Development of a Cocaine-Metabolizing Enzyme for Drug Overdose Treatment

描述（由申请人提供）：开发一种真正有效的抗可卡因药物非常具有挑战性，特别是用于治疗可卡因过量。目前还没有FDA批准的抗可卡因药物。通过给予丁酰胆碱酯酶（BChE）来促进可卡因代谢已被认为是一种有前途的可卡因滥用治疗策略。然而，这种血浆酶对天然存在的（-）-可卡因的催化活性很低。我们最近的综合计算-实验工作发现了人类BChE的高活性突变体，称为可卡因水解酶（CoHs），与野生型BChE相比，对可卡因的催化效率提高了1,000倍以上。体内实验表明，我们发现的CoCh是开发抗可卡因药物的有希望的候选者，特别是用于过量治疗。在这个拟议的项目中，我们专注于选择和优化最有前途的CocH作为一种新的治疗药物，通过各种计算机模拟，体外和体内方法的组合使用，用于可卡因过量治疗。具体目标是：（2）通过使用目标1中制备的CoOH材料，在体内表征CoOH的效力、生物/循环半衰期和免疫原性;（3）设计、制备和表征不仅具有高体内效力，而且具有更高的热稳定性和更长的循环半衰期而没有免疫原性。完成这项拟议的研究将导致最有前途的CocH实体的鉴定和开发，该实体在体内具有高的保护和拯救作用，高稳定性和足够长的生物半衰期，无免疫原性。在本研究中优化的CocH实体预计作为人类可卡因过量治疗的外源性酶是高度有效和安全的。

DESCRIPTION (provided by applicant): Development of a truly effective anti-cocaine medication has been very challenging, particularly for treatment of cocaine overdose. There is still no FDA-approved anti-cocaine medication. Enhancing cocaine metabolism by administration of butyrylcholinesterase (BChE) has been recognized as a promising treatment strategy for cocaine abuse. However, the catalytic activity of this plasma enzyme is low against the naturally occurring (-) - cocaine. Our recent integrated computational-experimental effort has led to discovery of high-activity mutants of human BChE, known as cocaine hydrolases (CocHs), with >1,000-fold improved catalytic efficiency against cocaine compared to wild-type BChE. In vivo evidences indicate that our discovered CocHs are promising candidates for development of an anti-cocaine medication, especially for the overdose treatment. In this proposed project, we focus on the selection and optimization of the most promising CocH as a novel therapeutic candidate for cocaine overdose treatment through a combined use of various in silico, in vitro, and in vivo approaches. The specific aims are: (1) To prepare and characterize the discovered CocHs in vitro for their catalytic activity and stability; (2) To characterize the CocHs in vivo for their potency, biological/circulatory half-lives, and immunogenicity by using the CocH materials prepared in Aim 1; (3) To design, prepare, and characterize new CocH entities that have not only a high in vivo potency, but also a higher thermal stability and a longer circulatory half-life without immunogenicity. Accomplishment of this proposed investigation will result in the identification and development of the most promising CocH entity that has a high in vivo potency in the protective and rescuing effects, a high stability, and a sufficiently long biological half-life without immunogenicity. The CocH entity optimized in this investigation is expected to be highly effective and safe as an exogenous enzyme for cocaine overdose treatment in humans.