)和诱导型(INOS)。在正常情况下，NO在动态平衡中起关键作用。在卒中中，由nNOS过量产生的NO会导致显著的神经毒性，而eNOS产生的NO有利于通过血管扩张和抑制血小板聚集来恢复血液流动，nNOS是治疗中风的良好分子靶点，但抑制剂必须对nNOS亚型具有明显的选择性，以便它们不会阻断eNOS产生的NO的积极作用。本研究的假设是：1)对nNOS和eNOS异构体的比较结构研究将确定差异，从而可以针对nNOS的异构体特异性抑制nNOS；2)了解天然NOS抑制剂的结构基础可能会提供新的NOS抑制策略。NNOS抑制的结构基础将通过高分辨率X射线结晶学研究来探索，涉及三个具体目标：1)确定nNOS催化血红素结构域的三维结构；2)表征和比较与nNOS和eNOS主域结合的nNOS特定异构体衍生物；以及3)确定天然蛋白抑制剂aveolin-1的NOS识别位点。这些研究将为nNOS亚型特异性药物设计提供基本的科学框架。

DESCRIPTION (provided by applicant): Nitric oxide (NO) is a gaseous messenger synthesized from L-arginine and molecular oxygen by three structurally distinct nitric oxide synthases (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Under normal conditions, NO plays key roles in homeostasis. In stroke, NO overproduction by nNOS results in significant neurotoxicity, while NO generated by eNOS is beneficial to restoring blood flow via vasodilation and inhibition of platelet aggregation, nNOS is an excellent molecular target for stroke therapy, but inhibitors must be clearly selective for the nNOS isoform such that they do not block the positive effects resulting from NO production by eNOS. The hypotheses underlying this research project are that 1) comparative structural study of the nNOS and eNOS isoforms will identify differences, which can be targeted for isoform specific inhibition of nNOS and 2) an understanding of the structural basis of inhibition of a natural NOS inhibitor may provide novel NOS inhibition strategies. The structural basis of nNOS inhibition will be probed by high resolution x-ray crystallographic studies addressing three Specific Aims: 1) the determination of the three dimensional structure of the catalytic heme domain of nNOS, 2) characterization and comparison of nNOS specific isoform derivatives bound to the nNOS and eNOS home domain, and 3) determination of the NOS recognition site of a natural protein inhibitor, caveolin-1. These studies will provide the basic science framework for nNOS isoform specific drug design.