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Mechanisms and kinetics of human arylamineN-acetyltransferase 1 inhibition by disulfiramFlorence Malka, Julien Dairou, Nilusha Ragunathan, Jean-Marie Dupret andFernando Rodrigues-LimaUniversite´Paris Diderot-Paris 7, Unite´de Biologie Fonctionnelle et Adaptative (BFA), CNRS Equipe d’Accueil Conventione´e (EAC) 7059,Laboratoire des Re´ponses Mole´culaires et Cellulaires aux Xe´nobiotiques, 75013, Paris, FranceIntroductionDisulfiram (DS) or tetraethylthiuram disulfide (TTD)(AntabuseÒ) has been used clinically in the treatmentof chronic alcoholism since 1948 [1] (Fig. 1). DS actsby irreversibly inhibiting the hepatic aldehyde dehydro-genase, leading to the accumulation of acetaldehydesafter alcohol ingestion [2]. The combined intake of DSand ethanol provokes unpleasant reactions (nauseaand vomiting), which are the basis of the therapeuticuse of DS. Several studies have shown that DS inhibitshepatic aldehyde dehydrogenase through covalentmodification of an active site cysteine residue [3]. Newpotential therapeutic uses, in particular for human can-cers and fungal infections, have been reported recentlyfor DS [4], indicating that the clinical applications ofthis drug are broader than previously thought. Never-theless, the mechanisms underlying these effects of DSremain poorly understood.DS is known to react with protein thiols to formmixed disulfides or covalent adducts [3]. However,many thiol-containing proteins do not react with DS,indicating that covalent modification by this drugshows marked specificity [5]. In addition to aldehydedehydrogenase, other enzymes have been reported tobe targeted by DS [6–8]. In particular, certain xeno-biotic-metabolizing enzymes (XMEs), such ascytochrome-P450 enzymes (CYP2E1) and glutathioneKeywordsarylamine N-acetyltransferase; cancer; drugtarget; inhibition; kineticsCorrespondenceF. Rodrigues-Lima, Universite´ParisDiderot-Paris 7, Unit of Functional andAdaptative Biology (BFA) – CNRS EAC7059, 75013 Paris, FranceFax: +33 1 57 27 83 29Tel: +33 1 57 27 83 32E-mail: fernando.rodrigues-lima@univ-paris-diderot.fr(Received 20 April 2009, revised 28 May2009, accepted 1 July 2009)doi:10.1111/j.1742-4658.2009.07189.xDisulfiram has been used for decades to treat alcoholism. Its therapeuticeffect is thought to be mediated by the irreversible inhibition of aldehydedehydrogenase. Recent reports have indicated new therapeutic uses ofdisulfiram, in particular in human cancers. Although the biochemical mech-anisms that underlie these effects remain largely unknown, certain enzymesinvolved in cancer processes have been reported to be targeted by disulfi-ram. Arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizingenzyme that biotransforms aromatic amine drugs and carcinogens. In addi-tion to its role in xenobiotic metabolism, several studies have suggestedthat NAT1 is involved in other physiological and ⁄ or pathological pro-cesses, such as folate metabolism or cancer progression. In this report, weprovide evidence that human NAT1 is a new enzymatic target of disulfi-ram. We found that disulfiram at clinically relevant concentrations impairsthe activity of endogenous NAT1 in human cancer cells. Further mechanis-tic and kinetic studies indicated that disulfiram reacts irreversibly with theactive site cysteine residue of NAT1, leading to its rapid inhibition(IC50= 3.3 ± 0.1 lm and ki=6· 104m)1Æmin)1).AbbreviationsAcCoA, acetyl-coenzyme A; DS, disulfiram; GSH, reduced glutathione; GST, glutathione S-transferase; IC50,half-maximal inhibitoryconcentration; NAT, arylamine N-acetyltransferase; NAT1, arylamine N-acetyltransferase 1; PAS, p-aminosalicylic acid; PNPA,p-nitrophenylacetate; TTD, tetraethylthiuram disulfide; XME, xenobiotic-metabolizing enzyme.4900 FEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBSS-transferase (GST), are impaired by DS with subse-quent effects on xenobiotic metabolism in vivo [9–11].Arylamine N-acetyltransferases (NATs) are phase 2XMEs that catalyse the transfer of an acetyl groupfrom acetyl-coenzyme A (AcCoA) to the nitrogen oroxygen atom of primary arylamines, hydrazines andtheir N-hydroxylated metabolites [12]. NATs plays akey role in the detoxification and ⁄ or activation ofnumerous drugs and carcinogens [13]. In humans, twofunctional isoforms, NAT1 and NAT2, have beendescribed [14]. Although their protein sequences aresimilar (81% identical), their kinetic selectivity andefficiency for aromatic substrates and tissue distribu-tion differ markedly (NAT2 is found mainly in theliver and intestinal epithelium, whereas NAT1 showswidespread expression) [15,16]. Both NAT isoformsare affected by genetic polymorphisms, which can bea potential source of pharmacological and ⁄ or patho-logical susceptibility [17]. In addition to the geneticmechanisms that govern NAT expression and activity,recent data have shown that NAT enzymes can beaffected by environmental chemicals, such as drugs orpollutants [18]. More specifically, human NAT1 hasbeen shown to be impaired by oxidants [19–21] andby certain therapeutic drugs, such as cisplatin [22]and acetaminophen [23]. Several recent studies haveindicated that NAT1 may contribute to increased can-cer risk and carcinogenesis [24–26], suggesting thatthis XME could be relevant for cancer treatment[27,28].The identification and characterization of the molec-ular targets of DS are of prime importance in order tounderstand the pharmacological and ⁄ or toxicologicaleffects of this therapeutic compound. In this study, weshow that NAT1 is inhibited by therapeutic concentra-tions of DS in vitro and in human cancer cells. Mecha-nistic and kinetic analyses indicate that DS reacts withthe active site cysteine residue of NAT1, leading to theirreversible inhibition of the enzyme, with a half-maxi-mal inhibitory concentration (IC50) of 3.3 ± 0.1 lmand a second-order inhibition rate constant (ki)of6 · 104m)1Æmin)1. Our work shows that NAT1 is anew molecular target of DS.ResultsDS impairs the activity of NAT1 in human cancercells at clinically relevant concentrationsDS is well known to inhibit the activity of liver alde-hyde dehydrogenase [2]. However, recent reports haveshown that DS may also react with other pro-teins ⁄ enzymes, in particular in cancer cells [4,7,8,29].The exposure of human lung cancer cells NCI-H292 totherapeutically relevant concentrations of DS( 30 lm) [4] led to the dose-dependent inhibition ofNAT1 (Fig. 2). Similar results were obtained withanother human lung cancer cell line (A549) (data notshown). These data suggest that NAT1 may be a newcellular target of DS.Therapeutically relevant concentrationsof DS inhibit recombinant human NAT1 in anirreversible and dose-dependent mannerTo investigate the molecular mechanisms underlyingthe DS-dependent inhibition of NAT1, we carried outfurther biochemical and kinetic analyses using recom-binant purified NAT1. To test whether DS inhibitsNAT1 directly, the recombinant enzyme was incubatedwith different clinically relevant concentrations of thedrug and its residual activity was measured. As shownin Figure 3, NAT1 was inhibited in a dose-dependentmanner by DS. Complete inhibition was observed with0% 0 15 30 45 20% 40% 60% 80% 100% 120% ***NAT activity (% of control) [DS] (µM) Fig. 2. Inhibition of endogenous NAT1 in human cancer cells byclinically relevant concentrations of DS. NCI-H292 cells wereexposed to different concentrations of DS in NaCl ⁄ Pifor 30 min at37 °C. NAT1 activity was measured by HPLC in total cell extracts.Extracts from untreated cells were used as controls. Enzyme activi-ties were normalized with respect to protein concentration. Errorbars indicate the standard deviations (*P < 0.05). Similar resultswere obtained with A549 cells (data not shown). An activity of100% corresponds to a specific activity towards 2-aminofluorene of10 ± 1 nmolÆmin)1Æmg)1.NSSSNSDisulfiram (DS) or tetraethylthiuram disulfide (TTD) Fig. 1. Chemical structure of DS.F. Malka et al. Inhibition of NAT1 functions by disulfiramFEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBS 4901concentrations as low as 8 lm and the IC50value wasestimated to be equal to 3.3 ± 0.1 lm.To test whether the DS-dependent inhibition ofNAT1 was irreversible, dialysis experiments were car-ried out as described by Butcher et al. [30]. DS-treatedNAT1 showed a residual activity of only 12 ± 1%and 3 ± 1% for dialysed and undialysed proteins,respectively [100% activity corresponded to a specificactivity towards p-aminosalicylic acid (PAS) of98 ± 7 nmolÆmin)1Æmg)1], suggesting the irreversibleinhibition of NAT1 by DS. Dialysis had no significanteffect on NAT1 activity, as also reported by Butcheret al. [30].We then investigated whether the DS-dependentinhibition of NAT1 could be reversed by reducingagents. As shown in Table 1, after inhibition by DS,NAT1 activity could not be restored by physiologicalconcentrations of reduced glutathione (GSH) or byhigh concentrations of the non-physiological reductantdithiothreitol. These data suggest that the DS-depen-dent inhibition of NAT1 is unlikely to be a result ofthe formation of a mixed disulfide, but rather of theformation of a stable DS adduct.DS reacts with NAT1 thiol groups at therapeuticconcentrationsDS is a drug that reacts with thiols, and most of itseffects are probably a result of its affinity for sulfhy-dryl groups in target proteins [3–5]. To test whethertherapeutic concentrations of DS react with the NAT1cysteine residue, we incubated the purified enzyme withDS in the concentration range shown to inhibit NAT1(Fig. 3). Free unmodified cysteine residues in theenzyme were labelled with fluorescein-conjugatediodoacetamide and detected in western blots, asdescribed previously [31]. As shown in Figure 4, DSreacted with NAT1 cysteine residues in a dose-depen-dent manner, as indicated by the disappearance of thefluorescein signal. This dose-dependent modificationwas correlated with the dose-dependent inhibition of0% 0 2 3 4 5 20% 40% 60% 80% 100% 120% NAT1 activity (% of control) [DS] (µM)****Fig. 3. Dose-dependent inhibition of recombinant human NAT1 byDS. NAT1 was incubated with clinically relevant concentrations ofDS in 25 mM Tris ⁄ HCl, pH 7.5, 1 mM EDTA for 30 min at 37 °C.NAT1 activity was then determined. Errors bars indicate standarddeviations (*P < 0.05). The results are presented as a percentageof control activity (100% activity corresponds to a specific activitytowards PAS of 98 ± 7 nmolÆmin)1Æmg)1).Table 1. Effects of reducing agents on DS-dependent inhibition ofNAT1.Conditions % of control activityaNAT1 + DS 0.5 ± 0.7NAT1 + DS + GSH (1 mM) 1.2 ± 0.9NAT1 + DS + GSH (2 mM) 1.3 ± 0.6NAT1 + DS + GSH (5 mM) 1.9 ± 1.4NAT1 + DS 0.4 ± 0.04NAT1 + DS + DTT (1 mM) 0.9 ± 0.1NAT1 + DS + DTT (2 mM) 1.4 ± 0.3NAT1 + DS + DTT (5 mM) 1.6 ± 0.3a100% activity corresponded to a specific activity towards PAS of98 ± 7 nmolÆmin)1Æmg)1.Fig. 4. Detection of the DS-dependent modification of NAT1 cyste-ine residues. NAT1 was incubated with DS in 25 mM Tris ⁄ HCl, pH7.5, 1 mM EDTA for 30 min at 37 °C. The reaction mixture wasincubated with fluorescein-conjugated iodoacetamide for 10 min at37 °C. Samples were then subjected to SDS-PAGE under reducingconditions, followed by western blotting using an anti-fluoresceinIgG (anti-fluorescein) or an anti-6 · His-tag IgG (anti-6 · Histag). Forthe control (Ct), NAT1 was not treated with DS. Quantification ofthe signals was carried out usingIMAGE J software (http://rsbweb.nih.gov/ij/). The fluorescein intensity was normalized with respectto the anti-6 · His-tag signal.Inhibition of NAT1 functions by disulfiram F. Malka et al.4902 FEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBSNAT1 by DS (Fig. 2), suggesting that the processesare linked.DS-dependent inhibition of NAT1 involvesinteractions with the active siteAmong the five cysteine residues present in thehuman NAT1 protein, one is localized in the enzymeactive site and is required for catalysis [32]. To testwhether the DS-dependent inhibition of NAT1 is aresult of reaction at the active site cysteine residue,we carried out protection experiments in the presenceof the physiological acetyl donor AcCoA, asdescribed by Liu et al. [33]. This approach has beenlargely used to identify whether the inhibition ofNAT enzymes by chemical compounds involves inter-actions with the active site [33,34]. The incubation ofNAT1 with DS (8 lm) caused 93 ± 2% inhibition ofthe enzyme, whereas the presence of AcCoA at 1 and2mm decreased the extent of inhibition to 35 ± 5%and 2 ± 4%, respectively (100% activity corre-sponded to a specific activity towards PAS of98 ± 7 nmolÆmin)1Æmg)1). These results suggest thatthe DS-dependent inhibition of NAT1 is causedby irreversible reaction with the active site cysteineresidue.Kinetic analysis of the DS-dependent inhibitionof NAT1We further analysed the inhibition of NAT1 by DS bycarrying out time course inhibition of the enzyme atdifferent DS concentrations. As shown in Figure 5A,incubation of the enzyme with DS led to a monoexpo-nential time-dependent loss of activity, indicating thatthe inhibition reaction obeyed apparent first-orderkinetics. The apparent first-order inhibition constants(kobs) were calculated for each concentration of DS.The plot of kobsas a function of DS concentration fit-ted well to a line passing at the origin (r2= 0.99)(Fig. 5B), indicating that the inhibition of NAT1 byDS occurs through a single-step bimolecular reaction.The second-order rate constant for the inhibition ofNAT1 by DS (ki) was deduced from the slope of kobsas a function of the DS concentration [DS], and was6 · 104m)1Æmin)1. The order of the reaction of DSwith NAT1 (n) can be deduced from the equationkobs= ki[DS]n, and can be calculated by plotting lnkobsas a function of ln [DS] (Fig. 5C). Linear regres-sion of these data indicated that they fitted well to aline (r2= 0.99) with a slope (n) equal to 0.99, suggest-ing that NAT1 inhibition by DS occurs through a1 : 1 stoichiometry.0.00.0 2.5 5.0 7.50.20.40.60.81.0Relative residual activityTime (min) DS 0 µM DS 4 µM DS 6 µM DS 8µM 0.00.10.20.30.40.50.6[DS] (µM)–1.6–1.2–0.8R2 = 0.997R2 = 0.999–0.41.0 1.5024682.0 2.5ln (kobs) kobs (min–1)ln ([DS])ABCFig. 5. Kinetic analysis of the DS-induced inhibition of NAT1 activ-ity. (A) NAT1 was incubated with various concentrations of DS in25 mM Tris ⁄ HCl, pH 7.5, 1 mM EDTA at 37 °C. At various short-timeintervals, aliquots were removed and assayed for residual activity.Plots of the relative residual activity as a function of time are shownand the data were found to fit well to a monoexponential time-dependent process. The error bars indicate standard deviations. Anactivity of 100% corresponds to a specific activity towards PAS of98 ± 7 nmolÆmin)1Æmg)1. (B) The apparent first-order inhibition con-stant (kobs) was calculated for each DS concentration and plotted.The second-order inhibition constant (ki) was determined from theslope and was 61 · 103M)1Æmin)1. The error bars indicate standarddeviations. (C) To determine the stoichiometry of the reaction of DSwith NAT1, the natural logarithm (ln) of kobswas plotted as a func-tion of ln [DS]. The slope was 0.99, indicating a 1 : 1 stoichiometry.The error bars indicate standard deviations.F. Malka et al. Inhibition of NAT1 functions by disulfiramFEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBS 4903Overall, our results suggest that DS-dependent inhi-bition of NAT1 is caused by an irreversible single step.This inhibition occurs in a competitive mannerthrough the modification of the active site catalyticcysteine residue of NAT1 by DS.DiscussionDS is an inhibitor of aldehyde dehydrogenase and iscurrently being used clinically for the treatment ofalcoholism. Recent data suggest that DS could havenew therapeutic uses, particularly in cancer [4]. Inter-estingly, DS was found to inhibit enzymes that havebeen associated with cancer progression, such as DNAtopoisomerases, matrix metalloproteinases and protea-some [6–8]. The biological effects of DS are now con-sidered to involve different cellular pathways [4].Therefore, the deciphering of new targets of DS mayhelp us to understand the therapeutic and toxicologicaleffects of this drug. In this article, we provide evidencethat the human XME NAT1 is a new target of DS.We found that endogenous NAT1 expressed by twohuman cancer cell lines was readily inhibited byshort-time exposure (30 min) to clinically relevantconcentrations of DS [4]. Interestingly, several studieshave associated NAT1 activity with an increased riskof cancer [13]. In addition, recent data support thesuggestion that NAT1 may play a role in breast cancerprogression [16,27]. NAT1 expression has also beenshown to be increased by androgens in human prostatecancer cells, which may have pathological implications[25]. Overall, these studies suggest that NAT1 is a can-cer-associated XME that could be targeted for cancertreatment [16,27].Mechanistic and kinetic analyses were carried out tobetter understand the molecular basis for the DS-dependent inhibition of NAT1 activity in cells. Ourdata indicate that recombinant NAT1 was irreversiblyinhibited by low clinically relevant concentrations ofDS ( 30 lm) with an IC50value equal to3.3 ± 0.1 lm. Recombinant aldehyde dehydrogenaseand DNA topoisomerases have been reported to beinhibited in vitro in a similar manner with IC50valuesclose to 35 lm [6,35]. IC50values ranging from five tohundreds of micromoles have been reported for differ-ent GST isoforms [10]. Kinetic analysis has also shownthat the DS-dependent inhibition of NAT1 occursrapidly with a second-order rate constant of6 · 104m)1Æmin)1. Overall, these data suggest that DShas an inhibitory potency against NAT1, similar tothat of known enzymatic DS targets.DS is known to react with thiol groups, leading tothe formation of mixed disulfides or covalent adductson target proteins [3]. The inhibition of targetenzymes, such as aldehyde dehydrogenase, by DSoften occurs through the modification of an active sitecysteine residue [3]. Accordingly, our data (iodoaceta-mide labelling, AcCoA protection assay and stoichi-ometric analysis) indicate that the DS-dependentinhibition of NAT1 is probably a result of the modifi-cation of the cysteine residue present in the enzymeactive site. Reducing agents, such as GSH or dith-iothreitol, used at high concentrations (up to 5 mm),were unable to restore NAT1 activity. This suggeststhat the DS-dependent inhibition of NAT1 is unlikelyto depend on the formation of a mixed disulfidewhich is readily reduced by 1 mm dithiothreitol atneutral pH, with subsequent recovery of enzymaticactivity, as observed for aldehyde dehydrogenase [3].Indeed, contrary to aldehyde dehydrogenase, NAT1does not possess two vicinal thiols in its active site[36] and cannot thus be inhibited by the DS-depen-dent formation of an intramolecular mixed disulfide[3]. Therefore, the DS-dependent inhibition of NAT1is probably the result of the formation of a stable DSadduct that could be inaccessible to displacement bythiol reagents [37]. Cisplatin, an anticancer drug, hasbeen reported recently to irreversibly inhibit NAT1(ki= 700 m)1Æmin)1) in vivo and in vitro. This inhibi-tion also occurs through the formation of an adductwith the NAT1 active site cysteine, which cannot bereduced by reducing agents [22]. Interestingly, thereaction of DS with NAT1 occurs 87 times faster(ki= 6.104m)1Æmin)1) than the reaction of cisplatinwith the enzyme, thus supporting the suggestion thatNAT1 could be an in vivo target of DS. The humanNAT2 isoform shares a similar structure and mecha-nism of action to human NAT1 [38]. This isoform isthus likely to be inhibited by DS through the modifi-cation of its catalytic cysteine. Further studies areneeded, however, to address whether DS reacts withNAT1 and NAT2 in a similar manner. NAT2 metab-olizes several aromatic amine drugs, such as isoniazid.The inhibition of NAT2 by DS could lead todrug–drug interactions as defects in NAT2 activityare associated with isoniazid hepatotoxicity [39].DS has been used for decades to treat alcoholism,and its therapeutic activity is thought to be mediatedthrough the irreversible inhibition of aldehyde dehy-drogenase. However, DS has been shown recently tohave new potential therapeutic applications [4].Accordingly, the biochemical mechanisms and cellularpathways that underlie the action of DS have alsobegun to emerge with the identification of new pro-tein targets of this drug [6–8]. Among them, XMEssuch as CYP 2E1 and certain GST isoforms haveInhibition of NAT1 functions by disulfiram F. Malka et al.4904 FEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBSbeen shown to be inhibited by DS, with subsequenteffects in vivo on xenobiotic metabolism [9–11]. Ourdata clearly indicate that the XME NAT1 could be anew target of DS. In addition to its role in xenobi-otic metabolism, there is increasing evidence to sug-gest that NAT1 may also be involved in otherphysiological and ⁄ or pathological processes, such asfolate metabolism [40,41] and cancer progression[26,27]. The overexpression of NAT1 in normal lumi-nal epithelial breast cells induced two of the hallmarktraits of cancer, i.e. enhanced growth and resistanceto certain therapeutic cytotoxic drugs used in cancertreatment (etoposide) [42]. Recent studies fromMinchin et al. [25] have shown that NAT1 is inducedby androgens in human prostate cancer cells, withpossible implications for cancer risk. The increasingevidence for an association of NAT1 with carcino-genesis suggests that its inhibition could be used incancer therapy. The synthesis of small molecules thatinhibit NAT1 in breast cancer cells has been reportedrecently [28]. The molecular mechanisms that underliethe anti-cancer activity of DS remain poorly under-stood. The DS-dependent inhibition of proteasomeand inactivation of ATF ⁄ CREB transcription factorhave been suggested to mediate DS anti-tumoralactivity [8,29]. However, the anti-cancer effects of DSare probably a result of multiple mechanisms thatcould act synergistically [4]. The DS-dependentimpairment of NAT1 could be one of these mecha-nisms.Experimental proceduresMaterialsPAS, p-nitrophenylacetate (PNPA), AcCoA, DS (or TTD),GSH and fluorescein-conjugated iodoacetamide were pur-chased from Sigma (St-Quentin-Fallavier, France). Cellculture reagents were from Invitrogen (Cergy-Pontoise,France). Anti-fluorescein Fab¢ fragments conjugated toperoxidase, monoclonal antibodies directed against His tagand anti-mouse IgG were obtained from Roche (Meylan,France). The Bradford protein assay kit was purchasedfrom Bio-Rad (Marne la Coquette, France). All otherreagents were obtained from Sigma, or Euromedex(Souffelweyersheim, France).Cell culture and exposure to DSHuman A549 lung carcinoma cells [43] and human NCI-H292 pulmonary mucoepidermoid carcinoma cells [44] weregrown in DMEM ⁄ F12 medium supplemented with 10%(v ⁄ v) fetal bovine serum, penicillin (100 UÆmL)1) and strep-tomycin (100 lgÆmL)1). Cell monolayers (100 mm petridishes) were washed with NaCl ⁄ Piand exposed to differentconcentrations of DS in 10 mL of NaCl ⁄ Pifor 30 min at37 °C. Controls were performed in the absence of DS. Onexposure, cells were washed with NaCl ⁄ Piand resuspendedin NaCl ⁄ Picontaining 0.2% Triton X-100 supplementedwith protease inhibitors. Cells were sonicated and centri-fuged for 15 min at 13 000 g. The supernatants wereremoved, their protein concentration determined andassayed for NAT1 activity using 2-aminofluorene.Expression and purification of recombinanthuman NAT1Human NAT1 was expressed as a 6 · His-tagged protein inEscherichia coli BL21 (DE3) cells transformed with apET28a-based plasmid, as described previously [31]. Onpurification on nickel-agarose beads, recombinant NAT1was reduced by incubation with 10 mm dithiothreitol for10 min at 4 °C and dialysed against 25 mm Tris ⁄ HCl, pH7.5. Purity was assessed by SDS-PAGE and protein concen-trations were determined using the Bradford reagent follow-ing the manufacturer’s instructions with bovine serumalbumin as a standard.Enzyme assaysRecombinant NAT1 enzyme activity was determined spec-trophotometrically using PNPA as the acetyl donor andPAS as the arylamine substrate [45]. Briefly, treated oruntreated samples containing NAT1 enzyme were assayedin a reaction mixture containing PAS (final concentration,500 lm)in25mm Tris ⁄ HCl, pH 7.5, 1 mm EDTA. Reac-tions were started by the addition of PNPA (final concen-tration, 2 mm). In all reaction mixtures, the finalconcentration of NAT1 was 115 nm. The reaction mixtureswas incubated for up to 15 min at 37 °C, and the reactionwas then quenched by the addition of SDS (final concentra-tion, 2%). P-Nitrophenol, generated by the NAT1-medi-ated hydrolysis of PNPA in the presence of PAS, wasquantified by measuring the absorbance at 410 nm [Biotek(Colmar, France) microplate reader]. For the controls, weomitted the enzyme or PAS. All enzyme reactions were per-formed in triplicate in conditions in which the initial rateswere linear. Enzymes activities are shown as percentages ofcontrol NAT1 activity.NAT1 activity was measured in cell extracts usingreverse-phase HPLC, as described previously [14]. Samples(25 lL) were mixed with the aromatic amine substrate2-aminofluorene (final concentration, 1 mm) in assay buffer(25 mm Tris ⁄ HCl, pH 7.5) at 37 °C. AcCoA (final concen-tration, 1 mm) was added to start the reaction, and thesamples were incubated at 37 °C for various periods of time(up to 20 min). The reaction was quenched by the additionof 200 lL of ice-cold aqueous perchlorate (15% w ⁄ v), andF. Malka et al. Inhibition of NAT1 functions by disulfiramFEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBS 4905proteins were recovered by centrifugation for 5 min at12 000 g;20lL of the supernatant was injected onto a C18reverse-phase HPLC column. All assays were performedunder initial reaction rate conditions. Enzyme activitieswere normalized according to the protein concentration ofcellular extracts determined using a Bio-Rad protein assaykit.Reaction of recombinant NAT1 with DSWe assessed the effect of DS on NAT1 enzyme activity bythe incubation of purified NAT1 samples (final concentra-tion of 75 lgÆmL)1with a specific activity towards PAS of98 ± 7 nmolÆmin)1Æmg)1) with various concentrations ofDS (up to a final concentration of 8 lm )in25mmTris ⁄ HCl, pH 7.5, 1 mm EDTA for 30 min. Mixtures werethen assayed for NAT1 activity as described above.To test whether the reaction of DS with NAT1 was irre-versible, the recombinant enzyme was incubated with DS(final concentration, 8 lm), and the mixture was dialysedagainst 25 mm Tris ⁄ HCl, pH 7.5, 1 mm EDTA, for 4 h at4 °C. Control assays were performed with untreated NAT1and gave 100% NAT1 activity.To test whether the DS-dependent inhibition of NAT1activity could be reversed by reducing agents, NAT1 (finalconcentration, 75 lgÆmL)1; specific activity towards PAS of98 nmolÆmin)1Æmg)1) was first inhibited by DS (final con-centration, 8 lm) for 20 min at 37 °C. The mixture wasthen incubated for 10 min at 37 °C with various concentra-tions of GSH or dithiothreitol (final concentrations up to5mm). A NAT1 enzyme assay was then carried out. Assaysperformed in these conditions, but without DS, gave 100%NAT1 activity.In substrate (AcCoA) protection experiments, NAT1(final concentration, 75 lgÆmL)1; specific activity towardsPAS of 98 ± 7 nmolÆmin)1Æmg)1) was incubated with DS(final concentration, 8 lm) in the presence of various con-centrations of AcCoA (final concentrations up to 2 mm)for 30 min at 37 °Cin25mm Tris ⁄ HCl, pH 7.5, 1 mmEDTA. Samples were then assayed. Assays performed inthese conditions with AcCoA alone gave 100% NAT1activity.For the kinetic analysis of DS-mediated NAT1 inhibi-tion, NAT1 (final concentration, 75 lgÆmL)1; specific activ-ity towards PAS of 98 ± 775 nmolÆmin)1Æmg)1) wasincubated with different concentrations of DS (final concen-tration up to 8 lm)at37°Cin25mm Tris ⁄ HCl, pH 7.5,1mm EDTA. At various time intervals, aliquots wereremoved and assayed for residual activity. The equation forthe rate of inhibition of recombinant NAT1 by DS can berepresented as – d[NAT1] ⁄ dt = ki[NAT1][DS], where[NAT1] is the concentration of active enzyme and kiis thesecond-order inhibition rate constant. The apparentfirst-order inhibition rate constants (kobs= ki[DS]) can becalculated for each DS concentration from the slope of thenatural logarithm of the percentage residual activity plottedagainst time. The second-order rate constant was deter-mined from the slope of kobsplotted against DS concen-tration.Fluorescein-conjugated iodoacetamide labellingof NAT1 cysteine residuesPurified NAT1 (final concentration, 75 lgÆmL)1; specificactivity towards PAS of 98 ± 7 nmolÆ min)1Æmg)1) was pre-incubated with or without (control, Ct) various concentra-tions of DS (up to a final concentration of 8 lm)in25mmTris ⁄ HCl, pH 7.5, 1 mm EDTA for 30 min at 37 °C. Sam-ples were incubated with fluorescein-conjugated iodoaceta-mide (final concentration, 20 lm) for 10 min at 37 °C.Samples were then analysed by SDS-PAGE under reducingconditions, followed by western blotting, using anti-fluores-cein Fab¢ fragments conjugated to peroxidase. Sampleswere also analysed by western blotting with monoclonalantibodies directed against 6 · His tag.Protein determination, SDS-PAGE and westernblottingProtein concentrations were determined using a Bradfordassay (Bio-Rad). Samples were combined with reducingSDS sample buffer and separated by SDS-PAGE. Gelswere stained with Coomassie Brilliant Blue R-250. Todetect proteins labelled by fluorescein-conjugated iodoaceta-mide in western blots, anti-fluorescein Fab¢ fragments con-jugated to horseradish peroxidase (1 : 50 000) were used.To control protein loading, the same membrane wasstripped by incubation for 1 h at 37 °C with stripping buf-fer (20 mm Tris ⁄ HCl, pH 7.5, 20% SDS, 2 mm dithiothrei-tol) and probed with anti-monoclonal anti-His IgG(1 : 10 000).Statistical analysisThe data are the means ± standard deviation of at leasttwo independent experiments performed in triplicate, unlessotherwise stated. One-way analysis of variance (anova) wasperformed and followed by Student’s t-test (unpairedand paired) between two groups using statview 5.0 (SASInstitute Inc., Cary, NC, USA).AcknowledgementsThis work was supported by Agence Franc¸ aise deSe´curite´Sanitaire de l’Environnement et du Travail(AFSSET), Association pour la Recherche sur le Can-cer (ARC), Leg Poix (Chancellerie des Universite´sdeParis), Association Franc¸ aise contre les MyopathiesInhibition of NAT1 functions by disulfiram F. Malka et al.4906 FEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBS(AFM) and Universite´Paris Diderot-Paris 7. Wethank Emile Petit for growing the cells.References1 Hald J & Jacobsen E (1948) A drug sensitizing theorganism to ethyl alcohol. Lancet 2, 1001–1004.2 Lipsky JJ, Shen ML & Naylor S (2001) In vivo inhibi-tion of aldehyde dehydrogenase by disulfiram. 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Exp Cell Res 243, 359–366.44 Steiner P, Joynes C, Bassi R, Wang S, Tonra JR,Hadari YR & Hicklin DJ (2007) Tumor growth inhibi-tion with cetuximab and chemotherapy in non-small celllung cancer xenografts expressing wild-type andmutated epidermal growth factor receptor. Clin CancerRes 13, 1540–1551.45 Mushtaq A, Payton M & Sim E (2002) The C-terminusof arylamine N-acetyl transferase from Salmonellatyphimurium controls enzymic activity. J Biol Chem17, 12175–12181.Inhibition of NAT1 functions by disulfiram F. Malka et al.4908 FEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBS . Mechanisms and kinetics of human arylamine N-acetyltransferase 1 inhibition by disulfiram Florence Malka, Julien Dairou,. DS-dependent inhibition of NAT1.Conditions % of control activityaNAT1 + DS 0.5 ± 0.7NAT1 + DS + GSH (1 mM) 1. 2 ± 0.9NAT1 + DS + GSH (2 mM) 1. 3 ± 0.6NAT1 +
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