title: Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification.
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 ∼12 min at 37 °C. The previously designed T172R/G173Q mutant (denoted as enzyme E172-173) with an improved in vitro half-life of ∼6 h at 37 °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 °C to >100 days, but also significantly improved the catalytic efficiency against cocaine by ∼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 ∼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.
authors.affiliation: Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky , 789 South Limestone Street, Lexington, Kentucky 40536, United States.
authors.forename: Lei
authors.initials: L
authors.lastname: Fang
authors.forename: K Martin
authors.initials: KM
authors.lastname: Chow
authors.forename: Shurong
authors.initials: S
authors.lastname: Hou
authors.forename: Liu
authors.initials: L
authors.lastname: Xue
authors.forename: Xiabin
authors.initials: X
authors.lastname: Chen
authors.forename: David W
authors.initials: DW
authors.lastname: Rodgers
authors.forename: Fang
authors.initials: F
authors.lastname: Zheng
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002384:Catalysis; D003042:Cocaine / Q000493:pharmacokinetics*; D015195:Drug Design; D051379:Mice; D056004:Molecular Dynamics Simulation; D009154:Mutation
chemical_list: D002265:Carboxylic Ester Hydrolases; C019921:cocaine esterase; D003042:Cocaine