NITRIC OXIDE SYNTHASE: ENZYMOLOGY AND METABOLIC ROLE

此资助申请是一个竞争性更新的RO 1从一个 一位富有成效、信誉卓著的调查员，将 对新型异构体选择性的开发和应用的先前研究 一氧化氮（NO）是一氧化氮合酶（NOS）抑制剂。 NO合酶（NOS）将L-精氨酸氧化为L-瓜氨酸， 在不同生理环境中介导细胞功能的关键作用。 已经鉴定了三种NOS同工型，包括内皮型（eNOS;或 III）、神经元（nNOS;或I型）和诱导型（iNOS;或II型）NOS。 虽然NOS已被证明有助于正常的生理功能， 包括调节血管张力、神经传递等， 过量的NO产生也可具有病理生理学效应。在3个NO中 iNOS同工型，iNOS已被最一致地确定为有助于高表达。 病理环境中NO的产生率，包括脓毒性休克、血管性 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种动物模型中，用 同种型选择性NOS抑制剂已显示出调节疾病的严重程度， 提示这些药物在临床竞技场中的潜在治疗作用。 然而，NOS拮抗剂临床应用的主要限制之一是， NOS非选择性抑制可能破坏正常生理功能 并产生不利影响。因此，迫切需要 开发高度特异性、异构体选择性NOS抑制剂。的PI 建议设计，合成和测试新的NOS抑制剂， 靶向iNOS和nNOS同种型。基本假设是“更好的NOS 抑制剂可以更有效地设计使用最近开发的 关于NOS亚型的结构，NOS反应机制， 以及NOS与其辅因子、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，并可以包含结构修饰， 在结合和对催化剂的敏感性方面实现异构体选择性 activation.这些抑制剂的设计将纳入新的信息， NOS活性机制和不同NOS的底物特异性 异构体，以及最近发表的数据NOS结构从x射线 结晶学PI建议通过研究这些新的抑制剂， 对体外人NOS同工型结合和活性的选择性影响，以及 如果成功的话，这些药物将在啮齿动物模型中进一步研究。 该提案包括三个具体目标。在目标1中，PI将确定 乙烯基-L-NIO，一种以前设计的NOS抑制剂， 并通过PI测试，选择性和不可逆地抑制nNOS同种型。 基于这些对相对nNOS选择性的潜在机制的研究， 在乙烯基-L-NIO的情况下，PI将设计乙烯基-L-NIO的类似物，其甚至可以具有 更高的nNOS选择性。在目标2中，PI将筛选已知和新化合物 能抑制NOS产生NO和超氧化物。目标#3，新 异构体选择性L-精氨酸拮抗剂将在一个 试图开发与iNOS同种型优先结合的试剂。 虽然在上一个赠款期间启动，但这些项目的发展 代理人取得了有限的成功。目前的战略是扩大这些 利用高分辨率X射线晶体学的新信息进行研究 eNOS和iNOS亚型结构的研究。PI也会开发新的代理人 来自Glaxo-Wellcome的基于亚砜亚胺的iNOS选择性抑制剂，以进一步 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.