title: A thermally stable form of bacterial cocaine esterase: a potential therapeutic agent for treatment of cocaine abuse.
abstract: Rhodococcal cocaine esterase (CocE) is an attractive potential treatment for both cocaine overdose and cocaine addiction. CocE directly degrades cocaine into inactive products, whereas traditional small-molecule approaches require blockade of the inhibitory action of cocaine on a diverse array of monoamine transporters and ion channels. The usefulness of wild-type (wt) cocaine esterase is hampered by its inactivation at 37 degrees C. Herein, we characterize the most thermostable form of this enzyme to date, CocE-L169K/G173Q. In vitro kinetic analyses reveal that CocE-L169K/G173Q displays a half-life of 2.9 days at 37 degrees C, which represents a 340-fold improvement over wt and is 15-fold greater than previously reported mutants. Crystallographic analyses of CocE-L169K/G173Q, determined at 1.6-A resolution, suggest that stabilization involves enhanced domain-domain interactions involving van der Waals interactions and hydrogen bonding. In vivo rodent studies reveal that intravenous pretreatment with CocE-L169K/G173Q in mice provides protection from cocaine-induced lethality for longer time periods before cocaine administration than wt CocE. Furthermore, intravenous administration (pretreatment) of CocE-L169K/G173Q prevents self-administration of cocaine in a time-dependent manner. Termination of the in vivo effects of CoCE seems to be dependent on, but not proportional to, its clearance from plasma as its half-life is approximately 2.3 h and similar to that of wt CocE (2.2 h). Taken together these data suggest that CocE-L169K/G173Q possesses many of the properties of a biological therapeutic for treating cocaine abuse but requires additional development to improve its serum half-life.
authors.affiliation: Department of Pharmacology, University of Michigan Medical School, 1301 Medical Sciences Research Building III, 1150 W Medical Center Drive, Ann Arbor, MI 48109-0600, USA.
authors.forename: Remy L
authors.initials: RL
authors.lastname: Brim
authors.forename: Mark R
authors.initials: MR
authors.lastname: Nance
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Youngstrom
authors.forename: Diwahar
authors.initials: D
authors.lastname: Narasimhan
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
authors.forename: John J G
authors.initials: JJ
authors.lastname: Tesmer
authors.forename: Roger K
authors.initials: RK
authors.lastname: Sunahara
authors.forename: James H
authors.initials: JH
authors.lastname: Woods
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000493:pharmacokinetics / Q000627:therapeutic use*; D019970:Cocaine-Related Disorders / Q000188:drug therapy*; D003461:Crystallography; D004795:Enzyme Stability; D006207:Half-Life; D008297:Male; D051379:Mice; D051381:Rats; D017207:Rats, Sprague-Dawley; D012054:Reinforcement, Psychology; D012240:Rhodococcus / Q000201:enzymology*; D013696:Temperature
chemical_list: D002265:Carboxylic Ester Hydrolases; C019921:cocaine esterase