Activation Mechanism of Soluble Guanylate Cyclase

一氧化氮（NO）信号传导对于几种生理功能是必不可少的，并且在细胞中功能障碍。 在这种信号级联中，涉及多种疾病，如勃起功能障碍、心脏病、 疾病、神经变性、中风、高血压和胃肠道疾病。可溶性 鸟苷酸环化酶（sGC）是NO的主要受体。NO在两个水平调节sGC， 这与NO信号传导的药理学观察一致， NO的两步激活机制。 神经元信号传导、心脏功能、血管张力和血管舒张对于适当的功能是至关重要的， 但NO两步活化的机理还没有被彻底研究。一 已经出现了通过sGC进行NO信号传导的新范例。了解sGC如何切换 从低到高的激活状态是这个新范例的核心。此外，sGC已成为 用于治疗两种形式的肺动脉高压的治疗靶点：慢性 血栓栓塞性肺动脉高压和肺动脉高压与FDA 批准的Adempas®。我们的具体目标包括：（i）NO如何激活sGC？，(ii)是什么 全长sGC的结构架构以及结构域间的相互作用是什么， 有助于sGC？的激活机制，和（iii）的作用机制是什么 Adempas®（riociguat）和sGC的相关刺激剂。实验方法将包括 以下生物化学方法：酶动力学、克隆、表达、纯化和 sGC野生型和定点突变体的表征，电子显微镜结构 方法，氢氘交换和肽图谱。这是一个中心目标， 建议开发一个完整的分子水平的观点之间的复杂关系NO， Adempas®和sGC等药物。这项工作的扩展到生理功能将 为认识和治疗人类NO信号转导障碍提供了理论依据 疾病

Nitric oxide (NO) signaling is essential to several physiological functions, and dysfunction in the in this signaling cascade is implicated in multiple diseases such as erectile dysfunction, heart disease, neurodegeneration, stroke, hypertension, and gastrointestinal disease. Soluble guanylate cyclase (sGC) is the primary receptor for NO. NO regulates sGC at two levels and this is consistent with pharmacological observations of NO signaling that are consistent with a two-step activation mechanism by NO. The amplitude and duration of these effects of NO in neuronal signaling, cardiac function, vascular tone and vasodilation are vital to proper function, but the mechanism for the two-step activation by NO has not been thoroughly investigated. A new paradigm for NO signaling through sGC has emerged. Understanding how sGC switches from a low to high activation state is central to this new paradigm. In addition, sGC has become a therapeutic target for the treatment of two forms of pulmonary hypertension: chronic thromboembolic pulmonary hypertension and pulmonary arterial hypertension with the FDA approved Adempas®. Our specific aims include: (i) How does NO activate sGC?, (ii) What is the structural architecture of full-length sGC and what are the inter-domain interactions that contribute to the activation mechanism of sGC?, and (iii) What is the mechanism of action of Adempas® (riociguat) and related stimulators of sGC. Experimental approaches will include the following biochemical methods: enzyme kinetics, cloning, expression, purification and characterization of wild type and site-directed mutants of sGC, electron microscopy structural methods, hydrogen-deuterium exchange and peptide mapping. It is a central goal of this proposal to develop a complete molecular level view of the complex relationship between NO, drugs like Adempas® and sGC. The extension of this work into physiological function will provide a rational basis for the understanding and treatment of NO signaling disorders in human disease.