Exogenous Chemical Probes of TLR-Mediated Neuroinflammation

描述(申请人提供)：经过多年的忽视，神经胶质细胞终于在药物开发商的雷达上注册了。在过去的七年中，有证据表明，神经胶质细胞被阿片类药物(如吗啡、美沙酮、度冷丁和羟考酮)激活，这种阿片诱导的神经胶质反应抑制阿片类药物的镇痛，导致阿片类药物耐受和依赖的形成。一篇文献已将阿片类药物的疗效和副作用与其对中枢神经系统(CNS)内神经胶质细胞的影响联系起来。 Toll样受体4(TLR4)介导的通路。在最近的一份报告中，阴氏实验室提供了第一个直接证据，表明吗啡通过与TLR4辅助蛋白MD-2结合，诱导TLR4/MD-2二聚化，并随后以类似于经典TLR4配体脂多糖(内毒素)的方式激活TLR4信号，从而产生神经炎症效应。此外，吗啡仅通过与MD-2的内毒素结合口袋结合来诱导神经炎症，这表明破坏必要的TLR4/MD-2相互作用足以抑制吗啡诱导的神经炎症。本提案的总体目标是验证TLR4/MD-2复合体作为一个可行的靶点，通过外源性化学探针抑制吗啡诱导的神经炎症。我们的中心假设是，通过阻断TLR4/MD-2复合体的形成，阿片类药物诱导的炎症反应也可以被抑制，从而增强吗啡的止痛作用。这项拟议的研究具有重要意义，因为它有望验证TLR4/MD-2复合体作为改善阿片类药物镇痛效果的新靶点。这项拟议的研究具有创新性，因为它是第一个试图调节阿片类药物诱导的胶质细胞激活的药物发现方法，而之前几乎所有的研究都集中在神经元上。这些研究建立在一个拥有专业知识的强大合作团队的基础上，该团队优化了TLR4激活的原子细节与阿片类药物使用的宏观疼痛管理低效之间的有效桥梁。AIM 1和AIM 2是两种独立的平行方法，旨在获得TLR4/MD-2相互作用的高度特异性抑制剂。我们的目标是开发以前发现的MD-2多肽和T5342126衍生物，作为TLR4/MD-2复合体的有效、选择性抑制剂，并具有所需的PK/PD特性。目的3将验证工作假设，即通过破坏TLR4/MD-2复合体，阿片类药物诱导的神经炎症也可以被阻断，从而增强急性阿片类药物的镇痛作用。来自AIMS 1和AIMS 2的优化抑制剂将在体外使用各种细胞系、体外小胶质细胞以及动物模型进行测试。拟议的研究如果成功，预计将产生重大的新结果：第一，这些结果将进一步了解这种机制 临床上相关的阿片类药物引起的神经炎症。其次，TLR4/MD-2复合体将被验证为开发神经炎症抑制药的可行靶点。第三，从拟议的研究中确定的TLR4途径的装订多肽和小分子拮抗剂将作为潜在候选药物的原型。

DESCRIPTION (provided by applicant): After years of neglect, glial cells are finally registering on drug developers' radar. Evidence has accrued over the last seven years that glia are activated by opioids (e.g. morphine, methadone, meperidine, and oxycodone) and that this opioid-induced glial response suppresses opioid analgesia, resulting in the development of opioid tolerance and dependence. A literature has developed linking opiate efficacy and side effects to their influence on glial cells within the central nervous system (CNS) via the signaling pathway mediated by toll-like receptor 4 (TLR4). In a recent report, the Yin lab has provided the first direct evidence that morphine creates its neuroinflammatory effects by binding to the TLR4 accessory protein, MD-2, and inducing TLR4/MD-2 dimerization and subsequent TLR4 signaling activation in a similar fashion to the classical TLR4 ligand, lipopolysaccharide (LPS; endotoxin). Further, morphine induces neuroinflammation solely through its binding in a specific LPS-binding pocket of MD-2, indicating that disruption of the essential TLR4/MD-2 interactions is sufficient to suppress morphine-induced neuroinflammation. The overall objective of the current proposal is to validate the TLR4/MD-2 complex as a feasible target to suppress morphine-induced neuroinflammation by exogenous chemical probes. Our central hypothesis is that by blocking the formation of the TLR4/MD-2 complex, opioid-induced inflammatory response can also be suppressed and thereby enhance morphine analgesia. The proposed research is significant because it is expected to validate the TLR4/MD-2 complex as a novel target for improving the analgesic efficacy of opioids. The proposed research is innovative because it is the first drug discovery approach attempting to regulate opioid-induced glial activation while almost all previous research focused on neurons. The studies are built on a strong collaborative team with expertise that optimizes its chance to effectively bridge the atomic details of TLR4 activation with the macroscopic pain management inefficiencies of opioid use. Aim 1 and Aim 2 are two independent, parallel approaches that aim to attain highly specific inhibitors of the TLR4/MD-2 interactions. We aim to develop previously identified MD-2 peptides and T5342126 derivatives as potent, selective inhibitors of the TLR4/MD-2 complex with desired PK/PD properties. Aim 3 will test the working hypothesis that by disrupting the TLR4/MD- 2 complex, opioid-induced neuroinflammation can also be blocked, thus enhancing acute opioid analgesia. The optimized inhibitors from Aims 1 and 2 will be tested in vitro using various cell lines, in ex vivo microglia, as well as in animal models. The proposed studies, if successful, are projected to yield significant novel outcome: First, the results will further the understanding of the mechanism of clinically relevant opioid-induced neuroinflammation. Second, the TLR4/MD-2 complex will be validated as a feasible target for developing neuroinflammation suppressant. Third, the stapled peptide and small-molecule antagonists of the TLR4 pathway identified from the proposed research will serve as prototypes for potential drug candidates.