Zheng F

General

Full name : Zheng Fang

First name : Fang

Mail : Molecular Modelling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536

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Country : USA

Email : fzhen2@email.uky.edu

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References (65)

Title : In vitro and in vivo stability of a highly efficient long-acting cocaine hydrolase - Shang_2024_Sci.Rep_14_10952
Author(s) : Shang L , Wei H , Deng J , Stewart MJ , LeSaint JE , Kyomuhangi A , Park S , Maul EC , Zhan CG , Zheng F
Ref : Sci Rep , 14 :10952 , 2024
Abstract : It is recognized as a promising therapeutic strategy for cocaine use disorder to develop an efficient enzyme which can rapidly convert cocaine to physiologically inactive metabolites. We have designed and discovered a series of highly efficient cocaine hydrolases, including CocH5-Fc(M6) which is the currently known as the most efficient cocaine hydrolase with both the highest catalytic activity against (-)-cocaine and the longest biological half-life in rats. In the present study, we characterized the time courses of protein appearance, pH, structural integrity, and catalytic activity against cocaine in vitro and in vivo of a CocH5-Fc(M6) bulk drug substance produced in a bioreactor for its in vitro and in vivo stability after long-time storage under various temperatures (-80, -20, 4, 25, or 37 degreesC). Specifically, all the tested properties of the CocH5-Fc(M6) protein did not significantly change after the protein was stored at any of four temperatures including -80, -20, 4, and 25 degreesC for~18 months. In comparison, at 37 degreesC, the protein was less stable, with a half-life of~82 days for cocaine hydrolysis activity. Additionally, the in vivo studies further confirmed the linear elimination PK profile of CocH5-Fc(M6) with an elimination half-life of~9 days. All the in vitro and in vivo data on the efficacy and stability of CocH5-Fc(M6) have consistently demonstrated that CocH5-Fc(M6) has the desired in vitro and in vivo stability as a promising therapeutic candidate for treatment of cocaine use disorder.
ESTHER : Shang_2024_Sci.Rep_14_10952
PubMedSearch : Shang_2024_Sci.Rep_14_10952
PubMedID: 38740850

Title : Long-lasting blocking of interoceptive effects of cocaine by a highly efficient cocaine hydrolase in rats - Wei_2024_Sci.Rep_14_927
Author(s) : Wei H , LeSaint JE , Jin Z , Zhan CG , Zheng F
Ref : Sci Rep , 14 :927 , 2024
Abstract : Cocaine dependence is a serious world-wide public health problem without an FDA-approved pharmacotherapy. We recently designed and discovered a highly efficient long-acting cocaine hydrolase CocH5-Fc(M6). The present study examined the effectiveness and duration of CocH5-Fc(M6) in blocking interoceptive effects of cocaine by performing cocaine discrimination tests in rats, demonstrating that the duration of CocH5-Fc(M6) in blocking cocaine discrimination was dependent on cocaine dose and CocH5-Fc(M6) plasma concentration. Particularly, a dose of 3 mg/kg CocH5-Fc(M6) effectively attenuated discriminative stimulus effects of 10 mg/kg cocaine, cumulative doses of 10 and 32 mg/kg cocaine, and cumulative doses of 10, 32 and 56 mg/kg cocaine by <= 20% for 41, 19, and 10 days, and completely blocked the discriminative stimulus effects for 30, 13, and 5 days with corresponding threshold plasma CocH5-Fc(M6) concentrations of 15.9, 72.2, and 221 nM, respectively, under which blood cocaine concentration was negligible. Additionally, based on the data obtained, cocaine discrimination model is more sensitive than the locomotor activity to reveal cocaine effects and that CocH5-Fc(M6) itself has no long-term toxicity regarding behavioral activities such as lever pressing and food consumption in rats, further demonstrating that CocH5-Fc(M6) has the desired properties as a promising therapeutic candidate for prevenance of cocaine dependence.
ESTHER : Wei_2024_Sci.Rep_14_927
PubMedSearch : Wei_2024_Sci.Rep_14_927
PubMedID: 38195724

Title : Human Butyrylcholinesterase Mutants for (-)-Cocaine Hydrolysis: A Correlation Relationship between Catalytic Efficiency and Total Hydrogen Bonding Energy with an Oxyanion Hole - Zheng_2023_J.Phys.Chem.B__
Author(s) : Zheng F , Hou S , Xue L , Yang W , Zhan CG
Ref : J Phys Chem B , : , 2023
Abstract : 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 (k(cat)/K(M) = 2.5 x 10(9) 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.
ESTHER : Zheng_2023_J.Phys.Chem.B__
PubMedSearch : Zheng_2023_J.Phys.Chem.B__
PubMedID: 38063500

Title : Recovery of dopaminergic system after cocaine exposure and impact of a long-acting cocaine hydrolase - Deng_2022_Addict.Biol_27_e13179
Author(s) : Deng J , Zhang T , Zheng X , Shang L , Zhan CG , Zheng F
Ref : Addict Biol , 27 :e13179 , 2022
Abstract : Dysregulation of dopamine transporters (DAT) within the dopaminergic system is an important biomarker of cocaine exposure. Depending on cocaine amount in-taken, one-time exposure in rats could lead to most (>95% of total) of DAT translocating to plasma membrane of the dopaminergic neurons compared to normal DAT distribution (~5.7% on the plasma membrane). Without further cocaine exposure, the time course of striatal DAT distribution, in terms of intracellular and plasma membrane fractions of DAT, represents a recovery process of the dopaminergic system. In this study, we demonstrated that after an acute cocaine exposure of 20 mg/kg (i.p.), the initial recovery process from days 1 to 15 in rats was relatively faster (from >95% on day 1 to ~35.4% on day 15). However, complete recovery of the striatal DAT distribution may take about 60 days. In another situation, with repeated cocaine exposures for once every other day for a total of 17 doses of 20 mg/kg cocaine (i.p.) from days 0 to 32, the complete recovery of striatal DAT distribution may take an even longer time (about 90 days), which represents a consequence of chronic cocaine use. Further, we demonstrated that a highly efficient Fc-fused cocaine hydrolase, CocH5-Fc(M6), effectively blocked cocaine-induced hyperactivity and DAT trafficking with repeated cocaine exposures by maintaining a plasma CocH5-Fc(M6) concentration <=58.7 +/- 2.9 nM in rats. The cocaine hydrolase protected dopaminergic system and helped the cocaine-altered DAT distribution to recover by preventing the dopaminergic system from further damage by cocaine.
ESTHER : Deng_2022_Addict.Biol_27_e13179
PubMedSearch : Deng_2022_Addict.Biol_27_e13179
PubMedID: 35754103

Title : Development of a Highly Efficient Long-Acting Cocaine Hydrolase Entity to Accelerate Cocaine Metabolism - Zheng_2022_Bioconjug.Chem__
Author(s) : Zheng F , Jin Z , Deng J , Chen X , Zheng X , Wang G , Kim K , Shang L , Zhou Z , Zhan CG
Ref : Bioconjug Chem , : , 2022
Abstract : It is particularly challenging to develop a truly effective pharmacotherapy for cocaine use disorder (CUD) treatment. Accelerating cocaine metabolism via hydrolysis at cocaine benzoyl ester using an efficient cocaine hydrolase (CocH) is known as a promising pharmacotherapeutic approach to CUD treatment. Preclinical and clinical studies on our first CocH (CocH1), in its human serum albumin-fused form known as TV-1380, have demonstrated the promise of a general concept of CocH-based pharmacotherapy for CUD treatment. However, the biological half-life of TV-1380 (t(1/2) = 8 h in rats, associated with t(1/2) = 43-77 h in humans) is not long enough for practical treatment of cocaine dependence, which requires enzyme injection for no more than once weekly. Through protein fusion of a human butyrylcholinesterase mutant (denoted as CocH5) with a mutant (denoted as Fc(M6)) of Fc from human IgG1, we have designed, prepared, and tested a new fusion protein (denoted as CocH5-Fc(M6)) for its pharmacokinetic profile and in vivo catalytic activity against (-)-cocaine. CocH5-Fc(M6) represents the currently most efficient long-acting cocaine hydrolase with both the highest catalytic activity against (-)-cocaine and the longest elimination half-life (t(1/2) = 229 +/- 5 h) in rats. As a result, even at a single modest dose of 3 mg/kg, CocH5-Fc(M6) can significantly and effectively accelerate the metabolism of cocaine in rats for at least 60 days. In addition, -70 nM CocH5-Fc(M6) in plasma was able to completely block the toxicity and physiological effects induced by intraperitoneal injection of a lethal dose of cocaine (60 mg/kg).
ESTHER : Zheng_2022_Bioconjug.Chem__
PubMedSearch : Zheng_2022_Bioconjug.Chem__
PubMedID: 35767675

Title : Effects of alcohol on metabolism and toxicity of cocaine in rats - Shang_2022_Toxicol.Rep_9_1586
Author(s) : Shang L , Zheng X , Zhang T , Deng J , Zhan CG , Zheng F
Ref : Toxicol Rep , 9 :1586 , 2022
Abstract : As most cocaine users drink alcohol, it is interesting to understand how a non-lethal dose of alcohol affects the metabolism and toxicity of cocaine. In this study, we examined the correlation between dose-dependent toxicity and the metabolism/pharmacokinetic (PK) profile of cocaine with or without alcohol (ethanol, 1sg/kg) co-administration in rats. The cocaine toxicity in rats with or without alcohol co-administration is characterized by not only the commonly used LD(50), but also the average times for the appearance of convulsion and death as well as total toxicity level (TTL) in the blood. All these data have consistently demonstrated that co-administration of alcohol increased cocaine toxicity, and that the alcohol-enhanced toxicity of cocaine is mainly attributed to the observed two additional metabolites (cocaethylene and norcocaethylene - products of chemical reactions of cocaine with alcohol catalyzed by metabolic enzymes carboxylesterase-1 and liver microsomal cytochrome P450 3A4) that are more toxic than cocaine itself. So, evaluation of the substance TTL should account for the blood levels of not only cocaine itself, but also its all toxic metabolites. In addition, for rats died of a lethal dose of cocaine (60 or 100smg/kg) combined with 1sg/kg alcohol, we also determined the TTL at the time of death, demonstrating that death would occur once the TTL reached a threshold (~16smicroM).
ESTHER : Shang_2022_Toxicol.Rep_9_1586
PubMedSearch : Shang_2022_Toxicol.Rep_9_1586
PubMedID: 36518391

Title : Catalytic activities of cocaine hydrolases against the most toxic cocaine metabolite norcocaethylene - Zheng_2020_Org.Biomol.Chem__
Author(s) : Zheng X , Chen X , Zhang T , Zhan M , Zhan CG , Zheng F
Ref : Org Biomol Chem , : , 2020
Abstract : 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 (kcat and KM) 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 (kcat = 9.5 min-1, KM = 11.7 muM, and kcat/KM = 8.12 x 105 M-1 min-1) to that against (-)-cocaine (kcat = 4.1 min-1, KM = 4.5 muM, and kcat/KM = 9.1 x 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 (kcat = 210 min-1, KM = 6.6 muM, and kcat/KM = 3.18 x 107 M-1 min-1). It has been demonstrated that E12-7 as an exogenous enzyme can efficiently metabolize norcocaethylene in rats.
ESTHER : Zheng_2020_Org.Biomol.Chem__
PubMedSearch : Zheng_2020_Org.Biomol.Chem__
PubMedID: 32101217

Title : Efficient Cocaine Degradation by Cocaine Esterase-Loaded Red Blood Cells - Rossi_2020_Front.Physiol_11_573492
Author(s) : Rossi L , Pierige F , Agostini M , Bigini N , Termopoli V , Cai Y , Zheng F , Zhan CG , Landry DW , Magnani M
Ref : Front Physiol , 11 :573492 , 2020
Abstract : Recombinant bacterial cocaine esterase (CocE) represents a potential protein therapeutic for cocaine use disorder treatment. Unfortunately, the native enzyme was highly unstable and the corresponding mutagenized derivatives, RBP-8000 and E196-301, although improving in vitro thermo-stability and in vivo half-life, were a partial solution to the problem. For cocaine use disorder treatment, an efficient cocaine-metabolizing enzyme with a longer residence time in circulation would be needed. We investigated in vitro the possibility of developing red blood cells (RBCs) loaded with RBP-8000 and E196-301 as a biocompatible system to metabolize cocaine for a longer period of time. RBP 8000 stability within human RBCs is limited (approximately 50% residual activity after 1 h at 37degC) and not different as for the free enzyme, while both free and encapsulated E196-301 showed a greater thermo-stability. By reducing cellular glutathione content during the loading procedure, in order to preserve the disulfide bonds opportunely created to stabilize the enzyme dimer structure, it was possible to produce an encapsulated protein maintaining 100% stability at least after 4 h at 37degC. Moreover, E196-301-loaded RBCs were efficiently able to degrade cocaine in a time- and concentration-dependent manner. The same stability results were obtained when murine RBCs were used paving the way to preclinical investigations. Thus, our in vitro data show that E196-301-loaded RBCs could act as efficient bioreactors in degrading cocaine to non-toxic metabolites to be possibly considered in substance-use disorder treatments. This approach should now be investigated in a preclinical model of cocaine use disorder to evaluate if further protein modifications are needed to further improve long term enzyme stability.
ESTHER : Rossi_2020_Front.Physiol_11_573492
PubMedSearch : Rossi_2020_Front.Physiol_11_573492
PubMedID: 33013487

Title : Identification of ovalbumin-derived peptides as multi-target inhibitors of AChE, BChE and BACE1 - Yu_2020_J.Sci.Food.Agric__
Author(s) : Yu Z , Dong W , Wu S , Shen J , Zhao W , Ding L , Liu J , Zheng F
Ref : J Sci Food Agric , : , 2020
Abstract : BACKGROUND: Alzheimer's disease (AD) is a kind of progressive neurodegenerative disease that occurs to the elderly. But there is no ideal treatment for AD. Thus, the purpose of this study is to identify anti-AD peptides from ovalbumin. RESULTS: The potential tripeptides IEK, LYR and CIK were selected for molecular docking. The '-CDOCKER_Energy' value of the best docking position of the tripeptide IEK, LYR and CIK interacting with acetylcholinesterase (AChE) were - 93.8119, -86.9556 and - 73.6370 kcal/mol, respectively. The '-CDOCKER_Energy' values for interaction with butyrylcholinesterase (BChE) were - 96.6386, -80.8392 and - 87.4341 kcal/mol, respectively. Most importantly, the '-CDOCKER_Energy' values for interaction with beta-site amyloid precursor protein cleavage enzyme1 (BACE1) were - 85.5903, -71.3342 and - 68.4290 kcal/mol, respectively. Overall, in vitro assays results demonstrated that peptide CIK exhibited impressive inhibitory activities against AChE, BChE, and BACE1, with the IC50 value of 6.76, 7.72, and 34.48 muM, respectively. Especially, CIK can be contacted with some peripheral anion sites (PAS) and catalytic sites on AChE, BChE and BACE1. CONCLUSION: Tripeptide CIK can effectively inhibit the activities of AChE, BChE and BACE1. Therefore, tripeptide CIK has the potential to effectively treat AD. This article is protected by copyright. All rights reserved.
ESTHER : Yu_2020_J.Sci.Food.Agric__
PubMedSearch : Yu_2020_J.Sci.Food.Agric__
PubMedID: 31997357

Title : Structure-based virtual screening leading to discovery of highly selective butyrylcholinesterase inhibitors with solanaceous alkaloid scaffolds - Zhou_2019_Chem.Biol.Interact_13ChEPon_308_372
Author(s) : Zhou S , Yuan Y , Zheng F , Zhan CG
Ref : Chemico-Biological Interactions , 308 :372 , 2019
Abstract : According to recent research advance, it is interesting to identify new, potent and selective inhibitors of human butyrylcholinesterase (BChE) for therapeutic treatment of both the Alzheimer's disease (AD) and heroin abuse. In this study, we carried out a structure-based virtual screening followed by in vitro activity assays, with the goal to identify new inhibitors that are selective for BChE over acetylcholinesterase (AChE). As a result, a set of new, selective inhibitors of human BChE were identified from natural products with solanaceous alkaloid scaffolds. The most active one of the natural products (compound 1) identified has an IC50 of 16.8nM against BChE. It has been demonstrated that the desirable selectivity of these inhibitors for BChE over AChE is mainly controlled by three key residues in the active site cavity, i.e. residues Q119, A277, and A328 in BChE versus the respective residues Y124, W286, and Y337 in AChE. Based on this structural insight, future rational design of new, potent and selective BChE inhibitors may focus on these key structural differences in the active site cavity.
ESTHER : Zhou_2019_Chem.Biol.Interact_13ChEPon_308_372
PubMedSearch : Zhou_2019_Chem.Biol.Interact_13ChEPon_308_372
PubMedID: 31152736

Title : Reengineering of Albumin-Fused Cocaine Hydrolase CocH1 (TV-1380) to Prolong Its Biological Half-Life - Cai_2019_AAPS.J_22_5
Author(s) : Cai Y , Zhou S , Jin Z , Wei H , Shang L , Deng J , Zhan CG , Zheng F
Ref : AAPS J , 22 :5 , 2019
Abstract : 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 +/- 2 h) compared to the corresponding C-terminal HSA-fused CocH1, i.e., CocH1-HSA (TV-1380 with t1/2 = 5-8 h), in rats. Further, the N-terminal HSA-fused CocH1 proteins with a linker have further prolonged biological half-lives: t1/2 = 17 +/- 2 h for both HSA-EAAAK-CocH1 and HSA-PAPAP-CocH1, and t1/2 = 18 +/- 3 h for HSA-(PAPAP)2-CocH1. These N-terminal HSA-fused CocH1 proteins may serve as more promising protein drug candidates for cocaine addiction treatment.
ESTHER : Cai_2019_AAPS.J_22_5
PubMedSearch : Cai_2019_AAPS.J_22_5
PubMedID: 31754920

Title : Development of a long-acting Fc-fused cocaine hydrolase with improved yield of protein expression - Chen_2019_Chem.Biol.Interact_13ChEPon_306_89
Author(s) : Chen X , Deng J , Zheng X , Zhang J , Zhou Z , Wei H , Zhan CG , Zheng F
Ref : Chemico-Biological Interactions , 306 :89 , 2019
Abstract : 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 approximately 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+/-7h in rats, but also a high yield of protein expression. Particularly, Fc(M3)-(PAPAP)2-CocH3 has a approximately 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.
ESTHER : Chen_2019_Chem.Biol.Interact_13ChEPon_306_89
PubMedSearch : Chen_2019_Chem.Biol.Interact_13ChEPon_306_89
PubMedID: 30986387

Title : Dimerization of human butyrylcholinesterase expressed in bacterium for development of a thermally stable bioscavenger of organophosphorus compounds - Cai_2019_Chem.Biol.Interact_13ChEPon_310_108756
Author(s) : Cai Y , Zhou S , Stewart MJ , Zheng F , Zhan CG
Ref : Chemico-Biological Interactions , 310 :108756 , 2019
Abstract : Human butyrylcholinesterase (BChE) is a widely distributed plasma enzyme. For decades, numerous research efforts have been directed at engineering BChE as a bioscavenger of organophosphorus insecticides and chemical warfare nerve agents. However, it has been a grand challenge to cost-efficiently produce BChE in large-scale. Recently reported studies have successfully designed a truncated BChE mutant (with amino-acid substitutions on 47 residues that are far away from the catalytic site), denoted as BChE-M47 for convenience, which can be expressed in E. coli without loss of its catalytic activity. In this study, we aimed to dimerize the truncated BChE mutant protein expressed in a prokaryotic system (E. coli) in order to further improve its thermal stability by introducing a pair of cross-subunit disulfide bonds to the BChE-M47 structure. Specifically, the E377C/A516C mutations were designed and introduced to BChE-M47, and the obtained new protein entity, denoted as BChE-M48, with a pair of cross-subunit disulfide bonds indeed exists as a dimer with significantly improved thermostability and unaltered catalytic activity and reactivity compared to BChE-M47. These results provide a new strategy for optimizing protein stability for production in a cost-efficient prokaryotic system. Our enzyme, BChE-M48, has a half-life of almost one week at a 37 degrees C, suggesting that it could be utilized as a highly stable bioscavenger of OP insecticides and chemical warfare nerve agents.
ESTHER : Cai_2019_Chem.Biol.Interact_13ChEPon_310_108756
PubMedSearch : Cai_2019_Chem.Biol.Interact_13ChEPon_310_108756
PubMedID: 31325422

Title : Assessment of phthalate ester residues and distribution patterns in Baijiu raw materials and Baijiu - Dong_2019_Food.Chem_283_508
Author(s) : Dong W , Guo R , Sun X , Li H , Zhao M , Zheng F , Sun J , Huang M , Wu J
Ref : Food Chem , 283 :508 , 2019
Abstract : Phthalate esters (PAEs) are harmful to human health and have been repeatedly identified in Baijiu samples. In our study, the distribution and degradation characteristics of 14 PAEs in Baijiu raw materials (BRMs) and Baijiu during distillation were detected using QuEChERS or vortex-assisted surfactant-enhanced-emulsification liquid-liquid micro-extraction (VSLLME) methods coupled with gas chromatography-mass spectrometry. The same five PAEs were detected in all tested samples, values ranged from 0.003 to 0.292 mg/kg; however, higher concentrations existed in BRMs compared to Baijiu samples. Using multivariate statistical analysis, detailed distinctions between different varieties of Baijiu and BRMs and separation-related PAE markers were revealed. PAEs concentration during Baijiu distillation showed a decreasing trend. The highest concentrations detected in distillate heads, were 1.6-, 2.3-, and 8.1-fold higher than those in heart1, heart2, and tail distillates, respectively. These findings revealed that PAEs may migrate from BRMs; moreover, that PAEs content can be regulated by distillation.
ESTHER : Dong_2019_Food.Chem_283_508
PubMedSearch : Dong_2019_Food.Chem_283_508
PubMedID: 30722905

Title : Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality - Wei_2018_Proc.Natl.Acad.Sci.U.S.A_115_E4151
Author(s) : Wei C , Yang H , Wang S , Zhao J , Liu C , Gao L , Xia E , Lu Y , Tai Y , She G , Sun J , Cao H , Tong W , Gao Q , Li Y , Deng W , Jiang X , Wang W , Chen Q , Zhang S , Li H , Wu J , Wang P , Li P , Shi C , Zheng F , Jian J , Huang B , Shan D , Shi M , Fang C , Yue Y , Li F , Li D , Wei S , Han B , Jiang C , Yin Y , Xia T , Zhang Z , Bennetzen JL , Zhao S , Wan X
Ref : Proc Natl Acad Sci U S A , 115 :E4151 , 2018
Abstract : Tea, one of the world's most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to approximately 0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred approximately 30 to 40 and approximately 90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.
ESTHER : Wei_2018_Proc.Natl.Acad.Sci.U.S.A_115_E4151
PubMedSearch : Wei_2018_Proc.Natl.Acad.Sci.U.S.A_115_E4151
PubMedID: 29678829
Gene_locus related to this paper: camsi-a0a4s4dr18 , camsi-a0a4s4etg9 , camsi-a0a4s4e3j5 , camsi-a0a4s4d2s5 , camsi-a0a4s4duc4 , camsi-a0a4v3wr80 , camsi-a0a4v3wpu4

Title : Catalytic Reaction Mechanism for Drug Metabolism in Human Carboxylesterase-1: Cocaine Hydrolysis Pathway - Yao_2018_Mol.Pharm_15_3871
Author(s) : Yao J , Chen X , Zheng F , Zhan CG
Ref : Mol Pharm , 15 :3871 , 2018
Abstract : Carboxylesterase-1 (CE-1) is a crucial enzyme responsible for metabolism/activation/inactivation of xenobiotics (therapeutic agents, prodrugs, abused drugs, and organophosphorus nerve agents etc.) and also involved in many other biological processes. In this study, we performed extensive computational modeling and simulations to understand the fundamental reaction mechanism of cocaine hydrolysis catalyzed by CE-1, revealing that CE-1-catalyzed cocaine hydrolysis follows a novel reaction pathway with only two reaction steps: a single-step acylation process and a single-step deacylation process. In the transition states of both single-step processes, the cocaine NH group joins the oxyanion hole to form an additional hydrogen bond with the negatively charged carbonyl oxygen atom of the cocaine. Thus, the transition states are stabilized by both intermolecular and intramolecular hydrogen bonds with the methyl ester of cocaine, specifically the carbonyl oxygen atom. The rate-limiting transition state is associated with the acylation process, and the activation free energy barrier was predicted to be 20.1 kcal/mol. Further, in vitro experimental kinetic analysis was performed for human CE-1-catalyzed cocaine hydrolysis. For CE-1-catalyzed cocaine hydrolysis, the computationally predicted free energy barrier (20.1 kcal/mol) is reasonably close to the experimentally derived turnover number ( kcat = 0.058 min(-1)), indicating the reasonability of the computational results. The obtained novel mechanistic insights are expected to benefit not only CE-1 related rational drug discovery but also future research on the catalytic mechanism of other esterases.
ESTHER : Yao_2018_Mol.Pharm_15_3871
PubMedSearch : Yao_2018_Mol.Pharm_15_3871
PubMedID: 30095924

Title : Kinetic characterization of cholinesterases and a therapeutically valuable cocaine hydrolase for their catalytic activities against heroin and its metabolite 6-monoacetylmorphine - Kim_2018_Chem.Biol.Interact_293_107
Author(s) : Kim K , Yao J , Jin Z , Zheng F , Zhan CG
Ref : Chemico-Biological Interactions , 293 :107 , 2018
Abstract : As the most popularly abused one of opioids, heroin is actually a prodrug. In the body, heroin is hydrolyzed/activated to 6-monoacetylmorphine (6-MAM) first and then to morphine to produce its toxic and physiological effects. It has been known that heroin hydrolysis to 6-MAM and morphine is accelerated by cholinesterases, including acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE). However, there has been controversy over the specific catalytic activities and functional significance of the cholinesterases, which requires for the more careful kinetic characterization under the same experimental conditions. Here we report the kinetic characterization of AChE, BChE, and a therapeutically promising cocaine hydrolase (CocH1) for heroin and 6-MAM hydrolyses under the same experimental conditions. It has been demonstrated that AChE and BChE have similar kcat values (2100 and 1840 min(-1), respectively) against heroin, but with a large difference in KM (2170 and 120muM, respectively). Both AChE and BChE can catalyze 6-MAM hydrolysis to morphine, with relatively lower catalytic efficiency compared to the heroin hydrolysis. CocH1 can also catalyze hydrolysis of heroin (kcat=2150 min(-1) and KM=245muM) and 6-MAM (kcat=0.223 min(-1) and KM=292muM), with relatively larger KM values and lower catalytic efficiency compared to BChE. Notably, the KM values of CocH1 against both heroin and 6-MAM are all much larger than previously reported maximum serum heroin and 6-MAM concentrations observed in heroin users, implying that the heroin use along with cocaine will not drastically affect the catalytic activity of CocH1 against cocaine in the CocH1-based enzyme therapy for cocaine abuse.
ESTHER : Kim_2018_Chem.Biol.Interact_293_107
PubMedSearch : Kim_2018_Chem.Biol.Interact_293_107
PubMedID: 30080993

Title : Metabolic Enzymes of Cocaine Metabolite Benzoylecgonine - Chen_2016_ACS.Chem.Biol_11_2186
Author(s) : Chen X , Zheng X , Zhan M , Zhou Z , Zhan CG , Zheng F
Ref : ACS Chemical Biology , 11 :2186 , 2016
Abstract : 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.
ESTHER : Chen_2016_ACS.Chem.Biol_11_2186
PubMedSearch : Chen_2016_ACS.Chem.Biol_11_2186
PubMedID: 27224254

Title : Effects of a cocaine hydrolase engineered from human butyrylcholinesterase on metabolic profile of cocaine in rats - Chen_2016_Chem.Biol.Interact_259_104
Author(s) : Chen X , Zheng X , Zhou Z , Zhan CG , Zheng F
Ref : Chemico-Biological Interactions , 259 :104 , 2016
Abstract : Accelerating cocaine metabolism through enzymatic hydrolysis at cocaine benzoyl ester is recognized as a promising therapeutic approach for cocaine abuse treatment. Our more recently designed A199S/F227A/S287G/A328W/Y332G mutant of human BChE, denoted as cocaine hydrolase-3 (CocH3), has a considerably improved catalytic efficiency against cocaine and has been proven active in blocking cocaine-induced toxicity and physiological effects. In the present study, we have further characterized the effects of CocH3 on the detailed metabolic profile of cocaine in rats administrated intravenously (IV) with 5 mg/kg cocaine, demonstrating that IV administration of 0.15 mg/kg CocH3 dramatically changed the metabolic profile of cocaine. Without CocH3 administration, the dominant cocaine-metabolizing pathway in rats was cocaine methyl ester hydrolysis to benzoylecgonine (BZE). With the CocH3 administration, the dominant cocaine-metabolizing pathway in rats became cocaine benzoyl ester hydrolysis to ecgonine methyl ester (EME), and the other two metabolic pathways (i.e. cocaine methyl ester hydrolysis to BZE and cocaine oxidation to norcocaine) became insignificant. The CocH3-catalyzed cocaine benzoyl ester hydrolysis to EME was so efficient such that the measured maximum blood cocaine concentration ( approximately 38 ng/ml) was significantly lower than the threshold blood cocaine concentration ( approximately 72 ng/ml) required to produce any measurable physiological effects.
ESTHER : Chen_2016_Chem.Biol.Interact_259_104
PubMedSearch : Chen_2016_Chem.Biol.Interact_259_104
PubMedID: 27154495

Title : Potential anti-obesity effects of a long-acting cocaine hydrolase - Zheng_2016_Chem.Biol.Interact_259_99
Author(s) : Zheng X , Deng J , Zhang T , Yao J , Zheng F , Zhan CG
Ref : Chemico-Biological Interactions , 259 :99 , 2016
Abstract : A long-acting cocaine hydrolase, known as CocH3-Fc(M3), engineered from human butyrylcholinesterase (BChE) was tested, in this study, for its potential anti-obesity effects. Mice on a high-fat diet gained significantly less body weight when treated weekly with 1 mg/kg CocH3-Fc(M3) compared to control mice, though their food intake was similar. There is no correlation between the average body weight and the average food intake, which is consistent with the previously reported observation in BChE knockout mice. In addition, molecular modeling was carried out to understand how ghrelin binds with CocH3, showing that ghrelin binds with CocH3 in a similar mode as ghrelin binding with wild-type human BChE. The similar binding structure mode explains why CocH3 has a similar catalytic activity against ghrelin.
ESTHER : Zheng_2016_Chem.Biol.Interact_259_99
PubMedSearch : Zheng_2016_Chem.Biol.Interact_259_99
PubMedID: 27163854

Title : Free energy profiles of cocaine esterase-cocaine binding process by molecular dynamics and potential of mean force simulations - Zhang_2016_Chem.Biol.Interact_259_142
Author(s) : Zhang Y , Huang X , Han K , Zheng F , Zhan CG
Ref : Chemico-Biological Interactions , 259 :142 , 2016
Abstract : The combined molecular dynamics (MD) and potential of mean force (PMF) simulations have been performed to determine the free energy profile of the CocE)-(+)-cocaine binding process in comparison with that of the corresponding CocE-(-)-cocaine binding process. According to the MD simulations, the equilibrium CocE-(+)-cocaine binding mode is similar to the CocE-(-)-cocaine binding mode. However, based on the simulated free energy profiles, a significant free energy barrier ( approximately 5 kcal/mol) exists in the CocE-(+)-cocaine binding process whereas no obvious free energy barrier exists in the CocE-(-)-cocaine binding process, although the free energy barrier of approximately 5 kcal/mol is not high enough to really slow down the CocE-(+)-cocaine binding process. In addition, the obtained free energy profiles also demonstrate that (+)-cocaine and (-)-cocaine have very close binding free energies with CocE, with a negligible difference ( approximately 0.2 kcal/mol), which is qualitatively consistent with the nearly same experimental KM values of the CocE enzyme for (+)-cocaine and (-)-cocaine. The consistency between the computational results and available experimental data suggests that the mechanistic insights obtained from this study are reasonable.
ESTHER : Zhang_2016_Chem.Biol.Interact_259_142
PubMedSearch : Zhang_2016_Chem.Biol.Interact_259_142
PubMedID: 27163853

Title : Unexpected Reaction Pathway for butyrylcholinesterase-catalyzed inactivation of hunger hormone ghrelin - Yao_2016_Sci.Rep_6_22322
Author(s) : Yao J , Yuan Y , Zheng F , Zhan CG
Ref : Sci Rep , 6 :22322 , 2016
Abstract : Extensive computational modeling and simulations have been carried out, in the present study, to uncover the fundamental reaction pathway for butyrylcholinesterase (BChE)-catalyzed hydrolysis of ghrelin, demonstrating that the acylation process of BChE-catalyzed hydrolysis of ghrelin follows an unprecedented single-step reaction pathway and the single-step acylation process is rate-determining. The free energy barrier (18.8 kcal/mol) calculated for the rate-determining step is reasonably close to the experimentally-derived free energy barrier (~19.4 kcal/mol), suggesting that the obtained mechanistic insights are reasonable. The single-step reaction pathway for the acylation is remarkably different from the well-known two-step acylation reaction pathway for numerous ester hydrolysis reactions catalyzed by a serine esterase. This is the first time demonstrating that a single-step reaction pathway is possible for an ester hydrolysis reaction catalyzed by a serine esterase and, therefore, one no longer can simply assume that the acylation process must follow the well-known two-step reaction pathway.
ESTHER : Yao_2016_Sci.Rep_6_22322
PubMedSearch : Yao_2016_Sci.Rep_6_22322
PubMedID: 26922910

Title : Kinetic characterization of a cocaine hydrolase engineered from mouse butyrylcholinesterase - Chen_2015_Biochem.J_466_243
Author(s) : Chen X , Huang X , Geng L , Xue L , Hou S , Zheng X , Brimijoin S , Zheng F , Zhan CG
Ref : Biochemical Journal , 466 :243 , 2015
Abstract : 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.
ESTHER : Chen_2015_Biochem.J_466_243
PubMedSearch : Chen_2015_Biochem.J_466_243
PubMedID: 25486543
Gene_locus related to this paper: human-BCHE

Title : Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification - Fang_2014_ACS.Chem.Biol_9_1764
Author(s) : Fang L , Chow KM , Hou S , Xue L , Chen X , Rodgers DW , Zheng F , Zhan CG
Ref : ACS Chemical Biology , 9 :1764 , 2014
Abstract : 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.
ESTHER : Fang_2014_ACS.Chem.Biol_9_1764
PubMedSearch : Fang_2014_ACS.Chem.Biol_9_1764
PubMedID: 24919140
Gene_locus related to this paper: rhosm-cocE

Title : Modeling in vitro inhibition of butyrylcholinesterase using molecular docking, multi-linear regression and artificial neural network approaches - Zheng_2014_Bioorg.Med.Chem_22_538
Author(s) : Zheng F , Zhan M , Huang X , Abdul Hameed MD , Zhan CG
Ref : Bioorganic & Medicinal Chemistry , 22 :538 , 2014
Abstract : Butyrylcholinesterase (BChE) has been an important protein used for development of anti-cocaine medication. Through computational design, BChE mutants with approximately 2000-fold improved catalytic efficiency against cocaine have been discovered in our lab. To study drug-enzyme interaction it is important to build mathematical model to predict molecular inhibitory activity against BChE. This report presents a neural network (NN) QSAR study, compared with multi-linear regression (MLR) and molecular docking, on a set of 93 small molecules that act as inhibitors of BChE by use of the inhibitory activities (pIC50 values) of the molecules as target values. The statistical results for the linear model built from docking generated energy descriptors were: r(2)=0.67, rmsd=0.87, q(2)=0.65 and loormsd=0.90; the statistical results for the ligand-based MLR model were: r(2)=0.89, rmsd=0.51, q(2)=0.85 and loormsd=0.58; the statistical results for the ligand-based NN model were the best: r(2)=0.95, rmsd=0.33, q(2)=0.90 and loormsd=0.48, demonstrating that the NN is powerful in analysis of a set of complicated data. As BChE is also an established drug target to develop new treatment for Alzheimer's disease (AD). The developed QSAR models provide tools for rationalizing identification of potential BChE inhibitors or selection of compounds for synthesis in the discovery of novel effective inhibitors of BChE in the future.
ESTHER : Zheng_2014_Bioorg.Med.Chem_22_538
PubMedSearch : Zheng_2014_Bioorg.Med.Chem_22_538
PubMedID: 24290065

Title : Kinetic characterization of human butyrylcholinesterase mutants for the hydrolysis of cocaethylene - Hou_2014_Biochem.J_460_447
Author(s) : Hou S , Zhan M , Zheng X , Zhan CG , Zheng F
Ref : Biochemical Journal , 460 :447 , 2014
Abstract : 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 muM and kcat/KM=4.40x105 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 muM and kcat/KM=3.79x108 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.
ESTHER : Hou_2014_Biochem.J_460_447
PubMedSearch : Hou_2014_Biochem.J_460_447
PubMedID: 24870023

Title : GW1929 inhibits alpha7 nAChR expression through PPARgamma-independent activation of p38 MAPK and inactivation of PI3-K\/mTOR: The role of Egr-1 - Hahn_2014_Cell.Signal_26_730
Author(s) : Hahn SS , Tang Q , Zheng F , Zhao S , Wu J
Ref : Cell Signal , 26 :730 , 2014
Abstract : Studies demonstrated that peroxisome proliferator-activated receptor gamma (PPARgamma) ligands reduce nicotine-induced non small cell lung carcinoma (NSCLC) cell growth through inhibition of nicotinic acetylcholine receptor (nAChR) mediated signaling pathways. However, the mechanisms by which PPARgamma ligands inhibited nAChR expression remain elucidated. Here, we show that GW1929, a synthetic PPARgamma ligand, not only inhibited but also antagonized the stimulatory effect of acetylcholine on NSCLC cell proliferation. Interestingly, GW1929 inhibited alpha7 nAChR expression, which was not blocked by GW9662, an antagonist of PPARgamma, or by PPARgamma siRNA, but was abrogated by the p38 MPAK inhibitor SB239063. GW1929 reduced the promoter activity of alpha7 nAChR and induced early growth response-1 (Egr-1) protein expression, which was overcame by SB239063, but enhanced by inhibitors of PI3-K and mTOR. Silencing of Egr-1 blocked, while overexpression of Egr-1 enhanced, the effect of GW1929 on alpha7 nAChR expression and promoter activity. Finally, GW1929 induced Egr-1 bound to specific DNA areas in the alpha7 nAChR gene promoter. Collectively, these results demonstrate that GW1929 not only inhibits but also antagonizes Ach-induced NSCLC cell growth by inhibition of alpha7 nAChR expression through PPARgamma-independent signals that are associated with activation of p38 MPAK and inactivation of PI3-K/mTOR, followed by inducing Egr-1 protein and Egr-1 binding activity in the alpha7 nAChR gene promoter. By downregulation of the alpha7 nAchR, GW1929 blocks cholinergic signaling and inhibits NSCLC cell growth.
ESTHER : Hahn_2014_Cell.Signal_26_730
PubMedSearch : Hahn_2014_Cell.Signal_26_730
PubMedID: 24412748

Title : Kinetic characterization of high-activity mutants of human butyrylcholinesterase for the cocaine metabolite norcocaine - Zhan_2014_Biochem.J_457_197
Author(s) : Zhan M , Hou S , Zhan CG , Zheng F
Ref : Biochemical Journal , 457 :197 , 2014
Abstract : 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 muM and kcat/Km=1.87x105 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 muM and kcat/Km=2.01x108 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.
ESTHER : Zhan_2014_Biochem.J_457_197
PubMedSearch : Zhan_2014_Biochem.J_457_197
PubMedID: 24125115

Title : A highly efficient cocaine-detoxifying enzyme obtained by computational design - Zheng_2014_Nat.Commun_5_3457
Author(s) : Zheng F , Xue L , Hou S , Liu J , Zhan M , Yang W , Zhan CG
Ref : Nat Commun , 5 :3457 , 2014
Abstract : 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.
ESTHER : Zheng_2014_Nat.Commun_5_3457
PubMedSearch : Zheng_2014_Nat.Commun_5_3457
PubMedID: 24643289
Gene_locus related to this paper: human-BCHE

Title : Amino-acid mutations to extend the biological half-life of a therapeutically valuable mutant of human butyrylcholinesterase - Fang_2014_Chem.Biol.Interact_214C_18
Author(s) : Fang L , Hou S , Xue L , Zheng F , Zhan CG
Ref : Chemico-Biological Interactions , 214C :18 , 2014
Abstract : 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.
ESTHER : Fang_2014_Chem.Biol.Interact_214C_18
PubMedSearch : Fang_2014_Chem.Biol.Interact_214C_18
PubMedID: 24582612
Gene_locus related to this paper: human-BCHE

Title : Joint neurodevelopmental and behavioral effects of nonylphenol and estradiol on F1 male rats - Jie_2013_Int.J.Environ.Health.Res_23_321
Author(s) : Jie Y , Fan QY , Binli H , Biao Z , Zheng F , Jianmei L , Jie X
Ref : Int J Environ Health Res , 23 :321 , 2013
Abstract : The purpose of present study is to examine whether gestational exposure of two major environmental endocrine-disrupting chemicals, nonylphenol (NP) and estradiol (E2), would affect nervous system development of offspring rats and explore the joint effects of NP and E2. After impregnation, dams were assigned to seven groups. The first and second groups received gavage with NP at dose levels of 50 mg/kg/day (NP-L) and 100 mg/kg/day (NP-H); the third and fourth groups were gavaged with E2 at dose levels of 10 mug/kg/day (E2-L) and 20 mug/kg/day (E2-H); the fifth and sixth groups were gavaged with joint NP and E2 [NP 50 mg/kg/day + E2 10 mug/kg/day (NP-E2-L) and NP 100 mg/kg/day+E2 20 mug/kg/day (NP-E2-H)] dissolved in groundnut oil; and the seventh group was orally administered with groundnut oil alone (vehicle control; 2 ml/kg/day), respectively, daily from gestational days 9 to 15 (transplacental exposures). Compared to the control, exclusive NP and E2 treatment groups, joint exposure to NP-E2-L and NP-E2-H has both produced a significant decrease in mean litter size and number of live pups per litter in dams; Offspring rats spent more time to perform cliff-drop aversion reflex, surface righting reflex, air righting reflex, auditory startle, and visual placing; In Morris water maze task, an increased escape latency was presented in offspring rats; In step-down avoidance test, offspring rats jointly exposed to NP and E2 spent more reaction time. Decrease in acetylcholinesterase activity and increase in choline acetyltransferase activity were observed in the hippocampus of offspring rats. Gestational joint exposure to NP and E2 might induce nervous development impairment of offspring rats. Moreover, additive toxic effects of NP and E2 on nervous development have been identified among offspring rats as well.
ESTHER : Jie_2013_Int.J.Environ.Health.Res_23_321
PubMedSearch : Jie_2013_Int.J.Environ.Health.Res_23_321
PubMedID: 23289371

Title : Catalytic activities of a cocaine hydrolase engineered from human butyrylcholinesterase against (+)- and (-)-cocaine - Xue_2013_Chem.Biol.Interact_203_57
Author(s) : Xue L , Hou S , Yang W , Fang L , Zheng F , Zhan CG
Ref : Chemico-Biological Interactions , 203 :57 , 2013
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 [(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.
ESTHER : Xue_2013_Chem.Biol.Interact_203_57
PubMedSearch : Xue_2013_Chem.Biol.Interact_203_57
PubMedID: 22917637

Title : Preparation and in vivo characterization of a cocaine hydrolase engineered from human butyrylcholinesterase for metabolizing cocaine - Xue_2013_Biochem.J_453_447
Author(s) : Xue L , Hou S , Tong M , Fang L , Chen X , Jin Z , Tai HH , Zheng F , Zhan CG
Ref : Biochemical Journal , 453 :447 , 2013
Abstract : 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=3.1 muM) specifically for cocaine, and the mutant was proven effective in protecting mice from acute cocaine toxicity of a lethal dose of cocaine (180 mg/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 in vivo 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 1 min 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.
ESTHER : Xue_2013_Biochem.J_453_447
PubMedSearch : Xue_2013_Biochem.J_453_447
PubMedID: 23849058

Title : Substrate selectivity of high-activity mutants of human butyrylcholinesterase - Hou_2013_Org.Biomol.Chem_11_7477
Author(s) : Hou S , Xue L , Yang W , Fang L , Zheng F , Zhan CG
Ref : Org Biomol Chem , 11 :7477 , 2013
Abstract : 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.
ESTHER : Hou_2013_Org.Biomol.Chem_11_7477
PubMedSearch : Hou_2013_Org.Biomol.Chem_11_7477
PubMedID: 24077614

Title : A model of glycosylated human butyrylcholinesterase - Fang_2013_Mol.Biosyst_10_348
Author(s) : Fang L , Zheng F , Zhan CG
Ref : Mol Biosyst , 10 :348 , 2013
Abstract : Human butyrylcholinesterase (BChE) and its mutants have shown great potential in treating cocaine overdose and addiction. In order to effectively suppress cocaine reward in the brain for a long period of time after an exogenous cocaine hydrolase administration, the therapeutic enzyme should have not only a high catalytic efficiency against cocaine, but also a sufficiently long circulation time. It has been known that PEGylation (covalent attachment of polyethylene glycol) modification of a therapeutic protein can prolong the biological half-life of the protein without affecting its biological function. However, the asparagine-linked glycans on the surface of glycosylated BChE may interfere with the PEGylation modification. In this study, we built a three-dimensional (3D) model of glycosylated human BChE to investigate the influence of glycans on the PEGylation modification. Glycans did not change the overall stability of the BChE structure, but could increase the flexibility of some local structures. For further evaluating the accessibility of the PEGylation reaction sites, particularly lysine residues, on the protein surface, we calculated the Solvent Accessible Surface Areas (SASAs) of these residues. The results indicate that some lysine residues show a significant decrease in SASA due to the direct or indirect influence of their surrounding glycans. The results also indicate that PEGylation reaction agents with smaller functional groups could have a better chance to react with lysine residues. This investigation provides a structural basis for rational engineering of human BChE and its mutants as therapeutic candidates.
ESTHER : Fang_2013_Mol.Biosyst_10_348
PubMedSearch : Fang_2013_Mol.Biosyst_10_348
PubMedID: 24327294

Title : Cocaine esterase-cocaine binding process and the free energy profiles by molecular dynamics and potential of mean force simulations - Huang_2012_J.Phys.Chem.B_116_3361
Author(s) : Huang X , Zhao X , Zheng F , Zhan CG
Ref : J Phys Chem B , 116 :3361 , 2012
Abstract : The combined molecular dynamics (MD) and potential of mean force (PMF) simulations have been performed to determine the free energy profiles for the binding process of (-)-cocaine interacting with wild-type cocaine esterase (CocE) and its mutants (T172R/G173Q and L119A/L169K/G173Q). According to the MD simulations, the general protein-(-)-cocaine binding mode is not affected by the mutations; e.g.. the benzoyl group of (-)-cocaine is always bound in a subsite composed of aromatic residues W151, W166, F261, and F408 and hydrophobic residue L407, while the carbonyl oxygen on the benzoyl group of (-)-cocaine is hydrogen-bonded with the oxyanion-hole residues Y44 and Y118. According to the PMF-calculated free energy profiles for the binding process, the binding free energies for (-)-cocaine with the wild-type, T172R/G173Q, and L119A/L169K/G173Q CocEs are predicted to be -6.4, -6.2, and -5.0 kcal/mol, respectively. The computational predictions are supported by experimental kinetic data, as the calculated binding free energies are in good agreement with the experimentally derived binding free energies, i.e., -7.2, -6.7, and -4.8 kcal/mol for the wild-type, T172R/G173Q, and L119A/L169K/G173Q, respectively. The reasonable agreement between the computational and experimental data suggests that the PMF simulations may be used as a valuable tool in new CocE mutant design that aims to decrease the Michaelis-Menten constant of the enzyme for (-)-cocaine.
ESTHER : Huang_2012_J.Phys.Chem.B_116_3361
PubMedSearch : Huang_2012_J.Phys.Chem.B_116_3361
PubMedID: 22385120

Title : Modeling of pharmacokinetics of cocaine in human reveals the feasibility for development of enzyme therapies for drugs of abuse - Zheng_2012_PLoS.Comput.Biol_8_e1002610
Author(s) : Zheng F , Zhan CG
Ref : PLoS Comput Biol , 8 :e1002610 , 2012
Abstract : A promising strategy for drug abuse treatment is to accelerate the drug metabolism by administration of a drug-metabolizing enzyme. The question is how effectively an enzyme can actually prevent the drug from entering brain and producing physiological effects. In the present study, we have developed a pharmacokinetic model through a combined use of in vitro kinetic parameters and positron emission tomography data in human to examine the effects of a cocaine-metabolizing enzyme in plasma on the time course of cocaine in plasma and brain of human. Without an exogenous enzyme, cocaine half-lives in both brain and plasma are almost linearly dependent on the initial cocaine concentration in plasma. The threshold concentration of cocaine in brain required to produce physiological effects has been estimated to be 0.22+/-0.07 microM, and the threshold area under the cocaine concentration versus time curve (AUC) value in brain (denoted by AUC2(infinity)) required to produce physiological effects has been estimated to be 7.9+/-2.7 microM.min. It has been demonstrated that administration of a cocaine hydrolase/esterase (CocH/CocE) can considerably decrease the cocaine half-lives in both brain and plasma, the peak cocaine concentration in brain, and the AUC2(infinity). The estimated maximum cocaine plasma concentration which a given concentration of drug-metabolizing enzyme can effectively prevent from entering brain and producing physiological effects can be used to guide future preclinical/clinical studies on cocaine-metabolizing enzymes. Understanding of drug-metabolizing enzymes is key to the science of pharmacokinetics. The general insights into the effects of a drug-metabolizing enzyme on drug kinetics in human should be valuable also in future development of enzyme therapies for other drugs of abuse.
ESTHER : Zheng_2012_PLoS.Comput.Biol_8_e1002610
PubMedSearch : Zheng_2012_PLoS.Comput.Biol_8_e1002610
PubMedID: 22844238

Title : Crystal structure of FAS thioesterase domain with polyunsaturated fatty acyl adduct and inhibition by dihomo-gamma-linolenic acid - Zhang_2011_Proc.Natl.Acad.Sci.U.S.A_108_15757
Author(s) : Zhang W , Chakravarty B , Zheng F , Gu Z , Wu H , Mao J , Wakil SJ , Quiocho FA
Ref : Proc Natl Acad Sci U S A , 108 :15757 , 2011
Abstract : Human fatty acid synthase (hFAS) is a homodimeric multidomain enzyme that catalyzes a series of reactions leading to the de novo biosynthesis of long-chain fatty acids, mainly palmitate. The carboxy-terminal thioesterase (TE) domain determines the length of the fatty acyl chain and its ultimate release by hydrolysis. Because of the upregulation of hFAS in a variety of cancers, it is a target for antiproliferative agent development. Dietary long-chain polyunsaturated fatty acids (PUFAs) have been known to confer beneficial effects on many diseases and health conditions, including cancers, inflammations, diabetes, and heart diseases, but the precise molecular mechanisms involved have not been elucidated. We report the 1.48 A crystal structure of the hFAS TE domain covalently modified and inactivated by methyl gamma-linolenylfluorophosphonate. Whereas the structure confirmed the phosphorylation by the phosphonate head group of the active site serine, it also unexpectedly revealed the binding of the 18-carbon polyunsaturated gamma-linolenyl tail in a long groove-tunnel site, which itself is formed mainly by the emergence of an alpha helix (the "helix flap"). We then found inhibition of the TE domain activity by the PUFA dihomo-gamma-linolenic acid; gamma- and alpha-linolenic acids, two popular dietary PUFAs, were less effective. Dihomo-gamma-linolenic acid also inhibited fatty acid biosynthesis in 3T3-L1 preadipocytes and selective human breast cancer cell lines, including SKBR3 and MDAMB231. In addition to revealing a novel mechanism for the molecular recognition of a polyunsaturated fatty acyl chain, our results offer a new framework for developing potent FAS inhibitors as therapeutics against cancers and other diseases.
ESTHER : Zhang_2011_Proc.Natl.Acad.Sci.U.S.A_108_15757
PubMedSearch : Zhang_2011_Proc.Natl.Acad.Sci.U.S.A_108_15757
PubMedID: 21908709
Gene_locus related to this paper: human-FASN

Title : Design, synthesis and interaction at the vesicular monoamine transporter-2 of lobeline analogs: potential pharmacotherapies for the treatment of psychostimulant abuse - Crooks_2011_Curr.Top.Med.Chem_11_1103
Author(s) : Crooks PA , Zheng G , Vartak AP , Culver JP , Zheng F , Horton DB , Dwoskin LP
Ref : Curr Top Med Chem , 11 :1103 , 2011
Abstract : The vesicular monoamine transporter-2 (VMAT2) is considered as a new target for the development of novel therapeutics to treat psychostimulant abuse. Current information on the structure, function and role of VMAT2 in psychostimulant abuse are presented. Lobeline, the major alkaloidal constituent of Lobelia inflata, interacts with nicotinic receptors and with VMAT2. Numerous studies have shown that lobeline inhibits both the neurochemical and behavioral effects of amphetamine in rodents, and behavioral studies demonstrate that lobeline has potential as a pharmacotherapy for psychostimulant abuse. Systematic structural modification of the lobeline molecule is described with the aim of improving selectivity and affinity for VMAT2 over neuronal nicotinic acetylcholine receptors and other neurotransmitter transporters. This has led to the discovery of more potent and selective ligands for VMAT2. In addition, a computational neural network analysis of the affinity of these lobeline analogs for VMAT2 has been carried out, which provides computational models that have predictive value in the rational design of VMAT2 ligands and is also useful in identifying drug candidates from virtual libraries for subsequent synthesis and evaluation.
ESTHER : Crooks_2011_Curr.Top.Med.Chem_11_1103
PubMedSearch : Crooks_2011_Curr.Top.Med.Chem_11_1103
PubMedID: 21050177

Title : Enzyme-therapy approaches for the treatment of drug overdose and addiction -
Author(s) : Zheng F , Zhan CG
Ref : Future Med Chem , 3 :9 , 2011
PubMedID: 21428822

Title : Human butyrylcholinesterase-cocaine binding pathway and free energy profiles by molecular dynamics and potential of mean force simulations - Huang_2011_J.Phys.Chem.B_115_11254
Author(s) : Huang X , Zheng F , Zhan CG
Ref : J Phys Chem B , 115 :11254 , 2011
Abstract : In the present study, we have performed combined molecular dynamics and potential of mean force (PMF) simulations to determine the enzyme-substrate (ES) binding pathway and the corresponding free energy profiles for wild-type butyrylcholinesterase (BChE) binding with (-)/(+)-cocaine and for the A328W/Y332G mutant binding with (-)-cocaine. According to the PMF simulations, for each ES binding system, the substrate first binds with the enzyme at a peripheral anionic site around the entrance of the active-site gorge to form the first ES complex (ES1-like) during the binding process. Further evolution from the ES1-like complex to the nonprereactive ES complex is nearly barrierless, with a free energy barrier lower than 1.0 kcal/mol. So, the nonprereactive ES binding process should be very fast. The rate-determining step of the entire ES binding process is the subsequent evolution from the nonprereactive ES complex to the prereactive ES complex. Further accounting for the entire ES binding process, the PMF-based simulations qualitatively reproduced the relative order of the experimentally derived binding free energies (DeltaG(bind)), although the simulations systematically overestimated the magnitude of the binding affinity and systematically underestimated the differences between the DeltaG(bind) values. The obtained structural and energetic insights into the entire ES binding process provide a valuable base for future rational design of high-activity mutants of BChE as candidates for an enzyme therapy for cocaine overdose and abuse.
ESTHER : Huang_2011_J.Phys.Chem.B_115_11254
PubMedSearch : Huang_2011_J.Phys.Chem.B_115_11254
PubMedID: 21902185

Title : Design, preparation, and characterization of high-activity mutants of human butyrylcholinesterase specific for detoxification of cocaine - Xue_2011_Mol.Pharmacol_79_290
Author(s) : Xue L , Ko MC , Tong M , Yang W , Hou S , Fang L , Liu J , Zheng F , Woods JH , Tai HH , Zhan CG
Ref : Molecular Pharmacology , 79 :290 , 2011
Abstract : 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.
ESTHER : Xue_2011_Mol.Pharmacol_79_290
PubMedSearch : Xue_2011_Mol.Pharmacol_79_290
PubMedID: 20971807

Title : Free energy perturbation simulation on transition states and high-activity mutants of human butyrylcholinesterase for (-)-cocaine hydrolysis - Yang_2010_J.Phys.Chem.B_114_10889
Author(s) : Yang W , Pan Y , Fang L , Gao D , Zheng F , Zhan CG
Ref : J Phys Chem B , 114 :10889 , 2010
Abstract : A unified computational approach based on free energy perturbation (FEP) simulations of transition states has been employed to calculate the mutation-caused shifts of the free energy change from the free enzyme to the rate-determining transition state for (-)-cocaine hydrolysis catalyzed by the currently most promising series of mutants of human butyrylcholinesterase (BChE) that contain the A199S/A328W/Y332G mutations. The FEP simulations were followed by Michaelis-Menten kinetics analysis determining the individual k(cat) and K(M) values missing for the A199S/F227A/A328W/Y332G mutant in this series. The calculated mutation-caused shifts of the free energy change from the free enzyme to the rate-determining transition state are in good agreement with the experimental kinetic data, demonstrating that the unified computational approach based on the FEP simulations of the transition states may be valuable for future computational design of new BChE mutants with a further improved catalytic efficiency against (-)-cocaine.
ESTHER : Yang_2010_J.Phys.Chem.B_114_10889
PubMedSearch : Yang_2010_J.Phys.Chem.B_114_10889
PubMedID: 20677742

Title : Design of high-activity mutants of human butyrylcholinesterase against (-)-cocaine: structural and energetic factors affecting the catalytic efficiency - Zheng_2010_Biochemistry_49_9113
Author(s) : Zheng F , Yang W , Xue L , Hou S , Liu J , Zhan CG
Ref : Biochemistry , 49 :9113 , 2010
Abstract : 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 approximately 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.
ESTHER : Zheng_2010_Biochemistry_49_9113
PubMedSearch : Zheng_2010_Biochemistry_49_9113
PubMedID: 20886866

Title : Reaction pathway and free energy profile for prechemical reaction step of human butyrylcholinesterase-catalyzed hydrolysis of (-)-cocaine by combined targeted molecular dynamics and potential of mean force simulations - Huang_2010_J.Phys.Chem.B_114_13545
Author(s) : Huang X , Pan Y , Zheng F , Zhan CG
Ref : J Phys Chem B , 114 :13545 , 2010
Abstract : Combined targeted molecular dynamics (TMD) and potential of mean force (PMF) simulations have been carried out to uncover the detailed pathway and determine the corresponding free energy profile for the structural transformation from the nonprereactive butyrylcholinesterase (BChE)-(-)-cocaine binding to the prereactive BChE-(-)-cocaine binding associated with the (-)-cocaine rotation in the binding pocket of BChE. It has been shown that the structural transformation involves two transition states (TS1(rot) and TS2(rot)). TS1(rot) is mainly associated with the deformation of the nonprereactive complex, whereas TS2(rot) is mainly associated with the formation of the prereactive complex. It has also been demonstrated that the A328W/Y332G mutation significantly reduces the steric hindrance for (-)-cocaine rotation in the binding pocket of BChE and, thus, decreases the free energy barrier for the structural transformation from the nonprereactive binding to the prereactive binding. The calculated relative free energy barriers are all consistent with available experimental kinetic data. The new mechanistic insights obtained and the novel computational protocol tested in this study should be valuable for future computational design of high-activity mutants of BChE. The general computational strategy and approach based on the combined TMD and PMF simulations may be also valuable in computational studies of detailed pathways and free energy profiles for other similar mechanistic problems involving ligand rotation or another type of structural transformation in the binding pocket of a protein.
ESTHER : Huang_2010_J.Phys.Chem.B_114_13545
PubMedSearch : Huang_2010_J.Phys.Chem.B_114_13545
PubMedID: 20883001

Title : A novel selective muscarinic acetylcholine receptor subtype 1 antagonist reduces seizures without impairing hippocampus-dependent learning - Sheffler_2009_Mol.Pharmacol_76_356
Author(s) : Sheffler DJ , Williams R , Bridges TM , Xiang Z , Kane AS , Byun NE , Jadhav S , Mock MM , Zheng F , Lewis LM , Jones CK , Niswender CM , Weaver CD , Lindsley CW , Conn PJ
Ref : Molecular Pharmacology , 76 :356 , 2009
Abstract : Previous studies suggest that selective antagonists of specific subtypes of muscarinic acetylcholine receptors (mAChRs) may provide a novel approach for the treatment of certain central nervous system (CNS) disorders, including epileptic disorders, Parkinson's disease, and dystonia. Unfortunately, previously reported antagonists are not highly selective for specific mAChR subtypes, making it difficult to definitively establish the functional roles and therapeutic potential for individual subtypes of this receptor subfamily. The M(1) mAChR is of particular interest as a potential target for treatment of CNS disorders. We now report the discovery of a novel selective antagonist of M(1) mAChRs, termed VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl)piperazin-1-yl)propyl)-benzo[c][1,2,5]thiadiazole-4 sulfonamide]. Equilibrium radioligand binding and functional studies demonstrate a greater than 75-fold selectivity of VU0255035 for M(1) mAChRs relative to M(2)-M(5). Molecular pharmacology and mutagenesis studies indicate that VU0255035 is a competitive orthosteric antagonist of M(1) mAChRs, a surprising finding given the high level of M(1) mAChR selectivity relative to other orthosteric antagonists. Whole-cell patch-clamp recordings demonstrate that VU0255035 inhibits potentiation of N-methyl-D-aspartate receptor currents by the muscarinic agonist carbachol in hippocampal pyramidal cells. VU0255035 has excellent brain penetration in vivo and is efficacious in reducing pilocarpine-induced seizures in mice. We were surprised to find that doses of VU0255035 that reduce pilocarpine-induced seizures do not induce deficits in contextual freezing, a measure of hippocampus-dependent learning that is disrupted by nonselective mAChR antagonists. Taken together, these data suggest that selective antagonists of M(1) mAChRs do not induce the severe cognitive deficits seen with nonselective mAChR antagonists and could provide a novel approach for the treatment certain of CNS disorders.
ESTHER : Sheffler_2009_Mol.Pharmacol_76_356
PubMedSearch : Sheffler_2009_Mol.Pharmacol_76_356
PubMedID: 19407080

Title : Inactivation of dipeptidyl peptidase IV attenuates the virulence of Streptococcus suis serotype 2 that causes streptococcal toxic shock syndrome - Ge_2009_Curr.Microbiol_59_248
Author(s) : Ge J , Feng Y , Ji H , Zhang H , Zheng F , Wang C , Yin Z , Pan X , Tang J
Ref : Curr Microbiol , 59 :248 , 2009
Abstract : Di-peptidyl peptidase IV (DPP IV), originally recognized as CD26 in eukaryotic cells, is distributed widely in microbial pathogens, including Streptococcus suis (S. suis), an emerging zoonotic agent. However, the role of DPP IV in S. suis virulence remains unclear. Here, we identified a dpp IV homologue from highly invasive isolate of S. suis 2 (SS2) causing streptococcal toxic shock syndrome (STSS). Enzymatic assays reproduced its enzymatic activity of dpp IV protein product as a functional DPP IV, and ELISA analysis demonstrated that SS2 DPP IV can interact with human fibronectin. An isogenic SS2 mutant of dpp IV, Delta dpp IV, was obtained by homologous recombination. Experimental animal infection suggested that an inactivation of dpp IV attenuates greatly its high virulence of Chinese virulent strains of SS2. Functional complementation can restore this defect in SS2 pathogenicity. To our knowledge, it may confirm, for the first time, that DPP IV contributes to SS2 virulence.
ESTHER : Ge_2009_Curr.Microbiol_59_248
PubMedSearch : Ge_2009_Curr.Microbiol_59_248
PubMedID: 19484301

Title : Discovery and development of a potent and highly selective small molecule muscarinic acetylcholine receptor subtype I (mAChR 1 or M1) antagonist in vitro and in vivo probe - Weaver_2009_Curr.Top.Med.Chem_9_1217
Author(s) : Weaver CD , Sheffler DJ , Lewis LM , Bridges TM , Williams R , Nalywajko NT , Kennedy JP , Mulder MM , Jadhav S , Aldrich LA , Jones CK , Marlo JE , Niswender CM , Mock MM , Zheng F , Conn PJ , Lindsley CW
Ref : Curr Top Med Chem , 9 :1217 , 2009
Abstract : This article describes the discovery and development of the first highly selective, small molecule antagonist of the muscarinic acetylcholine receptor subtype I (mAChR1 or M(1)). An M(1) functional, cell-based calcium-mobilization assay identified three distinct chemical series with initial selectivity for M(1) versus M(4). An iterative parallel synthesis approach was employed to optimize all three series in parallel, which led to the development of novel microwave-assisted chemistry and provided important take home lessons for probe development projects. Ultimately, this effort produced VU0255035, a potent (IC(50) = 130 nM) and selective (>75-fold vs. M(2)-M(5) and > 10 microM vs. a panel of 75 GPCRs, ion channels and transporters) small molecule M(1) antagonist. Further profiling demonstrated that VU0255035 was centrally penetrant (Brain(AUC)/Plasma(AUC) of 0.48) and active in vivo, rendering it acceptable as both an in vitro and in vivo MLSCN/ MLPCN probe molecule for studying and dissecting M(1) function.
ESTHER : Weaver_2009_Curr.Top.Med.Chem_9_1217
PubMedSearch : Weaver_2009_Curr.Top.Med.Chem_9_1217
PubMedID: 19807667

Title : Recent progress in protein drug design and discovery with a focus on novel approaches to the development of anti-cocaine medications - Zheng_2009_Future.Med.Chem_1_515
Author(s) : Zheng F , Zhan CG
Ref : Future Med Chem , 1 :515 , 2009
Abstract : Cocaine is highly addictive and no anti-cocaine medication is currently available. Accelerating cocaine metabolism, producing biologically inactive metabolites, is recognized as an ideal anti-cocaine medication strategy, especially for the treatment of acute cocaine toxicity. However, currently known wild-type enzymes have either too low a catalytic efficiency against the abused cocaine, in other words (-)-cocaine, or the in vivo half-life is too short. Novel computational strategies and design approaches have been developed recently to design and discover thermostable or high-activity mutants of enzymes based on detailed structures and catalytic/inactivation mechanisms. The structure- and mechanism-based computational design efforts have led to the discovery of high-activity mutants of butyrylcholinesterase and thermostable mutants of cocaine esterase as promising anti-cocaine therapeutics. The structure- and mechanism-based computational strategies and design approaches may be used to design high-activity and/or thermostable mutants of many other proteins that have clear therapeutic potentials and to design completely new therapeutic enzymes.
ESTHER : Zheng_2009_Future.Med.Chem_1_515
PubMedSearch : Zheng_2009_Future.Med.Chem_1_515
PubMedID: 20161378

Title : First-Principles Determination of Molecular Conformations of Indolizidine (-)-235B' in Solution - Zheng_2009_Theor.Chem.Acc_124_269
Author(s) : Zheng F , Dwoskin LP , Crooks PA , Zhan CG
Ref : Theor Chem Acc , 124 :269 , 2009
Abstract : Indolizidine (-)-235B' is a particularly interesting natural product, as it is the currently known, most potent and subtype-selective open-channel blocker of the alpha4beta2 nicotinic acetylcholine receptor (nAChR). In the current study, extensive first-principles electronic structure calculations have been carried out in order to determine the stable molecular conformations and their relative free energies of the protonated and deprotonated states of (-)-235B' in the gas phase, in chloroform, and in aqueous solution. The (1)H and (13)C NMR chemical shifts calculated using the computationally determined dominant molecular conformation of the deprotonated state are all consistent with available experimental NMR spectra of (-)-235B' in chloroform, which suggests that the computationally determined molecular conformations are reasonable. Our computational results reveal for the first time that two geminal H atoms on carbon-3 (C3) of (-)-235B' have remarkably different chemical shifts (i.e. 3.24 and 2.03 ppm). The computational results help one to better understand and analyze the experimental (1)H NMR spectra of (-)-235B'. The finding of remarkably different chemical shifts of two C3 geminal H atoms in a certain molecular conformation of (-)-235B' may also be valuable in analysis of NMR spectra of other related ring-containing compounds. In addition, the pK(a) of (-)-235B' in aqueous solution is predicted to be ~9.7. All of the computational results provide a solid basis for future studies of the microscopic and phenomenological binding of various receptor proteins with the protonated and deprotonated structures of this unique open-channel blocker of alpha4beta2 nAChRs. This computational study also demonstrates how one can appropriately use computational modeling and spectroscopic analysis to address the structural and spectroscopic problems that cannot be addressed by experiments alone.
ESTHER : Zheng_2009_Theor.Chem.Acc_124_269
PubMedSearch : Zheng_2009_Theor.Chem.Acc_124_269
PubMedID: 20161506

Title : QSAR study on maximal inhibition (Imax) of quaternary ammonium antagonists for S-(-)-nicotine-evoked dopamine release from dopaminergic nerve terminals in rat striatum - Zheng_2009_Bioorg.Med.Chem_17_4477
Author(s) : Zheng F , McConnell MJ , Zhan CG , Dwoskin LP , Crooks PA
Ref : Bioorganic & Medicinal Chemistry , 17 :4477 , 2009
Abstract : Maximal inhibition (I(max)) of the agonist effect is an important pharmacological property of inhibitors that interact with multiple receptor subtypes that are activated by the same agonist and which elicit the same functional response. This report represents the first QSAR study on a set of 66 mono- and bis-quaternary ammonium salts that act as antagonists at neuronal nicotinic acetylcholine receptors mediating nicotine-evoked dopamine release, conducted using multi-linear regression (MLR) and neural network (NN) analysis with the maximal inhibition (I(max)) values of the antagonists as target values. The statistical results for the generated MLR model were: r(2)=0.89, rmsd=9.01, q(2)=0.83 and loormsd=11.1; the statistical results for the generated NN model were: r(2)=0.89, rmsd=8.98, q(2)=0.83 and loormsd=11.2. The maximal inhibition values of the compounds exhibited a good correlation with the predictions made by the QSAR models developed, which provide a basis for rationalizing selection of compounds for synthesis in the discovery of effective and selective second generation inhibitors of nAChRs mediating nicotine-evoked dopamine release.
ESTHER : Zheng_2009_Bioorg.Med.Chem_17_4477
PubMedSearch : Zheng_2009_Bioorg.Med.Chem_17_4477
PubMedID: 19477134

Title : Computational neural network analysis of the affinity of N-n-alkylnicotinium salts for the alpha4beta2* nicotinic acetylcholine receptor - Zheng_2009_J.Enzyme.Inhib.Med.Chem_24_157
Author(s) : Zheng F , Zheng G , Deaciuc AG , Zhan CG , Dwoskin LP , Crooks PA
Ref : J Enzyme Inhib Med Chem , 24 :157 , 2009
Abstract : Based on an 85 molecule database, linear regression with different size datasets and an artificial neural network approach have been used to build mathematical relationships to fit experimentally obtained affinity values (K(i)) of a series of mono- and bis-quaternary ammonium salts from [(3)H]nicotine binding assays using rat striatal membrane preparations. The fitted results were then used to analyze the pattern among the experimental K(i) values of a set of N-n-alkylnicotinium analogs with increasing n-alkyl chain length from 1 to 20 carbons. The affinity of these N-n-alkylnicotinium compounds was shown to parabolically vary with increasing numbers of carbon atoms in the n-alkyl chain, with a local minimum for the C(4) (n-butyl) analogue. A decrease in K(i) value between C(12) and C(13) was also observed. The statistical results for the best neural network fit of the 85 experimental K(i) values are r(2) = 0.84, rmsd = 0.39; r(cv)(2) = 0.68, and loormsd = 0.56. The generated neural network model with the 85 molecule training set may also be of value for future predictions of K(i) values for new virtual compounds, which can then be identified, subsequently synthesized, and tested experimentally.
ESTHER : Zheng_2009_J.Enzyme.Inhib.Med.Chem_24_157
PubMedSearch : Zheng_2009_J.Enzyme.Inhib.Med.Chem_24_157
PubMedID: 18629679

Title : Free-energy perturbation simulation on transition states and redesign of butyrylcholinesterase - Yang_2009_Biophys.J_96_1931
Author(s) : Yang W , Pan Y , Zheng F , Cho H , Tai HH , Zhan CG
Ref : Biophysical Journal , 96 :1931 , 2009
Abstract : It is recognized that an ideal anti-cocaine treatment is to accelerate cocaine metabolism by producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., butyrylcholinesterase (BChE)-catalyzed hydrolysis of cocaine. BChE mutants with a higher catalytic activity against (-)-cocaine are highly desired for use as an exogenous enzyme in humans. To develop a rational design for high-activity mutants, we carried out free-energy perturbation (FEP) simulations on various mutations of the transition-state structures in addition to the corresponding free-enzyme structures by using an extended FEP procedure. The FEP simulations on the mutations of both the free-enzyme and transition-state structures allowed us to calculate the mutation-caused shift of the free-energy change from the free enzyme (BChE) to the transition state, and thus to theoretically predict the mutation-caused shift of the catalytic efficiency (k(cat)/K(M)). The computational predictions are supported by the kinetic data obtained from the wet experiments, demonstrating that the FEP-based computational design approach is promising for rational design of high-activity mutants of an enzyme. One of the BChE mutants designed and discovered in this study has an approximately 1800-fold improved catalytic efficiency against (-)-cocaine compared to wild-type BChE. The high-activity mutant may be therapeutically valuable.
ESTHER : Yang_2009_Biophys.J_96_1931
PubMedSearch : Yang_2009_Biophys.J_96_1931
PubMedID: 19254552

Title : Modeling binding modes of alpha7 nicotinic acetylcholine receptor with ligands: the roles of Gln117 and other residues of the receptor in agonist binding - Huang_2008_J.Med.Chem_51_6293
Author(s) : Huang X , Zheng F , Stokes C , Papke RL , Zhan CG
Ref : Journal of Medicinal Chemistry , 51 :6293 , 2008
Abstract : Extensive molecular docking, molecular dynamics simulations, and binding free energy calculations have been performed to understand how alpha7-specific agonists of nicotinic acetylcholine receptor (nAChR), including AR-R17779 (1), GTS-21 (4), and 4-OH-GTS-21 (5), interact with the alpha7 receptor, leading to important new insights into the receptor-agonist binding. In particular, the cationic head of 4 and 5 has favorable hydrogen bonding and cation-pi interactions with residue Trp149. The computational results have also led us to better understand the roles of Gln117 and other residues in the receptor binding with agonists. The computational predictions are supported by data obtained from wet experimental tests. The new insights into the binding and structure-activity relationship obtained from this study should be valuable for future rational design of more potent and selective agonists of the alpha7 receptor.
ESTHER : Huang_2008_J.Med.Chem_51_6293
PubMedSearch : Huang_2008_J.Med.Chem_51_6293
PubMedID: 18826295

Title : Structure-and-mechanism-based design and discovery of therapeutics for cocaine overdose and addiction - Zheng_2008_Org.Biomol.Chem_6_836
Author(s) : Zheng F , Zhan CG
Ref : Org Biomol Chem , 6 :836 , 2008
Abstract : (-)-Cocaine is a widely abused drug and there is currently no available anti-cocaine therapeutic. Promising agents, such as anti-cocaine catalytic antibodies and high-activity mutants of human butyrylcholinesterase (BChE), for therapeutic treatment of cocaine overdose have been developed through structure-and-mechanism-based design and discovery. In particular, a unique computational design strategy based on the modeling and simulation of the rate-determining transition state has been developed and used to design and discover desirable high-activity mutants of BChE. One of the discovered high-activity mutants of BChE has a approximately 456-fold improved catalytic efficiency against (-)-cocaine. The encouraging outcome of the structure-and-mechanism-based design and discovery effort demonstrates that the unique computational design approach based on transition state modeling and simulation is promising for rational enzyme redesign and drug discovery. The general approach of the structure-and-mechanism-based design and discovery may be used to design high-activity mutants of any enzyme or catalytic antibody.
ESTHER : Zheng_2008_Org.Biomol.Chem_6_836
PubMedSearch : Zheng_2008_Org.Biomol.Chem_6_836
PubMedID: 18292872

Title : Rational design of an enzyme mutant for anti-cocaine therapeutics - Zheng_2008_J.Comput.Aided.Mol.Des_22_661
Author(s) : Zheng F , Zhan CG
Ref : J Comput Aided Mol Des , 22 :661 , 2008
Abstract : (-)-Cocaine is a widely abused drug and there is no available anti-cocaine therapeutic. The disastrous medical and social consequences of cocaine addiction have made the development of an effective pharmacological treatment a high priority. An ideal anti-cocaine medication would be to accelerate (-)-cocaine metabolism producing biologically inactive metabolites. The main metabolic pathway of cocaine in body is the hydrolysis at its benzoyl ester group. Reviewed in this article is the state-of-the-art computational design of high-activity mutants of human butyrylcholinesterase (BChE) against (-)-cocaine. The computational design of BChE mutants have been based on not only the structure of the enzyme, but also the detailed catalytic mechanisms for BChE-catalyzed hydrolysis of (-)-cocaine and (+)-cocaine. Computational studies of the detailed catalytic mechanisms and the structure-and-mechanism-based computational design have been carried out through the combined use of a variety of state-of-the-art techniques of molecular modeling. By using the computational insights into the catalytic mechanisms, a recently developed unique computational design strategy based on the simulation of the rate-determining transition state has been employed to design high-activity mutants of human BChE for hydrolysis of (-)-cocaine, leading to the exciting discovery of BChE mutants with a considerably improved catalytic efficiency against (-)-cocaine. One of the discovered BChE mutants (i.e., A199S/S287G/A328W/Y332G) has a approximately 456-fold improved catalytic efficiency against (-)-cocaine. The encouraging outcome of the computational design and discovery effort demonstrates that the unique computational design approach based on the transition-state simulation is promising for rational enzyme redesign and drug discovery.
ESTHER : Zheng_2008_J.Comput.Aided.Mol.Des_22_661
PubMedSearch : Zheng_2008_J.Comput.Aided.Mol.Des_22_661
PubMedID: 17989928

Title : Most efficient cocaine hydrolase designed by virtual screening of transition states - Zheng_2008_J.Am.Chem.Soc_130_12148
Author(s) : Zheng F , Yang W , Ko MC , Liu J , Cho H , Gao D , Tong M , Tai HH , Woods JH , Zhan CG
Ref : Journal of the American Chemical Society , 130 :12148 , 2008
Abstract : Cocaine is recognized as the most reinforcing of all drugs of abuse. There is no anticocaine medication available. The disastrous medical and social consequences of cocaine addiction have made the development of an anticocaine medication a high priority. It has been recognized that an ideal anticocaine medication is one that accelerates cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., cocaine hydrolysis catalyzed by plasma enzyme butyrylcholinesterase (BChE). However, wild-type BChE has a low catalytic efficiency against the abused cocaine. Design of a high-activity enzyme mutant is extremely challenging, particularly when the chemical reaction process is rate-determining for the enzymatic reaction. Here we report the design and discovery of a high-activity mutant of human BChE by using a novel, systematic computational design approach based on transition-state simulations and activation energy calculations. The novel computational design approach has led to discovery of the most efficient cocaine hydrolase, i.e., a human BChE mutant with an approximately 2000-fold improved catalytic efficiency, promising for therapeutic treatment of cocaine overdose and addiction as an exogenous enzyme in human. The encouraging discovery resulted from the computational design not only provides a promising anticocaine medication but also demonstrates that the novel, generally applicable computational design approach is promising for rational enzyme redesign and drug discovery.
ESTHER : Zheng_2008_J.Am.Chem.Soc_130_12148
PubMedSearch : Zheng_2008_J.Am.Chem.Soc_130_12148
PubMedID: 18710224

Title : Molecular cloning, and characterization of a modular acetyl xylan esterase from the edible straw mushroom Volvariella volvacea - Ding_2007_FEMS.Microbiol.Lett_274_304
Author(s) : Ding S , Cao J , Zhou R , Zheng F
Ref : FEMS Microbiology Letters , 274 :304 , 2007
Abstract : A new Volvariella volvacea gene encoding an acetyl xylan esterase (designated as Vvaxe1) was cloned and expressed in Pichia pastoris. The cDNA contained an ORF of 1047 bp encoding 349 amino acids with a calculated mass of 39 990 Da. VvAXE1 is a modular enzyme consisting of an N-terminal signal peptide, a catalytic domain, and a cellulose-binding domain. The amino acid sequence of the enzyme exhibited a high degree of similarity to cinnamoyl esterase B from Penicillium funiculosum, and acetyl xylan esterases from Aspergillus oryzae, Penicillium purpurogenum, and Aspergillus ficuum. Recombinant acetyl xylan esterase released acetate from several acetylated substrates including beta-d-xylose tetraacetate and acetylated xylan. No activity was detectable on p-nitrophenyl acetate. Enzyme-catalyzed hydrolysis of 4-methylumbelliferyl acetate was maximal at pH 8.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 307.7 microM and a V(max) value of 24 733 IU micromol(-1) protein. ReAXE1 also exhibited a capacity to bind to Avicel and H(3)PO(4) acid-swollen cellulose.
ESTHER : Ding_2007_FEMS.Microbiol.Lett_274_304
PubMedSearch : Ding_2007_FEMS.Microbiol.Lett_274_304
PubMedID: 17623028

Title : Computational neural network analysis of the affinity of lobeline and tetrabenazine analogs for the vesicular monoamine transporter-2 - Zheng_2007_Bioorg.Med.Chem_15_2975
Author(s) : Zheng F , Zheng G , Deaciuc AG , Zhan CG , Dwoskin LP , Crooks PA
Ref : Bioorganic & Medicinal Chemistry , 15 :2975 , 2007
Abstract : Back-propagation artificial neural networks (ANNs) were trained on a dataset of 104 VMAT2 ligands with experimentally measured log(1/K(i)) values. A set of related descriptors, including topological, geometrical, GETAWAY, aromaticity, and WHIM descriptors, was selected to build nonlinear quantitative structure-activity relationships. A partial least squares (PLS) regression model was also developed for comparison. The nonlinearity of the relationship between molecular descriptors and VMAT2 ligand activity was demonstrated. The obtained neural network model outperformed the PLS model in both the fitting and predictive ability. ANN analysis indicated that the computed activities were in excellent agreement with the experimentally observed values (r(2)=0.91, rmsd=0.225; predictive q(2)=0.82, loormsd=0.316). The generated models were further tested by use of an external prediction set of 15 molecules. The nonlinear ANN model has r(2)=0.93 and root-mean-square errors of 0.282 compared with the experimentally measured activity of the test set. The stability test of the model with regard to data division was found to be positive, indicating that the generated model is predictive. The modeling study also reflected the important role of atomic distribution in the molecules, size, and steric structure of the molecules when they interact with the target, VMAT2. The developed models are expected to be useful in the rational design of new chemical entities as ligands of VMAT2 and for directing synthesis of new molecules in the future.
ESTHER : Zheng_2007_Bioorg.Med.Chem_15_2975
PubMedSearch : Zheng_2007_Bioorg.Med.Chem_15_2975
PubMedID: 17331733

Title : A glimpse of streptococcal toxic shock syndrome from comparative genomics of S. suis 2 Chinese isolates - Chen_2007_PLoS.One_2_e315
Author(s) : Chen C , Tang J , Dong W , Wang C , Feng Y , Wang J , Zheng F , Pan X , Liu D , Li M , Song Y , Zhu X , Sun H , Feng T , Guo Z , Ju A , Ge J , Dong Y , Sun W , Jiang Y , Yan J , Yang H , Wang X , Gao GF , Yang R , Yu J
Ref : PLoS ONE , 2 :e315 , 2007
Abstract : BACKGROUND: Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen, causing more than 200 cases of severe human infection worldwide, with the hallmarks of meningitis, septicemia, arthritis, etc. Very recently, SS2 has been recognized as an etiological agent for streptococcal toxic shock syndrome (STSS), which was originally associated with Streptococcus pyogenes (GAS) in Streptococci. However, the molecular mechanisms underlying STSS are poorly understood. METHODS AND FINDINGS: To elucidate the genetic determinants of STSS caused by SS2, whole genome sequencing of 3 different Chinese SS2 strains was undertaken. Comparative genomics accompanied by several lines of experiments, including experimental animal infection, PCR assay, and expression analysis, were utilized to further dissect a candidate pathogenicity island (PAI). Here we show, for the first time, a novel molecular insight into Chinese isolates of highly invasive SS2, which caused two large-scale human STSS outbreaks in China. A candidate PAI of approximately 89 kb in length, which is designated 89K and specific for Chinese SS2 virulent isolates, was investigated at the genomic level. It shares the universal properties of PAIs such as distinct GC content, consistent with its pivotal role in STSS and high virulence. CONCLUSIONS: To our knowledge, this is the first PAI candidate from S. suis worldwide. Our finding thus sheds light on STSS triggered by SS2 at the genomic level, facilitates further understanding of its pathogenesis and points to directions of development on some effective strategies to combat highly pathogenic SS2 infections.
ESTHER : Chen_2007_PLoS.One_2_e315
PubMedSearch : Chen_2007_PLoS.One_2_e315
PubMedID: 17375201
Gene_locus related to this paper: strsu-a4vws4 , strsu-q302y4 , strsy-a4vus4 , strsy-a4vwf6

Title : Modeling subtype-selective agonists binding with alpha4beta2 and alpha7 nicotinic acetylcholine receptors: effects of local binding and long-range electrostatic interactions - Huang_2006_J.Med.Chem_49_7661
Author(s) : Huang X , Zheng F , Chen X , Crooks PA , Dwoskin LP , Zhan CG
Ref : Journal of Medicinal Chemistry , 49 :7661 , 2006
Abstract : The subtype-selective binding of 14 representative agonists with alpha4beta2 and alpha7 nicotinic acetylcholine receptors (nAChRs) has been studied by performing homology modeling, molecular docking, geometry optimizations, and microscopic and phenomenological binding free energy calculations. All of the computational results demonstrate that the subtype selectivity of the agonists binding with alpha4beta2 and alpha7 7 nAChRs is affected by both local binding and long-range electrostatic interactions between the receptors and the protonated structures of the agonists. The effects of the long-range electrostatic interactions are mainly due to the distinct difference in the net charge of the ligand-binding domain between the two nAChR subtypes. For the alpha4beta2-selective agonists examined, the microscopic binding modes with the alpha4beta2 nAChR are very similar to the corresponding modes with the alpha7 nAChR, and therefore, the subtype selectivity of these agonists binding with alpha4beta2 and alpha7 nAChRs is dominated by the long-range electrostatic interactions. For the alpha7-selective agonists, their microscopic binding modes with the alpha7 nAChR are remarkably different from those with the alpha4beta2 nAChR so that the local binding (including the hydrogen bonding and cation-pi interactions) with the alpha7 nAChR is much stronger than that with the alpha4beta2 nAChR. The calculated phenomenological binding free energies are in good agreement with available experimental data for the relative binding free energies concerning the subtype selectivity of agonists binding with the two different nAChR subtypes. The fundamental insights obtained in the present study should be valuable for future rational design of potential therapeutic agents targeted to specific nAChR subtypes.
ESTHER : Huang_2006_J.Med.Chem_49_7661
PubMedSearch : Huang_2006_J.Med.Chem_49_7661
PubMedID: 17181149

Title : QSAR modeling of mono- and bis-quaternary ammonium salts that act as antagonists at neuronal nicotinic acetylcholine receptors mediating dopamine release - Zheng_2006_Bioorg.Med.Chem_14_3017
Author(s) : Zheng F , Bayram E , Sumithran SP , Ayers JT , Zhan CG , Schmitt JD , Dwoskin LP , Crooks PA
Ref : Bioorganic & Medicinal Chemistry , 14 :3017 , 2006
Abstract : Back-propagation artificial neural networks (ANNs) were trained on a dataset of 42 molecules with quantitative IC50 values to model structure-activity relationships of mono- and bis-quaternary ammonium salts as antagonists at neuronal nicotinic acetylcholine receptors (nAChR) mediating nicotine-evoked dopamine release. The ANN QSAR models produced a reasonable level of correlation between experimental and calculated log(1/IC50) (r2=0.76, r(cv)2=0.64). An external test for the models was performed on a dataset of 18 molecules with IC50 values >1 microM. Fourteen of these were correctly classified. Classification ability of various models, including self-organizing maps (SOM), for all 60 molecules was also evaluated. A detailed analysis of the modeling results revealed the following relative contributions of the used descriptors to the trained ANN QSAR model: approximately 44.0% from the length of the N-alkyl chain attached to the quaternary ammonium head group, approximately 20.0% from Moriguchi octanol-water partition coefficient of the molecule, approximately 13.0% from molecular surface area, approximately 12.6% from the first component shape directional WHIM index/unweighted, approximately 7.8% from Ghose-Crippen molar refractivity, and 2.6% from the lowest unoccupied molecular orbital energy. The ANN QSAR models were also evaluated using a set of 13 newly synthesized compounds (11 biologically active antagonists and two biologically inactive compounds) whose structures had not been previously utilized in the training set. Twelve among 13 compounds were predicted to be active which further supports the robustness of the trained models. Other insights from modeling include a structural modification in the bis-quinolinium series that involved replacing the 5 and/or 8 as well as the 5' and/or 8' carbon atoms with nitrogen atoms, predicting inactive compounds. Such data can be effectively used to reduce synthetic and in vitro screening activities by eliminating compounds of predicted low activity from the pool of candidate molecules for synthesis. The application of the ANN QSAR model has led to the successful discovery of six new compounds in this study with experimental IC50 values of less than 0.1 microM at nAChR subtypes responsible for mediating nicotine-evoked dopamine release, demonstrating that the ANN QSAR model is a valuable aid to drug discovery.
ESTHER : Zheng_2006_Bioorg.Med.Chem_14_3017
PubMedSearch : Zheng_2006_Bioorg.Med.Chem_14_3017
PubMedID: 16431111

Title : Modeling multiple species of nicotine and deschloroepibatidine interacting with alpha4beta2 nicotinic acetylcholine receptor: from microscopic binding to phenomenological binding affinity - Huang_2005_J.Am.Chem.Soc_127_14401
Author(s) : Huang X , Zheng F , Crooks PA , Dwoskin LP , Zhan CG
Ref : Journal of the American Chemical Society , 127 :14401 , 2005
Abstract : A variety of molecular modeling, molecular docking, and first-principles electronic structure calculations were performed to study how the alpha4beta2 nicotinic acetylcholine receptor (nAChR) binds with different species of two typical agonists, (S)-(-)-nicotine and (R)-(-)-deschloroepibatidine, each of which is distinguished by different free bases and protonation states. On the basis of these results, predictions were made regarding the corresponding microscopic binding free energies. Hydrogen-bonding and cation-pi interactions between the receptor and the respective ligands were found to be the dominant factors differentiating the binding strengths of different microscopic binding species. The calculated results and analyses demonstrate that, for each agonist, all the species are interchangeable and can quickly achieve a thermodynamic equilibrium in solution and at the nAChR binding site. This allows quantitation of the equilibrium concentration distributions of the free ligand species and the corresponding microscopic ligand-receptor binding species, their pH dependence, and their contributions to the phenomenological binding affinity. The predicted equilibrium concentration distributions, pK(a) values, absolute phenomenological binding affinities, and their pH dependence are all in good agreement with available experimental data, suggesting that the computational strategy from the microscopic binding species and affinities to the phenomenological binding affinity is reliable for studying alpha4beta2 nAChR-ligand binding. This should provide valuable information for future rational design of drugs targeting nAChRs. The general strategy of the "from-microscopic-to-phenomenological" approach for studying interactions of alpha4beta2 nAChRs with (S)-(-)-nicotine and (R)-(-)-deschloroepibatidine may also be useful in studying other types of ligand-protein interactions involving multiple molecular species of a ligand and in associated rational drug design.
ESTHER : Huang_2005_J.Am.Chem.Soc_127_14401
PubMedSearch : Huang_2005_J.Am.Chem.Soc_127_14401
PubMedID: 16218635

Title : M2 muscarinic acetylcholine receptor knock-out mice show deficits in behavioral flexibility, working memory, and hippocampal plasticity - Seeger_2004_J.Neurosci_24_10117
Author(s) : Seeger T , Fedorova I , Zheng F , Miyakawa T , Koustova E , Gomeza J , Basile AS , Alzheimer C , Wess J
Ref : Journal of Neuroscience , 24 :10117 , 2004
Abstract : Muscarinic acetylcholine receptors are known to play key roles in facilitating cognitive processes. However, the specific roles of the individual muscarinic receptor subtypes (M1-M5) in learning and memory are not well understood at present. In the present study, we used wild-type (M2+/+) and M2 receptor-deficient (M2-/-) mice to examine the potential role of M2 receptors in learning and memory and hippocampal synaptic plasticity. M2-/- mice showed significant deficits in behavioral flexibility and working memory in the Barnes circular maze and the T-maze delayed alternation tests, respectively. The behavioral deficits of M2-/- mice were associated with profound changes in neuronal plasticity studied at the Schaffer-CA1 synapse of hippocampal slices. Strikingly, short-term potentiation (STP) was abolished, and long-term potentiation (LTP) was drastically reduced after high-frequency stimulation of M2-/- hippocampi. Treatment of M2-/- hippocampal slices with the GABA(A) receptor antagonist, bicuculline, restored STP and significantly increased LTP. Whole-cell recordings from CA1 pyramidal cells demonstrated a much stronger disinhibition of GABAergic than glutamatergic transmission in M2-/- hippocampi, which was particularly prominent during stimulus trains. Increased strength of GABAergic inhibition is thus a likely mechanism underlying the impaired synaptic plasticity observed with M2-/- hippocampi. Moreover, the persistent enhancement of excitatory synaptic transmission in CA1 pyramidal cells induced by the transient application of a low concentration of a muscarinic agonist (referred to as LTP(m)) was totally abolished in M2-/- mice. Because impaired muscarinic cholinergic neurotransmission is associated with Alzheimer's disease and normal aging processes, these findings should be of considerable therapeutic relevance.
ESTHER : Seeger_2004_J.Neurosci_24_10117
PubMedSearch : Seeger_2004_J.Neurosci_24_10117
PubMedID: 15537882

Title : Fundamental reaction mechanism for cocaine hydrolysis in human butyrylcholinesterase - Zhan_2003_J.Am.Chem.Soc_125_2462
Author(s) : Zhan CG , Zheng F , Landry DW
Ref : Journal of the American Chemical Society , 125 :2462 , 2003
Abstract : Butyrylcholinesterase (BChE)-cocaine binding and the fundamental pathway for BChE-catalyzed hydrolysis of cocaine have been studied by molecular modeling, molecular dynamics (MD) simulations, and ab initio calculations. Modeling and simulations indicate that the structures of the prereactive BChE/substrate complexes for (-)-cocaine and (+)-cocaine are all similar to that of the corresponding prereactive BChE/butyrylcholine (BCh) complex. The overall binding of BChE with (-)-cocaine and (+)-cocaine is also similar to that proposed with butyrylthiocholine and succinyldithiocholine, i.e., (-)- or (+)-cocaine first slides down the substrate-binding gorge to bind to Trp-82 and stands vertically in the gorge between Asp-70 and Trp-82 (nonprereactive complex) and then rotates to a position in the catalytic site within a favorable distance for nucleophilic attack and hydrolysis by Ser-198 (prereactive complex). In the prereactive complex, cocaine lies horizontally at the bottom of the gorge. The fundamental catalytic hydrolysis pathway, consisting of acylation and deacylation stages similar to those for ester hydrolysis by other serine hydrolases, was proposed on the basis of the simulated prereactive complex and confirmed theoretically by ab initio reaction coordinate calculations. Both the acylation and deacylation follow a double-proton-transfer mechanism. The calculated energetic results show that within the chemical reaction process the highest energy barrier and Gibbs free energy barrier are all associated with the first step of deacylation. The calculated ratio of the rate constant (k(cat)) for the catalytic hydrolysis to that (k(0)) for the spontaneous hydrolysis is approximately 9.0 x 10(7). The estimated k(cat)/k(0) value of approximately 9.0 x 10(7) is in excellent agreement with the experimentally derived k(cat)/k(0) value of approximately 7.2 x 10(7) for (+)-cocaine, whereas it is approximately 2000 times larger than the experimentally derived k(cat)/k(0) value of approximately 4.4 x 10(4) for (-)-cocaine. All of the results suggest that the rate-determining step of the BChE-catalyzed hydrolysis of (+)-cocaine is the first step of deacylation, whereas for (-)-cocaine the change from the nonprereactive complex to the prereactive complex is rate-determining and has a Gibbs free energy barrier higher than that for the first step of deacylation by approximately 4 kcal/mol. A further analysis of the structural changes from the nonprereactive complex to the prereactive complex reveals specific amino acid residues hindering the structural changes, providing initial clues for the rational design of BChE mutants with improved catalytic activity for (-)-cocaine.
ESTHER : Zhan_2003_J.Am.Chem.Soc_125_2462
PubMedSearch : Zhan_2003_J.Am.Chem.Soc_125_2462
PubMedID: 12603134