Soluble guanylyl cyclase: mechanisms of activation and modulation

描述(申请人提供)：可溶性鸟苷酸环化酶(SGC)是一种关键的信号蛋白，当被一氧化氮激活时，它刺激第二信使cGMP的产生。这种cGMP信号通路在包括血压调节在内的许多心血管过程中是重要的，因此sGC作为治疗高血压、心力衰竭、动脉粥样硬化、勃起功能障碍、血栓形成和肾脏纤维化等心血管疾病的药物靶点引起了人们的极大关注。几种靶向sGC的化合物正处于临床前或临床试验阶段。申请人通过测定4个不同结构域的晶体结构，包括与sGC激活剂BAY 58-2667和一氧化氮的络合物，帮助理解鸟苷酸环化酶的结构。该应用程序的首要目标是进一步解开sGC的信号错综复杂，从而深入了解如何利用或绕过这一信号机制，以便可以通过药物利用这些信息来调节sGC。拟议的研究具有高度协作和多学科性质，涉及诸如X射线结晶学、电子顺磁共振(EPR)、等温量热(ITC)、诱变和细胞生物学/活性测量等技术。这3个具体目标是：特定目标1：验证sGC H-NOX结构域激活涉及血红素口袋部分扭曲的假设。还将探讨sGC激活剂的激活情况，以进行比较，并描绘这些血红素模拟物的作用模式。特定目的2：验证sGC的螺旋卷曲(CC)结构域是平行二聚体的假设，并且该结构域不是静态的，并且在激活过程中经历构象变化。具体目的3：验证sGC的N端负责结合一组化学上不同的sGC刺激物的假设；该sGC区域包括H-NOX和PAS/H-NOXA结构域(S)。这项拟议的研究将导致对sGC如何发挥作用以及GC激活剂/调节剂如何工作的新的分子见解，这将增强我们对心血管过程的理解，并可能导致开发治疗心血管疾病的新药。

DESCRIPTION (provided by applicant): The soluble guanylyl cyclase (sGC) is a key signaling protein that stimulates the production of the second messenger cGMP when activated by nitric oxide. This cGMP signaling pathway is important for a number of cardiovascular processes including blood pressure regulation and sGC has therefore attracted considerable attention as a pharmaceutical drug target to treat cardiovascular diseases such as hypertension, heart failure, atherosclerosis, erectile dysfunction, thrombosis, and renal fibrosis. Several compounds targeting sGC are in pre-clinical or clinical trial stages. The applicant has aided the structural understanding of guanylyl cyclases by determining crystal structures of 4 different domains including complexes with the sGC activators BAY 58-2667 and nitric oxide. The overarching goal of this application is to further unravel the signaling intricacies of sGC that could yield insights into how to either utilize or bypass this signaling mechanism such that this information can be pharmaceutically exploited to modulate sGC. The proposed research is of a highly collaborative and multi-disciplinary nature involving techniques such as X-ray crystallography, electron paramagnetic resonance (EPR), isothermal calorimetry (ITC), mutagenesis, and cell biology/activity measurements. The 3 Specific Aims are: Specific Aim 1: To test the hypothesis that the activation of the H-NOX domain of sGC involves a partial distortion of the heme pocket. The activation by sGC activators will also be probed for comparison and to delineate the mode of action of these heme mimetics. Specific Aim 2: To test the hypothesis that the coiled-coiled (CC) domain of sGC is a parallel dimer and that this domain is not static and undergoes conformational changes during the activation process. Specific Aim 3: To test the hypothesis that the N-terminal half of sGC is responsible for binding sets of chemically distinct sGC stimulators; this sGC region includes the H-NOX and PAS/H-NOXA domain(s). The proposed research will lead to new molecular insights into how sGC functions and how GC activators/modulators work and this will enhance our understanding of cardiovascular processes and could lead to the development of new drugs to treat cardiovascular diseases.