Long-acting cocaine hydrolase for addiction therapy

Xiabin Chen

Liu Xue

Shurong Hou

Zhenyu Jin

Ting Zhang

Fang Zheng

Chang Zhan

Supplementary Material

Supplementary File

Click here to view.^{(331K, pdf)}

^{Molecular Modeling and Biopharmaceutical Center, College of Pharmacy, University of Kentucky, Lexington, KY, 40536;}

^{Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, 40536}

^{To whom correspondence may be addressed. Email:}ude.yku@nahz or ude.yku.liame@2nehzf.

Edited by Joanna S. Fowler, Brookhaven National Laboratory, Upton, NY, and approved November 30, 2015 (received for review September 4, 2015)

Author contributions: F.Z. and C.-G.Z. designed research; X.C., L.X., S.H., Z.J., and T.Z. performed research; X.C., T.Z., F.Z., and C.-G.Z. analyzed data; and X.C., F.Z., and C.-G.Z. wrote the paper.

Edited by Joanna S. Fowler, Brookhaven National Laboratory, Upton, NY, and approved November 30, 2015 (received for review September 4, 2015)

Significance

It is essential for a truly effective addiction medication to block the drug's physiological effects effectively without affecting normal functions of the brain and other critical organs such as the heart and while still preventing relapse during abstinence. Most popularly used pharmacological approaches to addiction treatment, including all currently available addiction therapies, either affect normal functions of brain receptors/transporters or are unable to prevent relapse. The long-acting enzyme approach may provide a novel, truly promising therapy capable of effectively blocking the physiological and toxic effects of cocaine without affecting normal functions of the brain and other critical organs and prevent relapse during abstinence. New insights obtained in this study also may be valuable in guiding development of other therapeutic proteins.

Keywords: enzyme therapy, cocaine addiction, drug abuse, protein engineering

Significance

Abstract

Cocaine abuse is a world-wide public health and social problem without a US Food and Drug Administration-approved medication. An ideal anticocaine medication would accelerate cocaine metabolism, producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Our recent studies have led to the discovery of the desirable, highly efficient cocaine hydrolases (CocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of the CNS. Preclinical and clinical data have demonstrated that these CocHs are safe for use in humans and are effective for accelerating cocaine metabolism. However, the actual therapeutic use of a CocH in cocaine addiction treatment is limited by its short biological half-life (e.g., 8 h or shorter in rats). Here we demonstrate a novel CocH form, a catalytic antibody analog, which is a fragment crystallizable (Fc)-fused CocH dimer (CocH-Fc) constructed by using CocH to replace the Fab region of human IgG1. The CocH-Fc not only has a high catalytic efficiency against cocaine but also, like an antibody, has a considerably longer biological half-life (e.g., ∼107 h in rats). A single dose of CocH-Fc was able to accelerate cocaine metabolism in rats even after 20 d and thus block cocaine-induced hyperactivity and toxicity for a long period. Given the general observation that the biological half-life of a protein drug is significantly longer in humans than in rodents, the CocH-Fc reported in this study could allow dosing once every 2–4 wk, or longer, for treatment of cocaine addiction in humans.

Abstract

As is well known, cocaine is one of the most reinforcing abused drugs, stimulating the reward pathway of the brain and teaching the user to take it again (1 –3). Despite decades of effort, the classical pharmacodynamic approach of using small molecules to block or counteract the drug’s neuropharmacological actions has not proven successful for cocaine, because it would be extremely difficult to antagonize cocaine’s physiological effects without affecting normal functions of the CNS (4). In principle, pharmacological treatment for a drug of abuse can be pharmacodynamic or pharmacokinetic (5). Most current medications for other drugs of abuse use the classical pharmacodynamics approach, using small molecules to block or counteract the drug’s neuropharmacological actions at one or more neuronal binding sites. The inherent difficulties of antagonizing cocaine in the CNS led to the development of protein-based pharmacokinetic approaches with biologics such as monoclonal antibodies, vaccines that produce antibodies in the body, and enzymes (4, 6). The pharmacokinetic approach with an efficient enzyme is recognized as the most promising treatment strategy for cocaine overdose and addiction (4, 7 –9). Unlike the stoichiometric binding of an antibody with drug, one enzyme molecule can degrade multiple drug molecules, depending on the turnover number (catalytic rate constant, k_cat) and Michaelis–Menten constant (K_m). In humans, the principal metabolic enzyme of cocaine is a plasma enzyme known as butyrylcholinesterase (BChE), producing biologically inactive metabolites. Unfortunately, the catalytic efficiency (k_cat/K_m) of wild-type BChE against (-)-cocaine (which is the naturally occurring enantiomer of cocaine) is too low (k_cat = 4.1/min and K_m = 4.5 µM) (10) to be effective for cocaine metabolism. A mutant of human BChE with a considerably improved catalytic efficiency against (–)-cocaine is greatly desired.

Through structure- and mechanism-based computational modeling and simulation, we have successfully designed and identified human BChE mutants, recognized as true cocaine hydrolases (CocHs) in the literature (9) when they have at least 1,000-fold improved catalytic efficiency against (-)-cocaine compared with wild-type human BChE (11 –14). The first of our designed CocHs, known as “CocH1” (the A199S/S287G/A328W/Y332G mutant of human BChE) (11, 15), truncated after amino acid 529, was fused with human serum albumin (HSA) to prolong the biological t_1/2 (9). This HSA-fused BChE mutant is also known as “Albu-CocH,” “Albu-CocH1,” “AlbuBChE,” or “TV-1380” (Fig. 1B) in the literature (7 –9, 16). TV-1380 has been proven safe and effective for use in animals and humans (7, 8), but its actual therapeutic value for cocaine addiction treatment is still limited by the moderate biological t_1/2, which is ∼8 h in rats (9) and 43–77 h in humans (7). [In general, the biological t_1/2 of a therapeutic protein is significantly longer in humans than in rats (9).] A biological t_1/2 of 43–77 h in humans might be adequate for a twice-weekly therapy, depending on the dose of the enzyme used. In addition, our more recently designed and identified CocHs (12 –14) are significantly more active than TV-1380 against (-)-cocaine. Further engineering a more active CocH with a biological t_1/2 longer than that of TV-1380 is highly desired.

An external file that holds a picture, illustration, etc.
Object name is pnas.1517713113fig01.jpg

Fig. 1.

Protein structures and their catalytic parameters for cocaine hydrolysis. (A) Human BChE. (B) Albu-CocH1 (or TV-1380). (C) CAb 15A10. (D) CocH3-Fc.

Our current design strategy for enzyme engineering is based on the observation that an antibody such as human IgG has a very long biological t_1/2, because the fragment crystallizable (Fc) region of IgG can bind with the neonatal Fc receptor (FcRn) in the acidic environment of the endosome and later is transported to the cell surface where, upon exposure to a neutral pH, IgG is released back into the main bloodstream (17). In comparison, a recombinant enzyme such as BChE usually has a very short biological t_1/2, and an antibody usually has no catalytic activity. Using a stable analog of the transition state for cocaine hydrolysis, Landry et al. (1) successfully generated the first anticocaine catalytic antibody (CAb). Further effort generated the most active anticocaine CAb (Fig. 1C), known as “15A10” (k_cat = 2.3/min and K_m = 220 µM) (18, 19), whose catalytic activity against (-)-cocaine is still significantly lower than that of wild-type BChE. It has been difficult to improve the CAb activity further because a CAb, unlike an enzyme, can help stabilize the transition state only for nonenzymatic cocaine hydrolysis; a mechanistic study (20) indicates there is no formation of a covalent bond or breaking between the substrate and CAb during the reaction process.

We aimed to design a long-acting CocH form that has both the long biological t_1/2 of an antibody and the high catalytic activity of a CocH against (-)-cocaine. For this purpose, starting from human IgG1 (a dimer), which has both the Fc region (constant) and Fab region (variable), as seen in Fig. 1C, we may replace the Fab region by a CocH for each subunit of the dimeric IgG1 to construct a catalytic antibody analog. Technically, the C terminus of CocH3 [the A199S/F227A/S287G/A328W/Y332G mutant (12) of human BChE, full-length or truncated after amino acid 529 to delete the tetramerization domain] was fused with the N terminus of the hinge region linked with Fc. CocH3 has a significantly higher catalytic efficiency against (-)-cocaine (12) than does CocH1 or TV-1380. The obtained dimeric CocH3-Fc fusion enzyme depicted in Fig. 1D is highly efficient for cocaine hydrolysis. Obviously, the CocH3-Fc is different from but is structurally similar to antibody IgG1, containing Fc that can bind with FcRn in the acidic environment to prolong the biological t_1/2. Further possible mutations on the Fc region of CocH3-Fc also were examined so that the CocH3-Fc enzyme would have the longest possible biological t_1/2. Various CocH3-Fc forms, including Fc mutants, were proposed, prepared, and tested for their actual in vitro and in vivo profiles. In addition, Albu-CocH1 or TV-1380 also was prepared and tested for comparison. As shown below, the optimized CocH3-Fc indeed has both a significantly higher catalytic activity against cocaine and a much longer biological t_1/2 than TV-1380.

Acknowledgments

This work was supported in part by NIH Grants UH2 {"type":"entrez-nucleotide","attrs":{"text":"DA041115","term_id":"79228097"}}DA041115, R01 DA035552, R01 DA032910, R01 DA013930, and R01 DA025100 and by National Science Foundation Grant CHE-1111761.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1517713113/-/DCSupplemental.

Footnotes

References

1. Landry DW, Zhao K, Yang GX, Glickman M, Georgiadis TMAntibody-catalyzed degradation of cocaine. Science. 1993;259(5103):1899–1901.[PubMed][Google Scholar]
2. Milton AL, Everitt BJ. Neuroscience. Wiping drug memories. Science. 2012;336(6078):167–168.[PubMed]
3. Ersche KD, et al Abnormal brain structure implicated in stimulant drug addiction. Science. 2012;335(6068):601–604.[PubMed][Google Scholar]
4. Zheng F, Zhan C-GAre pharmacokinetic approaches feasible for treatment of cocaine addiction and overdose? Future Med Chem. 2012;4(2):125–128.[Google Scholar]
5. Gorelick DAPharmacokinetic approaches to treatment of drug addiction. Expert Rev Clin Pharmacol. 2008;1(2):277–290.[PubMed][Google Scholar]
6. Skolnick P, White D, Acri JBEditorial: Emerging targets for stimulant use disorders: Where to invest in an era of constrained resources? CNS Neurol Disord Drug Targets. 2015;14(6):691.[PubMed][Google Scholar]
7. Cohen-Barak O, et al Safety, pharmacokinetics, and pharmacodynamics of TV-1380, a novel mutated butyrylcholinesterase treatment for cocaine addiction, after single and multiple intramuscular injections in healthy subjects. J Clin Pharmacol. 2015;55(5):573–583.[Google Scholar]
8. Shram MJ, et al Assessment of pharmacokinetic and pharmacodynamic interactions between albumin-fused mutated butyrylcholinesterase and intravenously administered cocaine in recreational cocaine users. J Clin Psychopharmacol. 2015;35(4):396–405.[PubMed][Google Scholar]
9. Brimijoin S, et al A cocaine hydrolase engineered from human butyrylcholinesterase selectively blocks cocaine toxicity and reinstatement of drug seeking in rats. Neuropsychopharmacology. 2008;33(11):2715–2725.[Google Scholar]
10. Sun H, Pang Y-P, Lockridge O, Brimijoin SRe-engineering butyrylcholinesterase as a cocaine hydrolase. Mol Pharmacol. 2002;62(2):220–224.[PubMed][Google Scholar]
11. Pan Y, et al Computational redesign of human butyrylcholinesterase for anticocaine medication. Proc Natl Acad Sci USA. 2005;102(46):16656–16661.[Google Scholar]
12. Zheng F, et al Most efficient cocaine hydrolase designed by virtual screening of transition states. J Am Chem Soc. 2008;130(36):12148–12155.[Google Scholar]
13. Xue L, et al Design, preparation, and characterization of high-activity mutants of human butyrylcholinesterase specific for detoxification of cocaine. Mol Pharmacol. 2011;79(2):290–297.[Google Scholar]
14. Zheng F, et al A highly efficient cocaine-detoxifying enzyme obtained by computational design. Nature Commun. 2014;5:3457.[Google Scholar]
15. Yang W, Xue L, Fang L, Chen X, Zhan C-GCharacterization of a high-activity mutant of human butyrylcholinesterase against (-)-cocaine. Chem Biol Interact. 2010;187(1-3):148–152.[Google Scholar]
16. Willyard CPharmacotherapy: Quest for the quitting pill. Nature. 2015;522(7557):S53–S55.[PubMed][Google Scholar]
17. Roopenian DC, Akilesh SFcRn: The neonatal Fc receptor comes of age. Nat Rev Immunol. 2007;7(9):715–725.[PubMed][Google Scholar]
18. Larsen NA, et al Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10. Biochemistry. 2004;43(25):8067–8076.[PubMed][Google Scholar]
19. Yang G, et al Anti-cocaine catalytic antibodies: A synthetic approach to improved antibody diversity. J Am Chem Soc. 1996;118(25):5881–5890.[PubMed][Google Scholar]
20. Pan Y, Gao D, Zhan C-GModeling the catalysis of anti-cocaine catalytic antibody: Competing reaction pathways and free energy barriers. J Am Chem Soc. 2008;130(15):5140–5149.[Google Scholar]
21. Pan Y, Muzyka JL, Zhan C-GModel of human butyrylcholinesterase tetramer by homology modeling and dynamics simulation. J Phys Chem B. 2009;113(18):6543–6552.[Google Scholar]
22. Maxwell LJ, Singh JAAbatacept for rheumatoid arthritis: A Cochrane systematic review. J Rheumatol. 2010;37(2):234–245.[PubMed][Google Scholar]
23. Rigby MR, et al Alefacept provides sustained clinical and immunological effects in new-onset type 1 diabetes patients. J Clin Invest. 2015;125(8):3285–3296.[Google Scholar]
24. Fang L, et al Rational design, preparation, and characterization of a therapeutic enzyme mutant with improved stability and function for cocaine detoxification. ACS Chem Biol. 2014;9(8):1764–1772.[Google Scholar]
25. Xue L, et al Preparation and in vivo characterization of a cocaine hydrolase engineered from human butyrylcholinesterase for metabolizing cocaine. Biochem J. 2013;453(3):447–454.[Google Scholar]
26. Sun H, Shen ML, Pang Y-P, Lockridge O, Brimijoin SCocaine metabolism accelerated by a re-engineered human butyrylcholinesterase. J Pharmacol Exp Ther. 2002;302(2):710–716.[PubMed][Google Scholar]
27. Hou S, Zhan M, Zheng X, Zhan C-G, Zheng FKinetic characterization of human butyrylcholinesterase mutants for the hydrolysis of cocaethylene. Biochem J. 2014;460(3):447–457.[Google Scholar]
28. Smith M, Garner D, Niemann JTPharmacologic interventions after an LD50 cocaine insult in a chronically instrumented rat model: Are beta-blockers contraindicated? Ann Emerg Med. 1991;20(7):768–771.[PubMed][Google Scholar]
29. Bedford JA, Turner CE, Elsohly HNComparative lethality of coca and cocaine. Pharmacol Biochem Behav. 1982;17(5):1087–1088.[PubMed][Google Scholar]
30. Mets B, et al A catalytic antibody against cocaine prevents cocaine’s reinforcing and toxic effects in rats. Proc Natl Acad Sci USA. 1998;95(17):10176–10181.[Google Scholar]
31. Braman J, Papworth C, Greener ASite-directed mutagenesis using double-stranded plasmid DNA templates. Methods Mol Biol. 1996;57:31–44.[PubMed][Google Scholar]
32. Committee on Care and Use of Laboratory Animals (1996) Guide for the Care and Use of Laboratory Animals (Natl Inst Health, Bethesda), DHHS Publ No (NIH) 85-23.
33. Lockridge O, Schopfer LM, Winger G, Woods JHLarge-scale purification of butyrylcholinesterase from human plasma suitable for injection into monkeys; a potential new therapeutic for protection against cocaine and nerve agent toxicity. J Med Chem Biol Radiol Defense. 2005;3:1–20.[Google Scholar]
34. Mercolini L, et al Quantitative analysis of cocaine in human hair by HPLC with fluorescence detection. J Pharm Biomed Anal. 2008;48(2):456–461.[PubMed][Google Scholar]
35. Ko MC, et al Cocaine esterase: Interactions with cocaine and immune responses in mice. J Pharmacol Exp Ther. 2007;320(2):926–933.[PubMed][Google Scholar]