2,4-Diaminopyrimidines as potent inhibitors of Trypanosoma brucei and identification of molecular targets by a chemical proteomics approach

Background

There is an urgent need to develop new, safe and effective treatments for human African trypanosomiasis (HAT) because current drugs have extremely poor safety profiles and are difficult to administer. Here we report the discovery of 2,4-diaminopyrimidines, exemplified by 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid phenylamide (SCYX-5070), as potent inhibitors of Trypanosoma brucei and the related trypanosomatid protozoans Leishmania spp.

Methodology/Principal Findings

In this work we show that loss of T. brucei viability following SCYX-5070 exposure was dependent on compound concentration and incubation time. Pulse incubation of T. brucei with SCYX-5070 demonstrates that a short period of exposure (10–12 hrs) is required to produce irreversible effects on survival or commit the parasites to death. SCYX-5070 cured an acute trypanosomiasis infection in mice without exhibiting signs of compound related acute or chronic toxicity. To identify the molecular target(s) responsible for the mechanism of action of 2,4-diaminopyrimidines against trypanosomatid protozoa, a representative analogue was immobilized on a solid matrix (sepharose) and used to isolate target proteins from parasite extracts. Mitogen-activated protein kinases (MAPKs) and cdc2-related kinases (CRKs) were identified as the major proteins specifically bound to the immobilized compound, suggesting their participation in the pharmacological effects of 2,4-diaminopyrimidines against trypanosomatid protozoan parasites.

Conclusions/Significance

Results show that 2,4-diaminopyrimidines have a good in vitro and in vivo pharmacological profile against trypanosomatid protozoans and that MAPKs and CRKs are potential molecular targets of these compounds. The 2,4-diminipyrimidines may serve as suitable leads for the development of novel treatments for HAT.     

Diagnostic Accuracy of Loopamp Trypanosoma brucei Detection Kit for Diagnosis of Human African Trypanosomiasis in Clinical Samples

Background

Molecular methods have great potential for sensitive parasite detection in the diagnosis of human African trypanosomiasis (HAT), but the requirements in terms of laboratory infrastructure limit their use to reference centres. A recently developed assay detects the Trypanozoon repetitive insertion mobile element (RIME) DNA under isothermal amplification conditions and has been transformed into a ready-to-use kit format, the Loopamp Trypanosoma brucei. In this study, we have evaluated the diagnostic performance of the Loopamp Trypanosoma brucei assay (hereafter called LAMP) in confirmed T.b. gambiense HAT patients, HAT suspects and healthy endemic controls from the Democratic Republic of the Congo (DRC).

Methodology/Principal findings

142 T.b. gambiense HAT patients, 111 healthy endemic controls and 97 HAT suspects with unconfirmed status were included in this retrospective evaluation. Reference standard tests were parasite detection in blood, lymph or cerebrospinal fluid. Archived DNA from blood of all study participants was analysed in duplicate with LAMP. Sensitivity of LAMP in parasitologically confirmed cases was 87.3% (95% CI 80.9–91.8%) in the first run and 93.0% (95% CI 87.5–96.1%) in the second run. Specificity in healthy controls was 92.8% (95% CI 86.4–96.3%) in the first run and 96.4% (95% CI 91.1–98.6%) in the second run. Reproducibility was excellent with a kappa value of 0.81.

Conclusions/Significance

In this laboratory-based study, the Loopamp Trypanosoma brucei Detection Kit showed good diagnostic accuracy and excellent reproducibility. Further studies are needed to assess the feasibility of its routine use for diagnosis of HAT under field conditions.     

Preclinical Assessment of the Treatment of Second-Stage African Trypanosomiasis with Cordycepin and Deoxycoformycin

Background

There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.).

Methodology/Principal Findings

Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug.

Conclusions/Significance

Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.     

Identification of peptide mimotopes of Trypanosoma brucei gambiense variant surface glycoproteins

Background

The current antibody detection tests for the diagnosis of gambiense human African trypanosomiasis (HAT) are based on native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. These native VSGs are difficult to produce, and contain non-specific epitopes that may cause cross-reactions. We aimed to identify mimotopic peptides for epitopes of T.b. gambiense VSGs that, when produced synthetically, can replace the native proteins in antibody detection tests.

Methodology/Principal Findings

PhD.-12 and PhD.-C7C phage display peptide libraries were screened with mouse monoclonal antibodies against the predominant VSGs LiTat 1.3 and LiTat 1.5 of T.b. gambiense. Thirty seven different peptide sequences corresponding to a linear LiTat 1.5 VSG epitope and 17 sequences corresponding to a discontinuous LiTat 1.3 VSG epitope were identified. Seventeen of 22 synthetic peptides inhibited the binding of their homologous monoclonal to VSG LiTat 1.5 or LiTat 1.3. Binding of these monoclonal antibodies to respectively six and three synthetic mimotopic peptides of LiTat 1.5 and LiTat 1.3 was significantly inhibited by HAT sera (p<0.05).

Conclusions/Significance

We successfully identified peptides that mimic epitopes on the native trypanosomal VSGs LiTat 1.5 and LiTat 1.3. These mimotopes might have potential for the diagnosis of human African trypanosomiasis but require further evaluation and testing with a large panel of HAT positive and negative sera.     

Quantifying the Association between Bovine and Human Trypanosomiasis in Newly Affected Sleeping Sickness Areas of Uganda

Background

Uganda has active foci of both chronic and acute HAT with the acute zoonotic form of disease classically considered to be restricted to southeast Uganda, while the focus of the chronic form of HAT was confined to the northwest of the country. Acute HAT has however been migrating from its traditional disease focus, spreading rapidly to new districts, a spread linked to movement of infected cattle following restocking. Cattle act as long-term reservoirs of human infective T. b. rhodesiense showing few signs of morbidity, yet posing a significant risk to human health. It is important to understand the relationship between infected cattle and infected individuals so that an appropriate response can be made to the risk posed to the community from animals infected with human pathogens in a village setting.

Methodology/Principal Findings

This paper examines the relationship between human T. b. rhodesiense infection and human infective and non-human T. brucei s.l. circulating in cattle at village level in Kaberamaido and Dokolo Districts, Uganda. The study was undertaken in villages that had reported a case of sleeping sickness in the six months prior to sample collection and those villages that had never reported a case of sleeping sickness.

Conclusions and Significance

The sleeping sickness status of the villages had a significant effect with higher odds of infection in cattle from case than from non-case villages for T. brucei s.l. (OR: 2.94, 95%CI: 1.38–6.24). Cattle age had a significant effect (p<0.001) on the likelihood of T. brucei s.l. infection within cattle: cattle between 18–36 months (OR: 3.51, 95%CI: 1.63–7.51) and cattle over 36 months (OR: 4.20, 95%CI: 2.08–8.67) had significantly higher odds of T. brucei s. l. infection than cattle under 18 months of age. Furthermore, village human sleeping sickness status had a significant effect (p<0.05) on the detection of T. b. rhodesiense in the village cattle herd, with significantly higher likelihood of T. b. rhodesiense in the village cattle of case villages (OR: 25, 95%CI: 1.2–520.71). Overall a higher than average T. brucei s.l. prevalence (>16.3%) in a village herd over was associated with significantly higher likelihood of T. b. rhodesiense being detected in a herd (OR: 25, 95%CI: 1.2–520.71).     

Socio-Economic and Cultural Determinants of Human African Trypanosomiasis at the Kenya – Uganda Transboundary

Background

Kenya and Uganda have reported different Human African Trypanosomiasis incidences in the past more than three decades, with the latter recording more cases. This cross-sectional study assessed the demographic characteristics, tsetse and trypanosomiasis control practices, socio-economic and cultural risk factors influencing Trypanosoma brucei rhodesiense (T.b.r.) infection in Teso and Busia Districts, Western Kenya and Tororo and Busia Districts, Southeast Uganda. A conceptual framework was postulated to explain interactions of various socio-economic, cultural and tsetse control factors that predispose individuals and populations to HAT.

Methods

A cross-sectional household survey was conducted between April and October 2008. Four administrative districts reporting T.b.r and lying adjacent to each other at the international boundary of Kenya and Uganda were purposely selected. Household data collection was carried out in two villages that had experienced HAT and one other village that had no reported HAT case from 1977 to 2008 in each district. A structured questionnaire was administered to 384 randomly selected household heads or their representatives in each country. The percent of respondents giving a specific answer was reported. Secondary data was also obtained on socio-economic and political issues in both countries.

Results

Inadequate knowledge on the disease cycle and intervention measures contributed considerable barriers to HAT, and more so in Uganda than in Kenya. Gender-associated socio-cultural practices greatly predisposed individuals to HAT. Pesticides-based crop husbandry in the 1970''s reportedly reduced vector population while vegetation of coffee and banana''s and livestock husbandry directly increased occurrence of HAT. Livestock husbandry practices in the villages were strong predictors of HAT incidence. The residents in Kenya (6.7%) applied chemoprophylaxis and chemotherapeutic controls against trypanosomiasis to a larger extent than Uganda (2.1%).

Conclusion

Knowledge on tsetse and its control methods, culture, farming practice, demographic and socio-economic variables explained occurrence of HAT better than landscape features.     

Human and Animal Trypanosomes in C?te d'Ivoire Form a Single Breeding Population

Background

Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and contributes to the related veterinary disease, Nagana. T. brucei is segregated into three subspecies based on host specificity, geography and pathology. T. b. brucei is limited to animals (excluding some primates) throughout sub-Saharan Africa and is non-infective to humans due to trypanolytic factors found in human serum. T. b. gambiense and T. b. rhodesiense are human infective sub-species. T. b. gambiense is the more prevalent human, causing over 97% of human cases. Study of T. b. gambiense is complicated in that there are two distinct groups delineated by genetics and phenotype. The relationships between the two groups and local T. b. brucei are unclear and may have a bearing on the evolution of the human infectivity traits.

Methodology/Principal Findings

A collection of sympatric T. brucei isolates from Côte d’Ivoire, consisting of T. b. brucei and both groups of T. b. gambiense have previously been categorized by isoenzymes, RFLPs and Blood Incubation Infectivity Tests. These samples were further characterized using the group 1 specific marker, TgSGP, and seven microsatellites. The relationships between the T. b. brucei and T. b. gambiense isolates were determined using principal components analysis, neighbor-joining phylogenetics, STRUCTURE, F_ST, Hardy-Weinberg equilibrium and linkage disequilibrium.

Conclusions/Significance

Group 1 T. b. gambiense form a clonal genetic group, distinct from group 2 and T. b. brucei, whereas group 2 T. b. gambiense are genetically indistinguishable from local T. b. brucei. There is strong evidence for mating within and between group 2 T. b. gambiense and T. b. brucei. We found no evidence to support the hypothesis that group 2 T. b. gambiense are hybrids of group 1 and T. b. brucei, suggesting that human infectivity has evolved independently in groups 1 and 2 T. b. gambiense.     

Revisiting the immune trypanolysis test to optimise epidemiological surveillance and control of sleeping sickness in West Africa

Background

Because of its high sensitivity and its ease of use in the field, the card agglutination test for trypanosomiasis (CATT) is widely used for mass screening of sleeping sickness. However, the CATT exhibits false-positive results (i) raising the question of whether CATT-positive subjects who are negative in parasitology are truly exposed to infection and (ii) making it difficult to evaluate whether Trypanosoma brucei (T.b.) gambiense is still circulating in areas of low endemicity. The objective of this study was to assess the value of the immune trypanolysis test (TL) in characterising the HAT status of CATT-positive subjects and to monitor HAT elimination in West Africa.

Methodology/Principal Findings

TL was performed on plasma collected from CATT-positive persons identified within medical surveys in several West African HAT foci in Guinea, Côte d''Ivoire and Burkina Faso with diverse epidemiological statuses (active, latent, or historical). All HAT cases were TL+. All subjects living in a nonendemic area were TL−. CATT prevalence was not correlated with HAT prevalence in the study areas, whereas a significant correlation was found using TL.

Conclusion and Significance

TL appears to be a marker for contact with T.b. gambiense. TL can be a tool (i) at an individual level to identify nonparasitologically confirmed CATT-positive subjects as well as those who had contact with T.b. gambiense and should be followed up, (ii) at a population level to identify priority areas for intervention, and (iii) in the context of HAT elimination to identify areas free of HAT.     

Genetic control of resistance to Trypanosoma brucei brucei infection in mice

Background

Trypanosoma brucei brucei infects livestock, with severe effects in horses and dogs. Mouse strains differ greatly in susceptibility to this parasite. However, no genes controlling these differences were mapped.

Methods

We studied the genetic control of survival after T. b. brucei infection using recombinant congenic (RC) strains, which have a high mapping power. Each RC strain of BALB/c-c-STS/A (CcS/Dem) series contains a different random subset of 12.5% genes from the parental “donor” strain STS/A and 87.5% genes from the “background” strain BALB/c. Although BALB/c and STS/A mice are similarly susceptible to T. b. brucei, the RC strain CcS-11 is more susceptible than either of them. We analyzed genetics of survival in T. b. brucei-infected F₂ hybrids between BALB/c and CcS-11. CcS-11 strain carries STS-derived segments on eight chromosomes. They were genotyped in the F₂ hybrid mice and their linkage with survival was tested by analysis of variance.

Results

We mapped four Tbbr (Trypanosoma brucei brucei response) loci that influence survival after T. b. brucei infection. Tbbr1 (chromosome 3) and Tbbr2 (chromosome 12) have effects on survival independent of inter-genic interactions (main effects). Tbbr3 (chromosome 7) influences survival in interaction with Tbbr4 (chromosome 19). Tbbr2 is located on a segment 2.15 Mb short that contains only 26 genes.

Conclusion

This study presents the first identification of chromosomal loci controlling susceptibility to T. b. brucei infection. While mapping in F₂ hybrids of inbred strains usually has a precision of 40–80 Mb, in RC strains we mapped Tbbr2 to a 2.15 Mb segment containing only 26 genes, which will enable an effective search for the candidate gene. Definition of susceptibility genes will improve the understanding of pathways and genetic diversity underlying the disease and may result in new strategies to overcome the active subversion of the immune system by T. b. brucei.     

Phosphorylation-dependent protein interaction with Trypanosoma brucei 14-3-3 proteins that display atypical target recognition

Background

The 14-3-3 proteins are structurally conserved throughout eukaryotes and participate in protein kinase signaling. All 14-3-3 proteins are known to bind to evolutionally conserved phosphoserine-containing motifs (modes 1 and/or 2) with high affinity. In Trypanosoma brucei, 14-3-3I and II play pivotal roles in motility, cytokinesis and the cell cycle. However, none of the T. brucei 14-3-3 binding proteins have previously been documented.

Methodology/Principal Findings

Initially we showed that T. brucei 14-3-3 proteins exhibit far lower affinity to those peptides containing RSxpSxP (mode 1) and RxY/FxpSxP (mode 2) (where x is any amino acid residue and pS is phosphoserine) than human 14-3-3 proteins, demonstrating the atypical target recognition by T. brucei 14-3-3 proteins. We found that the putative T. brucei protein phosphatase 2C (PP2c) binds to T. brucei 14-3-3 proteins utilizing its mode 3 motif (–pS/pTx_1-2-COOH, where x is not Pro). We constructed eight chimeric PP2c proteins replacing its authentic mode 3 motif with potential mode 3 sequences found in Trypanosoma brucei genome database, and tested their binding. As a result, T. brucei 14-3-3 proteins interacted with three out of eight chimeric proteins including two with high affinity. Importantly, T. brucei 14-3-3 proteins co-immunoprecipitated with an uncharacterized full-length protein containing identified high-affinity mode 3 motif, suggesting that both proteins form a complex in vivo. In addition, a synthetic peptide derived from this mode 3 motif binds to T. brucei 14-3-3 proteins with high affinity.

Conclusion/Significance

Because of the atypical target recognition of T. brucei 14-3-3 proteins, no 14-3-3-binding proteins have been successfully identified in T. brucei until now whereas over 200 human 14-3-3-binding proteins have been identified. This report describes the first discovery of the T. brucei 14-3-3-binding proteins and their binding motifs. The high-affinity phosphopeptide will be a powerful tool to identify novel T. brucei 14-3-3-binding proteins.     

Genetic Diversity and Population Structure of Trypanosoma brucei in Uganda: Implications for the Epidemiology of Sleeping Sickness and Nagana

Background

While Human African Trypanosomiasis (HAT) is in decline on the continent of Africa, the disease still remains a major health problem in Uganda. There are recurrent sporadic outbreaks in the traditionally endemic areas in south-east Uganda, and continued spread to new unaffected areas in central Uganda. We evaluated the evolutionary dynamics underpinning the origin of new foci and the impact of host species on parasite genetic diversity in Uganda. We genotyped 269 Trypanosoma brucei isolates collected from different regions in Uganda and southwestern Kenya at 17 microsatellite loci, and checked for the presence of the SRA gene that confers human infectivity to T. b. rhodesiense.

Results

Both Bayesian clustering methods and Discriminant Analysis of Principal Components partition Trypanosoma brucei isolates obtained from Uganda and southwestern Kenya into three distinct genetic clusters. Clusters 1 and 3 include isolates from central and southern Uganda, while cluster 2 contains mostly isolates from southwestern Kenya. These three clusters are not sorted by subspecies designation (T. b. brucei vs T. b. rhodesiense), host or date of collection. The analyses also show evidence of genetic admixture among the three genetic clusters and long-range dispersal, suggesting recent and possibly on-going gene flow between them.

Conclusions

Our results show that the expansion of the disease to the new foci in central Uganda occurred from the northward spread of T. b. rhodesiense (Tbr). They also confirm the emergence of the human infective strains (Tbr) from non-infective T. b. brucei (Tbb) strains of different genetic backgrounds, and the importance of cattle as Tbr reservoir, as confounders that shape the epidemiology of sleeping sickness in the region.     

Adenosine Kinase of T. b. rhodesiense Identified as the Putative Target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine Using Chemical Proteomics

Background

Human African trypanosomiasis (HAT), a major parasitic disease spread in Africa, urgently needs novel targets and new efficacious chemotherapeutic agents. Recently, we discovered that 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) exhibits specific antitrypanosomal activity with an IC₅₀ of 1.0 µM on Trypanosoma brucei rhodesiense (T. b. rhodesiense), the causative agent of the acute form of HAT.

Methodology/Principal Findings

In this work we show adenosine kinase of T. b. rhodesiense (TbrAK), a key enzyme of the parasite purine salvage pathway which is vital for parasite survival, to be the putative intracellular target of compound 1 using a chemical proteomics approach. This finding was confirmed by RNA interference experiments showing that down-regulation of adenosine kinase counteracts compound 1 activity. Further chemical validation demonstrated that compound 1 interacts specifically and tightly with TbrAK with nanomolar affinity, and in vitro activity measurements showed that compound 1 is an enhancer of TbrAK activity. The subsequent kinetic analysis provided strong evidence that the observed hyperactivation of TbrAK is due to the abolishment of the intrinsic substrate-inhibition.

Conclusions/Significance

The results suggest that TbrAK is the putative target of this compound, and that hyperactivation of TbrAK may represent a novel therapeutic strategy for the development of trypanocides.     

In-silico Investigation of Antitrypanosomal Phytochemicals from Nigerian Medicinal Plants

Background

Human African trypanosomiasis (HAT), a parasitic protozoal disease, is caused primarily by two subspecies of Trypanosoma brucei. HAT is a re-emerging disease and currently threatens millions of people in sub-Saharan Africa. Many affected people live in remote areas with limited access to health services and, therefore, rely on traditional herbal medicines for treatment.

Methods

A molecular docking study has been carried out on phytochemical agents that have been previously isolated and characterized from Nigerian medicinal plants, either known to be used ethnopharmacologically to treat parasitic infections or known to have in-vitro antitrypanosomal activity. A total of 386 compounds from 19 species of medicinal plants were investigated using in-silico molecular docking with validated Trypanosoma brucei protein targets that were available from the Protein Data Bank (PDB): Adenosine kinase (TbAK), pteridine reductase 1 (TbPTR1), dihydrofolate reductase (TbDHFR), trypanothione reductase (TbTR), cathepsin B (TbCatB), heat shock protein 90 (TbHSP90), sterol 14α-demethylase (TbCYP51), nucleoside hydrolase (TbNH), triose phosphate isomerase (TbTIM), nucleoside 2-deoxyribosyltransferase (TbNDRT), UDP-galactose 4′ epimerase (TbUDPGE), and ornithine decarboxylase (TbODC).

Results

This study revealed that triterpenoid and steroid ligands were largely selective for sterol 14α-demethylase; anthraquinones, xanthones, and berberine alkaloids docked strongly to pteridine reductase 1 (TbPTR1); chromenes, pyrazole and pyridine alkaloids preferred docking to triose phosphate isomerase (TbTIM); and numerous indole alkaloids showed notable docking energies with UDP-galactose 4′ epimerase (TbUDPGE). Polyphenolic compounds such as flavonoid gallates or flavonoid glycosides tended to be promiscuous docking agents, giving strong docking energies with most proteins.

Conclusions

This in-silico molecular docking study has identified potential biomolecular targets of phytochemical components of antitrypanosomal plants and has determined which phytochemical classes and structural manifolds likely target trypanosomal enzymes. The results could provide the framework for synthetic modification of bioactive phytochemicals, de novo synthesis of structural motifs, and lead to further phytochemical investigations.     

Focus-specific clinical profiles in human African Trypanosomiasis caused by Trypanosoma brucei rhodesiense

Background

Diverse clinical features have been reported in human African trypanosomiasis (HAT) foci caused by Trypanosoma brucei rhodesiense (T.b.rhodesiense) giving rise to the hypothesis that HAT manifests as a chronic disease in South-East African countries and increased in virulence towards the North. Such variation in disease severity suggests there are differences in host susceptibility to trypanosome infection and/or genetic variation in trypanosome virulence. Our molecular tools allow us to study the role of host and parasite genotypes, but obtaining matched extensive clinical data from a large cohort of HAT patients has previously proved problematic.

Methods/Principal Findings

We present a retrospective cohort study providing detailed clinical profiles of 275 HAT patients recruited in two northern foci (Uganda) and one southern focus (Malawi) in East Africa. Characteristic clinical signs and symptoms of T.b.rhodesiense infection were recorded and the degree of neurological dysfunction determined on admission. Clinical observations were mapped by patient estimated post-infection time. We have identified common presenting symptoms in T.b.rhodesiense infection; however, marked differences in disease progression and severity were identified between foci. HAT was characterised as a chronic haemo-lymphatic stage infection in Malawi, and as an acute disease with marked neurological impairment in Uganda. Within Uganda, a more rapid progression to meningo-encephaltic stage of infection was observed in one focus (Soroti) where HAT was characterised by early onset neurodysfunction; however, severe neuropathology was more frequently observed in patients in a second focus (Tororo).

Conclusions/Significance

We have established focus-specific HAT clinical phenotypes showing dramatic variations in disease severity and rate of stage progression both between northern and southern East African foci and between Ugandan foci. Understanding the contribution of host and parasite factors in causing such clinical diversity in T.b.rhodesiense HAT has much relevance for both improvement of disease management and the identification of new drug therapy.     

Affinity Is an Important Determinant of the Anti-Trypanosome Activity of Nanobodies

Background

The discovery of Nanobodies (Nbs) with a direct toxic activity against African trypanosomes is a recent advancement towards a new strategy against these extracellular parasites. The anti-trypanosomal activity relies on perturbing the highly active recycling of the Variant-specific Surface Glycoprotein (VSG) that occurs in the parasite''s flagellar pocket.

Methodology/Principal Findings

Here we expand the existing panel of Nbs with anti-Trypanosoma brucei potential and identify four categories based on their epitope specificity. We modified the binding properties of previously identified Nanobodies Nb_An05 and Nb_An33 by site-directed mutagenesis in the paratope and found this to strongly affect trypanotoxicity despite retention of antigen-targeting properties. Affinity measurements for all identified anti-trypanosomal Nbs reveal a strong correlation between trypanotoxicity and affinity (K_D), suggesting that it is a crucial determinant for this activity. Half maximal effective (50%) affinity of 57 nM was calculated from the non-linear dose-response curves. In line with these observations, Nb humanizing mutations only preserved the trypanotoxic activity if the K_D remained unaffected.

Conclusions/Significance

This study reveals that the binding properties of Nanobodies need to be compatible with achieving an occupancy of >95% saturation of the parasite surface VSG in order to exert an anti-trypanosomal activity. As such, Nb-based approaches directed against the VSG target would require binding to an accessible, conserved epitope with high affinity.     

A Target-Based High Throughput Screen Yields Trypanosoma brucei Hexokinase Small Molecule Inhibitors with Antiparasitic Activity

Background

The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the γ-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay.

Methodology/Principal Findings

Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were ∼20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03≤EC₅₀<3 µM) with parasite specificity of the compounds being demonstrated using insect stage T. brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics.

Conclusions/Significance

The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.     

Quantifying the burden of rhodesiense sleeping sickness in Urambo District, Tanzania

Background

Human African trypanosomiasis is a severely neglected vector-borne disease that is always fatal if untreated. In Tanzania it is highly focalised and of major socio-economic and public health importance in affected communities.

Objectives

This study aimed to estimate the public health burden of rhodesiense HAT in terms of DALYs and financial costs in a highly disease endemic area of Tanzania using hospital records.

Materials and Methods

Data was obtained from 143 patients admitted in 2004 for treatment for HAT at Kaliua Health Centre, Urambo District. The direct medical and other indirect costs incurred by individual patients and by the health services were calculated. DALYs were estimated using methods recommended by the Global Burden of Disease Project as well as those used in previous rhodesiense HAT estimates assuming HAT under reporting of 45%, a figure specific for Tanzania.

Results

The DALY estimate for HAT in Urambo District with and without age-weighting were 215.7 (95% CI: 155.3–287.5) and 281.6 (95% CI: 209.1–362.6) respectively. When 45% under-reporting was included, the results were 622.5 (95% CI: 155.3–1098.9) and 978.9 (95% CI: 201.1–1870.8) respectively. The costs of treating 143 patients in terms of admission costs, diagnosis, hospitalization and sleeping sickness drugs were estimated at US$ 15,514, of which patients themselves paid US$ 3,673 and the health services US$ 11,841. The burden in terms of indirect non-medical costs for the 143 patients was estimated at US$ 9,781.

Conclusions

This study shows that HAT imposes a considerable burden on affected rural communities in Tanzania and stresses the urgent need for location- and disease-specific burden estimates tailored to particular rural settings in countries like Tanzania where a considerable number of infectious diseases are prevalent and, due to their focal nature, are often concentrated in certain locations where they impose an especially high burden.