Shane R. Wilkinson

A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes

Nifurtimox and benznidazole are the front-line drugs used to treat Chagas disease, the most important parasitic infection in the Americas. These agents function as prodrugs and must be activated within the parasite to have trypanocidal effects. Despite >40 years of research, the mechanism(s) of action and resistance have remained elusive. Here, we report that in trypanosomes, both drugs are activated by a NADH-dependent, mitochondrially localized, bacterial-like, type I nitroreductase (NTR), and that down-regulation of this explains how resistance may emerge. Loss of a single copy of this gene in Trypanosoma cruzi, either through in vitro drug selection or by targeted gene deletion, is sufficient to cause significant cross-resistance to a wide range of nitroheterocyclic drugs. In Trypanosoma brucei, loss of a single NTR allele confers similar cross-resistance without affecting growth rate or the ability to establish an infection. This potential for drug resistance by a simple mechanism has important implications, because nifurtimox is currently undergoing phase III clinical trials against African trypanosomiasis.

Trypanocidal drugs: mechanisms, resistance and new targets.

The protozoan parasites Trypanosoma brucei and Trypanosoma cruzi are the causative agents of African trypanosomiasis and Chagas disease, respectively. These are debilitating infections that exert a considerable health burden on some of the poorest people on the planet. Treatment of trypanosome infections is dependent on a small number of drugs that have limited efficacy and can cause severe side effects. Here, we review the properties of these drugs and describe new findings on their modes of action and the mechanisms by which resistance can arise. We further outline how a greater understanding of parasite biology is being exploited in the search for novel chemotherapeutic agents. This effort is being facilitated by new research networks that involve academic and biotechnology/pharmaceutical organisations, supported by public-private partnerships, and are bringing a new dynamism and purpose to the search for trypanocidal agents.

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.

The Trypanosoma cruzi Enzyme TcGPXI Is a Glycosomal Peroxidase and Can Be Linked to Trypanothione Reduction by Glutathione or Tryparedoxin

Trypanosoma cruziglutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites.

Trypanosoma cruzi expresses a plant-like ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum

In most aerobic organisms hemoperoxidases play a major role in H2O2-detoxification, but trypanosomatids have been reported to lack this activity. Here we describe the properties of an ascorbate-dependent hemoperoxidase (TcAPX) from the American trypanosome Trypanosoma cruzi. The activity of this plant-like enzyme can be linked to the reduction of the parasite-specific thiol trypanothione by ascorbate in a process that involves nonenzymatic interaction. The role of heme in peroxidase activity was demonstrated by spectral and inhibition studies. Ascorbate could saturate TcAPX activity indicating that the enzyme obeys Michaelis–Menten kinetics. Parasites that overexpressed TcAPX activity were found to have increased resistance to exogenous H2O2. To determine subcellular location an epitope-tagged form of TcAPX was expressed in T. cruzi, which was observed to colocalize with endoplasmic reticulum resident chaperone protein BiP. These findings identify an arm of the oxidative defense system of this medically important parasite. The absence of this redox pathway in the human host may be therapeutically exploitable.

Substoichiometric Levels of Cu2+ Ions Accelerate the Kinetics of Fiber Formation and Promote Cell Toxicity of Amyloid-β from Alzheimer Disease

A role for Cu2+ ions in Alzheimer disease is often disputed, as it is believed that Cu2+ ions only promote nontoxic amorphous aggregates of amyloid-β (Aβ). In contrast with currently held opinion, we show that the presence of substoichiometric levels of Cu2+ 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 Cu2+ ions at a physiological pH causes Aβ to approach its isoelectric point, thus inducing self-association and fiber formation. We further show that Cu2+ ions bound to Aβ are consistently more toxic to neuronal cells than Aβ in the absence of Cu2+ ions, whereas Cu2+ ions in the absence of Aβ are not cytotoxic. The degree of Cu-Aβ cytotoxicity correlates with the levels of Cu2+ ions that accelerate fiber formation. We note the effect appears to be specific for Cu2+ ions as Zn2+ ions inhibit the formation of fibers. An active role for Cu2+ ions in accelerating fiber formation and promoting cell death suggests impaired copper homeostasis may be a risk factor in Alzheimer disease.

Evidence that trypanothione reductase is an essential enzyme in Leishmania by targeted replacement of the tryA gene locus

Trypanothione reductase (TR), a flavoprotein oxidoreductase central to the unique thiol‐redox system that operates in trypanosomatid protozoa, has been proposed as a potential target for the chemotherapy of trypanosomatid infections. In this study, targeted gene replacement was used to obtain evidence that TR is an essential cellular component and that its physiological function is crucial for parasite survival. Precise replacement of the Leishmania donovani tryA gene encoding TR was only possible upon simultaneous expression of the tryA coding region from an episome; in its absence, attempted removal of the last tryA allele invariably led to the generation of an extra copy of tryA, seemingly as a result of selective chromosomal polysomy. Partial replacement mutants were drastically affected in their ability to survive inside cytokine‐activated macrophages in a murine model of Leishmania infection. As no compensatory mechanism for the partial loss of TR activity was observed in these mutants and as it was not possible to obtain viable Leishmania devoid of TR catalytic activity, specific inhibitors of this enzyme are likely to be useful anti‐leishmanial agents for chemotherapeutic use.

Mitochondrial superoxide radicals mediate programmed cell death in Trypanosoma cruzi: cytoprotective action of mitochondrial iron superoxide dismutase overexpression

Trypanosoma cruzi undergo PCD (programmed cell death) under appropriate stimuli, the mechanisms of which remain to be established. In the present study, we show that stimulation of PCD in T. cruzi epimastigotes by FHS (fresh human serum) results in rapid (<1 h) externalization of phosphatidylserine and depletion of the low molecular mass thiols dihydrotrypanothione and glutathione. Concomitantly, enhanced generation of oxidants was established by EPR and immuno-spin trapping of radicals using DMPO (5,5-dimethylpyrroline-N-oxide) and augmentation of the glucose flux through the pentose phosphate pathway. In the early period (<20 min), changes in mitochondrial membrane potential and inhibition of respiration, probably due to the impairment of ADP/ATP exchange with the cytosol, were observed, conditions that favour the generation of O2*-. Accelerated rates of mitochondrial O2*- production were detected by the inactivation of the redox-sensitive mitochondrial aconitase and by oxidation of a mitochondrial-targeted probe (MitoSOX). Importantly, parasites overexpressing mitochondrial FeSOD (iron superoxide dismutase) were more resistant to the PCD stimulus, unambiguously indicating the participation of mitochondrial O2*- in the signalling process. In summary, FHS-induced PCD in T. cruzi involves mitochondrial dysfunction that causes enhanced O(2)(*-) formation, which leads to cellular oxidative stress conditions that trigger the initiation of PCD cascades; moreover, overexpression of mitochondrial FeSOD, which is also observed during metacyclogenesis, resulted in cytoprotective effects.

TcGPXII, a glutathione-dependent Trypanosoma cruzi peroxidase with substrate specificity restricted to fatty acid and phospholipid hydroperoxides, is localized to the endoplasmic reticulum.

Until recently, it had been thought that trypanosomes lack glutathione peroxidase activity. Here we report the subcellular localization and biochemical properties of a second glutathione-dependent peroxidase from Trypanosoma cruzi (TcGPXII). TcGPXII is a single-copy gene which encodes a 16 kDa protein that appears to be specifically dependent on glutathione as the source of reducing equivalents. Recombinant TcGPXII was purified and shown to have peroxidase activity towards a narrow substrate range, restricted to hydroperoxides of fatty acids and phospholipids. Analysis of the pathway revealed that TcGPXII activity could be readily saturated by glutathione and that the peroxidase functioned by a Ping Pong mechanism. Enzyme reduction was shown to be the rate-limiting step in this pathway. Using immunofluorescence, TcGPXII was shown to co-localize with a homologue of immunoglobulin heavy-chain binding protein (BiP), a protein restricted to the endoplasmic reticulum and Golgi. As the smooth endoplasmic reticulum is the site of phospholipid and fatty acid biosynthesis, this suggests that TcGPXII may play a specific role in the T. cruzi oxidative defence system by protecting newly synthesized lipids from peroxidation.

Trypanocidal Activity of Nitroaromatic Prodrugs: Current Treatments and Future Perspectives

Chagas disease and African sleeping sickness are trypanosomal infections that represent important public health problems in Latin America and Africa, respectively. The restriction of these diseases to the poorer parts of the world has meant that they have been largely neglected and limited progress has been made in their treatment. The nitroheterocyclic prodrugs nifurtimox and benznidazole, in use against Chagas disease for >40 years, remain the only agents available for this infection. In the case of African sleeping sickness, nifurtimox has recently been added to the arsenal of medicines, with the nitroheterocycle fexinidazole currently under evaluation. For a long time, the cytotoxic mechanism of these drugs was poorly understood: nifurtimox was thought to act via production of superoxide anions and nitro radicals, while the mode of benznidazole action was more obscure. The trypanocidal activity of nitroheterocyclic drugs is now known to depend on a parasite type I nitroreductase (NTR). This enzyme is absent from mammalian cells, a difference that forms the basis for the drug selectivity. The role of this enzyme in drug activation has been genetically and biochemically validated. It catalyses the 2-electron reduction of nitroheterocyclic compounds within the parasite, producing toxic metabolites without significant generation of superoxide. Recognition that this enzyme is responsible for activation of nitroheterocyclic prodrugs has allowed screening for compounds that preferentially target the parasite. This approach has led to the identification of two new classes of anti-trypanosomal agents, nitrobenzylphosphoramide mustards and aziridinyl nitrobenzamides, and promises to yield new, safer, more effective drugs.