Unraveling the antitrypanosomal mechanism of benznidazole and related 2-nitroimidazoles: From prodrug activation to DNA damage.

Nitroheterocycles represent an important class of compound used to treat trypanosomiasis. They often function as prodrugs and can undergo type I nitroreductase (NTR1)-mediated activation before promoting their antiparasitic activities although the nature of these downstream effects has yet to be determined. Here, we show that in an NTR1-dependent process, benznidazole promotes DNA damage in the nuclear genome of Trypanosoma brucei, providing the first direct link between activation of this prodrug and a downstream trypanocidal mechanism. Phenotypic and protein expression studies revealed that components of the trypanosome's homologous recombination (HR) repair pathway (TbMRE11, γH2A, TbRAD51) cooperate to resolve the benznidazole-induced damage, indicating that the prodrug-induced lesions are most likely double stand DNA breaks, while the sequence/recruitment kinetics of these factors parallels that in other eukaryotes HR systems. When extended to other NTR1-activated 2-nitroimidazoles, some were shown to promote DNA damage. Intriguingly, the lesions induced by these required TbMRE11 and TbCSB activities to fix leading us to postulate that TbCSB may operate in systems other than the transcription-coupled nucleotide excision repair pathway. Understanding how existing trypanosomal drugs work will aid future drug design and help unlock novel reactions/pathways that could be exploited as targets for therapeutic intervention.

Challenges in Chagas Disease Drug Development.

The protozoan parasite Trypanosoma cruzi causes Chagas disease, an important public health problem throughout Latin America. Current therapeutic options are characterised by limited efficacy, long treatment regimens and frequent toxic side-effects. Advances in this area have been compromised by gaps in our knowledge of disease pathogenesis, parasite biology and drug activity. Nevertheless, several factors have come together to create a more optimistic scenario. Drug-based research has become more systematic, with increased collaborations between the academic and commercial sectors, often within the framework of not-for-profit consortia. High-throughput screening of compound libraries is being widely applied, and new technical advances are helping to streamline the drug development pipeline. In addition, drug repurposing and optimisation of current treatment regimens, informed by laboratory research, are providing a basis for new clinical trials. Here, we will provide an overview of the current status of Chagas disease drug development, highlight those areas where progress can be expected, and describe how fundamental research is helping to underpin the process.

Distinct activation mechanisms trigger the trypanocidal activity of DNA damaging prodrugs.

Quinone-based compounds have been exploited to treat infectious diseases and cancer, with such chemicals often functioning as inhibitors of key metabolic pathways or as prodrugs. Here, we screened an aziridinyl 1,4-benzoquinone (ABQ) library against the causative agents of trypanosomiasis, and cutaneous leishmaniasis, identifying several potent structures that exhibited EC50 values of <100 nM. However, these compounds also displayed significant toxicity towards mammalian cells indicating that they are not suitable therapies for systemic infections. Using anti-T. brucei ABQs as chemical probes, we demonstrated that these exhibit different trypanocidal modes of action. Many functioned as type I nitroreductase (TbNTR) or cytochrome P450 reductase (TbCPR) dependent prodrugs that, following activation, generate metabolites which promote DNA damage, specifically interstrand crosslinks (ICLs). Trypanosomes lacking TbSNM1, a nuclease that specifically repairs ICLs, are hypersensitive to most ABQ prodrugs, a phenotype exacerbated in cells also engineered to express elevated levels of TbNTR or TbCPR. In contrast, ABQs that contain substituent groups on the biologically active aziridine do not function as TbNTR or TbCPR-activated prodrugs and do not promote DNA damage. By unravelling how ABQs mediate their activities, features that facilitate the desired anti-parasitic growth inhibitory effects could be incorporated into new, safer compounds targeting these neglected tropical diseases.

TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei.

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.

Unravelling the role of SNM1 in the DNA repair system of Trypanosoma brucei.

All living cells are subject to agents that promote DNA damage. A particularly lethal lesion are interstrand cross-links (ICL), a property exploited by several anti-cancer chemotherapies. In yeast and humans, an enzyme that plays a key role in repairing such damage are the PSO2/SNM1 nucleases. Here, we report that Trypanosoma brucei, the causative agent of African trypanosomiasis, possesses a bona fide member of this family (called TbSNM1) with expression of the parasite enzyme able to suppress the sensitivity yeast pso2Δ mutants display towards mechlorethamine, an ICL-inducing compound. By disrupting the Tbsnm1 gene, we demonstrate that TbSNM1 activity is non-essential to the medically relevant T. brucei life cycle stage. However, trypanosomes lacking this enzyme are more susceptible to bi- and tri-functional DNA alkylating agents with this phenotype readily complemented by ectopic expression of Tbsnm1. Genetically modified variants of the null mutant line were subsequently used to establish the anti-parasitic mechanism of action of nitrobenzylphosphoramide mustard and aziridinyl nitrobenzamide prodrugs, compounds previously shown to possess potent trypanocidal properties while exhibiting limited toxicity to mammalian cells. This established that these agents, following activation by a parasite specific type I nitroreductase, produce metabolites that promote formation of ICLs leading to inhibition of trypanosomal growth.

Evaluating 5-Nitrothiazoles as Trypanocidal Agents.

The growth-inhibitory properties of a 5-nitrothiazole series were evaluated against Trypanosoma brucei. A subset of related compounds displayed the greatest potency toward the parasite while exhibiting little cytotoxic effect on mammalian cells, with this antiparasitic activity dependent on expression of a type I nitroreductase by the trypanosome. We conclude that the 5-nitrothiazole class of nitroheterocyclic drugs may represent a new lead in the treatment of human African trypanosomiasis.

Discovery of potent nitrotriazole-based antitrypanosomal agents: In vitro and in vivo evaluation.

3-Nitro-1H-1,2,4-triazole- and 2-nitro-1H-imidazole-based amides with an aryloxy-phenyl core were synthesized and evaluated as antitrypanosomal agents. All 3-nitrotriazole-based derivatives were extremely potent anti-Trypanosoma cruzi agents at sub nM concentrations and exhibited a high degree of selectivity for the parasite. The 2-nitroimidazole analogs were only moderately active against T. cruzi amastigotes and exhibited low selectivity. Both types of compound were active against Leishmania donovani axenic amastigotes with excellent selectivity for the parasite, whereas three 2-nitroimidazole-based analogs were also moderately active against infected macrophages. However, no compound demonstrated selective activity against Trypanosoma brucei rhodesiense. The most potent in vitro anti-T. cruzi compounds were tested in an acute murine model and reduced the parasites to an undetectable level after five days of treatment at 13 mg/kg/day. Such compounds are potential inhibitors of T. cruzi CYP51 and, being excellent substrates for the type I nitroreductase (NTR) which is specific to trypanosomatids, work as prodrugs and constitute a new generation of effective and more affordable antitrypanosomal agents.

An essential type I nitroreductase from Leishmania major can be used to activate leishmanicidal prodrugs.

Nitroaromatic prodrugs are used to treat a range of microbial infections with selectivity achieved by specific activation reactions. For trypanosomatid parasites, this is mediated by type I nitroreductases. Here, we demonstrate that the causative agent of leishmaniasis, Leishmania major, expresses an FMN-containing nitroreductase (LmNTR) that metabolizes a wide range of substrates, and based on electron donor and acceptor preferences, it may function as an NADH:quinone oxidoreductase. Using gene deletion approaches, we demonstrate that this activity is essential to L. major promastigotes, the parasite forms found in the insect vector. Intriguingly, LmNTR(+/-) heterozygote promastigote parasites could readily differentiate into infectious metacyclic cells but these were unable to establish infections in cultured mammalian cells and caused delayed pathology in mice. Furthermore, we exploit the LmNTR activity evaluating a library of nitrobenzylphosphoramide mustards using biochemical and phenotypic screens. We identify a subset of compounds that display significant growth inhibitory properties against the intracellular parasite form found in the mammalian hosts. The leishmanicidal activity was shown to be LmNTR-specific as the LmNTR(+/-) heterozygote promastigotes displayed resistance to the most potent mustards. We conclude that LmNTR can be targeted for drug development by exploiting its prodrug activating property or by designing specific inhibitors to block its endogenous function.

Evaluating aziridinyl nitrobenzamide compounds as leishmanicidal prodrugs.

Many of the nitroaromatic agents used in medicine function as prodrugs and must undergo activation before exerting their toxic effects. In most cases, this is catalyzed by flavin mononucleotide (FMN)-dependent type I nitroreductases (NTRs), a class of enzyme absent from higher eukaryotes but expressed by bacteria and several eukaryotic microbes, including trypanosomes and Leishmania. Here, we utilize this difference to evaluate whether members of a library of aziridinyl nitrobenzamides have activity against Leishmania major. Biochemical screens using purified L. major NTR (LmNTR) revealed that compounds containing an aziridinyl-2,4-dinitrobenzyl core were effective substrates for the enzyme and showed that the 4-nitro group was important for this activity. To facilitate drug screening against intracellular amastigote parasites, we generated leishmanial cells that expressed the luciferase reporter gene and optimized a mammalian infection model in a 96-well plate format. A subset of aziridinyl-2,4-dinitrobenzyl compounds possessing a 5-amide substituent displayed significant growth-inhibitory properties against the parasite, with the most potent agents generating 50% inhibitory concentrations of <100 nM for the intracellular form. This antimicrobial activity was shown to be LmNTR specific since L. major NTR(+/-) heterozygote parasites were slightly resistance to most aziridinyl dinitrobenzyl agents tested. When the most potent leishmanicidal agents were screened against the mammalian cells in which the amastigote parasites were propagated, no growth-inhibitory effect was observed at concentrations of up to 100 µM. We conclude that the aziridinyl nitrobenzamides represent a new lead structure that may have the potential to treat leishmanial infections.

Evaluating 5-nitrofurans as trypanocidal agents.

The nitroheterocycle nifurtimox, as part of a nifurtimox-eflornithine combination therapy, represents one of a limited number of treatments targeting Trypanosoma brucei, the causative agent of human African trypanosomiasis. The mode of action of this prodrug involves an initial activation reaction catalyzed by a type I nitroreductase (NTR), an enzyme found predominantly in prokaryotes, leading to the formation of a cytotoxic unsaturated open-chain nitrile metabolite. Here, we evaluate the trypanocidal activities of a library of other 5-nitrofurans against the bloodstream form of T. brucei as a preliminary step in the identification of additional nitroaromatic compounds that can potentially partner with eflornithine. Biochemical screening against the purified enzyme revealed that all 5-nitrofurans were effective substrates for T. brucei NTR (TbNTR), with the preferred compounds having apparent kcat/Km values approximately 50-fold greater than those of nifurtimox. For several compounds, in vitro reduction by this nitroreductase yielded products characterized by mass spectrometry as either unsaturated or saturated open-chain nitriles. When tested against the bloodstream form of T. brucei, many of the derivatives displayed significant growth-inhibitory properties, with the most potent compounds generating 50% inhibitory concentrations (IC50s) around 200 nM. The antiparasitic activities of the most potent agents were demonstrated to be NTR dependent, as parasites having reduced levels of the enzyme displayed resistance to the compounds, while parasites overexpressing TbNTR showed hypersensitivity. We conclude that other members of the 5-nitrofuran class of nitroheterocycles have the potential to treat human African trypanosomiasis, perhaps as an alternative partner prodrug to nifurtimox, in the next generation of eflornithine-based combinational therapies.

Benznidazole-resistance in Trypanosoma cruzi is a readily acquired trait that can arise independently in a single population.

Benznidazole is the frontline drug used against Trypanosoma cruzi, the causative agent of Chagas disease. However, treatment failures are often reported. Here, we demonstrate that independently acquired mutations in the gene encoding a mitochondrial nitroreductase (TcNTR) can give rise to distinct drug-resistant clones within a single population. Following selection of benznidazole-resistant parasites, all clones examined had lost one of the chromosomes containing the TcNTR gene. Sequence analysis of the remaining TcNTR allele revealed 3 distinct mutant genes in different resistant clones. Expression studies showed that these mutant proteins were unable to activate benznidazole. This correlated with loss of flavin mononucleotide binding. The drug-resistant phenotype could be reversed by transfection with wild-type TcNTR. These results identify TcNTR as a central player in acquired resistance to benznidazole. They also demonstrate that T. cruzi has a propensity to undergo genetic changes that can lead to drug resistance, a finding that has implications for future therapeutic strategies.

Dissecting the interstrand crosslink DNA repair system of Trypanosoma cruzi.

DNA interstrand crosslinks (ICLs) are toxic lesions that can block essential biological processes. Here we show Trypanosoma cruzi, the causative agent of Chagas disease, is susceptible to ICL-inducing compounds including mechlorethamine and novel nitroreductase-activated prodrugs that have potential in treating this infection. To resolve such lesions, cells co-opt enzymes from "classical" DNA repair pathways that alongside dedicated factors operate in replication-dependent and -independent mechanisms. To assess ICL repair in T. cruzi, orthologues of SNM1, MRE11 and CSB were identified and their function assessed. The T. cruzi enzymes could complement the mechlorethamine susceptibility phenotype displayed by corresponding yeast and/or T. brucei null confirming their role as ICL repair factors while GFP-tagged TcSNM1, TcMRE11 and TcCSB were shown to localise to the nuclei of insect and/or intracellular form parasites. Gene disruption demonstrated that while each activity was non-essential for T. cruzi viability, nulls displayed a growth defect in at least one life cycle stage with TcMRE11-deficient trypomastigotes also compromised in mammalian cell infectivity. Phenotyping revealed all nulls were more susceptible to mechlorethamine than controls, a trait complemented by re-expression of the deleted gene. To assess interplay, the gene disruption approach was extended to generate T. cruzi deficient in TcSNM1/TcMRE11 or in TcSNM1/TcCSB. Analysis demonstrated these activities functioned across two ICL repair pathways with TcSNM1 and TcMRE11 postulated to operate in a replication-dependent system while TcCSB helps resolve transcription-blocking lesions. By unravelling how T. cruzi repairs ICL damage, specific inhibitors targeting repair components could be developed and used to increase the potency of trypanocidal ICL-inducing compounds.

Nifurtimox activation by trypanosomal type I nitroreductases generates cytotoxic nitrile metabolites.

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.

Targeting the substrate preference of a type I nitroreductase to develop antitrypanosomal quinone-based prodrugs.

Nitroheterocyclic prodrugs are used to treat infections caused by Trypanosoma cruzi and Trypanosoma brucei. A key component in selectivity involves a specific activation step mediated by a protein homologous with type I nitroreductases, enzymes found predominantly in prokaryotes. Using data from determinations based on flavin cofactor, oxygen-insensitive activity, substrate range, and inhibition profiles, we demonstrate that NTRs from T. cruzi and T. brucei display many characteristics of their bacterial counterparts. Intriguingly, both enzymes preferentially use NADH and quinones as the electron donor and acceptor, respectively, suggesting that they may function as NADH:ubiquinone oxidoreductases in the parasite mitochondrion. We exploited this preference to determine the trypanocidal activity of a library of aziridinyl benzoquinones against bloodstream-form T. brucei. Biochemical screens using recombinant NTR demonstrated that several quinones were effective substrates for the parasite enzyme, having K(cat)/K(m) values 2 orders of magnitude greater than those of nifurtimox and benznidazole. In tests against T. brucei, antiparasitic activity mirrored the biochemical data, with the most potent compounds generally being preferred enzyme substrates. Trypanocidal activity was shown to be NTR dependent, as parasites with elevated levels of this enzyme were hypersensitive to the aziridinyl agent. By unraveling the biochemical characteristics exhibited by the trypanosomal NTRs, we have shown that quinone-based compounds represent a class of trypanocidal compound.

Activation of benznidazole by trypanosomal type I nitroreductases results in glyoxal formation.

Benznidazole, a 2-nitroimidazole, is the front-line treatment used against American trypanosomiasis, a parasitic infection caused by Trypanosoma cruzi. Despite nearly 40 years of use, the trypanocidal activity of this prodrug is not fully understood. It has been proposed that benznidazole activation leads to the formation of reductive metabolites that can cause a series of deleterious effects, including DNA damage and thiol depletion. Here, we show that the key step in benznidazole activation involves an NADH-dependent trypanosomal type I nitroreductase. This catalyzes an oxygen-insensitive reaction with the interaction of enzyme, reductant, and prodrug occurring through a ping-pong mechanism. Liquid chromatography/mass spectrometry (LC/MS) analysis of the resultant metabolites identified 4,5-dihydro-4,5-dihydroxyimidazole as the major product of a reductive pathway proceeding through hydroxylamine and hydroxy intermediates. The breakdown of this product released the reactive dialdehyde glyoxal, which, in the presence of guanosine, generated guanosine-glyoxal adducts. These experiments indicate that the reduction of benznidazole by type I nitroreductase activity leads to the formation of highly reactive metabolites and that the expression of this enzyme is key to the trypanocidal properties displayed by the prodrug.

Design, synthesis, and evaluation of potential prodrugs of DFMO for reductive activation.

A series of potential DFMO prodrugs was designed through the incorporation of 4-nitrobenzyl ester or carbamate groups for potential activation by trypanosomal nitroreductase. It was found that only modification of N(ε)-amino group of DFMO by 4-nitro-2-fluorobenzyloxycarbonyl resulted in significant trypanocidal activity and could serve as a lead for further investigation.

Synthesis and antikinetoplastid activity of a series of N,N'-substituted diamines.

A series of 25 N,N'-substituted diamines were prepared by controlled reductive amination of free aliphatic diamines with different substituted benzaldehydes. The library was screened in vitro for antiparasitic activity on the causative agents of human African trypanosomiasis, Chagas' disease and visceral leishmaniasis. The most potent compounds were derived from a subset of diamines that contained a 4-OBn substitution, having a 50% parasite growth inhibition in the submicromolar (against Trypanosoma cruzi) or nanomolar (against Trypanosoma brucei and Leishmania donovani) range. We conclude that members of this series of N,N'-substituted diamines provide new lead structures that have potential to treat trypanosomal and leishmanial infections.

Substoichiometric levels of Cu2+ ions accelerate the kinetics of fiber formation and promote cell toxicity of amyloid-{beta} from Alzheimer disease.

A role for Cu(2+) ions in Alzheimer disease is often disputed, as it is believed that Cu(2+) ions only promote nontoxic amorphous aggregates of amyloid-ß (Aß). In contrast with currently held opinion, we show that the presence of substoichiometric levels of Cu(2+) ions in fact doubles the rate of production of amyloid fibers, accelerating both the nucleation and elongation of fiber formation. We suggest that binding of Cu(2+) ions at a physiological pH causes Aß to approach its isoelectric point, thus inducing self-association and fiber formation. We further show that Cu(2+) ions bound to Aß are consistently more toxic to neuronal cells than Aß in the absence of Cu(2+) ions, whereas Cu(2+) ions in the absence of Aß are not cytotoxic. The degree of Cu-Aß cytotoxicity correlates with the levels of Cu(2+) ions that accelerate fiber formation. We note the effect appears to be specific for Cu(2+) ions as Zn(2+) ions inhibit the formation of fibers. An active role for Cu(2+) ions in accelerating fiber formation and promoting cell death suggests impaired copper homeostasis may be a risk factor in Alzheimer disease.

Synthesis and structure-activity relationships of nitrobenzyl phosphoramide mustards as nitroreductase-activated prodrugs.

A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to explore their structure-activity relationships as substrates of nitroreductases from Escherichia coli and trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in E. coli nitroreductase-expressing cells and in trypanosomatids expressing endogenous nitroreductases.

Structural insights into the catalytic mechanism of Trypanosoma cruzi GPXI (glutathione peroxidase-like enzyme I).

Current drug therapies against Trypanosoma cruzi, the causative agent of Chagas disease, have limited effectiveness and are highly toxic. T. cruzi-specific metabolic pathways that utilize trypanothione for the reduction of peroxides are being explored as potential novel therapeutic targets. In the present study we solved the X-ray crystal structure of one of the T. cruzi enzymes involved in peroxide reduction, the glutathione peroxidase-like enzyme TcGPXI (T. cruzi glutathione peroxidase-like enzyme I). We also characterized the wild-type, C48G and C96G variants of TcGPXI by NMR spectroscopy and biochemical assays. Our results show that residues Cys48 and Cys96 are required for catalytic activity. In solution, the TcGPXI molecule readily forms a Cys48-Cys96 disulfide bridge and the polypeptide segment containing Cys96 lacks regular secondary structure. NMR spectra of the reduced TcGPXI are indicative of a protein that undergoes widespread conformational exchange on an intermediate time scale. Despite the absence of the disulfide bond, the active site mutant proteins acquired an oxidized-like conformation as judged from their NMR spectra. The protein that was used for crystallization was pre-oxidized by t-butyl hydroperoxide; however, the electron density maps clearly showed that the active site cysteine residues are in the reduced thiol form, indicative of X-ray-induced reduction. Our crystallographic and solution studies suggest a level of structural plasticity in TcGPXI consistent with the requirement of the atypical two-cysteine (2-Cys) peroxiredoxin-like mechanism implied by the behaviour of the Cys48 and Cys96 mutant proteins.