NITRIC OXIDE SYNTHASE: ENZYMOLOGY AND METABOLIC ROLE

此授权申请是对以下项目RO1的竞争性续订 高效且久负盛名的调查员将延长他的 新型异构体选择性化合物的开发与应用研究进展 一氧化氮合酶(NOS)抑制。一氧化氮(NO)是在 一氧化氮合酶将L-精氨酸氧化成L-瓜氨酸 在不同的生理环境中调节细胞功能的关键作用。 已鉴定出三种一氧化氮合酶亚型，包括内皮(eNOS；或类型 Iii)、神经元型(nNOS；或I型)和诱导型(iNOS；或II型)NOS。 尽管一氧化氮合酶已被证明对正常的生理功能有贡献， 包括调节血管张力，神经传递等， 过量的NO产生也会产生病理生理效应。三个NO中的一个 INOS的异构体，一直被认为是导致高水平 病理环境中的一氧化氮生成率，包括感染性休克、血管 水肿、炎症引起的组织损伤、肿瘤血管生成等 疾病。在这些疾病的几个动物模型中，用 亚型选择性一氧化氮合酶抑制剂已被证明可以调节疾病的严重程度， 提示这些药物在临床领域具有潜在的治疗作用。 然而，一氧化氮合酶拮抗剂临床应用的主要限制之一是， 非选择性抑制一氧化氮合酶可能会扰乱正常的生理功能 并产生不利影响。因此，迫切需要 开发高特异性、异构体选择性的一氧化氮合酶抑制剂。《少年派》 建议设计、合成和测试新的一氧化氮合酶抑制剂 靶向iNOS和nNOS亚型。基本假设是“更好的NOS” 使用最近开发的药物可以更有效地设计抑制剂 关于一氧化氮合酶亚型结构的信息，关于一氧化氮合酶反应机理的信息， 以及一氧化氮合酶与辅因子、底物和产物的相互作用。 这些研究将主要集中在L精氨酸的类似物上，因为它很容易 运输到细胞中，并可以结合结构修改， 实现结合的异构体选择性和对催化剂的敏感性 激活。这些抑制剂的设计将纳入新的信息 不同类型一氧化氮合酶活性及底物专一性的作用机制 异构体，以及最近发表的关于一氧化氮合酶结构的X射线数据 结晶学。PI建议测试这些新的抑制剂，通过研究它们的 体外选择性对人一氧化氮合酶亚型结合和活性的影响 如果成功，这些药物将在体内用啮齿动物模型进行进一步研究。 这项建议包括3个具体目标。在目标1中，PI将确定 一氧化氮合酶抑制剂乙烯基-L-硝酸乙酯的作用机制 经PI检测，选择性和不可逆地抑制nNOS异构体。 基于这些对nNOS相对选择性的机制的研究 乙烯基-L-尼欧，PI将设计类似的乙烯基-L-尼欧，甚至可能 更高的nNOS选择性。在目标2中，PI将筛选已知的和新的化合物 能同时抑制一氧化氮合酶产生一氧化氮和超氧化物的物质。在目标3中，新的 选择性L-精氨酸拮抗剂的开发和研究 尝试开发优先结合iNOS异构体的试剂。 虽然在前一个赠款期间启动，但这些项目的发展 特工们取得的成功有限。目前的战略是将这些扩展 利用高分辨率X射线结晶学新信息的研究 内皮型一氧化氮合酶及其亚型结构的研究。PI还将利用新的特工 从葛兰素史克、亚磺胺为基础的iNOS选择性抑制剂，到进一步 开发这些药物的新类似物。

This grant application is a competitive renewal of an RO1 from a highly productive and well-established investigator that will extend his previous studies on the development and application of novel isoform-selective nitric oxide synthase (NOS) inhbitors.Nitric oxide (NO) is produced during the oxidation of L-arginine to L-citrulline by NO synthase (NOS), and plays a critical role in mediating cellular function in diverse physiologic settings. Three NOS isoforms have been identified, including endothelial (eNOS; or type III), neuronal (nNOS; or, type I) and inducible (iNOS; or, type II) NOS. Although NOS has been shown to contribute to normal physiologic functions, including the regulation of vascular tone, neurotransmission, and others, excessive NO production can also have pathophysiologic effects. Of the 3 NOS isoforms, iNOS has been most consistently identified as contributing to high rates of NO production in pathologic settings, including septic shock, vascular edema, tissue injury due to inflammation, tumor angiogenesis, and other diseases. In several animal models of these disorders, treatment with isoform-selective NOS inhibitors have been shown to modulate disease severity, suggesting a potential therapeutic role of these agents in the clinical arena. One of the major limitations in the clinical use of NOS antagonists, however, is that non-selective inhibition of NOS may disrupt normal physiologic function and have adverse effects. As a result, there is a critical need for the development of highly specific, isoform-selective NOS inhibitors. The PI proposes to design, synthesize and test new NOS inhibitors that selectively target the iNOS and nNOS isoforms. The basic hypothesis is that "better NOS inhibitors can be more efficiently designed using recently developed information on the structure of NOS isoforms, on the NOS reaction mechanism, and on the interactions of NOS with its cofactors, substrates and products." These studies will primarily focus on analogs of L-arginine since it is readily transported into cells and can incorporate structural modifications that achieve isoform selectivity in binding and susceptibility to catalytic activation. The design of these inhibitors will incorporate new information on mechanisms of NOS activity and substrate specificity of the different NOS isoforms, as well as recently published data on NOS structure from x-ray crystallography. The PI proposes to test these new inhibitors by studying their effects on human NOS isoform binding and activity in vitro for selectivity, and if successful, these agents will be further studied with rodent models in vivo. This proposal includes 3 specific aims. In aim #1, the PI will determine mechanisms by which vinyl-L-NIO, a NOS inhibitor that was previously designed and tested by the PI, selectively and irreversibly inhibits the nNOS isoform. Based on these studies of mechanisms underlying relative nNOS selectivity of vinyl-L-NIO, the PI will design analogs of vinyl-L-NIO that may have even greater nNOS selectivity. In Aim #2, the PI will screen known and new compounds that can inhibit both NO and superoxide generation by NOS. In Aim #3, new isoform-selective L-arginine antagonists will be developed and studied in an attempt to develop agents with preferential binding to the iNOS isoform. Although initiated during the previous grant period, the development of these agents met with limited success. The current strategy is to extend these studies by utilizing new information from high resolution x-ray crystallography studies on eNOS and iNOS isoform structure. The PI will also exploit new agents from Glaxo-Wellcome, sulfoximine-based iNOS -selective inhibitors, to further develop new analogs of these agents.