Xirong Zheng

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.

Kinetic characterization of a cocaine hydrolase engineered from mouse butyrylcholinesterase.

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 (k(cat)/K(M)) 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.

A quantitative LC–MS/MS method for simultaneous determination of cocaine and its metabolites in whole blood

HIGHLIGHTSA LC–MS/MS method to simultaneously detect cocaine and 9 metabolites is developed.The method is highly sensitive and reliable for all of the 10 compounds detected.Metabolic profile of cocaine in rats with co‐administration of alcohol is determined. ABSTRACT As new metabolic pathways of cocaine were recently identified, a high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed to simultaneously determine cocaine and nine cocaine‐related metabolites in whole blood samples. One‐step solid phase extraction was used to extract all of the ten compounds and corresponding internal standards from blood samples. All compounds and internal standards extracted were separated on an Atlantis T3 (100 Å, 3 &mgr;m, 2.1 mm × 150 mm I.D) column and detected in positive ion and high sensitivity mode with multiple reaction monitoring. This method was validated for its sensitivity, linearity, specificity, accuracy, precision, recovery, and stability. All of the ten compounds were quantifiable ranging from the lower limit of quantification (LLOQs) of ˜10 nM (1.9–3.2 ng/ml) to ˜1000 nM (190–320 ng/ml) without any interfering substance. Accuracy and precision were determined, and both of them were within the acceptance criteria of the United States (US) Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines. The recovery was above 66.7% for all compounds. Stability tests demonstrated the stability of compounds under different storage conditions in whole blood samples. The method was successfully applied to a pharmacokinetic study with co‐administration of cocaine and alcohol in rats.

Clinical Potential of an Enzyme-Based Novel Therapy for Cocaine Overdose

It is a grand challenge to develop a truly effective medication for treatment of cocaine overdose. The current available, practical emergence treatment for cocaine overdose includes administration of a benzodiazepine anticonvulsant agent (e.g. diazepam) and/or physical cooling with an aim to relieve the symptoms. The inherent difficulties of antagonizing physiological effects of drugs in the central nervous system have led to exploring protein-based pharmacokinetic approaches using biologics like vaccines, monoclonal antibodies, and enzymes. However, none of the pharmacokinetic agents has demonstrated convincing preclinical evidence of clinical potential for drug overdose treatment without a question mark on the timing used in the animal models. Here we report the use of animal models, including locomotor activity, protection, and rescue experiments in rats, of drug toxicity treatment with clinically relevant timing for the first time. It has been demonstrated that an efficient cocaine-metabolizing enzyme developed in our previous studies can rapidly reverse the cocaine toxicity whenever the enzyme is given to a living rat, demonstrating promising clinical potential of an enzyme-based novel therapy for cocaine overdose as a successful example in comparison with the commonly used diazepam.

Metabolic Enzymes of Cocaine Metabolite Benzoylecgonine

Cocaine is one of the most addictive drugs without a U.S. Food and Drug Administration (FDA)-approved medication. Enzyme therapy using an efficient cocaine-metabolizing enzyme is recognized as the most promising approach to cocaine overdose treatment. The actual enzyme, known as RBP-8000, under current clinical development for cocaine overdose treatment is our previously designed T172R/G173Q mutant of bacterial cocaine esterase (CocE). The T172R/G173Q mutant is effective in hydrolyzing cocaine but inactive against benzoylecgonine (a major, biologically active metabolite of cocaine). Unlike cocaine itself, benzoylecgonine has an unusually stable zwitterion structure resistant to further hydrolysis in the body and environment. In fact, benzoylecgonine can last in the body for a very long time (a few days) and, thus, is responsible for the long-term toxicity of cocaine and a commonly used marker for drug addiction diagnosis in pre-employment drug tests. Because CocE and its mutants are all active against cocaine and inactive against benzoylecgonine, one might simply assume that other enzymes that are active against cocaine are also inactive against benzoylecgonine. Here, through combined computational modeling and experimental studies, we demonstrate for the first time that human butyrylcholinesterase (BChE) is actually active against benzoylecgonine, and that a rationally designed BChE mutant can not only more efficiently accelerate cocaine hydrolysis but also significantly hydrolyze benzoylecgonine in vitro and in vivo. This sets the stage for advanced studies to design more efficient mutant enzymes valuable for the development of an ideal cocaine overdose enzyme therapy and for benzoylecgonine detoxification in the environment.

Design, synthesis, and discovery of 5-((1,3-diphenyl-1 H -pyrazol-4-yl)methylene)pyrimidine-2,4,6(1 H ,3 H ,5 H )-triones and related derivatives as novel inhibitors of mPGES-1

Human mPGES-1 has emerged as a promising target in exploring a next generation of anti-inflammatory drugs, as selective mPGES-1 inhibitors are expected to discriminatively suppress the production of induced PGE2 without blocking the normal biosynthesis of other prostanoids including homeostatic PGE2. Therefore, this therapeutic approach is believed to reduce the adverse effects associated with the application of traditional non-steroidal anti-inflammatory drugs (tNSAIDs) and selective COX-2 inhibitors (coxibs). Identified from structure-based virtue screening, the compound with (Z)-5-benzylidene-2-iminothiazolidin-4-one scaffold was used as lead in rational design of novel inhibitors. Besides, we further designed, synthesized, and evaluated 5-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-triones and structurally related derivatives for their in vitro inhibitory activities. According to in vitro activity assays, a number of these compounds were capable of inhibiting human mPGES-1, with the desirable selectivity for mPGES-1 over COX isozymes.

Structure-based discovery of mPGES-1 inhibitors suitable for preclinical testing in wild-type mice as a new generation of anti-inflammatory drugs

Human mPGES-1 is recognized as a promising target for next generation of anti-inflammatory drugs without the side effects of currently available anti-inflammatory drugs, and various inhibitors have been reported in the literature. However, none of the reported potent inhibitors of human mPGES-1 has shown to be also a potent inhibitor of mouse or rat mPGES-1, which prevents using the well-established mouse/rat models of inflammation-related diseases for preclinical studies. Hence, despite of extensive efforts to design and discover various human mPGES-1 inhibitors, the promise of mPGES-1 as a target for the next generation of anti-inflammatory drugs has never been demonstrated in any wild-type mouse/rat model using an mPGES-1 inhibitor. Here we report discovery of a novel type of selective mPGES-1 inhibitors potent for both human and mouse mPGES-1 enzymes through structure-based rational design. Based on in vivo studies using wild-type mice, the lead compound is indeed non-toxic, orally bioavailable, and more potent in decreasing the PGE2 (an inflammatory marker) levels compared to the currently available drug celecoxib. This is the first demonstration in wild-type mice that mPGES-1 is truly a promising target for the next generation of anti-inflammatory drugs.