Browsing by Author "Ochoa, Rodrigo"
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Item The Akt-like kinase of Leishmania panamensis: as a new molecular target for drug discovery(Acta Tropica, 2017) Tirado-Duarte, Didier; Marín-Villa, Marcel; Ochoa, Rodrigo; Blandón-Fuentes, Gustavo; Soares, Maurilio José; Robledo, Sara Maria; Varela-Miranda, Rubén E.The Akt-like kinase of Leishmania spp. is a cytoplasmic orthologous protein of the serine/threonine kinase B-PKB/human-Akt group, which is involved in the cellular survival of these parasites. By the application of a computational strategy we obtained two specific inhibitors of the Akt-like protein of L. panamensis (UBMC1 and UBMC4), which are predicted to bind specifically to the pleckstrin domain (PH) of the enzyme. We show that the Akt-like of Leishmania panamensis is phospho-activated in parasites under nutritional and thermic stress, this phosphorylation is blocked by the UBMC1 and UMBC2 and such inhibition leads to cell death. Amongst the effects caused by the inhibitors on the parasites we found high percentage of hypodiploidy and loss of mitochondrial membrane potential. Ultrastructural studies showed highly vacuolated cytoplasm, as well as shortening of the flagellum, loss of nuclear membrane integrity and DNA fragmentation. Altogether the presented results suggest that the cell death caused by UMBC1 and UMBC4 may be associated to an apoptosis-like process. The compounds present an inhibitory concentration (IC50) over intracellular amastigotes of L. panamensis of 9.2 ± 0.8 μM for UBMC1 and 4.6 ± 1.9 μM for UBMC4. The cytotoxic activity for UBMC1 and UBMC4 in human macrophages derived from monocytes (huMDM) was 29 ± 1.2 μM and >40 μM respectively. Our findings strongly support that the presented compounds can be plausible candidates as a new therapeutic alternative for the inhibition of specific kinases of the parasite.Item The Akt-like kinase of Leishmania panamensis: As a new molecular target for drug discovery(Elsevier B.V., 2018-01-01) Tirado-Duarte, Didier; Marín-Villa, Marcel; Ochoa, Rodrigo; Blandón-Fuentes, Gustavo; Soares, Maurílio José; Robledo, Sara María; Varela-M, R. E.The Akt-like kinase of Leishmania spp. is a cytoplasmic orthologous protein of the serine/threonine kinase B-PKB/human-Akt group, which is involved in the cellular survival of these parasites. By the application of a computational strategy we obtained two specific inhibitors of the Akt-like protein of L. panamensis (UBMC1 and UBMC4), which are predicted to bind specifically to the pleckstrin domain (PH) of the enzyme. We show that the Akt-like of Leishmania panamensis is phospho-activated in parasites under nutritional and thermic stress, this phosphorylation is blocked by the UBMC1 and UMBC2 and such inhibition leads to cell death. Amongst the effects caused by the inhibitors on the parasites we found high percentage of hypodiploidy and loss of mitochondrial membrane potential. Ultrastructural studies showed highly vacuolated cytoplasm, as well as shortening of the flagellum, loss of nuclear membrane integrity and DNA fragmentation. Altogether the presented results suggest that the cell death caused by UMBC1 and UMBC4 may be associated to an apoptosis-like process. The compounds present an inhibitory concentration (IC50) over intracellular amastigotes of L. panamensis of 9.2 ± 0.8 μM for UBMC1 and 4.6 ± 1.9 μM for UBMC4. The cytotoxic activity for UBMC1 and UBMC4 in human macrophages derived from monocytes (huMDM) was 29 ± 1.2 μM and >40 μM respectively. Our findings strongly support that the presented compounds can be plausible candidates as a new therapeutic alternative for the inhibition of specific kinases of the parasite. © 2017 The AuthorsItem Identification of a RAC/AKT-like gene in Leishmania parasites as a putative therapeutic target in leishmaniasis(BioMed Central Ltd., 2017-10-10) Varela-M, R. E.; Ochoa, Rodrigo; Muskus, Carlos Enrique; Muro, Antonio; Mollinedo, FaustinoBackground: Leishmaniasis is one of the world's most neglected diseases caused by at least 20 different species of the protozoan parasite Leishmania. Although new drugs have become recently available, current therapy for leishmaniasis is still unsatisfactory. A subgroup of serine/threonine protein kinases named as related to A and C protein kinases (RAC), or protein kinase B (PKB)/AKT, has been identified in several organisms including Trypanosoma cruzi parasites. PKB/AKT plays a critical role in mammalian cell signaling promoting cell survival and is a major drug target in cancer therapy. However, the role of protozoan parasitic PKB/AKT remains to be elucidated. Results: We have found that anti-human AKT antibodies recognized a protein of about 57 kDa in Leishmania spp. parasites. Anti-human phospho-AKT(Thr308) antibodies identified a protein in extracts from Leishmania spp. that was upregulated following parasite exposure to stressful conditions, such as nutrient deprivation or heat shock. Incubation of AKT inhibitor X with Leishmania spp. promastigotes under stressful conditions or with Leishmania-infected macrophages led to parasite cell death. We have identified and cloned a novel gene from Leishmania donovani named Ld-RAC/AKT-like gene, encoding a 510-amino acid protein of approximately 57.6 kDa that shows a 26.5% identity with mammalian AKT1. Ld-RAC/AKT-like protein contains major mammalian PKB/AKT hallmarks, including the typical pleckstrin, protein kinase and AGC kinase domains. Unlike mammalian AKT that contains key phosphorylation sites at Thr308 and Ser473 in the activation loop and hydrophobic motif, respectively, Ld-RAC/AKT-like protein has a Thr residue in both motifs. By domain sequence comparison, we classified AKT proteins from different origins in four major subcategories that included different parasites. Conclusions: Our data suggest that Ld-RAC/AKT-like protein represents a Leishmania orthologue of mammalian AKT involved in parasite stress response and survival, and therefore could become a novel therapeutic and druggable target in leishmaniasis therapy. In addition, following comparative sequence analyses, we found the RAC/AKT-like proteins from Leishmania constitute a subgroup by themselves within a general AKT-like protein family. © 2017 The Author(s).Item Identification of a RAC/AKT-like gene in Leishmania parasites as a putative therapeutic target in leishmaniasis.(Parasites and Vectors, 2018) Varela-M, Rubén E.; Ochoa, Rodrigo; Muskus, Carlos E.; Muro, Antonio; Mollinedo, FaustinoBackground Leishmaniasis is one of the world’s most neglected diseases caused by at least 20 different species of the protozoan parasite Leishmania. Although new drugs have become recently available, current therapy for leishmaniasis is still unsatisfactory. A subgroup of serine/threonine protein kinases named as related to A and C protein kinases (RAC), or protein kinase B (PKB)/AKT, has been identified in several organisms including Trypanosoma cruzi parasites. PKB/AKT plays a critical role in mammalian cell signaling promoting cell survival and is a major drug target in cancer therapy. However, the role of protozoan parasitic PKB/AKT remains to be elucidated. ResultsWe have found that anti-human AKT antibodies recognized a protein of about 57 kDa in Leishmania spp. parasites. Anti-human phospho-AKT(Thr308) antibodies identified a protein in extracts from Leishmania spp. that was upregulated following parasite exposure to stressful conditions, such as nutrient deprivation or heat shock. Incubation of AKT inhibitor X with Leishmania spp. promastigotes under stressful conditions or with Leishmania-infected macrophages led to parasite cell death. We have identified and cloned a novel gene from Leishmania donovani named Ld-RAC/AKT-like gene, encoding a 510-amino acid protein of approximately 57.6 kDa that shows a 26.5% identity with mammalian AKT1. Ld-RAC/AKT-like protein contains major mammalian PKB/AKT hallmarks, including the typical pleckstrin, protein kinase and AGC kinase domains. Unlike mammalian AKT that contains key phosphorylation sites at Thr308 and Ser473 in the activation loop and hydrophobic motif, respectively, Ld-RAC/AKT-like protein has a Thr residue in both motifs. By domain sequence comparison, we classified AKT proteins from different origins in four major subcategories that included different parasites. Conclusions Our data suggest that Ld-RAC/AKT-like protein represents a Leishmania orthologue of mammalian AKT involved in parasite stress response and survival, and therefore could become a novel therapeutic and druggable target in leishmaniasis therapy. In addition, following comparative sequence analyses, we found the RAC/AKT-like proteins from Leishmania constitute a subgroup by themselves within a general AKT-like protein family.Item Identification of potential kinase inhibitors within the pi3k/akt pathway of leishmania species(2021) Ochoa, Rodrigo; Ortega Pajares, Amaya; Castello, Florencia A.; Serral, Federico; Fernández Do Porto, Darío; Villa Pulgarin, Janny A.; Varela M, Rubén E.; Muskus, CarlosLeishmaniasis is a public health disease that requires the development of more effective treatments and the identification of novel molecular targets. Since blocking the PI3K/AKT pathway has been successfully studied as an effective anticancer strategy for decades, we examined whether the same approach would also be feasible in Leishmania due to their high amount and diverse set of annotated proteins. Here, we used a best reciprocal hits protocol to identify potential protein kinase homologues in an annotated human PI3K/AKT pathway. We calculated their ligandibility based on available bioactivity data of the reported homologues and modelled their 3D structures to estimate the druggability of their binding pockets. The models were used to run a virtual screening method with molecular docking. We found and studied five protein kinases in five different Leishmania species, which are AKT, CDK, AMPK, mTOR and GSK3 homologues from the studied pathways. The compounds found for different enzymes and species were analysed and suggested as starting point scaffolds for the design of inhibitors. We studied the kinases’ participation in protein–protein interaction networks, and the potential deleterious effects, if inhibited, were supported with the literature. In the case of Leishmania GSK3, an inhibitor of its human counterpart, prioritized by our method, was validated in vitro to test its anti-Leishmania activity and indirectly infer the presence of the enzyme in the parasite. The analysis contributes to improving the knowledge about the presence of similar signalling pathways in Leishmania, as well as the discovery of compounds acting against any of these kinases as potential molecular targets in the parasiteItem In Silico, In Vitro, and Pharmacokinetic Studies of UBMC-4, a Potential Novel Compound for Treating against Trypanosoma cruzi(MDPI, 2022-06) Bustamante, Christian; Díez Mejía, Andrés Felipe; Arbeláez, Natalia; Soares, Maurilio José; Robledo, Sara M.; Ochoa, Rodrigo; Varela M., Rubén E.; Marín Villa, MarcelThe lack of therapeutic alternatives for the treatment of Chagas disease, a neglected disease, drives the discovery of new drugs with trypanocidal activity. Consequently, we conducted in vitro studies using UBMC-4, a potential Trypanosoma cruzi AKT-like pleckstrin homology (PH) domain inhibitory compound found using bioinformatics tools. The half effective concentration (EC50) on intracellular amastigotes was determined at 1.85 ± 1 µM showing low cytotoxicity (LC50) > 40 µM on human cell lines tested. In order to study the lethal effect caused by the compound on epimastigotes, morphological changes were assessed by scanning and transmission electron microscopy. Progressive alterations such as flagellum inactivation, cell size reduction, nuclear structure alteration, condensation of chromatin towards the nuclear periphery, vacuole formation, and mitochondrial swelling with kinetoplast integrity loss were evidenced. In addition, apoptosis-like markers in T. cruzi were assessed by flow cytometry, demonstrating that the effect of UBMC-4 on T. cruzi AKT-like kinase reduced the tolerance to nutritional stress-triggered, apoptosis-like events, including DNA fragmentation, mitochondrial damage, and loss of plasma membrane integrity. After this, UBMC-4 was formulated for oral administration and pharmacokinetics were analyzed in a mouse model. Finally, upon oral administration of 200 mg/kg in mice, we found that a UBMC-4 plasma concentration remaining in circulation beyond 24 h after administration is well described by the two-compartment model. We conclude that UBMC-4 has an effective trypanocidal activity in vitro at low concentrations and this effect is evident in T. cruzi cell structures. In mice, UBMC-4 was well absorbed and reached plasma concentrations higher than the EC50, showing features that would aid in developing a new drug to treat Chagas disease. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Item Search of Allosteric Inhibitors and Associated Proteins of an AKT-like Kinase from Trypanosoma cruzi(International Journal of Molecular Sciences, 2018) Ochoa, Rodrigo; Rocha-Roa, Cristian; Marín-Villa, Marcel; Robledo, Sara M.; Varela-M, Rubén E.Proteins associated to the PI3K/AKT/mTOR signaling pathway are widely used targets for cancer treatment, and in recent years they have also been evaluated as putative targets in trypanosomatids parasites, such as Trypanosoma cruzi. Here, we performed a virtual screening approach to find candidates that can bind regions on or near the Pleckstrin homology domain of an AKT-like protein in T. cruzi. The compounds were also evaluated in vitro. The in silico and experimental results allowed us to identify a set of compounds that can potentially alter the intracellular signaling pathway through the AKT-like kinase of the parasite; among them, a derivative of the pyrazolopyridine nucleus with an IC50 of 14.25 ± 1.00 μM against amastigotes of T. cruzi. In addition, we built a protein–protein interaction network of T. cruzi to understand the role of the AKT-like protein in the parasite, and look for additional proteins that can be postulated as possible novel molecular targets for the rational design of compounds against T. cruzi.Item Search of allosteric inhibitors and associated proteins of an AKT-like kinase from trypanosoma cruzi(MDPI AG, 2018-12-12) Ochoa, Rodrigo; Rocha-Roa, Cristian; Marín-Villa, Marcel; Robledo, Sara María; Varela-M, R. E.Proteins associated to the PI3K/AKT/mTOR signaling pathway are widely used targets for cancer treatment, and in recent years they have also been evaluated as putative targets in trypanosomatids parasites, such as Trypanosoma cruzi. Here, we performed a virtual screening approach to find candidates that can bind regions on or near the Pleckstrin homology domain of an AKT-like protein in T. cruzi. The compounds were also evaluated in vitro. The in silico and experimental results allowed us to identify a set of compounds that can potentially alter the intracellular signaling pathway through the AKT-like kinase of the parasite; among them, a derivative of the pyrazolopyridine nucleus with an IC 50 of 14.25 ± 1.00 µM against amastigotes of T. cruzi. In addition, we built a protein–protein interaction network of T. cruzi to understand the role of the AKT-like protein in the parasite, and look for additional proteins that can be postulated as possible novel molecular targets for the rational design of compounds against T. cruzi. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.