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

拟议的研究补充了HEAL资助“发现和验证一种新的孤儿GPCR” 作为神经性疼痛治疗干预的靶点”（R 01 NS 113257）。神经性疼痛病状 非常难以治疗1 -4，迫切需要新的非麻醉性镇痛药。我们最近的工作 导致发现在细胞中孤儿G蛋白偶联受体160（oGPCR; GPR 160）的激活， 脊髓有助于神经性疼痛状态的发展5。因为没有小分子 GPR 160的拮抗剂，GPR 160信号传导的贡献是使用遗传和 免疫药理学方法。阻断GPR 160可阻断和逆转神经性疼痛，但对神经性疼痛无影响。 急性疼痛设置，这表明GPR 160在从急性到慢性疼痛的转变中是重要的。我们也 去乙酰化GPR 160，并将可卡因和安非他明调节的转录肽（CARTp）鉴定为 配体.5阻断脊髓中内源性CARTp信号传导可减弱神经性疼痛，而 鞘内注射CARTp通过GPR 160依赖性引起啮齿类动物疼痛超敏反应 细胞外信号调节激酶（ERK）和环AMP反应元件结合途径（CREB）。我们 这些发现首次将GPR 160去神经化，将其确定为神经性疼痛的决定因素， 治疗靶点与非阿片类药物为基础的小分子GPR 160拮抗剂，并提供见解，其 GPR 160从未被分离和生物化学表征， 理解CARTp与纯化蛋白质的直接相互作用从未做过。5在这里，我们建议 分离和生化表征GPR 160，目前被确定为待研究的 可药用基因组靶标，并建立GPR 160的生化表征方法 与CARTp激活剂相互作用。我们将对生化检测进行优化和规模化 用于未来高通量生物化学筛选的蛋白质生产，以鉴定 GPR 160激活。具体而言，我们将开发GPR 160表达系统和纯化方案。 并且将表征/优化受体的稳定性和溶解性以用于使用纳米盘的生物化学研究 approach.我们将使用纯化的蛋白质表征GPR 160和CARTp的直接相互作用， 互补结合测定，例如用荧光素标记的（FAM-）CART p的荧光偏振（FP 和使用未标记的CART β的表面等离子体共振（SPR）。这些研究对于确定 CARTp/GPR 160相互作用的分子机制，并启动新抑制剂的大规模筛选， 开发新的治疗方法。 影响：小分子GPR 160拮抗剂作为非成瘾性镇痛药的发现和开发 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.