Long-acting aldicarb hydrolase as a medical countermeasure for aldicarb poisoning

长效涕灭威水解酶作为涕灭威中毒的医学对策

• 批准号: 10724752
• 负责人: CHANG-GUO ZHAN
• 金额: $ 53.3万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10724752
• 关键词: Acetylcholinesterase; Acetylcholinesterase Inhibitors; Acute; Aerosols; Aftercare; Air; Aldicarb; Animal Model; Animals; Atropine; Binding; Biological Assay; Brain; Butyrylcholinesterase; Carbamates; Categories; Cavia; Chemical Warfare; Chemicals; Clinical; Cocaine; Cocaine use disorder; Computer Models; Control Groups; Convulsions; Corn Oil; Data; Development; Dose; Drug Metabolic Detoxication; Engineering; Enzymes; Evaluation; Exposure to; Future; Hour; Human Engineering; Hydrolase; Hydrolysis; Hyperactivity; In Vitro; Intoxication; Investigation; Lead; Lethal Dose 50; Libraries; Literature; Molecular; Mus; Oral; Organophosphorus Compounds; Outcome; Oximes; Paraoxon; Pesticides; Phosphorylation; Poisoning; Polymers; Proteins; Rattus; Reaction; Reporting; Research; Safety; Sarin; Security; Soman; Talc; Testing; Toxic effect; absorption; acute toxicity; aged; bioscavenger; blood-brain barrier crossing; cholinergic; clinical candidate; clinical development; computer studies; cyclosarin; dermal exposure; drug candidate; experience; experimental study; exposure route; immunogenicity; improved; in vivo; intraperitoneal; lead optimization; mass casualty; medical countermeasure; mortality; nerve agent; neurotoxicity; novel; pharmacokinetics and pharmacodynamics; physical property; preclinical development; programs; rational design; success; tabun

As one of the Chemicals of Concern (CoC) identified by the US Department of Homeland Security, aldicarb belongs to the Cholinergic Warfare and Pesticides category. As the most toxic carbamate pesticide – a potent, fast-acting inhibitor of acetylcholinesterase (AChE), aldicarb is readily absorbed from all routes of exposure, including oral and dermal exposure. In all species tested, the acute oral toxicities of aldicarb are similar. Due to its physical properties documented in literature, aldicarb could be used by terrorists to cause mass casualty. Organophosphorus (OP) warfare/pesticides and carbamate pesticides have a common mode of action for their neurotoxicity as AChE inhibitors. Currently available options for treating this type of poisoning, such as administration of atropine or co-administration of atropine and pralidoxime (2-PAM), have limited efficacy. There have been extensive efforts in development of improved options for treatment of OP poisoning. Relatively less studies have been carried out in development of aldicarb poisoning treatment. Reported studies indicated that 2-PAM had neither positive nor negative effects on survival in animal studies on aldicarb intoxication. This outcome is understandable, as 2-PAM was developed to reactivate phosphorylated AChE (an intermediate formed from the inhibition reaction of AChE with an OP), but not carbamylated AChE (an intermediate formed from the reaction of AChE with a carbamate like aldicarb). It is highly desired to develop a new, effective post- exposure treatment option for aldicarb poisoning. This investigation will focus on rational design and discovery of an engineered enzyme capable of rapidly and efficiently detoxifying aldicarb as a catalytic aldicarb bioscavenger for aldicarb poisoning. As an effective bioscavenger, it must be able to react with aldicarb more rapidly than AChE so as to protect AChE from reaction (carbamylation) with aldicarb. In preliminary studies, we have demonstrated that an Fc-fused cocaine hydrolase (Fc-CocH), developed previously in our lab for treatment of cocaine use disorders (CUDs), can more potently bind with aldicarb than with AChE and wild-type butyrylcholinesterase (BChE) and can also catalyze aldicarb hydrolysis. In further in vivo rescue experiment, this Fc-CocH protein powerfully rescued all mice that had been injected intraperitoneally (IP) with a lethal dose (~2 × LD50) of aldicarb. With the encouraging preliminary data, taking advantage of our positive experience in preclinical and clinical development of Fc-CocHs, we propose to first evaluate an in-house library of Fc-CocHs for their activities with aldicarb in order to select the one with the highest catalytic activity for aldicarb hydrolysis and the best overall in vivo profiles, followed by lead optimization to optimize its catalytic activity against aldicarb, substrate selectivity, post-exposure in vivo efficacy, and toxicity/immunogenicity profiles. Accomplishment of this investigation will deliver a couple of safe, highly efficient aldicarb hydrolases that are promising for treatment of aldicarb poisoning. The one with the best overall in vivo profiles will serve as a clinical candidate, and the second best will serve as a backup, for further preclinical and clinical development in the future.

涕灭威作为美国国土安全部确定的“关注化学品”之一， 属于胆碱能战争和杀虫剂类别。作为毒性最大的氨基甲酸酯杀虫剂-一种强效的， 涕灭威是乙酰胆碱酯酶（AChE）的速效抑制剂，很容易通过所有接触途径被吸收， 包括口腔和皮肤接触。在所有测试物种中，涕灭威的急性口服毒性相似。由于 涕灭威的物理特性在文献中有记载，恐怖分子可利用涕灭威造成大规模伤亡。 有机磷（OP）战剂/杀虫剂和氨基甲酸酯杀虫剂具有共同的作用模式， 神经毒性作为乙酰胆碱酯酶抑制剂。目前治疗这类中毒的方法有： 阿托品给药或阿托品和解磷定（2-PAM）联合给药的效果有限。那里 在开发用于治疗OP中毒的改进选择方面已经进行了广泛的努力。相对较少 已开展研究开发涕灭威中毒治疗方法。报告的研究表明， 2-在涕灭威中毒的动物研究中，PAM对存活率既无正面影响，也无负面影响。这 结果是可以理解的，因为开发2-PAM是为了重新激活磷酸化的AChE（中间体 由AChE与OP的抑制反应形成），但不包括氨甲酰化AChE（形成的中间体 乙酰胆碱酯酶与氨基甲酸酯（如涕灭威）的反应。我们迫切希望开发一种新的、有效的后- 涕灭威中毒的接触治疗选择。本次调查将集中在理性设计和发现 一种能够快速有效地使涕灭威解毒的工程酶， 涕灭威中毒的生物清除剂作为一种有效的生物清除剂，它必须能够与涕灭威发生更大的反应 以保护乙酰胆碱酯酶不与涕灭威反应（氨甲酰化）。在初步研究中，我们 我们已经证明，我们实验室以前开发的用于治疗的Fc融合可卡因水解酶（Fc-CocH） 与乙酰胆碱酯酶和野生型相比， 丁酰胆碱酯酶（BChE）也可催化涕灭威水解。在进一步的体内拯救实验中， Fc-CocH蛋白强有力地拯救了所有腹腔注射致死剂量（~ 2.5mg/kg）的小鼠。 × LD50）。有了令人鼓舞的初步数据，利用我们的积极经验， 在临床前和临床开发Fc-CocH的过程中，我们建议首先评估Fc-CocH的内部文库 为了筛选出对涕灭威水解具有最高催化活性的化合物， 以及最佳的总体体内特性，然后进行先导优化以优化其对涕灭威的催化活性， 底物选择性、暴露后体内功效和毒性/免疫原性特征。完成这一 研究将提供一对安全、高效的涕灭威水解酶，有望用于治疗 涕灭威中毒具有最佳整体体内概况的一个将作为临床候选物，而第二个将作为临床候选物。 best将作为未来进一步临床前和临床开发的备份。