Identification of allosteric molecules for DOR-KOR heteromer-mediated peripheral analgesia

目前临床上使用的止痛药以中枢神经系统内的u阿片受体为靶点，并能产生 危险的、往往是致命的不良反应，包括呼吸抑制和成瘾。重要的是，这些 止痛药是导致阿片类药物危机的主要原因，随着SARS的爆发，阿片类药物危机进一步恶化。 冠状病毒-2大流行。因此，迫切需要治疗疼痛的替代方法。这一点从 局麻药的疗效，抑制外周痛感觉神经元是治疗疼痛的一种有前途的方法 (伤害性感受器)，介导疼痛的神经传递。我们已经发现，增量阿片受体(DOR)-kappa阿片类物质 受体(KOR)异构体被表达，当被激活时，产生强烈而持久的抗伤害作用。 因此，DOR-KOR异构体可能是开发外周受限的一个有前途的目标 治疗伤害性疼痛的止痛药。在这个应用中，我们提出了一个多方面和高度协作的 鉴定选择性激活DOR-KOR异构体的类药物小分子的方法 痛觉感受器。我们开发选择性激活DOR-KOR异构体的药物的方法是 GPCR异构体的一个独特性质--异构体间变构的优势。蛋白原间变构是 其中异构体对中的一个原构体的邻位立体配体变构地改变了亲和力和/或 正构体配体对另一原体物的内在效力。我们已经证明了这种相互作用 作用于DOR-KOR异构体的配体发生变构作用。此外，我们还鉴定了一种配体，6‘- GNTI，它是伤害感受器中DOR和KOR的拮抗剂，但选择性地通过原构体间变构作用 激活DOR-KOR异构体，产生强烈的抗伤害作用。在这里，我们将利用这一内部- 原变构作用用于鉴定新的异构体选择性化合物。这个项目的具体目标是 使用1)基于结构多样性的化学合成策略来识别新的DOR-KOR异构体配体 2)类药物化合物化学文库的高通量筛选(HTS)；3)基于结构的 电子计算机筛选可用于商业用途的化合物文库。最初将使用以下内容识别命中 HeK细胞表达系统。从体外筛选中出现的潜在候选化合物将经历 ADME和受体选择性筛选。然后将筛选合适的化合物以寻找外周介导的 DOR-KOR异构体在大鼠伤害性感受器中的抗伤害效应。出现的化合物 在这个项目中(我们预计~5种化合物)将使用NIH BPN UG3/UH3进行进一步开发 通过启用IND的毒理学和第一阶段临床测试促进候选人的计划，以实现更安全和 有效治疗疼痛。我们还将获得有价值的结构-活性关系信息 将有助于未来设计DOR、KOR和DOR-KOR异构体的已确定的DOR和KOR配体 配基。这种方法的成功应用(蛋白间变构作用)也可以应用于其他GPCR。 异构体有助于我们进一步了解异构体在生理系统中的作用。

Current clinically prescribed analgesics target mu opioid receptors within the CNS and can produce dangerous and often lethal adverse effects, including respiratory depression and addiction. Importantly, these analgesics are principally responsible for precipitating the opioid crisis, which has only worsened with the SARS- CoV-2 pandemic. Thus, alternative approaches for treatment of pain are badly needed. As evidenced by the efficacy of local anesthetics, a promising approach to treat pain is to inhibit peripheral pain-sensing neurons (nociceptors) that mediate pain neurotransmission. We have found that delta opioid receptor (DOR)-kappa opioid receptor (KOR) heteromers are expressed and when activated produce strong and sustained antinociception. Thus, the DOR-KOR heteromer may be a promising target for development of peripherally-restricted analgesic drugs to treat nociceptive pain. In this application, we propose a multifaceted and highly collaborative approach to identify small, drug-like molecules that selectively activate the DOR-KOR heteromer expressed by nociceptors. Our approach to develop drugs that selectively activate the DOR-KOR heteromer is to take advantage of a unique property of GPCR heteromers – interprotomer allosterism. Interprotomer allosterism is where an orthosteric ligand for one of the protomers in the heteromeric pair allosterically alters the affinity and/or intrinsic efficacy of the orthosteric ligand for the other protomer. We have demonstrated that such interprotomer allosterism occurs for ligands acting at the DOR-KOR heteromer. Moreover, we have identified one ligand, 6’- GNTI, that is an antagonist at both DOR and KOR in nociceptors, but via interprotomer allosterism, selectively activates the DOR-KOR heteromer resulting in strong antinociception. Here we will take advantage of this inter- protomer allosterism to identify novel heteromer-selective compounds. The Specific Aims of this project are to identify novel DOR-KOR heteromer ligands using 1) a synthetic strategy based on structurally diverse chemical series; 2) high-throughput screening (HTS) of chemical libraries of drug-like compounds; and 3) structure-based in-silico screening of libraries of commercially-available chemical compounds. Hits will be initially identified using HEK cell expression systems. Potential candidate compounds that emerge from the in vitro screens will undergo ADME and receptor selectivity screening. Suitable compounds will then be screened for peripherally-mediated antinociceptive efficacy mediated by the DOR-KOR heteromer in nociceptors in rats. Compounds that emerge from this project (we expect ~5 compounds) will undergo further development using the NIH BPN UG3/UH3 program to advance candidates through IND-enabling toxicology and phase 1 clinical testing for safer and effective treatment of pain. We also will have obtained valuable structure-activity relationship information about the identified DOR and KOR ligands that will aid in the future design of DOR, KOR and DOR-KOR heteromer ligands. Successful application of this approach (interprotomer allosterism) could be applied for other GPCR heteromers to help further our understanding of the function of heteromers in physiological systems.