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• W2016292622 abstract "The prodrug nifurtimox has been used for more than 40 years to treat Chagas disease and forms part of a recently approved combinational therapy that targets West African trypanosomiasis. Despite this, its mode of action is poorly understood. Detection of reactive oxygen and nitrogen intermediates in nifurtimox-treated extracts led to the proposal that this drug induces oxidative stress in the target cell. Here, we outline an alternative mechanism involving reductive activation by a eukaryotic type I nitroreductase. Several enzymes proposed to metabolize nifurtimox, including prostaglandin F2α synthase and cytochrome P450 reductase, were overexpressed in bloodstream-form Trypanosoma brucei. Only cells with elevated levels of the nitroreductase displayed altered susceptibility to this nitrofuran, implying a key role in drug action. Reduction of nifurtimox by this enzyme was shown to be insensitive to oxygen and yields a product characterized by LC/MS as an unsaturated open-chain nitrile. This metabolite was shown to inhibit both parasite and mammalian cell growth at equivalent concentrations, in marked contrast to the parental prodrug. These experiments indicate that the basis for the selectivity of nifurtimox against T. brucei lies in the expression of a parasite-encoded type I nitroreductase. The prodrug nifurtimox has been used for more than 40 years to treat Chagas disease and forms part of a recently approved combinational therapy that targets West African trypanosomiasis. Despite this, its mode of action is poorly understood. Detection of reactive oxygen and nitrogen intermediates in nifurtimox-treated extracts led to the proposal that this drug induces oxidative stress in the target cell. Here, we outline an alternative mechanism involving reductive activation by a eukaryotic type I nitroreductase. Several enzymes proposed to metabolize nifurtimox, including prostaglandin F2α synthase and cytochrome P450 reductase, were overexpressed in bloodstream-form Trypanosoma brucei. Only cells with elevated levels of the nitroreductase displayed altered susceptibility to this nitrofuran, implying a key role in drug action. Reduction of nifurtimox by this enzyme was shown to be insensitive to oxygen and yields a product characterized by LC/MS as an unsaturated open-chain nitrile. This metabolite was shown to inhibit both parasite and mammalian cell growth at equivalent concentrations, in marked contrast to the parental prodrug. These experiments indicate that the basis for the selectivity of nifurtimox against T. brucei lies in the expression of a parasite-encoded type I nitroreductase. IntroductionAcross the tropics, 10 million people are infected by the parasites Trypanosoma cruzi and Trypanosoma brucei, the causative agents of Chagas disease and human African trypanosomiasis, respectively (1Brun R. Blum J. Chappuis F. Burri C. Lancet. 2010; 375: 148-159Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 2Rassi Jr., A. Rassi A. Marin-Neto J.A. Lancet. 2010; 375: 1388-1402Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar). They are responsible for 60,000 deaths per year and represent a major public health problem in regions of the world least able to deal with the associated economic burden. Their primary route of transmission is by blood-sucking insect vectors. However, other pathways, notably blood transfusion, organ transplantation, and illicit drug usage coupled with population migration, have resulted in both infections becoming problematic in non-endemic areas (3Bern C. Montgomery S.P. Clin. Infect. Dis. 2009; 49: e52-e54Crossref PubMed Scopus (437) Google Scholar, 4Gascon J. Bern C. Pinazo M.J. Acta Trop. 2010; 115: 22-27Crossref PubMed Scopus (438) Google Scholar, 5Gautret P. Clerinx J. Caumes E. Simon F. Jensenius M. Loutan L. Schlagenhauf P. Castelli F. Freedman D. Miller A. Bronner U. Parola P. Euro Surveill. 2009; 14 (pii=19327)Crossref PubMed Google Scholar). Chagas disease is an emerging problem with as many as 300,000 people in the United States infected by T. cruzi, and blood supplies are now routinely screened for the parasite. Current treatment of both infections is restricted to a series of drugs whose mode(s) of action are poorly understood (6Wilkinson S.R. Kelly J.M. Expert Rev. Mol. Med. 2009; 11: e31Crossref PubMed Scopus (173) Google Scholar). Establishing how existing therapies work could contribute to more effective treatment and aid drug development.The 5-nitrofuran drug nifurtimox has been used for more than 40 years to treat Chagas disease. Its use is controversial as it is toxic, reportedly carcinogenic, and of limited efficacy. Additionally, T. cruzi strains refractory to treatment have been isolated, a situation compounded by failure to complete the recommended drug schedules. Nifurtimox drug regimes can take up to 4 months and are frequently associated with unpleasant side effects. Despite these problems, nifurtimox, as part of the nifurtimox-eflornithine combination therapy, is now recommended for late-stage West African sleeping sickness (7Priotto G. Fogg C. Balasegaram M. Erphas O. Louga A. Checchi F. Ghabri S. Piola P. PLoS Clin. Trials. 2006; 1: e39Crossref PubMed Google Scholar, 8Priotto G. Kasparian S. Mutombo W. Ngouama D. Ghorashian S. Arnold U. Ghabri S. Baudin E. Buard V. Kazadi-Kyanza S. Ilunga M. Mutangala W. Pohlig G. Schmid C. Karunakara U. Torreele E. Kande V. Lancet. 2009; 374: 56-64Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) and is also undergoing assessment as a treatment for pediatric neuroblastoma (9Saulnier Sholler G.L. Kalkunte S. Greenlaw C. McCarten K. Forman E. J. Pediatr. Hematol. Oncol. 2006; 28: 693-695Crossref PubMed Scopus (33) Google Scholar, 10Saulnier Sholler G.L. Brard L. Straub J.A. Dorf L. Illeyne S. Koto K. Kalkunte S. Bosenberg M. Ashikaga T. Nishi R. J. Pediatr. Hematol. Oncol. 2009; 31: 187-193Crossref PubMed Scopus (34) Google Scholar).As with most nitroheterocyclic agents, nifurtimox functions as a prodrug and must undergo activation by nitroreduction. Two classes of enzyme can catalyze this process, the type I and type II nitroreductases (11Peterson F.J. Mason R.P. Hovsepian J. Holtzman J.L. J. Biol. Chem. 1979; 254: 4009-4014Abstract Full Text PDF PubMed Google Scholar). Type I nitroreductases are NAD(P)H-dependent, FMN binding proteins commonly found in bacteria but rare in eukaryotes. They mediate a two-electron reduction of the nitro group to generate a nitroso intermediate (Reaction 1) that rapidly undergoes reduction to a hydroxylamine derivative (Reaction 2):RNO2+2e−→RNORNO+2e−→RNHOHREACTION 1 AND 2For nitrofuran compounds, the hydroxylamine can then be processed further to generate either the amine, which is believed to be inert, or nitrenium cations that promote DNA breakage (12McCalla D.R. Reuvers A. Kaiser C. Cancer Res. 1971; 31: 2184-2188PubMed Google Scholar, 13Streeter A.J. Hoener B.A. Pharm. Res. 1988; 5: 434-436Crossref PubMed Scopus (23) Google Scholar, 14Beckett A.H. Robinson A.E. J. Med. Pharm. Chem. 1959; 1: 155-164Crossref PubMed Scopus (22) Google Scholar). Alternatively, fragmentation of the furan ring may occur, yielding open-chain nitriles (11Peterson F.J. Mason R.P. Hovsepian J. Holtzman J.L. J. Biol. Chem. 1979; 254: 4009-4014Abstract Full Text PDF PubMed Google Scholar, 15Gavin J.J. Ebetino F.F. Freedman R. Waterbury W.E. Arch. Biochem. Biophys. 1966; 113: 399-404Crossref PubMed Scopus (28) Google Scholar, 16Swaminathan S. Bryan G.T. Cancer Res. 1984; 44: 2331-2338PubMed Google Scholar). Because reduction by type I nitroreductase does not involve oxygen and does not result in the production of reactive oxygen species, this activity is said to be “oxygen-insensitive.”In contrast, the ubiquitous type II nitroreductases contain FMN or FAD as a co-factor, and their activity is “oxygen-sensitive.” They catalyze the one-electron reduction of a substrate, forming a nitro anion radical (Reaction 3) (17Mason R.P. Holtzman J.L. Biochemistry. 1975; 14: 1626-1632Crossref PubMed Scopus (165) Google Scholar). In the presence of oxygen, this radical undergoes futile cycling, resulting in the production of superoxide anions and regeneration of the parent nitro-compound (Reaction 4) (18Mason R.P. Holtzman J.L. Biochem. Biophys. Res. Commun. 1975; 67: 1267-1274Crossref PubMed Scopus (242) Google Scholar).RNO2+e−→RNO2.−RNO2.−+O2→RNO2+O2.−REACTION 3 AND 4Following observations that nifurtimox-treated trypanosomal extracts generated superoxide anions and nitro anion radicals, it was proposed that this compound mediated its activity through induction of oxidative stress in reactions catalyzed by type II nitroreductases (19Docampo R. Stoppani A.O. Arch. Biochem. Biophys. 1979; 197: 317-321Crossref PubMed Scopus (116) Google Scholar, 20Docampo R. Mason R.P. Mottley C. Muniz R.P. J. Biol. Chem. 1981; 256: 10930-10933Abstract Full Text PDF PubMed Google Scholar, 21Viodé C. Bettache N. Cenas N. Krauth-Siegel R.L. Chauvière G. Bakalara N. Périé J. Biochem. Pharmacol. 1999; 57: 549-557Crossref PubMed Scopus (150) Google Scholar). Several flavoproteins, including dihydrolipoamide dehydrogenase, cytochrome P450 reductase, and trypanothione reductase, were subsequently shown to mediate the one-electron reduction of nifurtimox in vitro (21Viodé C. Bettache N. Cenas N. Krauth-Siegel R.L. Chauvière G. Bakalara N. Périé J. Biochem. Pharmacol. 1999; 57: 549-557Crossref PubMed Scopus (150) Google Scholar, 22Schöneck R. Billaut-Mulot O. Numrich P. Ouaissi M.A. Krauth-Siegel R.L. Eur. J. Biochem. 1997; 243: 739-747Crossref PubMed Scopus (56) Google Scholar, 23Blumenstiel K. Schöneck R. Yardley V. Croft S.L. Krauth-Siegel R.L. Biochem. Pharmacol. 1999; 58: 1791-1799Crossref PubMed Scopus (92) Google Scholar). To date there is insufficient functional evidence to suggest that this occurs in vivo and that trypanosomes overexpressing trypanothione reductase display the same susceptibility to nifurtimox as control cells (24Kelly J.M. Taylor M.C. Smith K. Hunter K.J. Fairlamb A.H. Eur. J. Biochem. 1993; 218: 29-37Crossref PubMed Scopus (76) Google Scholar). The only experimental evidence for superoxide anion involvement in nifurtimox toxicity is indirect and comes from studies on the trypanosomal superoxide dismutase repertoire. T. brucei TbSODB1 null mutants displayed enhanced sensitivity to nifurtimox (25Prathalingham S.R. Wilkinson S.R. Horn D. Kelly J.M. Antimicrob. Agents Chemother. 2007; 51: 755-758Crossref PubMed Scopus (36) Google Scholar). The same pattern of redox cycling seen in T. cruzi is observed in mammalian cells, and the selectivity of this prodrug toward the parasite is not explained by this model. Despite these issues, oxidative stress resulting from type II nitroreductase activity has been generally accepted as the main trypanocidal mechanism of nifurtimox, although this has been questioned (26Boiani M. Piacenza L. Hernández P. Boiani L. Cerecetto H. González M. Denicola A. Biochem. Pharmacol. 2010; 79: 1736-1745Crossref PubMed Scopus (96) Google Scholar, 27Wilkinson S.R. Taylor M.C. Horn D. Kelly J.M. Cheeseman I. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5022-5027Crossref PubMed Scopus (316) Google Scholar, 28Baker N. Alsford S. Horn D. Mol. Biochem. Parasitol. 2011; 176: 55-57Crossref PubMed Scopus (94) Google Scholar).Recently, an alternative activation mechanism has been proposed following the identification of two trypanosomal enzymes that can catalyze the two-electron reduction of nifurtimox (27Wilkinson S.R. Taylor M.C. Horn D. Kelly J.M. Cheeseman I. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5022-5027Crossref PubMed Scopus (316) Google Scholar, 29Kubata B.K. Kabututu Z. Nozaki T. Munday C.J. Fukuzumi S. Ohkubo K. Lazarus M. Maruyama T. Martin S.K. Duszenko M. Urade Y. J. Exp. Med. 2002; 196: 1241-1251Crossref PubMed Scopus (101) Google Scholar). One, a prostaglandin F2α synthase, also known as the Old Yellow Enzyme, expressed by T. cruzi, can reduce the nitrofuran in vitro, but oxidoreductase activity only occurs under anaerobic conditions. The contribution, if any, that this enzyme makes to drug metabolism within the parasite has not been established. The second pathway involves a trypanosomal type I nitroreductase (NTR) 2The abbreviations used are: NTR, trypanosomal type I nitroreductase; ESI, electrospray ionization; TbCPR2 and TbCPR3, Trypanosoma brucei cytochrome P450 reductase 2 and 3, respectively; TbNTR, Trypanosoma brucei type I nitroreductase; TbPGS, Trypanosoma brucei prostaglandin F2α synthase; TcNTR, Trypanosoma cruzi type I nitroreductase. that displays characteristics shown by many of its bacterial homologues; the activity is oxygen-insensitive, the enzyme contains FMN as co-factor, and it can metabolize a wide range of nitro- and quinone-based compounds. Using T. cruzi and T. brucei lines with altered levels of this enzyme, a clear link between this nitroreductase activity and nifurtimox activation has been demonstrated (27Wilkinson S.R. Taylor M.C. Horn D. Kelly J.M. Cheeseman I. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5022-5027Crossref PubMed Scopus (316) Google Scholar). Null mutant/heterozygous cells display resistance to various nitroheterocyclic agents, including nifurtimox, whereas overexpression confers hypersensitivity. Additionally, T. cruzi selected for resistance to nifurtimox were found to have lost one copy of the chromosome containing the TcNTR gene.The aim of this work was to determine the role of NTRs in nifurtimox action. We show that although low levels of oxygen consumption can be detected during nifurtimox reduction, NTRs are predominantly oxygen-insensitive enzymes that catalyze the four-electron reduction of the nitrofuran under both aerobic and anaerobic conditions. This produces an unsaturated open-chain nitrile that displays equivalent cytotoxicity toward mammalian and parasite cells, unlike the parental prodrug.DISCUSSIONThe mode of action and basis of selectivity of the anti-trypanosomal compound nifurtimox has been an enigma since its introduction as a therapy for Chagas disease. In this article we provide evidence showing that trypanosomal type I nitroreductases catalyze reduction of this prodrug to an unsaturated open-chain nitrile derivative (FIGURE 4, FIGURE 5) and that the product, unlike the nitrofuran precursor, displays equivalent growth inhibitory properties against parasite and mammalian cells (Fig. 6). Together, these findings demonstrate that selective toxicity of nifurtimox may be attributable to type I nitroreductase expression by trypanosomes and indicate that this prodrug mediates a significant component of its anti-parasitic activity by a mechanism other than induction of oxidative stress.Activation of nitrofurans can occur by two routes, involving the two-electron reduction of the compound, reactions mediated by type I nitroreductases, or a one-electron pathway, as catalyzed by type II enzymes. Bacteria express both activities, but it is the type I system that underpins the selectivity of nitrofuran antibiotics (39McCalla D.R. Kaiser C. Green M.H. J. Bacteriol. 1978; 133: 10-16Crossref PubMed Google Scholar, 40Sastry S.S. Jayaraman R. Mol. Gen. Genet. 1984; 196: 379-380Crossref PubMed Scopus (14) Google Scholar, 41Whiteway J. Koziarz P. Veall J. Sandhu N. Kumar P. Hoecher B. Lambert I.B. J. Bacteriol. 1998; 180: 5529-5539Crossref PubMed Google Scholar). Prokaryotes selected for resistance to nitrofurazone invariably acquire mutations in their type I nitroreductase genes. Following activation, the precise antimicrobial action of these prodrugs is largely unknown, but it is believed to rely on production of toxic intermediate metabolites that ultimately trigger DNA damage and cell death (12McCalla D.R. Reuvers A. Kaiser C. Cancer Res. 1971; 31: 2184-2188PubMed Google Scholar, 13Streeter A.J. Hoener B.A. Pharm. Res. 1988; 5: 434-436Crossref PubMed Scopus (23) Google Scholar). In contrast, nitrofuran reduction in eukaryotic cells is generally mediated by enzymes with a type II activity, leading to nitro anion radical formation. In an aerobic environment, the radical then undergoes futile cycling, resulting in regeneration of the parental nitro-compound and production of reactive oxygen species (11Peterson F.J. Mason R.P. Hovsepian J. Holtzman J.L. J. Biol. Chem. 1979; 254: 4009-4014Abstract Full Text PDF PubMed Google Scholar, 17Mason R.P. Holtzman J.L. Biochemistry. 1975; 14: 1626-1632Crossref PubMed Scopus (165) Google Scholar, 19Docampo R. Stoppani A.O. Arch. Biochem. Biophys. 1979; 197: 317-321Crossref PubMed Scopus (116) Google Scholar, 36Moreno S.N. Mason R.P. Docampo R. J. Biol. Chem. 1984; 259: 6298-6305Abstract Full Text PDF PubMed Google Scholar). The latter may then induce a state of oxidative stress in the target cell. Mammalian cells do express enzymes that can mediate the two-electron reduction of nitroaromatic compounds (42Boland M.P. Knox R.J. Roberts J.J. Biochem. Pharmacol. 1991; 41: 867-875Crossref PubMed Scopus (106) Google Scholar, 43Wu K. Knox R. Sun X.Z. Joseph P. Jaiswal A.K. Zhang D. Deng P.S. Chen S. Arch. Biochem. Biophys. 1997; 347: 221-228Crossref PubMed Scopus (127) Google Scholar, 44Cenas N. Prast S. Nivinskas H. Sarlauskas J. Arnér E.S. J. Biol. Chem. 2006; 281: 5593-5603Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), but these activities are generally low in normal tissues and elevated in tumor cells, a difference that has been exploited in the development of anti-cancer therapies (45Jaiswal A.K. Free Radic. Biol. Med. 2000; 29: 254-262Crossref PubMed Scopus (380) Google Scholar). Whether such eukaryotic enzymes play a role in nitrofuran metabolism has not been addressed.The predominance of type II nitroreductases in eukaryotic cells, coupled with observations that nifurtimox-treated trypanosome extracts generated superoxide anions and nitro anion radicals, supported the proposal that parasite killing activity was mediated by the induction of oxidative stress. As a consequence, the presence of trypanosomal type I enzymes and their role in nitrofuran activation has been largely overlooked. However, following the identification of NTR and demonstration of its key role in nitroaromatic drug toxicity, the mechanism of nifurtimox action needs to be re-evaluated (27Wilkinson S.R. Taylor M.C. Horn D. Kelly J.M. Cheeseman I. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5022-5027Crossref PubMed Scopus (316) Google Scholar). Against this backdrop, we overexpressed prostaglandin F2α synthase and two cytochrome P450 reductases, enzymes reported to interact with nifurtimox (29Kubata B.K. Kabututu Z. Nozaki T. Munday C.J. Fukuzumi S. Ohkubo K. Lazarus M. Maruyama T. Martin S.K. Duszenko M. Urade Y. J. Exp. Med. 2002; 196: 1241-1251Crossref PubMed Scopus (101) Google Scholar, 34Portal P. Villamil S.F. Alonso G.D. De Vas M.G. Flawiá M.M. Torres H.N. Paveto C. Mol. Biochem. Parasitol. 2008; 160: 42-51Crossref PubMed Scopus (22) Google Scholar) and demonstrated that elevation of their levels in bloodstream-form T. brucei had no effect on toxicity (Fig. 1). In contrast, the central role of NTR in this process was clearly demonstrated, as parasites with elevated levels were hypersensitive to nifurtimox (Fig. 1) in agreement with our previous work (27Wilkinson S.R. Taylor M.C. Horn D. Kelly J.M. Cheeseman I. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5022-5027Crossref PubMed Scopus (316) Google Scholar, 32Hall B.S. Wu X. Hu L. Wilkinson S.R. Antimicrob. Agents Chemother. 2010; 54: 1193-1199Crossref PubMed Scopus (51) Google Scholar, 35Bot C. Hall B.S. Bashir N. Taylor M.C. Helsby N.A. Wilkinson S.R. Antimicrob. Agents Chemother. 2010; 54: 4246-4252Crossref PubMed Scopus (40) Google Scholar). Additionally, in a whole-genome “loss of function” screen using nifurtimox against an induced T. brucei RNAi library, the only hits generated targeted the TbNTR transcript (28Baker N. Alsford S. Horn D. Mol. Biochem. Parasitol. 2011; 176: 55-57Crossref PubMed Scopus (94) Google Scholar). Therefore, NTR remains the only trypanosomal enzyme with a confirmed role in nifurtimox activation within the parasite.The ability of NTR to reduce nitroheterocyclic compounds, including nifurtimox, under aerobic conditions indicates that this enzyme is a type I nitroreductase. Our observation that nifurtimox metabolism was slightly more efficient under anaerobic than aerobic conditions and the low level of oxygen consumption (Fig. 2) are fully consistent with the activity reported for Escherichia coli oxygen-insensitive enzymes, previously ascribed to protein impurities (11Peterson F.J. Mason R.P. Hovsepian J. Holtzman J.L. J. Biol. Chem. 1979; 254: 4009-4014Abstract Full Text PDF PubMed Google Scholar). The effect of oxygen on activity may reflect that certain intermediaries generated by type I catalysis are prone to oxidation (46Nivinskas H. Koder R.L. Anusevicius Z. Sarlauskas J. Miller A.F. Cenas N. Arch. Biochem. Biophys. 2001; 385: 170-178Crossref PubMed Scopus (58) Google Scholar). Some hydroxylamines can be oxidized when exposed to air. Alternatively, the type I/type II dichotomy may be less absolute than previously thought. In any case, it is clear that the predominant mode of NTR action is via a two-electron reduction pathway.Several different reaction products have been identified from the type I-mediated reduction of nitrofurans, including amines and open-chain nitriles (11Peterson F.J. Mason R.P. Hovsepian J. Holtzman J.L. J. Biol. Chem. 1979; 254: 4009-4014Abstract Full Text PDF PubMed Google Scholar, 15Gavin J.J. Ebetino F.F. Freedman R. Waterbury W.E. Arch. Biochem. Biophys. 1966; 113: 399-404Crossref PubMed Scopus (28) Google Scholar, 16Swaminathan S. Bryan G.T. Cancer Res. 1984; 44: 2331-2338PubMed Google Scholar). However, in many cases these are reported to be biologically inactive. Instead, prodrug toxicity is believed to occur via formation of protein and nucleic acid adducts with reactive intermediates such as the hydroxylamine or nitrenium ions (12McCalla D.R. Reuvers A. Kaiser C. Cancer Res. 1971; 31: 2184-2188PubMed Google Scholar, 13Streeter A.J. Hoener B.A. Pharm. Res. 1988; 5: 434-436Crossref PubMed Scopus (23) Google Scholar, 47Swaminathan S. Lower Jr., G.M. Bryan G.T. Cancer Res. 1982; 42: 4479-4484PubMed Google Scholar). Here, we demonstrated that the nitrile product generated from nifurtimox is toxic to bloodstream-form trypanosomes, displaying an IC50 value comparable with that of the parent compound (Fig. 6). Additionally, unlike nifurtimox itself, the metabolite also demonstrated significant growth inhibitory activity against a cultured mammalian cell line. For most nitrofurans, reduction to the open-chain nitrile is reported to go to completion, with the biologically inactive saturated form being the main end product. For nifurtimox reduction this is not the case, and the major open-chain nitrile formed is in the unsaturated state. One explanation for why other groups have not detected significant quantities of the unsaturated open-chain nitrile is that products were isolated after prolonged incubation (>48 h) or were derived from animals during pharmacological testing (15Gavin J.J. Ebetino F.F. Freedman R. Waterbury W.E. Arch. Biochem. Biophys. 1966; 113: 399-404Crossref PubMed Scopus (28) Google Scholar, 16Swaminathan S. Bryan G.T. Cancer Res. 1984; 44: 2331-2338PubMed Google Scholar), whereas in our system, LC/MS analysis of the nifurtimox-derived material was performed within 60 min of initiating the reaction. However, even after 24 h, the unsaturated open-chain nitrile product generated from nifurtimox was still the major peak in traces, with only small amounts of the saturated form detectable. This indicates that the unsaturated open-chain nitrile derived from this trypanocidal prodrug is relatively stable, and this stability may explain why nifurtimox is an effective anti-parasitic agent despite being a poor substrate for NTRs compared with other nitroheterocyclic compounds (32Hall B.S. Wu X. Hu L. Wilkinson S.R. Antimicrob. Agents Chemother. 2010; 54: 1193-1199Crossref PubMed Scopus (51) Google Scholar, 35Bot C. Hall B.S. Bashir N. Taylor M.C. Helsby N.A. Wilkinson S.R. Antimicrob. Agents Chemother. 2010; 54: 4246-4252Crossref PubMed Scopus (40) Google Scholar). As unsaturated open-chain nitriles have rarely been observed following nitroreduction, their role in nitrofuran toxicity has been overlooked, and the mechanism of action is unknown. The unsaturated open-chain nitrile derived from nifurtimox has the potential to function as a Michael acceptor and could react non-specifically with a range of cellular components. This may explain the pleiotropic effects of nifurtimox on trypanosomes, where treatment has been reported to inhibit various enzyme activities, modify thiol levels, and cause DNA damage (21Viodé C. Bettache N. Cenas N. Krauth-Siegel R.L. Chauvière G. Bakalara N. Périé J. Biochem. Pharmacol. 1999; 57: 549-557Crossref PubMed Scopus (150) Google Scholar, 48Maya J.D. Repetto Y. Agosín M. Ojeda J.M. Tellez R. Gaule C. Morello A. Mol. Biochem. Parasitol. 1997; 86: 101-106PubMed Google Scholar, 49Goijman S.G. Frasch A.C. Stoppani A.O. Biochem. Pharmacol. 1985; 34: 1457-1461Crossref PubMed Scopus (24) Google Scholar, 50Goijman S.G. Stoppani A.O. Biochem. Pharmacol. 1985; 34: 1331-1336Crossref PubMed Scopus (16) Google Scholar, 51Jockers-Scherübl M.C. Schirmer R.H. Krauth-Siegel R.L. Eur. J. Biochem. 1989; 180: 267-272Crossref PubMed Scopus (148) Google Scholar).We have now shown that nifurtimox reduction by a trypanosomal type I nitroreductase leads to formation of a toxic product. The precise mode of action of the resultant unsaturated open-chain nitrile product remains to be determined, but given that it is equally toxic to mammalian and parasite cells, it is plausible that the downstream targets are common to both host and pathogen. If this is the case, then the basis of nifurtimox selectivity is due to expression of the type I nitroreductase in the parasite. Understanding how nifurtimox exerts its trypanocidal effects may lead to the development of novel anti-parasitic drugs that utilize the bioreductive activity of this enzyme. IntroductionAcross the tropics, 10 million people are infected by the parasites Trypanosoma cruzi and Trypanosoma brucei, the causative agents of Chagas disease and human African trypanosomiasis, respectively (1Brun R. Blum J. Chappuis F. Burri C. Lancet. 2010; 375: 148-159Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 2Rassi Jr., A. Rassi A. Marin-Neto J.A. Lancet. 2010; 375: 1388-1402Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar). They are responsible for 60,000 deaths per year and represent a major public health problem in regions of the world least able to deal with the associated economic burden. Their primary route of transmission is by blood-sucking insect vectors. However, other pathways, notably blood transfusion, organ transplantation, and illicit drug usage coupled with population migration, have resulted in both infections becoming problematic in non-endemic areas (3Bern C. Montgomery S.P. Clin. Infect. Dis. 2009; 49: e52-e54Crossref PubMed Scopus (437) Google Scholar, 4Gascon J. Bern C. Pinazo M.J. Acta Trop. 2010; 115: 22-27Crossref PubMed Scopus (438) Google Scholar, 5Gautret P. Clerinx J. Caumes E. Simon F. Jensenius M. Loutan L. Schlagenhauf P. Castelli F. Freedman D. Miller A. Bronner U. Parola P. Euro Surveill. 2009; 14 (pii=19327)Crossref PubMed Google Scholar). Chagas disease is an emerging problem with as many as 300,000 people in the United States infected by T. cruzi, and blood supplies are now routinely screened for the parasite. Current treatment of both infections is restricted to a series of drugs whose mode(s) of action are poorly understood (6Wilkinson S.R. Kelly J.M. Expert Rev. Mol. Med. 2009; 11: e31Crossref PubMed Scopus (173) Google Scholar). Establishing how existing therapies work could contribute to more effective treatment and aid drug development.The 5-nitrofuran drug nifurtimox has been used for more than 40 years to treat Chagas disease. Its use is controversial as it is toxic, reportedly carcinogenic, and of limited efficacy. Additionally, T. cruzi strains refractory to treatment have been isolated, a situation compounded by failure to complete the recommended drug schedules. Nifurtimox drug regimes can take up to 4 months and are frequently associated with unpleasant side effects. Despite these problems, nifurtimox, as part of the nifurtimox-eflornithine combination therapy, is now recommended for late-stage West African sleeping sickness (7Priotto G. Fogg C. Balasegaram M. Erphas O. Louga A. Checchi F. Ghabri S. Piola P. PLoS Clin. Trials. 2006; 1: e39Crossref PubMed Google Scholar, 8Priotto G. Kasparian S. Mutombo W. Ngouama D. Ghorashian S. Arnold U. Ghabri S. Baudin E. Buard V. Kazadi-Kyanza S. Ilunga M. Mutangala W. Pohlig G. Schmid C. Karunakara U. Torreele E. Kande V. Lancet. 2009; 374: 56-64Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) and is also undergoing assessment as a treatment for pediatric neuroblastoma (9Saulnier Sholler G.L. Kalkunte S. Greenlaw C. McCarten K. Forman E. J. Pediatr. Hematol. Oncol. 2006; 28: 693-695Crossref PubMed Scopus (33) Google Scholar, 10Saulnier Sholler G.L. Brard L. Straub J.A. Dorf L. Illeyne S. Koto K. Kalkunte S. Bosenberg M. Ashikaga T. Nishi R. J. Pediatr. Hematol. Oncol. 2009; 31: 187-193Crossref PubMed Scopus (34) Google Scholar).As with most nitroheterocyclic agents, nifurtimox functions as a prodrug and must undergo activation by nitroreduction. Two classes of enzyme can catalyze this process, the type I and type II nitroreductases (11Peterson F.J. Mason R.P. Hovsepian J. Holtzman J.L. J. Biol. Chem. 1979; 254: 4009-4014Abstract Full Text PDF PubMed Google Scholar). Type I nitrored" @default.
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• W2016292622 title "Nifurtimox Activation by Trypanosomal Type I Nitroreductases Generates Cytotoxic Nitrile Metabolites" @default.
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