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Trypanosoma cruzi, the parasite causing Chagas disease, is a digenetic flagellated protist that infects mammals (including humans) and reduviid insect vectors. Therefore, T. cruzi must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard, it is well known that T. cruzi has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established has been the capability of T. cruzi to use glucose and amino acids to support the differentiation process occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities of using externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production. Additionally, we show that electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid (TCA) cycle, which can be used to feed anabolic pathways such as the de novo biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization into the mitochondrion diminishes the survival to severe starvation, and impairs metacyclogenesis., Author summary Trypanosoma cruzi is a protist parasite with a life cycle involving two types of hosts, a vertebrate one (which includes humans, causing Chagas disease) and an invertebrate one (kissing bugs, which vectorize the infection among mammals). In both hosts, the parasite faces environmental challenges such as sudden changes in the metabolic composition of the medium in which they develop, severe starvation, osmotic stress and redox imbalance, among others. Because kissing bugs feed infrequently in nature, an intriguing aspect of T. cruzi biology (it exclusively inhabits the digestive tube of these insects) is how they subsist during long periods of starvation. In this work, we show that this parasite performs a metabolic switch from glucose consumption to lipid oxidation, and it is able to consume lipids and the lipid-derived fatty acids from both internal origins as well as externally supplied compounds. When fatty acid oxidation is chemically inhibited by etomoxir, a very well-known drug that inhibits the translocation of fatty acids into the mitochondria, the proliferative insect stage of the parasites has dramatically diminished survival under severe metabolic stress and its differentiation into its infective forms is impaired. Our findings place fatty acids in the centre of the scene regarding their extraordinary resistance to nutrient-depleted environments.

During its complex life cycle, Trypanosoma cruzi colonizes different niches in its insect and mammalian hosts. This characteristic determined the types of parasites that adapted to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. These T. cruzi stages could be exposed to fatty acids originating from the degradation of the perimicrovillar membrane. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production via beta-oxidation. Lastly, we also show that fatty acids are degraded through beta-oxidation. Additionally, through beta-oxidation, electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid cycle, which can be used to feed other anabolic pathways such as the de novo biosynthesis of fatty acids.Author SummaryTrypanosoma cruzi is a protist parasite with a life cycle involving two types of hosts, a vertebrate one (which includes humans, causing Chagas disease) and an invertebrate one (kissing bugs, which vectorize the infection among mammals). In both hosts, the parasite faces environmental challenges such as sudden changes in the metabolic composition of the medium in which they develop, severe starvation, osmotic stress and redox imbalance, among others. Because kissing bugs feed infrequently in nature, an intriguing aspect of T. cruzi biology (it exclusively inhabits the digestive tube of these insects) is how they subsist during long periods of starvation. In this work, we show that this parasite performs a metabolic switch from glucose consumption to lipid oxidation, and it is able to consume lipids and the lipid-derived fatty acids from both internal origins as well as externally supplied compounds. When fatty acid oxidation is chemically inhibited by etomoxir, a very well-known drug that inhibits the translocation of fatty acids into the mitochondria, the proliferative insect stage of the parasites has dramatically diminished survival under severe metabolic stress and its differentiation into its infective forms is impaired. Our findings place fatty acids in the centre of the scene regarding their extraordinary resistance to nutrient-depleted environments.

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. Δ1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from Δ1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is an NADPH-dependent cytosolic enzyme with a Kmapp for P5C of 27.7 μM and with a higher expression in the insect-resident form of the parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Kiapp=45±0.7μM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model, cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite, and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase.

Pep5 (WELVVLGKL) is a fragment of cyclin D2 that exhibits a 2-fold increase in the S phase of the HeLa cell cycle. When covalently bound to a cell-penetrating peptide (Pep5-cpp), the nonapeptide induces cell death in several tumor cells, including breast cancer and melanoma cells. Additionally, Pep5-cpp reduces the in vivo tumor volume of rat glioblastoma. Chagas disease, which is caused by the flagellated parasite Trypanosoma cruzi, is a neglected disease that occurs mainly in the Americas, where it is considered an important public health issue. Given that there are only two options for treating the disease, it is exceptionally crucial to search for new molecules with potential pharmacological action against the parasites. In this study, we demonstrate that Pep5-cpp induces cell death in epimastigote, trypomastigote, and amastigote forms of T. cruzi The Pep5-cpp peptide was also able to decrease the percentage of infected cells without causing any detectable toxic effects in mammalian host cells. The infective, i.e., trypomastigote form of T. cruzi pretreated with Pep5-cpp was unable to infect LLC-MK2 monkey kidney cells. Also, Pep5-binding proteins were identified by mass spectrometry, including calmodulin-ubiquitin-associated protein, which is related to the virulence and parasitemia of T. cruzi Taken together, these data suggest that Pep5 can be used as a novel alternative for the treatment of Chagas disease.

Pep5 (WELVVLGKL) is a fragment of cyclin D2 that exhibits a 2-fold increase in the S phase of the HeLa cell cycle. When covalently bound to a cell-penetrating peptide (Pep5-cpp), the nonapeptide induces cell death in several tumor cells, including breast cancer and melanoma cells. Additionally, Pep5-cpp reduces the in vivo tumor volume of rat glioblastoma. Chagas disease, which is caused by the flagellated parasite Trypanosoma cruzi, is a neglected disease that occurs mainly in the Americas, where it is considered an important public health issue. Given that there are only two options for treating the disease, it is exceptionally crucial to search for new molecules with potential pharmacological action against the parasites. In this study, we demonstrate that Pep5-cpp induces cell death in epimastigote, trypomastigote, and amastigote forms of T. cruzi. The Pep5-cpp peptide was also able to decrease the percentage of infected cells without causing any detectable toxic effects in mammalian host cells. The infective, i.e., trypomastigote form of T. cruzi pretreated with Pep5-cpp was unable to infect LLC-MK(2) monkey kidney cells. Also, Pep5-binding proteins were identified by mass spectrometry, including calmodulin-ubiquitin-associated protein, which is related to the virulence and parasitemia of T. cruzi. Taken together, these data suggest that Pep5 can be used as a novel alternative for the treatment of Chagas disease.

Objective : This work aims at the synthesis of megazol analogs with antitrypanosomicidal activity. Chagas’disease is caused by Trypanosoma cruzi and is a debilitating disease that has both acute and chronic forms. Many South Americans suffer from the chronic form of Chagas’disease, and there is no treatment currently available. Methods : In the chemical part, classical techniques of heterocyclic synthesis as well as usual methods of identification were used. In the biological part the cell proliferation test was used in vitro and the IC 50. Results : We synthesized a series of derivatives of 2-(1-methyl-5-nitro-2-imidazolyl)-5-substituted-1,3,4-thiadiazoles where 1-acetyl, 1-propyl and 1-nonyl were used as the substituent (4,6,7). Derivatives without nitro group were also synthesized (3,12) along with thiosemicarbazones (8,9,10) and a 5-(5-nitro-2-furanyl)-1,3,4-thiadiazol-2-amine (11). These compounds were evaluated using an in vitro test where were measured the cell proliferation. The derivatives that obtained the best results underwent further tests, in which their IC50 was calculated. The data revealed that two compounds (4,6) were effective against the parasite (IC50= 0.354 µM; IC50= 2.13 µM) and besides that, obtained the same results as the positive control, antimycim and rotenone. All proposed structures were obtained in satisfactory yields and purities. Conclusions : In conclusion, the in vitro trypanocidal activity makes these compounds promising leads in the development of an effective therapeutic agent. However, this study must be completed by additional tests with in vitro amastigote/macrophage models or in vivo mouse models. Analyzing the amide derivatives, compounds (4) and (6) were the ones that presented the best results.

Trypanosoma cruzi, the etiological agent of Chagas disease, consumes glucose and amino acids depending on the environmental availability of each nutrient during its complex life cycle. For example, amino acids are the major energy and carbon sources in the intracellular stages of the T. cruzi parasite, but their consumption produces an accumulation of NH4+ in the environment, which is toxic. These parasites do not have a functional urea cycle to secrete excess nitrogen as low-toxicity waste. Glutamine synthetase (GS) plays a central role in regulating the carbon/nitrogen balance in the metabolism of most living organisms. We show here that the gene TcGS from T. cruzi encodes a functional glutamine synthetase; it can complement a defect in the GLN1 gene from Saccharomyces cerevisiae and utilizes ATP, glutamate and ammonium to yield glutamine in vitro. Overall, its kinetic characteristics are similar to other eukaryotic enzymes, and it is dependent on divalent cations. Its cytosolic/mitochondrial localization was confirmed by immunofluorescence. Inhibition by Methionine sulfoximine revealed that GS activity is indispensable under excess ammonium conditions. Coincidently, its expression levels are maximal in the amastigote stage of the life cycle, when amino acids are preferably consumed, and NH4+ production is predictable. During host-cell invasion, TcGS is required for the parasite to escape from the parasitophorous vacuole, a process sine qua non for the parasite to replicate and establish infection in host cells. These results are the first to establish a link between the activity of a metabolic enzyme and the ability of a parasite to reach its intracellular niche to replicate and establish host-cell infection., Author summary Trypanosoma cruzi, the agent that causes Chagas disease, has a complex life cycle, alternating between insects and mammals and thus facing environments with different metabolite compositions. T. cruzi can consume glucose and/or amino acids, depending on their availability. However, amino acid consumption produces excess ammonium, which must be eliminated in a non-toxic manner. Here, we show that the enzyme glutamine synthetase contributes to the management of excess ammonium and uses it to form the amino acid glutamine. During its life cycle, the parasite invades mammalian host cells and transiently becomes enclosed in a tight vacuole, where it differentiates into the amastigote, an amino acid consumer stage. Amastigotes must escape from the vacuole into the host-cell cytoplasm to initiate intracellular replication. In this work, we show that the inhibition of T. cruzi glutamine synthetase aborts parasite evasion from the vacuole. We propose that this enzyme contributes to the control of ammonium produced by parasite metabolism, as ammonium increases the internal pH of the parasitophorous vacuole, making the enzymes for the T. cruzi evasion process non-functional. This knowledge could be useful for designing new anti-T. cruzi drugs.

Trypanosoma cruzi, the aetiological agent of Chagas disease, can obtain L-glutamine (Gln) through the enzyme glutamine synthetase (GS) using glutamate (Glu) and ammonia as substrates. In this work, we show additional non-canonical roles for this amino acid: its involvement in ATP maintenance and parasite survival under severe metabolic stress conditions and its participation in the differentiation process occurring in the insect vector (metacyclogenesis). These roles are dependent on the supply of Gln from an extracellular source. We show that T. cruzi incorporates Gln through a saturable and specific transport system, which results in unusual stability at elevated temperatures. The activity was moderately higher at pH values between 6 and 7 and was sensitive to the dissipation of the H+ gradient at the plasma membrane. When analysed in the different life cycle stages, we found that Gln transport is developmentally regulated. In fact, Gln uptake and GS activity seem to be finely regulated at most stages: when GS activity is increased, transport is decreased and vice versa, with the exception of trypomastigotes, where both sources of Gln are diminished. This metabolic adaptation reflects the relevance of Gln in T. cruzi biology and the plasticity of these parasites to adjust their metabolism to changing environments.

Trypanosoma cruzi e Trypanosoma brucei são os agentes etiológicos da doença de Chagas e da doença do sono, respectivamente. Ambos são tripanossomatídeos, apresentam um ciclo de vida que alterna entre os hospedeiros mamíferos e os hospedeiros invertebrados e apresentam o metabolismo baseado no consumo de aminoácidos e/ou glicose, dependendo da disponibilidade de nutrientes no ambiente. Neste trabalho foi demonstrado a importância da glutamina (Gln) em diferentes aspectos da biologia do T. cruzi e a relevância da Gln e da enzima glutamina sintetase (GS) para formas sanguícolas de T. brucei. A Gln é transportada pelo T. cruzi e pelo T. brucei a partir do meio externo. Em T. cruzi foi demonstrado que esse transporte é realizado por um único sistema, saturável, específico, dependente de ATP e do gradiente de H+ na menbrana do parasita. Também foi demonstrado que a Gln é importante para replicação das formas amastigotas e epimastigotas, além de promover o processo de metaciclogênese. Tratamento com análogos estruturais da Gln dimuiu a proliferação do estágio epimastigota e também a diferenciação para tripomastigota metacíclico. Além do mais células infectadas e tratadas com os análogos apresentaram redução do número de tripomastigotas que eclodiram das células, demonstrando que a Gln também é importante para os estágios intracelulares. Em formas sanguícolas de T. brucei, a enzima GS é ativa, mas é incapaz de suprir a necessidade de Gln do parasita, fazendo com que seja completamente dependente do transporte a partir do meio externo. A Gln é importante para a proliferação formas sanguícolas e correta progressão do ciclo celular. Em meio sem Gln os parasitas são incapazes de manter a proliferação normalmente, sendo que este processo é dependente da concentração de Gln no meio externo. Também foi demonstrado que a Gln participa do processo de modificação pós-traducional de glutamilação da tubulina. Conclui-se portanto que a Gln é um aminoácido fundamental para sobrevivência do T. cruzi e do T. brucei. Trypanosoma cruzi and Trypanosoma brucei are the etiologic agent of Chagas disease and sleeping sickness, respectively. Both parasites are trypanosomatids that have a complex life cycle, which alternates between a mammalian host and insect vector. T. cruzi and T. brucei are able to use carbohydrates and amino acids as energy source, depending on availability of nutrients in the different environments that parasites go through in the life cycle. In this work we demonstrate that glutamine (Gln) is an important metabolite that participates in many biological processes in T. cruzi, and the relevance of the enzyme glutamine synthetase and Gln in bloodstream forms of T. brucei. T. cruzi and T. brucei are able to uptake Gln from the medium. T. cruzi incorporate Gln through a single and saturable transport system. Gln uptake system is dependent on ATP intracellular levels and H+ gradient and is a highly specific system. Also was demonstrated that Gln is important to replicative stages amastigote and epimastigote, and promotes the metacyclogenesis process. The treatment with Gln analogs impared the epimastigote replication and the differentiation from epimastigote to trypomastigote metacyclic. Moreover, analogs treatment in the infected cells decrease the number of trypomastigotes released from the cells, suggesting that Gln is important to intracellular development of T. cruzi. This work also demonstrates that the enzyme glutamine synthetase is active in bloodstream forms from T. brucei, but is not enough to produce the amount of Gln required by the parasite. T. brucei, bloodstream forms are completely dependent of Gln uptake from the medium. The proper proliferation rate and correct cell cycle progress are dependent of Gln concentration in the medium. Moreover Gln participates in the tubulin glutamylation process in bloodstream forms; this is a post translational modification that is important to microtubules dynamics and cytokinesis process. We concluded that Gln is a fundamental amino acid to maintenance of T. cruzi and T. brucei.

Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease that affects 5–6 million people in endemic areas of the Americas. Presently, chemotherapy relies on two compounds that were proposed as trypanocidal drugs four decades ago: nifurtimox and benznidazole. Both drugs are able to eliminate parasitemia and to avoid seroconversion in infected people when used in the acute phase; however, their use in the chronic phase (the time when the majority of cases are diagnosed) is limited due to their serious side effects. Memantine is a glutamate receptor antagonist in the central nervous system of mammals that has been used for the treatment of Alzheimer’s disease. Our group previously reported memantine as a trypanocidal drug that is able to induce apoptosis-like death in T. cruzi. In the present work, we further investigated the effects of memantine on the infection of RAW 264.7 macrophages and in vivo (in BALB/c mice). Here, we showed that memantine is able to diminish NO and Ca2+ entry in both LPS-activated and non-activated cells. These results, together with the fact that memantine was also able to reduce the infection of macrophages, led us to propose that this drug is able to activate a pro-oxidant non-NO-dependent cell defense mechanism. Finally, infected mice that were treated with memantine had diminished parasitemia, cardiac parasitic load, and inflammatory infiltrates. In addition, the treated mice had an increased survival rate. Taken together, these results indicate memantine to be a candidate drug for the treatment of Chagas disease., Author summary Chagas disease affects approximately 5 million people and is caused by the protist parasite Trypanosoma cruzi. Until now, there are no vaccines to prevent the human infection, and the therapy relies on the use of two drugs discovered more than 50 years ago, nifurtimox and benznidazole. Both drugs are efficient during the acute phase of the disease, however their efficacy in the chronic phase, when most of patients are diagnosed is controversial. In addition, both drugs are toxic, causing severe side effects during the treatment. For these reasons, new drugs against T. cruzi are urgently needed. In this work, we report a series of experiments supporting the repositioning of memantine, a drug used for treating Alzheimer´s disease, to treat the T. cruzi infection in an experimental infection model. Our data show that infected mice treated with memantine have diminished their parasitemia, cardiac parasitic load and inflammatory infiltrates and more importantly, they have diminished their mortality. Taken together, these results prompt memantine as a promising drug for treating Chagas disease.

Trypanosoma cruzi is the etiological agent of Chagas disease, affecting approximately 10 million people in the Americas and with some 40 million people at risk. The objective of this study was to evaluate the anti- T. cruzi activity of three new diamidines that have a 3,4-ethylenedioxy extension of the thiophene core, designated MB17, MB19, and MB38. All three diamidines exhibited dose-dependent inhibition of epimastigote replication. The mechanisms of action of these diamidines were investigated. Unlike MB17 and MB19, MB38 exhibited a significant increase in the number of annexin-propidium iodide double-labeled cells compared to levels in control parasites. As MB17 had shown a lower 50% inhibitory concentration (IC 50 ) against epimastigote growth, the mechanism of action of this drug was studied in more detail. MB17 triggered a decrease in the intracellular ATP levels. As a consequence, MB17 affected the genomic DNA and kinetoplast DNA (kDNA) and impaired the parasite cell cycle. Moreover, MB17 caused DNA fragmentation, with a more severe effect on kDNA than on nuclear DNA, resulting in dyskinetoplastic cells. MB17 was tested for toxicity and effectiveness for the treatment of infected CHO-K 1 cells, exhibiting a 50% cytotoxic concentration (CC 50 ) of 13.47 ± 0.37 μM and an IC 50 of 0.14 ± 0.12 μM against trypomastigote release. MB17 also diminished the infection index by 60% at 0.5 μM. In conclusion, despite belonging to the same family, these diamidines have different efficiencies. To summarize, MB17 was the most potent of these diamidines against epimastigotes, producing DNA damage preferentially in kDNA, impairing the parasite cell cycle, and decreasing the infection index and trypomastigote release from infected mammalian host cells, with a high selectivity index (SI) (T. cruzi .

Arginine kinase catalyzes the reversible transphosphorylation between ADP and phosphoarginine which plays a critical role in the maintenance of cellular energy homeostasis. Arginine kinase from the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, meets the requirements to be considered as a potential therapeutic target for rational drug design including being absent in its mammalian hosts. In this study a group of polyphenolic compounds was evaluated as potential inhibitors of arginine kinase using molecular docking techniques. Among the analyzed compounds with the lowest free binding energy to the arginine kinase active site (

Trypanosoma cruzi, the aetiological agent of Chagas's disease, metabolizes glucose, and after its exhaustion, degrades amino acids as energy source. Here, we investigate histidine uptake and its participation in energy metabolism. No putative genes for the histidine biosynthetic pathway have been identified in genome databases of T. cruzi, suggesting that its uptake from extracellular medium is a requirement for the viability of the parasite. From this assumption, we characterized the uptake of histidine in T. cruzi, showing that this amino acid is incorporated through a single and saturable active system. We also show that histidine can be completely oxidised to CO2. This finding, together with the fact that genes encoding the putative enzymes for the histidine - glutamate degradation pathway were annotated, led us to infer its participation in the energy metabolism of the parasite. Here, we show that His is capable of restoring cell viability after long-term starvation. We confirm that as an energy source, His provides electrons to the electron transport chain, maintaining mitochondrial inner membrane potential and O2 consumption in a very efficient manner. Additionally, ATP biosynthesis from oxidative phosphorylation was found when His was the only oxidisable metabolite present, showing that this amino acid is involved in bioenergetics and parasite persistence within its invertebrate host.

Este trabalho identificou a ocorrência de cistos de protozoários presentes em alface (Lactuca sativa) comercializada na feira livre e em supermercados de Santana do Ipanema, Alagoas. Aplicou-se um questionário investigativo com os consumidores na feira livre da cidade, em cinco pontos de comercialização de alface, o que demonstrou que 59% dos entrevistados ingerem hortaliças diariamente. A alface é o vegetal mais consumido, representando 48%, e 1/3 dos pesquisados utiliza somente água no asseio das hortaliças. As amostras de alface foram coletadas nos mesmos locais de aplicação do questionário e submetidas ao método de sedimentação espontânea (Hoffman et al.,1934). A partir das análises parasitológicas, constatou-se que em 60% das amostras foram identificados parasitas pertencentes ao grupo dos protozoários como Entamoeba histolytica e Giardia lamblia. Os resultados observados corroboram um indicativo de alto risco de contaminação por enteroparasitas pelos consumidores de alface da cidade de Santana do Ipanema.

A doença de Chagas, também conhecida como tripanossomíase americana é causada pelo protozoário flagelado Trypanosoma cruzi e afeta aproximadamente 10 milhões de pessoas em áreas endêmicas do México, America central e do Sul. A quimioterapia disponível atualmente limita-se a dois compostos: Nifurtimox e o Benzonidazol. Ambos os fármacos reduzem os sintomas da doença e a mortalidade das pessoas infectadas, quando utilizadas na fase aguda, mas sua eficácia na fase crônica é controversa. Além do mais apresentam vários efeitos colaterais. O T. cruzi é capaz de utilizar carboidratos e aminoácidos como fonte de energia e carbono. O nosso grupo tem estudado o envolvimento do glutamato na resistência do parasita ao estresse térmico, oxidativo e metabólico como também a importância do aminoácido GABA no metabolismo do T. cruzi, visto que este também é transportado pelo parasita. Nesse contexto, o objetivo desse trabalho foi avaliar a interferência de fármacos, que atuam via receptores ou transportadores de GABA e glutamato, no ciclo de vida do T. cruzi. Os fármacos selecionados foram: Vigabatrina, Pregabalina, MK-801, MTEP e Memantina. Os resultados demonstram que apenas Memantina apresentou um melhor efeito tripanocida. Inibiu a proliferação de formas epimastigotas, interferiu na metaciclogênese, como também afetou o metabolismo energético do parasita com a diminuição dos níveis de ATP, além de desencadear mecanismos que levam a apoptose das formas epimastigotas. Memantina também interferiu no ciclo intracelular do parasita, mais especificamente no estágio amastigota. Interessantemente as fases do ciclo do parasita que são mais afetadas, são as fases que requerem mais energia: estágios replicativos (amastigota e epimastigota), como também os processos de diferenciação e invasão celular. Apesar de apresentar valores de IC50 que podem ser considerados altos em relação aos valores descritos para formas epimastigotas tratadas com Benzonidazol, esses resultados mostram perspectivas promissoras, visto que o Memantina poderá ser utilizado como composto líder para o desenho de derivados com atividade tripanocida otimizada. Chagas disease, also known as American trypanosomiasis is caused by the flagellate protozoan Trypanosoma cruzi and affects approximately 10 million people in endemic areas of Mexico, Central America and South America, with about 25 million people living in areas at risk of infection. The currently available chemotherapy is limited to two compounds, which are: Nifurtimox and Benznidazole. Both drugs reduce symptoms of the disease and mortality of infected people when used in the acute phase, but its efficacy in chronic phase (phase in which the majority of cases are diagnosed) is still controversial. Moreover these drugs have several side effects. T. cruzi is able to utilize carbohydrate and amino acids as carbon and energy source. Our group has studied the involvement of glutamate in parasite resistance to thermal, oxidative and metabolic stress, as well as the importance of the amino acid GABA metabolism in T. cruzi. The aim of this study was to evaluate the interference of drugs, that act in GABA and glutamate transporters or receptors, in the life cycle of T. cruzi. The drugs selected were: vigabatrin, pregabalin, MK-801, memantine and MTEP. The results showed that only memantine has a better trypanocidal effect. Memantine inhibited the proliferation of epimastigotes, interfered in metacyclogenesis, and affected the energy metabolism of the parasite with decreased levels of ATP, and trigger mechanisms that lead to apoptosis of epimastigotes. Moreover interferes with intracellular cycle of the parasite, specifically in amastigote stage. Interesting that stages of the parasite that require more energy are more affected: replicative stages (epimastigote and amastigote) as well as the processes of differentiation and cell invasion. IC50 value is more than that described to epimastigotes treateds with Benznidazole, but Memantine could be used as leader compound for the design of drugs with optimized trypanocidal activity.

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and affects approximately 10 million people in endemic areas of Mexico and Central and South America. Currently available chemotherapies are limited to two compounds: Nifurtimox and Benznidazole. Both drugs reduce the symptoms of the disease and mortality among infected individuals when used during the acute phase, but their efficacy during the chronic phase (during which the majority of cases are diagnosed) remains controversial. Moreover, these drugs have several side effects. The aim of this study was to evaluate the effect of Memantine, an antagonist of the glutamate receptor in the CNS of mammals, on the life cycle of T. cruzi. Memantine exhibited a trypanocidal effect, inhibiting the proliferation of epimastigotes (IC50 172.6 µM). Furthermore, this compound interfered with metacyclogenesis (approximately 30% reduction) and affected the energy metabolism of the parasite. In addition, Memantine triggered mechanisms that led to the apoptosis-like cell death of epimastigotes, with extracellular exposure of phosphatidylserine, increased production of reactive oxygen species, decreased ATP levels, increased intracellular Ca2+ and morphological changes. Moreover, Memantine interfered with the intracellular cycle of the parasite, specifically the amastigote stage (IC50 31 µM). Interestingly, the stages of the parasite life cycle that require more energy (epimastigote and amastigote) were more affected as were the processes of differentiation and cell invasion., Author Summary Trypanosoma cruzi is a parasite transmitted to mammal hosts by insect vectors known as kissing bugs. This species can result pathogenic for humans, causing Chagas' disease in the Americas. Its treatment relies on two drugs discovered more than 40 years ago. Besides their toxicity, a main drawback of these drugs is the fact that they are highly efficient only during the acute phase of the infection. But due to the lack of specific symptoms, the acute phase of the infection is largely not diagnosed. In fact, most of patients are diagnosed in the chronic phase, where the treatments are not satisfactory. In view of that, it is urgent to look for new drugs with low toxicity and able to kill the parasite in chronic patients. On the basis of previous finding, we looked for drugs against glutamate recognizing surface molecules, keeping special attention on those that are already in use in humans for other purposes (this strategy is called drug repositioning, and allow to save time and money in clinical trials: several parameters such as toxicity, pharmacokinetics, side effects in humans are already known). Here we report that Memantine, a NMDA glutamate receptors antagonist already in use to treat Alzheimer's disease, presents interesting perspectives as a trypanocidal drug.