Explore the vast range of titles available.

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.

Chagas disease, caused by the protozoan intracellular parasite Trypanosoma cruzi , is a highly neglected tropical disease, causing significant morbidity and mortality in central and south America. Current treatments are inadequate, and recent clinical trials of drugs inhibiting CYP51 have failed, exposing a lack of understanding of how to translate laboratory findings to the clinic. Following these failures many new model systems have been developed, both in vitro and in vivo , that provide improved understanding of the causes for clinical trial failures. Amongst these are in vitro rate-of-kill (RoK) assays that reveal how fast compounds kill intracellular parasites. Such assays have shown clear distinctions between the compounds that failed in clinical trials and the standard of care. However, the published RoK assays have some key drawbacks, including low time-resolution and inability to track the same cell population over time. Here, we present a new, live-imaging RoK assay for intracellular T . cruzi that overcomes these issues. We show that the assay is highly reproducible and report high time-resolution RoK data for key clinical compounds as well as new chemical entities. The data generated by this assay allow fast acting compounds to be prioritised for progression, the fate of individual parasites to be tracked, shifts of mode-of-action within series to be monitored, better PKPD modelling and selection of suitable partners for combination therapy.

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.

Identifying how small molecules act to kill malaria parasites can lead to new “chemically validated” targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes Pf ACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and Pf ACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates Pf ACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of Pf ACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the Pf ACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability. Drug resistance to current antimalarials is rising and new drugs and targets are urgently needed. Here the authors identify Plasmodium falciparum acyl-CoA synthetase 10 as a new target whose inhibition leads to a decrease in triacylglycerols.

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.

An increase in treatment failures for malaria highlights an urgent need for new tools to understand and minimize the spread of drug resistance. We describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium spp, the causative agent of malaria. Saturation mutagenesis was used to generate a mutation library containing all conceivable mutations for a region of the antimalarial-binding site of a promising drug target, Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS). Screening of this high-coverage library with characterized Pf KRS inhibitors revealed multiple resistance-conferring substitutions including several clinically relevant mutations. Genetic validation of these mutations confirmed resistance of up to 100-fold and computational modeling dissected their role in drug resistance. We discuss potential applications of this data including the potential to design compounds that can bypass the most serious resistance mutations and future resistance surveillance.

Cryptosporidiosis is a leading cause of diarrheal-associated morbidity and mortality, predominantly affecting children under 5 years old in low-and-middle-income countries. There is no effective treatment and no vaccine. New therapeutics are emerging from drug discovery efforts. It is critical that mode of action studies are performed alongside drug discovery to ensure the best clinical outcomes. Unfortunately, technology to identify and validate drug targets for is severely lacking. We used lysyl-tRNA synthetase ( KRS) and DDD01510706 as a target-compound pair to develop both chemical and genetic tools for mode of action studies for . We adapted thermal proteome profiling (TPP) for , an unbiased approach for target identification. Using TPP we identified the molecular target of DDD01510706 and confirm that it is KRS. Genetic tools confirm that KRS is expressed throughout the life cycle and that this target is essential for parasite survival. Parasites genetically modified to over-express KRS or parasites with a mutation at the compound-binding site are resistant to treatment with DDD01510706. We leveraged these mutations to generate a second drug selection marker for genetic modification of , KRS . This second selection marker is interchangeable with the original selection marker, Neo , and expands the range of reverse genetic approaches available to study parasite biology. Due to the sexual nature of the life cycle, parental strains containing different drug selection markers can be crossed . Selection with both drug markers produces highly efficient genetic crosses (>99% hybrid progeny), paving the way for forward genetics approaches in .

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.

Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. New therapeutic options with diverse mechanisms of actions (MoAs) are required to consolidate progress toward control of this disease and combat drug resistance. Here, we describe the development of a scalable resistance library screen (RES-Seq) as a tool to facilitate the identification and prioritization of anti-leishmanial compounds acting via novel MoA. We have amassed a large collection of Leishmania donovani cell lines resistant to frontline drugs and compounds in the VL pipeline, with resistance-conferring mutations fully characterized. New phenotypic hits screened against this highly curated panel of resistant lines can determine cross-resistance and potentially shared MoA. The ability to efficiently identify compounds acting via previously established MoA is vital to maintain diversity within drug development portfolios. To expedite screening, short identifier DNA barcodes were introduced into resistant clones enabling pooling and simultaneous screening of multiple cell lines. Illumina sequencing of barcodes enables the growth kinetics and relative fitness of multiple cell lines under compound selection to be tracked. Optimal conditions allowing discrimination of resistant and sensitive clones were established (3× and 10× EC 50 for 3 days) and applied to screening of a complex library with VL preclinical and clinical drug candidates. RES-Seq is set to play an important role in ensuring that anti-leishmanial compounds exploiting diverse mechanisms of action are developed, ultimately providing options for future drug combination strategies. Visceral leishmaniasis (VL) remains the third largest parasitic killer worldwide, responsible for 20,000–30,000 deaths each year. Control and ultimate elimination of VL will require a range of therapeutic options with diverse mechanisms of action to combat drug resistance. One approach to ensure that compounds in development exploit diverse mechanisms of action is to screen them against highly curated cell lines resistant to drugs already in the VL pipeline. The identification of cross-resistant cell lines indicates that test compounds are likely acting via previously established mechanisms. Current cross-resistance screens are limited by the requirement to profile individual resistant cell lines one at a time. Here, we introduce unique DNA barcodes into multiple resistant cell lines to facilitate parallel profiling. Utilizing the power of Illumina sequencing, growth kinetics and relative fitness under compound selection can be monitored revolutionizing our ability to identify and prioritize compounds acting via novel mechanisms.

A series of 61 thiazolidine-2,4-diones bearing a styryl group at position 5 was synthesized in 2–5 steps and their structure was proved by elemental and spectral analyses. The compounds obtained were evaluated in vitro against the promastigote stage of the kinetoplastid parasite Leishmania infantum and the human HepG2 cell line, to determine selectivity indices and to compare their activities with those of antileishmanial reference drugs. The study of structure–activity relationships indicated the potential of some derivatives bearing a nitro group on the phenyl ring, especially when located at the meta position. Thus, among the tested series, compound 14c appeared as a hit compound with good antileishmanial activity (EC50 = 7 µM) and low cytotoxicity against both the hepatic HepG2 and macrophage THP-1 human cell lines (CC50 = 101 and 121 µM, respectively), leading to good selectivity indices (respectively, 14 and 17), in comparison with the reference antileishmanial drug compound miltefosine (EC50 = 3.3 µM, CC50 = 85 and 30 µM, SI = 26 and 9). Regarding its mechanism of action, among several possibilities, it was demonstrated that compound 14c is a prodrug bioactivated, predominantly by L. donovani nitroreductase 1, likely leading to the formation of cytotoxic metabolites that form covalent adducts in the parasite. Finally, compound 14c is lipophilic (measured CHI LogD7.7 = 2.85) but remains soluble in water (measured PBS solubility at pH7.4 = 16 µM), highlighting the antileishmanial potential of the nitrostyrylthiazolidine-2,4-dione scaffold.