Uncovering the roles of oxysterols in neuropathic pain

慢性神经病理性疼痛在美国困扰着1500万至2000万人。1确定了相关的分子机制 是开发新型非阿片类镇痛剂的关键。2我们最近发现G-αI蛋白偶联受体 (GPCR)GPR183转录本在神经后脊髓背角神经胶质细胞中的表达增加 3 GPR183的主要内源性配体是有效的 信号转导7α，25-二羟基胆固醇(7α，25-OHC)。4-6然而，对7-α，25-OHC的作用知之甚少。 OHC/GPR183比我们最近发表的更痛苦。3此外，商业GPR183拮抗剂非常 具有有限的化学性质，这对它们在体内的实用性提出了质疑。3，7为了解决这个问题，我们发起了 一种电子药物发现方法，筛选出550万种与 GPR183药效团模型，然后对接并排名最高的相似对接得分。3前16名 化合物在钙动员(FLIPR)试验中被测试以鉴定几个小分子选择性 以SAE-14.3SAE-14为例，对IC50在NM范围内的GPR183拮抗剂进行反筛选 在NIMH PDSP8上对GPC罗马没有明显的活性，超过300个受体。全身性SAE-14 给药逆转神经损伤所致的机械性和冷觉超敏(行为超敏) 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系统。 鞘内注射(i.th)模拟效应。注射SAE-14，确定脊髓为潜在的 GPR183拮抗剂作用。3此外，用I.TH概括了疼痛行为表型。注射7α，25- OHC或激活GαI/O连接的氟化类似物(百日咳毒素)，依赖GPR183的丝裂原激活 蛋白激酶(MAPK)信号(初步数据)。DH-SC中的MAPK信号在调控中起关键作用 神经炎性/伤害性基因与持续性疼痛敏感性。9，10 GPR183依赖 SAE-14处理的大鼠的DH-SC无偏转录分析支持MAPK信号转导 神经病理性疼痛(初步数据)。SAE-14缺乏足够的代谢稳定性和溶解/吸收能力 (初步数据)和SAE-14构效关系的研究对于优化和 发现可用药的GPR183拮抗剂。根据我们已公布的工作3和初步数据，我们 假设DH-SC中的7α，25-OHC/GPR183信令有助于发育和维护 神经病理性疼痛状态。目标1中的拟议研究将调查7-α，25-OHC/GPR183在丝裂原活化蛋白激酶中的作用 DHSC中的激活和神经炎症。然后在目标2中，我们将在以下基础上开发GPR183拮抗剂 SAE-14，并使用测试漏斗筛选，以寻找体内具有最佳稳定性和生物利用度的药物 AIM 3中啮齿动物神经病理性疼痛模型的药理学特征：我们的发现将进一步加深我们的理解 神经病理性疼痛中GPR183信号转导机制的研究及首个具有特性的小分子GPR183拮抗剂的鉴定 适用于非成瘾性止痛药的开发，解决了Hear倡议的一个关键任务。

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.