理想的可卡因滥用治疗，需要同时去除可卡因本身及其主要有毒代谢物苯甲酰芽子碱（benzoylecgonin，BE）的毒性作用。当前唯一位于临床试验阶段的候选药物RBP-8000，虽然可以快速水解可卡因，但是对BE没有任何催化活性。我们首次证实了人类丁酰胆碱酯酶（butyrylcholinesterase，BChE）是BE的代谢酶，可以催化BE水解成没有生物活性的芽子碱（ecgonine，ECG）和苯甲酸。但是野生BChE对BE的催化活性太低，无法快速的水解BE并除去其毒性。本项目旨在设计高活性的BE水解酶，同时表达各突变体，测定其催化活性和体内的作用能力。具体内容如下：1）利用已建立的计算机辅助设计模型，提高BChE对BE的催化效率；2）表达和纯化所设计的突变体，并测定其体外活性；3）开展体内动物实验，测定BE水解酶的生物半衰期，以及加速体内BE代谢的能力。

An ideal therapy for cocaine abuse should detoxify not only cocaine itself but also its main toxic metabolite benzoylecgonine (BE). RBP-8000, the only drug candidate in clinical trial for cocaine detoxification, is highly efficient against cocaine but inactive against BE. We demonstrated for the first time that human butyrylcholinesterase (BChE) is the metabolic enzyme of BE, catalyzing the hydrolysis of BE into biologically inactive metabolites ecgonine (ECG) and benzoic acid. However, the catalytic efficiency of wild-type BChE against BE is too low to be effective for BE metabolism or detoxification. The currently proposed investigation is focused on the design of highly active BE hydrolases, preparation and testing new enzymes for their catalytic activities and in vivo potencies. The specific aims are: 1) to improve the catalytic efficiency of BChE against BE, using our established computational modeling and simulation; 2) to express, purify, and test the designed mutants for their in vitro activities; 3) to characterize the BE hydrolases for their biological half-lives and in vivo potencies for metabolizing BE.