Susan Wyllie

Nitro drugs for the treatment of trypanosomatid diseases: past, present, and future prospects

Highlights • Two nitro drugs are currently used in the treatment of trypanosomatid diseases.• Several new nitroaromatics are being developed against the trypanosomatid diseases.• Many nitro drugs and drug candidates act as prodrugs which require bioactivation.• Nitroaromatics can have disparate mechanisms of action in trypanosomatid parasites.

Increased levels of thiols protect antimony unresponsive Leishmania donovani field isolates against reactive oxygen species generated by trivalent antimony

The current trend of antimony (Sb) unresponsiveness in the Indian subcontinent is a major impediment to effective chemotherapy of visceral leishmaniasis (VL). Although contributory mechanisms studied in laboratory-raised Sb-R parasites include an up-regulation of drug efflux pumps and increased thiols, their role in clinical isolates is not yet substantiated. Accordingly, our objectives were to study the contributory role of thiols in the generation of Sb unresponsiveness in clinical isolates. Promastigotes were isolated from VL patients who were either Sb responsive (n=2) or unresponsive (n=3). Levels of thiols as measured by HPLC and flow cytometry showed higher basal levels of thiols and a faster rate of thiol regeneration in Sb unresponsive strains as compared with sensitive strains. The effects of antimony on generation of reactive oxygen species (ROS) in normal and thiol-depleted conditions as also their H2O2 scavenging activity indicated that in unresponsive parasites, Sb-mediated ROS generation was curtailed, which could be reversed by depletion of thiols and was accompanied by a higher H2O2 scavenging activity. Higher levels of thiols in Sb-unresponsive field isolates from patients with VL protect parasites from Sb-mediated oxidative stress, thereby contributing to the antimony resistance phenotype.

The Anti-Trypanosome Drug Fexinidazole Shows Potential for Treating Visceral Leishmaniasis

Fexinidazole, a drug in clinical testing for African sleeping sickness, shows potential as an oral treatment for another neglected tropical disease. A New Job for an Old Drug Fever, fatigue, weight loss, and swelling of the spleen and liver are all symptoms of visceral leishmaniasis—a tropical disease that is also known as kala-azar or black fever. Caused by the protozoan parasite Leishmania donovani, which is transmitted to people through the bite of a sand fly, the disease is almost always fatal if untreated. Although several drugs exist, they are costly and not always safe, effective, or easy to administer. To address the need for better drugs, Wyllie et al. investigated the possibility of using fexinidazole to treat visceral leishmaniasis. This antiparasitic compound, developed decades ago, is now undergoing early clinical trials as an oral therapy for African sleeping sickness, a disease that is caused by a related protozoan parasite called Trypanosoma brucei. Fexinidazole’s mode of action is thought to involve a trypanosome nitroreductase; the finding that a closely related enzyme is encoded by the leishmania genome inspired Wyllie et al. to pursue fexinidazole as a therapy for visceral leishmaniasis. They found that the compound and two of its metabolites (which rapidly form in vivo) showed activity against both developmental stages of L. donovani in vitro. The metabolites were cytotoxic, killing all the parasites within 30 hours. For unclear reasons, only the metabolites were active against L. donovani grown in macrophages (the cells in which the parasite reproduces during infection). In a mouse model of visceral leishmaniasis, a daily oral dose of fexinidazole for 5 days almost completely suppressed infection—an activity that is comparable to that of drugs currently in clinical use against this deadly tropical disease. Visceral leishmaniasis kills more people than any other parasitic disease except malaria. The clinical trials of fexinidazole for African sleeping sickness have already shown that the drug is extremely safe. The discovery that it may also be a viable oral treatment for visceral leishmaniasis bodes well for those afflicted with this disease. Safer and more effective oral drugs are required to treat visceral leishmaniasis, a parasitic disease that kills 50,000 to 60,000 people each year in parts of Asia, Africa, and Latin America. Here, we report that fexinidazole, a drug currently in phase 1 clinical trials for treating African trypanosomiasis, shows promise for treating visceral leishmaniasis. This 2-substituted 5-nitroimidazole drug is rapidly oxidized in vivo in mice, dogs, and humans to sulfoxide and sulfone metabolites. Both metabolites of fexinidazole were active against Leishmania donovani amastigotes grown in macrophages, whereas the parent compound was inactive. Pharmacokinetic studies with fexinidazole (200 mg/kg) showed that fexinidazole sulfone achieves blood concentrations in mice above the EC99 (effective concentration inhibiting growth by 99%) value for at least 24 hours after a single oral dose. A once-daily regimen for 5 days at this dose resulted in a 98.4% suppression of infection in a mouse model of visceral leishmaniasis, equivalent to that seen with the drugs miltefosine and Pentostam, which are currently used clinically to treat this tropical disease. In African trypanosomes, the mode of action of nitro drugs involves reductive activation via a NADH (reduced form of nicotinamide adenine dinucleotide)–dependent bacterial-like nitroreductase. Overexpression of the leishmanial homolog of this nitroreductase in L. donovani increased sensitivity to fexinidazole by 19-fold, indicating that a similar mechanism is involved in both parasites. These findings illustrate the potential of fexinidazole as an oral drug therapy for treating visceral leishmaniasis.

Cross-Resistance to Nitro Drugs and Implications for Treatment of Human African Trypanosomiasis

ABSTRACT The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (∼27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay.

ABSTRACT Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.

Anti-trypanosomatid drug discovery: an ongoing challenge and a continuing need

The WHO recognizes human African trypanosomiasis, Chagas disease and the leishmaniases as neglected tropical diseases. These diseases are caused by parasitic trypanosomatids and range in severity from mild and self-curing to near invariably fatal. Public health advances have substantially decreased the effect of these diseases in recent decades but alone will not eliminate them. In this Review, we discuss why new drugs against trypanosomatids are required, approaches that are under investigation to develop new drugs and why the drug discovery pipeline remains essentially unfilled. In addition, we consider the important challenges to drug discovery strategies and the new technologies that can address them. The combination of new drugs, new technologies and public health initiatives is essential for the management, and hopefully eventual elimination, of trypanosomatid diseases from the human population.

Elevated levels of tryparedoxin peroxidase in antimony unresponsive Leishmania donovani field isolates

Enhancement of the anti-oxidant metabolism of Leishmania parasites, dependent upon the unique dithiol trypanothione, has been implicated in laboratory-generated antimony resistance. Here, the role of the trypanothione-dependent anti-oxidant pathway is studied in antimony-resistant clinical isolates. Elevated levels of tryparedoxin and tryparedoxin peroxidase, key enzymes in hydroperoxide detoxification, were observed in antimonial resistant parasites resulting in an increased metabolism of peroxides. These data suggest that enhanced anti-oxidant defences may play a significant role in clinical resistance to antimonials.

A comparative study of methylglyoxal metabolism in trypanosomatids

The glyoxalase system, comprising the metalloenzymes glyoxalase I (GLO1) and glyoxalase II (GLO2), is an almost universal metabolic pathway involved in the detoxification of the glycolytic byproduct methylglyoxal to d‐lactate. In contrast to the situation with the trypanosomatid parasites Leishmania major and Trypanosoma cruzi, this trypanothione‐dependent pathway is less well understood in the African trypanosome, Trypanosoma brucei. Although this organism possesses a functional GLO2, no apparent GLO1 gene could be identified in the T. brucei genome. The absence of GLO1 in T. brucei was confirmed by the lack of GLO1 activity in whole cell extracts, failure to detect a GLO1‐like protein on immunoblots of cell lysates, and lack of d‐lactate formation from methylglyoxal as compared to L. major and T. cruzi. T. brucei procyclics were found to be 2.4‐fold and 5.7‐fold more sensitive to methylglyoxal toxicity than T. cruzi and L. major, respectively. T. brucei also proved to be the least adept of the ‘Tritryp’ parasites in metabolizing methylglyoxal, producing l‐lactate rather than d‐lactate. Restoration of a functional glyoxalase system by expression of T. cruzi GLO1 in T. brucei resulted in increased resistance to methylglyoxal and increased conversion of methylglyoxal to d‐lactate, demonstrating that GLO2 is functional in vivo. Procyclic forms of T. brucei possess NADPH‐dependent methylglyoxal reductase and NAD+‐dependent l‐lactaldehyde dehydrogenase activities sufficient to account for all of the methylglyoxal metabolized by these cells. We propose that the predominant mechanism for methylglyoxal detoxification in the African trypanosome is via the methylglyoxal reductase pathway to l‐lactate.

Roles of Trypanothione S-Transferase and Tryparedoxin Peroxidase in Resistance to Antimonials

ABSTRACT The clinical value of antimonial drugs, the mainstay therapy for leishmaniasis, is now threatened by the emergence of acquired drug resistance, and a comprehensive understanding of the underlying mechanisms is required. Using the model organism Leishmania tarentolae, we have examined the role of trypanothione S-transferase (TST) in trivalent antimony [Sb(III)] resistance. TST has S-transferase activity with substrates such as chlorodinitrobenzene as well as peroxidase activity with alkyl and aryl hydroperoxides but not with hydrogen peroxide. Although S-transferase activity and TST protein levels were unchanged in Sb(III)-sensitive and -resistant lines, rates of metabolism of hydrogen peroxide, t-butyl hydroperoxide, and cumene hydroperoxide were significantly increased. Elevated peroxidase activities were shown to be both trypanothione and tryparedoxin dependent and were associated with the overexpression of classical tryparedoxin peroxidase (TryP) in the cytosol of L. tarentolae. The role of TryP in Sb(III) resistance was verified by overexpression of the recombinant Leishmania major protein in Sb(III)-sensitive promastigotes. An approximate twofold increase in the level of TryP activity in this transgenic cell line was accompanied by a significant decrease in sensitivity to Sb(III) (twofold; P < 0.001). Overexpression of an enzymatically inactive TryP failed to result in Sb(III) resistance. This indicates that TryP-dependent resistance is not due to sequestration of Sb(III) and suggests that enhanced antioxidant defenses may well be a key feature of mechanisms of clinical resistance to antimonial drugs.

Chemical Validation of Trypanothione Synthetase A POTENTIAL DRUG TARGET FOR HUMAN TRYPANOSOMIASIS

In the search for new therapeutics for the treatment of human African trypanosomiasis, many potential drug targets in Trypanosoma brucei have been validated by genetic means, but very few have been chemically validated. Trypanothione synthetase (TryS; EC 6.3.1.9; spermidine/glutathionylspermidine:glutathione ligase (ADP-forming)) is one such target. To identify novel inhibitors of T. brucei TryS, we developed an in vitro enzyme assay, which was amenable to high throughput screening. The subsequent screen of a diverse compound library resulted in the identification of three novel series of TryS inhibitors. Further chemical exploration resulted in leads with nanomolar potency, which displayed mixed, uncompetitive, and allosteric-type inhibition with respect to spermidine, ATP, and glutathione, respectively. Representatives of all three series inhibited growth of bloodstream T. brucei in vitro. Exposure to one of our lead compounds (DDD86243; 2 × EC50 for 72 h) decreased intracellular trypanothione levels to <10% of wild type. In addition, there was a corresponding 5-fold increase in the precursor metabolite, glutathione, providing strong evidence that DDD86243 was acting on target to inhibit TryS. This was confirmed with wild-type, TryS single knock-out, and TryS-overexpressing cell lines showing expected changes in potency to DDD86243. Taken together, these data provide initial chemical validation of TryS as a drug target in T. brucei.