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Neglected tropical diseases such as human African trypanosomiasis (HAT) are prevalent primarily in tropical climates and among populations living in poverty. Historically, the lack of economic incentive to develop new treatments for these diseases has meant that existing therapeutics have serious shortcomings in terms of safety, efficacy, and administration, and better therapeutics are needed. We now report a series of 3,5-disubstituted-7-azaindoles identified as growth inhibitors of Trypanosoma brucei, the parasite that causes HAT, through a high-throughput screen. We describe the hit-to-lead optimization of this series and the development and preclinical investigation of 29d, a potent anti-trypanosomal compound with promising pharmacokinetic (PK) parameters. This compound was ultimately not progressed beyond in vivo PK studies due to its inability to penetrate the blood-brain barrier (BBB), critical for stage 2 HAT treatments.

Protein-coding genes in the trypanosome genome are organized in polycistronic transcription units (PTUs). How RNA Polymerase II (Pol II) initiates PTU transcription has not been resolved and the current model favors initiation driven by chromatin epigenetic marks, rather than core-promoters. Here, we identify sequence-specific promoters by functional characterization of Pol II accumulation peaks identified by ChIP-Seq. Sequences located between oppositely orientated PTUs contained two independent and distinct promoters, each driving unidirectional transcription. Detailed analysis identified a 75-bp promoter necessary and sufficient to drive full reporter expression and containing functional motifs. Analysis of further promoters suggested activity and initiation is regulated and transcription-pattern is either focused or dispersed. In contrast to previous model of unregulated and promoter-independent transcription initiation, sequence-specific promoters determine the initiation of Pol II transcription of protein-coding genes in trypanosomes. These findings resolve the discrepancy between trypanosomes and other eukaryotes in how Pol II initiates protein-coding gene transcription and provide a new framework to events underlying eukaryotic transcription evolution.

Heterochromatin is essential in all eukaryotes to maintain genome integrity, long-term gene repression and to help chromosome segregation during mitosis. However, heterochromatin regions must be restricted by boundary elements to avoid its spreading over actively transcribed loci. In Plasmodium falciparum, facultative heterochromatin is important to regulate parasite virulence, antigenic variation and transmission. However, the underlying molecular mechanisms regulating repressive regions remain unknown. To investigate this topic, we chose the ap2-g gene, which forms a strictly delimited and independent heterochromatin island. Using electrophoretic motility shift assay (EMSA) we identified an ap2-g exon element at the 3’ end binding nuclear protein complexes. Upon replacement of this region by a gfp gene, we observed a shift in the heterochromatin boundary resulting in HP1 (Heterochromatin Protein 1) spreading over ∼2 additional kb downstream. We used this DNA element to purify candidate proteins followed by proteomic analysis. The identified complexes were found to be enriched in RNA-binding proteins, pointing to a potential role of RNA in the regulation of the ap2-g 3’ heterochromatin boundary. Our results provide insight into the unexplored topic of heterochromatin biology in P. falciparum and identify a DNA element within the master regulator of sexual commitment modulating heterochromatin spreading.

From a high-throughput screen of 42 444 knownhuman kinases inhibitors, a pyrazolo[1,5-b]pyridazine scaffold wasidentified to begin optimization for the treatment of human Africantrypanosomiasis. Previously reported data for analogous com-pounds against human kinases GSK-3β, CDK-2, and CDK-4 wereleveraged to try to improve the selectivity of the series, resulting in23awhich showed selectivity forT. b. bruceiover these threehuman enzymes. In parallel, properties known to influence theabsorption, distribution, metabolism, and excretion (ADME)profile of the series were optimized resulting in20gbeingprogressed into an efficacy study in mice. Though20gshowedtoxicity in mice, it also demonstrated CNS penetration in a PKstudy and significant reduction of parasitemia in four out of the sixmice., This work was supported by National Institutes of HealthGrants (R01AI114685 (M.P.P. and M.N.), R01AI082577(M.P.P.), R56AI099476 (M.P.P.), R01AI124046 (M.P.P.),R21AI127594 (M.P.P.), the Spanish Ministerio de Economía,Industria y Competitividad (M.N., Grant SAF2015-71444-P;D.G.P., Grant SAF2016-79957-R., and Subdirección Generalde Redes y Centros de Investigación Cooperativa (RICET)(M.N., Grant RD16/0027/0019; D.G.P., Grant RD16/0027/0014), Grant RTI2018-097210-B-100 (MINCIU-FEDER) toF.G. C.R.C. acknowledges grant support from the NIH-NIAID(Grant R21AI126296) and the Bill and Melinda GatesFoundation (Grant OPP1171488), as well as the technicalassistance of Brian M. Suzuki for screening adultS. mansoni.We are grateful to AstraZeneca for performing thein vitroADME experiments presented throughout and to CharlesRiver Labs for thein vitroADME data presented inTables S3−S5 in the Supporting Information. We thank GSK Tres Cantosopen lab foundation for running the PK studies discussed inthis publication. An academic license for ChemAxon (https://www.chemaxon.com) is gratefully acknowledged. We thankDr. Melissa Buskes for help in the preparation of thismanuscrip

From a high-throughput screen of 42 444 known human kinases inhibitors, a pyrazolo[1,5-b]pyridazine scaffold was identified to begin optimization for the treatment of human African trypanosomiasis. Previously reported data for analogous compounds against human kinases GSK-3β, CDK-2, and CDK-4 were leveraged to try to improve the selectivity of the series, resulting in 23a which showed selectivity for T. b. brucei over these three human enzymes. In parallel, properties known to influence the absorption, distribution, metabolism, and excretion (ADME) profile of the series were optimized resulting in 20g being progressed into an efficacy study in mice. Though 20g showed toxicity in mice, it also demonstrated CNS penetration in a PK study and significant reduction of parasitemia in four out of the six mice.

A kinase-targeting cell-based high-throughput screen (HTS) against Trypanosoma brucei was recently reported, and this screening set included the Published Kinase Inhibitor Set (PKIS). From the PKIS was identified 53 compounds with pEC(50) >= 6. Utilizing the published data available for the PKIS, a statistical analysis of these active antiparasitic compounds was performed, allowing identification of a set of human kinases having inhibitors that show a high likelihood for blocking T. brucei cellular proliferation in vitro. This observation was confirmed by testing other established inhibitors of these human kinases and by mining past screening campaigns at GlaxoSmithKline. Overall, although the parasite targets of action are not known, inhibitors of this set of human kinases displayed an enhanced hit rate relative to a random kinase-targeting HTS campaign, suggesting that repurposing efforts should focus primarily on inhibitors of these specific human kinases. We therefore term this statistical analysis-driven approach "preferred lead repurposing"., We acknowledge funding from The Tres Cantos Open Lab Foundation (TC-007) and the National Institutes of Health (R01AI082577, R01AI114685).

Summary African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human-infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance-associated protein (SRA protein), protects against ApoL1-mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA-mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well-characterized endosomal markers. By three-dimensional deconvolved immunofluorescence single-cell analysis, combined with double-labelling immunoelectron microscopy, we found that ≈ 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency.

OBJECTIVE To evaluate Trypanosoma brucei gambiense infection in peri-domestic livestock from Kogo and Mbini foci (Equatorial Guinea) in order to investigate its possible implication in the sleeping sickness transmission cycle in these hypoendemic foci. METHODS Samples from 698 domestic animals (goats, sheep and pigs) from trypanosomiasis-endemic localities of Kogo and Mbini foci were tested for animal trypanosomes and T. b. gambiense (group I) by species-specific polymerase chain reaction. RESULTS Trypanosoma brucei s.l., the predominant trypanosome species, was detected in 182 (52.6%) samples from Mbini and in 127 (36.1%) samples from Kogo. T. b. gambiense was only identified in seven (2%) of the Mbini samples and one co-infection (with T. vivax) was observed. CONCLUSION The occurrence of T. b. gambiense in peri-domestic livestock in Mbini and its absence in Kogo could explain the epidemiological differences between the two foci and could have significant implications for sleeping sickness control in Equatorial Guinea.

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2015.00765, Gambiense trypanosomiasis is considered an anthroponotic disease. Consequently, control programs are generally aimed at stopping transmission of Trypanosoma brucei gambiense (T. b. gambiense) by detecting and treating human cases. However, the persistence of numerous foci despite efforts to eliminate this disease questions this strategy as unique tool to pursue the eradication. The role of animals as a reservoir of T. b. gambiense is still controversial, but could partly explain maintenance of the infection at hypo-endemic levels. In the present study, we evaluated the presence of T. b. gambiense in wild animals in Equatorial Guinea. The infection rate ranged from 0.8% in the insular focus of Luba to more than 12% in Mbini, a focus with a constant trickle of human cases. The parasite was detected in a wide range of animal species including four species never described previously as putative reservoirs. Our study comes to reinforce the hypothesis that animals may play a role in the persistence of T. b. gambiense transmission, being particularly relevant in low transmission settings. Under these conditions the integration of sustained vector control and medical interventions should be considered to achieve the elimination of gambiense trypanosomiasis., We thank the hunter teams of Equatorial Guinea and the staff of the National Sleeping Sickness Control Programme. We wanted to extend our thanks to the authorities of the Ministry of Health and Social Welfare of Equatorial Guinea and the Spanish Agency for International Development Cooperation (AECID). We are grateful to Dr. Vincent Jamonneau (IRD) for providing valuable comments of the manuscript. J-MB is supported by a Miguel Servet Fellowship (CP09/00300) and funded by “Fondo de Investigación Sanitaria”(FIS) PI10/01128. AB and MN are granted by ISCIII -Subdirección General de Redes y Centros de InvestigaciónCooperativa (RICET) RD12/0018/0001 and RD12/0018/0015, respectively.

African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compounds against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chemical libraries using phenotypic and target-based approaches. Combinatorial chemistry libraries contain hundreds of thousands of compounds; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chemical diversity that can serve as excellent templates for the synthesis of novel, biologically active molecules. We report here a validated HTS platform for the screening of microbial extracts against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial extracts from the MEDINA Natural Products library. Tandem liquid chromatography-mass spectrometry showed that 48 extracts contain potentially new compounds that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product extracts against the above-mentioned kinetoplastid parasites.

© 2014 Diaz et al. In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs.

Trypanosoma brucei gambiense infection is widely considered an anthroponosis, although it has also been found in wild and domestic animals. Thus, fauna could act as reservoir, constraining the elimination of the parasite in hypo-endemic foci. To better understand the possible maintenance of T. b. gambiense in local fauna and investigate the molecular mechanisms underlying adaptation, we generated adapted cells lines (ACLs) by in vitro culture of the parasites in different mammalian sera. Using specific antibodies against the Variant Surface Glycoproteins (VSGs) we found that serum ACLs exhibited different VSG variants when maintained in pig, goat or human sera. Although newly detected VSGs were independent of the sera used, the consistent appearance of different VSGs suggested remodelling of the co-transcribed genes at the telomeric Expression Site (VSG-ES). Thus, Expression Site Associated Genes (ESAGs) sequences were analysed to investigate possible polymorphism selection. ESAGs 6 and 7 genotypes, encoding the transferrin receptor (TfR), expressed in different ACLs were characterised. In addition, we quantified the ESAG6/7 mRNA levels and analysed transferrin (Tf) uptake. Interestingly, the best growth occurred in pig and human serum ACLs, which consistently exhibited a predominant ESAG7 genotype and higher Tf uptake than those obtained in calf and goat sera. We also detected an apparent selection of specific ESAG3 genotypes in the pig and human serum ACLs, suggesting that other ESAGs could be involved in the host adaptation processes. Altogether, these results suggest a model whereby VSG-ES remodelling allows the parasite to express a specific set of ESAGs to provide selective advantages in different hosts. Finally, pig serum ACLs display phenotypic adaptation parameters closely related to human serum ACLs but distinct to parasites grown in calf and goat sera. These results suggest a better suitability of swine to maintain T. b. gambiense infection supporting previous epidemiological results. Figures 12 Citation: Cordon-Obras C, Cano J, González-Pacanowska D, Benito A, Navarro M, et al. (2013) Trypanosoma brucei gambiense Adaptation to Different Mammalian Sera Is Associated with VSG Expression Site Plasticity. PLoS ONE 8(12): e85072. doi:10.1371/journal.pone.0085072 Editor: Mauricio Martins Rodrigues, Federal University of São Paulo, Brazil Received: July 31, 2013; Accepted: November 21, 2013; Published: December 23, 2013 Copyright: © 2013 Cordon-Obras et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación, Plan Nacional (SAF2012-40029), Junta de Andalucia (CTS-5841), Fondo de Investigación Sanitaria (FIS) PI10/01128 and VI PN de I+D+I 2008-2011, ISCIII -Subdirección General de Redes y Centros de Investigación Cooperativa (RICET) RD12/0018/0001 and RD12/0018/0015. JMB is supported by a Miguel Servet Fellowship CP09/00300. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Background Sleeping sickness is spread over 36 Sub-Saharan African countries. In West and Central Africa, the disease is caused by Trypanosoma brucei gambiense, which produces a chronic clinical manifestation. The Luba focus (Bioko Island, Equatorial Guinea) has not reported autochthonous sleeping sickness cases since 1995, but given the complexity of the epidemiological cycle, the elimination of the parasite in the environment is difficult to categorically ensure. Methodology/Principal Findings The aim of this work is to assess, by a molecular approach (Polymerase Chain Reaction, PCR), the possible permanence of T. b. gambiense in the vector (Glossina spp.) and domestic fauna in order to improve our understanding of the epidemiological situation of the disease in an isolated focus considered to be under control. The results obtained show the absence of the parasite in peridomestic livestock but its presence, although at very low rate, in the vector. On the other hand, interesting entomological data highlight that an elevated concentration of tsetse flies was observed in two out of the ten villages considered to be in the focus. Conclusions These findings demonstrate that even in conditions of apparent control, a complete parasite clearance is difficult to achieve. Further investigations must be focused on animal reservoirs which could allow the parasites to persist without leading to human cases. In Luba, where domestic livestock are scarcer than other foci in mainland Equatorial Guinea, the epidemiological significance of wild fauna should be assessed to establish their role in the maintenance of the infection., Author Summary Sleeping sickness is a neglected disease with an important impact on public health of many countries of Sub-Saharan Africa. It is transmitted by tsetse fly bites (the vector) and mainly affects remote and rural populations. The chronic form, caused by Trypanosoma brucei gambiense, includes almost 90% of reported cases, and it is often misdiagnosed or lately detected after months or years of infection. Many efforts have been carried out to control the disease and interesting advances have been achieved. Although elimination is considered possible, there is an urgent need to understand the disease dynamic, especially in foci with very low rate (or absent) of infection for a long time. We performed a parasite screening in tsetse flies and livestock from Luba focus (Equatorial Guinea), considered to be “controlled” since 1995 (no human cases for fifteen years). The obtained results demonstrate that T. b. gambiense still remains in the environment and entomological data reveal high population density of the vector in some localities. This finding suggests that other intervention ways focused on control of vector populations, combined with the detection of human cases, could be necessary to achieve the total elimination of the parasite in hypoendemic foci.

[Background] Luba is one of the four historical foci of Human African Trypanosomiasis (HAT) on Bioko Island, in Equatorial Guinea. Although no human cases have been detected since 1995, T. b. gambiense was recently observed in the vector Glossina palpalis palpalis. The existence of cryptic species within this vector taxon has been previously suggested, although no data are available regarding the evolutionary history of tsetse flies populations in Bioko., [Methods] A phylogenetic analysis of 60 G. p. palpalis from Luba was performed sequencing three mitochondrial (COI, ND2 and 16S) and one nuclear (rDNA-ITS1) DNA markers. Phylogeny reconstruction was performed by Distance Based, Maximum Likelihood and Bayesian Inference methods., [Results] The COI and ND2 mitochondrial genes were concatenated and revealed 10 closely related haplotypes with a dominant one found in 61.1% of the flies. The sequence homology of the other 9 haplotypes compared to the former ranged from 99.6 to 99.9%. Phylogenetic analysis clearly clustered all island samples with flies coming from the Western African Clade (WAC), and separated from the flies belonging to the Central Africa Clade (CAC), including samples from Mbini and Kogo, two foci of mainland Equatorial Guinea. Consistent with mitochondrial data, analysis of the microsatellite motif present in the ITS1 sequence exhibited two closely related genotypes, clearly divergent from the genotypes previously identified in Mbini and Kogo., [Conclusions] We report herein that tsetse flies populations circulating in Equatorial Guinea are composed of two allopatric subspecies, one insular and the other continental. The presence of these two G. p. palpalis cryptic taxa in Equatorial Guinea should be taken into account to accurately manage vector control strategy, in a country where trypanosomiasis transmission is controlled but not definitively eliminated yet., This work has been supported by ‘Fondo de Investigacion Sanitaria (FIS)’ (PI10/01128) and by VI PN de I + D + I 2008–2011, ISCIII -Subdirección General de Redes y Centros de Investigación Cooperativa RD12/0018/0001 and RD12/0018/0015 (RICET). JMB is supported by Miguel Servet Fellowship CP09/00300.