Mutant Of Human Butyrylcholinesterase Research Articles

Butyrylcholinesterase with altered catalytic triad, acting as a promising bioscavenger against organophosphorus agents

Cholinesterases (ChEs) are irreversibly inhibited by organophosphorus compounds (OP) through phosphylation of their active site serine. OPs can be regarded as pseudo-substrates of ChEs in which water-mediated dephosphylation is very slow or impossible. Then, it has been attempted by genetic engineering to convert ChEs into OP hydrolases by speeding up dephosphylation rates. The knowledge of ChEs crystal structure and implementation of potent in silico methods allow rational computer design of new mutants. Such ChE mutants could be used as catalytic bioscavengers in medical counter-measures of OP poisoning. In the present work, using QM/MM MD, we designed a new mutant of human butyrylcholinesterase (BChE). This double mutant, Glu325Gly/Asn322Glu, alters the functioning of the catalytic triad (Ser198.His438.Glu325 in wild-type enzyme), making a new one Ser198.His438.Glu322. Calculations with echothiophate as a model OP showed that the new catalytic triad is stable and capable of hydrolysing the phosphorylated serine. Thus, self-reactivation of the mutated enzyme is possible. Although the calculated dephosphylation rate constant is low, it is expected that the introduction of a third mutation will lead to a much faster turnover.

Human Butyrylcholinesterase Mutants for (-)-Cocaine Hydrolysis: A Correlation Relationship between Catalytic Efficiency and Total Hydrogen Bonding Energy with an Oxyanion Hole.

A combined computational and experimental study has been carried out to explore and test a quantitative correlation relationship between the relative catalytic efficiency (RCE) of human butyrylcholinesrase (BChE) mutant-catalyzed hydrolysis of substrate (-)-cocaine and the total hydrogen bonding energy (tHBE) of the carbonyl oxygen of the substrate with the oxyanion hole of the enzyme in the modeled transition-state structure (TS1), demonstrating a satisfactory linear correlation relationship between ln(RCE) and tHBE. The satisfactory correlation relationship has led us to computationally predict and experimentally confirm new human BChE mutants that have a further improved catalytic activity against (-)-cocaine, including the most active one (the A199S/F227S/S287G/A328W/Y332G mutant) with a 2790-fold improved catalytic efficiency (kcat/KM = 2.5 × 109 min-1 M-1) compared to the wild-type human BChE. Compared to the reference mutant (the A199S/S287G/A328W/Y332G mutant) tested in the reported clinical development of an enzyme therapy for cocaine dependence treatment, this new mutant (with a newly predicted additional F227S mutation) has an improved catalytic efficiency against (-)-cocaine by ∼2.6-fold. The good agreement between the computational and experimental ln(RCE) values suggests that the obtained correlation relationship is robust for computational prediction. A similar correlation relationship could also be explored in studying BChE or other serine hydrolases/esterases with an oxyanion hole stabilizing the carbonyl oxygen in the rate-determining reaction step of the enzymatic hydrolysis of other substrates.

Reengineering of Albumin-Fused Cocaine Hydrolase CocH1 (TV-1380) to Prolong Its Biological Half-Life.

Therapeutic treatment of cocaine toxicity or addiction is a grand medical challenge. As a promising therapeutic strategy for treatment of cocaine toxicity and addiction to develop a highly efficient cocaine hydrolase (CocH) capable of accelerating cocaine metabolism to produce physiologically/biologically inactive metabolites, our previously designed A199S/S287G/A328W/Y332G mutant of human butyrylcholinesterase (BChE), known as cocaine hydrolase-1 (CocH1), possesses the desirably high catalytic activity against cocaine. The C-terminus of CocH1, truncated after amino acid #529, was fused to human serum albumin (HSA) to extend the biological half-life. The C-terminal HSA-fused CocH1 (CocH1-HSA), known as Albu-CocH1, Albu-CocH, AlbuBChE, Albu-BChE, or TV-1380 in literature, has shown favorable preclinical and clinical profiles. However, the actual therapeutic value of TV-1380 for cocaine addiction treatment is still limited by the short half-life. In this study, we designed and tested a new type of HSA-fused CocH1 proteins, i.e., N-terminal HSA-fused CocH1, with or without a linker between the HSA and CocH1 domains. It has been demonstrated that the catalytic activity of these new fusion proteins against cocaine is similar to that of TV-1380. However, HSA-CocH1 (without a linker) has a significantly longer biological half-life (t1/2 = 14 ± 2h) compared to the corresponding C-terminal HSA-fused CocH1, i.e., CocH1-HSA (TV-1380 with t1/2 = 5-8h), in rats. Further, the N-terminal HSA-fused CocH1 proteins with a linker have further prolonged biological half-lives: t1/2 = 17 ± 2h for both HSA-EAAAK-CocH1 and HSA-PAPAP-CocH1, and t1/2 = 18 ± 3h for HSA-(PAPAP)2-CocH1. These N-terminal HSA-fused CocH1 proteins may serve as more promising protein drug candidates for cocaine addiction treatment.

A new sensitive spectrofluorimetric method for measurement of activity and kinetic study of cholinesterases

A new spectrofluorimetric method more sensitive than the Ellman method was developed for determination of both acetylcholinesterase and butyrylcholinesterase activity and for kinetic analysis of these enzymes and their mutants. Two selected mutants of human butyrylcholinesterase (E197Q and E197G) were included in this work. As for the Ellman's method, substrates are thiocholine esters, but the chromogenic reagent, DTNB (dithio-bisnitro benzoic acid) is replaced by a fluorogenic probe, “Calbiochem Probe IV”, (3-(7-Hydroxy-2-oxo-2H-chromen-3-ylcarbamoyl)acrylic acid methylester).Compared to the classical Ellman's method, the sensitivity of this new spectrofluorimetric assay is 2 orders of magnitude higher. The method allows measurement of activity in media containing <10−11 M of cholinesterase active sites at low substrate concentrations, either under first order conditions, [S] << Km, or under conditions where kinetics obeys the Michaelis-Menten model, i.e. at [S] < 1 mM for wild-type enzymes. The method adapted to titration plate reader assays is suitable for clinical and toxicological routine analyses, for high throughput screening of novel cholinesterase mutants and screening of inhibitor libraries of pharmacological interest.

Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad

A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 μs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.

Development of a long-acting Fc-fused cocaine hydrolase with improved yield of protein expression

Human butyrylcholinesterase (BChE) is known as a safe and effective protein for detoxification of organophosphorus (OP) nerve agents. Its rationally designed mutants with considerably improved catalytic activity against cocaine, known as cocaine hydrolases (CocHs), are recognized as the most promising drug candidates for the treatment of cocaine abuse. However, it is a grand challenge to efficiently produce active recombinant BChE and CocHs with a sufficiently long biological half-life. In the present study, starting from a promising CocH, known as CocH3 (i.e. A199S/F227A/S287G/A328W/Y332G mutant of human BChE), which has a ~2000-fold improved catalytic activity against cocaine compared to wild-type BChE, we designed an N-terminal fusion protein, Fc(M3)-(PAPAP)2-CocH3, which was constructed by fusing Fc of human IgG1 to the N-terminal of CocH3 and further optimized by inserting a linker between the two protein domains. Without lowering the enzyme activity, Fc(M3)-(PAPAP)2-CocH3 expressed in Chinese hamster ovary (CHO) cells has not only a long biological half-life of 105 ± 7 h in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP)2-CocH3 has a ~21-fold increased protein expression yield in CHO cells compared to CocH3 under the same experimental conditions. Given the observations that Fc(M3)-(PAPAP)2-CocH3 has not only a high catalytic activity against cocaine and a long biological half-life, but also a high yield of protein expression, this new protein entity reported in this study would be a more promising candidate for therapeutic treatment of cocaine overdose and addiction.

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.

Kinetic characterization of human butyrylcholinesterase mutants for the hydrolysis of cocaethylene.

It is known that the majority of cocaine users also consume alcohol. Alcohol can react with cocaine to produce a significantly more cytotoxic compound, cocaethylene. Hence a truly valuable cocaine-metabolizing enzyme as treatment for cocaine abuse/overdose should be efficient for not only cocaine itself, but also cocaethylene. The catalytic parameters (kcat and KM) of human BChE (butyrylcholinesterase) and two mutants (known as cocaine hydrolases E14-3 and E12-7) for cocaethylene are characterized in the present study, for the first time, in comparison with those for cocaine. On the basis of the obtained kinetic data, wild-type human BChE has a lower catalytic activity for cocaethylene (kcat=3.3 min(-1), KM=7.5 μM and kcat/KM=4.40 × 10(5) M(-1)·min(-1)) compared with its catalytic activity for (-)-cocaine. E14-3 and E12-7 have a considerably improved catalytic activity against cocaethylene compared with the wild-type BChE. E12-7 is identified as the most efficient enzyme for hydrolysing cocaethylene in addition to its high activity for (-)-cocaine. E12-7 has an 861-fold improved catalytic efficiency for cocaethylene (kcat=3600 min(-1), KM=9.5 μM and kcat/KM=3.79 × 10(8) M(-1)·min(-1)). It has been demonstrated that E12-7 as an exogenous enzyme can indeed rapidly metabolize cocaethylene in rats. Further kinetic modelling has suggested that E12-7 with an identical concentration as that of the endogenous BChE in human plasma can effectively eliminate (-)-cocaine, cocaethylene and norcocaine in simplified kinetic models of cocaine abuse and overdose associated with the concurrent use of cocaine and alcohol.

Amino-acid mutations to extend the biological half-life of a therapeutically valuable mutant of human butyrylcholinesterase

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 ∼7 to ∼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.

Kinetic characterization of high-activity mutants of human butyrylcholinesterase for the cocaine metabolite norcocaine

It has been known that cocaine produces its toxic and physiological effects through not only cocaine itself, but also norcocaine formed from cocaine oxidation catalysed by microsomal CYP (cytochrome P450) 3A4 in the human liver. The catalytic parameters (kcat and Km) of human BChE (butyrylcholinesterase) and its three mutants (i.e. A199S/S287G/A328W/Y332G, A199S/F227A/S287G/A328W/E441D and A199S/F227A/S287G/A328W/Y332G) for norcocaine have been characterized in the present study for the first time and compared with those for cocaine. On the basis of the obtained kinetic data, wild-type human BChE has a significantly lower catalytic activity for norcocaine (kcat=2.8 min(-1), Km=15 μM and kcat/Km=1.87 × 10(5) M(-1)·min(-1)) compared with its catalytic activity for (-)-cocaine. The BChE mutants examined in the present study have considerably improved catalytic activities against both cocaine and norcocaine compared with the wild-type enzyme. Within the enzymes examined in the present study, the A199S/F227A/S287G/A328W/Y332G mutant (CocH3) is identified as the most efficient enzyme for hydrolysing both cocaine and norcocaine. CocH3 has a 1080-fold improved catalytic efficiency for norcocaine (kcat=2610 min(-1), Km=13 μM and kcat/Km=2.01 × 10(8) M(-1)·min(-1)) and a 2020-fold improved catalytic efficiency for cocaine. It has been demonstrated that CocH3 as an exogenous enzyme can rapidly metabolize norcocaine, in addition to cocaine, in rats. Further kinetic modelling has suggested that CocH3 with an identical concentration with that of the endogenous BChE in human plasma can effectively eliminate both cocaine and norcocaine in a simplified kinetic model of cocaine abuse.

Preparation and in vivo characterization of a cocaine hydrolase engineered from human butyrylcholinesterase for metabolizing cocaine

Cocaine is a widely abused drug without an FDA (Food and Drug Administration)-approved medication. It has been recognized that an ideal anti-cocaine medication would accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. human BChE (butyrylcholinesterase)-catalysed hydrolysis. However, the native human BChE has a low catalytic activity against cocaine. We recently designed and discovered a BChE mutant (A199S/F227A/S287G/A328W/Y332G) with a high catalytic activity (kcat=5700 min-1, Km=3.1μM) specifically for cocaine, and the mutant was proven effective in protecting mice from acute cocaine toxicity of a lethal dose of cocaine (180mg/kg of body weight, LD100). Further characterization in animal models requires establishment of a high-efficiency stable cell line for the BChE mutant production at a relatively larger scale. It has been extremely challenging to develop a high-efficiency stable cell line expressing BChE or its mutant. In the present study, we successfully developed a stable cell line efficiently expressing the BChE mutant by using a lentivirus-based repeated-transduction method. The scaled-up protein production enabled us to determine for the first time the invivo catalytic activity and the biological half-life of this high-activity mutant of human BChE in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant (administered to mice 1min prior to cocaine) can quickly metabolize cocaine and completely eliminate cocaine-induced hyperactivity in rodents, implying that the BChE mutant may be developed as a promising therapeutic agent for cocaine abuse treatment.

Determination of binding points of methylene blue and cationic phenoxazine dyes on human butyrylcholinesterase

In this study, the binding points of MethB and two structurally-related cationic phenoxazine dyes [meldola blue (MB) and nile blue (NB)] to human butyrylcholinesterase (BChE) were investigated by molecular docking and site directed mutagenesis. The comparative inhibitory effects of MethB, MB and NB on recombinant wild type BChE and six human BChE mutants were spectrophotometrically studied. Kinetic analyses yielded the following information: MethB and MB were found to cause nonlinear inhibition of all recombinant BChEs except Y332A, compatible with a multi-site binding model. On the other hand, MethB and MB caused linear mixed inhibition of Y332A mutant, compatible with a single binding mode. Comparing the inhibitory effects in aspect of Ki values with recombinant wild type BChE (Ki=0.042μM), MethB was found to be ∼30, 80 and 270-fold less effective as an inhibitor of Y332A, F329A and T120F, respectively. NB caused nonlinear inhibition of all recombinant BChEs. The inhibitory effect of NB on Y332A mutant was ∼370-fold lower, compared to recombinant wild type BChE (Ki=0.006μM). Considering both kinetic and molecular docking results together, it was concluded that threonine 120, phenylalanine 329 and tyrosine 332 are critical amino acids in binding of cationic phenoxazine/phenothiazine structured ligands to human BChE.

Substrate selectivity of high-activity mutants of human butyrylcholinesterase.

Cocaine is one of the most addictive drugs, and there is still no FDA (Food and Drug Administration)-approved medication specific for cocaine abuse. A promising therapeutic strategy is 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. However, the native BChE has a low catalytic efficiency against the abused cocaine, i.e. (-)-cocaine. Our recently designed and discovered A199S/F227A/S287G/A328W/Y332G mutant and other mutants of human BChE have a considerably improved catalytic efficiency against (-)-cocaine. In the present study, we carried out both computational modeling and experimental kinetic analysis on the catalytic activities of these promising new BChE mutants against other known substrates, including neurotransmitter acetylcholine (ACh), acetylthiocholine (ATC), butyrylthiocholine (BTC), and (+)-cocaine, in comparison with the corresponding catalytic activity against (-)-cocaine. Both the computational modeling and kinetic analysis have consistently revealed that all the examined amino acid mutations only considerably improve the catalytic efficiency of human BChE against (-)-cocaine, without significantly improving the catalytic efficiency of the enzyme against any of the other substrates examined. In particular, all the examined BChE mutants have a slightly lower catalytic efficiency against neurotransmitter ACh compared to the wild-type BChE. This observation gives us confidence in developing an anti-cocaine enzyme therapy by using one of these BChE mutants, particularly the A199S/F227A/S287G/A328W/Y332G mutant.

Why Does the G117H Mutation Considerably Improve the Activity of Human Butyrylcholinesterase against Sarin? Insights from Quantum Mechanical/Molecular Mechanical Free Energy Calculations

Human butyrylcholinesterase (BChE) is recognized as the most promising bioscavenger for organophosphorus (OP) warfare nerve agents. The G117H mutant of human BChE has been identified as a potential catalytic bioscavenger with a remarkably improved activity against OP nerve agents such as sarin, but it still does not satisfy the clinical use. For further design of the higher-activity mutants against OP nerve agents, it is essential to understand how the G117H mutation improves the activity. The reaction mechanisms and the free energy profiles for spontaneous reactivation of wild-type BChE and its G117H mutant phosphorylated by sarin have been explored, in this study, by performing first-principles quantum mechanical/molecular mechanical free energy calculations, and the remarkable role of the G117H mutation on the activity has been elucidated. For both the wild-type and G117H mutant enzymes, H438 acts as a general base to initiate the spontaneous reactivation that consists of two reaction steps: the nucleophilic attack at the phosphorus by a water molecule and decomposition of the pentacoordinated phosphorus intermediate. The calculated overall free energy barriers, i.e., 30.2 and 23.9 kcal/mol for the wild type and G117H mutant, respectively, are in good agreement with available experimental kinetic data. On the basis of the calculated results, the mutated residue (H117 in the G117H mutant) cannot initiate the spontaneous reactivation as a general base. Instead, it skews the oxyanion hole and makes the phosphorus more open to the nucleophilic water molecule, resulting in a remarkable change in the rate-determining step and significantly improved catalytic activity of human BChE.

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

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 (kcat/KM) against (+)- and (−)-cocaine, and indeed has a catalytic efficiency (kcat/KM=1.84×109M−1min−1) against (−)-cocaine comparable to that (kcat/KM=1.37×109M−1min−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.

Reaction Pathway and Free Energy Profile for Cocaine Hydrolase-Catalyzed Hydrolysis of (-)-Cocaine.

Reaction pathway of (-)-cocaine hydrolysis catalyzed by our recently discovered most efficient cocaine hydrolase, which is the A199S/F227A/S287G/A328W/Y332G mutant of human butyrylcholinesterase (BChE), and the corresponding free energy profile have been studied by performing first-principles pseudobond quantum mechanical/molecular mechanical (QM/MM)-free energy (FE) calculations. Based on the QM/MM-FE results, the catalytic hydrolysis process consists of four major reaction steps, including the nucleophilic attack on carbonyl carbon of (-)-cocaine benzoyl ester by hydroxyl group of S198, dissociation of (-)-cocaine benzoyl ester, nucleophilic attack on carbonyl carbon of (-)-cocaine benzoyl ester by water, and finally the dissociation between (-)-cocaine benzoyl group and S198 of the enzyme. The second reaction step is rate-determining. The calculated free energy barrier associated with the transition state for the rate-determining step is ~15.0 kcal/mol, which is in excellent agreement with the experimentally-derived activation free energy of ~14.7 kcal/mol. The mechanistic insights obtained from the present study will be valuable for rational design of more active cocaine hydrolase against (-)-cocaine. In particular, future efforts aiming at further increasing the catalytic activity of the enzyme against (-)-cocaine should focus on stabilization of the transition state for the second reaction step in which the benzoyl ester of (-)-cocaine dissociates.

Accelerating Cocaine Metabolism as an Approach to the Treatment of Cocaine Abuse and Toxicity

One pharmacokinetic approach to the treatment of cocaine abuse and toxicity involves the development of compounds that can be safely administered to humans and that accelerate the metabolism of cocaine to inactive components. Catalytic antibodies have been developed and shown to accelerate cocaine metabolism, but their catalytic efficiency for cocaine is relatively low. Mutations of human butyrylcholinesterase and a bacterial cocaine esterase found in the soil of coca plants have also been developed. These compounds accelerate cocaine metabolism and antagonize the behavioral and toxic effects of cocaine in animal models. Of these two approaches, the human butyrylcholinesterase mutants show the most immediate promise as they would not be expected to evoke an immune response in humans.

X-ray crystallographic snapshots of reaction intermediates in the G117H mutant of human butyrylcholinesterase, a nerve agent target engineered into a catalytic bioscavenger

OPs (organophosphylates) exert their acute toxicity through inhibition of acetylcholinesterase, by phosphylation of the catalytic serine residue. Engineering of human butyrylcholinesterase, by substitution of a histidine residue for the glycine residue at position 117, led to the creation of OP hydrolase activity. However, the lack of structural information and poor understanding of the hydrolytic mechanism of the G117H mutant has hampered further improvements in the catalytic activity. We have solved the crystallographic structure of the G117H mutant with a variety of ligands in its active site. A sulfate anion bound to the active site suggested the positioning for an OP prior to phosphylation. A fluoride anion was found in the active site when NaF was added to the crystallization buffer. In the fluoride complex, the imidazole ring from the His117 residue was substantially shifted, adopting a relaxed conformation probably close to that of the unliganded mutant enzyme. Additional X-ray structures were obtained from the transient covalent adducts formed upon reaction of the G117H mutant with the OPs echothiophate and VX [ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl](methyl)phosphinate]. The position of the His117 residue shifted in response to the introduction of these adducts, overlaying the phosphylserine residue. These structural data suggest that the dephosphylation mechanism involves either a substantial conformational change of the His117 residue or an adjacent nucleophilic substitution by water.

Design, preparation, and characterization of high-activity mutants of human butyrylcholinesterase specific for detoxification of cocaine.

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 (-)-cocaine but also certain selectivity for (-)-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 (-)-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 (-)-cocaine but also the desirable selectivity for (-)-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 cocaine-induced 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.

Design of High-Activity Mutants of Human Butyrylcholinesterase against (−)-Cocaine: Structural and Energetic Factors Affecting the Catalytic Efficiency

The present study was aimed to explore the correlation between the protein structure and catalytic efficiency of butyrylcholinesterase (BChE) mutants against (-)-cocaine by modeling the rate-determining transition state (TS1), i.e., the transition state for the first step of chemical reaction process, of (-)-cocaine hydrolysis catalyzed by various mutants of human BChE in comparison with the wild type. Molecular modeling of the TS1 structures revealed that mutations on certain nonactive site residues can indirectly affect the catalytic efficiency of the enzyme against (-)-cocaine through enhancing or weakening the overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. Computational insights and predictions were supported by the catalytic activity data obtained from wet experimental tests on the mutants of human BChE, including five new mutants reported for the first time. The BChE mutants with at least ∼1000-fold improved catalytic efficiency against (-)-cocaine compared to the wild-type BChE are all associated with the TS1 structures having stronger overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. The combined computational and experimental data demonstrate a reasonable correlation relationship between the hydrogen-bonding distances in the TS1 structure and the catalytic efficiency of the enzyme against (-)-cocaine.