title: Preparation and in vivo characterization of a cocaine hydrolase engineered from human butyrylcholinesterase for metabolizing cocaine.
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 μM) 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.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, USA.
authors.forename: Liu
authors.initials: L
authors.lastname: Xue
authors.forename: Shurong
authors.initials: S
authors.lastname: Hou
authors.forename: Min
authors.initials: M
authors.lastname: Tong
authors.forename: Lei
authors.initials: L
authors.lastname: Fang
authors.forename: Xiabin
authors.initials: X
authors.lastname: Chen
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Jin
authors.forename: Hsin-Hsiung
authors.initials: HH
authors.lastname: Tai
authors.forename: Fang
authors.initials: F
authors.lastname: Zheng
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
mesh_terms: D000818:Animals; D002091:Butyrylcholinesterase / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002460:Cell Line; D003042:Cocaine / Q000378:metabolism*; D019970:Cocaine-Related Disorders; D006224:Cricetinae; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D009043:Motor Activity / Q000187:drug effects; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D002091:Butyrylcholinesterase; D003042:Cocaine