Department of Chemistry, Chemistry of Drug Development, University of Turku, Vatselankatu 2, 20500, Turku, Finland.
Division of Cardiology, Department of Internal Medicine, University of Texas Medical School At Houston, 1941 East Road, Houston, TX, 77054, USA.
Sci Rep. 2020 Oct 15;10(1):17469. doi: 10.1038/s41598-020-74611-x.
Cardiovascular diseases are the number one death worldwide. Nitric oxide (NO)-NO-sensitive (soluble) guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway regulates diverse set of important physiological functions, including maintenance of cardiovascular homeostasis. Resting and activated sGC enzyme converts guanosine triphosphate to an important second messenger cGMP. In addition to traditional NO generators, a number of sGC activators and stimulators are currently in clinical trials aiming to support or increase sGC activity in various pathological conditions. cGMP-specific phosphodiesterases (PDEs), which degrade cGMP to guanosine monophosphate, play key role in controlling the cGMP level and the strength or length of the cGMP-dependent cellular signaling. Thus, PDE inhibitors also have clear clinical applications. Here, we introduce a homogeneous quenching resonance energy transfer (QRET) for cGMP to monitor both sGC and PDE activities using high throughput screening adoptable method. We demonstrate that using cGMP-specific antibody, sGC or PDE activity and the effect of small molecules modulating their function can be studied with sub-picomole cGMP sensitivity. The results further indicate that the method is suitable for monitoring enzyme reactions also in complex biological cellular homogenates and mixture.
心血管疾病是全球头号死因。一氧化氮（NO）-NO 敏感（可溶性）鸟苷酸环化酶（sGC）-环鸟苷酸（cGMP）途径调节着包括维持心血管稳态在内的多样化重要生理功能。静止和激活的 sGC 酶将三磷酸鸟苷转化为重要的第二信使 cGMP。除了传统的 NO 生成剂外，目前还有许多 sGC 激活剂和刺激剂正在临床试验中，旨在支持或增加各种病理条件下的 sGC 活性。cGMP 特异性磷酸二酯酶（PDEs）将 cGMP 降解为单磷酸鸟苷，在控制 cGMP 水平和 cGMP 依赖性细胞信号的强度或长度方面发挥关键作用。因此，PDE 抑制剂也具有明确的临床应用。在这里，我们引入了一种用于 cGMP 的均相淬灭共振能量转移（QRET），以采用高通量筛选可接受的方法来监测 sGC 和 PDE 的活性。我们证明，使用 cGMP 特异性抗体，可以以亚皮摩尔 cGMP 的灵敏度研究 sGC 或 PDE 活性以及调节其功能的小分子的作用。结果还表明，该方法适用于监测复杂的生物细胞匀浆和混合物中的酶反应。
