Argyriou Aikaterini I, Makrynitsa Garyfallia I, Dalkas Georgios, Georgopoulou Dimitra A, Salagiannis Konstantinos, Vazoura Vassiliki, Papapetropoulos Andreas, Topouzis Stavros, Spyroulias Georgios A
Department of Pharmacy, University of Patras, GR-26504, Patras, Greece.
Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, 26504, Patras, Greece.
Curr Res Struct Biol. 2021 Nov 18;3:324-336. doi: 10.1016/j.crstbi.2021.11.003. eCollection 2021.
The gasotransmitter nitric oxide (NO) is a critical endogenous regulator of homeostasis, in major part via the generation of cGMP (cyclic guanosine monophosphate) from GTP (guanosine triphosphate) by NO's main physiological receptor, the soluble guanylate cyclase (sGC). sGC is a heterodimer, composed of an α1 and a β1 subunit, of which the latter contains the heme-nitric oxide/oxygen (H-NOX) domain, responsible for NO recognition, binding and signal initiation. The NO/sGC/cGMP axis is dysfunctional in a variety of diseases, including hypertension and heart failure, especially since oxidative stress results in heme oxidation, sGC unresponsiveness to NO and subsequent degradation. As a central player in this axis, sGC is the focus of intense research efforts aiming to develop therapeutic molecules that enhance its activity. A class of drugs named sGC "activators" aim to replace the oxidized heme of the H-NOX domain, thus stabilizing the enzyme and restoring its activity. Although numerous studies outline the pharmacology and binding behavior of these compounds, the static 3D models available so far do not allow a satisfactory understanding of the structural basis of sGC's activation mechanism by these drugs. Herein, application NMR describes different conformational states during the replacement of the heme by a sGC activators. We show that the two sGC activators (BAY 58-2667 and BAY 60-2770) significantly decrease the conformational plasticity of the recombinant H-NOX protein domain of sp. cyanobacterium, rendering it a lot more rigid compared to the heme-occupied H-NOX. NMR methodology also reveals, for the first time, a surprising bi-directional competition between reduced heme and these compounds, pointing to a highly dynamic regulation of the H-NOX domain. This competitive, bi-directional mode of interaction is also confirmed by monitoring cGMP generation in A7r5 vascular smooth muscle cells by these activators. We show that, surprisingly, heme's redox state impacts differently the bioactivity of these two structurally similar compounds. In all, by NMR-based and functional approaches we contribute unique experimental insight into the dynamic interaction of sGC activators with the H-NOX domain and its dependence on the heme redox status, with the ultimate goal to permit a better design of such therapeutically important molecules.
气体递质一氧化氮（NO）是体内稳态的关键内源性调节因子，主要通过其主要生理受体可溶性鸟苷酸环化酶（sGC）将三磷酸鸟苷（GTP）生成环磷酸鸟苷（cGMP）来实现。sGC是一种异二聚体，由α1和β1亚基组成，其中后者包含血红素 - 一氧化氮/氧（H-NOX）结构域，负责NO的识别、结合和信号启动。NO/sGC/cGMP轴在包括高血压和心力衰竭在内的多种疾病中功能失调，特别是因为氧化应激导致血红素氧化、sGC对NO无反应并随后降解。作为该轴的核心参与者，sGC是旨在开发增强其活性的治疗分子的大量研究工作的重点。一类名为sGC“激活剂”的药物旨在替代H-NOX结构域中氧化的血红素，从而稳定酶并恢复其活性。尽管众多研究概述了这些化合物的药理学和结合行为，但迄今为止可用的静态三维模型尚不能令人满意地理解这些药物激活sGC的机制的结构基础。在此，应用核磁共振（NMR）描述了sGC激活剂取代血红素过程中的不同构象状态。我们表明，两种sGC激活剂（BAY 58-2667和BAY 60-2770）显著降低了蓝藻重组H-NOX蛋白结构域的构象可塑性，使其比结合血红素的H-NOX更加刚性。NMR方法还首次揭示了还原血红素与这些化合物之间惊人的双向竞争，表明H-NOX结构域具有高度动态调节。这些激活剂在A7r5血管平滑肌细胞中监测cGMP生成也证实了这种竞争性的双向相互作用模式。我们表明，令人惊讶的是，血红素的氧化还原状态对这两种结构相似的化合物的生物活性有不同的影响。总之，通过基于NMR和功能的方法，我们为sGC激活剂与H-NOX结构域的动态相互作用及其对血红素氧化还原状态的依赖性提供了独特的实验见解，最终目标是更好地设计此类具有治疗重要性的分子。
