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1-30-2018 High Activity Mutants of Cocaine Esterase for Cocaine Hydrolysis Chang-Guo Zhan University of Kentucky, [email protected]
Fang Zheng University of Kentucky, [email protected]
Lei Fang University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits oy u.
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Recommended Citation Zhan, Chang-Guo; Zheng, Fang; and Fang, Lei, "High Activity Mutants of Cocaine Esterase for Cocaine Hydrolysis" (2018). Pharmaceutical Sciences Faculty Patents. 172. https://uknowledge.uky.edu/ps_patents/172
This Patent is brought to you for free and open access by the Pharmaceutical Sciences at UKnowledge. It has been accepted for inclusion in Pharmaceutical Sciences Faculty Patents by an authorized administrator of UKnowledge. For more information, please contact [email protected]. I IIIII IIIIIIII Ill lllll lllll US009879240B2lllll lllll lllll lllll lllll lllll 111111111111111111 c12) United States Patent (IO) Patent No.: US 9,879,240 B2 Zhan et al. (45) Date of Patent: Jan.30,2018
(54) HIGH ACTIVITY MUTANTS OF COCAINE Beadle, B. M., and Shoichet, B. K. (2002) Structural bases of ESTERASE FOR COCAINE HYDROLYSIS stability-function tradeoffs in enzymes. J. Mo!. Biol. 321, 285- 296. (71) Applicant: University of Kentucky Research Tokuriki, N., Stricher, F., Serrano, L., and Tawrik, D. S. (2008) How Foundation, Lexington, KY (US) protein stability and new functions trade off PLoS Comput. Biol. 4, el000002. (72) Inventors: Chang-Guo Zhan, Lexington, KY Thomas, V. L., McReynolds, A. C., and Shoichetb, B. K. (2010) (US); Fang Zheng, Lexington, KY Structural bases for stability-function tradeoffs in antibiotic resis (US); Lei Fang, Lexington, KY (US) tance. J. Mo!. Biol. 396, 47-59. Brim, R. L., Nance, M. R, Youngstrom, D. W., Narasimhan, D., (73) Assignee: University of Kentucky Research Zhan, C.-G., Tesmer, J. J., Sunahara, R. K., and Woods, J. H. (2010) A thermally stable form of bacterial cocaine esterase: A potential Foundation, Lexington, KY (US) therapeutic agent for treatment of cocaine abuse. Mo!. Pharmacol. 77, 593-600. ( *) Notice: Subject to any disclaimer, the term ofthis Collins, G. T., Brim, R. L., Narasimhan, D., Ko, M.-C., Sunahara, patent is extended or adjusted under 35 R. K., Zhan, C.-G., and Woods, J. H. (2009) Cocaine esterase U.S.C. 154(b) by 173 days. prevents cocaine-induced toxicity and the ongoing intravenous self-administration of cocaine in rats. J. Pharmacol. Exp. They. 331, (21) Appl. No.: 14/924,181 445-455. Narasimhan, D., Nance, M. R., Gao, D., Ko, M.-C., Macdonald, J., Tamburi, P., Yoon, D., Landry, D. M., Woods, J. H., Zhan, C.-G., (22) Filed: Oct. 27, 2015 Tesmer, J. J. G., and Sunahara, R K. (2010) Structural analysis of thermostabilizing mutations of cocaine esterase. Protein. Eng. Des. (65) Prior Publication Data Se!. 23, 537-547. Narasimhan, D., Collins, G. T., Nance, M. R., Nichols, J., Edwald, US 2016/0122732 Al May 5, 2016 E., Chan, J., Ko, M.-C., Woods, J. H., Tesmer, J. J. G., and Sunahara, R. K. (2011) Subunit stabilization and polyethylene glycolation of Related U.S. Application Data cocaine esterase improves in vivo residence time. Mo!. Pharmacal. (60) Provisional application No. 62/073,562, filed on Oct. 80, 1056-1065. 31, 2014. Collins, G. T., Zaks, M. E., Cunningham, A. R., St, Clair, C., Nichols, J., Narasimhan, D., Ko, M.-C., Sunahara, R. K., and Woods, J. H. (2011) Effects of a long-acting mutant bacterial (51) Int. Cl. cocaine esterase on acute cocaine toxicity in rats. Drug Alcohol A61K 38/00 (2006.01) Depend. 118, 158-165. C12N 9/18 (2006.01) Collins, G. T., Narasimhan, D., Cunningham, A. R., Zaks, M. E., A61K 38/46 (2006.01) Nichols, J., Ko, M.-C., Sunahara, R. K., and Woods, J. H. (2012) Longlasting effects of a PEGylated mutant cocaine esterase (CocE) (52) U.S. Cl. on the reinforcing and discriminative stimulus effects of cocaine in CPC ...... C12N 9/18 (2013.01); A61K 38/465 rats. Neuropsychopharmacology 37, 1092-1103. (2013.01); Cl2Y 301/01084 (2013.01) Collins, G. T., Brim, II L., Noon, K. R., Narasimhan, D., Lukacs, N. (58) Field of Classification Search W., Sunahara, R. K., Woods, J. H., and Ko, M.-C. (2012) Repeated CPC ...... A61K 38/00; A61K 38/465; C12N 9/18; administration of a mutant cocaine esterase: Effects on plasma cocaine levels, cocaine-induced cardiovascular activity, and C12Y 301/01084 immune responses in rhesus monkeys. J. Pharmacol. Exp. Ther. 342, See application file for complete search history. 205-213. Yang, W., Pan, Y., Zheng, F., Cho, H., Tai, H.-H., and Zhan, C.G. (56) References Cited (2009) Free-energy perturbation simulation on transition states and redesign of butyrylcholinesterase. Biophys. J. 96, 1931-1938. PUBLICATIONS (Continued)
Carrera, M. R. A., Kaufmann, G. F., Mee, J. M., Meijler, M. M., Primary Examiner - Kagnew H Gebreyesus Koob, G. F., and Janda, K. D. (2004) Treating cocaine addiction (74) Attorney, Agent, or Firm - Stites & Harbison, with viruses. Proc. Natl. Acad. Sci. U.S.A. 101, 10416-10421. PLLC; Mandy Wilson Decker Meijler, M. M., Kaufmann, G. F., Q, L., Mee, J. M., Coyle, A. R., Moss, J. A., Wirsching, P., Matsushita, M., and Janda, K. D. (2005) (57) ABSTRACT Fluorescent cocaine probes: A tool forthe selection and engineering The Bacterial cocaine esterase (CocE) mutants disclosed of therapeutic antibodies. J. Am. Chem. Soc. 127, 2477-2484. Larsen, N. A., Turner, J. M., Stevens, J., Rosser, S. J., Basran, A., herein each have enhanced catalytic efficiency for ( -) Lerner, R. A., Bruce, N. C., and Wilson, I. A. (2002) Crystal cocaine, as compared to CocE mutants in the prior art, structure of a bacterial cocaine esterase. Nat. Struct. Mo!. Biol. 9, including CocE mutant E172-173. The presently-disclosed 17-21. subject matter further includes a pharmaceutical composi Ko, M.-C., Bowen, L .D., Narasimhan, D., Berlin, A. A., Lukacs, N. tion including a mutant of bacterial cocaine hydrolase, as W., Sunahara, R K., Cooper, Z. D., and Woods, J. H. (2007) Cocaine described herein, and a suitable pharmaceutical carrier. The esterase: Interactions with cocaine and immune responses in mice. presently-disclosed subject matter further includes a method J. Pharmacol. Exp. Ther. 320, 326-933. of treating a cocaine-induced condition comprising admin Gao, D., Narasimhan, D. L., Macdonald, J., Ko, M.-C., Landry, D. istering to an individual an effective amount of a mutant of W., Woods, J. H., Sunahara, R. K., and Zhan, C.-G. (2009) Thermostable variants of cocaine esterase for long-time protection bacterial cocaine hydrolase variant, as disclosed herein, to against cocaine toxicity. Mo!. Pharmacol. 75, 318-323. accelerate cocaine metabolism and produce biologically Shoichet, B. K., Baase, W. A., Kurold, R, and Matthews, B. W. inactive metabolites. ( 1995) A relationship between protein stability and protein function. Proc. Natl. Acad. Sci. U.S.A. 92, 452-456. 14 Claims, 5 Drawing Sheets US 9,879,240 B2 Page 2
(56) References Cited
PUBLICATIONS
Hou, S., Xue, L, Yang, W., Fang, L., Zheng, F., and Zhan, C.-G. (2013) Substrate selectivity of high-activity mutants of human butyrylcholinesterase. Org. BiotnoL Chem. 11, 7477-7485. Pan, Y., Gao, D., Yang, W., Cho, H., Yang, G., Tai, H.-H., and Zhan, C.-G. (2005) Computational redesign of human butyrylcholinesterase for anticocaine medication. Proc. Natl. Acad. Sci. U.S.A. 102, 16656-16661. Zheng, F., Yang, W., Xue, L., Hou, S., Liu, J., and Zhan, C.-G. (2010) Design of high-activity mutants of human butyrylcholinesterase against (--)-cocaine: structural and energetic factors affecting the catalytic efficiency. Biochemistry 49, 9113- 9119. Gao, D., and Zhan, C.-G. (2006) Modeling evolution of hydrogen bonding and stabilization of transition states in the process of cocaine hydrolysis catalyzed by human butyrylcholinesterase. Pro teins 62, 99-110. Hamza, A, Cho, H., Tai, H. H., and Zhan, C.-G. (2005) Molecular dynamics simulation of cocaine binding with human butyrylcholinesterase and its mutants. J. Phys. Chem. B 109, 4776- 4782. Pan, Y., Gao, D., Yang, W., Cho, H., and Zhan, C.-G. (2007) Free energy perturbation (FEP) simulation on the transition states of cocaine hydrolysis catalyzed by human butyrylcholinesterase and its mutants. J. Am. Chem. Soc. 129, 13537-13543. Zheng, F., Yang, W., Ko, M.-C., Liu, J., Cho, H., Gao, D., Tong, M., Tai, H.-H., Woods, J. H., and Zhan, C.-G. (2008) Most efficient cocaine hydrolase designed by virtual screening of transition states. J. Am. Chem. Soc. 130, 12148-12155. U.S. Patent Jan.30,2018 Sheet 1 of 5 US 9,879,240 B2
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� E196-JO! '*' PEGyfate FIG. 6 U.S. Patent Jan.30,2018 Sheet 5 of 5 US 9,879,240 B2 ...... ·f.Uft!l...... YU�1l .·.(:(,ct) l>..�· ...... • ."·····.·····" ..········.·····". t, t;} N W 4!) ·�� FIGS. 7}\-B dfatdkk 1 �······ ····� 4: .. ·····� FIG. 8 US 9,879,240 B2 1 2 HIGH ACTIVITY MUTANTS OF COCAINE manly recognized "stability-function trade-off' theory/hy ESTERASE FOR COCAINE HYDROLYSIS pothesis, (14) protein residues that contribute to catalysis or ligand binding are not optimal for protein stability and, thus, RELATED APPLICATIONS there is a balance between the stability and function. Indeed, 5 extensive studies (14, 17-22) demonstrate that thermostabi This application claims priority from U.S. Provisional lizing mutations of enzymes decrease the catalytic activities Application Ser. No. 62/073,562 filed Oct. 31, 2014, the of those enzymes and that mutations improving the catalytic entire disclosure of which is incorporated herein by this activities decrease the thermal stability. Nevertheless, some reference. CocE mutants with an improved thermal stability have 10 successfully been designed and discovered in recently GOVERNMENT SUPPORT CLAUSE reported studies, (11, 23-26) and these thermostablemutants do not decrease, or only slightly decrease, the catalytic This invention was made with Govermnent support under efficiency(k calKM) of CocE against cocaine. Further animal NIH number NIH ROI DA025100. The Government has behavior studies (27-29) reveal that these CocE mutants are certain rights in this invention. 15 promising in development of an enzyme therapy for cocaine TECHNICAL FIELD abuse. Notably, one of the reported thermostable mutants of The presently-disclosed subject matter relates to thera CocE, i.e. the Tl 72R/G 173Q mutant (known as drug RBP- peutic enzymes, such as a mutant of bacterial cocaine 20 8000, with Clinica!Trials.gov Identifier of NCT01846481 in hydrolase, in particular, cocaine esterase (CocE) polypeptide clinical development) designed through computational mod variant(s) with amino acid substitutions relative to wild type eling and simulations (11) has been advanced to the ran CocE. domized, double-blind, placebo controlled clinical trial phase II for cocaine overdose treatment. The Tl 72R/G 173Q INTRODUCTION 25 mutant (denoted as enzyme El 72-173 here for convenience) was designed through introducing favorable noncovalent Cocaine overdose and addiction have resulted in serious forces including a hydrogen bond between domains I and II medical and social problems in modem society. (1) So far, of the protein. (11) This CocE mutant has an in vitro half-life there is no anticocaine medication approved by the Food and of about 6 hours at 37° C. without decreasing the catalytic Drug Administration (FDA). (2, 3) Cocaine causes physi 30 activity of CocE against cocaine. (11) The half-life of about ological effects by binding with the dopamine transporter 6 hours at 37° C. is long enough for cocaine overdose and, thus, blocking dopamine reuptake. The disastrous treatment, because one just needs to use the enzyme to medical and social consequences of cocaine abuse have rapidly detoxify cocaine. However, for cocaine addiction made the development of an anticocaine medication a high treatment, it is desirable to have a highly efficient cocaine- priority. However, despite decades of efforts, the classical 35 metabolizing enzyme in the body with a residence time as pharmacodynamic approach has failed to yield a truly useful long as possible. With a highly efficiently cocaine-metabo small-molecule receptor/transporter antagonist. The alterna lizing enzyme in the body, whenever a cocaine abuser uses tive pharmacokineticapproach is to interfere with the deliv cocaine again, the enzyme would rapidly metabolize cocaine ery of cocaine to its receptors and/or accelerate its metabo so that the cocaine abuser would not feel the stimulant lism in the body. (2, 4-8) It would be an ideal to develop an 40 effects of cocaine. exogenous enzyme which can accelerate cocaine metabo To further develop an improved therapeutic enzyme for lism and produce biologically inactive metabolites. cocaine abuse treatment, one would like to both extend the Bacterial cocaine esterase (CocE) has been recognized as half-life of E172-173 at 37° C. and improve the catalytic the most efficient natural enzyme for hydrolyzing the natu efficiency against cocaine. It has been shown (11, 15, 26) rally occurring(- )-cocaine. (9) No other natural esterase has 45 that the thermal stability of El 72-173 at 37° C. can be a catalytic activity for cocaine comparable to that of CocE. enhanced by extra mutations on E172-173. However, none Studies have shown that CocE can help to prevent extreme of the reported extra mutations on El 72-173 improved the cocaine toxicity and can even prevent the lethal effects of catalytic efficiency against cocaine. cocaine in some subjects. (10) However, a major obstacle to the clinical application of CocE is the thermoinstability of 50 SUMMARY wild-type CocE with a half-life of only about 12 minutes at physiological temperature (37° C.). (11) It is highly desir The presently-disclosed subject matter meets some or all able to develop thermostablemutants of CocE for therapeu- of the above-identified needs, as will become evident to tic treatment of cocaine abuse (overdose and addiction). In those of ordinary skill in the art after a study of information fact, thermal stability is a well-known common problem in 55 provided in this document. protein drug development. (11) In general, the more ther This S=ary describes several embodiments of the mally stable a protein drug, the longer shelf half-life the presently-disclosed subject matter, and in many cases lists protein drug can have. variations and permutations of these embodiments. Generally speaking, the thermal stability of a protein can This Summary is merely exemplary of the numerous and be improved by enhancing the weak interactions inside the 60 varied embodiments. Mention of one or more representative enzyme through either noncovalent forces, such as hydrogen features of a given embodiment is likewise exemplary. Such bonds, (12) or covalent linkage, such as disulfide bonds. (13) an embodiment can typically exist with or without the Particularly for an enzyme, besides improving its stability, it feature(s) mentioned; likewise, those features can be applied is also important to maintain the catalytic activity of the to other embodiments of the presently-disclosed subject enzyme. However, it is much more challenging to engineer 65 matter, whether listed in this Summary or not. To avoid an enzyme with an improved stability without decreasing the excessive repetition, this Summary does not list or suggest catalytic activity. (14-16) In general, according to the com- all possible combinations of such features. US 9,879,240 B2 3 4 Here, a rationally-designed new mutant of El 72-173 is tively), domain I is shown in red, domain II is shown in reported, which has not only considerably extended the in green, and domain III is shown in yellow. (FIG. lC) Key ° vitro half-life at 37 C., but also significantly improved the residues L196 (alb) and 1301 (alb) shown in ball and sticks catalytic efficiency against cocaine. The new CocE mutant on the dimer interface. (i.e. the Tl 72R/G173Q/Ll 96C/1301 C mutant of CocE, 5 FIGS. 2A-2C show plots of measured initial reaction rates denoted as enzyme E196-301 for convenience) was modi (represented in µM min-1 per nM enzyme at 37° C., with fied further via PEGylation in order to extend the in vivo error bars) versus the substrate concentration for ( -)-cocaine residence time of the enzyme. The PEGylated E196-301 was hydrolysis catalyzed by CocE mutants: (FIG. 2A) Tl 72R/ used to fully protect mice from a lethal dose of cocaine (180 G173Q (FIG. 28) T172R/G173Q/G4C/S10; (FIG. 2C) 0 mg/kg, LD100) for at least 3 days, indicating that it might be 1 Tl 72R/G1 73Q/Ll 96C/1301 C. a more promising enzyme candidate for development of FIG. 3 provides a plot of the remaining enzyme activity novel anticocaine therapeutics. of the Tl 72R/Gl 73Q/L196C/I301C CocE against cocaine Hence, the presently-disclosed subject matter includes a versus the time of the enzyme incubation at 37° C. The mutant of bacterial cocaine hydrolase. The presently-dis catalytic activity of the incubated enzyme was assayed after closed subject matter includes a new mutant of El 72-173. In 15 some instances, the mutant enzyme has one or more cross 0, 2, 9, 12, 31, and 100 days. subunit disulfide bond. In some embodiments, the one FIGS. 4A-4D depict a crystal structure of the CocE cross-subunit disulfide bond is a bond between C196a and mutant dimer. (FIG. 4A) Ribbons representation of the C301b, or C301a and C196b. In some preferred embodi homodimeric molecule generated by applying 2-fold crys ments, the enzyme has a pair of cross-subunit disulfide 20 tallographic symmetry. The side chains of the cysteine bonds at C196a and C301b, and C301a and C196b. residues forming intersubunit disulfide bonds are shown in In some embodiments, the mutant of bacterial cocaine a space filling representation. (FIG. 48) View of the dimer hydrolase provides improved stability and/or improved cata rotated 90° about a horizontal axis. (FIG. 4C) Fo-Fc electron lytic activity against cocaine. In some embodiments, the density (green, 3.5 sigma contour) calculated with the rig mutant of bacterial cocaine hydrolase provides improved 25 idly placed (before refinement) molecular replacement stability and/or improved catalytic activity against cocaine. model having residues 196 and 301 altered to glycines. The In some embodiments, the mutant bacterial cocaine hydro final refinedmodel in a stick representation is superimposed lase includes at least one polyethylene glycol polymer chain on the map. (FIG. 4D) Final SIGMAA-weighted 2Fo-Fc attached thereto. In some embodiments, the pegylation electron density map (blue, 1.0 sigma cutoff)in the region of improves stability of the enzyme. In some preferred embodi- 30 the disulfide bond with the final model shown in a stick ments, the hydrolase is conjugated to a maleimide linked representation. polyethylene glycol. In some embodiments, the PEG mol FIG. 5 illustrates the in vivo effectiveness of E196-301 ecule is branched. (black squares) and the PEGylated E196-301 (red triangles) The presently disclosed subject matter further includes a in the protection of mice from cocaine-induced lethality. A pharmaceutical composition that includes a bacterial 35 single dose (30 mg/kg) of E196-301 (PEGylated or unPE cocaine hydrolase variant and a suitable pharmaceutical earner. Gylated) was administered (i.v.) 1 min before the first i.p. = The presently-disclosed subject matter further includes a administration of 180 mg/kg cocaine (n 5). The mice were method of treating a cocaine-induced condition, which challenged daily with 180 mg/kg cocaine until no mouse includes administering to an individual an effective amount 40 survived. E196-301 refers to the T172R/G173Q/L196C/ of bacterial cocaine hydrolase variant or a pharmaceutical I301C mutant of CocE. composition comprising a bacterial cocaine hydrolase vari FIG. 6 illustrates backbone superposition between the ant, as described herein, to accelerate cocaine metabolism X-ray crystal structures of E172-173 and E196-301. E172- and produce biologically inactive metabolites. In some 173 is represented in green ribbons, and E196-301 is rep embodiments, the bacterial cocaine hydrolase variant exhib- 45 resented in red ribbons. The black arrow indicates the shift its a one-hundred-fold or more increase in cocaine hydro direction. Here, El 72-173 represents Tl 72R/G173Q CocE, lysis catalytic efficiencycompared to compared to El 72-E73 and El 96-301 refers to Tl 72R/G173Q/Ll 96C/I301 C CocE. mutant hydrolase, which is currently in clinical trials for FIGS. 7A-7B show the time-dependence of important treatment of cocaine overdose. H ...0 distances (relevant to hydrogen bonds) from the 50 MD-simulated El 72-173 and E196-301 structures. Y44HH- BRIEF DESCRIPTION OF THE DRAWINGS CocO represents the distance between the hydroxyl hydro gen (denoted as HH) of the Y44 side chain and the carbonyl The novel features of the subject matter of the present oxygen ( denoted as CocO) of (-)-cocaine benzoyl ester. disclosure are set forth with particularity in the following Y118H-Coc0 refers to the distance between hydrogen (H) description and in the appended sample claims. A better 55 of the Yll 8 backbone and the carbonyl oxygen (CocO) of understanding of the features and advantages of the (-)-cocaine benzoyl ester. El 72-173 refers to Tl 72R/ presently disclosed subject matter will be obtained by ref G173Q CocE, and E196-301 refers to T172R/G173Q/ erence to the following detailed description that sets forth L196C/I301C CocE. illustrative embodiments, in which the principles of the FIG. 8 is an illustration of intermonomer disulfide bonds invention(s) are used, and the accompanying drawings. 60 in the CocE mutant (E196-301) dimer refined in space group FIGS. lA-lC present a modeled E172-173 dimer struc P65. The complete process of structure determination was ture. (FIG. lA) The time-dependent Ca-Ca distances carried out in a space group with lower symmetry than the between L196 and 1301 in the MD-simulated dimer struc true space group (P6522) in order to assess the effectsof the ture of El 72-173. The letters a and b indicated after the dimer being located on a crystallographic two-fold axis. residue numbers refer to subunits a and b, respectively. (FIG. 65 Noncrystallographic symmetry restraints were not used dur 18) The modeled El 72-173 dimer structure shown in rib ing refinement. The panels show the two, now not strictly bons (with a and b referring to subunits a and b, respec- identical, disulfide bonds for the final model (Rwork----0.17, US 9,879,240 B2 5 6 Rfree-0.21; yellow carbons) superimposed on the identical effective amount of a butyrylcholinesterase polypeptide disulfide bonds in the model refined on the crystallographic variant, as disclosed herein, to lower blood cocaine concen two-fold axis (cyan carbons). tration. While the following terms used herein are believed to be BRIEF DESCRIPTION OF THE SEQUENCE well understood by one of ordinary skill in the art, defini LISTING tions are set forth to facilitate explanation of the presently disclosed subject matter. SEQ ID NO: 1 is a nucleotide sequence encoding wild The terms "polypeptide", "protein", and "peptide", which type cocaine esterase (CocE) polypeptide of SEQ ID NO: 2. are used interchangeably herein, refer to a polymer of the SEQ ID NO: 2 is the amino acid of wild type CocE. 10 protein amino acids, or amino acid analogs, regardless of its SEQ ID NO: 3 is a nucleotide sequence encoding the size or function. Although "protein" is oftenused in refer CocE polypeptide variant of SEQ ID NO: 4; ence to relatively large polypeptides, and "peptide" is often SEQ ID NO: 4 is an amino acid sequence encoding the used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term "polypeptide" as CocE polypeptide variant having the following amino acid 15 used herein refers to peptides, polypeptides, and proteins, substitutions, as compared to wild type CocE: Tl 72R, unless otherwise noted. The terms "protein", "polypeptide", Gl 73Q, L196C, and I301C. and "peptide" are used interchangeably herein when refer ring to a gene product. Thus, exempl polypeptides DESCRIPTION OF EXEMPLARY ary include gene products, naturally occurring proteins, EMBODIMENTS 20 homologs, orthologs, paralogs, fragments and other equiva lents, variants, and analogs of the foregoing. The details of one or more embodiments of the presently The term "variant" or "mutant" refers to an amino acid disclosed subject matter are set forth in this document. sequence that is different from the reference polypeptide by Modifications to embodiments described in this document, one or more amino acids, e.g., one or more amino acid and other embodiments, will be evident to those of ordinary 25 substitutions. For example a CocE polypeptide variant dif skill in the art after a study of the information provided in fers from wild-type CocE by one or more amino acid this document. The information provided in this document, substitutions, i.e., mutations. and particularly the specific details of the described exem The terms "polypeptide fragment" or "fragment", when plary embodiments, is provided primarily for clearness of used in reference to a reference polypeptide, refers to a understanding and no unnecessary limitations are to be 30 polypeptide in which amino acid residues are deleted as understood therefrom. In case of conflict, the specification of compared to the reference polypeptide itself, but where the this document, including definitions, will control. remaining amino acid sequence is usually identical to the The presently-disclosed subject matter is illustrated by corresponding positions in the reference polypeptide. Such specific but non-limiting examples throughout this descrip deletions can occur at the amino-terminus, carboxy-terminus tion. The examples may include compilations of data that are 35 of the reference polypeptide, or alternatively both. For representative of data gathered at various times during the example, CocE polypeptide fragment can have 1, 2, 3, 4, 5, course of development and experimentation related to the 6, 7, 8, 9, or 10 fewer amino acids than a full-length present invention(s). Each example is provided by way of wild-type CocE polypeptide. CocE polypeptide fragments explanation of the present disclosure and is not a limitation are also inclusive of fragments of CocE polypeptide vari- thereon. In fact, it will be apparent to those skilled in the art 40 ants. that various modifications and variations can be made to the The term "cocaine" may refer to any and all forms of teachings of the present disclosure without departing from cocaine, including smoked, heated, inhaled, or injected the scope of the disclosure. For instance, features illustrated cocaine. Further, in some embodiments, the term "cocaine" or described as part of one embodiment can be used with refers to any substance obtained, isolated and/or derived another embodiment to yield a still further embodiment. 45 from the leaves of a coca plant. And in certain embodiments, All references to singular characteristics or limitations of the term "(-)-cocaine" refers to methyl (1R,2R,3S,5S)-3- the present disclosure shall include the corresponding plural (benzoyloxy )-8-methy1-8-azabicyclo [3 .2 .1]octane-2-car characteristic(s) or limitation(s) and vice versa, unless oth boxy late. erwise specified or clearly implied to the contrary by the Unless otherwise indicated, the term "administering" is context in which the reference is made. 50 inclusive of all means known to those of ordinary skill in the All combinations of method or process steps as used art for providing a preparation to a subject, including admin herein can be performed in any order, unless otherwise istration by inhalation, nasal administration, topical admin specified or clearly implied to the contrary by the context in istration, intravaginal administration, ophthalmic adminis which the referenced combination is made. tration, intraaural administration, intracerebral The presently-disclosed subject matter includes cocaine 55 administration, intravitreous administration, intracameral hydrolase (CocE) polypeptide variants. In some embodi administration, posterior sub-Tenon administration, poste ments, the CocE polypeptide variant is a mutant of El 72- rior juxtascleral administration, subretinal administration, 173. The CocE polypeptide variants disclosed herein each suprachoroidal administration, cell-based administration or have enhanced catalytic efficiency for (-)-cocaine, as com production, rectal administration, and parenteral administra- pared to CocE mutants in the prior art, including CocE 60 tion, including injectable such as intravenous administra mutant E 1 72-173. tion, intra-arterial administration, intramuscular administra The presently-disclosed subject matter further includes a tion, and/or subcutaneous administration. Administration pharmaceutical composition including a butyrylcholinest can be continuous or intermittent. A preparation can be erase polypeptide variant, as described herein, and a suitable administered therapeutically; that is, administered to treat an pharmaceutical carrier.The presently-disclosed subject mat 65 existing condition of interest. A preparation can be admin ter further includes a method of treating a cocaine-induced istered prophylactically; that is, administered for prevention condition comprising administering to an individual an of a condition of interest. US 9,879,240 B2 7 8 In some embodiments a subject will be administered an presently-disclosed subject matter belongs. Although any effective amount of at least one enzyme, compound and/or methods, devices, and materials similar or equivalent to composition provided in the present disclosure. In this those described herein can be used in the practice or testing respect,the term "effectiveamount" refers to an amount that of the presently-disclosed subject matter, representative is sufficient to achieve the desired result or to have an effect methods, devices, and materials are now described. on an undesired condition. For example, a "therapeutically Following long-standing patent law convention,the terms effective amount" refers to an amount that is sufficient to "a","an", and "the" refer to "one or more" when used in this achieve the desired therapeutic result or to have an effect on application, including the claims. Thus, for example, refer undesired symptoms, but is generally insufficient to cause ence to "an enzyme" includes a plurality of such enzymes, adverse side effects. The specific therapeutically effective 10 and so forth. dose level for any particular patient will depend upon a Unless otherwise indicated, all numbers expressing quan variety of factors including the disorder being treated and tities of ingredients, properties such as reaction conditions, the severity of the disorder; the specific composition and so forth used in the specification and claims are to be employed; the age,body weight,general health,sex and diet understood as being modified in all instances by the term of the patient; the time of administration; the route of 15 "about". Accordingly, unless indicated to the contrary, the administration; the rate of excretion of the specific com numerical parameters set forth in this specification and pound employed; the duration of the treatment; drugs used claims are approximations that can vary depending upon the in combination or coincidental with the specific compound desired properties sought to be obtained by the presently employed and like factors well known in the medical arts. disclosed subject matter. For example, it is well within the skill of the art to start doses 20 As used herein, the term "about," when referring to a of a compound at levels lower than those required to achieve value or to an amount of mass, weight, time, volume, the desired therapeutic effect and to gradually increase the concentration or percentage is meant to encompass varia dosage until the desired effect is achieved. If desired, the tions of in some embodiments ±50%, in some embodiments effective daily dose can be divided into multiple doses for ±40%, in some embodiments ±30%, in some embodiments purposes of administration. Consequently,single dose com- 25 ±20%, in some embodiments ±10%, in some embodiments positions can contain such amounts or submultiples thereof ±5%, in some embodiments ±1 %, in some embodiments to make up the daily dose. The dosage can be adjusted by the ±0.5%, and in some embodiments ±0.1 % from the specified individual physician in the event of any contraindications. amount, as such variations are appropriate to perform the Dosage can vary, and can be administered in one or more disclosed method. dose administrations daily,for one or several days. Guidance 30 As used herein, ranges can be expressed as from "about" can be found in the literature for appropriate dosages for one particular value, and/or to "about" another particular given classes of pharmaceutical products. In further various value. It is also understood that there are a number of values aspects, a preparation can be administered in a "prophylac disclosed herein,and that each value is also herein disclosed tically effective amount"; that is, an amount effective for as "about" that particular value in addition to the value itself. prevention of a disease or condition. 35 For example, if the value "10" is disclosed, then "about 10" Additionally, the terms "subject" or "subject in need is also disclosed. It is also understood that each unit between thereof' refer to a target of administration, which optionally two particular units are also disclosed. For example, if 10 displays symptoms related to a particular disease,pathologi- and 15 are disclosed, then 11, 12, 13, and 14 are also cal condition, disorder, or the like. The subject of the herein disclosed. disclosed methods can be a vertebrate, such as a mammal, a 40 As will be recognized by one of ordinary skill in the art, fish, a bird, a reptile, or an amphibian. Thus, the subject of the terms "reduce", "reducer", "reduction", "reducing", the herein disclosed methods can be a human, non-human "suppression," "suppressing," "suppressor," "inhibition," primate,horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea "inhibiting" or "inhibitor" do not refer to a complete elimi pig or rodent. The term does not denote a particular age or nation of angiogenesis in all cases. Rather,the skilled artisan sex. Thus, adult and newborn subjects, as well as fetuses, 45 will understand that the term "reducing", "suppressing" or whether male or female, are intended to be covered. A "inhibiting" refers to a reduction or decrease in a particular patient refers to a subject afflictedwith a disease or disorder. condition. Such reduction or decrease can be determined The term "subject" includes human and veterinary subjects. relative to a control. In some embodiments, the reduction or In some embodiments the subject in need thereof will be decrease relative to a control can be about a 1, 2, 3, 4, 5, 6, suffering or will have been diagnosed cocaine addiction 50 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and/or related diseases, disorders, pathologies, or condi 24, 25,26,27,28, 29,30,31,32, 33,34,35,36, 37,38,39, tions. 40, 41,42,43,44, 45,46,47,48, 49, 50, 51, 52, 53, 54, 55, As used herein, the terms "treatment" or "treating" relate 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, to any treatment of a condition of interest, including but not 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, limited to prophylactic treatment and therapeutic treatment. 55 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% As such, the termstreatment or treating include, but are not decrease. limited to: preventing a condition of interest or the devel As described herein, the presently-disclosed subject mat opment of a condition of interest; inhibiting the progression ter further includes pharmaceutical compositions compris of a condition of interest; arresting or preventing the devel ing at least one enzyme described herein together with a opment of a condition of interest; reducing the severity of a 60 pharmaceutically acceptable carrier. condition of interest; ameliorating or relieving symptoms The term "pharmaceutically acceptable carrier" refers to associated with a condition of interest; and causing a regres sterile aqueous or nonaqueous solutions, dispersions, sus sion of the condition of interest or one or more of the pensions or emulsions, as well as sterile powders for recon symptoms associated with the condition of interest. stitution into sterile injectable solutions or dispersions just Unless defined otherwise, all technical and scientific 65 prior to use. Proper fluidity can be maintained, for example, terms used herein have the same meaning as commonly by the use of coating materials such as lecithin, by the understood by one of ordinary skill in the art to which the maintenance of the required particle size in the case of US 9,879,240 B2 9 10 dispersions and by the use of surfactants. These composi Preparations for oral administration can be suitably formu tions can also contain adjuvants such as preservatives, lated to give controlled release of the active compound. For wetting agents, emulsifying agents and dispersing agents. buccal administration the compositions can take the form of Prevention of the action of microorganisms can be ensured tablets or lozenges formulated in conventional manner. by the inclusion of various antibacterial and antifungal The compositions can be formulated as eye drops. For agents such as paraben, chlorobutanol, phenol, sorbic acid example, the pharmaceutically acceptable carrier may com and the like. It can also be desirable to include isotonic prise saline solution or other substances used to formulate agents such as sugars, sodium chloride and the like. Pro eye drop, optionally with other agents. Thus, eye drop longed absorption of the injectable pharmaceutical form can formulations permit for topical administration directly to the be brought about by the inclusion of agents, such as alumi 10 eye of a subject. num monostearate and gelatin, which delay absorption. The compositions can also be formulated as a preparation Injectable depot forms are made by forming microencapsule for implantation or injection. Thus, for example, the com matrices of the drug in biodegradable polymers such as pounds can be formulated with suitable polymeric or hydro polylactide-polyglycolide, poly(orthoesters) and poly(anhy phobic materials (e.g., as an emulsion in an acceptable oil) drides ). Depending upon the ratio of drug to polymer and the 15 or ion exchange resins, or as sparingly soluble derivatives nature of the particular polymer employed, the rate of drug (e.g., as a sparingly soluble salt). The compounds can also release can be controlled. Depot injectable formulations are be formulated in rectal compositions, creams or lotions, or also prepared by entrapping the drug in liposomes or micro transdermal patches. emulsions which are compatible with body tissues. The The presently-disclosed subject matter further includes a injectable formulations can be sterilized, for example, by 20 kit that can include an enzyme and/or a pharmaceutical filtration through a bacterial-retaining filter or by incorpo composition as described herein, packaged together with a rating sterilizing agents in the form of sterile solid compo device useful for administration of the compound or com sitions which can be dissolved or dispersed in sterile water position. As will be recognized by those or ordinary skill in or other sterile injectable media just prior to use. Suitable the art, the appropriate administration-aiding device will inert carriers can include sugars such as lactose. 25 depend on the formulation ofthe compound or composition Suitable formulations include aqueous and non-aqueous that is selected and/or the desired administration site. For sterile injection solutions that can contain antioxidants, example, if the formulation of the compound or composition buffers, bacteriostats, bactericidal antibiotics and solutes is appropriate for injection in a subject, the device could be that render the formulation isotonic with the bodily fluids of a syringe. For another example, if the desired administration the intended recipient; and aqueous and non-aqueous sterile 30 site is cell culture media, the device could be a sterile suspensions, which can include suspending agents and pipette. thickening agents. The presently-disclosed subject matter includes mutants The compositions can take such forms as suspensions, of bacterial cocaine hydrolase. The CocE polypeptide vari solutions or emulsions in oily or aqueous vehicles, and can ants disclosed herein each have enhanced catalytic efficiency contain formulatory agents such as suspending, stabilizing 35 for ( -)-cocaine, as compared to CocE variant presently in and/or dispersing agents. Alternatively, the active ingredient trials. The presently-disclosed subject matter further can be in powder form for constitution with a suitable includes a pharmaceutical composition including a bacterial vehicle, e.g., sterile pyrogen-free water, before use. cocaine hydro lase mutant, as described herein, and a suitable The formulations can be presented in unit-dose or multi pharmaceutical carrier.The presently-disclosed subject mat dose containers, for example sealed ampoules and vials, and 40 ter further includes a method of treating a cocaine-induced can be stored in a frozen or freeze-dried (lyophilized) condition comprising administering to an individual an condition requiring only the addition of sterile liquid carrier effective amount of a bacterial cocaine hydrolase mutant, as immediately prior to use. disclosed herein, to accelerate cocaine metabolism and pro For oral administration, the compositions can take the duce biologically inactive metabolites. form of, for example, tablets or capsules prepared by a 45 In some embodiments, the present disclosure provides a conventional technique with pharmaceutically acceptable CocE polypeptide variant for treatment of cocaine abuse. excipients such as binding agents ( e.g., pregelatinized maize Moreover, the CocE polypeptide variant(s) of the present starch, polyvinylpyrrolidone or hydroxypropyl methylcellu disclosure may have (i) an improved in vitro and/or in vivo lose); fillers (e.g., lactose, microcrystalline cellulose or half-life and/or (ii) an improved catalytic activity and/or calcium hydrogen phosphate); lubricants (e.g., magnesium 50 catalytic efficiency, as compared to therapeutic enzymes stearate, talc or silica); disintegrants (e.g., potato starch or previously known in the art. sodium starch glycolate); or wetting agents (e.g., sodium Some embodiments of the present disclosure provide a lauryl sulphate). The tablets can be coated by methods mutant of bacterial cocaine hydrolase. And in some embodi known in the art. ments, the mutant of bacterial cocaine hydrolase provides Liquid preparations for oral administration can take the 55 improved stability and/or improved catalytic activity against form of, for example, solutions, syrups or suspensions, or cocame. they can be presented as a dry product for constitution with In certain embodiments, the CocE polypeptide variant of water or other suitable vehicle before use. Such liquid the present disclosure comprises a Tl 72R/G173Q mutant. In preparations can be prepared by conventional techniques some embodiments, the enzyme of the present disclosure with pharmaceutically acceptableadditives such as suspend- 60 comprises a T172R/G173Q/L196C/1301C mutant of ing agents (e.g., sorbitol syrup, cellulose derivatives or cocaine esterase (CocE). In a particular embodiment, the hydrogenated edible fats); emulsifying agents (e.g. lecithin CocE polypeptide variant of the present disclosure com or acacia); non-aqueous vehicles (e.g., almond oil, oily prises El 72-173 enzyme. And in some embodiments, the esters, ethyl alcohol or fractionated vegetable oils); and CocE polypeptide variant(s) of the present disclosure com preservatives (e.g., methyl or propyl-p-hydroxybenzoates or 65 prises E196-301. Furthermore, in some embodiments, the sorbic acid). The preparations can also contain buffer salts, enzyme(s) of the present disclosure may be modified by flavoring, coloring and sweetening agents as appropriate. PEGylation. And in certain embodiments, treatment and/or US 9,879,240 B2 11 12 modification of an enzyme via PEGylation extends the in cross-subunit disulfide bonds that stabilize the dimer struc vivo residence time of the enzyme. ture. The cross-subunit disulfide bonds were confirmed by Numerous derivatives of PEG and methods for making X-ray diffraction. The designed L196C/1301C mutations them and conjugating them to an enzyme are known in the have not only considerably extended the in vitro half-life at ° art and are suitable for use in the present invention. In some 3 7 C. to >100 days, but also significantly improved the preferred embodiments, the PEG contains maleimide which catalytic efficiency against cocaine by about 150%. reacts selectively with thiol residues in a Michael addition In addition, the thermostable E196-301 can be PEGylated reaction. Other functionalized PEGs can be used in embodi to significantly prolong the residence time in a subject. For ments, and adjusted according to desired properties of the example, the PEGylated E196-301 can fully protect mice PEGylated enzyme, for example in vivo residence time. 10 from a lethal dose of cocaine (180 mg/kg, LDlOO) for at In some embodiments, the enzyme(s) of the present least 3 days, with an average protection time of about 94 disclosure may have an in vitro and/or in vivo half-life of hours. This is the longest in vivo protection of mice from the between about 1 hour and about 1 year and/or of any amount lethal dose of cocaine demonstrated within all studies using of time within that range. an exogenous enzyme reported so far. Hence, El 96-301 may Additionally, in certain embodiments, the enzyme(s) of 15 become a valuable therapeutic enzyme for cocaine abuse the present disclosure may have an in vitro and/or in vivo treatment, and it demonstrates that a general design strategy half-life of between about 4 and about 8 hours, between and protocol to simultaneously improve both the stability about 5 and about 7 hours or between about 5.5 and about and function are feasible for rational protein drug design. 6.5 hours. And in some embodiments, the enzyme(s) of the Accordingly, in some embodiments, an enzyme of the present disclosure have an in vitro and/or in vivo half-life of 20 present disclosure may be pegylated. In other words, in about 6 hours. Moreover, any such half-life is or can be some embodiments, at least one chain of polyethylene maintained at physiological temperature in a human subject. glycol (PEG) polymer may be attached to the enzyme of In other words, in certain embodiments, the enzyme(s) of the interest. And in certain embodiments, the pegylation (pro present disclosure may have an in vitro and/or in vivo cess via which the enzyme is pegylated) of an enzyme may half-life of between about 4 and about 8 hours at a tern- 25 improve the enzyme's residence time in a subject. perature at or around physiological temperature in a human Further, in some embodiments, the present disclosure is subject. Thus, in some embodiments, the enzyme(s) of the directed to a method of protecting a subject from an over present disclosure have an in vitro and/or in vivo half-life of dose and/or a lethal dose of cocaine. The method includes at between about 4 and about 8 hours between about 36.5° C. least the step of administering an amount of at least one and about 37.5° C., which is normal human body tempera- 30 enzyme of the present disclosure to a subject. In some ture. embodiments, administration of an effective amount of an Meanwhile, in other embodiments, the enzyme(s) of the enzyme of the present disclosure can protect a subject from present disclosure may have an in vitro and/or in vivo and overdose and/or a lethal dose of cocaine for between half-life of greater than about 100 days. And in some about 24 and about 100 hours. In certain embodiments, embodiments, the enzyme(s) of the present disclosure have 35 administration of an effective amount of an enzyme of the an in vitro and/or in vivo half-life of between about 1 and present disclosure protects a subject from overdose and/or about 150 days. Moreover, any such half-life is or can be lethal dose of cocaine for at least about 48 hours, at least maintained at physiological temperature in a human subject. about 72 hours, and/or at least about 96 hours. In other words, in certain embodiments, the enzyme(s) of the In some embodiments, the enzyme of the present disclo- present disclosure may have a half-life of greater than about 40 sure comprises a dimer. And in certain embodiments, the 100 days at a temperature at or around physiological tem enzyme of the present disclosure has been modified to perature in a human subject. Thus, in some embodiments, include disulfide bonds between the two subunits of the the enzyme(s) of the present disclosure have an in vitro dimer. Indeed, in certain embodiments, disulfide bonds are and/or in vivo half-life of greater than or equal to about 100 introduced between the subunits of an enzyme of the present days at about 36.5° C. and about 37.5° C. and/or at a normal 45 disclosure. These disulfide bonds are referred to as cross and/or an average human body temperature. subunit disulfide bonds. In certain embodiments, the present disclosure further Additionally, in some embodiments, the enzyme of the provides a method of treating, preventing and/or reducing present disclosure includes at least one mutation that substance abuse, such as cocaine abuse and/or cocaine increases the size of the active site cavity of the enzyme. And addiction. In the present disclosure, "substance abuse" may 50 in certain embodiments, the at least one mutation improves include, for example, craving, drug seeking, and/or self the catalytic activity of the enzyme. administration. The method comprises at least the step of The presently-disclosed subject matter is illustrated by the administering a CocE polypeptide variant provided in the following specific but non-limiting examples. The following present disclosure to a subject. In some embodiments, the examples may include compilations of data that are repre- subject is in need of treatment. 55 sentative of data gathered at various times during the course Cocaine esterase is known as the most efficient natural of development and experimentation related to the present enzyme for cocaine hydrolysis. A major obstacle to the invention. clinical application of wild-type CocE is the thermoinsta bility with a half-life of only about 12 minutes at 37° C. The EXAMPLES previously designed Tl 72R/G 173Q mutant (denoted as 60 enzyme E172-173) with an improved in vitro half-life of Mutant Design: Insights from Molecular Modeling. about 6 hours at about 37° C. is currently in clinical trial The inventors of the present disclosure first aimed to Phase II for cocaine overdose treatment. stabilize the dimer structure of El 72-173 and then analyzed Through molecular modeling and dynamics simulation, the possibly stabilized dimer structure and estimated how the inventors of the present disclosure have designed and 65 the activity of the dimer would change. The computational characterized a new mutant of El 72-173 with extra L196C/ design strategy relied on the molecular dynamics (MD) 1301C mutations (denoted as enzyme E196-301) to produce simulated dimer structure of El72-173 and the idea that the US 9,879,240 B2 13 14 dimer structure can be stabilized by introducing disulfide lar modeling and X-ray structural analysis (see below) of the bonds between the two subunits of the dimer. So, the dimer structure of the Tl72R/G 173Q/1, 196C/1301C mutant inventors carried out a sufficiently long MD simulation (50 also suggested that the extra L196C/1301C mutations could ns) on the E172-173 dimer structure in order to obtain a slightly increase the size of the active site cavity of the dynamically stable dimer structure. To search for appropri enzyme and, thus, might improve the catalytic activity. ate mutational sites to introduce the cross-subunit disulfide In Vitro Characterization of the Designed Tl72R/G 173Q/ bonds, a self-developed script was used to scan the key L196C/1301 C Mutant. internuclear distances between the Ca atoms of the residues Based on the computational insights, the inventors carried on the dimer interface from the collected snapshots of the out wet experimental tests, including site-directed mutagen- MD trajectory. Essentially, each pair of residues from dif lO esis, protein expression, purification, and enzyme activity ferent subunits was evaluated computationally for the simu assays on the Tl72R/Gl 73Q and Tl72R/Gl 73Q/1,196C/ lated Ca-Ca distance. If the simulated Ca-Ca distance was 1301C mutants of CocE. For comparison, the inventors of within 7 A, the pair of the residues would be checked the present disclosure also prepared and characterized the manually for further evaluation of the detailed interactions. 15 Tl72R/G 1730/G4C/S lOC mutant which has been known to The most hopeful pair of residues may be mutated to have a pair of cross-subunit disulfide bonds. (26) All of the cysteine for introducing possible cross-subunit disulfide mutants were expressed similarly well. To minimize the bond(s). possible systematic experimental errors of the kinetic data, Based on the analysis of the MD trajectory, L196C/1301C all of the three mutants were simultaneously prepared and mutations satisfied all of the structural requirements. Sum 20 characterized under the same experimental conditions, marized in Table 1 are the maximum, minimum, and average which allowed for fair comparison of their catalytic activity values of the Ca-Ca distances associated with the stable against (-)-cocaine. Michaelis-Menten kinetics of the enzy MD trajectory (10 to SO ns) in comparison with the corre matic hydrolysis of (-)-cocaine was determinedby perform sponding Ca-Ca distances in the X-ray crystal structure. As ing the sensitive radiometric assays using [3H](-)-cocaine seen in Table 1, the detailed analysis of the MD trajectory 25 (labeled on its benzene ring) with varying concentrations of predicted that extra L196C/1301C mutations on E172-173 the substrate. Depicted in FIG. 2 are the measured kinetic may introduce the desirable cross-subunit disulfide bonds data, and summarized in Table 2 are the kinetic parameters between the two subunits of the El72-173 dimer. Depicted determined at 37° C. in FIG. lA are the simulated time-dependent Ca-Ca dis As seen in Table 2, kcat=2600 min-1, KM=2.9 µM, and tances for the pair of residues. Depicted in FIG. 18 is the = 30 kcat/KM 9.2xl08 min-1 M-1 for the Tl72R/G 173Q mutant MD-simulated El72-173 dimer structure consisting of sub- under the current clinical development. Compared to the units a and b. FIG. lC is the detailed information about the Tl72R/G 1 73Q mutant, the Tl72R/G 1 73Q/G4C/S1 OC MD-simulated El72-173 dimer structure concerning this mutant has a slightly smaller kcat value (2340 min-1) and a important pair of residues. The interface between the two slightly smaller KM value (2.1 µM). Overall, the catalytic subunits is mainly composed of some residues from all of 35 efficiency (kcat/KM) changed about 20% (from 9.2x108 the three domains (I, II, and III) in the form of a-helices, min-1 M-1 to 1.lx109 min-1 M-1). Interestingly, the new �-sheets, and loops. L196 is located on one a-helix (E184- mutant (Tl72R/G 173Q/Ll 96C/I301 C) designed in the pres Nl 97) in domain II, 1301 is located on a loop in domain 1. ent study has both a significantly increased kcat value (3450 min-1) and a significantly smaller KM value (1.5 µM). As a TABLE 1 = 40 result, the catalytic efficiency (kcat/KM 2.3x109 min-1 Maximwn, Minimum, and Average Ca-Ca Distances between Key M-1) of the T172R/G173Q/L196C/I301C mutant has a, Residues in the El 72-173 Dimer Obtained from the 50 ns MD -150% improvement from that (kcat/KM=9.2x108 min-1 Simulation in Comparison with the Corresponding Ca-Ca M-1) of the Tl72R/Gl 73Q mutant under the currentclinical Distances in the X-ray Crystal Structure development. Ca-Ca distances (A) 45 TABLE 2 El 72-173 structure L196a-1301b 1301a-L196b Kinetic Parameters Determined for (-)-Cocaine Hydrolysis Crystal structure of El72-173 a 7.29 7.20 Catalyzed by the Tl 721t/Gl 73Q, Tl 72R/Gl73Q/G4C/S10C, MD-simulated El 72-173 Maximum 10.85 7.19 and Tl 72R/G 1730/L196C/1301 C Mutants of CocEa structure (50 ns MDl Minimum 5.16 4.51 50 Average 7.15 5.44 kca/KM 1 1 1 CocE mutant kca, (min- ) KM (µM) (min- M- ) aT172RIG173QCocE (E172-173) crystal structure (PDB ID: 3!2F). hpully relaxed MD simulation of the El 72-173 dimer structure starting from the crystal T172R/G173Q 2600 2.9 9.2 x 108 structure. Tl 72R/Gl 73Q/G4C/S10C 2340 2.1 1.1 x 109 Tl 72R/Gl 73Q/L196C/1301C 3450 1.5 2.3 x 109 According to the locations of these residues, it is highly 55 possible to introduce a pair of cross-subunit disulfide bonds (C196a-C301 b and C301a-C196b) through the L196C/ Based on the encouraging kinetic data, the purified pro 1301C mutations on E172-173. The computationally tein of the Tl72R/G 173Q/Ll 96C/I301 C mutant was tested designed new mutant, i.e. the Tl72R/G 173Q/Ll 96C/1301 C for the thermal stability at 37° C. For this purpose, the mutant (denoted as enzyme E196-301 for convenience), 60 enzyme was incubated at 37° C., and the catalytic activity of may have a pair of cross-subunit disulfide bonds: one the incubated enzyme against cocaine was assayed at dif between C196 of subunit a (C196a) and C301 of subunit b ferent time points. (C301b ), and the other between C301 of subunit a (C301a) As seen in FIG. 3, the enzyme showed a relatively faster and C196 of subunit b (C196b). decrease of the activity ( around 20%) during the first a few It is also interesting to note that residue 1301 is on a loop, 65 days, compared to the last 90 days. The relatively faster implying that the loop flexibility may help to form the decrease of the activity during the first a few days is likely desirable cross-subunit disulfide bonds. In addition, molecu- due to the possibility that certain percentage of the mutant US 9,879,240 B2 15 16 protein molecules had not yet formed the expected cross cess.A slightly larger active site could better accommodate subunit disulfide bonds before the thermal stability test. such a large substrate like cocaine and, thus, make the Although the cross-subunit disulfide bonds were expected to enzyme more active against cocaine; the similar type of form spontaneously (as no extra oxidation reagent was structure-activity correlation was noted for previously employed in this study to facilitate the disulfide bond 5 designed mutants of human butyrylcholinesterase (BChE). formation), a small percentage of the new mutant (Tl 72R/ (31) The BChE mutants with a slightly larger active site G173Q/L196C/I301C) molecules did not really form the have a significantly improved catalytic efficiency against cross-subunit disulfide bonds. Those Tl 72R/ cocaine without changing substrate specificity. (31, 32) Gl 73Q1L196C/I301C mutant molecules without the cross To further understand why E196-301 has an improved subunit disulfide bonds could lose the activity more rapidly, 10 catalytic activity against cocaine compared to El 72-173, the like the Tl 72R/G 173Q mutant which has an in vitro half-life present inventors performed MD simulations on the transi of -6ha t 37° C. (11) However, after the first few days, the tion state (TS 1) for the initial reaction step of (-)-cocaine enzyme activity decreased very slowly. Within the last 90 hydrolysis catalyzed by El 72-173 and E196-301.Previous days, the enzyme activity decreased for only, -15%.Overall, computational studies 33 on the catalytic reaction mecha- the enzyme still retained more than 60% of the enzyme 15 nism for wild-type CocE-catalyzed hydrolysis of (-)-co activity after incubation at 37° C.for 100 days, indicating caine revealed that the CocE-catalyzed cocaine hydrolysis is that the in vitro half-life of the Tl 72R/Gl 73Q/L196C/I301C initialized by the nucleophilic attack on the carbonyl carbon mutant at 37° C. should be longer than 100 days. of (-)-cocaine benzoyl ester by the hydroxyl oxygen of Confirmation of the Cross-Subunit Disulfide Bonds. Serl! 7, and that the transition state is stabilized by hydrogen With the encouraging data about the significant improve 20 bonding of the carbonyl oxygen of cocaine benzoyl ester ment in both the catalytic activity and thermal stability at 3 7° with the hydroxyl group ofY 44 side chain and the NH group C., the inventors of the present disclosure have determined of the Yll 8 backbone. (33) The (-)-cocaine hydrolysis the crystal structure of the T172R/G173Q/L196C/I301C catalyzed by El 72-173 or El 96-301 is expected to follow mutant in order to directly confirm the formation of the the same catalytic reaction mechanism, as the residues #196 cross-subunit disulfide bonds between L196C and I301C. 25 and #301 are all far away from the active site.Based on the The CocE mutant crystallized with one monomer in the established mechanistic understanding, the stronger the asymmetric unit, but a symmetry related molecule forms an hydrogen bonding of the carbonyl oxygen of the substrate extensive interface, burying over 1900 A of solvent-acces withY 44 side chain and Yl 18backbone, the more active the sible surface area as calculated by the PISA server.(30) The enzyme. dimer formed by these two molecules (FIGS. 4A and B) 30 The general strategy and protocol for performing MD corresponds to previously reported structures of unlinked simulation on a transition state of enzymatic reaction using (PDB 3PUH) and covalently linked (PDB 3PUI) CocE the classical force field have been described in detail else dimers. (2 6) Thus, the expected homodimer was formed in where.(34, 35) Based on the protoco 1,34 the lengths of the the crystals.Initial difference maps using the rigidly placed transition bonds (i.e., the covalent bonds that gradually form (without refinement) CocE mutant structure with glycine 35 or break during the reaction step associated with the tran residues substituted at residue positions 196 and 301 showed sition state) in the transition state are restrained according to strong positive Fo-Fc difference electron density for the the previous QM/MM reaction-coordinate calculations, cysteine side chains at the dimer interface (FIG. 4C) con assuming that the these bond lengths do not significantly sistent with disulfide bond formation. Subsequent refine change after themutations. The transition-state modeling in ment of the model with cysteine residues introduced at these 40 the present study was based on the inventors' QM/MM positions and no disulfide restraints gave well-defined optimized TS! structure33 for CocE-catalyzed hydrolysis of weighted 2Fo-Fc density and excellent geometry consistent (-)-cocaine. It is reasonable to assume that the transition with disulfide bond formation across the subunits (FIG. 4D, bond lengths in the TS! structure will not significantly see FIGS.4A and B).The sulfur-to-sulfurdistance is 2.0 A change after the Tl 72R/G 173Q or Tl 72R/G 1730JL196C/ and the dihedral angle is 94 °. 45 1301C mutations. So, MD simulations were carried out on In summary, the crystal structure of the enzyme unam the TS! structures corresponding to E172-173 and E196- biguously confirms the presence of the engineered intersub 301, with the transition bond lengths restrained. unit disulfide bonds. Since the dimer axis correspondsto a Depicted in FIG. 7 are the simulated time-dependent crystallographic 2-fold axis (space group P6522), the struc H ...0 distances (relevant to the hydrogen bonds) in ture was also refined in a lower symmetry group (P 65), 50 El 72-173 and El 96-301 during the MD simulations for 50 which places a disulfide-linked dimer in the asymmetric unit ns. The detailed analysis of the key H . . . 0 distances and does not therefore impose symmetry on the model.This between enzyme residues and cocaine is summarized in refinement resulted in geometry for the two disulfide bonds Table 3.In El 72-173, the H ...0 distance between CocO that is nearly identical to that present in the dimer on the and Y44HH was 3.02 A in maximum, 1.44 A in minimum, crystallographic 2-fold axis (FIG.8), eliminating the possi- 55 and 1.87 A in average, while the H ...0 distance between bility of any artifact from this placement. CocO and Y114H was 3.14 A in maximum, 1.61 A in Structure-Activity Correlation. minimum, and 2.16A in average.In E196-301, the H ...0 Superposition between the X-ray crystal structures of distance between Y44HH and CocO was 3.17 A in maxi El 72-173 (representing the Tl 72R/G 173Q mutant) and mum, 1.44 A in minimum, and 1.80 A in average, while the E196-301 (representing the T172R/G173WL196C/1301C 60 H ...0 distance between CocO and Y114H was 2.89A in mutant) revealed a root-mean-square deviation (RMSD) maximum, 1.61 A in minimum, and 2.13 A in average. value of 0.299 A, indicating the high similarity of the two According to these simulated H ...0 distances, the H ... protein structures.However, the superposition also revealed O distances of the two hydrogen bonds in E196-301 are all a slight shift of two a-helices (H2 and H3 in domain II) in shorter than the correspondingones in El 72-173, suggesting E196-301 compared to that in EI72-173 (see FIG. 6). The 65 that cocaine has the stronger hydrogen bonding with E196- shift created a slightly enlarged active-site cavity in E196- 301 compared to that with E172-173 in the TS! structure. 301, which could possibly favor the catalytic reaction pro- The enhanced hydrogen bonding helps to stabilize the US 9,879,240 B2 17 18 transition state (TS 1) structure during the catalytic reaction in vivo protection of mice from a lethal dose of cocaine (180 process and, thus, lower the energy barrier, which explains mg/kg, LI3100) demonstrated so far within all in vivo the improved catalytic activity of the new mutant. studies using an exogenous enzyme. In Vivo Protection of Mice Against Cocaine-Induced It should be pointed out that the in vivo studies described Lethality. above are a simplified animal model (using a high dose of For development of an effective cocaine abuse treatment enzyme and high doses of cocaine for convenience of animal using a cocaine-metabolizing enzyme, it is highly desired to behavior observation) to show the long residence time of the have a long residence time of the enzyme in the body. To PEGylated El 96-301. As discussed earlier in this report, the have a long residence time in the body, the enzyme must be long residence time of the enzyme is crucial for an effective thermostable at 37 ° C. for a sufficiently long time. So, it is 10 enzyme-based cocaine addiction treatment. For practical a necessary condition, but not a sufficient condition, for an cocaine addiction treatment using a cocaine-metabolizing enzyme having a long residence time in the body to have a enzyme in humans, the cocaine doses are much lower (a long in vitro half-life of the enzyme at 37° C. An enzyme typical cocaine addiction dose is -1 mg/kg, which is much may be eliminated rapidly from the body, even if it is very lower than the lethal dose of 180 mg/kg used in the animal thermostable at 37° C. In this consideration, PEGylation is 15 model) and, correspondingly, the required dose of the a popularly used strategy to prevent the possible rapid enzyme for effective cocaine metabolism may be less than elimination of a protein from the body. Hence, the inventors about 30 mg/kg. further engineered E196-301 through the PEGylation modi Concluding Remarks. fication. Activity assays confirmedthat the PEGylated E196- Molecular dynamics simulation and subsequent structural 301 completely maintained its activity against cocaine as 20 analysis on the dimer structure of a therapeutic cocaine that of El 96-301. metabolizing enzyme, i.e. El72-173 (which is the Tl72R/ G 173Q mutant of CocE), led to the prediction that the extra TABLE 3 L196C/1301 C mutations on El72-173 can produce cross subunit disulfide bonds in the dimer. The formation of Summary of the MD-Simulated Key Distances (in A) between 25 cross-subunit disulfide bonds were expected to stabilize the the Hydrogen Atoms of Key Residue and the Carbonyl dimer structure and also improve the catalytic activity Oxygen of (-)-Cocaine Benzoyl Ester in the Rate-Determining Transition-State Structures of CocE against cocaine. Following the computational prediction, in vitro experi Distances (A) mental studies have demonstrated that the computationally 30 designed new CocE mutant (Tl 72R/G173Q/Ll 96C/1301C), Hydrogen bond Maximum Minimum Average i.e. E196-301, indeed has a significantly improved catalytic Y44HH-Coc0b E172-173a 3.02 1.44 1.87 efficiency against cocaine and a considerably extended in E196-301a 3.17 1.44 1.80 vitro half-life (>100 days) at 37° C. Y118H-Cococ E172-173a 3.14 1.61 2.16 E196-301a 2.89 1.61 2.13 The predicted cross-subunit disulfide bonds in the E196- 35 301 dimer structure were confirmed by X-ray diffraction.In aE172-173 represents T172RIG173Q CocE, and E196-301 refers to T172RIG173Q/ addition, in vivo studies in mice demonstrated that the L196C/!301 C CocE. by44HH-Coc0 represents the distance between the hydroxyl hydrogen (denoted as HH) PEGylated E196-301 can fully protect mice from a lethal of the Y44 side chain and the carbonyl oxygen (denoted as CocO) of(-)-cocaine benzoyl ester. dose of cocaine (180 mg/kg, LD 100) for at least 3 days, with 'Yl 18H-Coc0 refers to the distance betweenthe hydrogen (H) ofthe Yl 18 backbone and the average protection time being about 94 hours. All of the the carbonyl oxygen (CocO) of (-)-cocaine benzoyl ester. 40 data suggests that the currently designed enzyme E196-301 To test the ability of E196-301 (unPEGylated, unless with improved thermal stability and catalytic activity against specified otherwise) and the PEGylated E196-301 in pro cocaine is more valuable than the existing therapeutic tecting mice (n= 5) from a lethal dose of cocaine, E196-301 enzyme El 72-173, which is under clinical trial phase II for or the PEGylated E16-301 was administered intravenously cocaine overdose treatment. The encouraging outcomes of (at a single dose of 30 mg/kg) 1 min before the first 45 this study also suggest that the structure-and-mechanism intraperitoneal administration of 180 mg/kg cocaine based computational design and integrated computational (LDlOO). experimental approach are promising for rational protein Depicted in FIG. 5 are the data for the in vivo protection drug design. The general computational protein design strat of mice provided by E196-301 and the PEGylated E196-301 egy and approach to simultaneously improve both the pro- against the cocaine-induced lethality. As seen in FIG. 5, 50 tein stability and function may also be valuable for engi E196-301 protected the mice from death after the first neering other proteins. Intermonomer disulfide bonds in the injection of 180 mg/kg cocaine but lost the efficacy at the CocE mutant (E196-301) dimer refined in space group P65 second injection of 180 mg/kg cocaine 24 hours later. The is shown in FIG. 8. PEGylated E196-301 was able to fully protect the mice Material and Methods (n= 5) for at least 72 hours from the acute toxicity of a lethal 55 Computational Methods Used for the Mutant Design. dose of cocaine (180 mg/kg, LDlOO): no mouse died after For the molecular dynamics (MD) simulations on the the fourth cocaine challenge at 72 hours, three mice died CocE dimer structures, the starting structure of the El72- after the fifth cocaine challenge at 96 hours, and the remain 173 dimer was the X-ray crystal structure ( deposited in the ing two mice died after the final (sixth) cocaine challenge at Protein Data Bank) at 2.0 A resolution (PDB ID: 3I2F) (26) 120 hours (see FIG. 5). According to the data depicted in 60 and the starting structure of the E196-301 dimer was the FIG. 5, the PEGylated E196-301 can protect the three mice X-ray crystal structure determined in the present study. In (60%) with the protection time (tp) being between 72 and 96 order to simulate the TSl structures for the enzymatic h: 72 h SEQUENCES SEQ ID NO, 1 1 90 ATGGTGGACGGGAATTACAGTGTTGCCTCGAACGTGATGGTTCCGATGCGTGATGGGGTGCGTCTGGCGGTCGACCTGTACCGACCAGAT 91 180 GCTGATGGACCTGTTCCGGTCCTGCTGGTTCGCAACCCATACGACAAGTTCGACGTGTTCGCGTGGTCGACGCAGTCGACAAACTGGCTT 181 270 GAGTTCGTGCGTGATGGCTATGCCGTGGTCATTCAAGACACGCGTGGCTTGTTCGCATCGGAAGGTGAGTTCGTCCCGCACGTTGACGAC 271 360 GAAGCTGACGCCGAGGATACGTTGAGCTGGATTCTGGAACAAGCGTGGTGCGACGGCAATGTGGGCATGTTCGGCGTTTCGTACTTGGGT 360 450 GTGACCCAGTGGCAGGCCGCCGTATCCGGCGTTGGTGGGCTGAAGGCGATCGCGCCGTCCATGGCGTCGGCGGACTTGTACCGCGCCCCG 450 540 TGGTACGGCCCTGGTGGTGCGCTTTCAGTCGAGGCGCTGTTGGGCTGGTCAGCTCTCATAGGTACTGGGCTCATCACGTCGAGGTCTGAC 541 630 GCCCGGCCCGAAGACGCAGCCGACTTCGTCCAACTCGCAGCAATTCTCAATGACGTCGCTGGCGCGGCGTCGGTCACGCCCCTGGCCGAG 631 720 CAACCGCTTCTGGGCCGACTGATTCCGTGGGTGATCGATCAGGTTGTCGATCACCCCGACAACGATGAATCATGGCAGTCCATTAGCTTG 721 810 TTTGAACGACTCGGCGGGTTGGCAACACCGGCCTTGATCACGGCTGGGTGGTACGACGGGTTCGTCGGCGAATCGTTGCGCACTTTCGTT 811 900 GCGGTCAAGGACAATGCCGACGCACGTTTGGTTGTCGGCCCTTGGAGTCACAGCAACCTCACTGGTCGGAATGCGGACCGGAAGTTCGGC 901 990 ATTGCCGCGACCTACCCGATTCAAGAAGCCACCACGATGCACAAGGCATTCTTCGACCGGCACCTCCGCGGCGAGACCGATGCACTCGCA 991 1080 GGCGTCCCCAAAGTGCGGCTGTTCGTAATGGGCATCGATGAGTGGCGTGACGAAACGGACTGGCCACTGCCGGACACGGCGTATACGCCC 1081 1170 TTCTATCTTGGAGGTAGCGGGGCTGCGAATACCTCCACGGGTGGTGGAACACTGTCGACGTCGATTTCCGGAACTGAATCTGCTGACACC 1171 1260 TACCTGTATGATCCGGCCGATCCCGTGCCTTCGCTCGGGGGGACGCTGCTGTTCCACAACGGAGACAACGGACCCGCCGACCAACGTCCC 1261 1350 ATTCATGACCGGGACGACGTGTTGTGTTACAGCACTGAGGTATTGACCGACCCGGTGGAAGTAACCGGCACCGTCTCCGCCCGGCTGTTC 1351 1440 GTGTCGTCATCAGCGGTGGACACTGATTTCACCGCCAAACTTGTCGACGTATTTCCCGACGGTCGCGCGATCGCGCTGTGTGACGGGATC 1441 1530 GTGCGGATGCGGTACCGCGAGACGTTGGTCAATCCAACCTTGATCGAAGCGGGCGAAATCTACGAGGTTGCTATCGACATGCTTGCAACC 1531 1620 TCGAATGTATTCCTGCCAGGGCATCGCATCATGGTCCAAGTATCAAGTAGCAACTTCCCGAAATACGACCGCAATTCGAATACCGGCGGA US 9,879,240 B2 27 28 -continued SEQUENCES 1621 1710 GTAATCGCACGGGAACAGCTCGAAGAGATGTGCACCGCCGTGAACCGCATTCACCGAGGACCTGAGCATCCCAGCCACATTGTGCTGCCG 1711 ATTATCAAGCGA SEQ ID NO, 2 1 60 MVDGNYSVASNVMVPMRDGVRLAVDLYRPDADGPVPVLLVRNPYDKFDVFAWSTQSTNWL 61 120 EFVRDGYAVVIQDTRGLFASEGEFVPHVDDEADAEDTLSWILEQAWCDGNVGMFGVSYLG 121 180 VTQWQAAVSGVGGLKAIAPSMASADLYRAPWYGPGGALSVEALLGWSALIGTGLITSRSD 181 240 ARPEDAADFVQLAAI�NDVAGAASVTPLAEQPLLGRLIPWVIDQVVDHPDNDESWQSISL 241 300 FERLGGLATPALITAGWYDGFVGESLRTFVAVKDNADARLVVGPWSHSNLTGRNADRKFG 301 360 lAATYPIQEATTMHKAFFDRHLRGETDALAGVPKVRLFVMGIDEWRDETDWPLPDTAYTP 361 420 FYLGGSGAANTSTGGGTLSTSISGTESADTYLYDPADPVPSLGGTLLFHNGDNGPADQRP 421 480 IHDRDDVLCYSTEVLTDPVEVTGTVSARLFVSSSAVDTDFTAKLVDVFPDGRAIALCDGI 481 540 VRMRYRETLVNPTLIEAGEIYEVAIDMLATSNVFLPGHRIMVQVSSSNFPKYDRNSNTGG 541 VIAREQLEEMCTAVNRIHRGPEHPSHIVLPIIKR Note, Residues labeled in red are, T172, G173, L196, and 1301 SEQ ID NO, 3 T172R/G173Q/L196C/I301C CocE 1 90 ATGGTGGACGGGAATTACAGTGTTGCCTCGAACGTGATGGTTCCGATGCGTGATGGGGTGCGTCTGGCGGTCGACCTGTACCGACCAGAT 91 180 GCTGATGGACCTGTTCCGGTCCTGCTGGTTCGCAACCCATACGACAAGTTCGACGTGTTCGCGTGGTCGACGCAGTCGACAAACTGGCTT 181 270 GAGTTCGTGCGTGATGGCTATGCCGTGGTCATTCAAGACACGCGTGGCTTGTTCGCATCGGAAGGTGAGTTCGTCCCGCACGTTGACGAC 271 360 GAAGCTGACGCCGAGGATACGTTGAGCTGGATTCTGGAACAAGCGTGGTGCGACGGCAATGTGGGCATGTTCGGCGTTTCGTACTTGGGT 360 450 GTGACCCAGTGGCAGGCCGCCGTATCCGGCGTTGGTGGGCTGAAGGCGATCGCGCCGTCCATGGCGTCGGCGGACTTGTACCGCGCCCCG 450 540 TGGTACGGCCCTGGTGGTGCGCTTTCAGTCGAGGCGCTGTTGGGCTGGTCAGCTCTCATAGGTCGCCAGCTCATCACGTCGAGGTCTGAC 541 630 GCCCGGCCCGAAGACGCAGCCGACTTCGTCCAACTCGCAGCAATTTGCAATGACGTCGCTGGCGCGGCGTCGGTCACGCCCCTGGCCGAG 631 720 CAACCGCTTCTGGGCCGACTGATTCCGTGGGTGATCGATCAGGTTGTCGATCACCCCGACAACGATGAATCATGGCAGTCCATTAGCTTG 721 810 TTTGAACGACTCGGCGGGTTGGCAACACCGGCCTTGATCACGGCTGGGTGGTACGACGGGTTCGTCGGCGAATCGTTGCGCACTTTCGTT 811 900 GCGGTCAAGGACAATGCCGACGCACGTTTGGTTGTCGGCCCTTGGAGTCACAGCAACCTCACTGGTCGGAATGCGGACCGGAAGTTCGGC 901 990 TGCGCCGCGACCTACCCGATTCAAGAAGCCACCACGATGCACAAGGCATTCTTCGACCGGCACCTCCGCGGCGAGACCGATGCACTCGCA 991 1080 GGCGTCCCCAAAGTGCGGCTGTTCGTAATGGGCATCGATGAGTGGCGTGACGAAACGGACTGGCCACTGCCGGACACGGCGTATACGCCC 1081 1170 TTCTATCTTGGAGGTAGCGGGGCTGCGAATACCTCCACGGGTGGTGGAACACTGTCGACGTCGATTTCCGGAACTGAATCTGCTGACACC 1171 1260 TACCTGTATGATCCGGCCGATCCCGTGCCTTCGCTCGGGGGGACGCTGCTGTTCCACAACGGAGACAACGGACCCGCCGACCAACGTCCC 1261 1350 ATTCATGACCGGGACGACGTGTTGTGTTACAGCACTGAGGTATTGACCGACCCGGTGGAAGTAACCGGCACCGTCTCCGCCCGGCTGTTC US 9,879,240 B2 29 30 -continued SEQUENCES 1351 1440 GTGTCGTCATCAGCGGTGGACACTGATTTCACCGCCAAACTTGTCGACGTATTTCCCGACGGTCGCGCGATCGCGCTGTGTGACGGGATC 1441 1530 GTGCGGATGCGGTACCGCGAGACGTTGGTCAATCCAACCTTGATCGAAGCGGGCGAAATCTACGAGGTTGCTATCGACATGCTTGCAACC 1531 1620 TCGAATGTATTCCTGCCAGGGCATCGCATCATGGTCCAAGTATCAAGTAGCAACTTCCCGAAATACGACCGCAATTCGAATACCGGCGGA 1621 1710 GTAATCGCACGGGAACAGCTCGAAGAGATGTGCACCGCCGTGAACCGCATTCACCGAGGACCTGAGCATCCCAGCCACATTGTGCTGCCG 1711 ATTATCAAGCGA SEQ ID NO, 4 T172R/G173Q/L196C/1301C CocE 1 60 MVDGNYSVASNVMVPMRDGVRLAVDLYRPDADGPVPVLLVRNPYDKFDVFAWSTQSTNWL 61 120 EFVRDGYAVVIQDTRGLFASEGEFVPHVDDEADAEDTLSWILEQAWCDGNVGMFGVSYLG 121 180 VTQWQAAVSGVGGLKAIAPSMASADLYRAPWYGPGGALSVEALLGWSALIGgQLITSRSD 181 240 ARPEDAADFVQLAAifNDVAGAASVTPLAEQPLLGRLIPWVIDQVVDHPDNDESWQSISL 241 300 FERLGGLATPALITAGWYDGFVGESLRTFVAVKDNADARLVVGPWSHSNLTGRNADRKFG 301 360 fAATYPIQEATTMHKAFFDRHLRGETDALAGVPKVRLFVMGIDEWRDETDWPLPDTAYTP 361 420 FYLGGSGAANTSTGGGTLSTSISGTESADTYLYDPADPVPSLGGTLLFHNGDNGPADQRP 421 480 IHDRDDVLCYSTEVLTDPVEVTGTVSARLFVSSSAVDTDFTAKLVDVFPDGRAIALCDGI 481 540 VRMRYRETLVNPTLIEAGEIYEVAIDMLATSNVFLPGHRIMVQVSSSNFPKYDRNSNTGG 541 VIAREQLEEMCTAVNRIHRGPEHPSHIVLPIIKR Note, Residues labeled in red are, R172, Q173, C196, and C301 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS, 4 <210> SEQ ID NO 1 <211> LENGTH, 1722 <212> TYPE, DNA <213> ORGANISM, Rhodococcus sp. MBl <400> SEQUENCE, 1 atggtggacg ggaattacag tgttgcctcg aacgtgatgg ttccgatgcg tgatggggtg 60 cgtctggcgg tcgacctgta ccgaccagat gctgatggac ctgttccggt cctgctggtt 120 cgcaacccat acgacaagtt cgacgtgttc gcgtggtcga cgcagtcgac aaactggctt 180 gagttcgtgc gtgatggcta tgccgtggtc attcaagaca cgcgtggctt gttcgcatcg 240 gaaggtgagt tcgtcccgca cgttgacgac gaagctgacg ccgaggatac gttgagctgg 300 attctggaac aagcgtggtg cgacggcaat gtgggcatgt tcggcgtttc gtacttgggt 360 gtgacccagt ggcaggccgc cgtatccggc gttggtgggc tgaaggcgat cgcgccgtcc 420 atggcgtcgg cggacttgta ccgcgccccg tggtacggcc ctggtggtgc gctttcagtc 480 gaggcgctgt tgggctggtc agctctcata ggtactgggc tcatcacgtc gaggtctgac 540 gcccggcccg aagacgcagc cgacttcgtc caactcgcag caattctcaa tgacgtcgct 600 ggcgcggcgt cggtcacgcc cctggccgag caaccgcttc tgggccgact gattccgtgg 660 gtgatcgatc aggttgtcga tcaccccgac aacgatgaat catggcagtc cattagcttg 720 US 9,879,240 B2 31 32 -continued tttgaacgac tcggcgggtt ggcaacaccg gccttgatca cggctgggtg gtacgacggg 780 ttcgtcggcg aatcgttgcg cactttcgtt gcggtcaagg acaatgccga cgcacgtttg 840 gttgtcggcc cttggagtca cagcaacctc actggtcgga atgcggaccg gaagttcggc 900 attgccgcga cctacccgat tcaagaagcc accacgatgc acaaggcatt cttcgaccgg 960 cacctccgcg gcgagaccga tgcactcgca ggcgtcccca aagtgcggct gttcgtaatg 1020 ggcatcgatg agtggcgtga cgaaacggac tggccactgc cggacacggc gtatacgccc 1080 ttctatcttg gaggtagcgg ggctgcgaat acctccacgg gtggtggaac actgtcgacg 1140 tcgatttccg gaactgaatc tgctgacacc tacctgtatg atccggccga tcccgtgcct 1200 tcgctcgggg ggacgctgct gttccacaac ggagacaacg gacccgccga ccaacgtccc 1260 attcatgacc gggacgacgt gttgtgttac agcactgagg tattgaccga cccggtggaa 1320 gtaaccggca ccgtctccgc ccggctgttc gtgtcgtcat cagcggtgga cactgatttc 1380 accgccaaac ttgtcgacgt atttcccgac ggtcgcgcga tcgcgctgtg tgacgggatc 1440 gtgcggatgc ggtaccgcga gacgttggtc aatccaacct tgatcgaagc gggcgaaatc 1500 tacgaggttg ctatcgacat gcttgcaacc tcgaatgtat tcctgccagg gcatcgcatc 1560 atggtccaag tatcaagtag caacttcccg aaatacgacc gcaattcgaa taccggcgga 1620 gtaatcgcac gggaacagct cgaagagatg tgcaccgccg tgaaccgcat tcaccgagga 1680 cctgagcatc ccagccacat tgtgctgccg attatcaagc ga 1722 <210> SEQ ID NO 2 <211> LENGTH, 574 <212> TYPE, PRT <213> ORGANISM, Rhodococcus sp. MBl <400> SEQUENCE, 2 Met Val Asp Gly Asn Tyr Ser Val Ala Ser Asn Val Met Val Pro Met 1 5 10 15 Arg Asp Gly Val Arg Leu Ala Val Asp Leu Tyr Arg Pro Asp Ala Asp 20 25 30 Gly Pro Val Pro Val Leu Leu Val Arg Asn Pro Tyr Asp Lys Phe Asp 35 40 45 Val Phe Ala Trp Ser Thr Gln Ser Thr Asn Trp Leu Glu Phe Val Arg 50 55 60 Asp Gly Tyr Ala Val Val Ile Gln Asp Thr Arg Gly Leu Phe Ala Ser 65 70 75 80 Glu Gly Glu Phe Val Pro His Val Asp Asp Glu Ala Asp Ala Glu Asp 85 90 95 Thr Leu Ser Trp Ile Leu Glu Gln Ala Trp Cys Asp Gly Asn Val Gly 100 105 110 Met Phe Gly Val Ser Tyr Leu Gly Val Thr Gln Trp Gln Ala Ala Val 115 120 125 Ser Gly Val Gly Gly Leu Lys Ala Ile Ala Pro Ser Met Ala Ser Ala 130 135 140 Asp Leu Tyr Arg Ala Pro Trp Tyr Gly Pro Gly Gly Ala Leu Ser Val 145 150 155 160 Glu Ala Leu Leu Gly Trp Ser Ala Leu Ile Gly Thr Gly Leu Ile Thr 165 170 175 Ser Arg Ser Asp Ala Arg Pro Glu Asp Ala Ala Asp Phe Val Gln Leu 180 185 190 US 9,879,240 B2 33 34 -continued Ala Ala Ile Leu Asn Asp Val Ala Gly Ala Ala Ser Val Thr Pro Leu 195 200 205 Ala Glu Gln Pro Leu Leu Gly Arg Leu Ile Pro Trp Val Ile Asp Gln 210 215 220 Val Val Asp His Pro Asp Asn Asp Glu Ser Trp Gln Ser Ile Ser Leu 225 230 235 240 Phe Glu Arg Leu Gly Gly Leu Ala Thr Pro Ala Leu Ile Thr Ala Gly 245 250 255 Trp Tyr Asp Gly Phe Val Gly Glu Ser Leu Arg Thr Phe Val Ala Val 260 265 270 Lys Asp Asn Ala Asp Ala Arg Leu Val Val Gly Pro Trp Ser His Ser 275 280 285 Asn Leu Thr Gly Arg Asn Ala Asp Arg Lys Phe Gly Ile Ala Ala Thr 290 295 300 Tyr Pro Ile Gln Glu Ala Thr Thr Met His Lys Ala Phe Phe Asp Arg 305 310 315 320 His Leu Arg Gly Glu Thr Asp Ala Leu Ala Gly Val Pro Lys Val Arg 325 330 335 Leu Phe Val Met Gly Ile Asp Glu Trp Arg Asp Glu Thr Asp Trp Pro 340 345 350 Leu Pro Asp Thr Ala Tyr Thr Pro Phe Tyr Leu Gly Gly Ser Gly Ala 355 360 365 Ala Asn Thr Ser Thr Gly Gly Gly Thr Leu Ser Thr Ser Ile Ser Gly 370 375 380 Thr Glu Ser Ala Asp Thr Tyr Leu Tyr Asp Pro Ala Asp Pro Val Pro 385 390 395 400 Ser Leu Gly Gly Thr Leu Leu Phe His Asn Gly Asp Asn Gly Pro Ala 405 410 415 Asp Gln Arg Pro Ile His Asp Arg Asp Asp Val Leu Cys Tyr Ser Thr 420 425 430 Glu Val Leu Thr Asp Pro Val Glu Val Thr Gly Thr Val Ser Ala Arg 435 440 445 Leu Phe Val Ser Ser Ser Ala Val Asp Thr Asp Phe Thr Ala Lys Leu 450 455 460 Val Asp Val Phe Pro Asp Gly Arg Ala Ile Ala Leu Cys Asp Gly Ile 465 470 475 480 Val Arg Met Arg Tyr Arg Glu Thr Leu Val Asn Pro Thr Leu Ile Glu 485 490 495 Ala Gly Glu Ile Tyr Glu Val Ala Ile Asp Met Leu Ala Thr Ser Asn 500 505 510 Val Phe Leu Pro Gly His Arg Ile Met Val Gln Val Ser Ser Ser Asn 515 520 525 Phe Pro Lys Tyr Asp Arg Asn Ser Asn Thr Gly Gly Val Ile Ala Arg 530 535 540 Glu Gln Leu Glu Glu Met Cys Thr Ala Val Asn Arg Ile His Arg Gly 545 550 555 560 Pro Glu His Pro Ser His Ile Val Leu Pro Ile Ile Lys Arg 565 570 <210> SEQ ID NO 3 <211> LENGTH, 1722 <212> TYPE, DNA <213> ORGANISM, Artificial Sequence <220> FEATURE, <223> OTHER INFORMATION, Synthesized US 9,879,240 B2 35 36 -continued <400> SEQUENCE, 3 atggtggacg ggaattacag tgttgcctcg aacgtgatgg ttccgatgcg tgatggggtg 60 cgtctggcgg tcgacctgta ccgaccagat gctgatggac ctgttccggt cctgctggtt 120 cgcaacccat acgacaagtt cgacgtgttc gcgtggtcga cgcagtcgac aaactggctt 180 gagttcgtgc gtgatggcta tgccgtggtc attcaagaca cgcgtggctt gttcgcatcg 240 gaaggtgagt tcgtcccgca cgttgacgac gaagctgacg ccgaggatac gttgagctgg 300 attctggaac aagcgtggtg cgacggcaat gtgggcatgt tcggcgtttc gtacttgggt 360 gtgacccagt ggcaggccgc cgtatccggc gttggtgggc tgaaggcgat cgcgccgtcc 420 atggcgtcgg cggacttgta ccgcgccccg tggtacggcc ctggtggtgc gctttcagtc 480 gaggcgctgt tgggctggtc agctctcata ggtcgccagc tcatcacgtc gaggtctgac 540 gcccggcccg aagacgcagc cgacttcgtc caactcgcag caatttgcaa tgacgtcgct 600 ggcgcggcgt cggtcacgcc cctggccgag caaccgcttc tgggccgact gattccgtgg 660 gtgatcgatc aggttgtcga tcaccccgac aacgatgaat catggcagtc cattagcttg 720 tttgaacgac tcggcgggtt ggcaacaccg gccttgatca cggctgggtg gtacgacggg 780 ttcgtcggcg aatcgttgcg cactttcgtt gcggtcaagg acaatgccga cgcacgtttg 840 gttgtcggcc cttggagtca cagcaacctc actggtcgga atgcggaccg gaagttcggc 900 tgcgccgcga cctacccgat tcaagaagcc accacgatgc acaaggcatt cttcgaccgg 960 cacctccgcg gcgagaccga tgcactcgca ggcgtcccca aagtgcggct gttcgtaatg 1020 ggcatcgatg agtggcgtga cgaaacggac tggccactgc cggacacggc gtatacgccc 1080 ttctatcttg gaggtagcgg ggctgcgaat acctccacgg gtggtggaac actgtcgacg 1140 tcgatttccg gaactgaatc tgctgacacc tacctgtatg atccggccga tcccgtgcct 1200 tcgctcgggg ggacgctgct gttccacaac ggagacaacg gacccgccga ccaacgtccc 1260 attcatgacc gggacgacgt gttgtgttac agcactgagg tattgaccga cccggtggaa 1320 gtaaccggca ccgtctccgc ccggctgttc gtgtcgtcat cagcggtgga cactgatttc 1380 accgccaaac ttgtcgacgt atttcccgac ggtcgcgcga tcgcgctgtg tgacgggatc 1440 gtgcggatgc ggtaccgcga gacgttggtc aatccaacct tgatcgaagc gggcgaaatc 1500 tacgaggttg ctatcgacat gcttgcaacc tcgaatgtat tcctgccagg gcatcgcatc 1560 atggtccaag tatcaagtag caacttcccg aaatacgacc gcaattcgaa taccggcgga 1620 gtaatcgcac gggaacagct cgaagagatg tgcaccgccg tgaaccgcat tcaccgagga 1680 cctgagcatc ccagccacat tgtgctgccg attatcaagc ga 1722 <210> SEQ ID NO 4 <211> LENGTH, 574 <212> TYPE, PRT <213> ORGANISM, Artificial Sequence <220> FEATURE, <223> OTHER INFORMATION, Synthesized <400> SEQUENCE, 4 Met Val Asp Gly Asn Tyr Ser Val Ala Ser Asn Val Met Val Pro Met 1 5 10 15 Arg Asp Gly Val Arg Leu Ala Val Asp Leu Tyr Arg Pro Asp Ala Asp 20 25 30 Gly Pro Val Pro Val Leu Leu Val Arg Asn Pro Tyr Asp Lys Phe Asp 35 40 45 US 9,879,240 B2 37 38 -continued Val Phe Ala Trp Ser Thr Gln Ser Thr Asn Trp Leu Glu Phe Val Arg 50 55 60 Asp Gly Tyr Ala Val Val Ile Gln Asp Thr Arg Gly Leu Phe Ala Ser 65 70 75 80 Glu Gly Glu Phe Val Pro His Val Asp Asp Glu Ala Asp Ala Glu Asp 85 90 95 Thr Leu Ser Trp Ile Leu Glu Gln Ala Trp Cys Asp Gly Asn Val Gly 100 105 110 Met Phe Gly Val Ser Tyr Leu Gly Val Thr Gln Trp Gln Ala Ala Val 115 120 125 Ser Gly Val Gly Gly Leu Lys Ala Ile Ala Pro Ser Met Ala Ser Ala 130 135 140 Asp Leu Tyr Arg Ala Pro Trp Tyr Gly Pro Gly Gly Ala Leu Ser Val 145 150 155 160 Glu Ala Leu Leu Gly Trp Ser Ala Leu Ile Gly Arg Gln Leu Ile Thr 165 170 175 Ser Arg Ser Asp Ala Arg Pro Glu Asp Ala Ala Asp Phe Val Gln Leu 180 185 190 Ala Ala Ile Cys Asn Asp Val Ala Gly Ala Ala Ser Val Thr Pro Leu 195 200 205 Ala Glu Gln Pro Leu Leu Gly Arg Leu Ile Pro Trp Val Ile Asp Gln 210 215 220 Val Val Asp His Pro Asp Asn Asp Glu Ser Trp Gln Ser Ile Ser Leu 225 230 235 240 Phe Glu Arg Leu Gly Gly Leu Ala Thr Pro Ala Leu Ile Thr Ala Gly 245 250 255 Trp Tyr Asp Gly Phe Val Gly Glu Ser Leu Arg Thr Phe Val Ala Val 260 265 270 Lys Asp Asn Ala Asp Ala Arg Leu Val Val Gly Pro Trp Ser His Ser 275 280 285 Asn Leu Thr Gly Arg Asn Ala Asp Arg Lys Phe Gly Cys Ala Ala Thr 290 295 300 Tyr Pro Ile Gln Glu Ala Thr Thr Met His Lys Ala Phe Phe Asp Arg 305 310 315 320 His Leu Arg Gly Glu Thr Asp Ala Leu Ala Gly Val Pro Lys Val Arg 325 330 335 Leu Phe Val Met Gly Ile Asp Glu Trp Arg Asp Glu Thr Asp Trp Pro 340 345 350 Leu Pro Asp Thr Ala Tyr Thr Pro Phe Tyr Leu Gly Gly Ser Gly Ala 355 360 365 Ala Asn Thr Ser Thr Gly Gly Gly Thr Leu Ser Thr Ser Ile Ser Gly 370 375 380 Thr Glu Ser Ala Asp Thr Tyr Leu Tyr Asp Pro Ala Asp Pro Val Pro 385 390 395 400 Ser Leu Gly Gly Thr Leu Leu Phe His Asn Gly Asp Asn Gly Pro Ala 405 410 415 Asp Gln Arg Pro Ile His Asp Arg Asp Asp Val Leu Cys Tyr Ser Thr 420 425 430 Glu Val Leu Thr Asp Pro Val Glu Val Thr Gly Thr Val Ser Ala Arg 435 440 445 Leu Phe Val Ser Ser Ser Ala Val Asp Thr Asp Phe Thr Ala Lys Leu 450 455 460 US 9,879,240 B2 39 40 -continued Val Asp Val Phe Pro Asp Gly Arg Ala Ile Ala Leu Cys Asp Gly Ile 465 470 475 480 Val Arg Met Arg Tyr Arg Glu Thr Leu Val Asn Pro Thr Leu Ile Glu 485 490 495 Ala Gly Glu Ile Tyr Glu Val Ala Ile Asp Met Leu Ala Thr Ser Asn 500 505 510 Val Phe Leu Pro Gly His Arg Ile Met Val Gln Val Ser Ser Ser Asn 515 520 525 Phe Pro Lys Tyr Asp Arg Asn Ser Asn Thr Gly Gly Val Ile Ala Arg 530 535 540 Glu Gln Leu Glu Glu Met Cys Thr Ala Val Asn Arg Ile His Arg Gly 545 550 555 560 Pro Glu His Pro Ser His Ile Val Leu Pro Ile Ile Lys Arg 565 570 20 We claim: 9. The CocE polypeptide variant of claim 5, wherein the 1. A cDNA molecule comprising the sequence of SEQ ID enzyme is conjugated to the polyethylene glycol polymer NO: 3. and the polyethylene glycol polymer maleimide-linked 2. A cDNA molecule comprising a nucleic acid sequence branched poly(ethylene glycol) (PEG). 25 which encodes a cocaine esterase (CocE) polypeptide vari 10. A pharmaceutical composition comprising the CocE ant, comprising the amino acid sequence of SEQ ID NO: 4. polypeptide variant of claim 4; and a suitable pharmaceuti 3_ : A cocain� esterase (CocE) polypeptide variant, com cally carrier. pnsmg the am1 0 acid sequence of SEQ ID NO: 2, including _ 1;1 11. The pharmaceutical composition comprising of claim the ammo acid sequences mutations: T172R, G173Q 10, and further comprising at least one polyethylene glycol L196C, and 1301C. 30 4 A cocaine esterase (CocE) polypeptide variant, com polymer chain attached thereto. _ : pnsmg the amino acid sequence of SEQ ID NO: 4, or the 12. A method of treating a cocaine-induced condition amino acid sequence encoded by the nucleic acid sequence comprising administering to an individual an effective of SEQ ID NO: 3. amount of the CocE polypeptide variant of claim 4 to accelerate cocaine metabolism and produce biologically 5. The CocE polypeptide variant of claim 4, comprising at 35 least one cross-subunit disulfide bond. inactive metabolites. 13. T e meth of claim 12, wherein said CocE polypep 6. The CocE polypeptide variant of claim 4, wherein the . � ?� at least one cross-subunit disulfide bond is a bond between tide vanant exhibits a one-hundred-fold or more increase in C196a and C301b, or C301a and C196b. cocaine hydrolysis catalytic efficiency as compared to a mutant that includes only the Tl 72R/G173Q mutations. 7. The CocE polypeptide variant of claim 4, having a 40 half-life of greater than about 100 days at 37° C. 14. The method of claim 12, wherein said CocE polypep 8. The CocE polypeptide variant of claim 4, further tide variant provides protection from a lethal dose of cocaine comprising at least one polyethylene glycol polymer chain for at least 72 hours. attached thereto. * * * * *