A screen for small molecule modulators of human GPCR MrgX1

描述(申请人提供)：该项目的目标是对具有止痒和抗慢性疼痛治疗意义的人类MRgX1的拮抗剂和激动剂进行大规模的小分子化合物筛选。背根神经节(DRG)的初级感觉神经元在疼痛和瘙痒的起始和中介中起着重要作用。我们已经证明，MRGS是一种G蛋白偶联受体家族，特异性地表达在DRG神经元上，既可以作为瘙痒受体，也可以作为慢性疼痛的内源性抑制剂。因此，调节MRG的激活为治疗慢性疼痛和瘙痒提供了一个独特和有前途的靶点。我们已经成功地对ChemBridge多样性文库(20,000个化合物)进行了中试筛选，使用了两步三加成方案来筛选人的MRgX1激动剂、拮抗剂和变构调节剂。建议中列出了试点筛选的结果。在目标I中，我们建议用与试点筛选相同的方法筛选更大和更多样化的化合物集合(即MLSMR文库)，以寻找人类MRgX1的激动剂、拮抗剂和变构调节剂。在AIM II中，我们将通过针对验证筛选、确证筛选、计数器筛选、替代筛选和正交试验的不同目的而优化的多个二次检测来确认来自一次筛选的阳性结果。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，通过钙成像和全细胞贴片记录来检测阳性HITS的有效性、效力和特异性。为二次化验设定了标准，以选择与药物相似的有效和特定的打击。在目标III中，我们将在小鼠行为测试中测试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止痒作用。我们已经生成、开发和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，我们在高通量筛选和药物化学方面也得到了我们的合作者约翰霍普金斯大学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计划的大力支持。这些活性化合物可能被用作治疗慢性疼痛和瘙痒的药物。

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.