Activation Mechanism of Soluble Guanylate Cyclase

一氧化氮(NO)信号对一些生理功能是必不可少的，而在 在这个信号级联中，涉及多种疾病，如勃起功能障碍、心脏 疾病、神经退行性变、中风、高血压和胃肠道疾病。可溶的 鸟苷环化酶(SGC)是NO的主要受体。NO在两个水平上调节sGC和 这与NO信号的药理学观察是一致的，与 NO的两步活化机制。NO的这些作用的幅度和持续时间 神经元信号、心脏功能、血管张力和血管扩张对正常功能至关重要， 但NO两步激活的机制还没有得到深入的研究。一个 通过sGC传递NO信号的新范式已经出现。了解sGC如何切换 从低激活状态到高激活状态是这一新范式的核心。此外，sgc已经成为 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)什么是 全长sGC的结构架构以及哪些域间相互作用 有助于sGC的激活机制？以及(Iii)作用机制是什么？ Adempas®(Riociguat)和sGC的相关刺激物。实验方法将包括 以下生化方法：酶动力学、克隆、表达、纯化和 SGC野生型和定点突变体的特性、电子显微镜结构 方法：氢-氚交换和多肽图谱。这是这一目标的中心目标 提议发展一个完整的分子水平的观点，以了解NO、 ADEMPAS®和sGC等药物。将这项工作扩展到生理功能将 为认识和治疗人类NO信号转导障碍提供合理依据 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.