Probing Opioid-Induced Glial Activation with Peptide Antagonists

描述（由申请人提供）：疼痛的药理学治疗长期以来受到阿片类药物不良副作用的限制：耐受性、依赖性和过量的可能性。有文献将阿片类药物的副作用与其对中枢神经系统（CNS）神经胶质细胞的影响联系起来。本文提出了一个研究阿片类药物介导的胶质细胞活化的作用，通过Toll样受体4（TLR 4）介导的信号通路。TLR 4是一种在神经胶质中表达但在CNS内的神经元中不表达的整合膜受体，其与其辅助蛋白髓样分化蛋白-2（MD-2）复合起作用。TLR 4/MD-2复合物对TLR 4信号转导至关重要。该提案的核心假设是，抑制TLR 4/MD-2的关联将阻碍TLR 4信号通路，从而防止阿片类药物诱导的神经胶质激活的负面副作用。通过选择性阻断TLR 4和MD-2之间的关键蛋白质-蛋白质相互作用，预计阿片耐受性和依赖性将减弱，从而增加当前疼痛药物治疗的功效。这种方法是创新的，因为它是第一个旨在抑制神经胶质介导的阿片类药物副作用的建议。此外，该研究预计将产生重大成果：（1）TLR 4/MD-2相互作用的抑制剂将成为开发药物的原型，以对抗阿片类药物的耐受成瘾和过量的副作用。TLR 4/MD-2相互作用还涉及其他病理学（例如脓毒症），因此将成为各种疾病的潜在靶标。(2)重要的是，TLR 4/MD-2相互作用的拮抗剂将阐明TLR 4途径对阿片诱导的神经胶质活化的贡献。这些抑制剂将有助于确定TLR 4途径本身的作用机制，揭示TLR 4识别其配体的分子特异性。拟议的研究建立在一个强大的合作团队基础上，该团队拥有一系列专业知识，包括蛋白质设计，生物化学和生物物理测定开发，X射线结构分析和疼痛管理动物模型。拟议的研究采用计算设计的肽衍生自MD-2的TLR 4-bidning区域，预计将与全长MD-2蛋白竞争并阻止TLR 4信号转导。这些肽将为小分子抑制剂提供起点。这项工作的意义在于它对临床和科学进步的影响。深入研究阿片类药物诱导胶质细胞活化的机制，将有助于我们了解阿片类药物耐受和成瘾的发展过程，为研究阿片类药物依赖和滥用提供新的视角。关于其对科学进步的影响，拟议的研究将阐明TLR 4激活的分子机制，这与许多相互关联的信号传导和免疫调节途径有关，对理解疼痛抑制至关重要。 公共卫生相关性：这项研究旨在揭示阿片类药物诱导的神经胶质激活机制，这既阻碍了阿片类药物有效控制疼痛的能力，也对药物成瘾和滥用的发展做出了重要贡献。将采用最先进的技术来定义，设计，创建和测试预计可防止阿片类药物诱导的神经胶质激活的新化学实体，从而优化阿片类药物镇痛，同时防止临床阿片类药物使用的负面后果。

DESCRIPTION (provided by applicant): The pharmacological treatment of pain has long been limited by the negative side effects of opioids: development of tolerance, dependence, and possibility of overdose. A literature has developed linking opiate side effects to their influence on glial cells of the central nervous system (CNS). Herein lies a proposal to investigate the role of an opiate-mediated glial activation, via the signaling pathway mediated by toll-like receptor 4 (TLR4). TLR4, an integral membrane receptor expressed in glia but no in neurons within the CNS, functions in complex with its accessory protein, Myeloid Differentiation protein-2 (MD-2). The TLR4/MD-2 complex is crucial to the TLR4-signaling transduction. The proposal's central hypothesis is that inhibition of the TLR4/MD-2 association will impede the TLR4-signaling pathway, thereby preventing the negative side effects from opiate-induced glial activation. By selectively blocking the critical protein-protein interactions between TLR4 and MD-2, opiate tolerance and dependence is predicted to attenuate, thereby increasing the efficacy of current pain pharmacotherapies. This approach is innovative, as it is the first proposal aimed at the inhibition of glial- mediated opioid side effects. Further, the research is expected to yield significant outcomes: (1) inhibitors of the TLR4/MD-2 interaction will be prototypes for the development of drugs to counteract opioid side effects from tolerance to addiction and overdose. The TLR4/MD-2 interaction is also implicated in other pathologies (e.g. sepsis), and will therefore serve potential targets for various diseases. (2) Importantly, antagonists of the TLR4/MD-2 interaction will elucidate the contribution of the TLR4 pathway to opiate-induced glial activation. The inhibitors will help determine the mechanism of action of the TLR4 pathway itself, shedding light on the molecular specificity with which TLR4 recognizes its ligands. The proposed studies are built on a strong collaborative team with a spectrum of expertise covering from protein design, biochemistry and biophysical assay development, x-ray structural analysis, and animal models for pain management. The proposed studies employ computationally designed peptides derived from the TLR4-bidning regions of MD-2, which is expected to compete with the full-length MD-2 protein and prevent the TLR4 signal transduction. These peptides will provide starting points for the small-molecule inhibitors. The significance of this work lies in its impacts on both clinical and scientific advancement. Dissecting the mechanism of opiate-induced glial activation will help us understand the development of opioid tolerance and addiction, as well as establish a novel angle from which to address drug dependence and abusing of these opiates. Regarding its impact on scientific advancement, the proposed studies will illuminate the molecular mechanism of TLR4 activation, which is relevant to a many interrelated signaling and immunomodulatory pathways, and crucial to the understanding of pain suppression. PUBLIC HEALTH RELEVANCE: The proposed research aims to unravel the mechanism of opioid-induced glial activation that both hinders the ability of opioids to effectively control pain and also importantly contributes to the development of drug addiction and abuse. State-of-the-art technologies will be employed to define, design, create, and test new chemical entities predicted to prevent opioid induced glial activation, thereby optimizing opioid analgesia while preventing negative consequences of clinical opioid use.