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1.  Activation of Bicyclic Nitro-drugs by a Novel Nitroreductase (NTR2) in Leishmania 
PLoS Pathogens  2016;12(11):e1005971.
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.
Author Summary
Visceral leishmaniasis (kala-azar) is a serious vector borne disease afflicting people, particularly in parts of Asia, Africa and Latin America. There are approximately 400,000 new cases and an estimated 40,000 deaths each year, making it the second biggest parasitic killer after malaria. We recently discovered that delamanid–an oral nitro-drug used for the treatment of tuberculosis–shows promise for the treatment of leishmaniasis with potential to provide a much needed alternative to the current unsatisfactory anti-leishmanial drugs. Understanding how a drug works is important for selecting the most appropriate partner drugs to be used to increase efficacy and decrease toxicity in patients, to minimise the risk of drug resistance emerging and in designing second generation drugs. Using a combination of biochemical and genetic approaches we have discovered a novel nitroreductase (NTR2) that is necessary and sufficient for the anti-leishmanial activity of delamanid and related experimental drugs containing a nitro-group attached to two fused rings. This enzyme is responsible for activating bicyclic nitro-compounds to form toxic products that kill the parasite. In contrast, the previously identified nitroreductase (NTR1), which specifically activates monocyclic drugs, is not involved in this process. This knowledge can be applied to develop novel treatments for this disease.
doi:10.1371/journal.ppat.1005971
PMCID: PMC5094698  PMID: 27812217
2.  Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase 
PLoS Neglected Tropical Diseases  2016;10(5):e0004714.
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.
Author Summary
There are few validated and fully characterised targets suitable for drug discovery against African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the biochemical properties of the bifunctional enzyme, dihydrofolate reductase–thymidylate synthase (DHFR-TS), and its susceptibility to a range of classical inhibitors normally used in the treatment of cancer, bacterial or protozoal infections. Some of these drugs are extremely potent against the isolated enzyme, but much less so against the intact trypanosome. We have found that modulating certain medium components can affect drug sensitivity, presumably by either competition for uptake and competition for the active site of DHFR-TS. In the case of one human TS inhibitor (raltitrexed) the inhibitor is more potent against the intact parasite. We propose that addition of extra glutamic acid residues not only improves retention in the cell, but also increases potency against TS, as it does in human cells.
doi:10.1371/journal.pntd.0004714
PMCID: PMC4866688  PMID: 27175479
3.  Drug resistance in eukaryotic microorganisms 
Nature microbiology  2016;1(7):16092.
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.
doi:10.1038/nmicrobiol.2016.92
PMCID: PMC5215055  PMID: 27572976
4.  The anti-tubercular drug delamanid as a potential oral treatment for visceral leishmaniasis 
eLife  null;5:e09744.
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.
DOI: http://dx.doi.org/10.7554/eLife.09744.001
eLife digest
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.
DOI: http://dx.doi.org/10.7554/eLife.09744.002
doi:10.7554/eLife.09744
PMCID: PMC4878867  PMID: 27215734
Leishmania donovani; Visceral leishmaniasis; drug repurposing; pharmacokinetics; pharmacodynamics; OPC-67683; Mouse; Other
5.  Discovery of a Quinoline-4-carboxamide Derivative with a Novel Mechanism of Action, Multistage Antimalarial Activity, and Potent in Vivo Efficacy 
Journal of Medicinal Chemistry  2016;59(21):9672-9685.
The antiplasmodial activity, DMPK properties, and efficacy of a series of quinoline-4-carboxamides are described. This series was identified from a phenotypic screen against the blood stage of Plasmodium falciparum (3D7) and displayed moderate potency but with suboptimal physicochemical properties and poor microsomal stability. The screening hit (1, EC50 = 120 nM) was optimized to lead molecules with low nanomolar in vitro potency. Improvement of the pharmacokinetic profile led to several compounds showing excellent oral efficacy in the P. berghei malaria mouse model with ED90 values below 1 mg/kg when dosed orally for 4 days. The favorable potency, selectivity, DMPK properties, and efficacy coupled with a novel mechanism of action, inhibition of translation elongation factor 2 (PfEF2), led to progression of 2 (DDD107498) to preclinical development.
doi:10.1021/acs.jmedchem.6b00723
PMCID: PMC5108032  PMID: 27631715
6.  The Role of Folate Transport in Antifolate Drug Action in Trypanosoma brucei* 
The Journal of Biological Chemistry  2016;291(47):24768-24778.
The aim of this study was to identify and characterize mechanisms of resistance to antifolate drugs in African trypanosomes. Genome-wide RNAi library screens were undertaken in bloodstream form Trypanosoma brucei exposed to the antifolates methotrexate and raltitrexed. In conjunction with drug susceptibility and folate transport studies, RNAi knockdown was used to validate the functions of the putative folate transporters. The transport kinetics of folate and methotrexate were further characterized in whole cells. RNA interference target sequencing experiments identified a tandem array of genes encoding a folate transporter family, TbFT1–3, as major contributors to antifolate drug uptake. RNAi knockdown of TbFT1–3 substantially reduced folate transport into trypanosomes and reduced the parasite's susceptibly to the classical antifolates methotrexate and raltitrexed. In contrast, knockdown of TbFT1–3 increased susceptibly to the non-classical antifolates pyrimethamine and nolatrexed. Both folate and methotrexate transport were inhibited by classical antifolates but not by non-classical antifolates or biopterin. Thus, TbFT1–3 mediates the uptake of folate and classical antifolates in trypanosomes, and TbFT1–3 loss-of-function is a mechanism of antifolate drug resistance.
doi:10.1074/jbc.M116.750422
PMCID: PMC5114424  PMID: 27703008
drug resistance; folate; RNA interference (RNAi); transport; Trypanosoma brucei; RIT-seq; antifolate; human African trypanosomiasis; methotrexate; uptake
7.  The N-myristoylome of Trypanosoma cruzi 
Scientific Reports  2016;6:31078.
Protein N-myristoylation is catalysed by N-myristoyltransferase (NMT), an essential and druggable target in Trypanosoma cruzi, the causative agent of Chagas’ disease. Here we have employed whole cell labelling with azidomyristic acid and click chemistry to identify N-myristoylated proteins in different life cycle stages of the parasite. Only minor differences in fluorescent-labelling were observed between the dividing forms (the insect epimastigote and mammalian amastigote stages) and the non-dividing trypomastigote stage. Using a combination of label-free and stable isotope labelling of cells in culture (SILAC) based proteomic strategies in the presence and absence of the NMT inhibitor DDD85646, we identified 56 proteins enriched in at least two out of the three experimental approaches. Of these, 6 were likely to be false positives, with the remaining 50 commencing with amino acids MG at the N-terminus in one or more of the T. cruzi genomes. Most of these are proteins of unknown function (32), with the remainder (18) implicated in a diverse range of critical cellular and metabolic functions such as intracellular transport, cell signalling and protein turnover. In summary, we have established that 0.43–0.46% of the proteome is N-myristoylated in T. cruzi approaching that of other eukaryotic organisms (0.5–1.7%).
doi:10.1038/srep31078
PMCID: PMC4974623  PMID: 27492267
8.  Trisubstituted Pyrimidines as Efficacious and Fast-Acting Antimalarials 
Journal of Medicinal Chemistry  2016;59(13):6101-6120.
In this paper we describe the optimization of a phenotypic hit against Plasmodium falciparum, based on a trisubstituted pyrimidine scaffold. This led to compounds with good pharmacokinetics and oral activity in a P. berghei mouse model of malaria. The most promising compound (13) showed a reduction in parasitemia of 96% when dosed at 30 mg/kg orally once a day for 4 days in the P. berghei mouse model of malaria. It also demonstrated a rapid rate of clearance of the erythrocytic stage of P. falciparum in the SCID mouse model with an ED90 of 11.7 mg/kg when dosed orally. Unfortunately, the compound is a potent inhibitor of cytochrome P450 enzymes, probably due to a 4-pyridyl substituent. Nevertheless, this is a lead molecule with a potentially useful antimalarial profile, which could either be further optimized or be used for target hunting.
doi:10.1021/acs.jmedchem.6b00028
PMCID: PMC4947981  PMID: 27314305
9.  A novel multiple-stage antimalarial agent that inhibits protein synthesis 
Nature  2015;522(7556):315-320.
Summary
There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. We describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the parasite, with good pharmacokinetic properties, and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along mRNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.
doi:10.1038/nature14451
PMCID: PMC4700930  PMID: 26085270
10.  Genomic and Proteomic Studies on the Mode of Action of Oxaboroles against the African Trypanosome 
PLoS Neglected Tropical Diseases  2015;9(12):e0004299.
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.
Author Summary
The mode of action of a new class of boron-containing chemicals (the oxaboroles), currently under development for the treatment of human African trypanosomiasis, is unknown. Here we identify a number of potential candidate proteins that could be involved either in the mode of action of these compounds or in the mechanism of resistance. This information could prove critical in protecting the compounds against resistance emerging in the field as well as opening up new avenues for drug discovery.
doi:10.1371/journal.pntd.0004299
PMCID: PMC4689576  PMID: 26684831
11.  Nitroheterocyclic drug resistance mechanisms in Trypanosoma brucei 
Objectives
The objective of this study was to identify the mechanisms of resistance to nifurtimox and fexinidazole in African trypanosomes.
Methods
Bloodstream-form Trypanosoma brucei were selected for resistance to nifurtimox and fexinidazole by stepwise exposure to increasing drug concentrations. Clones were subjected to WGS to identify putative resistance genes. Transgenic parasites modulating expression of genes of interest were generated and drug susceptibility phenotypes determined.
Results
Nifurtimox-resistant (NfxR) and fexinidazole-resistant (FxR) parasites shared reciprocal cross-resistance suggestive of a common mechanism of action. Previously, a type I nitroreductase (NTR) has been implicated in nitro drug activation. WGS of resistant clones revealed that NfxR parasites had lost >100 kb from one copy of chromosome 7, rendering them hemizygous for NTR as well as over 30 other genes. FxR parasites retained both copies of NTR, but lost >70 kb downstream of one NTR allele, decreasing NTR transcription by half. A single knockout line of NTR displayed 1.6- and 1.9-fold resistance to nifurtimox and fexinidazole, respectively. Since NfxR and FxR parasites are ∼6- and 20-fold resistant to nifurtimox and fexinidazole, respectively, additional factors must be involved. Overexpression and knockout studies ruled out a role for a putative oxidoreductase (Tb927.7.7410) and a hypothetical gene (Tb927.1.1050), previously identified in a genome-scale RNAi screen.
Conclusions
NTR was confirmed as a key resistance determinant, either by loss of one gene copy or loss of gene expression. Further work is required to identify which of the many dozens of SNPs identified in the drug-resistant cell lines contribute to the overall resistance phenotype.
doi:10.1093/jac/dkv376
PMCID: PMC4743696  PMID: 26581221
12.  The R Enantiomer of the Antitubercular Drug PA-824 as a Potential Oral Treatment for Visceral Leishmaniasis 
Antimicrobial Agents and Chemotherapy  2013;57(10):4699-4706.
The novel nitroimidazopyran agent (S)-PA-824 has potent antibacterial activity against Mycobacterium tuberculosis in vitro and in vivo and is currently in phase II clinical trials for tuberculosis (TB). In contrast to M. tuberculosis, where (R)-PA-824 is inactive, we report here that both enantiomers of PA-824 show potent parasiticidal activity against Leishmania donovani, the causative agent of visceral leishmaniasis (VL). In leishmania-infected macrophages, (R)-PA-824 is 6-fold more active than (S)-PA-824. Both des-nitro analogues are inactive, underlining the importance of the nitro group in the mechanism of action. Although the in vitro and in vivo pharmacological profiles of the two enantiomers are similar, (R)-PA-824 is more efficacious in the murine model of VL, with >99% suppression of parasite burden when administered orally at 100 mg kg of body weight−1, twice daily for 5 days. In M. tuberculosis, (S)-PA-824 is a prodrug that is activated by a deazaflavin-dependent nitroreductase (Ddn), an enzyme which is absent in Leishmania spp. Unlike the case with nifurtimox and fexinidazole, transgenic parasites overexpressing the leishmania nitroreductase are not hypersensitive to either (R)-PA-824 or (S)-PA-824, indicating that this enzyme is not the primary target of these compounds. Drug combination studies in vitro indicate that fexinidazole and (R)-PA-824 are additive whereas (S)-PA-824 and (R)-PA-824 show mild antagonistic behavior. Thus, (R)-PA-824 is a promising candidate for late lead optimization for VL and may have potential for future use in combination therapy with fexinidazole, currently in phase II clinical trials against VL.
doi:10.1128/AAC.00722-13
PMCID: PMC3811480  PMID: 23856774
13.  Homoserine and quorum-sensing acyl homoserine lactones as alternative sources of threonine: a potential role for homoserine kinase in insect-stage Trypanosoma brucei 
Molecular Microbiology  2014;95(1):143-156.
De novo synthesis of threonine from aspartate occurs via the β-aspartyl phosphate pathway in plants, bacteria and fungi. However, the Trypanosoma brucei genome encodes only the last two steps in this pathway: homoserine kinase (HSK) and threonine synthase. Here, we investigated the possible roles for this incomplete pathway through biochemical, genetic and nutritional studies. Purified recombinant TbHSK specifically phosphorylates L-homoserine and displays kinetic properties similar to other HSKs. HSK null mutants generated in bloodstream forms displayed no growth phenotype in vitro or loss of virulence in vivo. However, following transformation into procyclic forms, homoserine, homoserine lactone and certain acyl homoserine lactones (AHLs) were found to substitute for threonine in growth media for wild-type procyclics, but not HSK null mutants. The tsetse fly is considered to be an unlikely source of these nutrients as it feeds exclusively on mammalian blood. Bioinformatic studies predict that tsetse endosymbionts possess part (up to homoserine in Wigglesworthia glossinidia) or all of the β-aspartyl phosphate pathway (Sodalis glossinidius). In addition S. glossinidius is known to produce 3-oxohexanoylhomoserine lactone which also supports trypanosome growth. We propose that T. brucei has retained HSK and threonine synthase in order to salvage these nutrients when threonine availability is limiting.
doi:10.1111/mmi.12853
PMCID: PMC4460637  PMID: 25367138
15.  A comparative study of type I and type II tryparedoxin peroxidases in Leishmania major 
The FEBS journal  2007;274(21):5643-5658.
Summary
The genome of Leishmania major, the causative agent of cutaneous Leishmaniasis, contains three nearly identical genes encoding putative glutathione peroxidases, which only differ at their N- and C-termini. Since the gene homologues are essential in trypanosomes, they may also represent potential drug targets in leishmania. Recombinant protein for the shortest of these showed negligible peroxidase activity with glutathione as electron donor indicating that it is not a bone fide glutathione peroxidase. In contrast, high peroxidase activity was obtained with tryparedoxin indicating that these proteins belong to a new class of monomeric tryparedoxin-dependent peroxidases (TDPX) distinct from the classical decameric 2-Cys peroxiredoxins (TryP). Mass spectrometry studies revealed that oxidation of TDPX1 with peroxides results in formation of an intramolecular disulphide bridge between Cys35 and Cys83. Site-directed mutagenesis and kinetic studies showed that Cys35 is essential for peroxidase activity whereas Cys83 is essential for reduction by tryparedoxin. Detailed kinetic studies comparing TDPX1 and TryP1 showed that both enzymes obey saturation ping pong kinetics with respect to tryparedoxin and peroxide. Both enzymes show high affinity for tryparedoxin and broad substrate specificity for hydroperoxides. TDPX1 shows higher affinity towards hydrogen peroxide and cumene hydroperoxide than to t-butyl hydroperoxide whereas no specific substrate preference could be detected for TryP1. TDPX1 exhibits rate constants up to 8 × 104 M−1s−1 whereas TryP1 exhibits higher rate constants ~106 M−1s−1. All three TDPX proteins together constitute about 0.05 % of the Leishmania major promastigote protein content whereas the TryPs are ~40 times more abundant. Possible specific functions of TDPXs are discussed.
doi:10.1111/j.1742-4658.2007.06087.x
PMCID: PMC3430366  PMID: 17922848
Glutathione peroxidase; tryparedoxin peroxidase; peroxiredoxin; trypanothione; Leishmania
16.  Arsenic Exposure and Outcomes of Antimonial Treatment in Visceral Leishmaniasis Patients in Bihar, India: A Retrospective Cohort Study 
PLoS Neglected Tropical Diseases  2015;9(3):e0003518.
Background
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.
Methods
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.
Results
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.
Discussion/Conclusion
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.
Author Summary
The parasitic disease visceral leishmaniasis (VL) causes a significant burden of illness and death in India. The main drug used to treat VL, which is based on the chemical element antimony, stopped working well in about half of all patients in the late twentieth century. We hypothesised that arsenic exposure of the Indian population, through contaminated groundwater, was contributing to treatment failure with antimony based drugs. Arsenic and antimony are similar chemical elements and exposure of the parasite to arsenic within the liver of arsenic-exposed patients could allow the parasite to become resistant to treatment with antimony. Using a field-based questionnaire study we retrospectively evaluated whether arsenic exposure was linked to antimonial treatment failure in a cohort of 110 antimonial treated patients. No significant association was found, although this may be because the number of patients in the study was low as antimony use was officially discontinued in 2005 due to high rates of treatment failure. However, arsenic exposure was found to increase risk of mortality from VL particularly if death occurred more than 3 months after the symptoms of VL developed. More research into the relationship between arsenic exposure and mortality in VL is warranted.
doi:10.1371/journal.pntd.0003518
PMCID: PMC4346263  PMID: 25730310
17.  TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei 
Nucleic Acids Research  2014;43(Database issue):D637-D644.
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.
doi:10.1093/nar/gku944
PMCID: PMC4384016  PMID: 25300491
18.  Biochemical and genetic characterization of Trypanosoma cruzi N-myristoyltransferase 
Biochemical Journal  2014;459(Pt 2):323-332.
Co- and post-translational N-myristoylation is known to play a role in the correct subcellular localization of specific proteins in eukaryotes. The enzyme that catalyses this reaction, NMT (N-myristoyltransferase), has been pharmacologically validated as a drug target in the African trypanosome, Trypanosoma brucei. In the present study, we evaluate NMT as a potential drug target in Trypanosoma cruzi, the causative agent of Chagas’ disease, using chemical and genetic approaches. Replacement of both allelic copies of TcNMT (T. cruzi NMT) was only possible in the presence of a constitutively expressed ectopic copy of the gene, indicating that this gene is essential for survival of T. cruzi epimastigotes. The pyrazole sulphonamide NMT inhibitor DDD85646 is 13–23-fold less potent against recombinant TcNMT than TbNMT (T. brucei NMT), with Ki values of 12.7 and 22.8 nM respectively, by scintillation proximity or coupled assay methods. DDD85646 also inhibits growth of T. cruzi epimastigotes (EC50=6.9 μM), but is ~1000-fold less potent than that reported for T. brucei. On-target activity is demonstrated by shifts in cell potency in lines that over- and under-express NMT and by inhibition of intracellular N-myristoylation of several proteins in a dose-dependent manner. Collectively, our findings suggest that N-myristoylation is an essential and druggable target in T. cruzi.
The present study shows that N-myristoyltransferase is essential for growth of Trypanosoma cruzi, the parasite responsible for Chagas’ disease. The kinetic properties of the enzyme are described along with evidence that growth is specifically inhibited by blocking N-myristoylation in the parasite.
doi:10.1042/BJ20131033
PMCID: PMC3969225  PMID: 24444291
Chagas’ disease; click chemistry; drug target; N-myristoylation; Trypanosoma cruzi; validation; CAP5.5, cytoskeleton-associated protein 5.5; DIG, digoxigenin; DKO, double knockout; DMEM, Dulbecco’s modified Eagle’s medium; HYG, hygromycin phosphotransferase; NMT, N-myristoyltransferase; NMTOE, NMT overexpressor; PAC, puromycin N-acetyltransferase; RTH/FBS, RPMI 1640 medium supplemented with trypticase, haemin, Hepes and 10% heat-inactivated FBS; SKO, single knockout; TbNMT, Trypanosoma brucei NMT; TCEP, tris-(2-carboxyethyl)phosphine; TcNMT, Trypanosoma cruzi NMT; TcTryR, Trypanosoma cruzi trypanothione reductase; WT, wild-type
19.  Norspermidine Is Not a Self-Produced Trigger for Biofilm Disassembly 
Cell  2014;156(4):844-854.
Summary
Formation of Bacillus subtilis biofilms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the polyamine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 μM prevents B. subtilis biofilm formation, (3) endogenous norspermidine is present in biofilms at 50–80 μM, and (4) norspermidine prevents biofilm formation by condensing biofilm exopolysaccharide. In contrast, we find that, at concentrations up to 200 μM, exogenous norspermidine promotes biofilm formation. We find that norspermidine is absent in wild-type B. subtilis biofilms at all stages, and higher concentrations of exogenous norspermidine eventually inhibit planktonic growth and biofilm formation in an exopolysaccharide-independent manner. Moreover, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, confirming that norspermidine is not physiologically relevant to biofilm function in this species.
Graphical Abstract
Highlights
•Norspermidine is not found or synthesized in Bacillus subtilis biofilms•Exogenous norspermidine inhibits growth of wild-type cells•Exogenous norspermidine inhibits growth of exopolysaccharide-deficient cells•Lower levels of exogenous norspermidine promote biofilm formation
It has been reported that norspermidine is synthesized by the bacterium Bacillus subtilis and inhibits biofilm formation by condensing exopolysaccharide. Now it is shown that norspermidine is not synthesized by B. subtilis, and high levels of exogenous norspermidine inhibit cell growth in an exopolysaccharide-independent manner.
doi:10.1016/j.cell.2014.01.012
PMCID: PMC3969229  PMID: 24529384
20.  Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy 
Molecular microbiology  2013;90(2):10.1111/mmi.12376.
Summary
African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) – a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.
doi:10.1111/mmi.12376
PMCID: PMC3868941  PMID: 23980694
21.  Genome sequence of the human malaria parasite Plasmodium falciparum 
Nature  2002;419(6906):10.1038/nature01097.
The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host–parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.
doi:10.1038/nature01097
PMCID: PMC3836256  PMID: 12368864
22.  Comparison of a High-Throughput High-Content Intracellular Leishmania donovani Assay with an Axenic Amastigote Assay 
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.
doi:10.1128/AAC.02398-12
PMCID: PMC3697379  PMID: 23571538
23.  Allosteric Activation of Trypanosomatid Deoxyhypusine Synthase by a Catalytically Dead Paralog*♦ 
The Journal of Biological Chemistry  2013;288(21):15256-15267.
Background: Deoxyhypusine synthase (DHS) catalyzes the spermidine-dependent modification of translation factor eIF5A.
Results: Trypanosomatid DHS activity is increased 3000-fold by heterotetramer formation with a catalytically dead paralog, and both gene products are essential for parasite growth.
Conclusion: Trypanosomatid DHS is a complex between catalytically impaired and inactive DHS subunits.
Significance: This activation mechanism uniquely evolved for two independent enzymes within the trypanosomatid polyamine pathway.
Polyamine biosynthesis is a key drug target in African trypanosomes. The “resurrection drug” eflornithine (difluoromethylornithine), which is used clinically to treat human African trypanosomiasis, inhibits the first step in polyamine (spermidine) biosynthesis, a highly regulated pathway in most eukaryotic cells. Previously, we showed that activity of a key trypanosomatid spermidine biosynthetic enzyme, S-adenosylmethionine decarboxylase, is regulated by heterodimer formation with a catalytically dead paralog (a prozyme). Here, we describe an expansion of this prozyme paradigm to the enzyme deoxyhypusine synthase, which is required for spermidine-dependent hypusine modification of a lysine residue in the essential translation factor eIF5A. Trypanosoma brucei encodes two deoxyhypusine synthase paralogs, one that is catalytically functional but grossly impaired, and the other is inactive. Co-expression in Escherichia coli results in heterotetramer formation with a 3000-fold increase in enzyme activity. This functional complex is also present in T. brucei, and conditional knock-out studies indicate that both DHS genes are essential for in vitro growth and infectivity in mice. The recurrent evolution of paralogous, catalytically dead enzyme-based activating mechanisms may be a consequence of the unusual gene expression in the parasites, which lack transcriptional regulation. Our results suggest that this mechanism may be more widely used by trypanosomatids to control enzyme activity and ultimately influence pathogenesis than currently appreciated.
doi:10.1074/jbc.M113.461137
PMCID: PMC3663545  PMID: 23525104
Parasite Metabolism; Polyamines; Protozoan; Trypanosoma brucei; Trypanosome; Deoxyhypusine; Deoxyhypusine Synthase; eIF5A; Spermidine
24.  Assessing the Essentiality of Leishmania donovani Nitroreductase and Its Role in Nitro Drug Activation 
The nitroimidazole fexinidazole has potential as a safe and effective oral drug therapy for the treatment of visceral leishmaniasis. To date, nitroheterocyclics have not been used in the treatment of leishmaniasis, and relatively little is known about their mechanism of action. In African trypanosomes, nitro drugs are reductively activated by a type I nitroreductase (NTR), absent in mammalian cells. Modulation of nitroreductase levels in Trypanosoma brucei directly affected sensitivity to nitro compounds, with reduced concentrations of the enzyme leading to moderate nitro drug resistance. In view of the progression of fexinidazole into clinical development for visceral leishmaniasis, here we assess the essentiality of the nitroreductase in Leishmania donovani and the effect of modulating nitroreductase levels on susceptibility to fexinidazole. The failure to directly replace both endogenous copies of the NTR gene, except in the presence of an ectopic copy of the gene, suggests that the NTR gene is essential for the growth and survival of L. donovani promastigotes. Loss of a single chromosomal copy of the L. donovani NTR gene resulted in parasites that were mildly resistant (<2-fold) to the predominant in vivo metabolite of fexinidazole, while parasites overexpressing NTR were 18-fold more susceptible. These data confirm that Leishmania NTR plays a pivotal role in fexinidazole activation. Reliance on a single enzyme for prodrug activation may leave fexinidazole vulnerable to the emergence of drug resistance. However, the essentiality of the NTR in L. donovani promastigotes, combined with the limited resistance shown by NTR single knockout cells, suggests that the potential for the spread of NTR-based resistance to fexinidazole may be limited.
doi:10.1128/AAC.01788-12
PMCID: PMC3553740  PMID: 23208716
25.  A Static-Cidal Assay for Trypanosoma brucei to Aid Hit Prioritisation for Progression into Drug Discovery Programmes 
Human African Trypanosomiasis is a vector-borne disease of sub-Saharan Africa that causes significant morbidity and mortality. Current therapies have many drawbacks, and there is an urgent need for new, better medicines. Ideally such new treatments should be fast-acting cidal agents that cure the disease in as few doses as possible. Screening assays used for hit-discovery campaigns often do not distinguish cytocidal from cytostatic compounds and further detailed follow-up experiments are required. Such studies usually do not have the throughput required to test the large numbers of hits produced in a primary high-throughput screen. Here, we present a 384-well assay that is compatible with high-throughput screening and provides an initial indication of the cidal nature of a compound. The assay produces growth curves at ten compound concentrations by assessing trypanosome counts at 4, 24 and 48 hours after compound addition. A reduction in trypanosome counts over time is used as a marker for cidal activity. The lowest concentration at which cell killing is seen is a quantitative measure for the cidal activity of the compound. We show that the assay can identify compounds that have trypanostatic activity rather than cidal activity, and importantly, that results from primary high-throughput assays can overestimate the potency of compounds significantly. This is due to biphasic growth inhibition, which remains hidden at low starting cell densities and is revealed in our static-cidal assay. The assay presented here provides an important tool to follow-up hits from high-throughput screening campaigns and avoid progression of compounds that have poor prospects due to lack of cidal activity or overestimated potency.
Author Summary
Trypanosoma brucei is a protozoan parasite causing African sleeping sickness. Current treatments for this disease have significant limitations, underlining the need for better and safer drugs. To identify new chemical starting points for drug development, large compound collections are screened against the parasite. Such screens typically do not distinguish between compounds that slow the growth of the parasite and compounds that actually kill the parasite (cidal compounds). Here, we present the development of an assay to identify such compounds. The main advantage of our assay is that it marries a relatively high-throughput to increased understanding of mode of action. Many active compounds (hits) are usually identified in T. brucei primary screening campaigns, making it difficult to select which compounds should undergo further development. Our assay allows testing of all of the hits for cidal activity so that only the most promising compounds are progressed. We show that the starting cell density used in the T. brucei growth assay can have a significant effect on the shape of dose response curves, and that important information regarding the mode of action of a compound can remain hidden at low starting densities as used commonly in T. brucei screening assays.
doi:10.1371/journal.pntd.0001932
PMCID: PMC3510075  PMID: 23209868

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