A screen for small molecule modulators of human GPCR MrgX1

描述（由申请人提供）：本项目的目的是进行大规模筛选人MrgX1拮抗剂和激动剂的小分子化合物，分别具有止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。我们已经表明，Mrgs，一个家庭的G蛋白偶联受体特异性表达在DRG神经元，功能作为瘙痒受体以及内源性抑制剂的慢性疼痛。因此，调节Mrg激活为治疗慢性疼痛和瘙痒提供了独特且有前途的靶点。我们已经成功地进行了一个试点筛选ChemBridge多样性库（20，000化合物）使用两个阶段和三个添加方案的人MrgX1激动剂，拮抗剂和变构调节剂。提案中介绍了试点筛选的结果。在目的I中，我们建议使用与中试筛选相同的方案筛选更大和更多样化的化合物集合（即MLSMR文库），以获得人MrgX1的激动剂、拮抗剂和变构调节剂。在Aim II中，我们将通过多项二级检测确认初步筛选的阳性命中，这些检测已针对验证筛选、确证性筛选、计数筛选、替代筛选和正交试验的不同目的进行了优化。此外，我们将使用来自野生型和Mrg缺陷小鼠的天然DRG神经元，通过Ca2+成像以及全细胞贴片记录来监测阳性命中的功效、效力和特异性。设定二级测定的标准以选择具有药物相似性的有效和特异性命中。在目的III中，我们将在小鼠行为测定中测试MrgX1调节剂的效力和特异性。我们将检查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止痒作用。我们已经生成、开发并获得了拟议实验所需的所有试剂，并制定了所有实验程序。此外，我们的合作伙伴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的神经翻译项目在高通量筛选和药物化学方面都给予了大力支持。这些活性化合物可用作慢性疼痛和瘙痒的治疗药物。

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.