Probing Opioid-Induced Glial Activation with Peptide Antagonists

描述(申请人提供)：疼痛的药物治疗长期以来一直受到阿片类药物的负面副作用的限制：形成耐受性、依赖性和过量服药的可能性。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。在此提出了一项建议，即通过Toll样受体4(TLR4)介导的信号通路，研究阿片剂介导的神经胶质细胞激活的作用。TLR4是一种完整的膜受体，表达于神经胶质细胞，但不表达于中枢神经系统内的神经元，与其附属蛋白髓系分化蛋白-2(MD-2)形成复合体发挥作用。TLR4/MD-2复合体在TLR4信号转导中起着至关重要的作用。该提案的中心假设是，抑制TLR4/MD-2关联将阻碍TLR4信号通路，从而防止阿片剂诱导的胶质细胞激活的负面副作用。通过选择性地阻断TLR4和MD-2之间的关键蛋白质-蛋白质相互作用，阿片类药物的耐受性和依赖性被预测为减轻，从而提高了当前疼痛药物治疗的有效性。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提议。此外，这项研究预计将产生重大成果：(1)TLR4/MD-2相互作用的抑制剂将成为药物开发的原型，以对抗阿片类药物从耐受到成瘾和过量的副作用。TLR4/MD-2的相互作用也与其他病理机制(如败血症)有关，因此将成为各种疾病的潜在靶点。(2)重要的是，TLR4/MD-2相互作用的拮抗剂将阐明TLR4通路在阿片剂诱导的神经胶质细胞激活中的作用。这些抑制剂将有助于确定TLR4途径本身的作用机制，揭示TLR4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的基础上，该团队拥有涵盖蛋白质设计、生物化学和生物物理分析开发、X射线结构分析和疼痛管理动物模型的一系列专业知识。拟议的研究使用了来自MD-2的TLR4结合区域的计算设计的多肽，预计它将与全长MD-2蛋白竞争并阻止TLR4信号转导。这些多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。剖析阿片类药物诱导神经胶质细胞激活的机制将有助于我们了解阿片类药物耐受和成瘾的发展，并为解决阿片类药物的药物依赖和滥用问题提供一个新的视角。关于TLR4对科学进步的影响，建议的研究将阐明TLR4激活的分子机制，这与许多相互关联的信号和免疫调节途径有关，并对理解疼痛抑制至关重要。 公共卫生相关性：拟议的研究旨在揭示阿片类药物诱导的神经胶质激活的机制，该机制既阻碍阿片类药物有效控制疼痛的能力，也重要地促进药物成瘾和滥用的发展。将使用最先进的技术来定义、设计、创建和测试新的化学实体，预计将防止阿片类药物诱导的神经胶质激活，从而优化阿片类药物的镇痛，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.