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

作为美国国土安全部确定的值得关注的化学品(CoC)之一，敌百威 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--一种有效的， 速效乙酰胆碱酯酶(AChE)抑制剂--敌百威很容易从所有暴露途径中被吸收， 包括口腔和皮肤接触。在所有被测试的物种中，敌百威的急性口服毒性是相似的。由于 根据文献记载，敌百威的物理特性可能会被恐怖分子用来造成大规模伤亡。 有机磷(OP)战争/杀虫剂和氨基甲酸酯类杀虫剂有一种共同的行动模式， 作为AChE抑制剂的神经毒性。目前可用于治疗这种类型的中毒的选择，例如 服用阿托品或联合服用阿托品和解磷定(2-PAM)，疗效有限。那里 在开发治疗OP中毒的改进方案方面做出了广泛的努力。相对较少 目前已开展了关于敌敌畏中毒治疗方法的研究。报道的研究表明， 2-PAM对敌百威中毒动物的存活无积极或消极影响。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(一种中间体 由AChE与OP的抑制反应形成的)，但不是氨基甲酰化的AChE(形成的中间体 由于AChE与一种类似敌百威的氨基甲酸酯的反应)。人们迫切希望开发出一种新的、有效的后处理方法。 敌百威中毒的暴露治疗选择。这项调查将集中于合理的设计和发现 一种工程酶，能够快速、有效地将敌百威解毒，作为一种催化杀虫威 敌百威中毒的生物清洁剂。作为一种有效的生物清除剂，它必须能够与敌百威发生更多的反应 比AChE更快，以保护AChE不与敌百威反应(氨甲酰化)。在初步研究中，我们 已经证明了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) 在可卡因使用障碍(CUD)中，与敌百威的结合比与AChE和野生型的结合更有效 丁酰胆碱酯酶(BChE)，也能催化敌百威的水解酶。在进一步的活体救援实验中，这 Fc-Coch蛋白有效地挽救了所有已注射致死剂量(~2)的小鼠 ×LD50)。凭借令人鼓舞的初步数据，利用我们在 FC-CocHs的临床前和临床发展，我们建议首先评估FC-CocHs的内部文库 以选择具有最高催化活性的一种化合物 和最好的整体体内分布，其次是先导优化，以优化其对敌百威的催化活性， 底物选择性、体内暴露后的效力和毒性/免疫原性特征。实现这一点 研究将提供两种安全、高效的敌百威水解酶，有望用于治疗 敌百威中毒。体内整体情况最好的一个将作为临床候选，第二个 百思买将作为后备，为未来进一步的临床前和临床开发提供支持。

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.