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Publication Abstract from PubMed

No small molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis systemically has recently gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37 degrees C has limited its therapeutic potential. Crosslinking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we utilize structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulphide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newly modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37 degrees C , representing greater than a 4700-fold improvement over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 hours, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in Rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo.

Subunit Stabilization and PEGylation of Cocaine Esterase Improves In Vivo Residence Time.,Narasimhan D, Collins GT, Nance MR, Nichols J, Edwald E, Chan J, Ko MC, Woods JH, Tesmer JJ, Sunahara RK Mol Pharmacol. 2011 Sep 2. PMID:21890748^[4]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.