Inhibitor Report for: Cocaine

Cocaine is a widely abused and addictive drug without an FDA-approved medication. Our recently designed and discovered cocaine hydrolase, particularly E12-7 engineered from human butyrylcholinesterase (BChE), has the promise of becoming a valuable cocaine abuse treatment. An ideal anti-cocaine therapeutic enzyme should have not only a high catalytic efficiency against cocaine, but also a sufficiently long biological half-life. However, recombinant human BChE and the known BChE mutants have a much shorter biological half-life compared to the native human BChE. The present study aimed to extend the biological half-life of the cocaine hydrolase without changing its high catalytic activity against cocaine. Our strategy was to design possible amino-acid mutations that can introduce cross-subunit disulfide bond(s) and, thus, change the distribution of the oligomeric forms and extend the biological half-life. Three new BChE mutants (E364-532, E377-516, and E535) were predicted to have a more stable dimer structure with the desirable cross-subunit disulfide bond(s) and, therefore, a different distribution of the oligomeric forms and a prolonged biological half-life. The rational design was followed by experimental tests in vitro and in vivo, confirming that the rationally designed new BChE mutants, i.e. E364-532, E377-516, and E535, indeed had a remarkably different distribution of the oligomeric forms and prolonged biological half-life in rats from approximately 7 to approximately 13h without significantly changing the catalytic activity against (-)-cocaine. This is the first demonstration that rationally designed amino-acid mutations can significantly prolong the biological half-life of a high-activity enzyme without significantly changing the catalytic activity.

The hydrolysis of cocaine (benzoylecgonine methyl ester) to ecgonine methyl ester by human butyrylcholinesterase (BCHE; EC 3.1.1.8) has been shown previously to constitute an important means to detoxicate this material to pharmacologically inactive metabolites. The naturally occurring (-)-cocaine is hydrolyzed to ecgonine methyl ester approximately 2000 times slower than the unnatural (+)-cocaine isomer. In good agreement with previous studies, (-)-cocaine bound to human BCHE with relatively good affinity and competitively inhibited the hydrolysis of the spectrophotometric substrate butyrylthiocholine with a Ki value of 8.0 microM. Similarly, (+)-cocaine also showed relatively high affinity for the human BCHE and competitively inhibited butyrylthiocholine hydrolysis with a Ki value of 5.4 microM. The phosphonothiolates corresponding to the transition state analogs for both (-)- and (+)-cocaine hydrolysis were synthesized and tested as inhibitors of human BCHE-catalyzed hydrolysis of butyrylthiocholine. The phosphonothiolate corresponding to the transition state for (-)-cocaine hydrolysis was a competitive inhibitor with a Ki value of 55.8 microM. The phosphonothiolate corresponding to the transition state for (+)-cocaine hydrolysis gave a Ki value of 25.9 microM, but, in addition, it also showed irreversible inhibition with a ki of inactivation of 68.8 min-1 M-1. It is likely that the mechanism-based inhibitor described herein may find use as a mechanistic probe of butyrylcholinesterase action and also possibly aid in the purification of this class of esterases.

Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A199S/S227A/S287G/A328W/Y332G mutant) have been characterized for their catalytic activities against cocaine, i.e. naturally occurring (-)-cocaine, in comparison with the corresponding human BChE (hBChE) and an hBChE-based cocaine hydrolase (hCocH, i.e. the A199S/F227A/S287G/A328W/Y332G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (-)-cocaine by ~8- and ~2000-fold respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. According to the kinetic data, the catalytic efficiency (kcat/KM) of mBChE against (-)-cocaine is comparable with that of hBChE, but the catalytic efficiency of mCocH against (-)-cocaine is remarkably lower than that of hCocH by ~250-fold. The remarkable difference in the catalytic activity between mCocH and hCocH is consistent with the difference between the enzyme-(-)-cocaine binding modes obtained from molecular modelling. Further, both mBChE and hBChE demonstrated substrate activation for all of the examined substrates [(-)-cocaine, ACh and BTC] at high concentrations, whereas both mCocH and hCocH showed substrate inhibition for all three substrates at high concentrations. The amino-acid mutations have remarkably converted substrate activation of the enzymes into substrate inhibition, implying that the rate-determining step of the reaction in mCocH and hCocH might be different from that in mBChE and hBChE.

Butyrylcholinesterase (BChE) gene therapy is emerging as a promising concept for treatment of cocaine addiction. BChE levels after gene transfer can rise 1000-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. For months or years, gene transfer of a BChE mutated into a cocaine hydrolase (CocH) can maintain enzyme levels that destroy cocaine within seconds after appearance in the blood stream, allowing little to reach the brain. Rapid enzyme action causes a sharp rise in plasma levels of two cocaine metabolites, benzoic acid (BA) and ecgonine methyl ester (EME), a smooth muscle relaxant that is mildly hypotensive and, at best, only weakly rewarding. The present study, utilizing Balb/c mice, tested reward effects and cardiovascular effects of administering EME and BA together at molar levels equivalent to those generated by a given dose of cocaine. Reward was evaluated by conditioned place preference. In this paradigm, cocaine (20 mg/kg) induced a robust positive response but the equivalent combined dose of EME + BA failed to induce either place preference or aversion. Likewise, mice that had undergone gene transfer with mouse CocH (mCocH) showed no place preference or aversion after repeated treatments with a near-lethal 80 mg/kg cocaine dose. Furthermore, a single administration of that same high cocaine dose failed to affect blood pressure as measured using the noninvasive tail-cuff method. These observations confirm that the drug metabolites generated after CocH gene transfer therapy are safe even after a dose of cocaine that would ordinarily be lethal.

Cocaine is a widely abused and addictive drug without an FDA-approved medication. Our recently designed and discovered cocaine hydrolase, particularly E12-7 engineered from human butyrylcholinesterase (BChE), has the promise of becoming a valuable cocaine abuse treatment. An ideal anti-cocaine therapeutic enzyme should have not only a high catalytic efficiency against cocaine, but also a sufficiently long biological half-life. However, recombinant human BChE and the known BChE mutants have a much shorter biological half-life compared to the native human BChE. The present study aimed to extend the biological half-life of the cocaine hydrolase without changing its high catalytic activity against cocaine. Our strategy was to design possible amino-acid mutations that can introduce cross-subunit disulfide bond(s) and, thus, change the distribution of the oligomeric forms and extend the biological half-life. Three new BChE mutants (E364-532, E377-516, and E535) were predicted to have a more stable dimer structure with the desirable cross-subunit disulfide bond(s) and, therefore, a different distribution of the oligomeric forms and a prolonged biological half-life. The rational design was followed by experimental tests in vitro and in vivo, confirming that the rationally designed new BChE mutants, i.e. E364-532, E377-516, and E535, indeed had a remarkably different distribution of the oligomeric forms and prolonged biological half-life in rats from approximately 7 to approximately 13h without significantly changing the catalytic activity against (-)-cocaine. This is the first demonstration that rationally designed amino-acid mutations can significantly prolong the biological half-life of a high-activity enzyme without significantly changing the catalytic activity.

Cocaine esterase (CocE) is known as the most efficient natural enzyme for cocaine hydrolysis. The major obstacle to the clinical application of wild-type CocE is the thermoinstability with a half-life of only approximately 12 min at 37 degrees C. The previously designed T172R/G173Q mutant (denoted as enzyme E172-173) with an improved in vitro half-life of approximately 6 h at 37 degrees C is currently in clinical trial Phase II for cocaine overdose treatment. Through molecular modeling and dynamics simulation, we designed and characterized a promising new mutant of E172-173 with extra L196C/I301C mutations (denoted as enzyme E196-301) to produce cross-subunit disulfide bonds that stabilize the dimer structure. The cross-subunit disulfide bonds were confirmed by X-ray diffraction. The designed L196C/I301C mutations have not only considerably extended the in vitro half-life at 37 degrees C to >100 days, but also significantly improved the catalytic efficiency against cocaine by approximately 150%. In addition, the thermostable E196-301 can be PEGylated to significantly prolong the residence time in mice. The PEGylated E196-301 can fully protect mice from a lethal dose of cocaine (180 mg/kg, LD100) for at least 3 days, with an average protection time of approximately 94h. This is the longest in vivo protection of mice from the lethal dose of cocaine demonstrated within all studies using an exogenous enzyme reported so far. Hence, E196-301 may be developed to become a more valuable therapeutic enzyme for cocaine abuse treatment, and it demonstrates that a general design strategy and protocol to simultaneously improve both the stability and function are feasible for rational protein drug design.

Cocaine hydrolase gene transfer of mutated human butyrylcholinesterase (BChE) is evolving as a promising therapy for cocaine addiction. BChE levels after gene transfer can be 1,500-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. Because mutated BChE is approximately 70 % as efficient in hydrolyzing acetylcholine as wild-type enzyme, it is important to examine the impact on cholinergic function. Here, we focused on memory and cognition (Stone T-maze), basic neuromuscular function (treadmill endurance and grip strength), and coordination (Rotarod). BALB/c mice were given adeno-associated virus vector or helper-dependent adenoviral vector encoding mouse or human BChE optimized for cocaine. Age-matched controls received saline or luciferase vector. Despite high doses (up to 10(13) particles per mouse) and high transgene expression (1,000-fold above baseline), no deleterious effects of vector treatment were seen in neurobehavioral functions. The vector-treated mice performed as saline-treated and luciferase controls in maze studies and strength tests, and their Rotarod and treadmill performance decreased less with age. Thus, neither the viral vectors nor the large excess of BChE caused observable toxic effects on the motor and cognitive systems investigated. This outcome justifies further steps toward an eventual clinical trial of vector-based gene transfer for cocaine abuse.

INTRODUCTION: Addiction to cocaine is a major problem around the world, but especially in developed countries where the combination of wealth and user demand has created terrible social problems. Although only some users become truly addicted, those who are often succumb to a downward spiral in their lives from which it is very difficult to escape. From the medical perspective, the lack of effective and safe, non-addictive therapeutics has instigated efforts to develop alternative approaches for treatment, including anticocaine vaccines designed to block cocaine's pharmacodynamic effects. AREAS COVERED: This paper discusses the implications of cocaine pharmacokinetics for robust vaccine antibody responses, the results of human vaccine clinical trials, new developments in animal models for vaccine evaluation, alternative vaccine formulations and complementary therapy to enhance anticocaine effectiveness. EXPERT OPINION: Robust anti-cocaine antibody responses are required for benefit to cocaine abusers, but since any reasonably achievable antibody level can be overcome with higher drug doses, sufficient motivation to discontinue use is also essential so that the relative barrier to cocaine effects will be appropriate for each individual. Combining a vaccine with achievable levels of an enzyme to hydrolyze cocaine to inactive metabolites, however, may substantially increase the blockade and improve treatment outcomes.

Compared with naturally occurring enzymes, computationally designed enzymes are usually much less efficient, with their catalytic activities being more than six orders of magnitude below the diffusion limit. Here we use a two-step computational design approach, combined with experimental work, to design a highly efficient cocaine hydrolysing enzyme. We engineer E30-6 from human butyrylcholinesterase (BChE), which is specific for cocaine hydrolysis, and obtain a much higher catalytic efficiency for cocaine conversion than for conversion of the natural BChE substrate, acetylcholine (ACh). The catalytic efficiency of E30-6 for cocaine hydrolysis is comparable to that of the most efficient known naturally occurring hydrolytic enzyme, acetylcholinesterase, the catalytic activity of which approaches the diffusion limit. We further show that E30-6 can protect mice from a subsequently administered lethal dose of cocaine, suggesting the enzyme may have therapeutic potential in the setting of cocaine detoxification or cocaine abuse.

Cocaine addiction affects millions of people with disastrous personal and social consequences. Cocaine is one of the most reinforcing of all drugs of abuse, and even those who undergo rehabilitation and experience long periods of abstinence have more than 80% chance of relapse. Yet there is no FDA-approved treatment to decrease the likelihood of relapse in rehabilitated addicts. Recent studies, however, have demonstrated a promising potential treatment option with the help of the serum enzyme butyrylcholinesterase (BChE), which is capable of breaking down naturally occurring (-)-cocaine before the drug can influence the reward centers of the brain or affect other areas of the body. This activity of wild-type (WT) BChE, however, is relatively low. This prompted the design of variants of BChE which exhibit significantly improved catalytic activity against (-)-cocaine. Plants are a promising means to produce large amounts of these cocaine hydrolase variants of BChE, cheaply, safely with no concerns regarding human pathogens and functionally equivalent to enzymes derived from other sources. Here, in expressing cocaine-hydrolyzing mutants of BChE in Nicotiana benthamiana using the MagnICON virus-assisted transient expression system, and in reporting their initial biochemical analysis, we provide proof-of-principle that plants can express engineered BChE proteins with desired properties.

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 [(11)C](-)-cocaine and [(11)C](+)-cocaine tracers in human subjects, only [(11)C](-)-cocaine was observed in the brain, whereas no significant [(11)C](+)-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 (kcat/KM) against (+)- and (-)-cocaine, and indeed has a catalytic efficiency (kcat/KM=1.84x10(9)M(-1)min(-1)) against (-)-cocaine comparable to that (kcat/KM=1.37x10(9)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.

To address the problem of acute cocaine overdose, we undertook molecular engineering of butyrylcholinesterase (BChE) as a cocaine hydrolase so that modest doses could be used to accelerate metabolic clearance of this drug. Molecular modeling of BChE complexed with cocaine suggested that the inefficient hydrolysis (k(cat) = 4 min(-1)) involves a rotation toward the catalytic triad, hindered by Tyr332. To eliminate rotational hindrance and retain substrate affinity, we introduced two amino acid substitutions (Ala328Trp/Tyr332Ala). The resulting mutant BChE reduced cocaine burden in tissues, accelerated plasma clearance by 20-fold, and prevented cocaine-induced hyperactivity in mice. The enzyme's kinetic properties (k(cat) = 154 min(-1), K(M) = 18 microM) satisfy criteria suggested previously for treating cocaine overdose (k(cat) >120 min(-1), K(M) < 30 microM). This success demonstrates that computationally guided mutagenesis can generate functionally novel enzymes with clinical potential.

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(-1) versus 4.1 min(-1)) and only slightly increased K(M) (18 microM versus 4.5 microM). 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.

Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (-)-cocaine only one-two thousandth as fast as the unnatural (+)-stereoisomer. A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate structural factors underlying the stereochemical difference in catalysis. Here, we report two three-dimensional Michaelis-Menten complexes of BChE liganded with natural and unnatural cocaine molecules, respectively, that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser(198) for hydrolysis. Rotation of (-)-cocaine appears to be hindered by interactions of its phenyl ring with Phe(329) and Trp(430). These interactions do not occur with (+)-cocaine. Because the rate of (-)-cocaine hydrolysis is predicted to be determined mainly by the re-orientation step, it should not be greatly influenced by pH. In fact, measured rates of this reaction were nearly constant over the pH range from 5.5 to 8.5, despite large rate changes in hydrolysis of (+)-cocaine. Our models can explain why BChE hydrolyzes (+)-cocaine faster than (-)-cocaine, and they suggest that mutations of certain residues in the catalytic site could greatly improve catalytic efficiency and the potential for detoxication.

The hydrolysis of cocaine (benzoylecgonine methyl ester) to ecgonine methyl ester by human butyrylcholinesterase (BCHE; EC 3.1.1.8) has been shown previously to constitute an important means to detoxicate this material to pharmacologically inactive metabolites. The naturally occurring (-)-cocaine is hydrolyzed to ecgonine methyl ester approximately 2000 times slower than the unnatural (+)-cocaine isomer. In good agreement with previous studies, (-)-cocaine bound to human BCHE with relatively good affinity and competitively inhibited the hydrolysis of the spectrophotometric substrate butyrylthiocholine with a Ki value of 8.0 microM. Similarly, (+)-cocaine also showed relatively high affinity for the human BCHE and competitively inhibited butyrylthiocholine hydrolysis with a Ki value of 5.4 microM. The phosphonothiolates corresponding to the transition state analogs for both (-)- and (+)-cocaine hydrolysis were synthesized and tested as inhibitors of human BCHE-catalyzed hydrolysis of butyrylthiocholine. The phosphonothiolate corresponding to the transition state for (-)-cocaine hydrolysis was a competitive inhibitor with a Ki value of 55.8 microM. The phosphonothiolate corresponding to the transition state for (+)-cocaine hydrolysis gave a Ki value of 25.9 microM, but, in addition, it also showed irreversible inhibition with a ki of inactivation of 68.8 min-1 M-1. It is likely that the mechanism-based inhibitor described herein may find use as a mechanistic probe of butyrylcholinesterase action and also possibly aid in the purification of this class of esterases.

Butyrylcholinesterase [BCHE (acylcholine acyl hydrolase); EC 3.1.1.8] limits the access of drugs, including tacrine, to other proteins. The "atypical" BCHE variant, in which Asp70 at the rim of the active site gorge is substituted by glycine, displayed a more drastically weakened interaction with tacrine than with cocaine, dibucaine, succinylcholine, BW284c51 [1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide], or alpha-solanine. To delineate the protein domains that are responsible for this phenomenon, we mutated residues within the rim of the active site gorge, the region parallel to the peripheral site in the homologous enzyme acetylcholinesterase [AChE (acetylcholine acetyl hydrolase); EC 3.1.1.7], the oxyanion hole, and the choline-binding site. When expressed in microinjected Xenopus laevis oocytes, all mutant DNAs yielded comparable amounts of immunoreactive protein products. Most mutants retained catalytic activity close to that of wild-type BCHE and were capable of binding ligands. However, certain modifications in and around the oxyanion hole caused a dramatic loss in activity. The affinities for tacrine were reduced more dramatically than for all other ligands, including cocaine, in both oxyanion hole and choline-binding site mutants. Modified ligand affinities further demonstrated a peripheral site in residues homologous with those of AChE. BCHE mutations that prevented tacrine interactions also hampered its ability to bind other drugs and inhibitors, which suggests a partial overlap of the binding sites. This predicts that in addition to their genetic predisposition to adverse responses to tacrine, homozygous carriers of "atypical" BCHE will be overly sensitive to additional anticholinesterases and especially so when exposed to several anticholinesterases in combination.