Cocaine Metabolism Accelerated by a Re-Engineered Human Butyrylcholinesterase

Abstract

Plasma butyrylcholinesterase (BChE) is important in the metabolism of cocaine, but natural human BChE has limited therapeutic potential for detoxication because of low catalytic efficiency with cocaine. Here we report pharmacokinetics of cocaine in rats treated with A328W/Y332A BChE, an excellent cocaine hydrolase designed with the aid of molecular modeling. Compared with wild-type BChE, this enzyme hydrolyzes cocaine with 40-fold improved k cat (154 min Ϫ1 versus 4.1 min Ϫ1 ) and only slightly increased K M (18 M versus 4.5 M). In rats given this hydrolase (3 mg/kg i.v.) 10 min before cocaine challenge (6.8 mg/kg i.v.), cocaine half-life was reduced from 52 min to 18 min. Mirroring the reductions of plasma cocaine were large increases in benzoic acid, a product of BChE-mediated cocaine hydrolysis. All other pharmacokinetic parameters confirmed a large, dose-dependent acceleration of cocaine removal by the injected cocaine hydrolase. These results show that A328W/Y332A, an efficient cocaine hydrolase in vivo as well as in vitro, might promote cocaine detoxication in a clinical setting.

A Cocaine Hydrolase Engineered from Human Butyrylcholinesterase Selectively Blocks Cocaine Toxicity and Reinstatement of Drug Seeking in Rats

“…Our research began with molecular dynamics simulation and computer-based ligand docking to guide mutations (A328W/Y332A) that elevated the BChE rate constant for cocaine hydrolysis (k cat ) by a factor of 40 (Sun et al, 2001(Sun et al, , 2002a. The double mutant blocked cocaine-induced locomotor activity in mice, blunted pressor responses in rats, and reduced drug accumulation in brain tissue (Sun et al, 2002b;Gao and Brimijoin, 2004). Further mutations identified by Pancook et al (2003) using random mutagenesis raised k cat by another factor of 5.…”

Rapid and Robust Protection against Cocaine-Induced Lethality in Rats by the Bacterial Cocaine Esterase

“…or i.p. injection of an LD 50 dose of cocaine fails to alter time to peak plasma cocaine concentration or peak plasma concentration of cocaine (Sun et al, 2002), suggesting that this enzyme would fail to protect significantly against cocaine overdose.…”

“…One mutant, A328Y, demonstrated an improved k cat compared with BChe (k cat ϭ 10.2 min Ϫ1 and K M ϭ 9 M) (Xie et al, 1999). Another mutant, A328W/Y332A has been reported to have an increase of approximately 9-fold in catalytic efficiency (k cat /K M ) compared with the wild-type enzyme, with a k cat of 154 min Ϫ1 and K M of 18 M. This enzyme significantly reduced cocaine-half-life and peak cocaine-plasma levels in rats (Sun et al, 2002) and decreased cocaine-induced locomotor activity by 80% in mice (Duysen et al, 2002). In addition, this mutant both blocked and reversed the hypertensive effects of cocaine in rats when given as a pre-or post-treatment, respectively (Gao and Brimijoin, 2004).…”

Medical Defense Against Protein Toxin Weapons

“…Protein engineering of catalytic scavengers has shown increasing promise as a potential medical countermeasure for low-molecular-weight toxins (161,162), and it may become possible to develop catalysts that combine the binding specificity of antitoxins with protease activity to selectively catalyze the hydrolysis or inactivation of protein toxins. Design or development of irreversible protease inhibitors as prophylactic antitoxins also merits increased emphasis.…”