Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence Time

Abstract

No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37°C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-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°C, representing an improvement of more than 4700-fold 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 h, 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.

Long-Lasting Effects of a PEGylated Mutant Cocaine Esterase (CocE) on the Reinforcing and Discriminative Stimulus Effects of Cocaine in Rats

“…Importantly, just as these changes did not alter the enzymatic activity of PEG-CCRQ CocE in vitro , the DM (RQ) and PEG-CCRQ forms of CocE appeared to be equally effective at eliminating cocaine in vivo as both enzymes produced at least 10-fold rightward shifts in the reinforcing effects of cocaine when comparable doses of B3 mg/kg were administered immediately before the start of cocaine self-administration sessions (Collins et al, 2009). Nevertheless, just as the prolonged duration of PEG-CCRQ CocE's protective effects differentiated it from DM CocE in rodent models of acute cocaine toxicity (Collins et al, 2011b;Narasimhan et al, 2011) differences between these enzymes began to emerge when PEG-CCRQ CocE was evaluated against cocaine self-administration using a multiple dose procedure that allowed for the longitudinal evaluation of an animal's sensitivity to the reinforcing effects of cocaine. Unlike the effects of B3 mg/ kg DM CocE, which had completely dissipated by 24 h (Collins et al, 2009), pretreatment with 3.2 mg/kg PEG-CCRQ CocE produced a longer lasting change in cocaine self-administration with a three-fold rightward shift in the dose-response curve observed 24 h later, and baseline-like rates of responding not observed until 48 h after administration.…”

Nano-antidotes for drug overdose and poisoning

“…Natural cocaine esterases have also been modified by site-specific mutagenesis to confer good catalytic activity at body temperature. The most efficient mutant was sterically stabilized with two 40-kD PEG chains to increase its circulation time (56). The esterase exhibited a relatively long-lasting protective efficacy (pretreated 24 to 72 hours prior to drug intake, with 3.2 to 32 mg/kg enzyme) in rats overdosed with lethal doses of cocaine.…”

Repeated Administration of a Mutant Cocaine Esterase: Effects on Plasma Cocaine Levels, Cocaine-Induced Cardiovascular Activity, and Immune Responses in Rhesus Monkeys

“…Although these findings suggest that immunologic responses to repeated administrations of DM CocE are not likely to affect its therapeutic effectiveness, recent attempts to improve the duration and further reduce the immunogenicity of CocE have resulted in a polyethylene glycol-modified mutant form of CocE (PEG-CCRQ CocE) capable of reducing the reinforcing, cardiovascular, convulsant, and lethal effects of cocaine for up to 48 h without eliciting increases in anti-CocE antibodies (Narasimhan et al, 2011;Collins et al, 2012). It is noteworthy that even though self-administration studies in rats (Collins et al, 2009 suggest that human cocaine abusers would be able to surmount the protective effects of CocE (by ingesting approximately 10 times more cocaine), the capacity of CocE to rapidly metabolize large doses of cocaine (Ͼ100 mg/kg i.v.…”