Uncovering the roles of oxysterols in neuropathic pain

揭示氧甾醇在神经性疼痛中的作用

• 批准号: 10504409
• 负责人: Christopher K Arnatt
• 金额: $ 52.03万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10504409
• 关键词: Address; Analgesics; Astrocytes; Behavioral; Biochemical; Biological Assay; Biological Availability; Cell Lineage; Cells; Chronic; Coupled; Data; Development; Disease; Docking; Dose; Drug Kinetics; Extracellular Signal Regulated Kinases; Female; Genes; Helping to End Addiction Long-term; Hypersensitivity; In Situ; In Vitro; Injections; Ipsilateral; Knockout Mice; Ligands; Link; Maintenance; Medical; Metabolic; Microglia; Mission; Mitogen-Activated Protein Kinases; Modeling; Molecular; Motor; Mus; National Institute of Mental Health; Nervous System Trauma; Neuroglia; Neurotoxins; Nociception; Opioid; Opioid Receptor; Oral; Pain; Persistent pain; Persons; Pertussis Toxin; Pharmaceutical Chemistry; Pharmacologic Actions; Pharmacology; Property; Proteins; Publications; Publishing; Rattus; Reaction Time; Reflex action; Rewards; Rodent; Role; Signal Transduction; Signaling Molecule; Site; Solubility; Spinal; Spinal Cord; Spinal cord posterior horn; Stimulus; Structure-Activity Relationship; System; Testing; Therapeutic Intervention; Time; Tissues; Transcript; Trauma; Traumatic Nerve Injury; Withdrawal; absorption; allodynia; analog; antagonist; base; behavioral phenotyping; behavioral response; behavioral study; chemical property; chronic neuropathic pain; design; drug discovery; endogenous opioids; improved; in silico; in vivo; interdisciplinary approach; male; nerve injury; neuroinflammation; non-opioid analgesic; novel; p38 Mitogen Activated Protein Kinase; pain model; painful neuropathy; pharmacophore; receptor; release of sequestered calcium ion into cytoplasm; side effect; small molecule; targeted treatment; tool; transcriptomics

Chronic neuropathic pain afflicts 15-20 million people in the U.S.1 Defining the molecular mechanisms involved is key to developing novel non-opioid analgesics.2 We recently discovered that the Gαi protein-coupled receptor (GPCR) Gpr183 transcript increases in glial cells of the dorsal horn of the spinal cord (DH-SC) following nerve injury and contributes to ensuing neuropathic pain.3 The primary endogenous ligand for GPR183 is the potent signaling oxysterol 7α,25-dihydroxycholesterol (7α,25-OHC).4-6 However, little is known about the roles of 7α,25- OHC/GPR183 in pain beyond our recent publication.3 Moreover, commercial GPR183 antagonists are very limited and have chemical properties that raise questions about their in vivo utility.3,7 To address this, we initiated an in silico drug discovery approach that screened 5.5 million commercial compounds with similarities to a GPR183 pharmacophore model, then docked and ranked the highest similar docking scores.3 The top 16 compounds were tested in calcium mobilization (FLIPR) assays to identify several small-molecule selective GPR183 antagonists with IC50 values in the nM range, exemplified by SAE-14.3 SAE-14 was counter-screened against the GPCRome at NIMH PDSP8 with no significant activity for over 300 receptors. Systemic SAE-14 administration reversed mechano-and cold allodynia (behavioral hypersensitivities) induced by nerve injury in male and female rodents with no observable side effects or engaging the endogenous opioid system.3 These effects were mimicked by intrathecal (i.th.) injection of SAE-14, identifying the spinal cord as a potential site of GPR183 antagonist action.3 Moreover, pain behavioral phenotypes were recapitulated by i.th. injection of 7α,25- OHC or a fluorinated analog that activated Gαi/o-linked (pertussis toxin), GPR183-dependent mitogen-activated protein kinase (MAPK) signaling (preliminary data). MAPK signaling in the DH-SC is crucial in regulating neuroinflammatory/pro-nociceptive genes and maintaining persistent pain sensitization.9,10 GPR183-dependent MAPK signaling was supported by unbiased transcriptomic analysis of the DH-SC from SAE-14-treated rats with neuropathic pain (preliminary data). SAE-14 lacks sufficient metabolic stability and solubility/absorption (preliminary data) and structure-activity relationship studies on SAE-14 are necessary for optimization and discovery of druggable GPR183 antagonists. Based on our published work3 and preliminary data, we hypothesize that 7α,25-OHC/GPR183 signaling in the DH-SC contributes to the development and maintenance of neuropathic pain states. Proposed studies in Aim 1 will investigate the role of 7α,25-OHC/GPR183 on MAPK activation and neuroinflammation in the DH-SC. Then in Aim 2, we will develop GPR183 antagonists based on SAE-14 and use a testing funnel to screen to find those with optimal stability and bioavailability for in vivo pharmacological profiling in rodent neuropathic pain models in Aim 3. Our findings will further our understanding of GPR183 signaling in neuropathic pain and identify the first small molecule GPR183 antagonists with properties suitable for non-addictive analgesics development addressing a key mission of the HEAL initiative.

慢性神经性疼痛困扰着美国 15-2000 万人1 定义所涉及的分子机制 是开发新型非阿片类镇痛药的关键。2 我们最近发现 Gαi 蛋白偶联受体 (GPCR) 神经后脊髓背角 (DH-SC) 神经胶质细胞中 Gpr183 转录物增加 损伤并导致随后的神经性疼痛。3 GPR183 的主要内源性配体是有效的 信号氧甾醇 7α,25-二羟基胆固醇 (7α,25-OHC).4-6 然而，人们对 7α,25- 的作用知之甚少。 OHC/GPR183 在疼痛方面的作用超出了我们最近发表的文章3。此外，商业 GPR183 拮抗剂非常有效。 有限且其化学特性引发了对其体内效用的质疑。3,7 为了解决这个问题，我们发起了 一种计算机药物发现方法，筛选了 550 万种与药物相似的商业化合物 GPR183药效团模型，然后对接并排名相似对接分数最高的3名前16名 在钙动员 (FLIPR) 测定中对化合物进行了测试，以鉴定几种小分子选择性 IC50 值在 nM 范围内的 GPR183 拮抗剂，例如 SAE-14.3 SAE-14 进行了反筛选 与 NIMH PDSP8 上的 GPCRome 相比，对 300 多种受体没有显着活性。系统性 SAE-14 给药可逆转由神经损伤引起的机械性和冷性异常性疼痛（行为超敏反应） 雄性和雌性啮齿动物没有可观察到的副作用或参与内源性阿片类药物系统。3 这些 通过鞘内 (i.th) 注射 SAE-14 来模拟效果，将脊髓确定为潜在的 SAE-14 部位 GPR183 拮抗剂作用。3 此外，i.th 概括了疼痛行为表型。注射7α,25- OHC 或激活 Gαi/o 连接（百日咳毒素）、GPR183 依赖性促细胞分裂原激活的氟化类似物 蛋白激酶 (MAPK) 信号传导（初步数据）。 DH-SC 中的 MAPK 信号传导对于调节至关重要 神经炎症/促伤害基因并维持持续的疼痛敏化。9,10 GPR183 依赖性 MAPK 信号传导得到 SAE-14 治疗大鼠 DH-SC 的无偏转录组学分析的支持 神经性疼痛（初步数据）。 SAE-14 缺乏足够的代谢稳定性和溶解度/吸收 （初步数据）和SAE-14的构效关系研究对于优化和 发现可药物化的 GPR183 拮抗剂。根据我们发表的工作3和初步数据，我们 假设 DH-SC 中的 7α,25-OHC/GPR183 信号传导有助于发育和维持 神经性疼痛状态。目标 1 中拟议的研究将调查 7α,25-OHC/GPR183 对 MAPK 的作用 DH-SC 中的激活和神经炎症。然后在目标2中，我们将基于GPR183拮抗剂开发 SAE-14并使用测试漏斗筛选以找到体内具有最佳稳定性和生物利用度的化合物 目标 3 中啮齿类神经病理性疼痛模型的药理学分析。我们的发现将进一步加深我们的理解 神经性疼痛中 GPR183 信号传导的研究，并鉴定出第一个具有特性的小分子 GPR183 拮抗剂 适用于非成瘾性镇痛药的开发，以实现 HEAL 计划的关键使命。