Benznidazole treatment leads to DNA damage in Trypanosoma cruzi and the persistence of rare widely dispersed non-replicative amastigotes in mice.

Benznidazole is the front-line drug used to treat infections with Trypanosoma cruzi, the causative agent of Chagas disease. However, for reasons that are unknown, treatment failures are common. When we examined parasites that survived benznidazole treatment in mice using highly sensitive in vivo and ex vivo bioluminescence imaging, we found that recrudescence is not due to persistence of parasites in a specific organ or tissue that preferentially protects them from drug activity. Surviving parasites are widely distributed and located in host cells where the vast majority contained only one or two amastigotes. Therefore, infection relapse does not arise from a small number of intact large nests. Rather, persisters are either survivors of intracellular populations where co-located parasites have been killed, or amastigotes in single/low-level infected cells exist in a state where they are less susceptible to benznidazole. To better assess the nature of parasite persisters, we exposed infected mammalian cell monolayers to a benznidazole regimen that reduces the intracellular amastigote population to <1% of the pre-treatment level. Of host cells that remained infected, as with the situation in vivo, the vast majority contained only one or two surviving intracellular amastigotes. Analysis, based on non-incorporation of the thymidine analogue EdU, revealed these surviving parasites to be in a transient non-replicative state. Furthermore, treatment with benznidazole led to widespread parasite DNA damage. When the small number of parasites which survive in mice after non-curative treatment were assessed using EdU labelling, this revealed that these persisters were also initially non-replicative. A possible explanation could be that triggering of the T. cruzi DNA damage response pathway by the activity of benznidazole metabolites results in exit from the cell cycle as parasites attempt DNA repair, and that metabolic changes associated with non-proliferation act to reduce drug susceptibility. Alternatively, a small percentage of the parasite population may pre-exist in this non-replicative state prior to treatment.

Local association of Trypanosoma cruzi chronic infection foci and enteric neuropathic lesions at the tissue micro-domain scale.

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.

Incomplete Recruitment of Protective T Cells Is Associated with Trypanosoma cruzi Persistence in the Mouse Colon.

Trypanosoma cruzi is the etiological agent of Chagas disease. Following T cell-mediated suppression of acute-phase infection, this intracellular eukaryotic pathogen persists long-term in a limited subset 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 undertook a systematic analysis of the immunological microenvironments 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 lifelong 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.

Synthesis, antiproliferative and antitrypanosomal activities, and DNA binding of novel 6-amidino-2-arylbenzothiazoles.

A series of 6-amidinobenzothiazoles, linked via phenoxymethylene or directly to the 1,2,3-triazole ring with a p-substituted phenyl or benzyl moiety, were synthesised and evaluated in vitro against four human tumour cell lines and the protozoan parasite Trypanosoma brucei. The influence of the type of amidino substituent and phenoxymethylene linker on antiproliferative and antitrypanosomal activities was observed, showing that the imidazoline moiety had a major impact on both activities. Benzothiazole imidazoline 14a, which was directly connected to N-1-phenyl-1,2,3-triazole, had the most potent growth-inhibitory effect (IC50 = 0.25 µM) on colorectal adenocarcinoma (SW620), while benzothiazole imidazoline 11b, containing a phenoxymethylene linker, exhibited the best antitrypanosomal potency (IC90 = 0.12 µM). DNA binding assays showed a non-covalent interaction of 6-amidinobenzothiazole ligands, indicating both minor groove binding and intercalation modes of DNA interaction. Our findings encourage further development of novel structurally related 6-amidino-2-arylbenzothiazoles to obtain more selective anticancer and anti-HAT agents.

Shaving Is an Epiphenomenon of Type I and II Anti-CD20-Mediated Phagocytosis, whereas Antigenic Modulation Limits Type I Monoclonal Antibody Efficacy.

Rituximab is an anti-CD20 mAb used in the treatment of B cell malignancies. Loss of surface CD20 Ag from the surface of target cells is thought to be one mechanism governing resistance to rituximab, but how this occurs is not completely understood. Two explanations for this have been proposed: antigenic modulation whereby mAb:CD20 complexes are internalized in a B cell intrinsic process and shaving, in which mAb:CD20 complexes undergo trogocytic removal by effector cells, such as macrophages. However, there is conflicting evidence as to which predominates in clinical scenarios and hence the best strategies to overcome resistance. In this study, we investigated the relative importance of modulation and shaving in the downregulation of surface mAb:CD20. We used both murine and human systems and treated ex vivo macrophages with varying concentrations of non-FcγR-interacting beads to achieve differential macrophage saturation states, hence controllably suppressing further phagocytosis of target cells. We then monitored the level and localization of mAb:CD20 using a quenching assay. Suppression of phagocytosis with bead treatment decreased shaving and increased modulation, suggesting that the two compete for surface rituximab:CD20. Under all conditions tested, modulation predominated in rituximab loss, whereas shaving represented an epiphenomenon to phagocytosis. We also demonstrate that the nonmodulating, glycoengineered, type II mAb obinutuzumab caused a modest but significant increase in shaving compared with type II BHH2 human IgG1 wild-type mAb. Therefore, shaving may represent an important mechanism of resistance when modulation is curtailed, and glycoengineering mAb to increase affinity for FcγR may enhance resistance because of shaving.

Challenges in Chagas Disease Drug Development.

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.

Synthesis of diphenoxyadamantane alkylamines with pharmacological interest.

In this work, the synthesis and the pharmacological evaluation of diphenoxyadamantane alkylamines Ia-f and IIa-f is described. The new diphenoxy-substituted adamantanes share structural features present in trypanocidal and antitubercular agents. 1-Methylpiperazine derivative Ia is the most potent against T. brucei compound, whilst its hexylamine congener IIf exhibits a significant antimycobacterial activity.

Assessing the Effectiveness of Curative Benznidazole Treatment in Preventing Chronic Cardiac Pathology in Experimental Models of Chagas Disease.

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.

Synthesis, anti-bacterial and anti-protozoal activities of amidinobenzimidazole derivatives and their interactions with DNA and RNA.

Amidinobenzimidazole derivatives connected to 1-aryl-substituted 1,2,3-triazole through phenoxymethylene linkers 7a-7e, 8a-8e, and 9a-9e were designed and synthesised with the aim of evaluating their anti-bacterial and anti-trypanosomal activities and DNA/RNA binding affinity. Results from anti-bacterial evaluations of antibiotic-resistant pathogenic bacteria revealed that both o-chlorophenyl-1,2,3-triazole and N-isopropylamidine moieties in 8c led to strong inhibitory activity against resistant Gram-positive bacteria, particularly the MRSA strain. Furthermore, the non-substituted amidine and phenyl ring in 7a induced a marked anti-bacterial effect, with potency against ESBL-producing Gram-negative E. coli better than those of the antibiotics ceftazidime and ciprofloxacin. UV-Vis and CD spectroscopy, as well as thermal denaturation assays, indicated that compounds 7a and 8c showed also binding affinities towards ctDNA. Anti-trypanosomal evaluations showed that the p-methoxyphenyl-1,2,3-triazole moiety in 7b and 9b enhanced inhibitory activity against T. brucei, with 8b being more potent than nifurtimox, and having minimal toxicity towards mammalian cells.

Tetrasubstituted Imidazolium Salts as Potent Antiparasitic Agents against African and American Trypanosomiases.

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.

Host and parasite genetics shape a link between Trypanosoma cruzi infection dynamics and chronic cardiomyopathy.

Host and parasite diversity are suspected to be key factors in Chagas disease pathogenesis. Experimental investigation of underlying mechanisms is hampered by a lack of tools to detect scarce, pleiotropic infection foci. We developed sensitive imaging models to track Trypanosoma cruzi infection dynamics and quantify tissue-specific parasite loads, with minimal sampling bias. We used this technology to investigate cardiomyopathy caused by highly divergent parasite strains in BALB/c, C3H/HeN and C57BL/6 mice. The gastrointestinal tract was unexpectedly found to be the primary site of chronic infection in all models. Immunosuppression induced expansion of parasite loads in the gut and was followed by widespread dissemination. These data indicate that differential immune control of T. cruzi occurs between tissues and shows that the large intestine and stomach provide permissive niches for active infection. The end-point frequency of heart-specific infections ranged from 0% in TcVI-CLBR-infected C57BL/6 to 88% in TcI-JR-infected C3H/HeN mice. Nevertheless, infection led to fibrotic cardiac pathology in all models. Heart disease severity was associated with the model-dependent frequency of dissemination outside the gut and inferred cumulative heart-specific parasite loads. We propose a model of cardiac pathogenesis driven by periodic trafficking of parasites into the heart, occurring at a frequency determined by host and parasite genetics.

Biological factors that impinge on Chagas disease drug development.

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.

TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei.

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.

Synthesis and biological evaluation of N-cyanoalkyl-, N-aminoalkyl-, and N-guanidinoalkyl-substituted 4-aminoquinoline derivatives as potent, selective, brain permeable antitrypanosomal agents.

Current drugs against human African trypanosomiasis (HAT) suffer from several serious drawbacks. The search for novel, effective, brain permeable, safe, and inexpensive antitrypanosomal compounds is therefore an urgent need. We have recently reported that the 4-aminoquinoline derivative huprine Y, developed in our group as an anticholinesterasic agent, exhibits a submicromolar potency against Trypanosoma brucei and that its homo- and hetero-dimerization can result in to up to three-fold increased potency and selectivity. As an alternative strategy towards more potent smaller molecule anti-HAT agents, we have explored the introduction of ω-cyanoalkyl, ω-aminoalkyl, or ω-guanidinoalkyl chains at the primary amino group of huprine or the simplified 4-aminoquinoline analogue tacrine. Here, we describe the evaluation of a small in-house library and a second generation of newly synthesized derivatives, which has led to the identification of 13 side chain modified 4-aminoquinoline derivatives with submicromolar potencies against T. brucei. Among these compounds, the guanidinononyltacrine analogue 15e exhibits a 5-fold increased antitrypanosomal potency, 10-fold increased selectivity, and 100-fold decreased anticholinesterasic activity relative to the parent huprine Y. Its biological profile, lower molecular weight relative to dimeric compounds, reduced lipophilicity, and ease of synthesis, make it an interesting anti-HAT lead, amenable to further optimization to eliminate its remaining anticholinesterasic activity.

Limited Ability of Posaconazole To Cure both Acute and Chronic Trypanosoma cruzi Infections Revealed by Highly Sensitive In Vivo Imaging.

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.

Bioluminescence imaging of chronic Trypanosoma cruzi infections reveals tissue-specific parasite dynamics and heart disease in the absence of locally persistent infection.

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.

Synthesis, biological profiling and mechanistic studies of 4-aminoquinoline-based heterodimeric compounds with dual trypanocidal-antiplasmodial activity.

Dual submicromolar trypanocidal-antiplasmodial compounds have been identified by screening and chemical synthesis of 4-aminoquinoline-based heterodimeric compounds of three different structural classes. In Trypanosoma brucei, inhibition of the enzyme trypanothione reductase seems to be involved in the potent trypanocidal activity of these heterodimers, although it is probably not the main biological target. Regarding antiplasmodial activity, the heterodimers seem to share the mode of action of the antimalarial drug chloroquine, which involves inhibition of the haem detoxification process. Interestingly, all of these heterodimers display good brain permeabilities, thereby being potentially useful for late stage human African trypanosomiasis. Future optimization of these compounds should focus mainly on decreasing cytotoxicity and acetylcholinesterase inhibitory activity.

Evidence that transport of iron from the lysosome to the cytosol in African trypanosomes is mediated by a mucolipin orthologue.

Bloodstream-form Trypanosoma brucei acquire iron by receptor-mediated endocytosis of host transferrin. However, the mechanism(s) by which iron is then transferred from the lysosome to the cytosol are unresolved. Here, we provide evidence for the involvement of a protein (TbMLP) orthologous to the mammalian endolysosomal cation channel Mucolipin 1. In T. brucei, we show that this protein is localized to the single parasite lysosome. TbMLP null mutants could only be generated in the presence of an expressed ectopic copy, suggesting that the protein is essential. RNAi-mediated ablation resulted in a growth defect in vitro and led to a sevenfold increase in susceptibility to the iron-chelators deferoxamine and salicylhydroxamic acid. Conditional null mutants remained viable when the ectopic copy was repressed, but were hypersensitive to deferoxamine and displayed a growth defect similar to that observed following RNAi. The conditional nulls also retained virulence in vivo in the absence of the doxycycline inducer. These data provide strong evidence that TbMLP has a role in import of iron into the cytosol of African trypanosomes. They also indicate that even when expression is greatly reduced, there is sufficient protein, or an alternative mechanism, to provide the parasite with an adequate supply of cytosolic iron.

Synthesis and antiprotozoal activity of oligomethylene- and p-phenylene-bis(methylene)-linked bis(+)-huprines.

We have synthesized a series of dimers of (+)-(7R,11R)-huprine Y and evaluated their activity against Trypanosoma brucei, Plasmodium falciparum, rat myoblast L6 cells and human acetylcholinesterase (hAChE), and their brain permeability. Most dimers have more potent and selective trypanocidal activity than huprine Y and are brain permeable, but they are devoid of antimalarial activity and remain active against hAChE. Lead optimization will focus on identifying compounds with a more favourable trypanocidal/anticholinesterase activity ratio.

Dynamics of Trypanosoma cruzi infection in hamsters and novel association with progressive motor dysfunction.

Chagas disease is a zoonosis caused by the protozoan parasite Trypanosoma cruzi. Clinical outcomes range from long-term asymptomatic carriage to cardiac, digestive, neurological and composite presentations that can be fatal in both acute and chronic stages of the disease. Studies of T. cruzi in animal models, principally mice, have informed our understanding of the biological basis of this variability and its relationship to infection and host response dynamics. Hamsters have higher translational value for many human infectious diseases, but they have not been well developed as models of Chagas disease. We transposed a real-time bioluminescence imaging system for T. cruzi infection from mice into female Syrian hamsters (Mesocricetus auratus). This enabled us to study chronic tissue pathology in the context of spatiotemporal infection dynamics. Acute infections were widely disseminated, whereas chronic infections were almost entirely restricted to the skin and subcutaneous adipose tissue. Neither cardiac nor digestive tract disease were reproducible features of the model. Skeletal muscle had only sporadic parasitism in the chronic phase, but nevertheless displayed significant inflammation and fibrosis, features also seen in mouse models. Whereas mice had normal locomotion, all chronically infected hamsters developed hindlimb muscle hypertonia and a gait dysfunction resembling spastic diplegia. With further development, this model may therefore prove valuable in studies of peripheral nervous system involvement in Chagas disease.