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

分离 GPR160，用于激活机制的生化分析，并开发高通量筛选方法来鉴定小分子抑制剂

• 批准号: 10176852
• 负责人: DANIELA SALVEMINI
• 金额: $ 15.15万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10176852
• 关键词: Activation Analysis; Acute; Acute Pain; Address; Affect; Antibodies; Attenuated; Binding; Biochemical; Biological Assay; CARTPT gene; Chronic; Complex; Cyclic AMP Response Element; Development; Disease; Dissociation; Exposure to; Extracellular Signal Regulated Kinases; Fluorescein; Fluorescence Polarization; Future; G-Protein-Coupled Receptors; Genetic; Genome; Goals; Grant; Hypersensitivity; In Vitro; Injections; Injury; Intrathecal Injections; Investigation; Kinetics; Label; Ligands; Maintenance; Measurement; Measures; Medical; Membrane Proteins; Messenger RNA; Methods; Molecular; Nervous system structure; Neuraxis; Neurotoxins; Orphan; Pain; Pathway interactions; Peptide Signal Sequences; Peptides; Production; Proteins; Proteolysis; Protocols documentation; Research; Rodent; Signal Pathway; Signal Transduction; Small Interfering RNA; Solubility; Spinal Cord; Spinal cord posterior horn; Stimulus; Surface Plasmon Resonance; System; Tail; Testing; Therapeutic; Therapeutic Intervention; Trauma; Traumatic Nerve Injury; Validation; Work; allodynia; aqueous; base; chronic neuropathic pain; chronic pain; chronic pain patient; high throughput screening; inhibitor/antagonist; innovation; insight; miniaturize; nanodisk; non-opioid analgesic; novel; novel therapeutics; painful neuropathy; protein expression; receptor; scale up; screening; side effect; small molecule; small molecule inhibitor; targeted treatment; therapeutic target

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.

拟议的研究补充了HEAL资助“一种新的孤儿GPCR的发现和验证”