Isolation of GPR160 for biochemical analysis of the activation mechanism and development of a high throughput screening assay to identify small molecule inhibitors

这项拟议的研究是对Hear拨款的补充，即发现和验证一个新的孤儿GPCRs 作为神经病理性疼痛治疗干预的目标“(R01NS113257)，神经病理性疼痛状况 非常难以治疗1-4，迫切需要新的非麻醉性止痛药。我们最近的工作 导致发现孤儿G蛋白偶联受体160(oGPCR；GPR160)的激活在 脊髓有助于神经病理性疼痛状态的发展。因为没有小分子 GPR160的拮抗剂，GPR160信号的贡献被用遗传和 免疫药理学方法。阻断GPR160可阻断和逆转神经病理性疼痛，但对 在急性疼痛环境中，GPR160在从急性疼痛到慢性疼痛的过渡过程中起着重要作用。我们也 去孤儿的GPR160并鉴定可卡因和苯丙胺调节的转录肽(CARTp)是一种 5阻断脊髓内源性CARTp信号可减轻神经病理性疼痛，而 鞘内注射CARTp通过依赖GPR160诱发啮齿动物痛性过敏 细胞外信号调节激酶(ERK)和环磷酸腺苷反应元件结合通路(CREB)。我们的 首次发现使GPR160去孤儿，将其确定为神经病理性疼痛和潜在的决定因素 非阿片类小分子GPR160拮抗剂的治疗靶点，并对其 信号通路。5 GPR160从未被分离出来，生化特征和研究 了解CARTp与纯化蛋白的直接相互作用从未进行过。5在这里，我们建议 GPR160的分离和生化特性研究目前被确认为研究不足的 可药物基因组靶点的建立及GPR160生化特性的建立 与CARTp激活剂相互作用。实现生化检测小型化、优质化、规模化。 用于未来高通量生化筛选的蛋白质生产以确定有效的抑制物 GPR160激活。具体来说，我们将开发GPR160表达系统(S)和纯化方案(S) 并将使用纳米盘表征/优化用于生化研究的受体的稳定性和溶解性 接近。我们将使用纯化的蛋白质和CARTp来表征GPR160和CARTp的直接相互作用 荧光偏振(FP)与荧光素标记(FAM-)CARTp的互补结合分析 和使用未标记的CARTp的表面等离子体共振(SPR)。这些研究对于定义 CARTp/GPR160相互作用的分子机制及其对新抑制剂的大规模筛选 开发新的治疗方法。 影响：小分子GPR160拮抗剂作为非成瘾镇痛剂的发现和开发 预计将对慢性疼痛患者的治疗产生巨大影响。

The proposed research supplements the HEAL grant "Discovery and validation of a novel orphan GPCR as a target for therapeutic intervention in neuropathic pain" (R01NS113257). Neuropathic pain conditions are exceedingly difficult to treat1-4 and novel non-narcotic analgesics are desperately needed. Our recent work led to the discovery that activation of the orphan G protein-coupled receptor 160 (oGPCR; GPR160) in the spinal cord contributes to the development of neuropathic pain states5. Since there are no small molecule antagonists of GPR160, contribution of GPR160 signaling was unraveled using genetic and immunopharmacological approaches. Blocking GPR160 blocks and reverses neuropathic pain with no effect in acute pain settings, suggesting that GPR160 is important in the transition from acute to chronic pain. We also de-orphanized GPR160 and identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a ligand.5 Blocking endogenous CARTp signaling in the spinal cord attenuates neuropathic pain, whereas intrathecal injection of CARTp evokes painful hypersensitivity in rodents through GPR160-dependent extracellular signal-regulated kinase (ERK) and cyclic AMP response element-binding pathways (CREB). Our findings are the first to de-orphanize GPR160, identify it as a determinant of neuropathic pain and potential therapeutic target with non-opioid based small molecule GPR160 antagonists, and provide insights to its signaling pathways.5 GPR160 has never been isolated and biochemically characterized and studies to understand the direct interaction of CARTp with the purified protein have never been done.5 Here, we propose to isolate and biochemically characterize GPR160, currently identified as one of the Understudied Druggable Genome targets, and to establish methods for biochemical characterization of GPR160 interaction with CARTp activator. We will miniaturize and optimize biochemical assay and scale up protein production for future high throughput biochemical screening to identify potent inhibitors of GPR160 activation. Specifically, we will develop GPR160 expression system(s) and purification protocol(s) and will characterize/optimize stability and solubility of the receptor for biochemical studies using a nanodisc approach. We will characterize direct interaction of GPR160 and CARTp using purified proteins and complementary binding assays such as fluorescence polarization (FP) with fluorescein-labeled (FAM-) CARTp and surface plasmon resonance (SPR) using unlabeled CARTp. These studies are critical for defining the molecular mechanism of CARTp/GPR160 interactions and initiating large-scale screens for new inhibitors to develop novel therapeutics. Impact: Discovery and development of small molecule GPR160 antagonists as non-addictive analgesics is anticipated to have a huge impact on the treatment of chronic pain patients.