Development of a Cocaine-Metabolizing Enzyme for Drug Overdose Treatment

描述（由申请人提供）：开发真正有效的抗可卡因药物非常具有挑战性，特别是对可卡因过量的治疗。目前还没有fda批准的抗可卡因药物。通过施用丁基胆碱酯酶（BChE）来增强可卡因代谢已被认为是一种很有前途的治疗可卡因滥用的策略。然而，这种血浆酶对天然存在的(-)-可卡因的催化活性较低。我们最近的综合计算-实验工作导致发现了人类BChE的高活性突变体，称为可卡因水解酶（CocHs），与野生型BChE相比，对可卡因的催化效率提高了1000倍。体内证据表明，我们发现的CocHs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作为可卡因过量治疗的新候选药物，通过多种硅内、体外和体内方法的联合使用。具体目的是：(1)制备并表征所发现的CocHs的体外催化活性和稳定性；(2)利用Aim 1中制备的CocHs材料，在体内表征CocHs的效力、生物/循环半衰期和免疫原性；(3)设计、制备和表征新的CocH实体，这些实体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，没有免疫原性。这项研究的完成将导致鉴定和开发最有前途的CocH实体，该实体具有高体内保护和挽救作用的效力，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实体有望作为一种外源性酶用于人类可卡因过量治疗是非常有效和安全的。

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. PUBLIC HEALTH RELEVANCE: Accelerating cocaine metabolism has been recognized as a promising treatment strategy for anti-cocaine medication. Accomplishment of the proposed investigation will result in the identification of reliable candidates of a novel enzyme therapy for cocaine overdose.