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

乙酰胆碱酯酶复合物蛋白质-蛋白质相互作用作为抗有机磷诱导的神经毒性的药物靶点。

• 批准号: 10303546
• 负责人: Jeremy Wayne Chambers
• 金额: $ 22.13万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303546
• 关键词: Acetylcholinesterase; Acetylcholinesterase Inhibitors; Active Sites; Acute; Age; Agrochemicals; Antibodies; Biochemistry; Brain; Cell surface; Cells; Cessation of life; Chemical Weapons; Chlorpyrifos; Cholinesterases; Chronic; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Drug Targeting; Effectiveness; Enzyme Reactivation; Enzyme Stability; Enzymes; Exposure to; Fluorides; Foundations; Future; Goals; Health; Human; Impairment; In Vitro; Individual; Insecticides; Intramuscular Injections; Kinetics; Knowledge; Lead; Literature; Long-Evans Rats; Measures; Methods; Muscle; Muscle Contraction; Muscular Atrophy; Neurologic Effect; Neurons; Occupational; Organophosphates; Outcome; Oximes; Parathion; Pesticides; Pharmacology; Physiology; Protein Kinase; Protein Tyrosine Kinase; Proteins; Publishing; Recovery; Refractory; Research; Research Project Grants; Resistance; Risk; Role; Sarin; Seizures; Serine; Serum; Symptoms; Synapses; Therapeutic; Time; Toxic effect; United States; Ventilatory Depression; aged; base; blood-brain barrier permeabilization; cholinergic; drug development; drug discovery; effectiveness evaluation; enzyme activity; experimental study; fluorophosphate; improved; in vivo; inhibitor/antagonist; innovation; inorganic phosphate; insight; loss of function mutation; member; nerve agent; neuromuscular; neurotoxicity; neurotransmission; novel strategies; prevent; prospective; protein complex; protein degradation; protein protein interaction; subcutaneous; therapeutic target; toxic organophosphate insecticide exposure; translational impact; ubiquitin-protein ligase; weapons

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.

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、 Novichok）是乙酰胆碱酯酶（AChE）的有效抑制剂，可引发胆碱能危机，表现为 肌肉收缩、癫痫发作，极端暴露于特定的有机磷化合物如对硫磷和死亡。 沙林可以迅速（几分钟内）不可逆地抑制乙酰胆碱酯酶（AChE），导致酶“老化”，难以被抑制。 现有的 AChE 重激活剂，即 2-解磷定 (2-PAM)，是美国目前 OP 治疗的一部分 目前重新激活老化乙酰胆碱酯酶的策略存在血脑屏障不足等缺点。 渗透性和体内测试的功效有限，迫切需要识别和完善新颖的药物。 OP 暴露后挽救 AChE 活性的方法，以减轻 OP 的破坏性影响。 拟议研究的长期目标是开发针对急性和反复OP暴露的可行、持久的治疗方法。 在拯救老化的乙酰胆碱酯酶之前，我们的工作重点是生产能够烷基化的新型重激活剂 磷酸化酶使它们能够与 2-PAM 等肟反应以恢复酶功能。 拟议的研究具有创新性，因为它采用了旨在增加老化乙酰胆碱酯酶和 新生细胞内 AChE 的恢复，例如，抑制或丧失肌肉特异性蛋白激酶 (MuSK)。 使 AChE 复合物不稳定，导致酶降解，因此，目前的研究。 目的是评估突触 AChE 复合物中的靶向蛋白是否可以增加 AChE 周转率和 恢复最佳的 AChE 活性 追求这一目标的中心假设是抑制或降低。 与老化 AChE 相关的蛋白质将导致老化酶的释放，为新的 AChE 腾出空间。 重新填充突触，恢复最佳神经传递，并减轻 OP 暴露的影响。 拟议研究的基本原理是消除老化的 AChE 将减轻 OP 的痛苦影响 根据已发表的研究和初步研究，该假设将恢复正常的神经传递。 通过实现以下具体目标进行研究：（1）评估靶向的体外治疗益处 AChE 复合物中的蛋白质进行降解，以及 (2) 检查急性 MuSK 抑制对衰老的影响 AChE 水平和酶活性在第一个目标中，Pz-1（一种已知的 MuSK 抑制剂）在初步研究中显示。 诱导老化 AChE 的更新，将用于治疗暴露于二异丙氟 (DFP) 的 Long-Evans 大鼠， 一种已知可快速老化 AChE 的 OP。 DFP 神经毒性、AChE 水平和胆碱酯酶活性的测量。 回收率将用于确定 Pz-1 功效。在第二个目标中，包含 AChE 的单个蛋白质将被使用。 使用药理学方法选择性地靶向降解，以评估每种物质的贡献 蛋白质对人体细胞中老化的 AChE 的稳定性，因此可以将单个复杂成分评估为药物 这项研究意义重大，因为它将提供基本的机制见解。 突触后 AChE 复合物的稳定性和周转率，并发现治疗 OP 暴露的新靶点。