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Benzoylecgonine (BZE) is the major toxic metabolite of cocaine and is responsible for the long‐term cocaine‐induced toxicity owing to its long residence time in humans. BZE is also the main contaminant following cocaine consumption. Here, we identified the bacterial cocaine esterase (CocE) as a BZE‐metabolizing enzyme (BZEase), which can degrade BZE into biological inactive metabolites (ecgonine and benzoic acid). CocE was redesigned by a reactant‐state‐based enzyme design theory. An encouraging mutant denoted as BZEase2, presented a >400‐fold improved catalytic efficiency against BZE compared with wild‐type (WT) CocE. In vivo, a single dose of BZEase2 (1 mg kg−1, IV) could eliminate nearly all BZE within only two minutes, suggesting the enzyme has the potential for cocaine overdose treatment and BZE elimination in the environment by accelerating BZE clearance. The crystal structure of a designed BZEase was also determined. [ABSTRACT FROM AUTHOR]

Accelerating cocaine metabolism through enzymatic hydrolysis at cocaine benzoyl ester is recognized as a promising therapeutic approach for cocaine abuse treatment. Our more recently designed A199S/F227A/S287G/A328W/Y332G mutant of human BChE, denoted as cocaine hydrolase-3 (CocH3), has a considerably improved catalytic efficiency against cocaine and has been proven active in blocking cocaine-induced toxicity and physiological effects. In the present study, we have further characterized the effects of CocH3 on the detailed metabolic profile of cocaine in rats administrated intravenously (IV) with 5 mg/kg cocaine, demonstrating that IV administration of 0.15 mg/kg CocH3 dramatically changed the metabolic profile of cocaine. Without CocH3 administration, the dominant cocaine-metabolizing pathway in rats was cocaine methyl ester hydrolysis to benzoylecgonine (BZE). With the CocH3 administration, the dominant cocaine-metabolizing pathway in rats became cocaine benzoyl ester hydrolysis to ecgonine methyl ester (EME), and the other two metabolic pathways ( i.e . cocaine methyl ester hydrolysis to BZE and cocaine oxidation to norcocaine) became insignificant. The CocH3-catalyzed cocaine benzoyl ester hydrolysis to EME was so efficient such that the measured maximum blood cocaine concentration (∼38 ng/ml) was significantly lower than the threshold blood cocaine concentration (∼72 ng/ml) required to produce any measurable physiological effects. [ABSTRACT FROM AUTHOR]