3 Disulfiram Potential Therapeutic Uses for-human Cancers

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2/1/08 9:isulfiram, an old drug with new potential therapeutic uses for human cancers a..... (DOI: 10.1039/b504392a)

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Journals Molecular BioSystems Contents List DOI: 10.1039/b504392a

Mol. Biosyst., 2005, 1 , 127-134 DOI: 10.1039/b504392a Review Article

Molecular BioSystems

Disulfiram, an old drug with new potential therapeutic uses for

human cancers and fungal infections

Zuben E. Sauna, Suneet Shukla and Suresh V. Ambudkar*Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes ofHealth, DHHS, Bethesda, Maryland 20892-4256.E-mail: [email protected]; Fax: (301) 435-

8188; Tel: (301) 402-4178

Received 30th March 2005, Accepted 16th May 2005

First published on the web 26th May 2005

Disulfiram, a drug used to treat alcoholism, has recently been indicated to play a primary as well as anadjuvant role in the treatment of many cancers and in the reversal of fungal drug-resistance. This reviewdiscusses the molecular mechanism of action of disulfiram and its potential use in the treatment of humancancers and fungal infections.

Introduction

Disulfiram, or tetraethylthiuram disulfide, has been used for over half a century for alcohol aversiontherapy; its pharmacokinetics have been extensively studied and it has an excellent safety record. Recentreports indicate that disulfiram and other dithiocarbamates may have a significant potential in thetreatment of human cancers. Disulfiram has been reported to induce apoptosis, shows metal ion-dependent antineoplastic activity, and arrests angiogenesis. Disulfiram has also been shown to inhibit theactivating transcription factor/cyclic-AMP-responsive element binding protein, which is implicated in thegrowth and progression of melanomas. Moreover, recent studies show that disulfiram inhibits the activityof ABC drug transport proteins, which are responsible for the development of multiple drug resistance incancer and fungal cells. Thus, disulfiram may have an important role as an adjuvant in the chemotherapyof human cancers and in the treatment of drug-resistant fungal infections. In addition to empiricalevidence, the biochemical mechanisms and cellular pathways that underlie the action of disulfiram havealso begun to emerge. Cancer is a disease involving complex networked systems that would benefit fromtreatmentsthat intervenein multiple pathways. Drugs such as disulfiram with limited toxicity andmanageable side effects that perturb several biomolecules and pathways may provide useful strategies inthe treatment of complex diseases.

Circumventing multidrug resistance: lessons learned from generations of failure

Systemic chemotherapy is extensively used as the treatment of choice in cancers that are metastatic, i.e.approximately 50% of all cancers.1However, no more than 10% of patients are cured by chemotherapy.The phenomenon of multidrug resistance (MDR) is recognized as frustrating efforts in the clinic toformulate effective chemotherapy against several blood cancers,2as well as the solid tumors associatedwith breast,3ovarian4and lower gastrointestinal tract cancers.5This resistance of cells to chemically
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diverse drugs with multiple mechanisms of action is defined as MDR. The expression of P-glycoprotein(Pgp), a product of theMDR1(ABCB1) gene, was correlated with the degree of drug resistance in severalcell lines.6,7A large body of evidence has since accumulated to strongly implicate Pgp and other energy-dependent pumps, MDR-associated protein 1 (MRP1, ABCC1) and the mitoxantrone resistance-associated protein (MXR, ABCG2) that extrude chemotherapeutic agents from cells (for a recent reviewsee 8). Pgp, MRP1 and ABCG2 are all members of the ATP-binding cassette (ABC) superfamily oftransport proteins. The ABC family of transport proteins represents one of the largest families of proteinsin living organisms.9These membrane proteins utilize the energy derived from the hydrolysis of ATP forthe vectorial transport of a variety of substrates, which include ions, sugars, lipids, peptides and naturalproduct hydrophobic anticancer drugs, across the cell membrane.10The functional unit of an ABCtransporter is a nucleotide binding domain (NBD) and one transmembrane domain comprised of sixputative transmembrane -helices.11The NBD consists of the highly conserved Walker A and B motifsand the ABC signature motif. Pgp consists of two putative transmembrane domains, each consisting of sixhydrophobic transmembrane helices and one NBD or ATP-binding domain.12While Pgp has twomembrane spanning domains, MRP1 has three.13,14Unlike Pgp and MRP1, ABCG2 has only one NBDdomain and a single membrane spanning domain.15,16The functional unit of Pgp appears to be a monomerand both its halves are necessary for function,10whereas the minimal functional unit of ABCG2 seems tobe a dimer.17,18

Besides being a significant clinical problem in the treatment of human cancers, MDR-mediated by

ABC transport proteins is rapidly gaining clinical importance in the treatment of fungal infections.Clinical antifungal resistance is not a major problem in normal patients, but immunocompromisedindividuals frequently develop systemic infections with a high incidence of antifungal resistance whichdevelops during clinical therapy.19,20The clinical therapy of systemic candidiasis, caused by the fungalpathogen Candida albicans, involves the use of antifungal azoles such as fluconazole, ketoconazole,itraconazole or voriconazole but the fungal pathogen develops resistance to these drugs, particularly afterprolonged treatment. The active extrusion of these antifungal agents by ABC transporters like Candidadrug resistance protein-P (Cdr1p and Cdr2p) is one of the major mechanisms which has been implicatedfor the development of MDR in yeast.21

Most strategies for reversing MDR have focused on the modulation of Pgp activity by usingcompounds, which are referred to as chemosensitizers or MDR modulators. The strategy involves co-

administrating the chemosensitizer with an anticancer drug.22This impairs the Pgp function, resulting inenhanced intracellular anticancer drug accumulation. Compounds belonging to chemical classes asdiverse as calcium channel blockers, calmodulin inhibitors, coronary vasodilators, alkaloids, quinolines,hormones, cyclosporines, surfactants and antibiotics have been shown to act as chemosensitizers. 10,22,23

The first generation of Pgp chemosensitizers followed the discovery by Tsuruo and coworkers 24that thecalcium channel blocker, verapamil, enhances the intracellular accumulation of many anticancer drugs.Many other calcium channel blockers tested for their ability to reverse the MDR phenotype showedcomparable results in vitro.25Besides the calcium channel blockers, the immunosuppressant, cyclosporinA, remains one of the most effective first generation MDR modulators. The major limitation to theclinical use of most first generation MDR modulators was that they typically reversed MDR atconcentrations that made them toxic.22,23,25,26The development of second-generation MDR modulators

focused on more potent and considerably less toxic analogs of the first generation agents. The in vitroandpreclinical studies suggested that PSC 833 (Valspodar), an analog of cyclosporin A, was promising.1

However, though several Phase III protocols are in progress, overall the results have not been veryencouraging23and Novartis has discontinued development of this drug. In recent years, several MDRmodulators have been developed specifically for Pgp unlike the first and second-generationchemosensitizers, which were initially developed for other disease conditions. These third generationmodulators exhibit effective MDR reversal in the 100200 nM range and include thecyclopropyldibenzosuberane LY 335979,27,28the acridonecarboxamide GF 120918,29,30thediketopiperazine XR9576,31the diarylimidazole OC144-09332and the amido-keto-pipecolinate
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derivative, VX-71033,34(see Table 1for chemical structures of selected first, second, and third generationmodulators). These agents are currently used in various clinical trials. Nonetheless, despite sustainedefforts for over 20 years, there is still no effective MDR modulator in clinical use. The most importantreason that MDR modulators fail in clinical trials is the unmanageable toxicity and adverse effects ofdoses required to reverse drug resistance.

Table 1The chemical structures of selected P-glycoprotein modulators

First generation modulators

Second generation modulators

Third generation modulators
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Using the dithiocarbamate disulfiram to sensitize drug resistant tumors to

chemotherapy

Disulfiram, first synthesized in 1881, was used to accelerate the vulcanization of rubber.35It was only in

the 1930s that disulfiram found a medicinal use as a scabiescide and, subsequently, as a vermicide36because it was toxic to lower animal forms due to its ability to chelate copper; an essential component ofthe respiratory chain of these organisms. In 1948 it was proposed that disulfiram be used in the treatmentof chronic alcoholism as alcohol aversion therapy.37This has proved to be immensely successful anddisulfiram, under the trade name Antabuse, continues to be used clinically to this day. 38Moreover,disulfiram is finding increasing use in cocaine addiction.3941There are several excellent reviews on theuse of disulfiram in narcotic addiction and due to space constraints this aspect will not be addressed here.

There has been renewed interest in disulfiram in the last few years, particularly in elucidating themechanistic basis for its action in reversal of MDR.42,43We have seen above that several generations ofMDR modulators that have been extremely successful in vitrohave failed in the clinic. Disulfiram, as areversal agent for MDR, comes with a huge advantage. Chick has reviewed a vast literature on the

hepatotoxicity, CNS adverse effects, neuropathology and drug interactions of disulfiram in the clinic.44Though monitoring of patients on high doses of disulfiram (over 250 mg per day) is recommended, 38

there are few adverse effects associated with long term treatment. Moreover, acute disulfiram overdose isuncommon. In adults, clinical manifestations after acute overdose are rare with doses less than 3 g.44Infact, when disulfiram was first introduced, doses of 13 g per day were employed 38and somepractitioners have argued that the current doses are not sufficient for all patients and have routinely used300500 mg per day.45Hepatotoxicity is the most common and serious cause for concern duringtreatment with disulfiram. Reports from Denmark, where the prescription of disulfiram is the highest inthe world, indicate that over a 22 year period fatalities associated with disulfiram induced hepatotoxicitywere 1 per 30 000 patients.46Moreover, hepatotoxicity is generally reversible if disulfiram is stoppedprior to clinical manifestation,47which can be managed using liver function tests. The toxicity data thus

suggest that, if necessary, disulfiram could be used in cancer chemotherapy at doses considerably higherthan those used in alcohol aversion therapy. The pharmacokinetics of disulfiram have also beenextensively studied and provide some clues as to its success in clinical practice. There is >80%bioavailability after an oral dose and the elimination of disulfiram and its metabolites is a very slowprocess. Approximately 20% of the drug remains in the body for 12 weeks post-ingestion. 48Consideringthe relatively non-discriminate biochemical actions of disulfiram in chelating metal ions and modifyingcysteine residues,49the excellent tolerance and absence of serious adverse events and side effects mayappear surprising. However, these are well established and undisputed clinical facts. 38,44,46

The effect of disulfiram on Pgp-mediated MDR was first reported by Loo and Clarke. 42The most
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common approach taken to disabling the molecular pumps associated with MDR has been to developcompetitive or non-competitive inhibitors that prevent the pumps from effluxing the chemotherapeuticagent.1,22,26However, Loo and Clark found that disulfiram inhibited Pgp through a novel mechanism, byinhibiting the maturation of the transporter.42Disulfiram did not inhibit the synthesis of Pgp as the totalamount of immunoreactive Pgp was the same at all concentrations of disulfiram. However, the mature,glycosylated form of the protein at the cell surface was drastically reduced. This had a physiologicalconsequence. Drug-resistant cells treated with disulfiram showed sensitivity to colchicine and vinblastinecomparable to control cells not transfected with Pgp. Moreover, disulfiram, in addition to reducing thecell surface expression, also inhibited Pgp-mediated ATP hydrolysis. Several groups have demonstratedthat disulfiram inactivates alcohol dehydrogenase by chemically modifying cysteines in the active site ofthe enzyme49,50and it appears that disulfiram may interact with the two cysteines in the Walker Adomains of both ATP sites of Pgp.42However, we observed a very interesting phenomenon when wemeasured ATP hydrolysis in the presence of disulfiram after first protecting the ATP sites with excessATP. Instead of inhibition, we observed stimulation of ATP hydrolysis.43This suggested an interactionbetween the drugsubstrate binding site of Pgp and disulfiram, which was confirmed by monitoring thedisplacement of the photoaffinity substrate analog [125I]-iodoarylazidoprazosin (IAAP) and [3H]-azidopine. In addition to Pgp, disulfiram has also been shown to interact with two other ABC transporters,MRP1 and MRP4.43The interactions of disulfiram with these ABC transporters are complex and areoutlined in Fig. 1A. When the ATP sites are accessible, disulfiram interacts with cysteines at or near the

active site, inhibits ATP hydrolysis and inactivates the protein. It must be noted that unlike compoundssuch asN-ethylmaleimide (NEM) that covalently modify a single sulfhydryl, disulfiram can formdisulfide bridges across two cysteines that lie within a distance of approximately 8 ( Fig. 1B). When theATP sites are protected, disulfiram stimulates ATP hydrolysis by Pgp (and to a limited extent by MRP1)and there is direct evidence that disulfiram also competes for the substrate-binding site of Pgp. Thetreatment of Pgp with NEM has no effect on drugsubstrate binding, while both disulfiram and NEMinhibit the binding of [ -32P]-8-azidoATP to Pgp. Thus, inhibition of drugsubstrate binding occurs by amechanism distinct from the effects of cysteine modification by disulfiram. The evidence suggests thatdisulfiram is unique in that as a single chemical moiety it neutralizes Pgp-mediated MDR by threeindependent mechanisms at two different levels. It (i) arrests Pgp maturation, (ii) inhibits the transportfunction and (iii) obstructs the ATP hydrolysis that powers drug transport.
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Fig. 1 The biochemical basis of the action of disulfiram on the ABC drug

transporters Pgp and Cdr1p.(A) Disulfiram interacts with both thetransport-substrate and ATP-binding sites of Pgp. When the ATP sites areunoccupied, disulfiram modifies cysteine residues (disulfide bond formation)at or near the ATP sites (see B below), resulting in inhibition of ATPhydrolysis. When the ATP sites are protected with excess ATP (although ATP

bound to both sites is shown, experimental evidence suggests binding of onlyone ATP molecule possibly at the inter-phase of two ATP sites), disulfiram(depicted in its chemical structure) interacts with the transport-substrate sites(in the membrane domains) and stimulates ATP hydrolysis. (B) Mechanism ofmodification of cysteines at or near the active site by disulfiram and NEM.Disulfiram first interacts with a cysteine residue to form an unstable mixeddisulfide. If there are additional cysteine moieties in the vicinity (68 ) of theunstable intermediate, it reacts with the thiol group to form an intramoleculardisulfide bond (upper panel). On the other hand, NEM covalently modifies


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cysteine residue (lower panel).

Though ATP-dependent molecular pumps that extrude anticancer agents are the principal routes bywhich cancer cells evade chemotherapy, there are several other more specific mechanisms of drugresistance. 5-Fluorouracil (5-FU) is a significant component in the treatment of colorectal cancer51andother solid tumors. Cancer cells with high nuclear activity of the transcription factor NF- B areextremely resistant to 5-FU.52,535-FU treatment activates NF- B, which makes the cells resistant to

apoptosis.

53,54

It was demonstrated by Fernandez et al. that several dithiocarbamates, including disulfiram,inhibit NF- B and induce apoptosis in T-cells.55Recently, Wang et al.demonstrated that for the 5-FUresistant colorectal cell line H6305-FU treatment with a combination of disulfiram and 5-FU resulted inanIC50(5-FU) 83-fold lower than treatment with 5-FU alone.56At the molecular level, disulfiramstrongly inhibited NF- B nuclear translocation and the binding of NF- B to DNA,56activities that arecorrelated with the antiapoptotic properties of NF- B.5759All members of the NF- B family have an N-terminal Rel homology (RH) domain that plays a central role in NF- B dimerization, nucleartranslocation and DNA binding.60The RH domain contains several sulfhydryl groups that are part of theDNA binding domain58and disulfiram and its metabolite diethyldithio-carbamate target these residues.56

The resistance of colorectal cancers to 5-FU is a very significant clinical problem54and an approach thatinvolves induction of the inhibitor of NF- B (I B) viagene therapy has had some success.61,62However,

the obstacles to the clinical fruition of this approach are considerable and a small molecule approachusing disulfiram is considered the more viable strategy.56

Disulfiram as an antifungal agent

Diethyldithiocarbamate and the two different substituted analogs of this compound were evaluated fortheir anticandidial effect.63These compounds were tested for their in vitroinhibitory effect on the growthof Candidastrains and it was observed that sodium diethyldithio-carbamte (DDTC) and sodiumdimethylthiocarbamate (DMDTC) produced inhibitory effects comparable to amphotericin-B, a drugclinically used to treat candidiasis. The in vivoeffects of these dithiocarbamates were also encouragingwithN-methyl-D-glucamine dithiocarbamate (NMGDTC) being the most effective. Furthermore, the

effectiveness of the dithiocarbamates was considerably enhanced if the infected mice wereimmunosuppressed with cisplatin. Finally, combinatorial therapy using dithiocarbamates withamphotericin-B had advantages over the use of the either agent alone. A systematic study testing sixchemically synthesized dithiocarbamate derivatives on 40 clinical isolates of Candida64demonstrated thatDDTC, DMDT, and sodium pyrrolidene dithiocarbamate (PYRDTC) showed significant inhibitoryeffects. Based on early experience with the use of disulfiram as a scabiescide and vermicide, it wassuggested that the dithiocarbamates exert their antifungal action by chelating metals that are indispensablecomponents of the respiratory chains of lower organisms. To test this hypothesis, excess metal ions wereadded to chelate the dithiocarbamates. This resulted in only partial reversal of their antifungal activity,which suggests that additional inhibitory mechanisms for dithiocarbamates may exist. These couldinclude interaction with the ABC multidrug transporters like Cdr1p and Cdr2p, which have beenimplicated in the development of fungal drug resistance.65,66In a screen to determine the drug resistance

profile of the ABC transporters of the yeast pleiotropic drug resistance network in S. cerevisiae, it wasfound that the ABC-MDR transporters mediate resistance to classes of clinically and agriculturallyimportant fungicides and dithiocarbamates, which were identified as the compounds to which the Pdr5p,Snq2p or Yor1p, the yeast multidrug transporters, confer resistance. 66This suggests that the ABCmultidrug transporters from S. cerevisiaeinteract with dithiocarbamates and a direct interaction ofdisulfiram with the known ABC transporter in C. albicans, Cdr1p, was recently demonstrated.67

Cdr1p has been shown to be involved in the energy-dependent efflux of antifungal compounds likefluconazole, miconazole, itraconazole and nystatin. We showed that disulfiram can be used as an inhibitor
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of Cdr1p-mediated drug resistance.67Disulfiram inhibited the binding of both [125I]-IAAP and [3H]-azidopine to Cdr1p, suggesting a direct interaction with substrate binding sites of the transporter.Moreover, disulfiram inhibited Cdr1p-mediated ATP hydrolysis, which was reversed by dithiothreiotol.These studies suggest that fungal and mammalian ABC transport proteins are inhibited by disulfiram viacomparable mechanisms. The inhibition of the Cdr1p activity by disulfiram would suggest that it could beused in combination with antifungal agents to reverse the drug resistance mediated by Cdr1p. Therefore,we checked the ability of disulfiram to modulate the drug resistance mediated by Cdr1p in S. cerevisiae.We found that the presence of the non-toxic concentration of disulfiram with the other antifungal agents

made the cells more susceptible to the drugs, which indicates the potential usefulness of disulfiram inantifungal therapy to sensitize drug resistant cells.

Use of disulfiram as an anticancer agent

Reviewing the status of cancer research over the past 25 years, Hanahan and Weinberg 68describe thescientific literature as complex beyond measure but argue that new progress will now come from beingable to understand cancer in terms of a small number of underlying principles. They devote their essay tosketching a broad outline of such principles and identify six traits that most, if not all, cancers haveacquired: sustained angiogenesis, evading apoptosis, tissue invasion and metastasis, limitless replicativepotential, self sufficiency in growth signals and insensitivity to antigrowth signals. It is interesting that

disulfiram has been shown to directly impact at least three of these traitsinducing apoptosis, acting asan antiangiogenesis agent and preventing tissue invasiveness and metastasis (Fig. 2).6974This of courseis in addition to a very significant role in synergistically enhancing the potency of anticancer drugs bydisabling MDR pumps.42,43
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Fig. 2 Potential therapeutic targets for disulfiram in a cancer cell.Illustration showing some of theinteractions demonstrated between disulfiram and cellular mechanisms implicated in human cancers. Seetext for details of the mechanisms. DS, disulfiram; 5-FU, 5-fluorouracil.

Disulfiram-mediated apoptosis has been shown to occur in thymocytes,75smooth muscle cells76andhuman hepatoma cells.77There is evidence that disulfiram induces p53.78,79The modulation of p53 occursat an early stage in the apoptotic pathway, resulting in inhibition of bcl-279and subsequent apoptosis. Ithas also been suggested that cell death induced by disulfiram is the result of transport of the Cu2+ion intothe cell.75The apoptotic effects of disulfiram have, however, generated some controversy as severalgroups have found that dicarbamates can be inhibitors of apoptosis,74,8082as they inhibit TNF- , itself apotent inducer of apoptosis.73A recent report, however, claims that the two effects occur at widely

separated time scales,74suggesting that the metabolites of disulfiram may have a more significant role inthese processes than disulfiram alone.In addition to inducing apoptosis, disulfiram has been shown to directly inhibit the growth of cancer

cells both in vitroand in vivo70,83and divalent metal ions (especially Cu2+) have been shown to enhanceits antineoplastic activity.69Cysteine residues are located at critical DNA binding regions of transcriptionfactors8486and DNA transcription factor binding is severely disrupted when these cysteines are modifiedby the formation of mixed disulfides. It has been directly demonstrated that disulfiram mediates theinhibition of the activating transcription factor/cyclic-AMP-responsive element binding protein(ATF/CREB), which is potentiated by Cu2+ions.70This had the effect of arresting the growth of
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malignant melanoma, which are notoriously resistant to chemotherapeutic agents.87A recent reportillustrates the potential for this strategy, where the combination of oral zinc gluconate and disulfiraminduced reduction in hepatic metastases and clinical remission in a patient with stage IV metastatic ocularmelanoma.70This study also clearly shows that disulfiram is a safe drug, as continuous therapy for 53months had negligible side effects.

Finally, disulfiram has been suggested to show antiangiogenic activity.71,72,88The mechanismsunderlying disulfiram-mediated inhibition of angiogenesis have recently been elucidated by Shiah et al.71

Degradation of the extracellular matrix precedes angiogenesis and cell invasion, which in turn areprerequisites for cancer metastasis.89,90This degradation of the extracellular matrix is affected by thematrix metalloproteinases.91,92Disulfiram was shown to be an effective inhibitor of the matrixmetalloproteinases and blocked angiogenesis in vivoin ten day old chicken embryos.71While it has beenwell established that metalloproteinases-inhibitors prevent angiogenesis, tumor dissemination andmetastasis transition to the clinic has been fraught with difficulties. Two favored candidates were TIMPs(tissue inhibitors of metalloproteinases), which are naturally occurring proteins, 93and BB-94 (batimastat),a synthetic low molecular weight inhibitor.94However, TIMPs have an extremely short half-life in vivoand are not promising for clinical applications while BB-94 has very poor solubility and has to beinjected as a detergent emulsion.94

Conclusions and future outlook

Investigating the role of disulfiram in the treatment of human cancers may at first glance seem anunrealistic proposition. In an era of molecular modeling and rational drug design, we would beinvestigating a 125 year old compound developed for the vulcanization of rubber. Disulfiram has theadded disadvantage that its effects on biomolecules appear to be non-discriminatory. On the other hand,disulfiram has a 75 year history in medicine, is a commercially available product (as Antabuse ) with aknown pharmacology profile and over 50 years of clinical experience. The normal adult dose ofdisulfiram in the treatment of alcoholism is 500 mg per day, which translates to an approximateconcentration of 20 M in a 100 kg individual. This is a concentration that is sufficient to elicit the invitroresponses of disulfiram. The last decade has seen numerous reports suggesting disulfiram may playa very useful role in the treatment of human cancers. Disulfiram does have very significant advantages: it

is a drug with a very good safety record and has excellent bioavailability. Both these factors are of criticalimportance. Several MDR modulators have failed in the clinic due to unmanageable side effects.8,22,26,95

Similarly, the lack of bioavailability has limited the use of drugs that inhibit angiogenesis and tumorprogression.93,94Also, sulfhydryl-reacting agents, despite their apparently unspecific mode of action,provide useful drugs against human diseases96and disulfiram shows remarkably few side-effects and isnot toxic, even with prolonged use.44,45,70,97Recent investigations have applied a range of biochemicaltechniques to understand the mechanistic basis of the specific actions of disulfiram. However, as thisreview enumerates, the actions of disulfiram are extremely diverse and this drug may have multipletherapeutic targets. This ties in with the emerging paradigm in systems biology: human cells and tissuesare a complex networked system. Thus, a drug discovery approach (especially for complex diseases suchas cancer) necessitates identification of combinations of small molecules that perturb cellular networks in

a desired fashion.98100While combinations of synergistic compounds have been exploited for decades,these are generally limited to agents known to be effective in a therapeutic area or to agents with a clearrationale for the combination. Systematic discovery based on systems biology, however, advocates amuch wider combination space. Borisy et al.101have designed a robotic screening and informatics systemto explore such combinatorial spaces. They implemented this system to test the inhibition of fluconazoleresistant C. albicanswith 30 drugs in pair-wise combinations. It is interesting that of the 435 possibletwo-component combinations, only six effective combinations emerged, of which two had disulfiram asone of the agents.101Empirical studies such as these are complementary to the mechanistic studies thatelucidate the interaction of disulfiram with specific cellular and molecular pathways. Taken together,
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these studies argue for more animal research and clinical trials to achieve the full therapeutic promise ofthis fascinating compound.

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