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

项目摘要。 有机磷(OP)杀虫剂(例如毒死蜱、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)的有效抑制剂，可引发胆碱能危象 肌肉收缩，癫痫发作，在极端暴露下死亡。特殊的OP化合物，如对硫磷和 沙林可以迅速地(在几分钟内)不可逆转地抑制AChE，导致一种难以抵抗的“老化”酶 现有的AChE再激活剂，即2-磷定(2-PAM)，这是美国目前OP治疗的一部分 各州。目前重新激活老年AChE的策略存在缺陷，如血脑屏障不足 体内渗透性差，药效有限。因此，迫切需要识别和提炼小说 抢救OP暴露后AChE活性的方法以减轻OP的破坏性影响。长的- 拟议研究的长期目标是为急性和重复的OP暴露开发可行的、持久的治疗方法。 此前抢救老年AChE的努力主要集中在生产新类型的可烷化的复活剂 这种磷酸化的酶使它们能够与2-PAM等肟类化合物反应，恢复酶功能。这个 拟议的研究具有创新性，因为它采用了旨在增加老年人流动率和 新生细胞内AChE的恢复。例如，抑制或丢失肌肉特异性蛋白激酶(麝香) 破坏AChE复合体的稳定性，导致酶的降解。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。基于已发表的和初步的研究，这一假设将 通过开展以下具体目标进行研究：(1)评估靶向的体外治疗益处 AChE复合体中蛋白质的降解，以及(2)检测急性麝香抑制对老年人的影响 AChE水平和酶活性。在第一个目标中，初步研究表明，已知的麝香抑制剂PZ-1 为了诱导老年AChE的周转，将用于治疗暴露于二异丙基氟(DFP)的Long-Evans大鼠， 一种众所周知的衰老手术会很快引起疼痛。DFP神经毒性、乙酰胆碱酯酶水平和胆碱酯酶活性的测定 恢复率将用于确定PZ-1的疗效。在第二个目标中，组成AChE的个别蛋白质将 使用药理学方法选择性地作为降解物的目标，以评估每种药物的贡献 蛋白质对人体细胞衰老AChE稳定性的影响，因此单个复杂成分可作为药物进行评价 在未来研究中的目标。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. Organophosphate (OP) insecticides (e.g., chlorpyrifos, Naled, parathion) and chemical weapons (e.g., sarin, VX, Novichok) are potent inhibitors of acetylcholinesterase (AChE) that can trigger cholinergic crises presenting as muscle contractions, seizures, and in extreme exposures death. Particular OP compounds like parathion and sarin can rapidly (within minutes) irreversibly inhibit AChE, resulting in an “aged” enzyme that is refractory to existing AChE reactivators, namely 2-pralidoxime (2-PAM), which is part of the current OP treatment in the United States. Current strategies to reactivate aged AChE have shortcomings such as insufficient blood-brain barrier permeability and limited efficacy in vivo. Consequently, there is an urgent need to identify and refine novel approaches to rescue AChE activity following OP exposure to mitigate the damaging effects of OPs. The long- term goal of the proposed research is to develop viable, lasting treatments for acute and repeated OP exposures. Previous efforts to rescue aged AChE have focused on producing new classes of reactivators that will alkylate the phosphorylated enzyme allowing them to react with oximes like 2-PAM to recover enzyme function. 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. 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 hypothesis will be investigated by undertaking the following specific aims: (1) Evaluate the in vitro therapeutic benefit of targeting proteins in the AChE complex for degradation, and (2) Examine the impact of acute MuSK inhibition on aged AChE levels and enzyme activity. In the first aim, Pz-1, a known inhibitor of MuSK shown in preliminary studies to induce turnover of aged AChE, will be used to treat Long-Evans rats exposed to diisopropyl fluoride (DFP), an OP known to age AChE quickly. Measures of DFP neurotoxicity, AChE levels, and cholinesterase activity recovery will be used to determine Pz-1 efficacy. In the second aim, individual proteins comprising the AChE will be selectively targeted for degradation using pharmacological approaches to assess the contribution of each protein to aged AChE stability in human cells, so that individual complex components may be evaluated as drug targets in future 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.