Acetylcholinesterase Complex Protein-Protein Interactions as Drug Targets Against Organophosphate-induced Neurotoxicity.

项目摘要。 这份行政副刊将向一所暑期高中介绍环境健康科学研究。 实习生。有机磷(OP)杀虫剂(例如NALED、对硫磷)和化学武器(例如沙林、VX、 诺维乔克)是乙酰胆碱酯酶(AChE)的强效抑制剂，可引发肌肉中的胆碱能危机 宫缩，癫痫发作，在极端暴露下死亡。某些行动可能很快(在几分钟内)不可逆转地 抑制乙酰胆碱酯酶，产生一种对肟类复活剂难以产生的“老化”物种，如2-磷定(2-PAM)，它是 美国OP治疗的一部分，其疗效最近受到质疑。因此， 迫切需要找到新的方法来挽救暴露于OP后的AChE活动，以缓解 对神经和肌肉的破坏性影响。拟议研究的长期目标是发展 治疗急性和慢性OP暴露的可行、持久的治疗方法。此前拯救老年疼痛的努力 专注于生产新的再活化剂，使磷酸化的酶烷化，从而允许2-PAM等肟类化合物 以恢复酶功能，但这些化合物存在血脑屏障不足等缺点 体内渗透性差，药效有限。拟议的研究具有创新性，因为它采用了策略 旨在提高陈化AChE的周转率和新生细胞内AChE的恢复。例如，抑制 或肌肉特异性蛋白激酶(Musk)的丢失会破坏AChE复合体的稳定性，从而导致 这种酶。最近的研究表明，Dok7是AChE复合体中的一种接头蛋白，是 麝香的活性，更重要的是，AChE定位在肌肉表面。因此，目前的研究 目的：评估突触AChE复合体中的靶向蛋白是否可以增加AChE的转换率和 恢复最佳的AChE活性。追求这一目标的中心假设是抑制或贬低 与老化AChE相关的蛋白质会导致老化酶的释放，为新的AChE让路 重新填充突触，恢复最佳的神经传递，并减轻OP暴露的影响。这个 提出这项研究的理由是，消除老年疼痛将减轻OP的有害影响 毒性和恢复适当的神经传递。根据已发表的和初步的研究，这位高中实习生 将调查父母资助的特定目标1中的元素：评估靶向的体外治疗益处 AChE复合体中的蛋白质用于降解。在提议的项目中，高中实习生将使用小RNA 降低Dok7的蛋白质水平。同样，学生将使用一种模仿Dok7的复古逆转肽- 麝香相互作用部位破坏蛋白质-蛋白质相互作用的稳定，我们假设这将导致释放 这些研究将确定Dok7的可药性，并确定是否将其作为药物进行评估 发现性研究。这项研究具有重要意义，因为它将为 突触后AChE复合体的稳定性和周转，并发现治疗OP暴露的新靶点。

Project Summary. This administrative supplement will introduce environmental health sciences research to a summer high school intern. Organophosphate (OP) insecticides (e.g., Naled, parathion) and chemical weapons (e.g., sarin, VX, Novichok) are robust inhibitors of acetylcholinesterase (AChE) triggering cholinergic crises resulting in muscle contractions, seizures, and in extreme exposures death. Certain OPs can rapidly (within minutes) irreversibly inhibit AChE, producing an “aged” species refractory to oxime reactivators, like 2-pralidoxime (2-PAM), which is part of the OP treatment in the United States and whose efficacy has recently been questioned. Consequently, there is an urgent need to discover novel approaches to rescue AChE activity following OP exposure to mitigate the damaging neurological and muscular effects. The long-term goal of the proposed research is to develop viable, lasting treatments for acute and chronic OP exposures. Previous efforts to rescue aged AChE have focused on producing new reactivators that will alkylate the phosphorylated enzyme allowing oximes like 2-PAM to recover enzyme function, but these compounds have shortcomings such as insufficient blood-brain barrier permeability and limited efficacy in vivo. The proposed research is innovative because it employs strategies aimed at increasing the turnover aged AChE and recovery of nascent intracellular AChE. For example, inhibiting or loss of muscle-specific protein kinase (MuSK) destabilizes the AChE complex leading to the degradation of the enzyme. Recent studies demonstrate that Dok7, an adaptor protein in the AChE complex, is necessary for MuSK activity and more importantly, AChE localization on the muscle surface. Therefore, the current research objective is to assess whether targeting proteins in the synaptic AChE complex can increase AChE turnover and restore optimal AChE activity. The central hypothesis in pursuit of this objective is that inhibiting or degrading proteins associated with aged AChE will cause the release of the aged enzyme, making way for new AChE to repopulate the synapse, restore optimal neurotransmission, and mitigate the effects of OP exposures. The rationale for the proposed research is that eliminating aged AChE will alleviate the detrimental effects of OP toxicity and restore proper neurotransmission. Based on published and preliminary studies, the high school intern will investigate elements in Specific Aim 1 of the parent grant: Evaluate the in vitro therapeutic benefit of targeting proteins in the AChE complex for degradation. In the proposed project, the high school intern will use small RNAs to diminish the protein levels of Dok7. Similarly, the student will use a retro-inverso peptide mimicking the Dok7- MuSK interaction site to destabilize the protein-protein interaction, which we hypothesize will result in the release of aged AChE These studies will determine the druggability of Dok7 and determine if it will be evaluated as drug discovery studies. This research is significant because it will provide fundamental mechanistic insights into the stability and turnover of post-synaptic AChE complexes and unearth new targets to treat OP exposures.