Repurposing of terconazole as an anti Trypanosoma cruzi agent

Trypanosoma cruzi is the causative agent of Chagas disease, a parasitic infection endemic in Latin America. Currently there are no effective treatments for the chronic phase of the disease, when most patients are diagnosed, therefore the development of new drugs is a priority area. Several triazoles, used as fungicides, exhibit trypanocidal activity both in vitro and in vivo. The mechanism of action of such drugs, both in fungi and in T. cruzi, relies in the inhibition of ergosterol biosynthesis affecting the cell viability and growth. Among them, terconazole was the first triazole antifungal drug for human use. In this work, the trypanocidal activity of terconazole was evaluated using in vitro assays. In epimastigotes of two parasites strains from different discrete typing units (Y and Dm28c) the calculated IC50 were 25.7 μM and 21.9 μM, respectively. In trypomastigotes and amastigotes (the clinically relevant life-stages of T. cruzi) a higher drug susceptibility was observed with IC50 values of 4.6 μM and 5.9 μM, respectively. Finally, the molecular docking simulations suggest that terconazole inhibits the T. cruzi cytochrome P450 14-α-demethylase, interacting in a similar way that other triazole drugs. Drug repurposing to Chagas disease treatment is one of the recommended approach according to the criterion of international health organizations for their application in neglected diseases.Fil: Reigada, Chantal. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Martínez Sayé, Melisa Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Valera Vera, Edward Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Miranda, Mariana Reneé. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Pereira, Claudio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

New Enzymes as Potential Therapeutic Targets for Trypanosomiases and Leishmaniasis

Infections caused by the protozoan parasites Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp. are among the most relevant public health problems in the developing countries.Fil: Pereira, Claudio Alejandro. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Medicas; ArgentinaFil: Silber, Ariel. Universidade de Sao Paulo; BrasilFil: Gonzalez Rey, Elena. Instituto de Parasitología y Biomedicina “Lopez-Neyra”; Españ

Identification of Trypanosoma cruzi Polyamine Transport Inhibitors by Computational Drug Repurposing

Trypanosoma cruzi is the causative agent of Chagas disease, a parasitic infection endemic in Latin America. In T. cruzi the transport of polyamines is essential because this organism is unable to synthesize these compounds de novo. Therefore, the uptake of polyamines from the extracellular medium is critical for survival of the parasite. The anthracene-putrescine conjugate Ant4 was first designed as a polyamine transport probe in cancer cells. Ant4 was also found to inhibit the polyamine transport system and produced a strong trypanocidal effect in T. cruzi. Considering that Ant4 is not currently approved by the FDA, in this work we performed computer simulations to find trypanocidal drugs approved for use in humans that have structures and activities similar to Ant4. Through a similarity ligand-based virtual screening using Ant4 as reference molecule, four possible inhibitors of polyamine transport were found. Three of them, promazine, chlorpromazine and clomipramine, showed to be effective inhibitors of putrescine uptake, and also revealed a trypanocidal activity in epimastigotes (IC50 values of 69.0, 50.8 and 49.4 μM, respectively) and trypomastigotes (IC50 values of 3.5, 2.8 and 1.4 μM, respectively). Finally, molecular docking simulations suggest that the interactions between the T. cruzi polyamine transporter TcPAT12 and all the identified inhibitors occur in the same region of the protein. However, this location is different from the site occupied by the natural substrates. The value of this effort is that repurposing known drugs in the treatment of other pathologies, especially neglected diseases such as Chagas disease, significantly decreases the time and economic cost of implementation.Fil: Reigada, Chantal. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Martínez Sayé, Melisa Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Phanstiel, Otto. University Of Central Florida; Estados UnidosFil: Valera Vera, Edward Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Miranda, Mariana Reneé. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Pereira, Claudio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Molecular and functional characterization of a spermidine transporter (TcPAT12) from Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas' disease, is the only eukaryotic cell which lacks the ability to synthesize polyamines de novo. In this work, we describe for the first time the molecular and biochemical properties of a high-affinity spermidine transporter from T. cruzi. The transporter gene TcPAT12 was functionally expressed in Xenopus laevis oocytes, showing high levels of spermidine uptake. Similar apparent affinity constants for spermidine uptake were obtained when comparing T. cruzi epimastigotes and heterologous expressed TcPAT12 in X. laevis. In addition, TcPAT12 also transports putrescine and the amino acid l-arginine at lower rates than spermidine.Fil: Carrillo, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Canepa, Gaspar Exequiel. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Algranati, Israel David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Pereira, Claudio Alejandro. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The superfamily keeps growing: Identification in trypanosomatids of RibJ, the first riboflavin transporter family in protists

Background: Trypanosomatid parasites represent a major health issue affecting hundreds of million people worldwide, with clinical treatments that are partially effective and/or very toxic. They are responsible for serious human and plant diseases including Trypanosoma cruzi (Chagas disease), Trypanosoma brucei (Sleeping sickness), Leishmania spp. (Leishmaniasis), and Phytomonas spp. (phytoparasites). Both, animals and trypanosomatids lack the biosynthetic riboflavin (vitamin B2) pathway, the vital precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) cofactors. While metazoans obtain riboflavin from the diet through RFVT/SLC52 transporters, the riboflavin transport mechanisms in trypanosomatids still remain unknown. Methodology/Principal findings: Here, we show that riboflavin is imported with high affinity in Trypanosoma cruzi, Trypanosoma brucei, Leishmania (Leishmania) mexicana, Crithidia fasciculata and Phytomonas Jma using radiolabeled riboflavin transport assays. The vitamin is incorporated through a saturable carrier-mediated process. Effective competitive uptake occurs with riboflavin analogs roseoflavin, lumiflavin and lumichrome, and co-factor derivatives FMN and FAD. Moreover, important biological processes evaluated in T. cruzi (i.e. proliferation, metacyclogenesis and amastigote replication) are dependent on riboflavin availability. In addition, the riboflavin competitive analogs were found to interfere with parasite physiology on riboflavin-dependent processes. By means of bioinformatics analyses we identified a novel family of riboflavin transporters (RibJ) in trypanosomatids. Two RibJ members, TcRibJ and TbRibJ from T. cruzi and T. brucei respectively, were functionally characterized using homologous and/or heterologous expression systems. Conclusions/Significance: The RibJ family represents the first riboflavin transporters found in protists and the third eukaryotic family known to date. The essentiality of riboflavin for trypanosomatids, and the structural/biochemical differences that RFVT/SLC52 and RibJ present, make the riboflavin transporter -and its downstream metabolism- a potential trypanocidal drug target.Fil: Balcazar, Dario Emmanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología ; ArgentinaFil: Vanrell, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Romano, Patricia Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Pereira, Claudio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Bonomi, Hernán Ruy. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Carrillo, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología ; Argentin

Characterisation of a developmentally regulated amino acid transporter gene from Leishmania amazonensis

The metabolism of protozoan parasites of the Leishmania genus is strongly based on amino acid consumption, but little is known about amino acid uptake in these organisms. In the present work, we identified a Leishmania amazonensis gene (La-PAT1) encoding a putative amino acid transporter that belongs to the amino acid/auxin permease family, a group of H +/amino acid symporters. This single copy gene is upregulated in amastigotes, the life cycle stage found in the mammalian host. La-PAT1 putative orthologous sequences were identified in Leishmania infantum, Leishmania donovani, Leishmania major and Trypanosoma.Fil: Geraldo, Murilo V.. Universidade de Sao Paulo; BrasilFil: Silber, Ariel Mariano. Universidade de Sao Paulo; BrasilFil: Pereira, Claudio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Uliana, Silvia R.B.. Universidade de Sao Paulo; Brasi

Amino acid metabolic routes in Trypanosoma cruzi: Possible therapeutic targets against Chagas' disease

Chagas' disease is a zoonosis caused by the parasite Trypanosoma cruzi, a haematic protozoan, transmitted by insects from the Reduviidae family. This constitutes a relevant health and socio-economic problem in the Americas, with 11 - 18 million people infected, and approximately 100 million people at risk. The therapeutic possibilities rely into two drugs, nifurtimox® and benznidazole®, that were discovered more than thirty years ago, and are mainly successful during the acute phase of the disease. In the majority of the cases the disease is diagnosed in the chronic phase, when the therapy is inefficient and the probability of cure is low. In addition, these drugs are highly toxic, with systemic side effects on patients. Trypanosoma cruzi has a metabolism largely based on the consumption of amino acids, mainly proline, aspartate and glutamate, which constitute the main carbon and energy sources in the insect stage of the parasite life cycle. These amino acids also participate in the differentiation process of the replicative non-infective form (epimastigote) to the non-replicative infective form (trypomastigote). In particular, the participation of proline in the intracellular differentiation cycle, which occurs in the mammalian host, was recently demonstrated. In addition, an arginine kinase has been described in T. cruzi and T. brucei, which converts free arginine to phosphoarginine, a phosphagen with a role as an energy reservoir. Arginine kinase seems to be an essential component of energy management during stress conditions. Taken together, these data indicate that amino acid metabolism may provide multiple as yet unexplored targets for therapeutic drugs.Fil: Silber, Ariel Mariano. Universidade de Sao Paulo; BrasilFil: Coli, Walter. Universidade de Sao Paulo; BrasilFil: Ulrich, Henning. Universidade de Sao Paulo; BrasilFil: Manso Alves, Maria. Universidade de Sao Paulo; BrasilFil: Pereira, Claudio Alejandro. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

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.Fil: Pereira, Claudio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Reigada, Chantal. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Martínez Sayé, Melisa Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Di Girolamo, Fabio Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Miranda, Mariana Reneé. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentin

Laboratory techniques to obtain different forms of Trypanosoma cruzi: applications to wild-type and genetically modified parasites

Nowadays, there are no simple techniques for mimicking in vitro the life cycle of the kinetoplasmtid Trypanosoma cruzi Chagas, 1909, causative agent of Chagas disease, especially for parasite strains maintained as epimastigotes for many years. In the present study, we propose a method for obtaining metacyclic trypomastigotes, which were capable of infecting mammalian cells by simply lowering pH media. The collected amastigotes and trypomastigotes were differentiated into epimastigotes closing T. cruzi life cycle in vitro. Metacyclogenesis rates and infectivity were enhanced in cycled parasites. Finally, using this method, we were able to infect cells with transgenic parasites obtaining trypomastigotes and amastigotes using a neomycin-resistant cell line.Fil: Camara, Maria de Los Milagros. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Medicas; Argentina. Universidad de Buenos Aires; ArgentinaFil: Bouvier, Leon Alberto. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Medicas; Argentina. Universidad de Buenos Aires; ArgentinaFil: Miranda, Mariana Reneé. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Medicas; Argentina. Universidad de Buenos Aires; ArgentinaFil: Reigada, Chantal. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Medicas; Argentina. Universidad de Buenos Aires; ArgentinaFil: Pereira, Claudio Alejandro. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Medicas; Argentina. Universidad de Buenos Aires; Argentin

Proline and ions accumulation in four Passiflora species under water-saline stress

Water-saline stress is one of the factors limiting growth and crop production, with the induction of morphological, structural and metabolic changes in higher plants. This study aimed to evaluate the response of four species with ten accessions of Passiflora to saline stress and drought stress, and the accumulation of proline and content of cationic macronutrients and their relationships with sodium. The plants were grown under controlled conditions for seven months, watered with nutrient solution with additions of NaCl (100 mM) to achieve the desired conductivities (1.5, 2.5, 4.0, and 5.5 dS m-1) and constant substrate humidity of 100%, 66% and 33%. The concentrations of Na+, K+, Ca++, Mg++ and proline in the leaves were determined. Proline accumulation increased in salinized plants, especially in young leaves. Proline increase was evident in accessions tolerant and moderately tolerant to salinity. These results indicate that tolerance to salinity and water stress in Passiflora relates to an improved ability to exclude sodium and greater ability to accumulate proline for osmotic adjustment. The increased salinity of the nutrient solution increased sodium contents, relations Na+/Ca++, Na+/Mg++, Na+/K+, and decreased Ca++, Mg++ and K+ levels, reflecting the nutritional imbalance caused by progressive saline stress.Water-saline stress is one of the factors limiting growth and crop production, with the induction of morphological, structural and metabolic changes in higher plants. This study aimed to evaluate the response of four species with ten accessions of Passiflora to saline stress and drought stress, and the accumulation of proline and content of cationic macronutrients and their relationships with sodium. The plants were grown under controlled conditions for seven months, watered with nutrient solution with additions of NaCl (100 mM) to achieve the desired conductivities (1.5, 2.5, 4.0, and 5.5 dS m-1) and constant substrate humidity of 100%, 66% and 33%. The concentrations of Na+, K+, Ca++, Mg++ and proline in the leaves were determined. Proline accumulation increased in salinized plants, especially in young leaves. Proline increase was evident in accessions tolerant and moderately tolerant to salinity. These results indicate that tolerance to salinity and water stress in Passiflora relates to an improved ability to exclude sodium and greater ability to accumulate proline for osmotic adjustment. The increased salinity of the nutrient solution increased sodium contents, relations Na+/Ca++, Na+/Mg++, Na+/K+, and decreased Ca++, Mg++ and K+ levels, reflecting the nutritional imbalance caused by progressive saline stress