Enhancing cocaine metabolism by administration of cocaine esterases has been recognized as a promising treatment strategy for cocaine overdose and addiction. The esterase CocE is the most efficient native enzyme for metabolizing naturally occurring (-)-cocaine yet identified. Through catalysis of (-)-cocaine hydrolysis, CocE can both prevent and reverse extreme (-)- cocaine toxicity in rodent models and it has the potential to be developed into a chemically useful antagonist of the toxic and behavioral effects of (-)-cocaine. In order to optimize the efficacy of this potential anti-cocaine medication and minimize its possible side effects (particularly immunogenicity), we propose to improve the catalytic efficiency of CocE against (-)- cocaine. The higher the catalytic efficiency of the enzyme against (-)-cocaine, the lower the dose required to achieve therapeutic effectiveness and the decrease in dose can reduce the overall immunological response. Hence we will focus on the rational design, discovery, and preclinical testing of CocE mutants with an improved catalytic efficiency against (-)-cocaine. The rational design of high-activity mutants of CocE against (-)-cocaine requires a detailed understanding of the mechanism for CocE-catalyzed hydrolysis of cocaine. This mechanism can be understood by performing computational studies using the state-of-the-art computational techniques of molecular modeling, simulation, and calculation.
The specific aims i nclude: (1) Elucidation of the detailed mechanism and reaction coordinate and the corresponding free energy profiles for CocE-catalyzed hydrolysis of cocaine by performing quantum mechanical (QM) calculations, hybrid quantum mechanical/molecular mechanical (QM/MM) calculations, and molecular dynamics (MD) simulations, etc. (2) Design, discovery, and testing of CocE mutants with an improved catalytic efficiency against (-)-cocaine by using a recently developed novel computational design approach based on the transition state modeling and simulation to computationally evaluate a large number of hypothetical CocE mutants, followed by wet experimental tests including site-directed mutagenesis, protein expression and purification, and in vitro and in vivo activity tests. The long-term objective of this investigation will be to eventually develop an efficient anti-cocaine medication using a high-activity mutant of CocE.

Public Health Relevance

Enhancing cocaine metabolism by administration of cocaine esterase (CocE) has been recognized as a promising treatment strategy for cocaine overdose and addiction. The high-activity mutants of CocE to be designed and discovered in this project will eventually lead to an efficient anti-cocaine medication.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA025100-05
Application #
8286381
Study Section
Human Development Research Subcommittee (NIDA)
Program Officer
Shih, Ming L
Project Start
2008-08-15
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$549,174
Indirect Cost
$123,759
Name
University of Kentucky
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
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Hou, Shurong; Zhan, Max; Zheng, Xirong et al. (2014) Kinetic characterization of human butyrylcholinesterase mutants for the hydrolysis of cocaethylene. Biochem J 460:447-57
Zheng, Fang; Xue, Liu; Hou, Shurong et al. (2014) A highly efficient cocaine-detoxifying enzyme obtained by computational design. Nat Commun 5:3457
Qiao, Yan; Han, Keli; Zhan, Chang-Guo (2014) Reaction pathways and free energy profiles for cholinesterase-catalyzed hydrolysis of 6-monoacetylmorphine. Org Biomol Chem 12:2214-27
Fang, Lei; Hou, Shurong; Xue, Liu et al. (2014) Amino-acid mutations to extend the biological half-life of a therapeutically valuable mutant of human butyrylcholinesterase. Chem Biol Interact 214:18-25
Zheng, Fang; Zhan, Max; Huang, Xiaoqin et al. (2014) Modeling in vitro inhibition of butyrylcholinesterase using molecular docking, multi-linear regression and artificial neural network approaches. Bioorg Med Chem 22:538-49
Fang, Lei; Zheng, Fang; Zhan, Chang-Guo (2014) A model of glycosylated human butyrylcholinesterase. Mol Biosyst 10:348-54
Lu, Haiting; Huang, Xiaoqin; AbdulHameed, Mohamed Diwan M et al. (2014) Binding free energies for nicotine analogs inhibiting cytochrome P450 2A6 by a combined use of molecular dynamics simulations and QM/MM-PBSA calculations. Bioorg Med Chem 22:2149-56
Li, Dongmei; Huang, Xiaoqin; Lin, Jianping et al. (2013) Catalytic mechanism of cytochrome P450 for N-methylhydroxylation of nicotine: reaction pathways and regioselectivity of the enzymatic nicotine oxidation. Dalton Trans 42:3812-20

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