Developing Small-Molecule Probes for Opioid-Induced Glial Activation

描述（由申请人提供）：长期以来，疼痛的药物治疗一直受到阿片类药物副作用的限制：耐受性，依赖性和过量危险的发展。文献已经发展了阿片类药物的副作用与其对中枢神经系统（CNS）内神经胶质细胞的影响。这些效应已被证明是toll样受体4 （TLR4）介导的神经胶质激活的结果，这导致疼痛增强和阿片耐受性和依赖性。因此，迫切需要通过TLR4来理解阿片类药物的失调。目前这项提议的目的是优化从虚拟筛选中确定的小分子探针，这反过来将用于研究胶质细胞激活及其对阿片类药物有效性的影响。本研究的基本原理是优化的小分子TLR4抑制剂可以作为高度特异性的探针来研究阿片诱导的胶质细胞激活的分子机制。这项研究具有重要意义，因为优化的小分子药物将阻碍新疗法的发展，以提高阿片类药物的有效性和安全性。拟议的研究是创新的，因为它是第一个药物发现方法，试图调节阿片类药物诱导的胶质细胞激活。这些研究是建立在一个强大的专业合作团队的基础上的，该团队优化了其机会，有效地将TLR4激活的原子细节与阿片类药物使用的宏观疼痛管理效率低下联系起来。在Aim 1中，将进行广泛的构效关系研究，以优化从硅筛选中鉴定的先导化合物。建立的体外生物物理和细胞分析将用于评估合成的小分子药物在阻断TLR4激活方面的效力。TLR4在配合物中与小分子配体的结晶将在平行努力中尝试阐明TLR4抑制的结构几何。这些结果将揭示配体/受体结合中的分子识别，从而为第一代小分子抑制剂的优化提供见解。Aim 2将验证第二个工作假设，即通过抑制阿片类药物诱导的TLR4激活，也可以阻断胶质细胞的激活，从而增强镇痛作用，减少对阿片类药物的耐受性和依赖性。拟议的研究，如果成功，预计将产生显著的新结果：首先，结果将阐明临床相关阿片类药物诱导的胶质细胞激活的机制。其次，从本研究中发现的TLR4小分子拮抗剂将作为潜在候选药物的原型。这些抑制剂可能在临床治疗阿片类药物副作用方面有用，解决包括阿片类药物成瘾、耐受性和滥用在内的公共卫生问题。

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 danger of overdose. A literature has developed linking opiate side effects to their influence on glial cells within the central nervous system (CNS). These effects have been shown to result from toll-like receptor 4 (TLR4)-mediated glial activation, which causes both pain enhancement and opioid tolerance and dependence. As such, there is an urgent need to understand opioid dysregulation via TLR4. The objective of this current proposal is to optimize a small-molecule probe identified from virtual screening, which in turn will be used to study glial activation and its impact on opioid effectiveness. The rationale underlying this research is that optimized small molecule TLR4 inhibitors can serve as highly specific probes to study the molecular mechanism of opioid-induced glial activation. The proposed research is significant because the optimized small molecule agents will preclude the development of novel therapeutics to increase opiate efficacy and safety. The proposed research is innovative because it is the first drug discovery approach attempting to regulate opioid-induced glial activation. The studies are built on a strong collaborative team with expertise that optimizes its chance to effectively bridge the atomic detail of TLR4 activation with the macroscopic pain management inefficiencies of opioid use. In Aim 1, extensive structure-activity relationship studies will be carried out to optimize the lead compound indentified from in silico screening. Established in vitro biophysical and cellular assays will then be used to evaluate synthesized small molecule agents for their potency in blocking TLR4 activation. Crystallization of TLR4 in complex with the small-molecule ligands will be attempted in a parallel effort to elucidate the structural geometry of TLR4 inhibition. These results will shed light on the molecular recognition in the ligand/receptor association, thereby providing insights for the optimization of the first generation small molecule inhibitors. Aim 2 will test the second working hypothesis, that by inhibiting opioid-induced TLR4 activation, glial activation can also be blocked, thus enhancing analgesia as well as reduce tolerance and dependence on opioids. The proposed studies, if successful, are projected to yield significant novel outcome: First, the results will shed light on the mechanism of clinically relevant opioid-induced glial activation. Second, the small molecule antagonists of TLR4 identified from the proposed research will serve as prototypes for potential drug candidates. These inhibitors may be clinically useful in the treatment of opioid side effects, addressing a public heath issue that encompasses opioid addiction, tolerance, and abusing. 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.