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

项目摘要。 这个行政补充将介绍环境健康科学的研究，以夏季高中 intern.有机磷酸酯（OP）杀虫剂（例如，Naled，Naldion）和化学武器（例如，沙林毒气VX毒气 Novichok）是乙酰胆碱酯酶（AChE）的强效抑制剂，可触发胆碱能危机，导致肌肉 宫缩癫痫在极端暴露下死亡某些OP可以迅速（在几分钟内）不可逆地 抑制乙酰胆碱酯酶，产生一种对肟类再活化剂（如2-解磷定（2-PAM））不敏感的"老化"物质， 这是美国OP治疗的一部分，其疗效最近受到质疑。因此，委员会认为， 迫切需要发现新的方法来拯救OP暴露后的AChE活性， 对神经和肌肉的破坏性影响拟议研究的长期目标是开发 急性和慢性OP暴露的可行，持久的治疗。以前拯救老年乙酰胆碱酯酶的努力 专注于生产新的再活化剂，将烷基化的磷酸化酶，允许肟，如2-PAM 恢复酶功能，但这些化合物存在血脑屏障不足等缺点 渗透性和有限的体内功效。拟议的研究是创新的，因为它采用了战略 旨在增加老化AChE的周转和新生细胞内AChE的恢复。例如抑制 肌肉特异性蛋白激酶（MuSK）的缺失使AChE复合物不稳定，导致 酶最近的研究表明，Dok7是乙酰胆碱酯酶复合物中的衔接蛋白， MuSK活性，更重要的是，AChE在肌肉表面的定位。因此，目前的研究 目的是评估突触乙酰胆碱酯酶复合物中的靶向蛋白是否可以增加乙酰胆碱酯酶的周转， 恢复最佳AChE活性。追求这一目标的核心假设是， 与老化的乙酰胆碱酯酶相关的蛋白质将导致老化酶的释放，为新的乙酰胆碱酯酶让路， 重新填充突触，恢复最佳神经传递，并减轻OP暴露的影响。的 提出这项研究的理由是，消除老化的乙酰胆碱酯酶将减轻OP的有害影响 毒性和恢复正常的神经传递。根据已发表的和初步的研究，高中实习生 将研究母基金具体目标1中的要素：评估靶向的体外治疗益处 AChE复合物中的蛋白质进行降解。在提议的项目中，高中实习生将使用小RNA 来降低Dok7的蛋白水平类似地，学生将使用一种模仿Dok7的反向肽， MuSK相互作用位点，使蛋白质-蛋白质相互作用不稳定，我们假设这将导致释放 这些研究将确定Dok7的可药用性，并确定它是否将作为药物进行评估。 发现研究这项研究是重要的，因为它将提供基本的机械见解， 突触后乙酰胆碱酯酶复合物的稳定性和营业额，并发现新的目标，以治疗OP的曝光。

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.