Catalytic activities of a cocaine hydrolase engineered from human butyrylcholinesterase against (+)- and (−)-cocaine

Abstract

It can be argued that an ideal anti-cocaine medication would be one that accelerates cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma. However, wild-type BChE has a low catalytic efficiency against naturally occurring (−)cocaine. Interestingly, wild-type BChE has a much higher catalytic activity against unnatural (+)cocaine. According to available positron emission tomography (PET) imaging analysis using [11C](−)-cocaine and [11C](+)-cocaine tracers in human subjects, only [11C](−)-cocaine was observed in the brain, whereas no significant [11C](+)-cocaine signal was observed in the brain. The available PET data imply that an effective therapeutic enzyme for treatment of cocaine abuse could be an exogenous cocaine-metabolizing enzyme with a catalytic activity against (−)cocaine comparable to that of wild-type BChE against (+)-cocaine. Our recently designed A199S/F227A/S287G/A328W/Y332G mutant of human BChE has a considerably improved catalytic efficiency against (−)-cocaine and has been proven active in vivo. In the present study, we have characterized the catalytic activities of wild-type BChE and the A199S/F227A/S287G/A328W/Y332G mutant against both (+)- and (−)-cocaine at the same time under the same experimental conditions. Based on the obtained kinetic data, the A199S/F227A/S287G/A328W/Y332G mutant has a similarly high catalytic efficiency (kcat/KM) against (+)- and (−)-cocaine, and indeed has a catalytic efficiency (kcat/KM = 1.84×109 M−1 min−1) against (−)-cocaine comparable to that (kcat/KM = 1.37×109 M−1 min−1) of wild-type BChE against (+)-cocaine. Thus, the mutant may be used to effectively prevent (−)-cocaine from entering brain and producing physiological effects in the enzyme-based treatment of cocaine abuse.

Long-Term Blockade of Cocaine Self-Administration and Locomotor Activation in Rats by an Adenoviral Vector–Delivered Cocaine Hydrolase

“…Early work demonstrated that administration of BChE could slightly reduce the physiologic and behavioral effects of cocaine (see review by Gorelick, 1997). More recently, collective efforts to mutate BChE into an efficient cocaine hydrolase (CocH) progressively raised its catalytic efficiency by up to 4400-fold (Sun et al, 2001(Sun et al, , 2002aPan et al, 2005;Gao and Brimijoin, 2006;Zheng et al, 2008Zheng et al, , 2014Xue et al, 2013). A near-optimal enzyme rescued rats from a lethal 100 mg/kg injection of cocaine and blocked cocaine-primed reinstatement, a model of relapse into cocaine-seeking behavior (Brimijoin et al, 2008).…”

Long-Term Reduction of Cocaine Self-Administration in Rats Treated with Adenoviral Vector-Delivered Cocaine Hydrolase: Evidence for Enzymatic Activity

“…This study examined a potentially successful therapy involving long-term gene transfer of an enzyme that rapidly hydrolyzes cocaine to inactive metabolites. The enzyme, a 'cocaine hydrolase' (CocH) derived by multiple mutations of human butyrylcholinesterase (BChE; Zheng et al, 2008;Xue et al, 2013), is expected to reduce drug access to brain. If CocH were sustained at adequate levels, it might aid users who desire to become abstinent.…”

Reaction pathway for cocaine hydrolase-catalyzed hydrolysis of (+)-cocaine

“…To validate the computational data, we carried out experimental kinetic analysis on the CocH-catalyzed hydrolysis of (+)-cocaine; 75 in fact, the computational studies described in this report were actually completed far before the experimental study 75 , but the submission of this report for publication was delayed considerably due to some unexpected reason. The experimental kinetic analysis revealed that catalytic rate constant ( k cat = 8990 min −1 ) 75 for CocH-catalyzed hydrolysis of (+)-cocaine is indeed significantly larger than that for bacterial CocE-catalyzed hydrolysis of (+)-cocaine ( k cat = ~1078 min −1 ) 74 determined in the room temperature.…”

“…31, 32 However, the reaction pathway for CocH-catalyzed hydrolysis of (+)-cocaine remains to be uncovered. In the present study, we have employed the first-principles pseudobond quantum mechanical/molecular mechanical-free energy (QM/MM-FE) 33–36 calculations, which has been demonstrated to be a powerful tool in simulating a variety of enzymes, 25, 37–57 to reveal the reaction pathway for CocH-catalyzed hydrolysis of (+)-cocaine.…”