A screen for small molecule modulators of human GPCR MrgX1

描述（由申请人提供）：该项目的目标是对人类MrgX1拮抗剂和激动剂的小分子化合物进行大规模筛选，分别具有抗痒和抗慢性疼痛的治疗意义。背根神经节（DRG）的初级感觉神经元在引发和介导疼痛和瘙痒中起重要作用。我们已经证明，Mrgs，一个在DRG神经元中特异性表达的G蛋白偶联受体家族，作为瘙痒受体和慢性疼痛的内源性抑制剂发挥作用。因此，调节Mrg激活为治疗慢性疼痛和瘙痒提供了一个独特而有希望的靶点。我们已经成功地进行了ChemBridge多样性文库（20,000个化合物）的试点筛选，使用两阶段和三添加方案，用于人类MrgX1激动剂，拮抗剂和变构调节剂。在提案中提出了试点筛选的结果。在Aim I中，我们建议筛选一个更大、更多样化的化合物集合（即MLSMR库），以筛选人类MrgX1的激动剂、拮抗剂和变构调节剂，其方案与试点筛选中使用的方案相同。在Aim II中，我们将通过多个二级分析确认主筛选的阳性结果，这些二级分析已针对不同目的进行了优化，包括验证筛选、确认筛选、计数筛选、替代筛选和正交试验。此外，我们将通过Ca2+成像和全细胞贴片记录监测野生型和mrg缺陷小鼠的原生DRG神经元，研究阳性撞击的功效、效力和特异性。标准设置为二级分析，以选择有效的和特定的命中与药物相似。在第三阶段，我们将在小鼠行为实验中测试MrgX1调节剂的效力和特异性。我们将研究MrgX1激动剂和变构激动剂对慢性疼痛的抑制作用以及拮抗剂的抗痒作用。我们已经生成、开发和获得了所有实验所需的试剂，并制定了所有实验程序。此外，我们还得到了合作者约翰霍普金斯离子通道中心和约翰霍普金斯大学脑科学研究所神经翻译项目在高通量筛选和药物化学方面的大力支持。这些活性化合物可作为慢性疼痛和瘙痒的治疗药物加以利用。

DESCRIPTION (provided by applicant): The goal of this project is to carry out a large scale screen of small molecular compounds for both antagonists and agonists of human MrgX1 with therapeutic implications of anti-itch and anti-chronic pain, respectively. Primary sensory neurons in dorsal root ganglia (DRG) play essential roles in initiating and mediating pain and itch. We have shown that Mrgs, a family of G protein-coupled receptors specifically expressed in DRG neurons, function as itch receptors as well as endogenous inhibitors of chronic pain. Therefore, modulating Mrg activation offers a unique and promising target to treat chronic pain and itch. We have successfully carried out a pilot screen of ChemBridge Diversity library (20,000 compounds) using a two-stage and three-addition scheme for human MrgX1 agonists, antagonists, and allosteric modulators. The results of the pilot screen are presented in the proposal. In Aim I, we propose to screen a larger and more diverse compound collection (i.e. the MLSMR library) for agonists, antagonists, and allosteric modulators of human MrgX1 with the same protocol as used in the pilot screen. In Aim II, we will confirm positive hits from the primary screen by multiple secondary assays which have been optimized for different purposes of validation screen, confirmatory screen, counter screen, alternative screen, and orthogonal assay. In addition, we will examine the efficacy, potency, and specificity of positive hits using native DRG neurons from wild-type and Mrg-deficient mice monitored by Ca2+ imaging as well as whole-cell patch recordings. Criteria are set for the secondary assays to select potent and specific hits with drug likeness. In Aim III, we will test the potency and specificity of MrgX1 modulators in mouse behavioral assay. We will examine the inhibitory effects of MrgX1 agonists and allosteric agonists on chronic pain as well as the anti- itch effects of antagonists. We have generated, developed, and obtained all reagents necessary for the proposed experiments and have worked out all experimental procedures. In addition, we have great support from our collaborators Johns Hopkins Ion Channel Center and Neurotranslation program at Johns Hopkins University Brain Science Institute on both high throughput screen and medicinal chemistry. These active compounds may be exploited as a therapeutic remedy of chronic pain and itch.