human liver carboxylesterase hce-1: binding specificity...

HUMAN LIVER CARBOXYLESTERASE hCE-1: BINDING SPECIFICITY FOR COCAINE,HEROIN, AND THEIR METABOLITES AND ANALOGS

MONICA R. BRZEZINSKI, BENJAMIN J. SPINK, ROBERT A. DEAN, CLIFFORD E. BERKMAN, JOHN R. CASHMAN, AND

WILLIAM F. BOSRON

Departments of Biochemistry and Molecular Biology (M.R.B., B.J.S., R.A.D., W.F.B.), Pathology and Laboratory Medicine (R.A.D.), andMedicine (W.F.B.), Indiana University School of Medicine, Indianapolis, IN and Seattle Biomedical Research Institute,

Seattle, WA (C.E.B., J.R.C.)

(Received March 7, 1997; accepted June 5, 1997)

ABSTRACT:

Purified human liver carboxylesterase (hCE-1) catalyzes the hydro-lysis of cocaine to form benzoylecgonine, the deacetylation ofheroin to form 6-acetylmorphine, and the ethanol-dependenttransesterification of cocaine to form cocaethylene. In this study,the binding affinities of cocaine, cocaine metabolites and analogs,heroin, morphine, and 6-acetylmorphine for hCE-1 were evaluatedby measuring their kinetic inhibition constants with 4-methylum-belliferyl acetate in a rapid spectrophotometric assay. The natu-rally occurring (R)-(2)-cocaine isomer displayed the highest affin-ity of all cocaine and heroin analogs or metabolites. The pseudo- orallopseudococaine isomers of cocaine exhibited lower affinity in-

dicating that binding to the enzyme is stereoselective. The methylester, benzoyl, and N-methyl groups of cocaine play importantroles in binding because removal of these groups increased Ki

values substantially. Compounds containing a variety of hydropho-bic substitutions at the benzoyl group of cocaine bound to theenzyme with high affinity. The high Ki value obtained for cocaeth-ylene relative to cocaine is consistent with weaker binding to theesterase and a longer elimination half-life reported for the metab-olite. The spectrophotometric competitive inhibition assay usedhere represents an effective method to identify drug or environ-mental esters metabolized by carboxylesterases like hCE-1.

Carboxylesterases are hydrolytic enzymes involved in the metabo-lism of drug and environmental esters in a variety of mammaliantissues. They catalyze the conversion of mostly lipophilic ester sub-strates to more water-soluble carboxylic acids plus alcohol products,thereby facilitating their elimination (1). The broad substrate speci-ficity exhibited by these enzymes enables the cell to metabolize a widevariety of esters. However, the lack of specificity generally kcat results inrelatively highKM

1 values and low catalytic efficiencies (kcat/KM) forhydrolysis of drug or environmental esters. The low catalytic efficiencyis usually compensated by the presence of relatively large amounts ofthese enzymes in tissues, especially in the liver.

A carboxylesterase with broad substrate specificity was recentlypurified from human liver and partially characterized (2). The enzymeis named hCE-1 for human carboxylesterase-1 according to the sug-gestion of Kroetzet al. (3), although it also has been referred to asesterase 1 (4). The hCE-1 is involved in the metabolism of cocaine byhydrolyzing cocaine to benzoylecgonine (fig. 1) (4), a major urinarymetabolite of cocaine (5). In the presence of ethanol, hCE-1 alsocatalyzes the ethyl transesterification of cocaine forming the pharma-cologically active metabolite, cocaethylene (4). The same enzymaticstep may be responsible for the conversion of norcocaine to norcoca-

ethylene (fig. 1) and both compounds may be hydrolyzed by thisenzyme to benzoylnorecgonine. A second carboxylesterase namedhCE-2 in human liver catalyzes the hydrolysis of the benzoyl group ofcocaine (4, 6) and the 3- or 6-acetyl groups of heroin (7).

The hCE-1 enzyme exhibits broad substrate-specificity for esterhydrolysis. 4-methylumbelliferyl acetate (4) andp-nitrophenyl valer-ate (8) are used in simple spectrophotometric assays. Both hCE-1 anda highly homologous enzyme from rat called hydrolase A hydrolyzep-nitrophenyl butyrate (3, 9). They also exhibit fatty acid ethyl estersynthase activity by catalyzing the formation of ethyloleate from oleicacid and ethanol (2, 9). Additionally, the rat (10) and human enzymes(2) catalyze the ethanol-dependent transesterification of cocaine tococaethylene.

The substrate specificity of hCE-1 with cocaine metabolites, toxins,and other drug esters has not been studied. Recently Kamenduliset al.(7) demonstrated that the enzyme plays a role in the metabolism ofheroin by catalyzing its hydrolysis to 6-acetylmorphine. The bindingaffinities of heroin, cocaine, and structurally related compounds tohCE-1 were evaluated in this study using the steady-state kineticanalysis of alternative substrates or competitive inhibitors in a spec-trophotometric assay employing 4-methylumbelliferyl acetate as sub-strate. The assay provides an efficient method for identifying com-pounds that bind with high affinity to the enzyme and for predictingester drugs hydrolyzed by this carboxylesterase.

Materials and Methods

Materials. All chemicals were purchased from Sigma Chemical Co. (St.Louis, MO), except ultra pure DTT which was obtained from the United StatesBiochemical Corp. (Cleveland, OH). The Q Sepharose and Con A Sepharosechromatography resins used for enzyme purification were purchased fromPharmacia (Uppsala, Sweden) and DEAE-cellulose was from Whatman Bio-systems (Maidstone, UK). Solvents were prepared with H2O treated with a

This work was supported by RO1 DA06836 and fellowships to M.R.B. from T32AA07462.

1 Abbreviations used are: KM, Michaelis-Menten constant; hCE, human car-boxylesterase; kcat, turnover number; Ki, competitive inhibition constant; DEA,N,N-diisopropylethylamine; DTT, dithiothreitol; SDS-PAGE, sodium dodecyl sul-fate-polyacrylamide gel electrophoresis.

Send reprint requests to: Dr. William F. Bosron, Department of Biochemistryand Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive,MS 405, Indianapolis, IN 46202-5122, [email protected].

0090-9556/97/2509-1089–1096$02.00/0DRUG METABOLISM AND DISPOSITION Vol. 25, No. 9Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A.

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Milli-Q filtration system (Millipore, Bedford, MA). Enzyme samples wereconcentrated with Amicon ultrafiltration stirred cells (Beverly, MA) usingYM30 membranes.

Drugs and Related Compounds.Atropine sulfate, tropacocaine HCl, andR-(2)-cocaine HCl were obtained from Sigma Chemical. Heroin HCl mono-hydrate and benzoylecgonine propyl ester HCl were purchased from Alltech-Applied Science Laboratories (State College, PA). Benzoylecgonine and co-caethylene were prepared in our laboratory by a previously publishedprocedure (11). Norcocaine and norcocaethylene were also prepared in ourlaboratory by theN-demethylation of cocaine and cocaethylene, respectively(12). Purity of the synthesized compounds was determined by HPLC andGC/MS (13). Compounds #1 thru #7 were synthesized as described below. Thestructures of these compounds are shown in table 1. All other compounds wereobtained from the National Institute on Drug Abuse.

Synthetic Procedures. 3b-(Methylphenylphosphonodithionyloxy)-8-azabi-cyclo[3.2.1]octane-2b-carboxylic acid methyl ester (#1).Lawesson’s reagent (42mg, 104mmol) and compound #4 (70 mg, 189mmol) were dissolved in tolueneand refluxed 3 hr. The solvent was removedin vacuoand the product waspurified by preparative TLC (silica; MeOH:CH2Cl2:NH4OH (30%) 5:95:0.25,Rf 5 0.55) to give a pale yellow oil (31 mg, 43% yield).1H NMR (CDCl3):d1.62–1.90 (m, 3H), 1.98–2.21 (m, 5H), 2.24 (s, 3H), 2.43–2.66 (m, 1H), 3.02and 3.12 (m, 1H), 3.24–3.39 (m, 1H), 3.55 (br s, 1H), 3.71 and 3.77 (s, 3H),4.97–5.12 (m, 1H), 7.41–7.57 (m, 3H), 7.82–7.98 (m, 2H).31P NMR (CDCl3):d94.47, 94.59.

3b-(Methyl phenylphosphonothionyloxy)-8-azabicyclo[3.2.1]octane-2b-car-boxylic acid methyl ester (#2).(R)-Ecgonine methyl ester (500 mg, 2.51 mmol)and tetrazole (18 mg, 0.25 mmol) were dissolved in CH2Cl2 (10 mL) under anAr gas atmosphere. DEA (685mL, 5.52 mmol) was addedvia syringe followedby the addition of dichlorophenylphosphine sulfide (429ml, 2.76 mmol) andthe reaction stirred 3 hr. Methanol (167ml, 3.76 mmol) was added and thereaction stirred an additional 1 hr. The solvent was removedin vacuoand theproduct was purified by flash chromatography (silica gel; MeOH:CH2Cl2:NH4OH (30%) 10:90:0.4, Rf 5 0.55) to give a colorless oil (352 mg, 38%yield). 1H NMR (CDCl3): d1.48–1.70 (m, 3H), 1.82–2.12 (m, 2H), 2.16 (s,3H), 2.35 and 2.50 (dt, 1H,J 5 3.2, 11.7 Hz), 2.72 and 3.02 (s, 1H), 3.16 and

3.26 (s, 1H), 3.40 and 3.49 (s, 1H), 3.55 and 3.73 (s, 3H), 3.59 and 3.65 (d, 3H,J 5 13.8 Hz), 4.72–7.94 (m, 1H), 7.37–7.54 (m, 3H), 7.77–7.95 (m, 2H).31PNMR (CDCl3): d88.21, 88.95.

3b-(Diphenylphosphonyloxy)-8-azabicyclo[3.2.1]octane-2b-carboxylic acidmethyl ester (#3).(R)-Ecgonine methyl ester (100 mg, 503ml) and tetrazole (4mg, 51 ml) were dissolved in benzene (10 ml) at 4°C under an Ar gasatmosphere. DEA (90ml, 503 mmol) was addedvia syringe followed by theaddition of diphenylphosphinic chloride (96ml, 503 mmol). The reactionmixture was stirred 2.5 hr warming slowly to room temperature. The solventwas removedin vacuoand the product was purified by flash chromatography(silica gel; MeOH:CH2Cl2:NH4OH (30%) 10:90:0.15, Rf 5 0.4) to give acolorless oil (116 mg, 58% yield).1H NMR (CDCl3): d1.41–1.55 (m, 2H),1.61–1.71 (m, 1H), 1.88–2.10 (m, 2H), 2.14 (s, 3H), 2.45 (t, 1H,J 5 11.6 Hz),2.81 (s, 1H), 3.15 (s, 1H), 3.42 (s, 1H), 3.69 (s, 3H), 4.61–4.73 (m, 1H),7.34–7.54 (m, 6H), 7.68–7.75 (m, 2H), 7.78–7.86 (m, 2H). MS (FAB)m/z400(M1H).

3b-(Methylphenylphosphonothiolyloxy)-8-azabicyclo[3.2.1]octane-2b-car-boxylic acid methyl ester (#4).Potassium ethyl xanthate (560 mg, 345mmol)and compound #2 (116 mg, 314mmol) were dissolved in dry acetone andrefluxed 10 hr after which dimethyl sulfate (33ml, 345 mmol) was added andthe reaction mixture was refluxed an additional 0.5 hr. The solvent wasremovedin vacuoand the product was purified by flash chromatography (silicagel; MeOH:CH2Cl2 10:90, Rf 5 0.25) to give a pale yellow oil (70 mg, 61%yield). 1H NMR (CDCl3): d1.54–1.70 (m, 2H), 1.82 (br s, 1H), 1.92–2.16 (m,2H), 2.04 and 2.08 (d, 3H,J 5 13.7 Hz), 2.30–2.60 (m, 1H), 2.96 and 3.08 (s,1H), 3.20 and 3.26 (s, 1H), 3.49 (s, 1H), 3.73 and 3.65 (s, 3H), 4.78–4.92 (m,1H), 7.38–7.56 (m, 3H), 7.76–7.90 (m, 2H).31P NMR (CDCl3): d44.62,44.95. MS (FAB)m/z370 (M1H).

3b-(Methylphenylphosphononyloxy)-8-azabicyclo[3.2.1]octane-2b-carbox-ylic acid methyl ester (#5).(R)-Ecgonine methyl ester (300 mg, 1.51 mmol)and tetrazole (11 mg, 0.15 mmol) were dissolved in benzene (10 ml) andchilled to 4°C under an Ar gas atmosphere. DEA (564ml, 3.17 mmol) wasaddedvia syringe, followed by the addition of phenylphosphonic dichloride(224 mL, 1.59 mmol), and the reaction mixture was stirred 15 hr, slowlywarming to room temperature. Methanol (100mL, 2.27 mmol) was added andthe reaction stirred an additional 1 hr. The solvent was removedin vacuoand

FIG. 1. Metabolic pathway of cocaine.

Three types of reactions occur in the metabolism of cocaine: 1) hydrolysis, 2) transesterification, and 3)N-demethylation. The competitive inhibition constants(Ki) calculated from steady-state kinetic analyses (eqs. 1 and 2) are listed under each of the six compounds.

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the product was purified by flash chromatography (silica gel; MeOH:CH2Cl210:90, Rf 5 0.25) to give a colorless oil (231 mg, 43% yield).1H NMR(CDCl3): d1.45–1.70 (m, 2H), 1.78–2.14 (m, 3H), 2.17 (s, 3H), 2.37 and 2.53(dt, 1H,J 5 2.5, 11.8 Hz), 2.75 and 3.06 (m, 1H), 3.16 and 3.26 (s, 1H), 3.41and 3.50 (s, 1H), 3.60 and 3.75 (s, 3H), 3.66 and 3.70 (d, 3H,J 5 11.3 Hz),

4.58–4.76 (m, 1H), 7.40–7.50 (m, 2H), 7.50–7.58 (m, 1H), 7.72–7.88 (m,2H). 31P NMR (CDCl3): d19.83, 20.38. MS (FAB)m/z354 (M1H).

3b-(Phenylphosphonic acid)-8-azabicyclo[3.2.1]octane-2b-carboxylic acidmethyl ester (#6).To a solution of compound #5 (20 mg, 57mmol) inacetonitrile (100ml) under an Ar gas atmosphere was added trimethylsilyl

TABLE 1

Partition coefficients and binding affinities of C3-substituted cocaine derivatives

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bromide (12.2ml, 92 mmol). The reaction mixture was stirred 1.5 hr afterwhich the reaction was quenched with water (50ml). The solvent was removedin vacuoand the product purified as the ammonium salt by eluting the mixturefrom a C18 solid phase extraction column (0.5 g) with methanol containing 1%NH4OH (30%) to give a white solid (20 mg, 99% yield).1H NMR (CDCl3):d1.91–2.09 (m, 3H), 2.16–2.40 (m, 3H), 2.75 (s, 3H), 3.08 (dd, 1H,J 5 7.3Hz), 3.70 (s, 3H), 3.80 (s, 1H), 4.02 (d, 1H,J 5 6.3 Hz), 4.08–4.72 (m, 1H),7.31–7.44 (m, 3H), 7.74 (dd, 2H,J 5 7.7, 12.6 Hz).31P NMR (CDCl3): d16.77.

3b-(Methanesulfonyloxy)-8-azabicyclo[3.2.1]octane-2b-carboxylic acidmethyl ester (#7).Methanesulfonyl chloride (69ml, 0.89 mmol) was addeddropwise to a stirring mixture of (R)-ecgonine methyl ester hydrochloride (0.20g, 0.85 mmol) and triethylamine (0.24 ml, 1.7 mmol) in CH2Cl2 (5 ml) at 0°C.After several hours at room temperature, the reaction mixture was washed with10% Na2CO3, dried over Na2SO4, and evaporatedin vacuo. The yellow residuewas purified by silica gel chromatography (10% MeOH/CH2Cl2) to afford themesylate as a light yellow solid (0.12 g, 51% yield).1H NMR (CDCl3): d5.04(m, 1H), 3.92 (s, 3H), 3.71 (m, 1H), 3.46 (m, 1H), 3.21 (m, 1H), 3.19 (s, 3H),2.70 (m, 1H), 2.37 (s, 3H), 2.20–2.35 (m, 2H), 2.01–2.11 (m, 1H), 1.72–1.88(m, 2H). MS (EI)m/z277 (M1H).

Carboxylesterase Assays.Acetylesterase activity was measured by incu-bating approximately 0.7mg of purified enzyme with 0.2 to 0.5 mM 4-methy-lumbelliferyl acetate in 90 mM KH2PO4, 40 mM KCl, pH 7.3 (1-ml totalvolume) at 37°C. The 4-methylumbelliferyl acetate stock solutions (25 mM)were prepared in dimethyl sulfoxide. The formation of 4-methylumbelliferonewas monitored for 1.5 min in a Perkin-Elmer Lambda 6 double-beam spec-trophotometer at 350 nm. Rates of ester hydrolysis were calculated by linearregression of absorbanceversustime using the extinction coefficient 12.2 cm21

mM21 for 4-methylumbelliferone (4). The time course of product formationwas linear throughout the time of assay (correlation coefficient for linearregression 98%). Specific activity is expressed asmmol product formed permin per mg protein. Protein concentration was determined with the Bio-Radprotein assay using bovine serum albumin as a standard.

Purification of Human Liver Carboxylesterase (hCE-1). The DEAE-cellulose and Q Sepharose chromatography steps in the purification of enzymefrom 60 g of human liver are identical to those described by Brzezinskiet al.(2). However, the last three steps that included Superose 6, Polybuffer Ex-changer, and Phenyl Superose columns were substituted by affinity chroma-tography on Con A Sepharose (7). Fractions of hCE-1 obtained from the QSepharose column were pooled and the buffer exchanged for 20 mM Tris-Cl,0.5 M NaCl, 1 mM CaCl2, 1 mM MnSO4, 1 mM benzamidine, 1 mM EDTA,and 1 mM DTT at pH 7.4 (Buffer A). The sample was loaded onto a Con ASepharose 4B column (2.53 6 cm) and bound protein was eluted with a 260ml linear gradient of Buffer A to Buffer A1 0.5 M methyl-a-D-mannopyr-anoside, pH 7.4. Active fractions were pooled and the buffer exchanged for 50mM NaH2PO4, 1 mM benzamidine, 1 mM EDTA, pH 7.0. The purifiedenzyme was concentrated to 1.3 mg/ml, sterile-filtered, and stored at 4°C tomaintain stability. The purity of hCE-1 was examined by SDS-PAGE (14) andisoelectric focusing. Purified hCE-1 was not contaminated with hCE-2.

Ki Determinations. The affinities of cocaine, cocaine metabolites, heroin,and heroin metabolites in fig. 1 and table 2 for hCE-1 were examined bytreating these compounds as alternative substrates or inhibitors that are com-petitive with 4-methylumbelliferyl acetate in the spectrophotometric enzymeassay. The equation for competitive inhibition is:

1/v 5 ~KM/Vmax!~1 1 @I#/Ki!~1/@S#! 1 1/Vmax (1)

where [S]5 4-methylumbelliferyl acetate concentration,KM is the Michaelisconstant for 4-methylumbelliferyl acetate,Vmax is the maximal activity for4-methylumbelliferyl acetate hydrolysis, [I]5 competitive inhibitor concen-tration, andKi 5 inhibition or dissociation constant for the dead-end inhibitor.The steady-state kinetic expression for alternative substrates is:

1/v 5 ~KM/Vmax!~1 1 @B#/Kb!~1/@S#! 1 1/Vmax (2)

where [B] is the alternate substrate andKb is its Michaelis constant. Note thateqs. 1 and 2 are identical in form.

The enzyme concentration was adjusted so that the activity was approxi-mately 3.6 U/ml with no inhibitor added. The buffer-substrate-inhibitor mix-

ture was equilibrated in the cuvette at 37°C for 1.5 min prior to the addition ofenzyme. Assay buffer was used to make the inhibitor stock solutions.(1)-Cocaine was dissolved in DMSO and compound #4 contained 5% DMSO.For cocaine, heroin, and the metabolites in table 2 and fig. 1, competitiveinhibition constants (Ki) and alternative substrate Michaelis constants (Kb)were determined from data sets consisting of 56 points generated with four4-methylumbelliferyl acetate concentrations (0.5 mM, 0.4 mM, 0.3 mM, and0.2 mM) and six inhibitor or alternative substrate concentrations. Duplicateactivity readings were obtained for each set of conditions. At the lowestsubstrate and highest inhibitor concentrations, it was necessary to reduce theassay time to avoid nonlinearity owing to substrate depletion. The data col-lected for each compound were evaluated for fit to competitive, noncompeti-tive, and uncompetitive inhibition models (15) by nonlinear regression ofinhibition equations using programs written in SAS (SAS Institute, Inc., Cary,NC). Additional data sets were collected for each compound until theKi

values, defined by the 95% confidence interval, overlapped.To determine inhibition constants (Ki) of compounds in tables 1 and 3, a

series of inhibitor concentrations were used ranging from 0.125 mM to 10 mM.At least 12 data points were generated from duplicate assays. The 4-methy-lumbelliferyl acetate concentration, [S]5 0.5 mM, was used to calculateVmax

using the following equation:

v 5 Vmax@S#/~KM 1 @S#! (3)

whereKM for 4-methylumbelliferyl acetate was 660mM. Competitive inhibi-tion constants (Ki) were fit to eq. 1 with no less than six inhibitor concentra-tions.

The log octanol-water partition coefficient (logp) of selected compoundswas estimated by the atom/fragment contribution method (16). Starting withthe experimentally determined logp of cocaine, coefficients assigned to atomsand fragments were added or subtracted to obtain logp values for the cocainederivatives.

Results

The purification procedure for hCE-1 was modified and simplifiedfrom that previously described (2). After the Q Sepharose column, anaffinity chromatography step was incorporated which gave a higheryield and a more stable enzyme suitable for steady-state kineticstudies (7). Acetylesterase activity was measured at each step of thepurification using 4-methylumbelliferyl acetate as the substrate. The

TABLE 2

Structures of heroin, 6-acetylmorphine, and morphine; and binding affinityto hCE-1

Compound R R Ki (mM)

Heroin OCOCH3 OCOCH3 0.36-Acetylmorphine H OCOCH3 0.3Morphine H H .10

Ki values of heroin, 6-monoacetylmorphine, and morphine were determinedby their competitive inhibition of the hydrolysis of 4-methylumbelliferylac-etate with purified hCE-1 as described inMethods.


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specific activity was 5.5 U/mg for the pure enzyme based on the4-methylumbelliferyl acetate assay compared with 6.8 U/mg obtainedwith the original procedure (2). The yield of acetylesterase activitywas 31%, whereas the yield by the previous procedure was only 3%.The purified enzyme exhibited a single, dominant band at 59 kD by

SDS-PAGE analysis (fig. 2). The carboxylesterase was considerablymore stable after this purification scheme since no loss of activity wasobserved with storage at 4°C over a period of several months.

Inhibition constants were determined for a series of compoundsusing a single concentration of the substrate 4-methylumbelliferyl

TABLE 3

Potencies of cocaine and related compounds in inhibiting hCE-1-catalyzed hydrolysis of 4-methylumbelliferyl acetate


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acetate and a range of inhibitor concentrations. In the assay, thesubstrate concentration (500mM) was set slightly below itsKM value.The KM determined for 4-methylumbelliferyl acetate at pH 7.3 and37°C was 6606 50 mM. The Ki values for cocaine, tropacocaine,atropine, plus cocaine isomers and metabolites are listed in table 3along with the substituent groups present at positions R1 through R4of the tropane ring. The compounds can be divided into four groupsaccording to theKi values as follows: cocaine and cocaethylene (0.01to 0.1 mM); benzoylecgonine propyl ester, pseudococaine isomers2,3

and allopseudococaine (0.1 to 1.0 mM); benzoylecgonine, tropaco-caine, and atropine (1.0 to 10.0 mM); and (S)-(1)-cocaine3, ecgoninemethyl ester, and ecgonine (.10 mM).

In table 1, the inhibition constants and estimated log octanol-waterpartition coefficients for cocaine and a series of C3-substituted deriv-atives are listed. There is a direct relationship between hydrophobicityand binding affinity,i.e., the compounds with a more hydrophobicsubstituent at the C3 position bind hCE-1 with greater affinity. Thedata are represented graphically in fig. 3. Linear regression analysiswas applied to two different sets of compounds: the WIN and RTIcompounds, and cocaine plus compounds #1 thru #7. Compound #6was not included because theKi was. 10. This was probably causedby a negatively charged oxygen atom at the neutral pH of the assay.Compound #5 was not included because theKi was higher thanexpected, probably because of nonenzymatic hydrolysis of the com-pound in stock solution.

The mechanism of inhibition was examined for cocaine, five co-caine metabolites (fig. 1), heroin, and 6-acetylmorphine by varyingboth 4-methylumbelliferyl acetate and the inhibitor or alternativesubstrate concentrations. As shown by the example of the Dixon plotfor 6-acetylmorphine inhibition of 4-methylumbelliferyl acetate hy-drolysis (fig. 4), the inhibition was competitive with respect to thesubstrate. This was verified by comparing the fit of data to competi-tive, noncompetitive, and uncompetitive inhibition models. The com-petitive inhibition fit yielded the highestF-statistic. The SE ofKi was,6% of the value obtained in each case. Cocaine, heroin, and theirmetabolites can be divided into four groups according toKi values asfollows: cocaine (0.01 mM); cocaethylene, norcocaethylene, heroin,and 6-acetylmorphine (0.1 to 1 mM); norcocaine, benzoylecgonine,and benzoylnorecgonine (1.0 to 10 mM); and morphine (.10 mM).Modification at the methyl ester, benzoyl orN-methyl sites of cocainecauses substantial increases in the inhibition constant.

Discussion

A family of carboxylesterases has been isolated and characterizedin human (8, 17), rat (10, 18), and rabbit tissues (19, 20). Thenumerous isozymes share several common structural characteristicsincluding about 60 kD subunit weight, microsomal subcellular local-ization, and an acidic isoelectric point (21). Two human liver ester-ases, hCE-1 and hCE-2, have been purified and partially characterized(2, 6). These two esterases can be distinguished by their molecularproperties: hCE-1 is a 180 kDa trimer with an isoelectric point of 5.8while hCE-2 is a 60 kDa monomer with an isoelectric point of 4.9.These esterases exhibit different substrate specificities. For example,hCE-1 hydrolyzes the methyl ester group of cocaine while hCE-2hydrolyzes the benzoyl ester (4). Important information about struc-ture-activity relationships has been obtained by examination of theamino acid sequence alignment of these human esterases with otheresterases and lipases (6, 22). Regions around the catalytic triad,including the active-site serine, are conserved. However, there is highvariability in residues suspected to be in the substrate binding site thatcould account for differences in substrate specificity (22).

In the present study, we evaluated the affinities of various com-

2 (R)-(1)-pseudococaine is the C2 epimer of (R)-(2)-cocaine, which is found inthe leaves of Erythroxylon coca.

3 (S)-(1)-cocaine is the unnatural enantiomer of (R)-(2)-cocaine, and (S)-(2)-pseudococaine is the C2 epimer of (S)-(1)-cocaine.

FIG. 2. SDS-PAGE analysis of carboxylesterase hCE-1.

Samples of the esterase were separated by SDS-PAGE (10% polyacryl-amide gel) and detected by silver stain (32). An aliquot from the final Con ASepharose step is shown in lanes 1 (0.5mg) and 2 (0.1mg). The protein bandin lane 2 corresponded to a molecular weight of approximately 59,000 basedon a standard curve generated with high range SDS-PAGE markers.

FIG. 3. Correlation of cocaine derivative hydrophobicity with affinity forhCE-1.

Ki values were plotted against the estimated log octanol-water partitioncoefficients for compounds listed in table 1. Linear regression analysis wasapplied to two sets of data points. The slopes of the lines are20.12 (r2 5 0.83)and21.45 (r2 5 0.74), respectively. Compound #5 (E) was not included in thelinear regression.


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pounds for hCE-1 by examining their competition with hydrolysis ofthe substrate 4-methylumbelliferyl acetate in a spectrophotometricassay. The compounds in fig. 1 and table 2 were examined bysteady-state kinetic analysis, and they obeyed a simple competitiveinhibition model since the fit of data to this mechanism was signifi-cantly better than to noncompetitive or uncompetitive models. Twotypes of compounds were examined in the kinetic analysis: alternateester substrates and dead-end, competitive inhibitors. As shown byeqs. 1 and 2, the only mathematical difference between these twomodes of inhibition is the definition of the inhibition constant,Ki 5inhibitor dissociation constant andKb 5 Michaelis constant for thealternate substrate.

TheKi values listed in table 3 for the cocaine isomers, metabolites,and derivatives range from 0.01 mM for the naturally occurring(R)-(2)-cocaine to.10 mM for the unnatural enantiomer (S)-(1)-cocaine. Binding to hCE-1 appears to be stereoselective, since theKi

for (R)-(2)-cocaine was at least 20 times lower than values obtainedfor (1)- and (2)-pseudococaine and (6)-allopseudococaine (table 3).Interestingly, Gatley (23) indicated that both (S)-(1)-cocaine and(R)-(2)-cocaine had similar affinities for the serum cholinesterase,but the rate of hydrolysis of the benzoyl ester of (1)-cocaine wasseveral orders of magnitude faster compared with (2)-cocaine, thenatural isomer. This observation agrees with a study comparing thecytotoxic potential of (R)-(2)-cocaine to (S)-(1)-cocaine and (S)-(2)-pseudococaine in cultured rat hepatocytes, which indicates thatthe stereoselective differences in the rates of hydrolysis primarilyaccount for the cytotoxic potency of cocaine (24).

The kinetic data define, in part, structure-binding relationshipsbetween the cocaine-like molecules and hCE-1. There appears to be apreference for the methyl ester group at the R1 positionversusethylester or propyl ester groups, as indicated by the;10 fold greaterKi

for cocaethylene and benzoylecgonine propyl esterversuscocaine.Removing the ester at this position to form tropacocaine results in a240-fold greater inhibition constant.Ki values similar to tropacocainewere obtained with the antimuscarinic agent atropine (25). Ecgoninemethyl ester, the de-esterified derivative with a hydroxyl group at R3,has virtually no affinity for hCE-1 with aKi .10 mM. Hence, theester linkage at R3 is very important for binding to hCE-1. Bulkygroups at the C3 position display intermediate binding affinities(0.01–2.2 mM) as shown by the WIN and RTI compounds (table 1).Compounds #1 and #2 (table 1) that contain an ester-like phosphoro-thionate linkage show remarkably high affinity withKi values of 0.01mM and 0.03 mM, respectively. The phosphorothionate inhibitor (#2)might be useful as a ligand to purify hCE-1 by affinity chromatogra-phy. Removal of theN-methyl group decreases affinity of cocaine,benzoylecgonine, and cocaethylene to the carboxylesterase since theKi values were substantially higher for the demethylated derivativesnorcocaine, benzoylnorecgonine, and norcocaethylene (fig. 1).

Many of the cocaine isomers, metabolites, and structurally relatedcompounds included in this study were previously used to investigatebinding to the cocaine receptor. A study by Carrollet al. (26) dem-onstrated the cocaine binding site at the dopamine transporter isstereoselective; the potency of natural (R)-(2)-cocaine was 60–670fold greater than that of its isomers. TheKi values reported for thetransporter with cocaine, cocaethylene, and benzoylecgonine propylester differed by less than 2-fold while a nearly 2000-fold differencein values was obtained between cocaine and benzoylecgonine (27).The two RTI compounds listed in table 1 displayed enhanced phar-macological activity relative to cocaine and were approximately 80-fold more potent in binding to the receptor protein than cocaine (28).Hence, both the receptor and hCE-1 exhibit similar stereoselectivityfor cocaine isomers but differing specificities for other analogs.

The Ki values for heroin and 6-acetylmorphine were determinedbecause hCE-1 is one of three enzymes that catalyzes the deacetyla-tion of heroin to form 6-acetylmorphine (7). AKi of 0.3 mM wasdetermined for both heroin and 6-acetylmorphine (table 2) suggestingthe ester group at the 3 position contributes very little to enzymebinding. By contrast, the ester group at the 6 position was crucial tobinding capability, since theKi value for morphine was.10 mM(table 2).

The concurrent use of alcohol and cocaine is a common practiceamong drug abusers. In the presence of ethanol, hCE-1 catalyzes theethyl transesterification of cocaine to form the active metabolitecocaethylene. TheKM for cocaine was 1166 17 mM (2). TheKi forcocaethylene was 10-fold higher than the value obtained for cocaine.The lower affinity of cocaethylene for hCE-1 is consistent with thelonger elimination half-life reported for cocaethylene, which is 24%longer than that of cocaine in humans (29). Since cocaethylene ismore potent than cocaine in mediating lethality in mice (30, 31), thecombined use of cocaine and ethanol may present a greater health riskthan cocaine alone.

Carboxylesterases play an important role in cocaine eliminationsince benzoylecgonine and ecgonine methyl ester (fig. 1) are themajor metabolites that eventually appear in urine (5). Studies indicatethat a minor pathway involving cytochrome P450 is largely respon-sible for cocaine-induced hepatotoxicity (32). This route involves theN-demethylation of cocaine to norcocaine (fig. 1), which then under-goes subsequent oxidation reactions in the endoplasmic reticulum.The presence of ethanol or other drugs that disrupt the hCE-1-mediated hydrolysis of cocaine may alter the formation of the cyto-toxic metabolites of cocaine. The identification of combinations ofdrugs that can alter cocaine metabolism is of considerable interest.

FIG. 4. Dixon plot of 6-acetylmorphine data.

A representative Dixon plot generated with data from a single experiment isshown. In the enzyme assay, the rate of 4-methylumbelliferone formation wasmonitored in the presence of varying substrate and inhibitor concentrations, asdescribed inMethods.Symbols correspond to 1/observed rates and linescorrespond to 1/predicted rates. The predicted rates are from the nonlinearregression fit of data to the equation for competitive inhibition. At the point ofintersection, the y coordinate is equivalent to 1/Vmax and the absolute value ofthe x coordinate is equivalent toKi.


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The competitive, spectrophotometric assay used in this study repre-sents a fast and effective method for evaluating the binding affinitiesof a series of compounds to the enzyme.

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