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Tuberculosis remains one of the leading causes of death from a single infectious agent, surpassing both AIDS and malaria. In recent years, two bicyclic nitroimidazole drugs, delamanid and pretomanid have been approved to treat this airborne infection. This has spurred a renewed interest in developing new and improved nitroimidazole analogs. We have previously identified a new bicyclic heteroaromatic subclass, the nitroimidazopyrazinones, with substituted analogs showing promising activity against Mycobacterium tuberculosis under both aerobic and hypoxic environments. A second generation of nitroimidazopyrazinones with extended biaryl side chain also possessed good antiparasitic activity against Trypanosoma brucei brucei and Trypanosoma cruzi, suggesting the utility of this new scaffold for development into potential candidates against both tuberculosis and the kinetoplastid parasites which cause neglected tropical diseases. In this study, we further evaluated the properties of nitroimidazopyrazinone derivatives by assessing their selectivity against different mycobacterial species, measuring their reduction potential, and determining the kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn), which is the activating enzyme of delamanid and pretomanid in M. tuberculosis. We also conducted an in vivo evaluation of a lead compound, MCC8967 that demonstrated a favorable pharmacokinetic profile, with good oral bioavailability and efficacy in an acute M. tuberculosis infection model. Two other promising compounds MCC9481 and MCC9482, with good in vitro activity (IC50 = 0.016 and 0.10 µM, respectively) against T. cruzi, the causative agent for Chagas diseases, were similarly tested for in vivo activity. These compounds also exhibited good oral bioavailability, and transiently reduced the acute-stage parasite burden by >98‒99% at doses of 50 mg/kg once or twice daily, similar to benznidazole at 100 mg/kg once daily. Overall, we have demonstrated that active nitroimidazopyrazinones have potential to be developed as clinical candidates against both tuberculosis and Chagas disease.Author SummaryTuberculosis and parasitic infections continue to impose a significant threat to public health and economic growth worldwide. Most of the efforts to control these diseases still rely on drug treatments with limited effectiveness and significant side effects. There is now an urgent need to develop new treatments to combat these infections. Here, we report the in vitro and in vivo profile of a new bicyclic nitroimidazole subclass, namely nitroimidazopyrazinones, against mycobacteria and Trypanosoma cruzi. We found that derivatives with monocyclic side chains are selective against Mycobacterium tuberculosis, the causative agent of tuberculosis, but not active against other nontuberculosis mycobacteria. In an acute mouse model, they were able to reduce the bacterial load in lungs via oral administration. From a biochemistry perspective, we demonstrated that deazaflavin-dependent nitroreductase (Ddn) could act effectively on nitroimidazopyrazinones, indicating the potential of Ddn as an activating enzyme for these new compounds in M. tuberculosis. We also showed that derivatives with extended biaryl side chain were effective in suppressing infection in an acute T. cruzi infected murine model, with satisfactory oral bioavailability. These findings improve the understanding of the biological profile of nitroimidazopyrazinones for further development as potential antitubercular and antiparasitic agents.

Background Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and is a serious public health problem throughout Latin America. With 6 million people infected, there is a major international effort to develop new drugs. In the chronic phase of the disease, the parasite burden is extremely low, infections are highly focal at a tissue/organ level, and bloodstream parasites are only intermittently detectable. As a result, clinical trials are constrained by difficulties associated with determining parasitological cure. Even highly sensitive PCR methodologies can be unreliable, with a tendency to produce “false-cure” readouts. Improved diagnostic techniques and biomarkers for cure are therefore an important medical need. Methodology/Principal findings Using an experimental mouse model, we have combined a multiplex assay system and highly sensitive bioluminescence imaging to evaluate serological procedures for diagnosis of T. cruzi infections and confirmation of parasitological cure. We identified a set of three antigens that in the context of the multiplex serology system, provide a rapid, reactive and highly accurate read-out of both acute and chronic T. cruzi infection. In addition, we describe specific antibody responses where down-regulation can be correlated with benznidazole-mediated parasite reduction and others where upregulation is associated with persistent infection. One specific antibody (IBAG39) highly correlated with the bioluminescence flux and represents a promising therapy monitoring biomarker in mice. Conclusions/Significance Robust, high-throughput methodologies for monitoring the efficacy of anti-T. cruzi drug treatment are urgently required. Using our experimental systems, we have identified markers of infection or parasite reduction that merit assessing in a clinical setting for the longitudinal monitoring of drug-treated patients.

Trypanosoma cruzi is the etiological agent of Chagas disease. Following T cell mediated suppression of the acute phase infection, this intracellular eukaryotic pathogen persists long-term in a limited sub-set of tissues at extremely low-levels. The reasons for this tissue-specific chronicity are not understood. Using a dual bioluminescent:fluorescent reporter strain and highly sensitive tissue imaging that allows experimental infections to be monitored at single-cell resolution, we have undertaken a systematic analysis of the immunological micro-environments of rare parasitized cells in the mouse colon, a key site of persistence. We demonstrate that incomplete recruitment of T cells to a subset of colonic infection foci permits the occurrence of repeated cycles of intracellular parasite replication and differentiation to motile trypomastigotes at a frequency sufficient to perpetuate chronic infections. The life-long persistence of parasites in this tissue site continues despite the presence, at a systemic level, of a highly effective T cell response. Overcoming this low-level dynamic host:parasite equilibrium represents a major challenge for vaccine development.

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. The mechanism of pathogenesis is poorly understood and the lack of a robust, predictive animal model has held back research. We screened a series of mouse models using gastrointestinal tracer assays and in vivo infection imaging systems to discover a subset exhibiting chronic digestive transit dysfunction and significant retention of faeces in both sated and fasted conditions. The colon was a specific site of both tissue parasite persistence, delayed transit and dramatic loss of myenteric neurons as revealed by whole-mount immunofluorescence analysis. DCD mice therefore recapitulated key clinical manifestations of human disease. We also exploited dual reporter transgenic parasites to home in on locations of rare chronic infection foci in the colon by ex vivo bioluminescence imaging and then used fluorescence imaging in tissue microdomains to reveal co-localisation of infection and enteric nervous system lesions. This indicates that long-term T. cruzi-host interactions in the colon drive DCD pathogenesis, suggesting that the efficacy of anti-parasitic chemotherapy against chronic disease progression warrants further pre-clinical investigation., Author summary Chagas disease (American trypanosomiasis) is caused by the protozoan parasite Trypanosoma cruzi. Chagas disease has two types, the cardiac form and the digestive form; some patients have symptoms of both. How the parasite causes digestive disease is poorly understood. It is known that damage to the gut’s nervous system is an important factor, but it has been unclear exactly where and when this damage occurs during the course of an infection and also why only a subset of infected people suffer from this outcome. We studied infections in mice and found certain combinations of strains of parasites and mice that exhibited symptoms similar to human digestive Chagas patients, including a problem with peristalsis that localised specifically to the colon. Using parasites that were genetically engineered to emit both bioluminescent and fluorescent light, we tracked infections over time and were able to analyse rare infected cells deep within the muscle tissue of the wall of the colon. We found evidence of damaged neurons in the same location as these infection foci over 6 months after initial infection. Our results show that digestive Chagas disease probably develops as a result of chronic infection and inflammation, which potentially changes approaches to treatment.

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. The only treatments currently available are nitro-derivative drugs that are characterised by high toxicity and limited efficacy. Therefore, there is an urgent need for more effective, less toxic therapeutic agents. We have previously identified the potential for Mannich base derivatives as novel inhibitors of this parasite. To further explore this family of compounds, we synthesised a panel of 69 new analogues, based on multi-parametric structure-activity relationships, which allowed optimization of both anti-parasitic activity, physicochemical parameters and ADME properties. Additionally, we optimized our in vitro screening approaches against all three developmental forms of the parasite, allowing us to discard the least effective and trypanostatic derivatives at an early stage. We ultimately identified derivative 3c, which demonstrated excellent trypanocidal properties, and a synergistic mode of action against trypomastigotes in combination with the reference drug benznidazole. Both its druggability and low-cost production make this derivative a promising candidate for the preclinical, in vivo assays of the Chagas disease drug-discovery pipeline.

Trypanosoma cruzi is the etiological agent of Chagas disease. Following T cell mediated suppression of the acute phase infection, this intracellular eukaryotic pathogen persists in a limited sub-set of tissues at extremely low-levels. The reasons for this tissue-specific chronicity are not understood. Using a dual bioluminescent:fluorescent reporter strain, which allows experimental infections to be imaged at single-cell resolution, we have characterised the ‘hyper-local’ immunological microenvironment of rare parasitized cells in the mouse colon, a key site of persistence. We demonstrate that incomplete recruitment of T cells to infection foci permits the occurrence of repeated cycles of intracellular parasite replication and differentiation to motile trypomastigotes at a frequency sufficient to perpetuate chronic infections. The life-long persistence of parasites in this tissue site continues despite the presence, at a systemic level, of a highly effective T cell response. Overcoming this low-level dynamic host:parasite equilibrium represents a major challenge for vaccine development.

Made available in DSpace on 2022-04-29T08:45:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Hydroxymethylnitrofurazone (NFOH) is a therapeutic candidate for Chagas disease (CD). It has negligible hepatotoxicity in a murine model compared to the front-line drug benznidazole (BZN). Here, using Trypanosoma cruzi strains that express bioluminescent and/or fluorescent reporter proteins, we further investigated the in vitro and in vivo activity of NFOH to define whether the compound is trypanocidal or trypanostatic. The in vitro activity was assessed by exploiting the fluorescent reporter strain using wash-out assays and real-time microscopy. For animal experi-mentation, BALB/c mice were inoculated with the bioluminescent reporter strain and assessed by highly sensitive in vivo and ex vivo imaging. Cyclophosphamide treatment was used to promote parasite relapse in the chronic stage of infection. Our data show that NFOH acts by a trypanostatic mechanism, and that it is more active than BZN in vitro against the infectious trypomastigote form of Trypanosoma cruzi. We also found that it is more effective at curing experimental infections in the chronic stage, compared with the acute stage, a feature that it shares with BZN. Therefore, given its reduced toxicity, enhanced anti-trypomastigote activity, and curative properties, NFOH can be considered as a potential therapeutic option for Chagas disease, perhaps in combination with other trypanocidal agents. School of Pharmaceutical Sciences Sao Paulo State University (UNESP) Department of Infection Biology Faculty of Infectious and Tropical Diseases London School of Hygiene and Tropical Medicine, Keppel Street LAPEN—Laboratory of Design and Synthesis of Chemotherapeutic Agents Potentially Active on Neglected Diseases Department of Pharmacy School of Pharmaceutical Sciences University of Sao Paulo (USP) Advanced Research Center in Medicine School of Medicine Union of the Colleges of the Great Lakes (UNILAGO) School of Pharmaceutical Sciences Sao Paulo State University (UNESP) FAPESP: 2016/10847-9

Phenotypic screening of a 900 compound library of antitubercular nitroimidazole derivatives related to pretomanid against the protozoan parasite Trypanosoma cruzi (the causative agent for Chagas disease) identified several structurally diverse hits with an unknown mode of action. Following initial profiling, a first proof-of-concept in vivo study was undertaken, in which once daily oral dosing of a 7-substituted 2-nitroimidazooxazine analogue suppressed blood parasitemia to low or undetectable levels, although sterile cure was not achieved. Limited hit expansion studies alongside counter-screening of new compounds targeted at visceral leishmaniasis laid the foundation for a more in-depth assessment of the best leads, focusing on both drug-like attributes (solubility, metabolic stability and safety) and maximal killing of the parasite in a shorter timeframe. Comparative appraisal of one preferred lead (58) in a chronic infection mouse model, monitored by highly sensitive bioluminescence imaging, provided the first definitive evidence of (partial) curative efficacy with this promising nitroimidazooxazine class.

Chronic Trypanosoma cruzi infections are typically lifelong, with small numbers of parasites surviving in restricted tissue sites, which include the gastrointestinal tract. There is considerable debate about the replicative status of these persistent parasites and whether there is a role for dormancy in long-term infection. Here, we investigated T. cruzi proliferation in the colon of chronically infected mice using 5-ethynyl-2′deoxyuridine incorporation into DNA to provide ‘snapshots’ of parasite replication status. Highly sensitive imaging of the extremely rare infection foci, at single-cell resolution, revealed that parasites are three times more likely to be in S-phase during the acute stage than during the chronic stage. By implication, chronic infections of the colon are associated with a reduced rate of parasite replication. Despite this, very few host cells survived infection for more than 14 days, suggesting that T. cruzi persistence continues to involve regular cycles of replication, host cell lysis and re-infection. We could find no evidence for wide-spread dormancy in parasites that persist in this tissue reservoir.

Chronic Trypanosoma cruzi infections are typically life-long, with small numbers of parasites surviving in restricted tissue sites, which include the gastro-intestinal tract. There is considerable debate about the replicative status of these persistent parasites. Here, we investigated T. cruzi proliferation in the colon of chronically infected mice using 5-ethynyl-2’deoxyuridine incorporation into DNA to provide “snapshots” of parasite replication. Highly sensitive imaging of infection foci at single cell resolution revealed that parasites are three times more likely to be in S-phase during the acute stage than during the chronic stage. By implication, chronic infections are associated with a reduced rate of parasite replication. Despite this, very few host cells survive infection for >14 days, suggesting that T. cruzi persistence continues to involve regular cycles of replication, host cell lysis and re-infection. Therefore, long-term persistence in the colon is more likely to be associated with reduced proliferation than with dormancy.

New assay designs are needed to improve the predictive value of the Trypanosoma cruzi in vitro tests used as part of the Chagas' disease drug development pipeline. Here, we employed a green fluorescent protein (eGFP)-expressing parasite line and live high-content imaging to monitor the growth of T. cruzi amastigotes in mouse embryonic fibroblasts. A novel assay design allowed us to follow parasite numbers over 6 days, in four-hour intervals, while occupying the microscope for only 24 hours per biological replicate. Dose-response curves were calculated for each time point after addition of test compounds, revealing how EC50 values first decreased over the time of drug exposure, and then leveled off. However, we observed that parasite numbers could vary, even in the untreated controls, and at different sites in the same well, which caused variability in the EC50 values. To overcome this, we established that fold change in parasite number per hour is a more robust and informative measure of drug activity. This was calculated based on an exponential growth model for every biological sample. The net fold change per hour is the result of parasite replication, differentiation, and death. The calculation of this fold change enabled us to determine the tipping point of drug action, i.e. the time point when the death rate of the parasites exceeded the growth rate and the fold change dropped below 1, depending on the drug concentration and exposure time. This revealed specific pharmacodynamic profiles of the benchmark drugs benznidazole and posaconazole., Author summary Chagas' disease, caused by Trypanosoma cruzi, is a chronic debilitating infection occurring mostly in Latin America. There is an urgent need for new, well tolerated drugs. However, the latest therapeutic candidates have yielded disappointing outcomes in clinical trials, despite promising preclinical results. This demands new and more predictive in vitro assays. To address this, we have developed an assay design that enables the growth of T. cruzi intracellular forms to be monitored in real time, under drug pressure, for 6 days post-infection. This allowed us to establish the tipping point of drug action, when the death rate of the parasites exceeded the growth rate. The resulting pharmacodynamics profiles can provide robust and informative details on anti-chagasic candidates, as demonstrated for the benchmark drugs benznidazole and posaconazole.

The protozoan parasite Trypanosoma cruzi causes Chagas disease, an important public health problem throughout Latin America. Current therapeutic options are characterised by limited efficacy, long treatment regimens and frequent toxic side-effects. Advances in this area have been compromised by gaps in our knowledge of disease pathogenesis, parasite biology and drug activity. Nevertheless, several factors have come together to create a more optimistic scenario. Drug-based research has become more systematic, with increased collaborations between the academic and commercial sectors, often within the framework of not-for-profit consortia. High-throughput screening of compound libraries is being widely applied, and new technical advances are helping to streamline the drug development pipeline. In addition, drug repurposing and optimisation of current treatment regimens, informed by laboratory research, are providing a basis for new clinical trials. Here, we will provide an overview of the current status of Chagas disease drug development, highlight those areas where progress can be expected, and describe how fundamental research is helping to underpin the process.

Infections with Trypanosoma cruzi are usually life-long despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understand how T. cruzi avoids immune-mediated destruction. However, this is a major technical challenge because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing, ex vivo imaging, and confocal microscopy, which has allowed us to visualise infected host cells in murine tissue, with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of 200 parasites, that we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites found in large mega-nests within the muscle fibres. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission, and have implications for drug development.IMPORTANCETrypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence in experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon, and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a life-long infection, despite a vigorous immune response. It also informs drug-development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.

Chagas disease results from infection with the trypanosomatid parasite Trypanosoma cruzi. Progress in developing new drugs has been hampered by the long term and complex nature of the condition and by our limited understanding of parasite biology. Technical difficulties in assessing the parasite burden during the chronic stage of infection have also proven to be a particular challenge. In this context, the development of noninvasive, highly sensitive bioluminescence imaging procedures based on parasites that express a red-shifted luciferase has greatly enhanced our ability to monitor infections in experimental models. Applications of this methodology have led to new insights into tissue tropism and infection dynamics and have been a major driver in drug development. The system has been further modified by the generation of parasite reporter lines that express bioluminescent:fluorescent fusion proteins, an advancement that has allowed chronic infections in mice to be examined at a cellular level. By exploiting bioluminescence, to identify the rare sites of tissue infection, and fluorescence to detect T. cruzi at the level of individual host cells in histological sections, it has been possible to investigate the replication and differentiation status of parasites in vivo and to examine the cellular environment of infection foci. In combination, these data provide a framework for the detailed dissection of disease pathogenesis and drug activity.

We have previously described a number of lipophilic conformationally constrained spiro carbocyclic 2,6-diketopiperazine (2,6-DKP)-1-acetohydroxamic acids as potent anti-trypanosomal agents. In this report, we extend the SAR analysis in this class of compounds with respect to in vitro growth inhibition of Trypanosoma and Leishmania parasites. Introduction of bulky hydrophobic substituents at the vicinal position of the basic nitrogen atom in the spiro carbocyclic 2,6-DKP ring system can provide analogues which are potently active against bloodstream-form T. brucei and exhibit significant activities towards T. cruzi epimastogotes and L. infantum promastogotes and intracellular amastigotes. In particular, compounds possessing a benzyl or 4-chlorobenzyl substituent were found to be the most active growth inhibitors, with activities in the low nanomolar and low micromolar ranges for T. brucei and L. infantum, respectively. The benzyl substituted (S)-enantiomer was the most potent derivative against T. brucei (IC50 =6.8 nM), T. cruzi (IC50 =0.21 μΜ) and L. infantum promastigotes (IC50 =2.67 μM) and intracellular amastigotes (IC50 =2.60 μM). Moreover, the (R)-chiral benzyl substituted derivative and its racemic counterpart displayed significant activities against L. donovani. Importantly, the active compounds show high selectivity in comparison with two mammalian cell lines. This article is protected by copyright. All rights reserved.

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is the most common cause of cardiac-related deaths in endemic regions of Latin America. There is an urgent need for new safer treatments because current standard therapeutic options, benznidazole and nifurtimox, have significant side effects and are only effective in the acute phase of the infection with limited efficacy in the chronic phase. Phenotypic high content screening against the intracellular parasite in infected VERO cells was used to identify a novel hit series of 5-amino-1,2,3-triazole-4-carboxamides (ATC). Optimization of the ATC series gave improvements in potency, aqueous solubility, and metabolic stability, which combined to give significant improvements in oral exposure. Mitigation of a potential Ames and hERG liability ultimately led to two promising compounds, one of which demonstrated significant suppression of parasite burden in a mouse model of Chagas' disease.

Investigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge., Author summary Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is becoming an emerging threat in non-endemic countries and establishing new foci in endemic countries. The treatment available has not changed significantly in over 40 years. Therefore, there is an urgent need for a greater understanding of parasite biology and disease pathogenesis to identify new therapeutic targets and to maximise the efficient use of existing drugs. We have used genetically modified strains of T. cruzi carrying a bioluminescence/fluorescence dual reporter fusion gene to monitor parasite replication in vivo during both acute and chronic infections in a mouse model. Utilising TUNEL assays for mitochondrial DNA replication and EdU incorporation for total DNA replication, we have found that parasite division within infected cells is asynchronous in all phases of infection. Differentiation also appears to be uncoordinated, with replicating amastigotes co-existing with non-dividing trypomastigotes in the same host cell.

In this report, we extend the SAR analysis of a number of lipophilic guanylhydrazone analogues with respect to in vitro growth inhibition of Trypanosoma brucei and Trypanosoma cruzi. Sleeping sickness and Chagas disease, caused by the tropical parasites T. brucei and T. cruzi, constitute a significant socioeconomic burden in low-income countries of sub-Saharan Africa and Latin America, respectively. Drug development is underfunded. Moreover, current treatments are outdated and difficult to administer, while drug resistance is an emerging concern. The synthesis of adamantane-based compounds that have potential as antitrypanosomal agents is extensively reviewed. The critical role of the adamantane ring was further investigated by synthesizing and testing a number of novel lipophilic guanylhydrazones. The introduction of hydrophobic bulky substituents onto the adamantane ring generated the most active analogues, illustrating the synergistic effect of the lipophilic character of the C1 side chain and guanylhydrazone moiety on trypanocidal activity. The n-decyl C1-substituted compound G8 proved to be the most potent adamantane derivative against T. brucei with activity in the nanomolar range (EC50=90 nM). Molecular simulations were also performed to better understand the structure-activity relationships between the studied guanylhydrazone analogues and their potential enzyme target.

INTRODUCTION: Chagas disease, the most important parasitic infection in Latin America, is caused by the intracellular protozoan Trypanosoma cruzi. To treat this disease, only two nitroheterocyclic compounds with toxic side effects exist and frequent treatment failures are reported. Hence there is an urgent need to develop new drugs. Recently, metabolomics has become an efficient and cost-effective strategy for dissecting drug mode of action, which has been applied to bacteria as well as parasites, such as different Trypanosome species and forms. OBJECTIVES: We assessed if the metabolomics approach can be applied to study drug action of the intracellular amastigote form of T. cruzi in a parasite-host cell system. METHODS: We applied a metabolic fingerprinting approach (DI-MS and NMR) to evaluate metabolic changes induced by six different (candidate) drugs in a parasite-host cell system. In a second part of our study, we analyzed the impact of two drugs on polar metabolites, lipid and proteins to evaluate if affected pathways can be identified. RESULTS: Metabolic signatures, obtained by the fingerprinting approach, resulted in three different clusters. Two can be explained by already known of mode actions, whereas the three experimental drugs formed a separate cluster. Significant changes induced by drug action were observed in all the three metabolic fractions (polar metabolites, lipids and proteins). We identified a general impact on the TCA cycle, but no specific pathways could be attributed to drug action, which might be caused by a high percentage of common metabolome between a eukaryotic host cell and a eukaryotic parasite. Additionally, ion suppression effects due to differences in abundance between host cells and parasites may have occurred. CONCLUSION: We validated the metabolic fingerprinting approach to a complex host-cell parasite system. This technique can potentially be applied in the early stage of drug discovery and could help to prioritize early leads or reconfirmed hits for further development.

BackgroundThe long term and complex nature of Chagas disease in humans has restricted studies on vaccine feasibility. Animal models also have limitations due to technical difficulties in monitoring the extremely low parasite burden that is characteristic of chronic stage infections. Advances in imaging technology offer alternative approaches that circumvent these problems. Here, we describe the use of highly sensitive whole bodyin vivoimaging to assess the efficacy of recombinant viral vector vaccines and benznidazole-cured infections to protect mice from challenge withTrypanosoma cruzi.Methodology/Principal FindingsMice were infected withT. cruzistrains modified to express a red-shifted luciferase reporter. Using bioluminescence imaging, we assessed the degree of immunity to re-infection conferred after benznidazole-cure. Mice infected for 14 days or more, prior to the initiation of treatment, were highly protected from challenge with both homologous and heterologous strains (>99% reduction in parasite burden). Sterile protection against homologous challenge was frequently observed. This level of protection was considerably greater than that achieved with recombinant vaccines. It was also independent of the route of infection or size of the challenge inoculum, and was long-lasting, with no significant diminution in immunity after almost a year. When the primary infection was benznidazole-treated after 4 days (before completion of the first cycle of intracellular infection), the degree of protection was much reduced, an outcome associated with a minimalT. cruzi-specific IFN-γ+T cell response.Conclusions/SignificanceOur findings suggest that a protective Chagas disease vaccine must have the ability to eliminate parasites before they reach organs/tissues, such as the GI tract, where once established, they become largely refractory to the induced immune response.AUTHOR SUMMARYChagas disease, which is caused by the protozoan parasiteTrypanosoma cruzi, is a major public health problem throughout Latin America. Attempts to develop a vaccine have been hampered by technical difficulties in monitoring the extremely low parasite burden during the life-long chronic stage of infection. To circumvent these issues, we used highly sensitive bioluminescence imaging to assess the ability of recombinant viral vector vaccines and drug-cured infections to confer protection against experimental challenge in mice. We observed that drug-cured infections were much more effective than subunit vaccines, with many instances of sterile protection. Efficacy was independent of the route of infection or size of the challenge inoculum, and was undiminished after almost a year. In addition, drug-cured infections conferred a high level of cross-strain protection. The highly sensitive imaging procedures enabled us to visualise parasite distribution in mice where sterile protection was not achieved. This suggested that to confer sterile protection, vaccines must prevent the infection of organs/tissues that act as parasite reservoirs during the chronic stage. Once established at these sites, parasites become largely refractory to vaccine-induced elimination.

Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. Despite a global research effort, there have been no significant treatment advances for at least 40 years. Gaps in our knowledge of T. cruzi biology and pathogenesis have been major factors in limiting progress. In addition, the extremely low parasite burden during chronic infections has complicated the monitoring of both disease progression and drug efficacy, even in predictive animal models. To address these problems, we genetically modified T. cruzi to express a red-shifted luciferase. Mice infected with these highly bioluminescent parasites can be monitored by in vivo imaging, with exquisite sensitivity. However, a major drawback of bioluminescence imaging is that it does not allow visualization of host-parasite interactions at a cellular level. To facilitate this, we generated T. cruzi strains that express a chimeric protein that is both bioluminescent and fluorescent. Bioluminescence allows the tissue location of infection foci to be identified, and fluorescence can then be exploited to detect parasites in histological sections derived from excised tissue. In this article, we describe in detail the in vivo imaging and confocal microscopy protocols that we have developed for visualizing T. cruzi parasites expressing these dual-reporter fusion proteins. The approaches make it feasible to locate individual parasites within chronically infected murine tissues, to assess their replicative status, to resolve the nature of host cells, and to characterize their immunological context.

The synthesis and pharmacological evaluation of C1-substituted adamantane hydrazones, their C2-substituted isomers, and C1-substituted adamantane furanoic carboxamides is described. These new adamantane derivatives exhibited an interesting pharmacological profile in terms of trypanocidal activity and selectivity. The most active adduct with the best selectivity in this study was found to be the phenylacetoxy hydrazone 1 b (2-[4-(tricyclo[3.3.1.13,7 ]dec-1-yl)phenyl]-N'-[(5-nitrofuran-2-yl)methylene]acetohydrazide; EC50 =11±0.9 nm, SITb =770).

In chronic Trypanosoma cruzi infections, parasite burden is controlled by effective, but nonsterilising immune responses. Infected cells are difficult to detect because they are scarce and focally distributed in multiple sites. However, advances in detection technologies have established a link between parasite persistence and the pathogenesis of Chagas heart disease. Long-term persistence likely involves episodic reinvasion as well as continuous infection, to an extent that varies between tissues. The primary reservoir sites in humans are not definitively known, but analysis of murine models has identified the gastrointestinal tract. Here, we highlight that quantitative, spatial, and temporal aspects of T. cruzi infection are central to a fuller understanding of the association between persistence, pathogenesis, and immunity, and for optimising treatment.

Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. Despite a global research effort, there have been no significant treatment advances for at least 40 years. Gaps in our knowledge of T. cruzi biology and pathogenesis have been major factors in limiting progress. In addition, the extremely low parasite burden during chronic infections has complicated the monitoring of both disease progression and drug efficacy, even in predictive animal models. To address these problems, we genetically modified T. cruzi to express a red-shifted luciferase. Mice infected with these highly bioluminescent parasites can be monitored by in vivo imaging, with exquisite sensitivity. However, a major drawback of bioluminescence imaging is that it does not allow visualization of host-parasite interactions at a cellular level. To facilitate this, we generated T. cruzi strains that express a chimeric protein that is both bioluminescent and fluorescent. Bioluminescence allows the tissue location of infection foci to be identified, and fluorescence can then be exploited to detect parasites in histological sections derived from excised tissue. In this article, we describe in detail the in vivo imaging and confocal microscopy protocols that we have developed for visualizing T. cruzi parasites expressing these dual-reporter fusion proteins. The approaches make it feasible to locate individual parasites within chronically infected murine tissues, to assess their replicative status, to resolve the nature of host cells, and to characterize their immunological context.

Background The long term and complex nature of Chagas disease in humans has restricted studies on vaccine feasibility. Animal models also have limitations due to technical difficulties in monitoring the extremely low parasite burden that is characteristic of chronic stage infections. Advances in imaging technology offer alternative approaches that circumvent these problems. Here, we describe the use of highly sensitive whole body in vivo imaging to assess the efficacy of recombinant viral vector vaccines and benznidazole-cured infections to protect mice from challenge with Trypanosoma cruzi. Methodology/Principal findings Mice were infected with T. cruzi strains modified to express a red-shifted luciferase reporter. Using bioluminescence imaging, we assessed the degree of immunity to re-infection conferred after benznidazole-cure. Those infected for 14 days or more, prior to the onset of benznidazole treatment, were highly protected from challenge with both homologous and heterologous strains. There was a >99% reduction in parasite burden, with parasites frequently undetectable after homologous challenge. This level of protection was considerably greater than that achieved with recombinant vaccines. It was also independent of the route of infection or size of the challenge inoculum, and was long-lasting, with no significant diminution in immunity after almost a year. When the primary infection was benznidazole-treated after 4 days (before completion of the first cycle of intracellular infection), the degree of protection was much reduced, an outcome associated with a minimal T. cruzi-specific IFN-γ+ T cell response. Conclusions/Significance Our findings suggest that a protective Chagas disease vaccine must have the ability to eliminate parasites before they reach organs/tissues, such as the GI tract, where once established, they become largely refractory to the induced immune response., Author summary Chagas disease, which is caused by the protozoan parasite Trypanosoma cruzi, is a major public health problem throughout Latin America. Attempts to develop a vaccine have been hampered by technical difficulties in monitoring the extremely low parasite burden during the life-long chronic stage of infection. To circumvent these issues, we used highly sensitive bioluminescence imaging to assess the ability of recombinant viral vector vaccines and drug-cured infections to confer protection against experimental challenge in mice. We observed that drug-cured infections were much more effective than subunit vaccines. Efficacy was independent of the route of infection or size of the challenge inoculum, and was undiminished after almost a year. In addition, drug-cured infections conferred a high level of cross-strain protection. The highly sensitive imaging procedures enabled us to visualise parasite distribution in mice where full protection was not achieved. This suggested that to confer protection, vaccines must prevent the infection of organs/tissues that act as parasite reservoirs during the chronic stage. Once established at these sites, parasites become largely refractory to vaccine-induced elimination.

Chagasic heart disease develops in 30% of those infected with the protozoan parasite Trypanosoma cruzi, but can take decades to become symptomatic. Because of this, it has been difficult to assess the extent to which antiparasitic therapy can prevent the development of pathology. We sought to address this question using experimental murine models, exploiting highly sensitive bioluminescent imaging to monitor curative efficacy. Mice were inoculated with bioluminescent parasites and then cured in either the acute or chronic stage of infection with benznidazole. At the experimental endpoint (5 to 6 months postinfection), heart tissue was removed and assessed for inflammation and fibrosis, two widely used markers of cardiac pathology. Infection of BALB/c and C3H/HeN mice with distinct T. cruzi lineages resulted in greatly increased myocardial collagen content at a group level, indicative of fibrotic pathology. When mice were cured by benznidazole in the acute stage, the development of pathology was completely blocked. However, if treatment was delayed until the chronic stage, cardiac fibrosis was observed in the BALB/c model, although the protective effect was maintained in the case of C3H/HeN mice. These experiments therefore demonstrate that curative benznidazole treatment early in murine T. cruzi infections can prevent the development of cardiac fibrosis. They also show that treatment during the chronic stage can block pathology but the effectiveness varies between infection models. If these findings are extendable to humans, it implies that widespread chemotherapeutic intervention targeted at early-stage infections could play a crucial role in reducing Chagas disease morbidity at a population level.

Highlights • A straightforward method for optimising Trypanosoma cruzi transfection efficiency. • Facilitated by hydroxyurea-induced cell-cycle synchronization. • Applicable to both episomal and integrative-mediated transformation. • Reduces the time required to generate genetically modified cell lines. • Increases the number of stably transformed clones., The limited flexibility and time-consuming nature of the genetic manipulation procedures applicable to Trypanosoma cruzi continue to restrict the functional dissection of this parasite. We hypothesised that transformation efficiency could be enhanced if electroporation was timed to coincide with DNA replication. To test this, we generated epimastigote cultures enriched at the G1/S boundary using hydroxyurea-induced cell-cycle synchronisation, and then electroporated parasites at various time points after release from the cell-cycle block. We found a significant increase in transformation efficiency, with both episomal and integrative constructs, when cultures were electroporated 1 h after hydroxyurea removal. It was possible to generate genetically modified populations in less than 2 weeks, compared to the normal 4–6 weeks, with a 5 to 8-fold increase in the number of stably transformed clones. This straightforward optimisation step can be widely applied and should help streamline functional studies in T. cruzi.

The antifungal drug posaconazole has shown significant activity against Trypanosoma cruzi in vitro and in experimental murine models. Despite this, in a recent clinical trial it displayed limited curative potential. Drug testing is problematic in experimental Chagas disease because of difficulties in demonstrating sterile cure, particularly during the chronic stage of infection when parasite burden is extremely low and tissue distribution is ill defined. To better assess posaconazole efficacy against acute and chronic Chagas disease, we have exploited a highly sensitive bioluminescence imaging system which generates data with greater accuracy than other methods, including PCR-based approaches. Mice inoculated with bioluminescent T. cruzi were assessed by in vivo and ex vivo imaging, with cyclophosphamide-induced immunosuppression used to enhance the detection of relapse. Posaconazole was found to be significantly inferior to benznidazole as a treatment for both acute and chronic T. cruzi infections. Whereas 20 days treatment with benznidazole was 100% successful in achieving sterile cure, posaconazole failed in almost all cases. Treatment of chronic infections with posaconazole did however significantly reduce infection-induced splenomegaly, even in the absence of parasitological cure. The imaging-based screening system also revealed that adipose tissue is a major site of recrudescence in mice treated with posaconazole in the acute, but not the chronic stage of infection. This in vivo screening model for Chagas disease is predictive, reproducible and adaptable to diverse treatment schedules. It should provide greater assurance that drugs are not advanced prematurely into clinical trial.

Chagas disease is caused by the insect-transmitted protozoan Trypanosoma cruzi, and is the most important parasitic infection in Latin America. [...]

SUMMARY Chagas disease is caused by infection with the insect-transmitted protozoan Trypanosoma cruzi, and is the most important parasitic infection in Latin America. The current drugs, benznidazole and nifurtimox, are characterized by limited efficacy and toxic side-effects, and treatment failures are frequently observed. The urgent need for new therapeutic approaches is being met by a combined effort from the academic and commercial sectors, together with major input from not-for-profit drug development consortia. With the disappointing outcomes of recent clinical trials against chronic Chagas disease, it has become clear that an incomplete understanding of parasite biology and disease pathogenesis is impacting negatively on the development of more effective drugs. In addition, technical issues, including difficulties in establishing parasitological cure in both human patients and animal models, have greatly complicated the assessment of drug efficacy. Here, we outline the major questions that need to be addressed and discuss technical innovations that can be exploited to accelerate the drug development pipeline.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 5–8 million people in Latin America. Although the nitroheterocyclic compound benznidazole has been the front-line drug for several decades, treatment failures are common. Benznidazole is a pro-drug and is bio-activated within the parasite by the mitochondrial nitroreductase TcNTR-1, leading to the generation of reactive metabolites that have trypanocidal activity. To better assess drug action and resistance, we sequenced the genomes of T. cruzi Y strain (35.5 Mb) and three benznidazole-resistant clones derived from a single drug-selected population. This revealed the genome-wide accumulation of mutations in the resistant parasites, in addition to variations in DNA copy-number. We observed mutations in DNA repair genes, linked with increased susceptibility to DNA alkylating and inter-strand cross-linking agents. Stop-codon-generating mutations in TcNTR-1 were associated with cross-resistance to other nitroheterocyclic drugs. Unexpectedly, the clones were also highly resistant to the ergosterol biosynthesis inhibitor posaconazole, a drug proposed for use against T. cruzi infections, in combination with benznidazole. Our findings therefore identify the highly mutagenic activity of benznidazole metabolites in T. cruzi, demonstrate that this can result in multi-drug resistance, and indicate that vigilance will be required if benznidazole is used in combination therapy.

SUMMARYThe trypanosomes cause two neglected tropical diseases, Chagas disease in the Americas and African trypanosomiasis in sub-Saharan Africa. Over recent years a raft of molecular tools have been developed enabling the genetic dissection of many aspects of trypanosome biology, including the mechanisms underlying resistance to some of the current clinical and veterinary drugs. This has led to the identification and characterization of key resistance determinants, including transporters for the anti-Trypanosoma bruceidrugs, melarsoprol, pentamidine and eflornithine, and the activator of nifurtimox-benznidazole, the anti-Trypanosoma cruzidrugs. More recently, advances in sequencing technology, combined with the development of RNA interference libraries in the clinically relevant bloodstream form ofT. bruceihave led to an exponential increase in the number of proteins known to interact either directly or indirectly with the anti-trypanosomal drugs. In this review, we discuss these findings and the technological developments that are set to further revolutionise our understanding of drug-trypanosome interactions. The new knowledge gained should inform the development of novel interventions against the devastating diseases caused by these parasites.

The insect-transmitted protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, and infects 5–8 million people in Latin America. Chagas disease is characterised by an acute phase, which is partially resolved by the immune system, but then develops as a chronic life-long infection. There is a consensus that the front-line drugs benznidazole and nifurtimox are more effective against the acute stage in both clinical and experimental settings. However, confirmative studies have been restricted by difficulties in demonstrating sterile parasitological cure. Here, we describe a systematic study of nitroheterocyclic drug efficacy using highly sensitive bioluminescence imaging of murine infections. Unexpectedly, we find both drugs are more effective at curing chronic infections, judged by treatment duration and therapeutic dose. This was not associated with factors that differentially influence plasma drug concentrations in the two disease stages. We also observed that fexinidazole and fexinidazole sulfone are more effective than benznidazole and nifurtimox as curative treatments, particularly for acute stage infections, most likely as a result of the higher and more prolonged exposure of the sulfone derivative. If these findings are translatable to human patients, they will have important implications for treatment strategies.

Trimethylsilyl chloride is an efficient activating agent for azines in isocyanide-based reactions, which then proceed through a key insertion of the isocyanide into a N-Si bond. The reaction is initiated by N activation of the azine, followed by nucleophilic attack of an isocyanide in a Reissert-type process. Finally, a second equivalent of the same or a different isocyanide inserts into the N-Si bond leading to the final adduct. The use of distinct nucleophiles leads to a variety of α-substituted dihydroazines after a selective cascade process. Based on computational studies, a mechanistic hypothesis for the course of these reactions was proposed. The resulting products exhibit significant activity against Trypanosoma brucei and T. cruzi, featuring favorable drug-like properties and safety profiles.

Chagas disease and African sleeping sickness are trypanosomal infections that represent important public health problems in Latin America and Africa, respectively. The restriction of these diseases to the poorer parts of the world has meant that they have been largely neglected and limited progress has been made in their treatment. The nitroheterocyclic prodrugs nifurtimox and benznidazole, in use against Chagas disease for > 40 years, remain the only agents available for this infection. In the case of African sleeping sickness, nifurtimox has recently been added to the arsenal of medicines, with the nitroheterocycle fexinidazole currently under evaluation. For a long time, the cytotoxic mechanisms of these drugs were poorly understood: nifurtimox was thought to act via production of superoxide anions and nitro radicals, while the mode of benznidazole action was more obscure. The trypanocidal activity of nitroheterocyclic drugs is now known to depend on a parasite type I nitroreductase (NTR). This enzyme is absent from mammalian cells, a difference that forms the basis for the drug selectivity. The role of this enzyme in drug activation has been genetically and biochemically validated. It catalyses the 2-electron reduction of nitroheterocyclic compounds within the parasite, producing toxic metabolites without significant generation of superoxide. Recognition that this enzyme is responsible for activation of nitroheterocyclic prodrugs has allowed screening for compounds that preferentially target the parasite. This approach has led to the identification of two new classes of anti-trypanosomal agents, nitrobenzylphosphoramide mustards and aziridinyl nitrobenzamides, and promises to yield new, safer, more effective drugs.

We describe novel acetohydroxamic acid derivatives with potent activity against cultured bloodstream-form Trypanosoma brucei and selectivity indices of >1000. These analogues were derived from conformationally constrained, lipophilic, spiro carbocyclic 2,6-diketopiperazine (2,6-DKP) scaffolds by attaching acetohydroxamic acid moieties to the imidic nitrogen. Optimal activity was achieved by placing benzyl groups adjacent to the basic nitrogen of the 2,6-DKP core. S-Enantiomer 7d was the most active derivative against T. brucei (IC(50) = 6.8 nM) and T. cruzi (IC(50) = 0.21 μM).

Topoisomerase-II accumulates at centromeres during prometaphase, where it resolves the DNA catenations that represent the last link between sister chromatids. Previously, using approaches including etoposide-mediated topoisomerase-II cleavage, we mapped centromeric domains in trypanosomes, early branching eukaryotes in which chromosome segregation is poorly understood. Here, we show that in bloodstream form Trypanosoma brucei, RNAi-mediated depletion of topoisomerase-IIα, but not topoisomerase-IIβ, results in the abolition of centromere-localized activity and is lethal. Both phenotypes can be rescued by expression of the corresponding enzyme from T. cruzi. Therefore, processes which govern centromere-specific topoisomerase-II accumulation/activation have been functionally conserved within trypanosomes, despite the long evolutionary separation of these species and differences in centromeric DNA organization. The variable carboxyl terminal region of topoisomerase-II has a major role in regulating biological function. We therefore generated T. brucei lines expressing T. cruzi topoisomerase-II truncated at the carboxyl terminus and examined activity at centromeres after the RNAi-mediated depletion of the endogenous enzyme. A region necessary for nuclear localization was delineated to six residues. In other organisms, sumoylation of topoisomerase-II has been shown to be necessary for regulated chromosome segregation. Evidence that we present here suggests that sumoylation of the T. brucei enzyme is not required for centromere-specific cleavage activity.

Trypanosoma cruzi undergo PCD (programmed cell death) under appropriate stimuli, the mechanisms of which remain to be established. In the present study, we show that stimulation of PCD in T. cruzi epimastigotes by FHS (fresh human serum) results in rapid (

Metacyclogenesis in Trypanosoma cruzi involves the differentiation of replicating non-infective epimastigotes into non-replicating metacyclic trypomastigotes. This pre-adapts parasites for infection of the mammalian host and is characterised by several morphological changes and structural alterations to the nucleus, including nucleolar disaggregation. Experimental investigation of these developmental processes has been hampered by a lack of robust molecular markers. Here, we describe the precise temporal expression of the T. cruzi-specific protein Met-III, in the genome reference strain CL Brener. Expression is restricted to metacyclics in the insect stages of the life-cycle and is rapidly down-regulated following invasion of mammalian cells. Met-III localises to dispersed foci typical of the disassembled nucleolus in metacyclics and to the discrete single nucleolus of cells soon after macrophage invasion. To identify elements that target Met-III, we generated a series of tagged green fluorescent protein fusion proteins and examined their sub-nuclear location in transformed parasites. These experiments demonstrated that amino and carboxyl terminal fragments, characterised by clusters of basic residues, could independently mediate nucleolar sequestration. To investigate the function of Met-III, we used gene deletion. This showed that Met-III is not required for the development of metacyclic trypomastigotes and that null mutants can complete the life-cycle in vitro.

Benznidazole is the main drug used to treat Trypanosoma cruzi infections. However, frequent instances of treatment failure have been reported. To better understand potential resistance mechanisms, we analysed three clones isolated from a single parasite population that had undergone benznidazole-selection. These clones exhibited differing levels of benznidazole-resistance (varying between 9 and 26-fold), and displayed cross-resistance to nifurtimox (2 to 4-fold). Each clone had acquired a stop-codon-generating mutation in the gene which encodes the nitroreductase (TcNTR) that is responsible for activating nitroheterocyclic pro-drugs. In addition, one clone had lost a copy of the chromosome containing TcNTR. However, these processes alone are insufficient to account for the extent and diversity of benznidazole-resistance. It is implicit from our results that additional mechanisms must also operate and that T. cruzi has an intrinsic ability to develop drug-resistance by independent sequential steps, even within a single population. This has important implications for drug development strategies.

The cell surface of the epimastigote form of Trypanosoma cruzi is covered by glycoconjugates rich in galactose. The parasite cannot take up galactose through its hexose transporter, suggesting that the epimerisation of UDP-glucose to UDP-galactose may be the parasite's only route to this sugar. The T. cruzi UDP-glucose 4'-epimerase is encoded by the TcGALE gene. We were unable to make a CL-Brener strain T. cruzi epimastigote TcGALE-/- null mutant, suggesting that the gene is essential. Two TcGALE+/- single-allele knockout clones displayed aberrant morphology and haploid deficiency with respect to galactose metabolism. The morphological phenotypes included shortened flagella, increased incidence of spheromastigotes, agglutination and a novel walnut-like appearance. The reduced supply of UDP-galactose was manifest in the two clones as a six- or nine-fold reduction in the expression of galactopyranose-containing cell surface mucins and negligible or two-fold reduction in the expression of galactofuranose-containing glycoinositolphospholipids. The major loss of mucins as opposed to glycoinositolphospholipids may indicate that the latter are more important for basic parasite survival in culture. The apparent haploid deficiency suggests that epimerase levels are close to limiting, at least in the epimastigote form, and might be exploited as a potential drug target.

Imidazolium salts are privileged compounds in organic chemistry, and have valuable biological properties. Recent studies show that symmetric imidazolium salts with bulky moieties can display antiparasitic activity against T. cruzi. After developing a facile methodology for the synthesis of tetrasubstituted imidazolium salts from propargylamines and isocyanides, we screened a small library of these adducts against the causative agents of African and American trypanosomiases. These compounds display nanomolar activity against T. brucei and low (or sub) micromolar activity against T. cruzi, with excellent selectivity indexes and favorable molecular properties, thereby emerging as promising hits for the treatment of Chagas disease and sleeping sickness.

Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites.

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, one of the world’s major neglected infections. Although development of improved antiparasitic drugs is considered a priority, there have been no significant treatment advances in the past 40 years. Factors that have limited progress include an incomplete understanding of pathogenesis, tissue tropism, and disease progression. In addition, in vivo models, which allow parasite burdens to be tracked throughout the chronic stage of infection, have been lacking. To address these issues, we have developed a highly sensitive in vivo imaging system based on bioluminescent T. cruzi, which express a red-shifted luciferase that emits light in the tissue-penetrating orange-red region of the spectrum. The exquisite sensitivity of this noninvasive murine model has been exploited to monitor parasite burden in real time throughout the chronic stage, has allowed the identification of the gastrointestinal tract as the major niche of long-term infection, and has demonstrated that chagasic heart disease can develop in the absence of locally persistent parasites. Here, we review the parameters of the imaging system and describe how this experimental model can be incorporated into drug development programs as a valuable tool for assessing efficacy against both acute and chronic T. cruzi infections.

Background The neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in Trypanosoma cruzi, the causative agent. T. cruzi expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy. Methodology/Principal Findings To assess the importance of TcAPx in protecting T. cruzi from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle in vitro, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on T. cruzi replication, dissemination or tissue tropism in vivo. Conclusions/Significance TcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design., Author Summary The single-cell parasite Trypanosoma cruzi causes Chagas disease, one of the major neglected tropical infections. Throughout Latin America, this disease is an important cause of premature death due to heart failure, and it is currently an emerging public health problem in the US, Europe and across the world. There are only two drugs available to treat the infection, benznidazole and nifurtimox. Both have significant side effects and resistance/treatment failures are increasing. The T. cruzi vitamin-C dependent peroxidase (TcAPx) is an anti-oxidant enzyme which is absent from the mammalian host, and has been proposed as a potential drug target. Using genetic manipulation, we have deleted the genes for this enzyme from T. cruzi. The resulting null mutants were hypersensitive to hydrogen peroxide but displayed normal sensitivity to benznidazole. Using a bioluminescent model of infection, we could demonstrate that the null mutants were able to establish a similar infection to that of wild type parasites. Our results show that TcAPx should not be pursued as a target for drug design.

Summary Chronic Trypanosoma cruzi infections lead to cardiomyopathy in 20–30% of cases. A causal link between cardiac infection and pathology has been difficult to establish because of a lack of robust methods to detect scarce, focally distributed parasites within tissues. We developed a highly sensitive bioluminescence imaging system based on T. cruzi expressing a novel luciferase that emits tissue-penetrating orange-red light. This enabled long-term serial evaluation of parasite burdens in individual mice with an in vivo limit of detection of significantly less than 1000 parasites. Parasite distributions during chronic infections were highly focal and spatiotemporally dynamic, but did not localize to the heart. End-point ex vivo bioluminescence imaging allowed tissue-specific quantification of parasite loads with minimal sampling bias. During chronic infections, the gastro-intestinal tract, specifically the colon and stomach, was the only site where T. cruzi infection was consistently observed. Quantitative PCR-inferred parasite loads correlated with ex vivo bioluminescence and confirmed the gut as the parasite reservoir. Chronically infected mice developed myocarditis and cardiac fibrosis, despite the absence of locally persistent parasites. These data identify the gut as a permissive niche for long-term T. cruzi infection and show that canonical features of Chagas disease can occur without continual myocardium-specific infection.

The American trypanosome Trypanosoma cruzi is exposed to toxic oxygen metabolites that are generated by drug metabolism and immune responses in addition to those produced by endogenous processes. However, much remains to be resolved about the parasite oxidative defense system, including the mechanism(s) of peroxide reduction. Here we show that reduction of peroxides in T. cruzi is catalyzed by two distinct trypanothione-dependent enzymes. These were localized to the cytosol and mitochondrion. Both are members of the peroxiredoxin family of antioxidant proteins and are characterized by the presence of two conserved domains containing redox active cysteines. The role of these proteins in protecting T. cruzi from peroxide-mediated damage was demonstrated following overexpression of enzyme activity. The parasite-specific features of T. cruzi cytoplasmic peroxiredoxin and T. cruzi mitochondrial peroxiredoxin may be exploitable in terms of drug development.