Transforming Clinical Pain Control by Targeting a Novel Non-Neuronal Receptor

描述(申请人提供)：阿片类药物诱导的胶质细胞激活，损害了疼痛治疗，并导致药物成瘾和滥用的发展，通过Toll样受体-4(TLR4)下游的信号通路进行调节，TLR4是一种跨膜受体，与其辅助蛋白MD-2形成复合体发挥功能。由于目前的阿片类药物治疗未能在控制疼痛的同时避免负面后果，迫切需要了解通过TLR4实现的阿片类药物失调。目前该方案的中心假设是，阻断TLR-4/MD-2复合体的形成可以抑制阿片类药物诱导的胶质细胞激活，从而增强镇痛作用，减少阿片类药物的耐受和依赖。这项研究的基本原理是，已识别的TLR4抑制剂选择性地阻断TLR4和MD-2之间的关键蛋白质-蛋白质相互作用，将为研究TLR4介导的信号通路在胶质细胞激活中的作用提供有用的工具。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。拟议的高风险/高回报办法如果成功，预计将产生重大的新成果。首先，这一结果将有助于阐明临床上相关的阿片类药物诱导胶质细胞激活的机制。其次，如果成功，在拟议的研究中确定的TLR4/MD-2相互作用的多肽和模拟多肽的拮抗剂可以作为更多类药物小分子抑制剂的原型。这些抑制剂最终可能应用于新疗法的开发，以提高阿片类止痛药的临床疗效，治疗阿片成瘾和滥用，以及其他临床相关适应症。拟议的研究建立在PI和Co-PI在设计、合成和评价新型蛋白质相互作用抑制剂方面的互补强度的基础上，Co-PI在神经胶质生物学方面拥有广泛的专业知识，并将在已确定的抑制剂的动物试验中提供支持。在目标1中，将开发阻断TLR4/MD-2复合体形成的TLR4拮抗剂。这里的工作假设是，来自MD-2的TLR4结合区的构象紧张肽可以与全长的MD-2蛋白竞争，从而抑制TLR4/MD-2的相互作用。在目标2中，将测试第二个工作假设，即TLR4/MD-2相互作用的抑制剂可以非竞争性地拮抗阿片类药物以阻断TLR4介导的神经胶质激活。细胞分析和动物模型将被用来评估TLR4拮抗剂在体外和体内对胶质细胞激活的抑制作用。这项拟议的研究具有重要意义，因为它有望建立TLR4/MD-2蛋白质-蛋白质复合体作为预防和治疗阿片类药物滥用的新治疗靶点。关于它对科学进步的积极影响，这项工作将(1)提高对药物依赖和疼痛抑制的科学理解，(2)允许开发新一代疗法。公共卫生相关性：我们的目标是揭开阿片类药物诱导的神经胶质细胞激活的机制，这对药物成瘾和滥用的发展起重要作用。作为拟议研究的结果，我们期望更好地了解阿片类药物诱导神经胶质激活的分子机制，并阐明调节这种激活的新靶点。这项拟议工作的学术价值不仅在于它在化学生物学和蛋白质工程领域的科学进步，而且还在于它对临床应用的潜在影响。这项工作将(1)提高对药物依赖和疼痛抑制的科学理解；(2)允许开发新一代疗法。

DESCRIPTION (provided by applicant): Opioid-induced glial activation, which compromises pain treatment and contributes to the development of drug addiction and abuse, is regulated via a signaling pathway downstream of toll-like receptor-4 (TLR4), a membrane spanning receptor that functions in complex with its accessory protein MD-2. As current opioid pharmacotherapeutics have failed to control pain while avoiding the negative consequences, there is an urgent need to understand opioid dysregulation via TLR4. The central hypothesis of the current proposal is that disruption of the TLR-4/MD-2 complex formation can inhibit opioid-induced glial activation, thereby enhancing analgesia and reducing opioid tolerance and dependence. The rationale underlying the proposed research is that the identified TLR4 inhibitors, which selectively block the critical protein-protein interactions between TLR4 and MD-2, will provide a useful tool for investigating the role of the TLR4-mediated signaling pathway in glial activation. The proposed research is innovative because it is the first drug discovery approach attempting to regulate opioid-induced glial activation. The proposed high risk/high reward approach, if successful, is projected to yield significant novel outcomes. First, the results will shed light on the mechanism of the clinically relevant opioid-induced glial activation. Second, if successful, the peptide and peptidomimetic antagonists of the TLR4/MD-2 interactions identified in the proposed research can serve as prototypes for more drug-like small-molecule inhibitors. These inhibitors may eventually find application in the development of novel therapeutics to enhance the clinical efficacy of opioid analgesics and to treat opioid addiction and abuse, as well as other clinically relevant indications. The proposed studies are built on the complementary strength of the PI in the design, synthesis, and evaluation of novel protein-protein interaction inhibitors, and of the Co-PI, who has extensive expertise in glial neurobiology and will provide support in animal testing of the identified inhibitors. In Aim 1, antagonists of TLR4 that block the TLR4/MD-2 complex formation will be developed. The working hypothesis here is that conformationally strained peptides derived from the TLR4-binding region of MD-2 can compete with the full-length MD-2 protein and thereby inhibit the TLR4/MD-2 interaction. In Aim 2, the second working hypothesis, that the inhibitors of the TLR4/MD-2 interactions can non-competitively antagonize opioids to block TLR4-mediated glial activation, will be tested. Cellular assays and animal models will be used to evaluate the inhibition of glial activation by the TLR4 antagonists both in vitro and in vivo. The proposed research is significant because it is expected to establish the TLR4/MD-2 protein-protein complex as a novel therapeutic target for preventing and treating opioid abuse. Regarding its positive impact on scientific advancements, this work will (1) improve scientific understanding of drug dependence and pain suppression and (2) allow the development of a new generation of therapeutics. PUBLIC HEALTH RELEVANCE: We aim to unravel the mechanism of opioid-induced glial activation that importantly contributes to the development of drug addiction and abuse. As an outcome of the proposed investigations, we expect to attain a better understanding of the molecular mechanisms of opioid-induced glial activation, and to clarify novel targets to regulate such activation. The intellectual merit of the proposed work lies not only in its scientific advancements in the field of chemical biology and protein engineering, but also in its potential impact on clinical applications. This work will (1) improve scientific understanding of drug dependence and pain suppression; and (2) allow the development of a new generation of therapeutics.