Activities of the enantiomers of cocaine and some related compounds as substrates and inhibitors of plasma butyrylcholinesterase

doi:10.1016/0006-2952(91)90665-R

Biochemical Pharmacology

Volume 41, Issue 8, 15 April 1991, Pages 1249-1254

https://doi.org/10.1016/0006-2952(91)90665-R Get rights and content

Abstract

The behaviors of the enantiomers of cocaine (benzoylecgonine methyl ester) and related compounds with butyrylcholinesterase (BChE; EC 3.1.1.8) were investigated spectrophotometrically at 235 nm. The unnatural enantiomer, (+)-cocaine, was hydrolyzed by BChE (extinction coefficient 6.7 L·mmol⁻¹·cm⁻¹) at about half the rate of benzoylcholine, but over 2000 times faster than naturally occurring (−)-cocaine. This rapid hydrolysis of (+)-cocaine may account, in part, for its pharmacological inactivity. (+)-Norcocaine, (+)-benzoylecgonine, (−)-Ω-cocaine and tropacocaine were also substrates for BChE. Hydrolysis of (+)-cocaine was sensitive to several standard inhibitors of BChE, including those of competitive, carbamate and organophosphorus classes. Although (−)-cocaine was a poor substrate for debenzoylation, it was fairly good competitive inhibitor (K_i∼10μM) of the hydrolysis of other substrates. The cocaine metabolites (−)-norcocaine, (−)-benzoylecgonine and (−)-ecgonine methyl ester inhibited BChE with K_i values of 15, 76 and 1300 μM, respectively. (+)-Ω-Cocaine had K_i = 3 μM. p-Nitro and p-fluoro derivatives of cocaine and analogs with phenyl and p-fluorophenyl groups in place of the benzoyl ester linkage (WIN 35,065-2 and WIN 35,428) inhibited BChE comparably to (−)-cocaine itself. Both cocaine enantiomers were weak inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7) from human erythrocytes with similar K_i values (160–170 μM). Although it is unlikely that the inhibition of BChE is an important factor in the subjective effects of cocaine, it may have implications for the toxicity of cocaine to the fetus, since BChE appears in the development of the central nervous system before AChE, and has been suggested to function as an embryonic acetylcholinesterase.

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The (+)-enantiomer is quickly cleared from plasma (within seconds) before reaching the central nervous system (CNS), [11] while the active (-)-form remains in circulation for more than 45 minutes. This time-span is sufficient for the latter to reach the CNS, which has a maximum cocaine response profile within a minutes time frame. [12] Recently, this enzyme was been reengineered to perform (-)-cocaine detoxification, and its catalytic efficiency was increased more than 2000-fold. [13,14]
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A majority of cocaine users also consume alcohol. The concurrent use of cocaine and alcohol produces the pharmacologically active metabolites cocaethylene and norcocaethylene, in addition to norcocaine. Both cocaethylene and norcocaethylene are more toxic than cocaine itself. Hence, a truly valuable cocaine-metabolizing enzyme for cocaine abuse/overdose treatment should be effective for the hydrolysis of not only cocaine, but also its metabolites norcocaine, cocaethylene, and norcocaethylene. However, there has been no report on enzymes capable of hydrolyzing norcocaethylene (the most toxic metabolite of cocaine). The catalytic efficiency parameters (k_cat and K_M) of human butyrylcholinesterase (BChE) and two mutants (known as cocaine hydrolases E14-3 and E12-7) against norcocaethylene have been characterized in the present study for the first time, and they are compared with those against cocaine. According to the obtained kinetic data, wild-type human BChE showed a similar catalytic efficiency against norcocaethylene (k_cat = 9.5 min⁻¹, K_M = 11.7 μM, and k_cat/K_M = 8.12 × 10⁵ M⁻¹ min⁻¹) to that against (−)-cocaine (k_cat = 4.1 min⁻¹, K_M = 4.5 μM, and k_cat/K_M = 9.1 × 10⁵ M⁻¹ min⁻¹). E14-3 and E12-7 showed an improved catalytic activity against norcocaethylene compared to wild-type BChE. E12-7 showed a 39-fold improved catalytic efficiency against norcocaethylene (k_cat = 210 min⁻¹, K_M = 6.6 μM, and k_cat/K_M = 3.18 × 10⁷ M⁻¹ min⁻¹). It has been demonstrated that E12-7 as an exogenous enzyme can efficiently metabolize norcocaethylene in rats.
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Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) are related enzymes found across the animal kingdom. The critical role of acetylcholinesterase in neurotransmission has been known for almost a century, but a physiological role for butyrylcholinesterase is just now emerging. The cholinesterases have been deliberately targeted for both therapy and toxicity, with cholinesterase inhibitors being used in the clinic for a variety of disorders and conversely for their toxic potential as pesticides and chemical weapons. Non-catalytic functions of the cholinesterases (ChEs) participate in both neurodevelopment and disease. Manipulating either the catalytic activities or the structure of these enzymes can potentially shift the balance between beneficial and adverse effect in a wide number of physiological processes.
Actions of Butyrylcholinesterase Against Cocaine
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Accelerating cocaine metabolism producing physiologically and biologically inactive metabolites is recognized as an ideal approach to therapeutic treatment of cocaine overdose and addiction. Butyrylcholinesterase (BChE) in plasma is the principal cocaine-metabolizing enzyme in humans. Unfortunately, catalytic efficiency of wild-type BChE against naturally occurring, biologically active (–)-cocaine is too low to be effective. Unique computational enzyme redesign approaches have been developed and employed to rationally design and discover a variety of BChE mutants, known as cocaine hydrolases (CocHs), with at least 1000-fold improved catalytic efficiency compared to the wild-type BChE against (–)-cocaine. Preclinical studies revealed that these CocHs are promising for treatment of cocaine overdose and addiction. The first CocH (i.e., CocH1) is currently under clinical trial phase II for cocaine addiction treatment. Compared to CocH1, more recently designed and discovered CocHs have not only significantly higher catalytic efficiency against (–)-cocaine and its toxic metabolites, but also significantly longer biological half-lives.
Catalytic activities of a cocaine hydrolase engineered from human butyrylcholinesterase against (+)- and (-)-cocaine
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Citation Excerpt :
The kinetic parameters summarized in Table 1 reveal that the A199S/F227A/S287G/A328 W/Y332G mutant of human BChE 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. Due to the similar catalytic efficiency of the A199S/F227A/S287G/A328 W/Y332G mutant against (−)-cocaine and wild-type BChE against (+)-cocaine, and in light of the aforementioned PET data with (+)-cocaine in literature [14,17,18], one can reasonably hypothesize that the A199S/F227A/S287G/A328 W/Y332G mutant may be used to effectively prevent (−)-cocaine from entering brain and producing physiological effects. In particular, if one could repeat the PET imaging analysis on (−)-cocaine pharmacokinetics using the A199S/F227A/S287G/A328 W/Y332G mutant as an exogenous enzyme with a plasma concentration being the same as the endogenous BChE, the time course of the brain concentration of (−)-cocaine would be comparable to that of the brain concentration of (+)-cocaine without the exogenous enzyme (with the endogenous BChE only).
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/A328 W/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/A328 W/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/A328 W/Y332G mutant has a similarly high catalytic efficiency (k_cat/K_M) against (+)- and (−)-cocaine, and indeed has a catalytic efficiency (k_cat/K_M = 1.84 × 10⁹ M⁻¹ min⁻¹) against (−)-cocaine comparable to that (k_cat/K_M = 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.
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2010, Chemico-Biological Interactions
Cocaine addiction and overdose are a well-known public health problem. There is no approved medication available for cocaine abuse treatment. Our recently designed and discovered high-activity mutant (A199S/S287G/A328W/Y332G) of human butyrylcholinesterase (BChE) has been recognized to be worth exploring for clinical application in humans as a potential anti-cocaine medication. The catalytic rate constant (k_cat) and Michaelis–Menten constant (K_M) for (−)-cocaine hydrolysis catalyzed by A199S/S287G/A328W/Y332G BChE (without fusion with any other peptide) have been determined to be 3060 min⁻¹ and 3.1 μM, respectively, in the present study. The determined kinetic parameters reveal that the un-fused A199S/S287G/A328W/Y332G mutant has a ∼1080-fold improved catalytic efficiency (k_cat/K_M) against (−)-cocaine compared to the wild-type BChE. The ∼1080-fold improvement in the catalytic efficiency of the un-fused A199S/S287G/A328W/Y332G mutant is very close to the previously reported the ∼1000-fold improvement in the catalytic efficiency of the A199S/S287G/A328W/Y332G mutant fused with human serum albumin. These results suggest that the albumin fusion did not significantly change the catalytic efficiency of the BChE mutant while extending the plasma half-life. In addition, we have also examined the catalytic activities of the A199S/S287G/A328W/Y332G mutant against two other substrates, acetylthiocholine (ATC) and butyrylthiocholine (BTC). It has been shown that the A199S/S287G/A328W/Y332G mutations actually decreased the catalytic efficiencies of BChE against ATC and BTC, while considerably improving the catalytic efficiency of BChE against (−)-cocaine.

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Activities of the enantiomers of cocaine and some related compounds as substrates and inhibitors of plasma butyrylcholinesterase

Abstract

Trends Pharmacol Sci

Eur J Pharmacol

Neuropharmacology

Biochem Pharmacol

Biochem Pharmacol

Clin Chim Acta

Arch Biochem Biophys

Clin Chim Acta

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Clin Biochem

Pharmacol Biochem Behav

The pharmacology of cocaine related to its abuse

Pharmacol Rev

Cellular and molecular mechanisms of drug dependence

Science

Sodium sensitive cocaine binding to rat striatal membrane: Possible relationship to dopamine uptake sites

J. Neurochem

Cocaine receptors on dopamine transporters are related to self administration of cocaine

Science

Cocaine inhibits muscarinic cholinergic receptors in heart and brain

J Pharmacol Exp Ther

Local anesthetics

Konfiguration des Cocains und Derivate der Ecgoninsaure

Helv Chim Acta

Stereoselective effects of cocaine and a phenyltropane analog

J Pharmacol Exp Ther

Dopamine transporter: Solubilization from dog caudate nucleus

Synapse

Mapping cocaine binding sites in human and baboon brain in vivo

Synapse

Rapid stereoselective hydrolysis of (+)-cocaine in baboon plasma prevents its uptake in the brain: Implications for behavioral studies

J Neurochem

Hydrolysis of cocaine in human plasma by cholinesterase

Life Sci

Cocaine metabolism: Cocaine and norcocaine hydrolysis by liver and serum esterases

Clin Pharmacol Ther