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Mammalian cells synthesise tetrahydrobiopterin de novo , an essential cofactor for hydroxylation of aromatic amino acids, cleavage of ether lipids and the synthesis of nitric oxide. In contrast, kinetoplastid parasites are pterin auxotrophs and none of the above metabolic functions can account for the essential requirement of an unconjugated pterin for growth. Here we investigate the pterin requirements for growth and survival of two medically important parasites ( T. brucei and L. major ) in comparison with the model insect parasite , Crithidia fasciculata . The pterin concentration required to support 50% of maximum growth of each parasite was determined in defined pterin-free media for a variety of naturally occurring pterins. T. brucei and C. fasciculata showed an identical order of preference with the most active being 6-biopterin, followed by dihydrobiopterin > tetrahydrobiopterin > L-neopterin > sepiapterin. In contrast, L. major showed a pronounced growth preference (>200-fold) for the reduced pterins over the the oxidised forms 6-biopterin and L-neopterin. The unnatural isomers 7-biopterin or D-neopterin supported growth poorly, or not at all, in these organisms. Other pterins were inactive. HPLC analysis of pterins supporting growth established that these were metabolised to the tetrahydro-forms (>95%) with no evidence of further interconversion. In the absence of pterins, the parasites failed to grow and lost viability with <1% surviving beyond 5–14 days. Relatively high concentrations of folate or dihydrofolate (>500 nM) could support growth in the absence of unconjugated pterin and HPLC analysis identified pteridoxamine and 6-hydroxymethylpterin (as tetrahydro-form) in cell extracts. A common feature of pterins that support growth is the presence of at least one or more linear carbon substituents at position 6 of the pteridine ring with at least one hydroxyl group, ideally in the 1 S configuration. The possible essential roles of these important metabolites are discussed.

Drug discovery pipelines for the \"neglected diseases\" are now heavily populated with nitroheterocyclic compounds. Recently, the bicyclic nitro-compounds (R)-PA-824, DNDI-VL-2098 and delamanid have been identified as potential candidates for the treatment of visceral leishmaniasis. Using a combination of quantitative proteomics and whole genome sequencing of susceptible and drug-resistant parasites we identified a putative NAD(P)H oxidase as the activating nitroreductase (NTR2). Whole genome sequencing revealed that deletion of a single cytosine in the gene for NTR2 that is likely to result in the expression of a non-functional truncated protein. Susceptibility of leishmania was restored by reintroduction of the wild-type gene into the resistant line, which was accompanied by the ability to metabolise these compounds. Overexpression of NTR2 in wild-type parasites rendered cells hyper-sensitive to bicyclic nitro-compounds, but only marginally to the monocyclic nitro-drugs, nifurtimox and fexinidazole sulfone, known to be activated by a mitochondrial oxygen-insensitive nitroreductase (NTR1). Conversely, a double knockout NTR2 null cell line was completely resistant to bicyclic nitro-compounds and only marginally resistant to nifurtimox. Sensitivity was fully restored on expression of NTR2 in the null background. Thus, NTR2 is necessary and sufficient for activation of these bicyclic nitro-drugs. Recombinant NTR2 was capable of reducing bicyclic nitro-compounds in the same rank order as drug sensitivity in vitro. These findings may aid the future development of better, novel anti-leishmanial drugs. Moreover, the discovery of anti-leishmanial nitro-drugs with independent modes of activation and independent mechanisms of resistance alleviates many of the concerns over the continued development of these compound series.

Trypanosoma cruzi ( T . cruzi) is the causative agent of Chagas’ disease, a parasitic infection responsible for significant morbidity and mortality in Latin America. The current treatments have many serious drawbacks and new drugs are urgently required. In the UK, T . cruzi is classified by the Advisory Committee on Dangerous Pathogens (ACDP) as a Hazard Group 3 organism and strict safety practices must be adhered to when handling this pathogen in the laboratory. Validated inactivation techniques are required for safe T . cruzi waste disposal and removal from Containment Level 3 (CL3) facilities for storage, transportation and experimental analysis. Here we assess three T . cruzi . inactivation methods. These include three freeze-thaw cycles, chemical inactivation with Virkon disinfectant, and air drying on Whatman FTA cards (A, B, C, Elute) and on a Mitra microsampling device. After each treatment parasite growth was monitored for 4–6 weeks by microscopic examination. Three freeze-thaw cycles were sufficient to inactivate all T . cruzi CLBrener Luc life cycle stages and Silvio x10/7 A1 large epimastigote cell pellets up to two grams wet weight. Virkon treatment for one hour inactivated T . cruzi Silvio x10/7 subclone A1 and CLBrener Luc both in whole blood and cell culture medium when incubated at a final concentration of 2.5% Virkon, or at ≥1% Virkon when in tenfold excess of sample volume. Air drying also inactivated T . cruzi CLBrener Luc spiked blood when dried on FTA A, B or Elute cards for ≥30 minutes and on a Mitra Microsampler for two hours. However, T . cruzi CLBrener Luc were not inactivated on FTA C cards when dried for up to two hours. These experimentally confirmed conditions provide three validated T . cruzi inactivation methods which can be applied to other related ACDP Hazard Group 2–3 kinetoplastid parasites.

The Indian subcontinent is the only region where arsenic contamination of drinking water coexists with widespread resistance to antimonial drugs that are used to treat the parasitic disease visceral leishmaniasis. We have previously proposed that selection for parasite resistance within visceral leishmaniasis patients who have been exposed to trivalent arsenic results in cross-resistance to the related metalloid antimony, present in the pentavalent state as a complex in drugs such as sodium stibogluconate (Pentostam) and meglumine antimonate (Glucantime). To test this hypothesis, Leishmania donovani was serially passaged in mice exposed to arsenic in drinking water at environmentally relevant levels (10 or 100 ppm). Arsenic accumulation in organs and other tissues was proportional to the level of exposure and similar to that previously reported in human liver biopsies. After five monthly passages in mice exposed to arsenic, isolated parasites were found to be completely refractory to 500 μg⋅mL ⁻¹ Pentostam compared with the control passage group (38.5 μg⋅mL ⁻¹) cultured in vitro in mouse peritoneal macrophages. Reassessment of resistant parasites following further passage for 4 mo in mice without arsenic exposure showed that resistance was stable. Treatment of infected mice with Pentostam confirmed that resistance observed in vitro also occurred in vivo. We conclude that arsenic contamination may have played a significant role in the development of Leishmania antimonial resistance in Bihar because inadequate treatment with antimonial drugs is not exclusive to India, whereas widespread antimonial resistance is.

Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies. Emergence of resistance in eukaryotic microbial pathogens is a major concern. This Review discusses the challenges posed by eukaryotic pathogens, therapies used to target them, emergence of resistance and new approaches to sustaining existing therapies and developing new ones.

African sleeping sickness or human African trypanosomiasis, caused by Trypanosoma brucei spp., is responsible for ∼30,000 deaths each year. Available treatments for this disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease when the parasite has infected the central nervous system. Here we report the validation of a molecular target and the discovery of associated lead compounds with the potential to address this lack of suitable treatments. Inhibition of this target— T. brucei N -myristoyltransferase—leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high-affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N -myristoylation in trypanosomes. The compounds identified have promising pharmaceutical properties and represent an opportunity to develop oral drugs to treat this devastating disease. Our studies validate T. brucei N -myristoyltransferase as a promising therapeutic target for human African trypanosomiasis. A new antitrypanosomal African trypanosomiasis, or sleeping sickness, caused by Trypanosoma brucei spp., is responsible for some 30,000 deaths each year. The few treatments that are available tend to involve drugs with poor efficacy and safety profiles. N -myristoyltransferase (NMT), which adds myristate to the N-terminal glycine of many eukaryotic and microbial proteins, has been suggested as a possible target for drugs to treat trypanosomiasis and other parasitic diseases — if selectivity to protect the human enzyme can be achieved. A new study validates T. brucei NMT as a viable protein target for antitrypanosomals, and a potent inhibitor with drug-like properties has been identified. The compound, DDD85646, kills T. brucei in the bloodstream in mouse models. African sleeping sickness, caused by Trypanosoma brucei species, is responsible for some 30,000 human deaths each year. Available treatments are limited by poor efficacy and safety profiles. However, a new molecular target for potential treatments has now been identified. The protein target is T. brucei N -myristoyltransferase. In further experiments, lead compounds have been discovered that inhibit this protein, kill trypanosomes in vitro and in vivo , and can cure trypanosomiasis in mice.

There is an urgent requirement for safe, oral and cost-effective drugs for the treatment of visceral leishmaniasis (VL). We report that delamanid (OPC-67683), an approved drug for multi-drug resistant tuberculosis, is a potent inhibitor of Leishmania donovani both in vitro and in vivo. Twice-daily oral dosing of delamanid at 30 mg kg-1 for 5 days resulted in sterile cures in a mouse model of VL. Treatment with lower doses revealed a U-shaped (hormetic) dose-response curve with greater parasite suppression at 1 mg kg-1 than at 3 mg kg-1 (5 or 10 day dosing). Dosing delamanid for 10 days confirmed the hormetic dose-response and improved the efficacy at all doses investigated. Mechanistic studies reveal that delamanid is rapidly metabolised by parasites via an enzyme, distinct from the nitroreductase that activates fexinidazole. Delamanid has the potential to be repurposed as a much-needed oral therapy for VL. Better, safer, oral drugs are desperately needed for the treatment of visceral leishmaniasis, a parasitic infectious disease that causes an estimated 40,000 deaths a year, predominantly in South America, East Africa and the Indian subcontinent. The parasite that causes visceral leishmaniasis is transmitted between individuals by blood-sucking sandflies, and there are currently no vaccines that protect against the disease. In addition, all currently available drug treatments have serious limitations – they are expensive, toxic, have to be applied over a long period of time (mainly by injection) and may become ineffective as the parasites adapt to resist the drug. A cost-effective way to find a new treatment for a disease is to repurpose existing clinically approved drugs that are used to treat other diseases. Patterson, Wyllie et al. now report that a drug called delamanid, which was recently approved for the treatment of tuberculosis, can cure visceral leishmaniasis in mice. The drug worked when applied orally at doses that might be achievable in human patients, and can also kill parasites obtained from human patients. Patterson, Wyllie et al. also provide evidence that suggests that delamanid is processed in the parasites by an unknown enzyme. However, this enzyme is not the one that activates a different class of drugs that are used to treat visceral leishmaniasis. Future studies now need to identify the enzyme that is targeted by delamanid, and could investigate combinations of drugs that slow the emergence of resistant parasites and improve delamanid’s safety and effectiveness. Clinical trials are required to test how well delamanid treats visceral leishmaniasis in humans.

In the late twentieth century, emergence of high rates of treatment failure with antimonial compounds (SSG) for visceral leishmaniasis (VL) caused a public health crisis in Bihar, India. We hypothesize that exposure to arsenic through drinking contaminated groundwater may be associated with SSG treatment failure due to the development of antimony-resistant parasites. A retrospective cohort design was employed, as antimony treatment is no longer in routine use. The study was performed on patients treated with SSG between 2006 and 2010. Outcomes of treatment were assessed through a field questionnaire and treatment failure used as a proxy for parasite resistance. Arsenic exposure was quantified through analysis of 5 water samples from within and surrounding the patient's home. A logistic regression model was used to evaluate the association between arsenic exposure and treatment failure. In a secondary analysis survival curves and Cox regression models were applied to assess the risk of mortality in VL patients exposed to arsenic. One hundred and ten VL patients treated with SSG were analysed. The failure rate with SSG was 59%. Patients with high mean local arsenic level had a non-statistically significant higher risk of treatment failure (OR = 1.78, 95% CI: 0.7-4.6, p = 0.23) than patients using wells with arsenic concentration <10 μg/L. Twenty one patients died in our cohort, 16 directly as a result of VL. Arsenic levels ≥ 10 μg/L increased the risk of all-cause (HR 3.27; 95% CI: 1.4-8.1) and VL related (HR 2.65; 95% CI: 0.96-7.65) deaths. This was time dependent: 3 months post VL symptom development, elevated risks of all-cause mortality (HR 8.56; 95% CI: 2.5-29.1) and of VL related mortality (HR 9.27; 95% CI: 1.8-49.0) were detected. This study indicates a trend towards increased treatment failure in arsenic exposed patients. The limitations of the retrospective study design may have masked a strong association between arsenic exposure and selection for antimonial resistance in the field. The unanticipated strong correlation between arsenic exposure and VL mortality warrants further investigation.

SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using orthogonal chemo-proteomic and genomic approaches. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. Cell-cycle analysis of cells incubated with sub-lethal concentrations of an oxaborole identified a subtle but significant accumulation of G2 and >G2 cells. Given the possibility of compromised DNA fidelity, we investigated long-term exposure of T. brucei to oxaboroles by generating resistant cell lines in vitro. Resistance proved more difficult to generate than for drugs currently used in the field, and in one of our three cell lines was unstable. Whole-genome sequencing of the resistant cell lines revealed single nucleotide polymorphisms in 66 genes and several large-scale genomic aberrations. The absence of a simple consistent mechanism among resistant cell lines and the diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field. The combined genetic and chemical biology approaches have provided lists of candidates to be investigated for more detailed information on the mode of action of this promising new drug class.

Bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed.