Interaction of the electrophilic ketoprofenyl-glucuronide ...dmd.aspetjournals.org/content/dmd/early/2007/10/25/dmd.107.016808.full.pdf · Carboxylic acid-containing drugs are metabolized

DMD#16808

Interaction of the electrophilic ketoprofenyl-glucuronide and ketoprofenyl-

coenzyme A conjugates with cytosolic glutathione S-transferases

Sandra Osbild, Jérome Bour, Benoît Maunit, Cécile Guillaume, Carine Asensio, Jean-

François Muller, Patrick Netter, Gilbert Kirsch, Denyse Bagrel, Françoise Lapicque and

Eric Battaglia

Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, Institut

Jean Barriol FR CNRS 2843, Université Paul Verlaine-Metz, 57070 Metz,

France (SO, GK, DB, EB) ;

Laboratoire de Spectrométrie de Masse et de Chimie Laser, Institut Jean

Barriol FR CNRS 2843, Université Paul Verlaine-Metz, 57078 Metz

Technopole, France (JB, BM, JFM);

UMR 7561 CNRS-UHP, Physiopathologie et Pharmacologie Articulaires,

Faculté de Médecine-BP 184, F-54505 Vandoeuvre les Nancy, France (CG,

CA, PN, FL)

DMD Fast Forward. Published on October 25, 2007 as doi:10.1124/dmd.107.016808

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on October 25, 2007 as DOI: 10.1124/dmd.107.016808

at ASPE

T Journals on February 3, 2020

dmd.aspetjournals.org

Dow

nloaded from

http://dmd.aspetjournals.org/

DMD#16808

2

Running title: interaction of ketoprofen acylated metabolites with GST

Address correspondence to:

Eric Battaglia, Ph.D.

LIMBP, Université Paul Verlaine-Metz, rue Général Delestraint

57070 Metz, France

Phone : (33)3 87 37 84 08

Email : [email protected]

Text pages (including references): 32

# of tables: 0

# of figures: 6

# of references: 42

# of words in abstract: 198

# of words in introduction: 596

# of words in discussion: 1306

Abbreviations used: COX, cyclooxygenases, GST, glutathione S-transferases;

GSH, glutathione reduced form; GSSG, glutathione disulfide; CDNB, 1-chloro-

2,4-dinitrobenzene; NQO, 4-nitroquinoline N-oxide; KPF, ketoprofen (2-(3-

benzoylphenyl) propionic acid); KPF-SCoA, ketoprofenyl-acyl-Coenzyme A;

KPF-OG, ketoprofenyl-acylglucuronide; KPF-SG, ketoprofenyl-S-acyl-

glutathione; 4MU sulfate, 4-methylumbelliferone sulfate; NSAIDs, nonsteroidal

anti-inflammatory drugs.


at ASPE



Dow

nloaded from


DMD#16808

3

Abstract

Carboxylic acid-containing drugs are metabolized mainly through the formation of

glucuronide and coenzyme A esters. These conjugates have been suspected to be

responsible for the toxicity of several nonsteroidal anti-inflammatory drugs because of the

reactivity of the electrophilic ester bond. We investigated in the present study the

reactivity of ketoprofenyl-acylglucuronide (KPF-OG) and ketoprofenyl-acyl-Coenzyme A

(KPF-SCoA) towards cytosolic rat liver glutathione S-transferases (GST). We observed

that KPF-ScoA, but not KPF-OG inhibited the conjugation of 1-chloro-2,4-dinitrobenzene

and 4-nitroquinoline N-oxide catalyzed by both purified cytosolic rat liver GST and GST

from FAO and H5-6 rat hepatoma cell lines. Photoaffinity labeling with KPF-SCoA

suggested that the binding of this metabolite may overlap the binding site of 4-

methylumbelliferone sulfate. Furthermore, high performance liquid chromatography and

mass spectrometry analysis showed that both hydrolysis and transacylation reactions

were observed in the presence of GST and glutathione. The formation of KPF-S-acyl-

glutathione could be kinetically characterized (apparent KM = 196.0 + 70.6 µM). It is

concluded that KPF-SCoA is both a GST inhibitor and a substrate of a GST-dependent

transacylation reaction. The reactivity and inhibitory potency of thioester CoA derivatives

towards GST may have potential implications on the reported in vivo toxicity of some

carboxylic acid-containing drugs.


at ASPE



Dow

nloaded from


DMD#16808

4

The development of pharmacovigilance tools over the years has led to the

detection of an increasing number of reports of adverse effects of drugs

(reviewed by (Lee, 2003)). Among those, the proportion of carboxylic acid

drugs withdrawn from the market is strikingly elevated. Carboxylic acid-

containing non steroidal anti-inflammatory drugs (NSAIDs) are widely used in

medicine due to their analgesic, antipyretic and anti-inflammatory activities.

Most frequently, the adverse drug reactions resulted in injury to the liver, which

is central for the metabolism of xenobiotics. Consequently, It has been

proposed that protein-drug adducts resulting from the metabolism of some of

these carboxylic acid substances may be associated to the incidence of

adverse drug reactions occasionally observed (Boelsterli, 2002). 2-Aryl

propionic acid-containing NSAIDs are metabolized by the phase II detoxification

enzymes, UDP-glucuronosyltransferases. Although this mechanism is often

considered as a detoxifying system, a few examples of bioactivation through

glucuronidation have been described in the case of carboxylic acid-containing

drugs leading to the formation of electrophilic acylglucuronides (Ritter, 2000). In

addition to glucuronidation, xenobiotics bearing a carboxylic acid group can

undergo a conjugation reaction to coenzyme A, catalyzed by both mitochondrial

and microsomal acyl-CoA synthetases, and resulting in the formation of acyl-

CoA thioesters of the corresponding compounds. This is for instance the case

of the 2-arylpropionic acid drug Ketoprofen (KPF), which is both glucuronidated

(Sabolovic et al., 2004; Sakaguchi et al., 2004) and thio-esterified with CoA


at ASPE



Dow

nloaded from


DMD#16808

5

(Carabaza et al., 1996). The latter metabolite appears to be an obligatory

intermediate in the epimerization of chiral profen drugs, such as KPF, as well

as in the formation of glycine, taurine and carnitine conjugates (Olsen et al.,

2005). KPF, among other drugs, has been used as a model compound to study

the possible reactivity of acyl metabolites with cellular proteins. Thus, it was

previously observed that both of these KPF acyl derivatives are chemically

reactive species which can trigger the formation of adducts with various

proteins such as albumin (Presle et al., 1996), UDP-glucuronosyltransferases

(Terrier et al., 1999), the cyclooxygenase COX-2 (Levoin et al., 2004) and

glucose-6-phosphate dehydrogenase (Asensio et al., 2007).

We have used in the present study the main metabolites of KPF, namely

ketoprofenyl-acylglucuronides (KPF-OG) and ketoprofenyl-acyl-coenzyme A

(KPF-SCoA) as acyl metabolites model compounds from carboxylic acid-

containing NSAIDs (Fig.1), to gain further insights into the reactivity of these

electrophilic metabolites. A study of the interaction of KPF-OG and KPF-SCoA

with cytosolic GST was here undertaken. The choice of these enzymes as

putative target for these metabolites was dictated by previous reports showing

that (1) various electrophilic esters can be substrates for GST (Hayes et al.,

2005), including acyl glucuronides (Shore et al., 1995) and acyl CoA derivatives

(Grillo and Benet, 2002), (2) fatty acyl coA are potent inhibitors of recombinant

GST (Silva et al., 1999), and (3) several CoA esters of xenobiotics can react

with reduced glutathione (GSH), the cosubstrate of GST, leading to the

corresponding thioester derivatives (references therein). The inhibition potency


at ASPE



Dow

nloaded from


DMD#16808

6

of KPF-OG and KPF-SCoA towards cytosolic rat liver GST was evaluated and

the reactivity of KPF-SCoA in the presence of these enzymes was elucidated

and kinetically characterized in the present work. These effects were evaluated

on the conjugation of GSH to both CDNB which is catalyzed by all cytosolic

GST except GST Theta and therefore is considered as a diagnostic GST

substrate, and NQO which has been reported to be an efficient substrate of

GST Mu and Pi (Aceto et al., 1990). Taking advantage of the photoactivatable

properties of the benzophenone moiety, the two metabolites of KPF were used

as photoaffinity labels to study their interaction with GST.


at ASPE



Dow

nloaded from


DMD#16808

7

Materials and Methods

Materials. GSH was purchased from Acros (Noisy-le-Grand, France). Purified

GST from rat liver, (RS)-Ketoprofen [R,S-2-(3-benzoylphenyl) propionic acid],

S-Ketoprofen, and all others chemicals were purchased from Sigma Aldrich (St

Quentin Fallavier, France) and were of the highest available degree of purity.

Cell culture reagents were from Eurobio (Courtaboeuf, France). KPF-OG was

prepared using a previously published enzymatic method (Terrier et al., 1999).

The chemical synthesis of KPF-SCoA has been previously described (Levoin et

al., 2002). Synthesis of ketoprofenyl-S-acyl-glutathione was performed as

described below. The rat hepatoma Fao and H5-6 cell lines have been cloned

from the H4IIEC3 cell line (Pitot et al., 1964) derived from Reuber H35

hepatoma cells (Reuber, 1961).

Instrumentation. HPLC was performed on a Waters system (Milford, MA)

equipped with a Waters 510 pump and a Waters 996 photodiode array detector

at 254 nm. MALDI-TOF experiments were performed in positive ion mode and

Post Source Decay (PSD) with a Bruker Reflex IV instrument (Bruker-Franzen

Analytik GmbH, Bremen, Germany). For the MALDI-TOF MS sample

preparation, 1 µL of 2,5-dihydroxybenzoic acid matrix solution (0.1 M in equal

volumes of water/acetonitrile) was added to 1 µL of the sample, placed on the

sample plate and allowed to dry in an air stream. Ionization was achieved by

using a nitrogen laser (λ = 337 nm, pulse duration = 4 ns, output energy = 400

µJ, repetition rate = 5 Hz). A reflectron mode with a total acceleration voltage of


at ASPE



Dow

nloaded from


DMD#16808

8

20 kV and an extraction delay time of 200 ns was used for the mass

spectrometry analysis. The ion assignment was attained after external

calibration performed with polyethylene glycol 400 Na+ and K+ cationized ions.

Chemical synthesis of ketoprofenyl-S-acyl-glutathione. Ketoprofenyl-S-

acyl-glutathione (KPF-SG) was chemically synthesized using a method

modified from the synthesis of acyl-CoA-ketoprofen conjugates (Levoin et al.,

2002), by substituting GSH for CoASH. To a solution of KPF (61 mg, 0.24

mmol) in 5.5 mL anhydrous dichloromethane was added a solution of 2,6

lutidine (28 µL, 0.24 mmol) and ethyl chloroformate (23 µL, 0.24 µmol in 1.85

mL anydrous dichloromethane). The mixture was stirred for 1 hour at room

temperature under nitrogen atmosphere. The formation of the activated KPF

reaction intermediate was monitored by HPLC (retention time 6.4 min) as

described (Levoin et al., 2002). When the reaction was completed, the mixture

was concentrated to dryness and solubilized in tetrahydrofuran (4.5 mL). GSH

(46.2 mg, 0.15 mmole in 4.5 mL distilled water) was then added, the pH

adjusted to 6.2 with NaOH (5N), and the mixture stirred at room temperature for

2 hours under nitrogen atmosphere. The formation of KPF-SG was followed by

HPLC as described below. Finally, tetrahydrofuran was evaporated and the

aqueous layer extracted with hexane to remove unreacted products and

concentrated. KPF-SG from the aqueous phase was purified by semi-

preparative HPLC as described below. The residue was characterized by

MALDI TOF positive ion mass spectrum (see Fig. 5).


at ASPE



Dow

nloaded from


DMD#16808

9

Inhibition of purified rat liver GST activities. GST activities were determined

using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, essentially as

described by (Habig et al., 1974). The enzymatic activity of purified rat liver

cytosolic GST (1.23 µg proteins/mL) was assayed at 340 nm (Uvikon 941

spectrophotometer, Kontron Instruments, SECOMAM, Alès, France) in 0.1 M

sodium phosphate buffer (pH 6.5) at 30°C for 4 min using GSH (3 mM in 0.1 M

sodium phosphate buffer, pH 6.5) and CDNB (4 mM in DMSO). When 4-

nitroquinoline N-oxide (4NQO, 0.1 mM in dimethylsulfoxide) was used as the

acceptor substrate, the assay was performed as described by (Stanley and

Benson, 1988), except that GSH concentration was held at 2 mM (0.1 M

sodium phosphate buffer, pH 6.5). For inhibition experiments, the tested

substances (0.1 mM) were dissolved in milliQ® water (CoASH, KPF-OG and

KPF-SCoA) or in dimethylsulfoxide (KPF, 4-methylumbelliferone-sulfate) and

included in the GST assays. Solvents were added at 2% (v/v) in the assays and

did not affect enzyme activities (results not shown). The residual activity (%) in

the presence of the inhibitors was calculated, with control (2% solvent)

representing 100% activity.

Inhibition of GST in cell homogenates. The rat hepatoma cell lines FAO and

H5-6 were grown to subconfluency in DMEM medium supplemented with 10%

(v/v) heat-inactivated fetal bovine serum, 100 UI/mL penicillin G, 100 µg/mL

streptomycin and 0.25 µg/mL amphotericin B. These cells were cultured in 250

mL-Falcon® flasks (BD Biosciences, Bedford, MA) at 37°C in a humidified

atmosphere containing 5% CO2. Cells were scraped in Phosphate Buffered


at ASPE



Dow

nloaded from


DMD#16808

10

Saline and centrifuged (4°C, 500 g) for 5 min. The pellet was resuspended in

0.1 M ice-cold sodium phosphate buffer (pH 7.0), sonicated twice for 10

seconds (VibraCell, Fisher Bioblock Scientific, Illkirch, France) and centrifuged

(4°C , 2500 g) for 15 min. The supernatant was kept at –80°C until GST assay.

The inhibitory potency of KPF, KPF-SCoA and KPF-OG (0.1 mM) on GST

activities (CDNB and 4NQO) in the FAO and H5-6 cell homogenates was

evaluated as described for purified rat liver GST, but with a final protein

concentration of 0.153 mg/mL and 0.468 mg/mL (for initial rate conditions)

respectively, in the assay.

Photoaffinity labeling of purified rat liver GST with ketoprofenyl-CoA.

Purified cytosolic rat liver GST (0.16 mg/mL) were placed into 1.5 mL-

microtubes and incubated for 0-2 hours at 4°C under a 365 nm-UV lamp

(Spectroline, Polylabo, Strasbourg, France) in 0.1 M sodium phosphate buffer

(pH 7.4) and in the presence of KPF-SCoA (0.5 mM). UV-irradiated samples

were withdrawn at different irradiation times (0-2 h) and diluted 100 times in 0.1

M sodium phosphate buffer (pH 6.5) prior to determination of GST activity using

CDNB as acceptor substrate, as described above. The effect of UV irradiation

alone was evaluated by performing in parallel the experiment in the absence of

KPF-SCoA. The residual activity (%) was calculated from the enzyme activity in

the non-irradiated assay at t0 representing 100 % residual activity. GST activity

protection against photoinactivation by KPF-SCoA (0.25 mM) was studied in

the presence of GSH (4 mM in 0.1 M sodium phosphate buffer, pH 6.5), CDNB

(4 mM), 4MU sulfate (2 mM), estrone 3-sulfate (2 mM), and the corresponding


at ASPE



Dow

nloaded from


DMD#16808

11

solvent alone (1% v/v), using the experimental procedures described above

and an irradiation time of 2h.

Protein Assays. Protein concentrations of cell homogenates and purified GST

were determined according to (Lowry et al., 1951), using bovine serum albumin

as standard.

HPLC analysis. Reaction products resulting from the incubation of GST and/or

GSH with KPF-SCoA were analyzed by reverse-phase HPLC with UV detection

at 254 nm. 10 µL of samples were injected in a Lichrospher RP-18 column

(18.5 µm, 125 x 4 mm, Merck KGaA, Darmstadt, Germany). The mobile phase

consisted of 45 % of 9 mM sodium phosphate buffer (pH 5.5) and 55 %

methanol and isocratic elution was carried out at a flow rate of 1.5 mL/min.

Reactivity of KPF-SCoA with GSH in the presence of GST. GSH (3 mM in

0.1 M sodium phosphate buffer, pH 6.5) was incubated at 30°C in 0.1 M

sodium phosphate buffer (pH 6.5) in presence of KPF-SCoA (0.5 mM) and with

or without GST (2.34 mg proteins/mL in 0.1 M sodium phosphate buffer, pH

6.5). A sample (23.44 µg proteins) was withdrawn from the mixture as a

function of time (0-4 h) and was mixed to four volumes of methanol to stop the

reaction and precipitate the proteins. After centrifugation (5 minutes, 12000g,

4°C) supernatants were stored at -80°C until HPLC analysis. Controls were

also run in parallel under the same conditions but with KPF-SCoA or CoASH

alone (0.5 mM), GSSG (3 mM) in place of GSH, and heat-denaturated GST,

respectively.


at ASPE



Dow

nloaded from


DMD#16808

12

The KPF-SCoA-concentration dependence of the reaction catalyzed by GST

was studied by incubating GST (2.34 mg proteins/mL) and GSH (3 mM) with

KPF-SCoA (0.01 mM to 1 mM) in 0.1 M sodium phosphate buffer (pH 6.5) at

30°C during 45 minutes. The reaction mixtures were then treated and analyzed

by reverse-phase HPLC as described above. Controls without GST were run in

parallel to account for the uncatalyzed reaction.

Quantification of ketoprofenyl-S-acyl-glutathione. KPF-SG produced in the

presence of GST was quantified as follows. KPF-SG fractions eluted from the

HPLC column were collected and fully hydrolyzed in the presence of an equal

volume of NaOH (0.5 N) for 2 h at 40°C. The amount of KPF-SG was deduced

from the amount of KPF released, the latter being quantified using a KPF

standard curve and HPLC separation.

Statistical Analysis. Values are expressed as the mean in residual activities ±

standard deviations (S.D.) of at least two experiments performed, at least, in

triplicates.


at ASPE



Dow

nloaded from


DMD#16808

13

Results

Inhibition of GST by ketoprofenyl-SCoA and ketoprofenyl-glucuronides.

The effect of the two main acyl metabolites of ketoprofen was first evaluated on

purified rat liver cytosolic GST to find out if GST are possible targets of these

compounds (Fig. 2 A). KPF is a chiral NSAID which has been reported to inhibit

GST (Sadzuka et al., 1994). It was tested in the present study either as

racemates (as administered) or as the S-enantiomer (the form with anti-

inflammatory properties due to the potent inhibition of COX). KPF was found to

more selectively inhibit the conjugation of 4-NQO, an efficient substrate of GST

Mu and Pi. S-KPF inhibition was similar to that observed with the KPF racemic

mixture (results not shown). The inhibitory potency of KPF was lost once

conjugated to glucuronic acid (Fig. 2A). If CoASH alone did not significantly

affect the GST activity, KPF-SCoA could inhibit these enzymes (Fig. 2A).

Likewise, cytosolic GST activity from the FAO and H5-6 rat hepatocarcinoma

cell lines were significantly inhibited by KPF-ScoA, but not by the

acylglucuronide (Fig. 2B).

We next characterized the mode of inhibition of GST by KPF-SCoA. A

Lineweaver-Burk representation could not be performed because of the

relatively low amount of this metabolite available for this study. Therefore,

another approach was undertaken, using KPF-SCoA as a photoaffinity probe

combined with ligand protection experiments. It was indeed previously reported

that KPF can bind to proteins upon UV-irradiation through the benzophenone


at ASPE



Dow

nloaded from


DMD#16808

14

moiety (Bosca et al., 1994; Chuang et al., 1999). We observed in preliminary

experiments that KPF could photolabel GST (unpublished observation).

Furthermore, irradiation of purified rat liver GST in the presence of 0.5 mM

KPF-SCoA led to a time-dependent inactivation, reaching 72 % inactivation

after two hours (Fig. 3A). Conversely, no inhibition of the conjugation reaction

could be detected in the absence of irradiation in these experimental

conditions. No significant protection was observed when the labeling was

carried out in the presence of either GSH, CDNB (Fig. 3B) or 2 mM estrone 3-

sulfate (not shown), while preincubation of the enzymes with 2 mM 4MU sulfate

prior to labeling with 0.25 mM KPF-SCoA brought near full protection against

photoinactivation. KPF-OG photolabeling of GST under similar conditions did

not trigger any significant enzyme inactivation (data not shown). This suggests

that KPF-SCoA binds within the 4MU sulfate binding site.

KPF-SCoA reactivity in the presence of GST. Since KPF-SCoA (but not

KPF-OG) exerts an inhibitory effect towards GST, we next determined the

possible reactivity of the former metabolite with GSH and evaluated the

possibility of a reaction mediated by these enzymes. A representative reverse

phase HPLC chromatogram obtained after incubation of KPF-SCoA with GSH

at pH 6.5 in the presence purified rat liver GST is shown in Fig. 4. Several

species with different retention times were detected at 254 nm. The retention

times of ~2.8 min and 3.3 min matched those of authentic KPF-SCoA R- and S-

enantiomers and therefore were considered as unreacted metabolites. This

assignment was confirmed by mass spectrometric analysis with MALDI TOF


at ASPE



Dow

nloaded from


DMD#16808

15

positive ion mass spectrum in accordance with the structures of the acyl CoA

metabolites (data not shown). The compound eluted at ~5.4 min comigrated

with authentic KPF and the detection of the protonated molecular ion MH+ m/z

255.33 is consistent with the structure of KPF (data not shown). The structure

of the compound eluted at ~4.7 min could not be proposed on the basis of the

retention time from the HPLC analysis and was therefore further characterized

by mass spectrometry.

MS identification of 2-(3-benzoylphenyl) propionic acid-glutathione. A

transacylation product resulting from the reaction between GSH and KPF-

SCoA was postulated on the basis of the electrophilic properties of the thioester

bound, as well as previous reports on similar metabolites (Shore et al., 1995;

Grillo and Benet, 2002; Li et al., 2002; Olsen et al., 2002; Grillo et al., 2003; Li

et al., 2003a). Mass spectrometric analysis was performed to allow the

identification of the reaction products separated by reversed-phase HPLC.

Furthermore, authentic 2-(3-benzoylphenyl) propionic acid-glutathione (KPF-

SG) resulting from such postulated transacylation was chemically synthesized

by coupling ketoprofen to GSH using the mixed anhydride method (Levoin et

al., 2004) and compared to the enzymatic reaction products resulting from the

incubation of GST with KPF-SCoA and GSH. MALDI-TOF mass spectrometry

analysis of KPF-SG obtained from chemical and enzymatic synthesis are

shown in Fig. 5. A characteristic protonated molecular ion MH+ m/z 544 and the

potassium adduct ion m/z 582 was detected for the authentic KPF-SG resulting

from the chemical synthesis while the potassium adduct ion m/z 582 was not


at ASPE



Dow

nloaded from


DMD#16808

16

found in the enzymatic reaction products (Fig. 5A and 5B). The lack of the

potassium adduct may be due to the weak concentration of potassium in the

analyzed solution. Likewise, analysis by Post Source Decay (PSD) technique

provided a mass spectrum of the product KPF-SG obtained by chemical

method identical to that acquired during the mass spectrometric analysis of the

product obtained by the enzymatic method. The PSD spectrum of KPF-SG MH+

ion m/z 544 showed characteristic fragment ions m/z 500, 469, 415, 308, 274

and 237 (Fig. 5C) originating from the proposed cleavages depicted in this

Figure.

Kinetic studies of the hydrolytic and transacylation reactions catalyzed by

GST. We characterized the GST-mediated transacylation and KPF-SCoA

hydrolysis by evaluating the time dependence and saturation pattern of these

reactions. The co-incubation of KPF-SCoA and GSH resulted in a significant

uncatalyzed reaction which varied from 3.5 % to 13.9 % of the reaction

observed in the presence of GST as a function of the incubation time (Fig. 6A).

Therefore, the sole contribution of the enzyme-catalyzed reaction was

evaluated by deducing from the amount of the total reaction products that

measured in the absence of enzyme (Fig. 6A). Incubation of KPF-SCoA with

GSH and GST resulted in a time-dependent formation of KPF, the later being

linear for up to 4h (Fig. 6A), defining within these experimental conditions the

time frame of initial rate for the hydrolytic reaction. Moreover, the kinetics of

KPF-SG formation was linear for up to 1 h. An incubation time of 45 min was

accordingly chosen to evaluate the substrate saturation pattern of the


at ASPE



Dow

nloaded from


DMD#16808

17

enzymatic reactions (hydrolysis and transacylation). A saturation curve was

observed for KPF-SCoA concentrations ranging from 0.01 mM to 1mM (Fig.

6B). A double reciprocal plot allowed the determination of an apparent KM

towards KPF-SCoA of 196.0 + 70.6 µM for the transacylation reaction (Fig. 6C).

On the other hand, the hydrolysis reaction resulting in the formation of KPF and

CoASH from KPF-SCoA was too slow to allow the determination of kinetic

parameters for this reaction (data not shown). Since proteins contain multiple

nucleophilic amino acid residues, such as cysteinyl residues which could

account for the observed reactivity of KPF-SCoA (independently of the enzyme

integrity), we evaluated the hydrolytic and transacylation reactions after thermal

denaturation of the purified GST. The products were detected by HPLC at

levels similar to that found with the uncatalyzed reaction (data not shown).

Furthermore, the free thiol moiety of GSH appears to be essential for the

reaction since no products could be detected when glutathione disufide was

substituted for GSH (data not shown).


at ASPE



Dow

nloaded from


DMD#16808

18

Discussion

A number of carboxylic acid drugs have been withdrawn from the market after

observation of unacceptable levels of hepatotoxicity and these idiosyncratic

drug reactions have been hypothesized to be associated to the formation of

chemically reactive substances. Thus, various acyl glucuronides have been

shown to be electrophilic metabolites which can spontaneously react with

proteins both in vitro and in vivo leading to adduct formation (Sallustio et al.,

2000). This reactivity is not limited to proteins since electrophilic acyl

glucuronide conjugates have been reported to react with the tripeptide

glutathione, the cosubstrate for conjugation reaction carried out by GST (Shore

et al., 1995; Grillo and Hua, 2003). A second bioactivation pathway involves the

formation of electrophilic acyl CoA derivatives by acyl-CoA synthetases. Acyl

CoA derivatives of xenobiotics can react with proteins and GSH, resulting in

adduct formation through nucleophilic acyl substitution (Sidenius et al., 2004).

KPF has been previously reported to inhibit GST (Sadzuka et al., 1994). In the

present study, we characterized the inhibitory potency and reactivity towards

GST of the two main acylated metabolites of KPF, namely KPF-OG and KPF-

SCoA (Fig. 1). While KPF-OG did not affect GST activity, we observed that

KPF-SCoA was able to inhibit both CDNB and NQO conjugation to GSH

catalyzed by purified rat liver GST (Fig. 2). A similar inhibitory pattern was

found in homogenates from H5-6 and FAO rat hepatocarcinoma cells and the

inhibition potency of KPF-SCoA was similar to the one observed with KPF (Fig.


at ASPE



Dow

nloaded from


DMD#16808

19

2). The lower inhibitory potency observed in the presence of cytosolic extracts

compared to purified GST is likely to be due to the buffering effect of additional

proteins interacting with the two acyl metabolites of KPF. Various proteins such

as albumin (Li et al., 2003b), sulfotransferases (Tulik et al., 2002), COX (Levoin

et al., 2004), hepatocyte nuclear factor-4α (Hertz et al., 2001) and glucose-6-

phosphate dehydrogenase (Asensio et al., 2007) have indeed been reported to

interact with CoA thioester derivatives. It was previously found that long-chain

saturated fatty acyl-CoAs and peroxisome proliferator–CoA thioesters exert

high affinity towards GST (Silva et al., 1999). Our results provide another

example of carboxylic acid–containing compound able to bind to GST. We

observed that this binding resulted in the inhibition of the conjugation activity of

these enzymes.

Taking advantage of the ability of KPF to bind covalently to its protein targets

upon UV irradiation (Chuang et al., 1999), KPF-SCoA was used as a

photoaffinity label to partially characterize the GST binding site of this

metabolite (Fig. 3). An UV-dependent GST inactivation by KPF-SCoA was

detected as well as a major protection in the presence of 4MU sulfate, while

GSH, CDNB and estrone 3-sulfate did not exert any protective effect.

Sulfoconjugates of steroids have been reported to bind in a so-called “ligandin”

or “nonsubstrate” site in the alpha-class rat liver GST isozyme 1-1 (Barycki and

Colman, 1997). Conversely, aromatic sulfoconjugates such as benzyl sulfate

are GST substrates in the presence of GSH (Gillham, 1971). Interestingly,

estrone 3-sulfate was also tested in this same work and was found not to be a


at ASPE



Dow

nloaded from


DMD#16808

20

GST substrate. Therefore, our experiment using KPF-SCoA as photolabel

suggests that it may bind within the electrophilic substrate site (H-site). This is

further supported by the fact that KPF-SCoA behaves as a GST substrate as

shown by the detection of a GST-dependent transacylation reaction with GSH

(Fig. 5 and 6). The observation that the diagnostic GST substrate CDNB does

not protect against inactivation even though KPF-SCoA is itself a GST

substrate can be explained in view of some previous works. For instance, it has

been reported that ethacrynic acid, both a GST inhibitor and a GST substrate,

can bind to human GST P1-1 in a nonproductive and productive mode, which

suggests the existence of multiple binding sites and binding modes for GST

ligands (Oakley et al., 1997). KPF-SCoA binding mode and binding

stoechiometry may thus be distinct from that of CDNB.

The reactivity of KPF-SCoA as potential GST substrate has been evaluated.

The chemical reactivity of the electrophilic thioester bound for a variety of

xenobiotic-SCoA metabolites towards the sulfhydryl moiety of cysteine in both

low molecular weight GSH and proteins has been indeed previously reported

(Shore et al., 1995; Grillo and Benet, 2002; Li et al., 2002; Olsen et al., 2002;

Grillo et al., 2003; Li et al., 2003a). Furthermore, xenobiotic–SCoA were found

to be much more reactive towards GSH compared to acyl glucuronides (Olsen

et al., 2002). While (Silva et al., 1999) highlighted the absence of metabolism

and hydrolysis of the thioester derivatives, interestingly, a transacylation

reaction with GSH, enhanced in the presence of GST, was detected in the

present study. MALDI/TOF MS analysis of KPF-SG obtained by enzymatic


at ASPE



Dow

nloaded from


DMD#16808

21

synthesis led to the identification of the expected ions MH+ m/z 544 (Fig. 5B) in

accordance with the proposed structure. This profile was similar to that

obtained with KPF-SG chemically synthesized (Fig. 5A). Hence we concluded

that KPF-CoA was converted to KPF-SG in the presence of GST.

Using the hypolipidemic drug clofibric acid, Grillo and Benet (2002) have

reported that rat liver GST increase the rate of clofibryl-S-acyl-glutathione

formation from a mixture of clofibryl-S-acyl-CoA and GSH, but the kinetic

parameters of the catalytic reaction were not determined. In the present study,

an apparent KM = 196.0 + 70.6 µM was determined for the transacylation

reaction. This value is similar to those of CDNB-conjugating enzymes. GST

catalyze miscellaneous GSH–dependent reactions such as nucleophilic

aromatic substitution, Michael-type addition, double bond izomerisation,

hydroperoxide reduction (reviewed by (Mahajan and Atkins, 2005)). In addition

to the transacylation reaction, the hydrolysis of KPF-SCoA enhanced in the

presence of GST was detected, but this hydrolysis occurred at a lower rate

(Fig. 6A). A relation between the rate of spontaneous transacylation and

hydrolysis of CoA thioesters has been reported (Sidenius et al., 2004). The

amounts of reaction products (hydrolysis and transacylation) was reduced to

those observed in the absence of enzyme when GST were heat-inactivated

(results not shown), demonstrating that native enzymes contribute to these

reactions. The hydrolytic reaction of KPF-SCoA did not result in a saturation

pattern, which therefore precluded the determination of the kinetic parameters

for this reaction. Hydrolysis of xenobiotic-CoA metabolites has been previously


at ASPE



Dow

nloaded from


DMD#16808

22

reported but this reaction was not studied in the presence of enzymes (Li et al.,

2003a; Sidenius et al., 2004). Furthermore, the hydrolytic activity of GST on

GSH esters (reverse reaction of GSH conjugation) has been demonstrated

(Dietze et al., 1998; Ibarra et al., 2003). It is not established, to the best of our

knowledge, whether carboxylic acid-SCoA conjugates are directly hydrolyzed

by GST or are first transacylated by GSH prior to hydrolysis by GST (reverse

conjugation).

Glutathione thioesters of carboxylic acid containing drugs may be excreted

unchanged in the bile (Shore et al., 1995) or degraded prior to excretion in

urine. Thus, clofibryl-S-acyl-GSH has been shown to be further metabolized in

vivo and in vitro to S- to N-acyl-cysteine amide derivatives with the contribution

of γ-glutamyltransferase (Grillo and Benet, 2001). More recently, the

nonsteroidal anti-inflammatory carboxyl drug, zomepirac was found to be

bioactivated to its acyl CoA metabolite, an obligatory intermediate to the in vivo

formation of glycine, taurine and carnitine conjugates (Olsen et al., 2005). A γ-

glutamyl transferase-mediated degradation of clofibryl-S-acyl-GSH resulting in

the formation of S- and N-acyl-cysteine amide derivatives were detected in rat

both in vitro and in vivo (Grillo and Benet, 2001). Whether xenobiotic acyl-GSH

conjugates and their subsequent metabolites may be considered as a

detoxification products remains to be established.

In conclusion, we report for the first time that, unlike KPF-OG, KPF-SCoA is an

inhibitor of both purified and cell homogenate GST. KPF-SCoA is also

transacylated in the presence of GSH and hydrolyzed through reactions


at ASPE



Dow

nloaded from


DMD#16808

23

mediated by purified rat liver GST. The physiotoxicological consequences of

GST inhibition, transacylation and hydrolysis reactions in the presence of CoA

thioesters will deserve future investigations.


at ASPE



Dow

nloaded from


DMD#16808

24

Acknowledgment

We thank Nicolas Levoin (CNRS UMR 7561) for his help during the initiation of

the project and Jacques Magdalou (CNRS UMR 7561) for helpful discussion on

the manuscript. We also wish to thank a reviewer for his suggestion regarding

the binding of KPF-SCoA to GST.


at ASPE



Dow

nloaded from


DMD#16808

25

References Aceto A, Di Ilio C, Lo Bello M, Bucciarelli T, Angelucci S and Federici G (1990)

Differential activity of human, rat, mouse and bacteria glutathione transferase isoenzymes towards 4-nitroquinoline 1-oxide. Carcinogenesis 11:2267-2269.

Asensio C, Levoin N, Guillaume C, Guerquin MJ, Rouguieg K, Chretien F, Chapleur Y, Netter P, Minn A and Lapicque F (2007) Irreversible inhibition of glucose-6-phosphate dehydrogenase by the coenzyme A conjugate of ketoprofen: a key to oxidative stress induced by non-steroidal anti-inflammatory drugs? Biochem Pharmacol 73:405-416.

Barycki JJ and Colman RF (1997) Identification of the nonsubstrate steroid binding site of rat liver glutathione S-transferase, isozyme 1-1, by the steroid affinity label, 3beta-(iodoacetoxy)dehydroisoandrosterone. Arch Biochem Biophys 345:16-31.

Boelsterli UA (2002) Xenobiotic acyl glucuronides and acyl CoA thioesters as protein-reactive metabolites with the potential to cause idiosyncratic drug reactions. Curr Drug Metab 3:439-450.

Bosca F, Miranda MA, Carganico G and Mauleon D (1994) Photochemical and photobiological properties of ketoprofen associated with the benzophenone chromophore. Photochem Photobiol 60:96-101.

Carabaza A, Suesa N, Tost D, Pascual J, Gomez M, Gutierrez M, Ortega E, Montserrat X, Garcia AM, Mis R, Cabre F, Mauleon D and Carganico G (1996) Stereoselective metabolic pathways of ketoprofen in the rat: incorporation into triacylglycerols and enantiomeric inversion. Chirality 8:163-172.

Chuang VT, Kuniyasu A, Nakayama H, Matsushita Y, Hirono S and Otagiri M (1999) Helix 6 of subdomain III A of human serum albumin is the region primarily photolabeled by ketoprofen, an arylpropionic acid NSAID containing a benzophenone moiety. Biochim Biophys Acta 1434:18-30.

Dietze EC, Grillo MP, Kalhorn T, Nieslanik BS, Jochheim CM and Atkins WM (1998) Thiol ester hydrolysis catalyzed by glutathione S-transferase A1-1. Biochemistry 37:14948-14957.

Gillham B (1971) The reaction of aralkyl sulphate esters with glutathione catalysed by rat liver preparations. Biochem J 121:667-672.

Grillo MP and Benet LZ (2001) Interaction of gamma-glutamyltranspeptidase with clofibryl-S-acyl-glutathione in vitro and in vivo in rat. Chem Res Toxicol 14:1033-1040.

Grillo MP and Benet LZ (2002) Studies on the reactivity of clofibryl-S-acyl-CoA thioester with glutathione in vitro. Drug Metab Dispos 30:55-62.

Grillo MP and Hua F (2003) Identification of zomepirac-S-acyl-glutathione in vitro in incubations with rat hepatocytes and in vivo in rat bile. Drug Metab Dispos 31:1429-1436.

Grillo MP, Knutson CG, Sanders PE, Waldon DJ, Hua F and Ware JA (2003) Studies on the chemical reactivity of diclofenac acyl glucuronide with


at ASPE



Dow

nloaded from


DMD#16808

26

glutathione: identification of diclofenac-S-acyl-glutathione in rat bile. Drug Metab Dispos 31:1327-1336.

Habig WH, Pabst MJ and Jakoby WB (1974) Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 249:7130-7139.

Hayes JD, Flanagan JU and Jowsey IR (2005) Glutathione transferases. Annu Rev Pharmacol Toxicol 45:51-88.

Hertz R, Sheena V, Kalderon B, Berman I and Bar-Tana J (2001) Suppression of hepatocyte nuclear factor-4alpha by acyl-CoA thioesters of hypolipidemic peroxisome proliferators. Biochem Pharmacol 61:1057-1062.

Ibarra C, Grillo MP, Lo Bello M, Nucettelli M, Bammler TK and Atkins WM (2003) Exploration of in vitro pro-drug activation and futile cycling by glutathione S-transferases: thiol ester hydrolysis and inhibitor maturation. Arch Biochem Biophys 414:303-311.

Lee WM (2003) Drug-induced hepatotoxicity. N Engl J Med 349:474-485. Levoin N, Blondeau C, Guillaume C, Grandcolas L, Chretien F, Jouzeau JY,

Benoit E, Chapleur Y, Netter P and Lapicque F (2004) Elucidation of the mechanism of inhibition of cyclooxygenases by acyl-coenzyme A and acylglucuronic conjugates of ketoprofen. Biochem Pharmacol 68:1957-1969.

Levoin N, Chretien F, Lapicque F and Chapleur Y (2002) Synthesis and biological testing of Acyl-CoA-ketoprofen conjugates as selective irreversible inhibitors of COX-2. Bioorg Med Chem 10:753-757.

Li C, Benet LZ and Grillo MP (2002) Studies on the chemical reactivity of 2-phenylpropionic acid 1-O-acyl glucuronide and S-acyl-CoA thioester metabolites. Chem Res Toxicol 15:1309-1317.

Li C, Grillo MP and Benet LZ (2003a) In vitro studies on the chemical reactivity of 2,4-dichlorophenoxyacetyl-S-acyl-CoA thioester. Toxicol Appl Pharmacol 187:101-109.

Li C, Olurinde MO, Hodges LM, Grillo MP and Benet LZ (2003b) Covalent binding of 2-phenylpropionyl-S-acyl-CoA thioester to tissue proteins in vitro. Drug Metab Dispos 31:727-730.

Lowry OH, Rosebrough NJ, Farr AL and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275.

Mahajan S and Atkins WM (2005) The chemistry and biology of inhibitors and pro-drugs targeted to glutathione S-transferases. Cell Mol Life Sci 62:1221-1233.

Oakley AJ, Rossjohn J, Lo Bello M, Caccuri AM, Federici G and Parker MW (1997) The three-dimensional structure of the human Pi class glutathione transferase P1-1 in complex with the inhibitor ethacrynic acid and its glutathione conjugate. Biochemistry 36:576-585.

Olsen J, Bjornsdottir I, Tjornelund J and Honore Hansen S (2002) Chemical reactivity of the naproxen acyl glucuronide and the naproxen coenzyme A thioester towards bionucleophiles. J Pharm Biomed Anal 29:7-15.


at ASPE



Dow

nloaded from


DMD#16808

27

Olsen J, Li C, Bjornsdottir I, Sidenius U, Hansen SH and Benet LZ (2005) In vitro and in vivo studies on acyl-coenzyme A-dependent bioactivation of zomepirac in rats. Chem Res Toxicol 18:1729-1736.

Pitot HC, Peraino C, Morse PA, Jr. and Potter VR (1964) Hepatomas in Tissue Culture Compared with Adapting Liver in Vivo. Natl Cancer Inst Monogr 13:229-245.

Presle N, Lapicque F, Fournel-Gigleux S, Magdalou J and Netter P (1996) Stereoselective irreversible binding of ketoprofen glucuronides to albumin. Characterization of the site and the mechanism. Drug Metab Dispos 24:1050-1057.

Reuber MD (1961) A transplantable bile-secreting hepatocellular carcinoma in the rat. J Natl Cancer Inst 26:891-899.

Ritter JK (2000) Roles of glucuronidation and UDP-glucuronosyltransferases in xenobiotic bioactivation reactions. Chem Biol Interact 129:171-193.

Sabolovic N, Heydel JM, Li X, Little JM, Humbert AC, Radominska-Pandya A and Magdalou J (2004) Carboxyl nonsteroidal anti-inflammatory drugs are efficiently glucuronidated by microsomes of the human gastrointestinal tract. Biochim Biophys Acta 1675:120-129.

Sadzuka Y, Shimizu Y, Takino Y and Hirota S (1994) Protection against cisplatin-induced nephrotoxicity in the rat by inducers and an inhibitor of glutathione S-transferase. Biochem Pharmacol 48:453-459.

Sakaguchi K, Green M, Stock N, Reger TS, Zunic J and King C (2004) Glucuronidation of carboxylic acid containing compounds by UDP-glucuronosyltransferase isoforms. Arch Biochem Biophys 424:219-225.

Sallustio BC, Sabordo L, Evans AM and Nation RL (2000) Hepatic disposition of electrophilic acyl glucuronide conjugates. Curr Drug Metab 1:163-180.

Shore LJ, Fenselau C, King AR and Dickinson RG (1995) Characterization and formation of the glutathione conjugate of clofibric acid. Drug Metab Dispos 23:119-123.

Sidenius U, Skonberg C, Olsen J and Hansen SH (2004) In vitro reactivity of carboxylic acid-CoA thioesters with glutathione. Chem Res Toxicol 17:75-81.

Silva C, Loyola G, Valenzuela R, Garcia-Huidobro T, Monasterio O and Bronfman M (1999) High-affinity binding of fatty acyl-CoAs and peroxisome proliferator-CoA esters to glutathione S-transferases effect on enzymatic activity. Eur J Biochem 266:143-150.

Stanley JS and Benson AM (1988) The conjugation of 4-nitroquinoline 1-oxide, a potent carcinogen, by mammalian glutathione transferases. 4-Nitroquinoline 1-oxide conjugation by human, rat and mouse liver cytosols, extrahepatic organs of mice and purified mouse glutathione transferase isoenzymes. Biochem J 256:303-306.

Terrier N, Benoit E, Senay C, Lapicque F, Radominska-Pandya A, Magdalou J and Fournel-Gigleux S (1999) Human and rat liver UDP-glucuronosyltransferases are targets of ketoprofen acylglucuronide. Mol Pharmacol 56:226-234.


at ASPE



Dow

nloaded from


DMD#16808

28

Tulik GR, Chodavarapu S, Edgar R, Giannunzio L, Langland A, Schultz B and Beckmann JD (2002) Inhibition of bovine phenol sulfotransferase (bSULT1A1) by CoA thioesters. Evidence for positive cooperativity and inhibition by interaction with both the nucleotide and phenol binding sites. J Biol Chem 277:39296-39303.


at ASPE



Dow

nloaded from


DMD#16808

29

Footnote This work was supported in part by Région de Lorraine. SO is a recipient of a

fellowship from Ministère de la Recherche et de l’Enseignement Supérieur

(France).

Reprint request: Eric Battaglia, PhD. Laboratoire d’Ingénierie Moléculaire et

Biochimie Pharmacologique, Institut Jean Barriol FR CNRS 2843, Université

Paul Verlaine-Metz, 57070 Metz, France ; Tel : (+33)387378408 ; Fax :

(+33)387378423 ; Email : [email protected].


at ASPE



Dow

nloaded from


DMD#16808

30

Legends for figures

Figure 1. Structure of Ketoprofen, Ketoprofen-acylglucuronide and

Ketoprofen-acylCoenzyme A. * Chiral center.

Figure 2. Inhibitory effect of KPF and its acyl metabolites towards GST

activities of purified enzyme and rat hepatoma cell homogenates. A. The

inhibition of the activity of purified rat liver cytosolic GST was evaluated in the

presence of the indicated compounds (0.1 mM) using either CDNB (■) or

4NQO (■) as substrate, under initial rate conditions. B. The inhibitory effect of

KPF, KPF-SCoA and KPF-OG (0.1 mM) towards GST was evaluated using

4NQO as substrate on FAO (□) and H5.6 (■) rat hepatoma cells

homogenates. Residual activity (%) was expressed as the ratio between the

activity in the presence of the compounds and in the absence of compound

(solvent alone) x 100. * p<0.05 vs control (solvent alone) (n=3).

Figure 3. Photoaffinity labeling of purified rat liver GST by KPF-SCoA.

A. Time-dependent GST photoinactivation in the presence of KPF-SCoA. Rat

liver GST (0.16 mg/mL) was incubated in the presence of KPF-SCoA (0.5 mM)

and irradiated (■) or not (♦) at 365 nm for 0-120 min. The GST was also

irradiated in the absence of KPF-SCoA (□). The mixture was then diluted 100


at ASPE



Dow

nloaded from


DMD#16808

31

times in sodium phosphate buffer 0.1 M (pH 6.5) and residual activity was

calculated as described in Figure 2. B. Ligand protection against GST

inactivation by KPF-SCoA. The irradiation of rat liver GST was performed in the

presence KPF-SCoA as described in A but for a single time point (120 min).

GST was irradiated either in the presence of 0.25 mM KPF-SCoA alone or

combined with 2 mM of the GST ligands 4MU sulfate, GSH, or CDNB. The

mixture was subsequently diluted 100 times and residual activity was calculated

as described in Fig. 2, using the enzyme activity found in the absence of

irradiation as corresponding to 100% activity (n=3). * p<0.05.

Figure 4. Representative HPLC chromatogram of the products generated

upon incubation of KPF-SCoA with GSH and GST. GST (2.34 mg/mL) were

incubated in 0.1 M sodium phosphate buffer (pH 6.5) for 2 h at 30°C in the

presence of KPF-SCoA (0.5 mM) and GSH (3 mM) and analyzed by reverse-

phase HPLC as described in Material and Methods.

Figure 5. MALDI TOF positive ion mass spectra of KPF-SG formed from (A)

chemical synthesis and (B) enzymatic synthesis. (C) PSD analysis of KPF-SG

produced by cytosolic rat liver GST in the presence of KPF-CoA and GSH as

described in the legend to Fig. 4.


at ASPE



Dow

nloaded from


DMD#16808

32

Figure 6. Characterization of the transacylation and hydrolytic reactions

of KPF-SCoA catalyzed by purified rat liver GST. A. Time-course of the

transacylation and hydrolytic reactions. KPF-SCoA (0.5 mM) and GSH (3 mM)

were incubated with purified rat liver GST and the reaction products were

analyzed as a function of time by reverse-phase HPLC as described in Material

and Methods. Both GST-mediated transacylation leading to KPF-SG (■) and

hydrolysis reaction to KPF (●) are plotted after subtracting the products

obtained in the presence of GST to those obtained in the absence of the

enzymes. Control reactions performed in the same conditions but without GST

are also plotted for transacylation (□) and hydrolysis (○) reactions. B.

Representative Michaelis plot of the GST-dependent catalytic reaction leading

to the formation of the transacylation product KPF-SG. The experimental

conditions were as described in A, but with an incubation time of 45 min, and

with KPF-SCoA concentrations ranging from 0.01 to 1 mM. C. Lineweaver-

Burk plot of B (n=3).


at ASPE



Dow

nloaded from


Figure 1

N

N

N

NH2

N

O

OP

OH

O

O

OPO

OH

OH

O

HN

HN

O

OHO

OH

PHO

O

S

O

H3C

O

Ketoprofenyl-acylCoenzyme A

H

CH3COOHO

Ketoprofen

OC-OO

HO

OHHO

O

O

H3C

O

Ketoprofenyl-acylglucuronide

*

*

*

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

DM

D Fast Forw

ard. Published on October 25, 2007 as D

OI: 10.1124/dm

d.107.016808 at ASPET Journals on February 3, 2020 dmd.aspetjournals.org Downloaded from


0

50

100

Control

KPF

KPF-SCoACoASH

KPF-OGre

sid

ual

act

ivit

y (%

)

*

**

**

0

50

100

KPF

KPF-SCoA

KPF-OG

resi

du

al a

ctiv

ity

(%)

**

*

*

A

B

Figure 2




DM

D Fast Forw


OI: 10.1124/dm



0

25

50

75

100

0 30 60 90 120

Time (minutes)

resi

dual

act

ivity

(%)

UV - + + + +

KPF-SCoA + + + + +4MU sulfate - - + - -

GSH - - - + -CDNB - - - - +

A

0

25

50

75

100

resi

du

al a

ctiv

ity

(%)

*B

Figure 3




DM

D Fast Forw


OI: 10.1124/dm



Figure 4

Elution time (min)

A25

4 nm




DM

D Fast Forw


OI: 10.1124/dm



544 582

500 520 540 560 580 600 0

500

1000

1500

2000

2500

3000

inte

nsity

m/z

A. Chemical synthesis of KPF-SG

Mr = 543 MH + m/z 544 MK + m/z 582

O

S

H3C

O

HN

NH

O

OOH

H2N

O

O

OH

274

237

145

45469

415

500

130

308

[M+H-129]+

[M+H-44]+

145 193

237

274

308 371 415 469 500

544

100 200 300 400 500 0

2000

4000

6000

8000

10000

12000

inte

nsi

ty

m/z

C. PSD spectrum of MH+ m/z 544

KPF-SG

544

500 520 540 560 580 600 0

200

400

600

800

1000

1200

inte

nsity

m/z

B. Enzymatic synthesis of KPF-SG

Mr = 543 MH + m/z 544

Figure 5




DM

D Fast Forw


OI: 10.1124/dm



0

10

20

30

0 60 120 180 240

Time (min)

Pro

duct

s (p

mole

s)

0

0.1

0.2

0.3

0.4

0 0.25 0.5 0.75 1

[KPF-SCoA] (mM)

KP

F-S

G (pm

ole

s/m

in)

r2 = 0,989

0

10

20

30

40

0 25 50 75 100

1/KPF-SCoA (mM)-1

1/V

(pm

ole

s/m

in)-1

B

A

C

Figure 6




DM

D Fast Forw


OI: 10.1124/dm



Documents

Interaction of the electrophilic ketoprofenyl-glucuronide ...dmd.aspetjournals.org/content/dmd/early/2007/10/25/dmd.107.016808.full.pdf · Carboxylic acid-containing drugs are metabolized