Design, Preparation, and Characterization of High-Activity Mutants of Human Butyrylcholinesterase Specific for Detoxification of Cocaine

Abstract

Cocaine is a widely abused drug without a U.S. Food and Drug Administration-approved medication. There is a recognized, promising anticocaine medication to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway [i.e., cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma]. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against (Ϫ)-cocaine but also certain selectivity for (Ϫ)-cocaine over neurotransmitter acetylcholine (ACh), such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. The present study accounting for the mutation-caused changes of the catalytic activities of BChE against both (Ϫ)-cocaine and ACh by means of molecular modeling and site-directed mutagenesis has led to identification of three BChE mutants that have not only a considerably improved catalytic efficiency against (Ϫ)-cocaine but also the desirable selectivity for (Ϫ)-cocaine over ACh. Two representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg). Pretreatment with the BChE mutant (i.e., 1 min before cocaine administration) dose-dependently protected mice against cocaineinduced convulsions and lethality. In particular, all mice pretreated with the mutant (e.g., 0.02 mg or more of A199S/F227A/S287G/ A328W/E441D BChE) survived. The in vivo data reveal the primary factor (i.e., the relative catalytic efficiency), determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment.

Long-Lasting Effects of a PEGylated Mutant Cocaine Esterase (CocE) on the Reinforcing and Discriminative Stimulus Effects of Cocaine in Rats

“…In an attempt to improve upon BChE's catalytic efficiency, Zhan and colleagues have used site-directed mutagenesis to create mutant BChEs capable of hydrolyzing cocaine B450-2000 times more efficiently than native BChE (Pan et al, 2005;Zheng et al, 2008). These mutant BChEs have shown promising results when evaluated in rodents, effectively reducing the cardiovascular, lethal, and response-reinstating effects of cocaine (Brimijoin et al, 2008;Xue et al, 2010;Zheng et al, 2008). In addition, an albumin-fused mutant BChE (Albu-CocH) has been shown to decrease progressive ratio responding for cocaine (Carroll et al, 2011), suggesting that it is also capable of reducing the reinforcing effectiveness of cocaine.…”

Repeated Administration of a Mutant Cocaine Esterase: Effects on Plasma Cocaine Levels, Cocaine-Induced Cardiovascular Activity, and Immune Responses in Rhesus Monkeys

“…Through a series of site-directed mutagenesis studies, Zhan and colleagues identified mutant BChEs capable of hydrolyzing cocaine approximately 450 to 2000 times faster than native BChE (Pan et al, 2005;Zheng et al, 2008). In rats and mice, these mutant BChEs effectively reduced the cardiovascular, lethal, and abuse-related effects of cocaine (Brimijoin et al, 2008;Zheng et al, 2008;Carroll et al, 2011;Xue et al, 2011), suggesting that such enzymes may provide a viable strategy for treating cocaine toxicity and abuse in humans.…”

Long-acting cocaine hydrolase for addiction therapy

“…A biological t 1/2 of 43-77 h in humans might be adequate for a twice-weekly therapy, depending on the dose of the enzyme used. In addition, our more recently designed and identified CocHs (12)(13)(14) are significantly more active than TV-1380 against (-)-cocaine. Further engineering a more active CocH with a biological t 1/2 longer than that of TV-1380 is highly desired.…”

“…Through structure-and mechanism-based computational modeling and simulation, we have successfully designed and identified human BChE mutants, recognized as true cocaine hydrolases (CocHs) in the literature (9) when they have at least 1,000-fold improved catalytic efficiency against (-)-cocaine compared with wild-type human BChE (11)(12)(13)(14). The first of our designed CocHs, known as "CocH1" (the A199S/S287G/A328W/Y332G mutant of human BChE) (11,15), truncated after amino acid 529, was fused with human serum albumin (HSA) to prolong the biological t 1/2 (9).…”