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Review
. 2012 Feb;4(2):137-50.
doi: 10.4155/fmc.11.194.

Bacterial cocaine esterase: a protein-based therapy for cocaine overdose and addiction

Diwahar Narasimhan  1 , James H Woods, Roger K Sunahara
Affiliations
Review

Bacterial cocaine esterase: a protein-based therapy for cocaine overdose and addiction

Diwahar Narasimhan et al. Future Med Chem. 2012 Feb.

Abstract

Cocaine is highly addictive and there are no pharmacotherapeutic drugs available to treat acute cocaine toxicity or chronic abuse. Antagonizing an inhibitor such as cocaine using a small molecule has proven difficult. The alternative approach is to modify cocaine's pharmacokinetic properties by sequestering or hydrolyzing it in serum and limiting access to its sites of action. We took advantage of a bacterial esterase (CocE) that has evolved to hydrolyze cocaine and have developed it as a therapeutic that rapidly and specifically clears cocaine from the subject. Native enzyme was unstable at 37°C, thus limiting CocE's potential. Innovative computational methods based on the protein's structure helped elucidate its mechanism of destabilization. Novel protein engineering methodologies were applied to substantially improve its stability in vitro and in vivo. These improvements rendered CocE as a powerful and efficacious therapeutic to treat cocaine intoxication and lead the way towards developing a therapy for addiction.

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Conflict of interest statement

Financial & competing interests disclosure

The NIDA and the NIGMS had no further role in study design; the collection, analysis and interpretation of data; the writing of the report; or in the decision to submit the paper for publication. D Narasimhan, JH Woods and RK Sunahara are authors on patent PCT/US2008, ‘Thermostabilization of Proteins’. JH Woods and RK Sunahara have consulted for Reckitt Benckiser Corporation.

The authors have no other relevant affifiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Figures

Figure 1
Figure 1. Structure of CocE
CocE is composed of three distinct structural domains (domains I–III). Domain I contains the catalytic S117 and adopts a canonical α-/β-hydrolase fold. The locations of point mutations predicted by computational methods and tested for in vitro activity are shown as dark spheres. A DTT-carbonate adduct (DBC) is evident in all structures that contain DTT.
Figure 2
Figure 2. Comparison of in vitro and in vivo half-lives of wild-type CocE and stable mutants
(A) For in vitro half-life calculation, decay in the capacity to convert cocaine to ecgonine methyl ester and benzoic acid was measured at 37°C. 50 ng/ml of wt-CocE, T172R-CocE, RQ-CocE, L169K CocE, KQ-CocE or CCRQ-CocE were incubated at 37°C and the activity calculated over time. τ1/2 values were measured from the resulting curves. (B) For in vivo half-life calculation, wt-CocE and the mutants were pre-injected into rodents and at several time points postinjection a lethal dose of cocaine (180 mg/kg) was given, and time taken to reach 50% lethality was calculated and plotted. wt: Wild-type.
Figure 3
Figure 3. Structural analysis of stabilizing CocE mutants
The crystal structures of wild-type CocE (A, C & E) compared with T172R (B), G173Q (D) and L169K (F). The stabilizing effect of the mutants appears to result from enhanced interactions between the H2 and H3 of domain II or between subunits (T172R substitution), or from additional interdomain contacts (G173Q and L169K). The L169K side chain exhibits two conformations. Note that L169 is poorly ordered in the wild-type CocE structure and its side chain is solvent exposed, which is expected to be destabilizing. A DTT-carbonate adduct (DBC) is evident in all structures that contain DTT.
Figure 4
Figure 4. Cocaine esterase protected against cocaine-reinforced operant responding in Sprague–Dawley rats
Saline, 1-mg RQ-CocE or KQ-CocE was given as a pretreatment 1 min prior to cocaine self-administration sessions. Rats in the saline substitution condition (open bar) received no cocaine from nose pokes during the session.
See this image and copyright information in PMC

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Website

    1. National Institute on Drug Abuse. www.drugabuse.gov/infofacts/understand.html.

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