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Concept: Trypanosome

170

Novel drugs to treat human African trypanosomiasis (HAT) are still urgently needed despite the recent addition of nifurtimox-eflornithine combination therapy (NECT) to WHO Model Lists of Essential Medicines against second stage HAT, where parasites have invaded the central nervous system (CNS). The pharmacology of a potential orally available lead compound, N-methoxy-6-{5-[4-(N-methoxyamidino) phenyl]-furan-2-yl}-nicotinamidine (DB844), was evaluated in a vervet monkey model of second stage HAT, following promising results in mice. DB844 was administered orally to vervet monkeys, beginning 28 days post infection (DPI) with Trypanosoma brucei rhodesiense KETRI 2537. DB844 was absorbed and converted to the active metabolite 6-[5-(4-phenylamidinophenyl)-furanyl-2-yl]-nicotinamide (DB820), exhibiting plasma C(max) values of 430 and 190 nM for DB844 and DB820, respectively, after the 14th dose at 6 mg/kg qd. A 100-fold reduction in blood trypanosome counts was observed within 24 h of the third dose and, at the end of treatment evaluation performed four days post the last drug dose, trypanosomes were not detected in the blood or cerebrospinal fluid of any monkey. However, some animals relapsed during the 300 days of post treatment monitoring, resulting in a cure rate of 3/8 (37.5%) and 3/7 (42.9%) for the 5 mg/kg×10 days and the 6 mg/kg×14 days dose regimens respectively. These DB844 efficacy data were an improvement compared with pentamidine and pafuramidine both of which were previously shown to be non-curative in this model of CNS stage HAT. These data show that synthesis of novel diamidines with improved activity against CNS-stage HAT was possible.

Concepts: Central nervous system, Nervous system, Pharmacology, Trypanosoma brucei, African trypanosomiasis, Trypanosoma, Euglenozoa, Trypanosome

168

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.

Concepts: Pharmacology, Drug discovery, Chemical compound, Trypanosoma brucei, African trypanosomiasis, Euglenozoa, Trypanosome, High-throughput screening

161

The emphasis placed on the activities of mobile teams in the detection of gambiense human African trypanosomiasis (HAT) can at times obscure the major role played by fixed health facilities in HAT control and surveillance. The lack of consistent and detailed data on the coverage of passive case-finding and treatment further constrains our ability to appreciate the full contribution of the health system to the control of HAT.

Concepts: Parasitic diseases, Trypanosoma brucei, African trypanosomiasis, Major, Trypanosome, Tsetse fly, Sleeping sickness, ObsCure II

27

Trypanosoma brucei are extracellular kinetoplastid parasites transmitted by the bloodsucking tsetse fly. They are responsible for the fatal disease human African trypanosomiasis (HAT), also known as Sleeping Sickness. In late stage infection, trypanosomes cross the blood-brain barrier (BBB) and invade the central nervous system (CNS) invariably leading to coma and death if untreated. There is no available vaccine and current late stage HAT chemotherapy consists of either melarsoprol, which is highly toxic causing up to 8% of deaths, or nifurtimox-eflornithine combination therapy (NECT), which is costly and difficult to administer. There is therefore an urgent need to identify new late stage HAT drug candidates. Here we review how current imaging tools, ranging from fluorescent confocal microscopy of live immobilised cells in culture to whole animal imaging, are providing insight into T. brucei biology, parasite / host interplay, trypanosome CNS invasion and disease progression. We also consider how imaging tools can be used for candidate drug screening purposes that could lead to new chemotherapies. This article is protected by copyright. All rights reserved.

Concepts: Immune system, Trypanosoma brucei, African trypanosomiasis, Trypanosoma, Euglenozoa, Trypanosome, Tsetse fly, Trypanosomiasis

25

BackgroundAfrican animal trypanosomiasis (AAT) is considered to be one of the greatest constraints to livestock production and livestock-crop integration in most African countries. South-eastern Uganda has suffered for more than two decades from outbreaks of zoonotic Human African Trypanosomiasis (HAT), adding to the burden faced by communities from AAT. There is insufficient AAT and HAT data available (in the animal reservoir) to guide and prioritize AAT control programs that has been generated using contemporary, sensitive and specific molecular techniques. This study was undertaken to evaluate the burden that AAT presents to the small-scale cattle production systems in south-eastern Uganda.MethodsRandomised cluster sampling was used to select 14% (57/401) of all cattle containing villages across Tororo District. Blood samples were taken from all cattle in the selected villages between September-December 2011; preserved on FTA cards and analysed for different trypanosomes using a suite of molecular techniques. Generalized estimating equation and Rogen-Gladen estimator models were used to calculate apparent and true prevalences of different trypanosomes while intra cluster correlations were estimated using a 1-way mixed effect analysis of variance (ANOVA) in R statistical software version 3.0.2.ResultsThe prevalence of all trypanosome species in cattle was 15.3% (95% CI; 12.2-19.1) while herd level trypanosome species prevalence varied greatly between 0-43%. Trypanosoma vivax (17.4%, 95% CI; 10.6-16.8) and Trypanosoma brucei rhodesiense (0.03%) were respectively, the most, and least prevalent trypanosome species identified.ConclusionsThe prevalence of bovine trypanosomes in this study indicates that AAT remains a significant constraint to livestock health and livestock production. There is need to implement tsetse and trypanosomiasis control efforts across Tororo District by employing effective, cheap and sustainable tsetse and trypanosomiasis control methods that could be integrated in the control of other endemic vector borne diseases like tick-borne diseases.

Concepts: Trypanosoma brucei, African trypanosomiasis, Trypanosoma, Euglenozoa, Trypanosome, Tsetse fly, Sleeping sickness, Trypanosomiasis

24

The protozoan parasite Trypanosoma brucei causes the fatal illness human African trypanosomiasis (HAT). Standard of care medications currently used to treat HAT have severe limitations, and there is a need to find new chemical entities that are active against infections of T. brucei. Following a “drug repurposing” approach, we tested anti-trypanosomal effects of carbazole-derived compounds called “Curaxins”. In vitro screening of 26 compounds revealed 22 with nanomolar potency against axenically cultured bloodstream trypanosomes. In a murine model of HAT, oral administration of compound 1 cured the disease. These studies established 1 as a lead for development of drugs against HAT. Pharmacological time-course studies revealed the primary effect of 1 to be concurrent inhibition of mitosis coupled with aberrant licensing of S-phase entry. Consequently, polyploid trypanosomes containing 8C equivalent of DNA per nucleus and three or four kinetoplasts were produced. These effects of 1 on the trypanosome are reminiscent of “mitotic slippage” or endoreplication observed in some other eukaryotes.

Concepts: Pharmacology, Chromosome, Mitosis, Trypanosoma brucei, African trypanosomiasis, Trypanosoma, Euglenozoa, Trypanosome

16

African trypanosomiasis is a deadly neglected disease caused by the extracellular parasite Trypanosoma brucei. Current therapies are characterized by high drug toxicity and increasing drug resistance mainly associated with loss-of-function mutations in the transporters involved in drug import. The introduction of new antiparasitic drugs into therapeutic use is a slow and expensive process. In contrast, specific targeting of existing drugs could represent a more rapid and cost-effective approach for neglected disease treatment, impacting through reduced systemic toxicity and circumventing resistance acquired through impaired compound uptake. We have generated nanoparticles of chitosan loaded with the trypanocidal drug pentamidine and coated by a single domain nanobody that specifically targets the surface of African trypanosomes. Once loaded into this nanocarrier, pentamidine enters trypanosomes through endocytosis instead of via classical cell surface transporters. The curative dose of pentamidine-loaded nanobody-chitosan nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, this new formulation displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine as a result of mutations in the surface transporter aquaglyceroporin 2. We conclude that this new drug delivery system increases drug efficacy and has the ability to overcome resistance to some anti-protozoal drugs.

Concepts: Pharmacology, Antibiotic resistance, Trypanosoma brucei, African trypanosomiasis, Trypanosoma, Trypanosome, Trypanosoma cruzi, Trypanosomiasis

13

Genetic-modification strategies are currently being developed to reduce the transmission of vector-borne diseases, including African trypanosomiasis. For tsetse, the vector of African trypanosomiasis, a paratransgenic strategy is being considered: this approach involves modification of the commensal symbiotic bacteria Sodalis to express trypanosome-resistance-conferring products. Modified Sodalis can then be driven into the tsetse population by cytoplasmic incompatibility (CI) from Wolbachia bacteria. To evaluate the effectiveness of this paratransgenic strategy in controlling African trypanosomiasis, we developed a three-species mathematical model of trypanosomiasis transmission among tsetse, humans, and animal reservoir hosts. Using empirical estimates of CI parameters, we found that paratransgenic tsetse have the potential to eliminate trypanosomiasis, provided that any extra mortality caused by Wolbachia colonization is low, that the paratransgene is effective at protecting against trypanosome transmission, and that the target tsetse species comprises a large majority of the tsetse population in the release location.

Concepts: Mathematics, Symbiosis, Parasitism, African trypanosomiasis, Wolbachia, Trypanosome, Tsetse fly, Trypanosomiasis

10

Gambian sleeping sickness (human African trypanosomiasis, HAT) outbreaks are brought under control by case detection and treatment although it is recognised that this typically only reaches about 75% of the population. Vector control is capable of completely interrupting HAT transmission but is not used because it is considered too expensive and difficult to organise in resource-poor settings. We conducted a full scale field trial of a refined vector control technology to determine its utility in control of Gambian HAT.

Concepts: Sleep disorder, African trypanosomiasis, Vector, Trypanosome, Tsetse fly, Sleeping sickness, Sterile insect technique, Geoffrey Douglas Hale Carpenter

9

Using whole-cell phenotypic assays, the GlaxoSmithKline high-throughput screening (HTS) diversity set of 1.8 million compounds was screened against the three kinetoplastids most relevant to human disease, i.e. Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. Secondary confirmatory and orthogonal intracellular anti-parasiticidal assays were conducted, and the potential for non-specific cytotoxicity determined. Hit compounds were chemically clustered and triaged for desirable physicochemical properties. The hypothetical biological target space covered by these diversity sets was investigated through bioinformatics methodologies. Consequently, three anti-kinetoplastid chemical boxes of ~200 compounds each were assembled. Functional analyses of these compounds suggest a wide array of potential modes of action against kinetoplastid kinases, proteases and cytochromes as well as potential host-pathogen targets. This is the first published parallel high throughput screening of a pharma compound collection against kinetoplastids. The compound sets are provided as an open resource for future lead discovery programs, and to address important research questions.

Concepts: Drug discovery, Chemical compound, Trypanosoma, Euglenozoa, Pharmacy, Trypanosome, High-throughput screening, Drug discovery hit to lead