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反可卡因药物一直是非常具有挑战性的，特别是对于可卡因过量的治疗。目前还没有FDA批准的反可卡因药物。应用丁酰胆碱酯酶(BChE)促进可卡因代谢已被认为是治疗可卡因滥用的一种有前途的策略。然而，这种血浆酶对自然产生的(-)-可卡因的催化活性很低。我们最近集成的计算-实验工作导致发现了人BChE的高活性突变，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，我们发现的可卡因是开发抗可卡因药物的有希望的候选者，特别是在过量治疗方面。在这个拟议的项目中，我们专注于通过结合使用各种硅胶、体外和体内方法来选择和优化最有希望的可卡因过量治疗的新候选药物。其具体目标是：(1)体外制备和表征所发现的CocH的催化活性和稳定性；(2)利用Aim 1中制备的CocH材料，对其在体内的效力、生物/循环半衰期和免疫原性进行表征；(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无免疫原性的循环半衰期的新型CocH实体。这项研究的完成将导致最有希望的COCH实体的鉴定和开发，它在体内具有高保护和拯救效果、高稳定性和足够长的无免疫原性的生物半衰期。在这项研究中优化的Coch实体预计将作为一种用于人类可卡因过量治疗的外源性酶高效和安全。

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.