Department of Neuroscience, The Scripps Research Institute, Jupiter, FL, USA.
Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA.
SLAS Discov. 2021 Oct;26(9):1177-1188. doi: 10.1177/24725552211020679. Epub 2021 Jun 11.
Regulators of G protein signaling (RGS) proteins serve as critical regulatory nodes to limit the lifetime and extent of signaling via G protein-coupled receptors (GPCRs). Previously, approaches to pharmacologically inhibit RGS activity have mostly focused on the inhibition of GTPase activity by interrupting the interaction of RGS proteins with the G proteins they regulate. However, several RGS proteins are also regulated by association with binding partners. A notable example is the mammalian RGS7 protein, which has prominent roles in metabolic control, vision, reward, and actions of opioid analgesics. In vivo, RGS7 exists in complex with the binding partners type 5 G protein β subunit (Gβ5) and R7 binding protein (R7BP), which control its stability and activity, respectively. Targeting the whole RGS7/Gβ5/R7BP protein complex affords the opportunity to allosterically tune opioid receptor signaling following opioid engagement while potentially bypassing undesirable side effects. Hence, we implemented a novel strategy to pharmacologically target the interaction between RGS7/Gβ5 and R7BP. To do so, we searched for protein complex inhibitors using a time-resolved fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay that measures compound-mediated alterations in the FRET signal between RGS7/Gβ5 and R7BP. We performed two HTS campaigns, each screening ~100,000 compounds from the Scripps Drug Discovery Library (SDDL). Each screen yielded more than 100 inhibitors, which will be described herein.
G 蛋白信号转导调节因子（RGS）蛋白作为关键的调节节点，可限制 G 蛋白偶联受体（GPCR）信号的寿命和程度。此前，药理学抑制 RGS 活性的方法主要集中在通过中断 RGS 蛋白与它们调节的 G 蛋白的相互作用来抑制 GTP 酶活性。然而，几种 RGS 蛋白也受与其结合伙伴的相互作用的调节。哺乳动物 RGS7 蛋白就是一个显著的例子，它在代谢控制、视觉、奖励和阿片类镇痛药的作用中具有重要作用。在体内，RGS7 与结合伙伴 5 型 G 蛋白β亚基（Gβ5）和 R7 结合蛋白（R7BP）形成复合物，分别控制其稳定性和活性。针对整个 RGS7/Gβ5/R7BP 蛋白复合物提供了一种机会，可以在阿片类药物结合后，变构调节阿片受体信号，同时可能绕过不良的副作用。因此，我们采用了一种新的策略来药理学靶向 RGS7/Gβ5 和 R7BP 之间的相互作用。为此，我们使用基于时间分辨荧光共振能量转移（FRET）的高通量筛选（HTS）测定法，寻找蛋白复合物抑制剂，该测定法可测量化合物介导的 RGS7/Gβ5 和 R7BP 之间 FRET 信号的变化。我们进行了两次 HTS 实验，每次筛选 Scripps 药物发现库（SDDL）中的约 10 万个化合物。每次筛选都产生了 100 多种抑制剂，本文将对其进行描述。
