Identifying inhibitors of Trypanosoma cruzi nucleoside diphosphate kinase 1 as potential repurposed drugs for Chagas' disease.

Trypanosoma cruzi is the causative agent of Chagas' disease, an endemic and neglected disease. The treatment is limited to only two drugs, benznidazole (BZL) and nifurtimox (NFX), introduced more than fifty years ago and no new advances have been made since then. Nucleoside diphosphate kinases (NDPK) are key metabolic enzymes which have gained interest as drug targets of pathogen organisms. Taking advantage of the computer-assisted drug repurposing approaches, in the present work we initiate a search of potential T. cruzi nucleoside diphosphate kinase 1 (TcNDPK1) inhibitors over an âˆ¼ 12,000 compound structures database to find drugs targeted to this enzyme with trypanocidal activity. Four medicines were selected and evaluated in vitro, ketorolac (KET, an anti-inflamatory), dutasteride (DUT, used to treat benign prostatic hyperplasia), nebivolol and telmisartan (NEB and TEL, used to treat high blood pressure). The four compounds were weak inhibitors and presented different trypanocidal effect on epimastigotes, trypomastigotes and intracellular stages. NEB and TEL were the most active drugs with increased effect on intracellular stages, (IC50 = 2.25 µM and 13.21 µM respectively), and selectivity indexes of 13.01 and 8.59 respectively, showing comparable effect to BZL, the first line drug for Chagas' disease treatment. In addition, both presented positive interactions when combined with BZL. Finally, transgenic epimastigotes with increased expression of TcNDPK1 were more resistant to TEL and NEB, suggesting that TcNDPK1 is at least one of the molecular targets. In view of the results, NEB and TEL could be repurposed medicines for Chagas' disease therapy.

Revisiting trypanosomatid nucleoside diphosphate kinases.

BACKGROUND: An increasing amount of research has led to the positioning of nucleoside diphosphate kinases (NDPK/NDK) as key metabolic enzymes among all organisms. They contribute to the maintenance the intracellular di- and tri- phosphate nucleoside homeostasis, but they also are involved in widely diverse processes such as gene regulation, apoptosis, signal transduction and many other regulatory roles. OBJETIVE: Examine in depth the NDPKs of trypanosomatid parasites responsible for devastating human diseases (e.g., Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp.) which deserve special attention. METHODS: The earliest and latest advances in the topic were explored, focusing on trypanosomatid NDPK features, multifunctionality and suitability as molecular drug targets. FINDINGS: Trypanosomatid NDPKs appear to play functions different from their host counterparts. Evidences indicate that they would perform key roles in the parasite metabolism such as nucleotide homeostasis, drug resistance, DNA damage responses and gene regulation, as well as host-parasite interactions, infection, virulence and immune evasion, placing them as attractive pharmacological targets. MAIN CONCLUSIONS: NDPKs are very interesting multifunctional enzymes. In the present review, the potential of trypanosomatid NDPKs was highlighted, raising awareness of their value not only with respect to parasite biology but also as molecular targets.

Trypanocidal activity of the anthocyanidin delphinidin, a non-competitive inhibitor of arginine kinase.

Arginine kinase from Trypanosoma cruzi (TcAK) catalyzes the interconversion of arginine and phosphoarginine to maintain the ATP/ADP cell balance, and is involved in the parasites' energetic homeostasis and stress responses. Using virtual screening approaches, some plant-derived polyphenolic pigments, such as anthocyanidins, were predicted to inhibit TcAK activity. Here, it was demonstrated that the anthocyanidin delphinidin showed a non-competitive inhibition mechanism of TcAK (Ki arginine = 1.32 µM and Ki ATP = 500 µM). Molecular docking simulations predicted that delphinidin occupies part of the ATP/ADP pocket, more specifically the one that binds the ribose phosphate, and molecular dynamics simulations confirmed the amino acids involved in binding. Delphinidin exerted trypanocidal activity over T. cruzi trypomastigotes with a calculated IC50 of 19.51 µM. Anthocyanidins are low-toxicity natural products which can be exploited for the development of trypanocidal drugs with less secondary effects than those currently used for the treatment of Chagas disease.

Revisiting trypanosomatid nucleoside diphosphate kinases

BACKGROUND An increasing amount of research has led to the positioning of nucleoside diphosphate kinases (NDPK/NDK) as key metabolic enzymes among all organisms. They contribute to the maintenance the intracellular di- and tri- phosphate nucleoside homeostasis, but they also are involved in widely diverse processes such as gene regulation, apoptosis, signal transduction and many other regulatory roles. OBJETIVE Examine in depth the NDPKs of trypanosomatid parasites responsible for devastating human diseases (e.g., Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp.) which deserve special attention. METHODS The earliest and latest advances in the topic were explored, focusing on trypanosomatid NDPK features, multifunctionality and suitability as molecular drug targets. FINDINGS Trypanosomatid NDPKs appear to play functions different from their host counterparts. Evidences indicate that they would perform key roles in the parasite metabolism such as nucleotide homeostasis, drug resistance, DNA damage responses and gene regulation, as well as host-parasite interactions, infection, virulence and immune evasion, placing them as attractive pharmacological targets. MAIN CONCLUSIONS NDPKs are very interesting multifunctional enzymes. In the present review, the potential of trypanosomatid NDPKs was highlighted, raising awareness of their value not only with respect to parasite biology but also as molecular targets.

Effect of capsaicin on the protozoan parasite Trypanosoma cruzi.

Trypanosoma cruzi is the causative agent of Chagas disease. There are only two approved treatments, both of them unsuitable for the chronic phase, therefore the development of new drugs is a priority. Trypanosoma cruzi arginine kinase (TcAK) is a promising drug target since it is absent in humans and it is involved in cellular stress responses. In a previous study, possible TcAK inhibitors were identified through computer simulations resulting the best compounds capsaicin and cyanidin derivatives. Here, we evaluate the effect of capsaicin on TcAK activity and its trypanocidal effect. Although capsaicin produced a weak enzyme inhibition, it had a strong trypanocidal effect on epimastigotes and trypomastigotes (IC50 = 6.26 µM and 0.26 µM, respectively) being 20-fold more active on trypomastigotes than mammalian cells. Capsaicin was also active on the intracellular cycle reducing by half the burst of trypomastigotes at approximately 2 µM. Considering the difference between the concentrations at which parasite death and TcAK inhibition occur, other possible targets were predicted. Capsaicin is a selective trypanocidal agent active in nanomolar concentrations, with an IC50 57-fold lower than benznidazole, the drug currently used for treating Chagas disease.

A novel stage-specific glycosomal nucleoside diphosphate kinase from Trypanosoma cruzi.

Nucleoside diphosphate kinases (NDPK) are key enzymes involved in the intracellular nucleotide maintenance in all living organisms, especially in trypanosomatids which are unable to synthesise purines de novo. Four putative NDPK isoforms were identified in the Trypanosoma cruzi Chagas, 1909 genome but only two of them were characterised so far. In this work, we studied a novel isoform from T. cruzi called TcNDPK3. This enzyme presents an atypical N-terminal extension similar to the DM10 domains. In T. cruzi, DM10 sequences targeted other NDPK isoform (TcNDPK2) to the cytoskeleton, but TcNDPK3 was localised in glycosomes despite lacking a typical peroxisomal targeting signal. In addition, TcNDPK3 was found only in the bloodstream trypomastigotes where glycolytic enzymes are very abundant. However, TcNDPK3 mRNA was also detected at lower levels in amastigotes suggesting regulation at protein and mRNA level. Finally, 33 TcNDPK3 gene orthologs were identified in the available kinetoplastid genomes. The characterisation of new glycosomal enzymes provides novel targets for drug development to use in therapies of trypanosomatid associated diseases.

Cytosolic Trypanosoma cruzi nucleoside diphosphate kinase generates large granules that depend on its quaternary structure.

Nucleoside diphosphate kinase (NDPK) is a key enzyme in the control of cellular concentrations of nucleoside triphosphates, and has been shown to play important roles in many cellular processes. In this work we investigated the subcellular localization of the canonical NDPK1 from Trypanosoma cruzi (TcNDPK1), the etiological agent Chagas's Disease, and evaluated the effect of adding an additional weak protein-protein interaction domain from the green fluorescent protein (GFP). Immunofluorescence microscopy revealed that the enzyme from wild-type and TcNDPK1 overexpressing parasites has a cytosolic distribution, being the signal more intense around the nucleus. However, when TcNDPK1 was fused with dimeric GFP it relocalizes in non-membrane bounded granules also located adjacent to the nucleus. In addition, these granular structures were dependent on the quaternary structure of TcNDPK1 and GFP since mutations in residues involved in their oligomerization dramatically decrease the amount of granules. This phenomenon seems to be specific for TcNDPK1 since other cytosolic hexameric enzyme from T. cruzi, such as the NADP(+)-linked glutamate dehydrogenase, was not affected by the fusion with GFP. In addition, in parasites without GFP fusions granules could be observed in a subpopulation of epimastigotes under metacyclogenesis and metacyclic trypomastigotes. Organization into higher protein arrangements appears to be a singular feature of canonical NDPKs; however the physiological function of such structures requires further investigation.

[The mammalian TOR pathway is present in Trypanosoma cruzi. In silico reconstruction and possible functions]. / La vía de transducción de señales TOR de mamíferos está presente en Trypanosoma cruzi. Reconstrucción in silico y posibles funciones.

The mammalian TOR pathway ("Target Of Rapamycin") is a regulatory protein network involved in a wide range of processes including cell growth and differentiation, providing a functional switch between anabolic and catabolic cell metabolism. Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle with different morphological stages in various hosts. This life cycle implies that parasites have to deal with fluctuations in the extracellular medium that should be detected and counteracted adapting their metabolism. A candidate to be the mediator between the receptors / sensors of the environment and cellular adaptive response is the TOR pathway. In this paper we integrate the bibliographic data of the TOR pathway in trypanosomatids by in silico analysis (computer simulation of biological structures and processes) of the parasite's genome. Possible effectors and processes regulated by this metabolic pathway are also proposed. Given that the information on the mechanisms of signal transduction in trypanosomatids is scarce, we consider the model presented in this work may be a reference for future experimental work.

La vía de transducción de señales TOR de mamíferos está presente en Trypanosoma cruzi: Reconstrucción in silico y posibles funciones / The mammalian TOR pathway is present in Trypanosoma cruzi: In silico reconstruction and possible functions

La vía TOR ("Target Of Rapamycin") de mamíferos es una red proteica de regulación para una amplia gama de procesos involucrados en el crecimiento y la diferenciación celular, constituyendo un interruptor funcional entre el metabolismo anabólico y catabólico de la célula. El Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, tiene un ciclo de vida muy complejo con diferentes estadios morfológicos en varios hospedadores. Este ciclo de vida implica que los parásitos enfrentan grandes fluctuaciones en el medio extracelular que deben ser detectadas y a las cuales deben responder adaptando su metabolismo. Un candidato a ser el mediador entre los receptores/sensores del medio y la respuesta adaptativa celular es la vía TOR. En este trabajo integramos los datos bibliográficos de la vía TOR de organismos tripanosomátidos con un análisis in silico (simulación computacional de procesos o estructuras biológicas) del genoma del parásito. Se proponen además posibles efectores y procesos regulados por esta ruta metabólica. Teniendo en cuenta que existe muy poca información sobre los mecanismos de transducción de señales en tripanosomátidos, consideramos que el mapa presentado en este trabajo puede ser una referencia para futuros trabajos experimentales.(AU)

The mammalian TOR pathway ("Target Of Rapamycin") is a regulatory protein network involved in a wide range of processes including cell growth and differentiation, providing a functional switch between anabolic and catabolic cell metabolism. Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle with different morphological stages in various hosts. This life cycle implies that parasites have to deal with fluctuations in the extracellular medium that should be detected and counteracted adapting their metabolism. A candidate to be the mediator between the receptors / sensors of the environment and cellular adaptive response is the TOR pathway. In this paper we integrate the bibliographic data of the TOR pathway in trypanosomatids by in silico analysis (computer simulation of biological structures and processes) of the parasites genome. Possible effectors and processes regulated by this metabolic pathway are also proposed. Given that the information on the mechanisms of signal transduction in trypanosomatids is scarce, we consider the model presented in this work may be a reference for future experimental work.(AU)

La vía de transducción de señales TOR de mamíferos está presente en Trypanosoma cruzi: Reconstrucción in silico y posibles funciones / The mammalian TOR pathway is present in Trypanosoma cruzi: In silico reconstruction and possible functions

La vía TOR ("Target Of Rapamycin") de mamíferos es una red proteica de regulación para una amplia gama de procesos involucrados en el crecimiento y la diferenciación celular, constituyendo un interruptor funcional entre el metabolismo anabólico y catabólico de la célula. El Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, tiene un ciclo de vida muy complejo con diferentes estadios morfológicos en varios hospedadores. Este ciclo de vida implica que los parásitos enfrentan grandes fluctuaciones en el medio extracelular que deben ser detectadas y a las cuales deben responder adaptando su metabolismo. Un candidato a ser el mediador entre los receptores/sensores del medio y la respuesta adaptativa celular es la vía TOR. En este trabajo integramos los datos bibliográficos de la vía TOR de organismos tripanosomátidos con un análisis in silico (simulación computacional de procesos o estructuras biológicas) del genoma del parásito. Se proponen además posibles efectores y procesos regulados por esta ruta metabólica. Teniendo en cuenta que existe muy poca información sobre los mecanismos de transducción de señales en tripanosomátidos, consideramos que el mapa presentado en este trabajo puede ser una referencia para futuros trabajos experimentales.

The mammalian TOR pathway ("Target Of Rapamycin") is a regulatory protein network involved in a wide range of processes including cell growth and differentiation, providing a functional switch between anabolic and catabolic cell metabolism. Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle with different morphological stages in various hosts. This life cycle implies that parasites have to deal with fluctuations in the extracellular medium that should be detected and counteracted adapting their metabolism. A candidate to be the mediator between the receptors / sensors of the environment and cellular adaptive response is the TOR pathway. In this paper we integrate the bibliographic data of the TOR pathway in trypanosomatids by in silico analysis (computer simulation of biological structures and processes) of the parasite's genome. Possible effectors and processes regulated by this metabolic pathway are also proposed. Given that the information on the mechanisms of signal transduction in trypanosomatids is scarce, we consider the model presented in this work may be a reference for future experimental work.