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Disulfiram is a potent modulator of multidrug transporter Cdr1p of Candida albicans
BBRC Biochemical and Biophysical Research Communications 322 (2004) 520–525 www.elsevier.com/locate/ybbrc
Disulfiram is a potent modulator of multidrug transporter Cdr1p of Candida albicansq Suneet Shuklaa, Zuben E. Saunaa, Rajendra Prasadb, Suresh V. Ambudkara,* a
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4256, USA b Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India Received 13 July 2004
Abstract To find novel drugs for effective antifungal therapy in candidiasis, we examined disulfiram, a drug used for the treatment of alcoholism, for its role as a potential modulator of Candida multidrug transporter Cdr1p. We show that disulfiram inhibits the oligomycin-sensitive ATPase activity of Cdr1p and 2.5 mM dithiothreitol reverses this inhibition. Disulfiram inhibited the binding of photoaffinity analogs of both ATP ([a-32P]8-azidoATP; IC50 = 0.76 lM) and drug-substrates ([3H]azidopine and [125I]iodoarylazidoprazosin; IC50 12 lM) to Cdr1p in a concentration-dependent manner, suggesting that it can interact with both ATP and substrate-binding site(s) of Cdr1p. Furthermore, a non-toxic concentration of disulfiram (1 lM) increased the sensitivity of Cdr1p expressing Saccharomyces cerevisiae cells to antifungal agents (fluconazole, miconazole, nystatin, and cycloheximide). Collectively these results demonstrate that disulfiram reverses Cdr1p-mediated drug resistance by interaction with both ATP and substrate-binding sites of the transporter and may be useful for antifungal therapy. Published by Elsevier Inc. Keywords: ABC transporter; ATP hydrolysis; Antifungal agents; Multidrug resistance; Photoaffinity labeling; Yeast
Development of drug resistance in clinically relevant fungal pathogens poses an impediment to antifungal therapy. Various mechanisms that contribute towards the development of resistance in Candida have been proposed. They include overexpression of or mutations in the target enzyme of azoles, lanosterol 14a-demethylase, and overexpression of drug efflux pumps [1] belonging to the ATP-binding cassette (ABC) [2] and to the major facilitator superfamilies (MFS) of transporters [3,4]. Among ABC transporters, Cdr1p has been shown to play a key role in azole resistance in Candida albicans
q Abbreviations: ABC, ATP-binding cassette; IAAP, iodoarylazidoprazosin; MDR, multidrug resistance; MIC, minimum inhibitory concentration; P-gp, P-glycoprotein; PM; plasma membrane; YEPD, yeast extract peptone dextrose. * Corresponding author. Fax: +1 301 435 8188. E-mail address: [email protected] (S.V. Ambudkar).
0006-291X/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bbrc.2004.07.151
[5,6]. There is an urgent need for development of new effective modulators, which would inhibit the efflux functions of the multidrug resistance (MDR) pumps like Cdr1p. For instance, P-glycoprotein (P-gp)-mediated anticancer drug resistance in cancer cells can be reversed through the use of immunosuppressive agents such as cyclosporin A [7], FK506 [8], as well as the non-immunosuppressive cyclosporin analog PSC833 [9]. Disulfiram, also known as Antabuse, a drug clinically used for treating alcoholism, was found to be potentially useful to inhibit P-gp function [10] and has recently also been shown to act as a modulator for P-gp, as well as other multidrug transporters such as MRP1 and MRP4 [11]. The photoaffinity substrate analogs of P-gp, iodoarylazidoprazosin, azidopine, and iodoarylazidorhodamine 123 have been reported to bind to the drug-binding sites of Cdr1p from C. albicans [12,13]. Due to functional similarities between Cdr1p and
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human P-gp, we have in this study evaluated the potency of disulfiram, as a novel modulator of Cdr1p. We show here that disulfiram inhibits oligomycinsensitive ATP hydrolysis and the binding of the photoaffinity analogs of ATP and drug-substrates (IAAP and azidopine) to Cdr1p. We also show that the presence of disulfiram along with the antifungal agents increases the efficiency of the drugs and makes the cells more sensitive to these drugs. Thus, similar to its observed inhibitory effect on human P-gp and MRP1-mediated drug resistance [10,11], disulfiram also inhibits the Cdr1p-mediated drug resistance in Cdr1p overexpressing Saccharomyces cerevisiae cells. Based on these observations, we propose that disulfiram may have clinical significance in the treatment of infections caused by C. albicans.
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ing) and the photoaffinity analog was added. The samples were incubated for an additional 5 min under subdued light. The samples were then illuminated with a UV lamp assembly (PGC Scientifics, Gaithersburg, MD) fitted with two Black light (self-filtering) UV-A long wave-F15T8BLB tubes (365 nm) for 10 min at room temperature (21–23 °C) or on ice (for 8-azidoATP labeling). Following SDS–PAGE on an 8% Tris–glycine gel at constant voltage, gels were dried and exposed to Bio-Max MR film (Eastman Kodak, Rochester, NY) at 80 °C for 12–24 h. The radioactivity incorporated into the Cdr1p band was quantified using the STORM 860 phosphorimager system (Molecular Dynamics, Sunnyvale, CA) and the software ImageQuaNT as described previously [16]. ATPase assay. The PM (100 lg protein/ml) were incubated with the indicated concentrations of disulfiram and/or DTT at 37 °C for 5 min in 0.1 ml of a medium containing 59 mM Tris, pH 7.5, 7 mM MgCl2, and 20 lM oligomycin, where indicated. The samples were then brought to 30 °C. The assays were initiated by adding 5 mM ATP and the Cdr1p associated oligomycin-sensitive ATPase activity of the PM was measured as oligomycin-sensitive release of inorganic phosphate as described earlier [12].
Materials and methods Results Materials. Protease inhibitors, cycloheximide, miconazole, nystatin, and disulfiram, were obtained from Sigma Chemical (St. Louis, MO). [125I]Iodoarylazidoprazosin (IAAP) (2200 Ci/mmol) was purchased from Perkin–Elmer Life Sciences (Boston, MA). [a-32P]8AzidoATP (15–20 Ci/mmol) and 8-azidoATP were obtained from Affinity Labeling Technologies (Lexington, KY). [3H]Azidopine (60 Ci/mmol) was procured from Amersham Biosciences (Arlington Heights, IL). Fluconazole was kindly provided by Ranbaxy Laboratories (Gurgaon, India). Yeast strains and growth media. The S. cerevisiae strains used in this study are AD1-8u [14] (provided by Professor Richard D. Cannon, University of Otago, Dunedin, New Zealand) and PSCDR1-GFP [12] (AD1-8u derivative overexpressing Cdr1p-GFP). The yeast strains were cultured in YEPD broth (Bio101, Vista, CA). For agar plates 2% (w/v) bacto agar (Difco, BD Biosciences, NJ) was added to the medium. Drug susceptibility of S. cerevisiae. The sensitivity of S. cerevisiae cells to different drugs was determined by using spot assay. The following stock solutions were prepared: fluconazole 1 mg/ml (water), cycloheximide 0.1 mg/ml (water), nystatin 1 mg/ml (DMSO), miconazole 1 mg/ml (methanol), and disulfiram (1 mM) (ethanol). The yeast cells were grown overnight on YEPD plates and then resuspended in normal saline to an A600 of 0.1. Five microliters of fivefold serial dilutions of each strain was spotted on YEPD plates in the absence (control) and presence of the following drugs, fluconazole (2 lg/ ml), cycloheximide (30 ng/ml), miconazole (250 ng/ml), nystatin (500 ng/ml), and disulfiram (1 lM). Growth differences were recorded following incubation of the plates for 48 h at 30 °C. Growth was not affected by the presence of the solvents used for the drugs. Preparation of purified plasma membranes of S. cerevisiae cells. The purified plasma membranes (PM) of S. cerevisiae cells were prepared by using a sucrose discontinuous gradient as described [12]. The concentration of protein in the purified PM was determined by using Amido Black B as described [15]. Photoaffinity labeling of Cdr1p with [125I]IAAP, [3H]azidopine, and [a-32P]8-azidoATP. The PM (15 lg protein) was photoaffinity labeled with 3–6 nM [125I]IAAP (2200 Ci/mmol), 0.5 lM [3H]azidopine (60 Ci/mmol) or 10 lM [a-32P]8-azidoATP (10 lCi/nmol) and competed with drugs as described previously [12]. Briefly the PM were incubated with the indicated drug for 5 min at 37 °C in 0.1 ml of 50 mM Tris–HCl, pH 7.5, or in 0.1 ml ATPase assay buffer (59 mM Tris, pH 7.5, and 7 mM MgCl2) (for 8-azidoATP labeling). The samples were brought to room temperature or ice (for 8-azidoATP label-
To study the interaction of disulfiram with Cdr1p, we used a hyper-expression system, where Cdr1p is stably overexpressed from a genomic PDR5 locus in a S. cerevisiae mutant AD1-8u [12,14]. The AD1-8u was derived from a Pdr1-3 mutant strain with a gain of function mutation in the transcription factor Pdr1p, resulting in a constitutive hyperinduction of the PDR5 promoter [14]. We also tagged the GFP gene at the C-terminal end of CDR1 and demonstrated that GFP tagging and overexpression did not impair functional activity of the protein [12]. The overexpression of GFP-tagged Cdr1p in S. cerevisiae offered expression levels sufficient for biochemical characterization of the transporter. Disulfiram inhibits ATP hydrolysis by Cdr1p The oligomycin-sensitive ATP hydrolysis by purified PM from PSCDR1-GFP cells was measured in the presence or absence of disulfiram (100 lM) as described in Materials and methods. It was observed that the presence of 100 lM disulfiram inhibited the oligomycin-sensitive ATPase activity of Cdr1p by 65–75% compared to the control values (Fig. 1). When various concentrations ranging from 5 to 100 lM disulfiram were tested, the maximal inhibition of the ATPase activity was observed at 100 lM (data not shown). To understand the mechanism by which disulfiram inhibits ATP hydrolysis by Cdr1p, the ATPase activity was determined in the presence of DTT. Disulfiram inhibits activity of enzymes/ transporters by forming an intramolecular disulfide linkage between cysteines at or near the active site [17]. DTT can reduce these disulfide binds and reverse disulfirammediated inhibition of activity. We found that the simultaneous presence of 2.5 mM DTT and 100 lM disulfiram
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(30 lg protein) from AD1-8u and PSCDR1-GFP cells with 10 lM [a-32P]8-azidoATP (10 lCi/nmol) followed by UV irradiation led to the covalent incorporation of this analog into the protein. This labeling of Cdr1p with [a-32P]8-azidoATP was indeed inhibited by disulfiram (50 lM). Furthermore, this inhibition was concentration-dependent with an IC50 of 0.76 lM (Fig. 2B). Thus, disulfiram appears to interact directly with the ATPbinding sites of Cdr1p. Disulfiram inhibits the binding of IAAP and azidopine to Cdr1p Fig. 1. Effect of disulfiram in the presence and absence of DTT on the ATP hydrolysis by Cdr1p. The oligomycin-sensitive ATPase activity of Cdr1p in the presence of 100 lM disulfiram and/or 2.5 mM DTT was determined as described in Materials and methods. The Y-axis represents percent ATPase activity, alone (control) in the presence and absence of DTT and/or disulfiram (DS). The values are given as means ± standard deviation (error bars) for three independent experiments.
resulted in the reversal of the observed inhibition of ATP hydrolysis by disulfiram (Fig. 1). These data suggest that similar to its action in P-gp [11], disulfiram appears to inhibit ATPase activity of Cdr1p by modifying cysteine residues in or near the active site. Disulfiram inhibits the binding of [a-32P]8-azidoATP to Cdr1p Disulfiram could inhibit ATP hydrolysis either directly by blocking nucleotide binding or indirectly by interacting at the substrate-binding sites. To test the former hypothesis, we monitored the binding of [a-32P]8-azidoATP to Cdr1p in the presence of disulfiram. Fig. 2A shows that the incubation of the PM
To examine the effect of disulfiram on the substrate binding to Cdr1p, we used IAAP and azidopine, which are radiolabeled photoactive analogs of prazosin and dihydropyridine, respectively. These analogs have been used to determine the substrate interactions with P-gp [11,18,19] and Cdr1p [12]. We investigated the effect of disulfiram on the IAAP or azidopine binding, to PM from AD1-8u and PSCDR1-GFP as described in Materials and methods. We demonstrate in Figs. 3A and B that the binding of both [125I]IAAP and [3H]azidopine to Cdr1p was inhibited by disulfiram in a concentration-dependent manner following the Michaelis–Menten kinetics (Figs. 3C and D) with IC50 values of 12.4 and 12.3 lM, respectively. It should be noted that the IC50 values for inhibition by disulfiram of IAAP and azidopine binding to P-gp were 3.5–6.5 and 15.5 lM [11] as compared to 12.4 and 12.3 lM for Cdr1p, respectively. These data indicate that disulfiram inhibits the binding of the photoaffinity analogs to Cdr1p with a concentration range similar to that required for its inhibitory effect on human P-gp.
Fig. 2. Effect of disulfiram on [a-32P]8-azidoATP binding to Cdr1p. (A) The plasma membranes (50–75 lg protein) from AD1-8u (C) and PSCDR1-GFP (Cdr1p) were incubated in absence ( ) or presence (+) of disulfiram (50 lM) for 5 min at 37 °C in 0.1 ml of ATPase assay buffer and were photoaffinity labeled with 10 lM of [a-32P]8-azidoATP (10 lCi/nmol) as described in Materials and methods. (B) PM (0.5–0.75 mg protein/ml) from PSCDR1-GFP were incubated with the indicated concentration of disulfiram ranging from 0 to 50 lM for 5 min at 37 °C in 0.1 ml ATPase assay buffer and labeled with [a-32P]8-azidoATP as described in Materials and methods. The radioactivity incorporated into the Cdr1p band was quantified and the graph is plotted as described in Materials and methods. The experimental details are given in the graph and on the autoradiogram.
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Fig. 3. Disulfiram inhibits binding of [3H]azidopine and IAAP to Cdr1p in a concentration-dependent manner. The PM (0.50–1.0 mg protein/ml) from AD1-8u and PSCDR1-GFP cells were incubated with the increasing concentrations (0–100 lM) of disulfiram in 0.1 ml of 50 mM Tris–HCl, pH 7.5, for 5 min at 37 °C. The samples were brought to room temperature and (A) 0.5 lM [3H]azidopine or (B) 3–6 nM [125I]IAAP was added and incubated for an additional 5 min in the dark. The samples were processed and incorporation of radiolabel was quantitated as described in Materials and methods. The autoradiogram and the graphs represents the amounts of IAAP (A,C) and [3H]azidopine (B,D) incorporated in to Cdr1p in the presence of indicated concentration of disulfiram.
Disulfiram inhibits Cdr1p-mediated drug resistance in S. cerevisiae cells In order for disulfiram to be used clinically as a modulator in antifungal therapy, it is essential that it is able to reverse the drug resistance mediated by Cdr1p in the intact cells expressing the transporter. The ability of disulfiram to modulate drug resistance in the S. cerevisiae cells expressing Cdr1p was evaluated by using PSCDR1-GFP cells, which show resistance to antifungal drugs such as cycloheximide, fluconazole, miconazole, and nystatin [12]. The concentration of disulfiram, which could be safely used to study the Cdr1p-mediated drug resistance in these cells, was determined by a minimum inhibitory concentration (MIC) assay of disulfiram for AD1-8u (control) and PSCDR1-GFP (Cdr1p) cells using concentrations ranging from 0 to 12.5 lM. It was observed that both the control and the Cdr1p expressing cells showed an MIC80 value of 2.5 lM for disulfiram. Based on these results, a concentration of 1 lM disulfiram, which was non-toxic to both the Cdr1p (PSCDR1-GFP) as well as the control AD1-8u cells, was selected to be used in the following experiment. The control AD1-8u and PSCDR1-GFP cells were grown in the absence and presence of 1 lM disulfiram on a YEPD plate. As expected the presence of disulfiram (1 lM) did not
inhibit the growth of the cells (Fig. 4, left panels). In order to monitor the effect of disulfiram on the drug resistance of Cdr1p expressing cells, the control AD18u and the Cdr1p expressing (PSCDR1-GFP) cells were grown in the presence of only the indicated drug (cycloheximide 30 ng/ml, fluconazole 2 lg/ml, miconazole 250 ng/ml, and nystatin 500 ng/ml) or in the presence of both disulfiram (1 lM) and a given drug. It was observed that while Cdr1p expressing cells were able to grow in the presence of drug alone (Fig. 4, middle panels), the simultaneous presence of 1 lM disulfiram and the given drug inhibited the growth of Cdr1p expressing PSCDR1-GFP cells (Fig. 4, right panels) at all the dilutions. This was further confirmed by an MIC assay for these drugs in PSCDR1-GFP cells where, along with the varying concentrations of drugs (cycloheximide, fluconazole, miconazole, and nystatin), a constant non-toxic concentration of disulfiram (1 lM) was present. It was observed that the presence of disulfiram (1 lM) along with the drugs inhibited the growth of the Cdr1p expressing cells at all the concentrations, while disulfiram (1 lM) alone did not have any significant effect on the growth of the control AD1-8u cells (data not shown). The results of the spot assay and the MIC inhibition data together suggest that disulfiram indeed acts as a modulator of the Cdr1p function and sensitizes cells to drugs, which are substrates for Cdr1p.
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Fig. 4. Effect of disulfiram on Cdr1p-mediated drug resistance. The AD1-8u and PSCDR1-GFP cells were grown overnight on YEPD plates. The cells were then resuspended in normal saline to an A600 of 0.1. Five microliters of fivefold serial dilution of each strain was spotted onto YEPD plates in the absence (control) and presence of the following drugs, cycloheximide (Cyh, 30 ng/ml), fluconazole (Flu, 2 lg/ml), miconazole (Mic, 250 ng/ml), nystatin (Nys, 500 ng/ml), and disulfiram (DS, 1 lM). Growth differences were recorded following incubation of the plates for 48 h at 30 °C.
Discussion The emphasis of many studies on MDR in C. albicans is currently focused on drugs to reverse MDR and perhaps the most interesting targets are the efflux pumps such as Cdr1p and Cdr2p, which are major contributors to the development of resistance. The inhibition of these pumps would probably result in an increase in the intracellular concentration of drugs such as azoles, thereby increasing the effectiveness of those drugs. The aim of this study was to determine the potential of disulfiram as a modulator of Cdr1p in the antifungal therapy. The results of the present study reveal that disulfiram inhibits both ATP hydrolysis and the binding of the photoaffinity analog of ATP, 8-azidoATP, to Cdr1p. Disulfiram is known to act as a cysteine modifying agent and forms an intramolecular disulfide linkage with the cysteines present near the active site of the protein [17]. It has been shown earlier that disulfiram inhibits the ATPase activity of P-gp, MRP1, and MRP4 probably by inhibiting the formation of intramolecular disulfide linkage in the nucleotide-binding pocket of these proteins [11]. This could also be the likely reason for the inhibition of ATPase activity of Cdr1p by disulfiram. It should be noted that there are 23 cysteines present in Cdr1p. Among these, 11 cysteines are present in the predicted cytoplasmic domains of the protein [20]. There are five cysteines present in both the nucleotide-
binding domain (NBD1) and NBD2 of this protein. The presence of these cysteines in both the NBDs could probably result in disulfiram forming intramolecular disulfide linkages, which in turn would result in the loss of ATP binding. To address this issue, we used a strong reducing agent, DTT, which reduces the disulfide crosslinking between the two sulfhydryl groups. The presence of DTT reversed disulfiram-mediated inhibition of binding and hydrolysis of ATP. These observations suggest that disulfiram acts by forming disulfide bridges and that the cysteine residues in the ATP-binding domains (NBDs) of Cdr1p play a crucial role in the inhibition of Cdr1p ATPase activity by disulfiram. We used IAAP and azidopine that covalently label Cdr1p, to evaluate the effect of disulfiram on the binding of the substrates. It was observed that disulfiram also inhibits the binding of both IAAP and azidopine to Cdr1p in a concentration-dependent manner. We have shown previously that the binding of these two photoaffinity compounds to P-gp was also inhibited by disulfiram [11] and suggested that in addition to the interaction of disulfiram with the ATP sites of Cdr1p (described above), it also acts as an inhibitor that affects drug-binding sites. The binding of photoaffinity compounds to Cdr1p is specifically competed by its known substrates, i.e., IAAP binding is competed by nystatin while azidopine binding is competed out by miconazole [12]. Thus, our data suggest that disulfiram may also
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block the binding of other substrates such as miconazole and nystatin to the transporter. This inhibition of the binding of the antifungal drugs would probably result in an increase in the intracellular concentration of these drugs, thus making the drugs more effective in the therapy of candidiasis. To test this hypothesis, we checked the drug resistance profile of the Cdr1p expressing S. cerevisiae cells in the presence and absence of a non-toxic concentration of disulfiram (1 lM) and observed that the presence of disulfiram along with the antifungal drugs tested makes the cells more susceptible towards these agents (Fig. 4). The fact that the presence of disulfiram along with the other antifungal agents did not further inhibit the growth in the control AD1-8u cells suggests that the observed chemosensitization is mediated by the interaction of the disulfiram with Cdr1p. Thus, these data indicate that disulfiram inhibits the Cdr1p-mediated functions by both inhibiting ATP and substrate binding to the protein. Disulfiram has been approved as a drug and has been used for the treatment of alcoholism for over 50 years and has a well-documented pharmacology. Studies show that disulfiram has a low toxicity [21] and can be prescribed over a long period of time at a dose of 500 mg/ day. The values determined in this study for disulfiram-mediated reversal of Cdr1p function are achievable in a clinical setting and therefore may be useful in clinical practice. It is important to note that the effective concentration of disulfiram required for reversal of drug transport function of human P-gp and MRP1 and Cdr1p in S. cerevisiae is in the similar range ([11] and this work). In conclusion, our studies suggest that disulfiram can be used as a potential modulator of Cdr1pmediated drug resistance in C. albicans and may be exploited in the treatment of candidiasis.
Acknowledgments We thank Dr. Michael M. Gottesman for helpful discussions and encouragement. We also thank George Leiman for assistance in the preparation of the manuscript.
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Disulfiram is a potent modulator of multidrug transporter Cdr1p of Candida albicansq Suneet Shuklaa, Zuben E. Saunaa, Rajendra Prasadb, Suresh V. Ambudkara,* a
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4256, USA b Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India Received 13 July 2004
Abstract To find novel drugs for effective antifungal therapy in candidiasis, we examined disulfiram, a drug used for the treatment of alcoholism, for its role as a potential modulator of Candida multidrug transporter Cdr1p. We show that disulfiram inhibits the oligomycin-sensitive ATPase activity of Cdr1p and 2.5 mM dithiothreitol reverses this inhibition. Disulfiram inhibited the binding of photoaffinity analogs of both ATP ([a-32P]8-azidoATP; IC50 = 0.76 lM) and drug-substrates ([3H]azidopine and [125I]iodoarylazidoprazosin; IC50 12 lM) to Cdr1p in a concentration-dependent manner, suggesting that it can interact with both ATP and substrate-binding site(s) of Cdr1p. Furthermore, a non-toxic concentration of disulfiram (1 lM) increased the sensitivity of Cdr1p expressing Saccharomyces cerevisiae cells to antifungal agents (fluconazole, miconazole, nystatin, and cycloheximide). Collectively these results demonstrate that disulfiram reverses Cdr1p-mediated drug resistance by interaction with both ATP and substrate-binding sites of the transporter and may be useful for antifungal therapy. Published by Elsevier Inc. Keywords: ABC transporter; ATP hydrolysis; Antifungal agents; Multidrug resistance; Photoaffinity labeling; Yeast
Development of drug resistance in clinically relevant fungal pathogens poses an impediment to antifungal therapy. Various mechanisms that contribute towards the development of resistance in Candida have been proposed. They include overexpression of or mutations in the target enzyme of azoles, lanosterol 14a-demethylase, and overexpression of drug efflux pumps [1] belonging to the ATP-binding cassette (ABC) [2] and to the major facilitator superfamilies (MFS) of transporters [3,4]. Among ABC transporters, Cdr1p has been shown to play a key role in azole resistance in Candida albicans
q Abbreviations: ABC, ATP-binding cassette; IAAP, iodoarylazidoprazosin; MDR, multidrug resistance; MIC, minimum inhibitory concentration; P-gp, P-glycoprotein; PM; plasma membrane; YEPD, yeast extract peptone dextrose. * Corresponding author. Fax: +1 301 435 8188. E-mail address: [email protected] (S.V. Ambudkar).
0006-291X/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bbrc.2004.07.151
[5,6]. There is an urgent need for development of new effective modulators, which would inhibit the efflux functions of the multidrug resistance (MDR) pumps like Cdr1p. For instance, P-glycoprotein (P-gp)-mediated anticancer drug resistance in cancer cells can be reversed through the use of immunosuppressive agents such as cyclosporin A [7], FK506 [8], as well as the non-immunosuppressive cyclosporin analog PSC833 [9]. Disulfiram, also known as Antabuse, a drug clinically used for treating alcoholism, was found to be potentially useful to inhibit P-gp function [10] and has recently also been shown to act as a modulator for P-gp, as well as other multidrug transporters such as MRP1 and MRP4 [11]. The photoaffinity substrate analogs of P-gp, iodoarylazidoprazosin, azidopine, and iodoarylazidorhodamine 123 have been reported to bind to the drug-binding sites of Cdr1p from C. albicans [12,13]. Due to functional similarities between Cdr1p and
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human P-gp, we have in this study evaluated the potency of disulfiram, as a novel modulator of Cdr1p. We show here that disulfiram inhibits oligomycinsensitive ATP hydrolysis and the binding of the photoaffinity analogs of ATP and drug-substrates (IAAP and azidopine) to Cdr1p. We also show that the presence of disulfiram along with the antifungal agents increases the efficiency of the drugs and makes the cells more sensitive to these drugs. Thus, similar to its observed inhibitory effect on human P-gp and MRP1-mediated drug resistance [10,11], disulfiram also inhibits the Cdr1p-mediated drug resistance in Cdr1p overexpressing Saccharomyces cerevisiae cells. Based on these observations, we propose that disulfiram may have clinical significance in the treatment of infections caused by C. albicans.
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ing) and the photoaffinity analog was added. The samples were incubated for an additional 5 min under subdued light. The samples were then illuminated with a UV lamp assembly (PGC Scientifics, Gaithersburg, MD) fitted with two Black light (self-filtering) UV-A long wave-F15T8BLB tubes (365 nm) for 10 min at room temperature (21–23 °C) or on ice (for 8-azidoATP labeling). Following SDS–PAGE on an 8% Tris–glycine gel at constant voltage, gels were dried and exposed to Bio-Max MR film (Eastman Kodak, Rochester, NY) at 80 °C for 12–24 h. The radioactivity incorporated into the Cdr1p band was quantified using the STORM 860 phosphorimager system (Molecular Dynamics, Sunnyvale, CA) and the software ImageQuaNT as described previously [16]. ATPase assay. The PM (100 lg protein/ml) were incubated with the indicated concentrations of disulfiram and/or DTT at 37 °C for 5 min in 0.1 ml of a medium containing 59 mM Tris, pH 7.5, 7 mM MgCl2, and 20 lM oligomycin, where indicated. The samples were then brought to 30 °C. The assays were initiated by adding 5 mM ATP and the Cdr1p associated oligomycin-sensitive ATPase activity of the PM was measured as oligomycin-sensitive release of inorganic phosphate as described earlier [12].
Materials and methods Results Materials. Protease inhibitors, cycloheximide, miconazole, nystatin, and disulfiram, were obtained from Sigma Chemical (St. Louis, MO). [125I]Iodoarylazidoprazosin (IAAP) (2200 Ci/mmol) was purchased from Perkin–Elmer Life Sciences (Boston, MA). [a-32P]8AzidoATP (15–20 Ci/mmol) and 8-azidoATP were obtained from Affinity Labeling Technologies (Lexington, KY). [3H]Azidopine (60 Ci/mmol) was procured from Amersham Biosciences (Arlington Heights, IL). Fluconazole was kindly provided by Ranbaxy Laboratories (Gurgaon, India). Yeast strains and growth media. The S. cerevisiae strains used in this study are AD1-8u [14] (provided by Professor Richard D. Cannon, University of Otago, Dunedin, New Zealand) and PSCDR1-GFP [12] (AD1-8u derivative overexpressing Cdr1p-GFP). The yeast strains were cultured in YEPD broth (Bio101, Vista, CA). For agar plates 2% (w/v) bacto agar (Difco, BD Biosciences, NJ) was added to the medium. Drug susceptibility of S. cerevisiae. The sensitivity of S. cerevisiae cells to different drugs was determined by using spot assay. The following stock solutions were prepared: fluconazole 1 mg/ml (water), cycloheximide 0.1 mg/ml (water), nystatin 1 mg/ml (DMSO), miconazole 1 mg/ml (methanol), and disulfiram (1 mM) (ethanol). The yeast cells were grown overnight on YEPD plates and then resuspended in normal saline to an A600 of 0.1. Five microliters of fivefold serial dilutions of each strain was spotted on YEPD plates in the absence (control) and presence of the following drugs, fluconazole (2 lg/ ml), cycloheximide (30 ng/ml), miconazole (250 ng/ml), nystatin (500 ng/ml), and disulfiram (1 lM). Growth differences were recorded following incubation of the plates for 48 h at 30 °C. Growth was not affected by the presence of the solvents used for the drugs. Preparation of purified plasma membranes of S. cerevisiae cells. The purified plasma membranes (PM) of S. cerevisiae cells were prepared by using a sucrose discontinuous gradient as described [12]. The concentration of protein in the purified PM was determined by using Amido Black B as described [15]. Photoaffinity labeling of Cdr1p with [125I]IAAP, [3H]azidopine, and [a-32P]8-azidoATP. The PM (15 lg protein) was photoaffinity labeled with 3–6 nM [125I]IAAP (2200 Ci/mmol), 0.5 lM [3H]azidopine (60 Ci/mmol) or 10 lM [a-32P]8-azidoATP (10 lCi/nmol) and competed with drugs as described previously [12]. Briefly the PM were incubated with the indicated drug for 5 min at 37 °C in 0.1 ml of 50 mM Tris–HCl, pH 7.5, or in 0.1 ml ATPase assay buffer (59 mM Tris, pH 7.5, and 7 mM MgCl2) (for 8-azidoATP labeling). The samples were brought to room temperature or ice (for 8-azidoATP label-
To study the interaction of disulfiram with Cdr1p, we used a hyper-expression system, where Cdr1p is stably overexpressed from a genomic PDR5 locus in a S. cerevisiae mutant AD1-8u [12,14]. The AD1-8u was derived from a Pdr1-3 mutant strain with a gain of function mutation in the transcription factor Pdr1p, resulting in a constitutive hyperinduction of the PDR5 promoter [14]. We also tagged the GFP gene at the C-terminal end of CDR1 and demonstrated that GFP tagging and overexpression did not impair functional activity of the protein [12]. The overexpression of GFP-tagged Cdr1p in S. cerevisiae offered expression levels sufficient for biochemical characterization of the transporter. Disulfiram inhibits ATP hydrolysis by Cdr1p The oligomycin-sensitive ATP hydrolysis by purified PM from PSCDR1-GFP cells was measured in the presence or absence of disulfiram (100 lM) as described in Materials and methods. It was observed that the presence of 100 lM disulfiram inhibited the oligomycin-sensitive ATPase activity of Cdr1p by 65–75% compared to the control values (Fig. 1). When various concentrations ranging from 5 to 100 lM disulfiram were tested, the maximal inhibition of the ATPase activity was observed at 100 lM (data not shown). To understand the mechanism by which disulfiram inhibits ATP hydrolysis by Cdr1p, the ATPase activity was determined in the presence of DTT. Disulfiram inhibits activity of enzymes/ transporters by forming an intramolecular disulfide linkage between cysteines at or near the active site [17]. DTT can reduce these disulfide binds and reverse disulfirammediated inhibition of activity. We found that the simultaneous presence of 2.5 mM DTT and 100 lM disulfiram
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(30 lg protein) from AD1-8u and PSCDR1-GFP cells with 10 lM [a-32P]8-azidoATP (10 lCi/nmol) followed by UV irradiation led to the covalent incorporation of this analog into the protein. This labeling of Cdr1p with [a-32P]8-azidoATP was indeed inhibited by disulfiram (50 lM). Furthermore, this inhibition was concentration-dependent with an IC50 of 0.76 lM (Fig. 2B). Thus, disulfiram appears to interact directly with the ATPbinding sites of Cdr1p. Disulfiram inhibits the binding of IAAP and azidopine to Cdr1p Fig. 1. Effect of disulfiram in the presence and absence of DTT on the ATP hydrolysis by Cdr1p. The oligomycin-sensitive ATPase activity of Cdr1p in the presence of 100 lM disulfiram and/or 2.5 mM DTT was determined as described in Materials and methods. The Y-axis represents percent ATPase activity, alone (control) in the presence and absence of DTT and/or disulfiram (DS). The values are given as means ± standard deviation (error bars) for three independent experiments.
resulted in the reversal of the observed inhibition of ATP hydrolysis by disulfiram (Fig. 1). These data suggest that similar to its action in P-gp [11], disulfiram appears to inhibit ATPase activity of Cdr1p by modifying cysteine residues in or near the active site. Disulfiram inhibits the binding of [a-32P]8-azidoATP to Cdr1p Disulfiram could inhibit ATP hydrolysis either directly by blocking nucleotide binding or indirectly by interacting at the substrate-binding sites. To test the former hypothesis, we monitored the binding of [a-32P]8-azidoATP to Cdr1p in the presence of disulfiram. Fig. 2A shows that the incubation of the PM
To examine the effect of disulfiram on the substrate binding to Cdr1p, we used IAAP and azidopine, which are radiolabeled photoactive analogs of prazosin and dihydropyridine, respectively. These analogs have been used to determine the substrate interactions with P-gp [11,18,19] and Cdr1p [12]. We investigated the effect of disulfiram on the IAAP or azidopine binding, to PM from AD1-8u and PSCDR1-GFP as described in Materials and methods. We demonstrate in Figs. 3A and B that the binding of both [125I]IAAP and [3H]azidopine to Cdr1p was inhibited by disulfiram in a concentration-dependent manner following the Michaelis–Menten kinetics (Figs. 3C and D) with IC50 values of 12.4 and 12.3 lM, respectively. It should be noted that the IC50 values for inhibition by disulfiram of IAAP and azidopine binding to P-gp were 3.5–6.5 and 15.5 lM [11] as compared to 12.4 and 12.3 lM for Cdr1p, respectively. These data indicate that disulfiram inhibits the binding of the photoaffinity analogs to Cdr1p with a concentration range similar to that required for its inhibitory effect on human P-gp.
Fig. 2. Effect of disulfiram on [a-32P]8-azidoATP binding to Cdr1p. (A) The plasma membranes (50–75 lg protein) from AD1-8u (C) and PSCDR1-GFP (Cdr1p) were incubated in absence ( ) or presence (+) of disulfiram (50 lM) for 5 min at 37 °C in 0.1 ml of ATPase assay buffer and were photoaffinity labeled with 10 lM of [a-32P]8-azidoATP (10 lCi/nmol) as described in Materials and methods. (B) PM (0.5–0.75 mg protein/ml) from PSCDR1-GFP were incubated with the indicated concentration of disulfiram ranging from 0 to 50 lM for 5 min at 37 °C in 0.1 ml ATPase assay buffer and labeled with [a-32P]8-azidoATP as described in Materials and methods. The radioactivity incorporated into the Cdr1p band was quantified and the graph is plotted as described in Materials and methods. The experimental details are given in the graph and on the autoradiogram.
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Fig. 3. Disulfiram inhibits binding of [3H]azidopine and IAAP to Cdr1p in a concentration-dependent manner. The PM (0.50–1.0 mg protein/ml) from AD1-8u and PSCDR1-GFP cells were incubated with the increasing concentrations (0–100 lM) of disulfiram in 0.1 ml of 50 mM Tris–HCl, pH 7.5, for 5 min at 37 °C. The samples were brought to room temperature and (A) 0.5 lM [3H]azidopine or (B) 3–6 nM [125I]IAAP was added and incubated for an additional 5 min in the dark. The samples were processed and incorporation of radiolabel was quantitated as described in Materials and methods. The autoradiogram and the graphs represents the amounts of IAAP (A,C) and [3H]azidopine (B,D) incorporated in to Cdr1p in the presence of indicated concentration of disulfiram.
Disulfiram inhibits Cdr1p-mediated drug resistance in S. cerevisiae cells In order for disulfiram to be used clinically as a modulator in antifungal therapy, it is essential that it is able to reverse the drug resistance mediated by Cdr1p in the intact cells expressing the transporter. The ability of disulfiram to modulate drug resistance in the S. cerevisiae cells expressing Cdr1p was evaluated by using PSCDR1-GFP cells, which show resistance to antifungal drugs such as cycloheximide, fluconazole, miconazole, and nystatin [12]. The concentration of disulfiram, which could be safely used to study the Cdr1p-mediated drug resistance in these cells, was determined by a minimum inhibitory concentration (MIC) assay of disulfiram for AD1-8u (control) and PSCDR1-GFP (Cdr1p) cells using concentrations ranging from 0 to 12.5 lM. It was observed that both the control and the Cdr1p expressing cells showed an MIC80 value of 2.5 lM for disulfiram. Based on these results, a concentration of 1 lM disulfiram, which was non-toxic to both the Cdr1p (PSCDR1-GFP) as well as the control AD1-8u cells, was selected to be used in the following experiment. The control AD1-8u and PSCDR1-GFP cells were grown in the absence and presence of 1 lM disulfiram on a YEPD plate. As expected the presence of disulfiram (1 lM) did not
inhibit the growth of the cells (Fig. 4, left panels). In order to monitor the effect of disulfiram on the drug resistance of Cdr1p expressing cells, the control AD18u and the Cdr1p expressing (PSCDR1-GFP) cells were grown in the presence of only the indicated drug (cycloheximide 30 ng/ml, fluconazole 2 lg/ml, miconazole 250 ng/ml, and nystatin 500 ng/ml) or in the presence of both disulfiram (1 lM) and a given drug. It was observed that while Cdr1p expressing cells were able to grow in the presence of drug alone (Fig. 4, middle panels), the simultaneous presence of 1 lM disulfiram and the given drug inhibited the growth of Cdr1p expressing PSCDR1-GFP cells (Fig. 4, right panels) at all the dilutions. This was further confirmed by an MIC assay for these drugs in PSCDR1-GFP cells where, along with the varying concentrations of drugs (cycloheximide, fluconazole, miconazole, and nystatin), a constant non-toxic concentration of disulfiram (1 lM) was present. It was observed that the presence of disulfiram (1 lM) along with the drugs inhibited the growth of the Cdr1p expressing cells at all the concentrations, while disulfiram (1 lM) alone did not have any significant effect on the growth of the control AD1-8u cells (data not shown). The results of the spot assay and the MIC inhibition data together suggest that disulfiram indeed acts as a modulator of the Cdr1p function and sensitizes cells to drugs, which are substrates for Cdr1p.
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Fig. 4. Effect of disulfiram on Cdr1p-mediated drug resistance. The AD1-8u and PSCDR1-GFP cells were grown overnight on YEPD plates. The cells were then resuspended in normal saline to an A600 of 0.1. Five microliters of fivefold serial dilution of each strain was spotted onto YEPD plates in the absence (control) and presence of the following drugs, cycloheximide (Cyh, 30 ng/ml), fluconazole (Flu, 2 lg/ml), miconazole (Mic, 250 ng/ml), nystatin (Nys, 500 ng/ml), and disulfiram (DS, 1 lM). Growth differences were recorded following incubation of the plates for 48 h at 30 °C.
Discussion The emphasis of many studies on MDR in C. albicans is currently focused on drugs to reverse MDR and perhaps the most interesting targets are the efflux pumps such as Cdr1p and Cdr2p, which are major contributors to the development of resistance. The inhibition of these pumps would probably result in an increase in the intracellular concentration of drugs such as azoles, thereby increasing the effectiveness of those drugs. The aim of this study was to determine the potential of disulfiram as a modulator of Cdr1p in the antifungal therapy. The results of the present study reveal that disulfiram inhibits both ATP hydrolysis and the binding of the photoaffinity analog of ATP, 8-azidoATP, to Cdr1p. Disulfiram is known to act as a cysteine modifying agent and forms an intramolecular disulfide linkage with the cysteines present near the active site of the protein [17]. It has been shown earlier that disulfiram inhibits the ATPase activity of P-gp, MRP1, and MRP4 probably by inhibiting the formation of intramolecular disulfide linkage in the nucleotide-binding pocket of these proteins [11]. This could also be the likely reason for the inhibition of ATPase activity of Cdr1p by disulfiram. It should be noted that there are 23 cysteines present in Cdr1p. Among these, 11 cysteines are present in the predicted cytoplasmic domains of the protein [20]. There are five cysteines present in both the nucleotide-
binding domain (NBD1) and NBD2 of this protein. The presence of these cysteines in both the NBDs could probably result in disulfiram forming intramolecular disulfide linkages, which in turn would result in the loss of ATP binding. To address this issue, we used a strong reducing agent, DTT, which reduces the disulfide crosslinking between the two sulfhydryl groups. The presence of DTT reversed disulfiram-mediated inhibition of binding and hydrolysis of ATP. These observations suggest that disulfiram acts by forming disulfide bridges and that the cysteine residues in the ATP-binding domains (NBDs) of Cdr1p play a crucial role in the inhibition of Cdr1p ATPase activity by disulfiram. We used IAAP and azidopine that covalently label Cdr1p, to evaluate the effect of disulfiram on the binding of the substrates. It was observed that disulfiram also inhibits the binding of both IAAP and azidopine to Cdr1p in a concentration-dependent manner. We have shown previously that the binding of these two photoaffinity compounds to P-gp was also inhibited by disulfiram [11] and suggested that in addition to the interaction of disulfiram with the ATP sites of Cdr1p (described above), it also acts as an inhibitor that affects drug-binding sites. The binding of photoaffinity compounds to Cdr1p is specifically competed by its known substrates, i.e., IAAP binding is competed by nystatin while azidopine binding is competed out by miconazole [12]. Thus, our data suggest that disulfiram may also
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block the binding of other substrates such as miconazole and nystatin to the transporter. This inhibition of the binding of the antifungal drugs would probably result in an increase in the intracellular concentration of these drugs, thus making the drugs more effective in the therapy of candidiasis. To test this hypothesis, we checked the drug resistance profile of the Cdr1p expressing S. cerevisiae cells in the presence and absence of a non-toxic concentration of disulfiram (1 lM) and observed that the presence of disulfiram along with the antifungal drugs tested makes the cells more susceptible towards these agents (Fig. 4). The fact that the presence of disulfiram along with the other antifungal agents did not further inhibit the growth in the control AD1-8u cells suggests that the observed chemosensitization is mediated by the interaction of the disulfiram with Cdr1p. Thus, these data indicate that disulfiram inhibits the Cdr1p-mediated functions by both inhibiting ATP and substrate binding to the protein. Disulfiram has been approved as a drug and has been used for the treatment of alcoholism for over 50 years and has a well-documented pharmacology. Studies show that disulfiram has a low toxicity [21] and can be prescribed over a long period of time at a dose of 500 mg/ day. The values determined in this study for disulfiram-mediated reversal of Cdr1p function are achievable in a clinical setting and therefore may be useful in clinical practice. It is important to note that the effective concentration of disulfiram required for reversal of drug transport function of human P-gp and MRP1 and Cdr1p in S. cerevisiae is in the similar range ([11] and this work). In conclusion, our studies suggest that disulfiram can be used as a potential modulator of Cdr1pmediated drug resistance in C. albicans and may be exploited in the treatment of candidiasis.
Acknowledgments We thank Dr. Michael M. Gottesman for helpful discussions and encouragement. We also thank George Leiman for assistance in the preparation of the manuscript.
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