Mechanisms and kinetics of human arylamine N-acetyltransferase 1 inhibition by disulfiram Florence Malka, Julien Dairou, Nilusha Ragunathan, Jean-Marie Dupret and Fernando Rodrigues-Lima Universite ´ 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, France Introduction Disulfiram (DS) or tetraethylthiuram disulfide (TTD) (Antabuse Ò ) has been used clinically in the treatment of chronic alcoholism since 1948 [1] (Fig. 1). DS acts by irreversibly inhibiting the hepatic aldehyde dehydro- genase, leading to the accumulation of acetaldehydes after alcohol ingestion [2]. The combined intake of DS and ethanol provokes unpleasant reactions (nausea and vomiting), which are the basis of the therapeutic use of DS. Several studies have shown that DS inhibits hepatic aldehyde dehydrogenase through covalent modification of an active site cysteine residue [3]. New potential therapeutic uses, in particular for human can- cers and fungal infections, have been reported recently for DS [4], indicating that the clinical applications of this drug are broader than previously thought. Never- theless, the mechanisms underlying these effects of DS remain poorly understood. DS is known to react with protein thiols to form mixed disulfides or covalent adducts [3]. However, many thiol-containing proteins do not react with DS, indicating that covalent modification by this drug shows marked specificity [5]. In addition to aldehyde dehydrogenase, other enzymes have been reported to be targeted by DS [6–8]. In particular, certain xeno- biotic-metabolizing enzymes (XMEs), such as cytochrome-P450 enzymes (CYP2E1) and glutathione Keywords arylamine N-acetyltransferase; cancer; drug target; inhibition; kinetics Correspondence F. Rodrigues-Lima, Universite ´ Paris Diderot-Paris 7, Unit of Functional and Adaptative Biology (BFA) – CNRS EAC 7059, 75013 Paris, France Fax: +33 1 57 27 83 29 Tel: +33 1 57 27 83 32 E-mail: fernando.rodrigues-lima@ univ-paris-diderot.fr (Received 20 April 2009, revised 28 May 2009, accepted 1 July 2009) doi:10.1111/j.1742-4658.2009.07189.x Disulfiram has been used for decades to treat alcoholism. Its therapeutic effect is thought to be mediated by the irreversible inhibition of aldehyde dehydrogenase. Recent reports have indicated new therapeutic uses of disulfiram, in particular in human cancers. Although the biochemical mech- anisms that underlie these effects remain largely unknown, certain enzymes involved in cancer processes have been reported to be targeted by disulfi- ram. Arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine drugs and carcinogens. In addi- tion to its role in xenobiotic metabolism, several studies have suggested that NAT1 is involved in other physiological and ⁄ or pathological pro- cesses, such as folate metabolism or cancer progression. In this report, we provide evidence that human NAT1 is a new enzymatic target of disulfi- ram. We found that disulfiram at clinically relevant concentrations impairs the activity of endogenous NAT1 in human cancer cells. Further mechanis- tic and kinetic studies indicated that disulfiram reacts irreversibly with the active site cysteine residue of NAT1, leading to its rapid inhibition (IC 50 = 3.3 ± 0.1 lm and k i =6· 10 4 m )1 Æmin )1 ). Abbreviations AcCoA, acetyl-coenzyme A; DS, disulfiram; GSH, reduced glutathione; GST, glutathione S-transferase; IC 50, half-maximal inhibitory concentration; 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 FEBS S-transferase (GST), are impaired by DS with subse- quent effects on xenobiotic metabolism in vivo [9–11]. Arylamine N-acetyltransferases (NATs) are phase 2 XMEs that catalyse the transfer of an acetyl group from acetyl-coenzyme A (AcCoA) to the nitrogen or oxygen atom of primary arylamines, hydrazines and their N-hydroxylated metabolites [12]. NATs plays a key role in the detoxification and ⁄ or activation of numerous drugs and carcinogens [13]. In humans, two functional isoforms, NAT1 and NAT2, have been described [14]. Although their protein sequences are similar (81% identical), their kinetic selectivity and efficiency for aromatic substrates and tissue distribu- tion differ markedly (NAT2 is found mainly in the liver and intestinal epithelium, whereas NAT1 shows widespread expression) [15,16]. Both NAT isoforms are affected by genetic polymorphisms, which can be a potential source of pharmacological and ⁄ or patho- logical susceptibility [17]. In addition to the genetic mechanisms that govern NAT expression and activity, recent data have shown that NAT enzymes can be affected by environmental chemicals, such as drugs or pollutants [18]. More specifically, human NAT1 has been shown to be impaired by oxidants [19–21] and by certain therapeutic drugs, such as cisplatin [22] and acetaminophen [23]. Several recent studies have indicated that NAT1 may contribute to increased can- cer risk and carcinogenesis [24–26], suggesting that this 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 to understand the pharmacological and ⁄ or toxicological effects of this therapeutic compound. In this study, we show 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 with the active site cysteine residue of NAT1, leading to the irreversible inhibition of the enzyme, with a half-maxi- mal inhibitory concentration (IC 50 ) of 3.3 ± 0.1 lm and a second-order inhibition rate constant (k i )of 6 · 10 4 m )1 Æmin )1 . Our work shows that NAT1 is a new molecular target of DS. Results DS impairs the activity of NAT1 in human cancer cells at clinically relevant concentrations DS is well known to inhibit the activity of liver alde- hyde dehydrogenase [2]. However, recent reports have shown 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 to therapeutically relevant concentrations of DS ( 30 lm) [4] led to the dose-dependent inhibition of NAT1 (Fig. 2). Similar results were obtained with another human lung cancer cell line (A549) (data not shown). These data suggest that NAT1 may be a new cellular target of DS. Therapeutically relevant concentrations of DS inhibit recombinant human NAT1 in an irreversible and dose-dependent manner To investigate the molecular mechanisms underlying the DS-dependent inhibition of NAT1, we carried out further biochemical and kinetic analyses using recom- binant purified NAT1. To test whether DS inhibits NAT1 directly, the recombinant enzyme was incubated with different clinically relevant concentrations of the drug and its residual activity was measured. As shown in Figure 3, NAT1 was inhibited in a dose-dependent manner by DS. Complete inhibition was observed with 0% 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 by clinically relevant concentrations of DS. NCI-H292 cells were exposed to different concentrations of DS in NaCl ⁄ P i for 30 min at 37 °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. Error bars indicate the standard deviations (*P < 0.05). Similar results were obtained with A549 cells (data not shown). An activity of 100% corresponds to a specific activity towards 2-aminofluorene of 10 ± 1 nmolÆmin )1 Æmg )1 . N S S S N S Disulfiram (DS) or tetraethylthiuram disulfide (TTD) Fig. 1. Chemical structure of DS. F. Malka et al. Inhibition of NAT1 functions by disulfiram FEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBS 4901 concentrations as low as 8 lm and the IC 50 value was estimated to be equal to 3.3 ± 0.1 lm. To test whether the DS-dependent inhibition of NAT1 was irreversible, dialysis experiments were car- ried out as described by Butcher et al. [30]. DS-treated NAT1 showed a residual activity of only 12 ± 1% and 3 ± 1% for dialysed and undialysed proteins, respectively [100% activity corresponded to a specific activity towards p-aminosalicylic acid (PAS) of 98 ± 7 nmolÆmin )1 Æmg )1 ], suggesting the irreversible inhibition of NAT1 by DS. Dialysis had no significant effect on NAT1 activity, as also reported by Butcher et al. [30]. We then investigated whether the DS-dependent inhibition of NAT1 could be reversed by reducing agents. As shown in Table 1, after inhibition by DS, NAT1 activity could not be restored by physiological concentrations of reduced glutathione (GSH) or by high concentrations of the non-physiological reductant dithiothreitol. These data suggest that the DS-depen- dent inhibition of NAT1 is unlikely to be a result of the formation of a mixed disulfide, but rather of the formation of a stable DS adduct. DS reacts with NAT1 thiol groups at therapeutic concentrations DS is a drug that reacts with thiols, and most of its effects are probably a result of its affinity for sulfhy- dryl groups in target proteins [3–5]. To test whether therapeutic concentrations of DS react with the NAT1 cysteine residue, we incubated the purified enzyme with DS in the concentration range shown to inhibit NAT1 (Fig. 3). Free unmodified cysteine residues in the enzyme were labelled with fluorescein-conjugated iodoacetamide and detected in western blots, as described previously [31]. As shown in Figure 4, DS reacted with NAT1 cysteine residues in a dose-depen- dent manner, as indicated by the disappearance of the fluorescein signal. This dose-dependent modification was correlated with the dose-dependent inhibition of 0% 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 by DS. NAT1 was incubated with clinically relevant concentrations of DS in 25 m M Tris ⁄ HCl, pH 7.5, 1 mM EDTA for 30 min at 37 °C. NAT1 activity was then determined. Errors bars indicate standard deviations (*P < 0.05). The results are presented as a percentage of control activity (100% activity corresponds to a specific activity towards PAS of 98 ± 7 nmolÆmin )1 Æmg )1 ). Table 1. Effects of reducing agents on DS-dependent inhibition of NAT1. Conditions % of control activity a NAT1 + DS 0.5 ± 0.7 NAT1 + DS + GSH (1 m M) 1.2 ± 0.9 NAT1 + DS + GSH (2 m M) 1.3 ± 0.6 NAT1 + DS + GSH (5 m M) 1.9 ± 1.4 NAT1 + DS 0.4 ± 0.04 NAT1 + DS + DTT (1 m M) 0.9 ± 0.1 NAT1 + DS + DTT (2 m M) 1.4 ± 0.3 NAT1 + DS + DTT (5 m M) 1.6 ± 0.3 a 100% activity corresponded to a specific activity towards PAS of 98 ± 7 nmolÆmin )1 Æmg )1 . Fig. 4. Detection of the DS-dependent modification of NAT1 cyste- ine residues. NAT1 was incubated with DS in 25 m M Tris ⁄ HCl, pH 7.5, 1 m M EDTA for 30 min at 37 °C. The reaction mixture was incubated with fluorescein-conjugated iodoacetamide for 10 min at 37 °C. Samples were then subjected to SDS-PAGE under reducing conditions, followed by western blotting using an anti-fluorescein IgG (anti-fluorescein) or an anti-6 · His-tag IgG (anti-6 · Histag). For the control (Ct), NAT1 was not treated with DS. Quantification of the signals was carried out using IMAGE J software (http://rsbweb. nih.gov/ij/). The fluorescein intensity was normalized with respect to 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 FEBS NAT1 by DS (Fig. 2), suggesting that the processes are linked. DS-dependent inhibition of NAT1 involves interactions with the active site Among the five cysteine residues present in the human NAT1 protein, one is localized in the enzyme active site and is required for catalysis [32]. To test whether the DS-dependent inhibition of NAT1 is a result of reaction at the active site cysteine residue, we carried out protection experiments in the presence of the physiological acetyl donor AcCoA, as described by Liu et al. [33]. This approach has been largely used to identify whether the inhibition of NAT enzymes by chemical compounds involves inter- actions with the active site [33,34]. The incubation of NAT1 with DS (8 lm) caused 93 ± 2% inhibition of the enzyme, whereas the presence of AcCoA at 1 and 2mm decreased the extent of inhibition to 35 ± 5% and 2 ± 4%, respectively (100% activity corre- sponded to a specific activity towards PAS of 98 ± 7 nmolÆmin )1 Æmg )1 ). These results suggest that the DS-dependent inhibition of NAT1 is caused by irreversible reaction with the active site cysteine residue. Kinetic analysis of the DS-dependent inhibition of NAT1 We further analysed the inhibition of NAT1 by DS by carrying out time course inhibition of the enzyme at different DS concentrations. As shown in Figure 5A, incubation of the enzyme with DS led to a monoexpo- nential time-dependent loss of activity, indicating that the inhibition reaction obeyed apparent first-order kinetics. The apparent first-order inhibition constants (k obs ) were calculated for each concentration of DS. The plot of k obs as a function of DS concentration fit- ted well to a line passing at the origin (r 2 = 0.99) (Fig. 5B), indicating that the inhibition of NAT1 by DS occurs through a single-step bimolecular reaction. The second-order rate constant for the inhibition of NAT1 by DS (k i ) was deduced from the slope of k obs as a function of the DS concentration [DS], and was 6 · 10 4 m )1 Æmin )1 . The order of the reaction of DS with NAT1 (n) can be deduced from the equation k obs = k i [DS] n , and can be calculated by plotting ln k obs as a function of ln [DS] (Fig. 5C). Linear regres- sion of these data indicated that they fitted well to a line (r 2 = 0.99) with a slope (n) equal to 0.99, suggest- ing that NAT1 inhibition by DS occurs through a 1 : 1 stoichiometry. 0.0 0.0 2.5 5.0 7.5 0.2 0.4 0.6 0.8 1.0 Relative residual activity Time (min) DS 0 µ M DS 4 µM DS 6 µM DS 8µM 0.0 0.1 0.2 0.3 0.4 0.5 0.6 [DS] (µ M) –1.6 –1.2 –0.8 R 2 = 0.997 R 2 = 0.999 –0.4 1.0 1.5 02468 2.0 2.5 ln (k obs ) k obs (min –1 ) ln ([DS]) A B C Fig. 5. Kinetic analysis of the DS-induced inhibition of NAT1 activ- ity. (A) NAT1 was incubated with various concentrations of DS in 25 m M Tris ⁄ HCl, pH 7.5, 1 mM EDTA at 37 °C. At various short-time intervals, aliquots were removed and assayed for residual activity. Plots of the relative residual activity as a function of time are shown and the data were found to fit well to a monoexponential time- dependent process. The error bars indicate standard deviations. An activity of 100% corresponds to a specific activity towards PAS of 98 ± 7 nmolÆmin )1 Æmg )1 . (B) The apparent first-order inhibition con- stant (k obs ) was calculated for each DS concentration and plotted. The second-order inhibition constant (k i ) was determined from the slope and was 61 · 10 3 M )1 Æmin )1 . The error bars indicate standard deviations. (C) To determine the stoichiometry of the reaction of DS with NAT1, the natural logarithm (ln) of k obs was 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 disulfiram FEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBS 4903 Overall, our results suggest that DS-dependent inhi- bition of NAT1 is caused by an irreversible single step. This inhibition occurs in a competitive manner through the modification of the active site catalytic cysteine residue of NAT1 by DS. Discussion DS is an inhibitor of aldehyde dehydrogenase and is currently being used clinically for the treatment of alcoholism. Recent data suggest that DS could have new therapeutic uses, particularly in cancer [4]. Inter- estingly, DS was found to inhibit enzymes that have been associated with cancer progression, such as DNA topoisomerases, 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 may help us to understand the therapeutic and toxicological effects of this drug. In this article, we provide evidence that the human XME NAT1 is a new target of DS. We found that endogenous NAT1 expressed by two human cancer cell lines was readily inhibited by short-time exposure (30 min) to clinically relevant concentrations of DS [4]. Interestingly, several studies have associated NAT1 activity with an increased risk of cancer [13]. In addition, recent data support the suggestion that NAT1 may play a role in breast cancer progression [16,27]. NAT1 expression has also been shown to be increased by androgens in human prostate cancer cells, which may have pathological implications [25]. Overall, these studies suggest that NAT1 is a can- cer-associated XME that could be targeted for cancer treatment [16,27]. Mechanistic and kinetic analyses were carried out to better understand the molecular basis for the DS- dependent inhibition of NAT1 activity in cells. Our data indicate that recombinant NAT1 was irreversibly inhibited by low clinically relevant concentrations of DS ( 30 lm) with an IC 50 value equal to 3.3 ± 0.1 lm. Recombinant aldehyde dehydrogenase and DNA topoisomerases have been reported to be inhibited in vitro in a similar manner with IC 50 values close to 35 lm [6,35]. IC 50 values ranging from five to hundreds of micromoles have been reported for differ- ent GST isoforms [10]. Kinetic analysis has also shown that the DS-dependent inhibition of NAT1 occurs rapidly with a second-order rate constant of 6 · 10 4 m )1 Æmin )1 . Overall, these data suggest that DS has an inhibitory potency against NAT1, similar to that of known enzymatic DS targets. DS is known to react with thiol groups, leading to the formation of mixed disulfides or covalent adducts on target proteins [3]. The inhibition of target enzymes, such as aldehyde dehydrogenase, by DS often occurs through the modification of an active site cysteine residue [3]. Accordingly, our data (iodoaceta- mide labelling, AcCoA protection assay and stoichi- ometric analysis) indicate that the DS-dependent inhibition of NAT1 is probably a result of the modifi- cation of the cysteine residue present in the enzyme active site. Reducing agents, such as GSH or dith- iothreitol, used at high concentrations (up to 5 mm), were unable to restore NAT1 activity. This suggests that the DS-dependent inhibition of NAT1 is unlikely to depend on the formation of a mixed disulfide which is readily reduced by 1 mm dithiothreitol at neutral pH, with subsequent recovery of enzymatic activity, as observed for aldehyde dehydrogenase [3]. Indeed, contrary to aldehyde dehydrogenase, NAT1 does 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 NAT1 is probably the result of the formation of a stable DS adduct that could be inaccessible to displacement by thiol reagents [37]. Cisplatin, an anticancer drug, has been reported recently to irreversibly inhibit NAT1 (k i = 700 m )1 Æmin )1 ) in vivo and in vitro. This inhibi- tion also occurs through the formation of an adduct with the NAT1 active site cysteine, which cannot be reduced by reducing agents [22]. Interestingly, the reaction of DS with NAT1 occurs 87 times faster (k i = 6.10 4 m )1 Æmin )1 ) than the reaction of cisplatin with the enzyme, thus supporting the suggestion that NAT1 could be an in vivo target of DS. The human NAT2 isoform shares a similar structure and mecha- nism of action to human NAT1 [38]. This isoform is thus likely to be inhibited by DS through the modifi- cation of its catalytic cysteine. Further studies are needed, however, to address whether DS reacts with NAT1 and NAT2 in a similar manner. NAT2 metab- olizes several aromatic amine drugs, such as isoniazid. The inhibition of NAT2 by DS could lead to drug–drug interactions as defects in NAT2 activity are associated with isoniazid hepatotoxicity [39]. DS has been used for decades to treat alcoholism, and its therapeutic activity is thought to be mediated through the irreversible inhibition of aldehyde dehy- drogenase. However, DS has been shown recently to have new potential therapeutic applications [4]. Accordingly, the biochemical mechanisms and cellular pathways that underlie the action of DS have also begun to emerge with the identification of new pro- tein targets of this drug [6–8]. Among them, XMEs such as CYP 2E1 and certain GST isoforms have Inhibition of NAT1 functions by disulfiram F. Malka et al. 4904 FEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBS been shown to be inhibited by DS, with subsequent effects in vivo on xenobiotic metabolism [9–11]. Our data clearly indicate that the XME NAT1 could be a new 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 other physiological and ⁄ or pathological processes, such as folate metabolism [40,41] and cancer progression [26,27]. The overexpression of NAT1 in normal lumi- nal epithelial breast cells induced two of the hallmark traits of cancer, i.e. enhanced growth and resistance to certain therapeutic cytotoxic drugs used in cancer treatment (etoposide) [42]. Recent studies from Minchin et al. [25] have shown that NAT1 is induced by androgens in human prostate cancer cells, with possible implications for cancer risk. The increasing evidence for an association of NAT1 with carcino- genesis suggests that its inhibition could be used in cancer therapy. The synthesis of small molecules that inhibit NAT1 in breast cancer cells has been reported recently [28]. The molecular mechanisms that underlie the anti-cancer activity of DS remain poorly under- stood. The DS-dependent inhibition of proteasome and inactivation of ATF ⁄ CREB transcription factor have been suggested to mediate DS anti-tumoral activity [8,29]. However, the anti-cancer effects of DS are probably a result of multiple mechanisms that could act synergistically [4]. The DS-dependent impairment of NAT1 could be one of these mecha- nisms. Experimental procedures Materials PAS, p-nitrophenylacetate (PNPA), AcCoA, DS (or TTD), GSH and fluorescein-conjugated iodoacetamide were pur- chased from Sigma (St-Quentin-Fallavier, France). Cell culture reagents were from Invitrogen (Cergy-Pontoise, France). Anti-fluorescein Fab¢ fragments conjugated to peroxidase, monoclonal antibodies directed against His tag and anti-mouse IgG were obtained from Roche (Meylan, France). The Bradford protein assay kit was purchased from Bio-Rad (Marne la Coquette, France). All other reagents were obtained from Sigma, or Euromedex (Souffelweyersheim, France). Cell culture and exposure to DS Human A549 lung carcinoma cells [43] and human NCI- H292 pulmonary mucoepidermoid carcinoma cells [44] were grown 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 petri dishes) were washed with NaCl ⁄ P i and exposed to different concentrations of DS in 10 mL of NaCl ⁄ P i for 30 min at 37 °C. Controls were performed in the absence of DS. On exposure, cells were washed with NaCl ⁄ P i and resuspended in NaCl ⁄ P i containing 0.2% Triton X-100 supplemented with protease inhibitors. Cells were sonicated and centri- fuged for 15 min at 13 000 g. The supernatants were removed, their protein concentration determined and assayed for NAT1 activity using 2-aminofluorene. Expression and purification of recombinant human NAT1 Human NAT1 was expressed as a 6 · His-tagged protein in Escherichia coli BL21 (DE3) cells transformed with a pET28a-based plasmid, as described previously [31]. On purification on nickel-agarose beads, recombinant NAT1 was reduced by incubation with 10 mm dithiothreitol for 10 min at 4 °C and dialysed against 25 mm Tris ⁄ HCl, pH 7.5. Purity was assessed by SDS-PAGE and protein concen- trations were determined using the Bradford reagent follow- ing the manufacturer’s instructions with bovine serum albumin as a standard. Enzyme assays Recombinant NAT1 enzyme activity was determined spec- trophotometrically using PNPA as the acetyl donor and PAS as the arylamine substrate [45]. Briefly, treated or untreated samples containing NAT1 enzyme were assayed in 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 final concentration of NAT1 was 115 nm. The reaction mixtures was incubated for up to 15 min at 37 °C, and the reaction was 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, was quantified by measuring the absorbance at 410 nm [Biotek (Colmar, France) microplate reader]. For the controls, we omitted the enzyme or PAS. All enzyme reactions were per- formed in triplicate in conditions in which the initial rates were linear. Enzymes activities are shown as percentages of control NAT1 activity. NAT1 activity was measured in cell extracts using reverse-phase HPLC, as described previously [14]. Samples (25 lL) were mixed with the aromatic amine substrate 2-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 the samples were incubated at 37 °C for various periods of time (up to 20 min). The reaction was quenched by the addition of 200 lL of ice-cold aqueous perchlorate (15% w ⁄ v), and F. Malka et al. Inhibition of NAT1 functions by disulfiram FEBS Journal 276 (2009) 4900–4908 ª 2009 The Authors Journal compilation ª 2009 FEBS 4905 proteins were recovered by centrifugation for 5 min at 12 000 g;20lL of the supernatant was injected onto a C18 reverse-phase HPLC column. All assays were performed under initial reaction rate conditions. Enzyme activities were normalized according to the protein concentration of cellular extracts determined using a Bio-Rad protein assay kit. Reaction of recombinant NAT1 with DS We assessed the effect of DS on NAT1 enzyme activity by the incubation of purified NAT1 samples (final concentra- tion of 75 lgÆmL )1 with a specific activity towards PAS of 98 ± 7 nmolÆmin )1 Æmg )1 ) with various concentrations of DS (up to a final concentration of 8 lm )in25mm Tris ⁄ HCl, pH 7.5, 1 mm EDTA for 30 min. Mixtures were then 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 dialysed against 25 mm Tris ⁄ HCl, pH 7.5, 1 mm EDTA, for 4 h at 4 °C. Control assays were performed with untreated NAT1 and gave 100% NAT1 activity. To test whether the DS-dependent inhibition of NAT1 activity could be reversed by reducing agents, NAT1 (final concentration, 75 lgÆmL )1 ; specific activity towards PAS of 98 nmolÆmin )1 Æmg )1 ) was first inhibited by DS (final con- centration, 8 lm) for 20 min at 37 °C. The mixture was then incubated for 10 min at 37 °C with various concentra- tions of GSH or dithiothreitol (final concentrations up to 5mm). A NAT1 enzyme assay was then carried out. Assays performed in these conditions, but without DS, gave 100% NAT1 activity. In substrate (AcCoA) protection experiments, NAT1 (final concentration, 75 lgÆmL )1 ; specific activity towards PAS of 98 ± 7 nmolÆmin )1 Æmg )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 mm EDTA. Samples were then assayed. Assays performed in these conditions with AcCoA alone gave 100% NAT1 activity. 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 )1 Æmg )1 ) was incubated 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 were removed and assayed for residual activity. The equation for the rate of inhibition of recombinant NAT1 by DS can be represented as – d[NAT1] ⁄ dt = k i [NAT1][DS], where [NAT1] is the concentration of active enzyme and k i is the second-order inhibition rate constant. The apparent first-order inhibition rate constants (k obs = k i [DS]) can be calculated for each DS concentration from the slope of the natural logarithm of the percentage residual activity plotted against time. The second-order rate constant was deter- mined from the slope of k obs plotted against DS concen- tration. Fluorescein-conjugated iodoacetamide labelling of NAT1 cysteine residues Purified NAT1 (final concentration, 75 lgÆmL )1 ; specific activity towards PAS of 98 ± 7 nmolÆ min )1 Æmg )1 ) was pre- incubated with or without (control, Ct) various concentra- tions of DS (up to a final concentration of 8 lm)in25mm Tris ⁄ 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 reducing conditions, followed by western blotting, using anti-fluores- cein Fab¢ fragments conjugated to peroxidase. Samples were also analysed by western blotting with monoclonal antibodies directed against 6 · His tag. Protein determination, SDS-PAGE and western blotting Protein concentrations were determined using a Bradford assay (Bio-Rad). Samples were combined with reducing SDS sample buffer and separated by SDS-PAGE. Gels were stained with Coomassie Brilliant Blue R-250. To detect 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 was stripped 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 analysis The data are the means ± standard deviation of at least two independent experiments performed in triplicate, unless otherwise stated. One-way analysis of variance (anova) was performed and followed by Student’s t-test (unpaired and paired) between two groups using statview 5.0 (SAS Institute Inc., Cary, NC, USA). Acknowledgements This work was supported by Agence Franc¸ aise de Se ´ curite ´ Sanitaire de l’Environnement et du Travail (AFSSET), Association pour la Recherche sur le Can- cer (ARC), Leg Poix (Chancellerie des Universite ´ sde Paris), Association Franc¸ aise contre les Myopathies Inhibition 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. We thank Emile Petit for growing the cells. References 1 Hald J & Jacobsen E (1948) A drug sensitizing the organism to ethyl alcohol. Lancet 2, 1001–1004. 2 Lipsky JJ, Shen ML & Naylor S (2001) In vivo inhibi- tion of aldehyde dehydrogenase by disulfiram. Chem Biol Interact 130–132, 93–102. 3 Veverka KA, Johnson KL, Mays DC, Lipsky JJ & Naylor S (1997) Inhibition of aldehyde dehydrogenase by disulfiram and its metabolite methyl diethylthiocar- bamoyl-sulfoxide. 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Mechanisms and kinetics of human arylamine N-acetyltransferase 1 inhibition by disulfiram Florence Malka, Julien Dairou,. DS-dependent inhibition of NAT1. Conditions % of control activity a NAT1 + DS 0.5 ± 0.7 NAT1 + DS + GSH (1 m M) 1. 2 ± 0.9 NAT1 + DS + GSH (2 m M) 1. 3 ± 0.6 NAT1 +