title: Amino-acid mutations to extend the biological half-life of a therapeutically valuable mutant of human butyrylcholinesterase.
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 ∼7 to ∼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.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, United States.
authors.forename: Lei
authors.initials: L
authors.lastname: Fang
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, United States.
authors.forename: Shurong
authors.initials: S
authors.lastname: Hou
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, United States.
authors.forename: Liu
authors.initials: L
authors.lastname: Xue
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, United States.
authors.forename: Fang
authors.initials: F
authors.lastname: Zheng
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, United States. Electronic address: zhan@uky.edu.
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
mesh_terms: D019943:Amino Acid Substitution; D055162:Biocatalysis; D001704:Biopolymers / Q000737:chemistry / Q000235:genetics; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003042:Cocaine / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D056004:Molecular Dynamics Simulation; D009154:Mutation
chemical_list: D001704:Biopolymers; D002091:Butyrylcholinesterase; D003042:Cocaine
keywords: Cholinesterase; Cocaine; Enzyme therapy; Molecular modeling