A thermally stable form of bacterial cocaine esterase: a potential therapeutic agent for treatment of cocaine abuse.
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.
Brim
Remy L
RL
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.
Nance
Mark R
MR
Youngstrom
Daniel W
DW
Narasimhan
Diwahar
D
Zhan
Chang-Guo
CG
Tesmer
John J G
JJ
Sunahara
Roger K
RK
Woods
James H
JH
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
D002265:Carboxylic Ester Hydrolases; C019921:cocaine esterase