Mecanismo de acción de drogas tripanocidas

Fil:Moreno, Silvia N. J.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

A glycosylphosphatidylinositol-anchored carbonic anhydrase-related protein of Toxoplasma gondii is important for rhoptry biogenesis and virulence

Carbonic anhydrase-related proteins (CARPs) have previously been described as catalytically inactive proteins closely related to α-carbonic anhydrases (α-CAs). These CARPs are found in animals (both vertebrates and invertebrates) and viruses as either independent proteins or domains of other proteins. We report here the identification of a new CARP (TgCA_RP) in the unicellular organism Toxoplasma gondii that is related to the recently described η-class CA found in Plasmodium falciparum. TgCA_RP is posttranslationally modified at its C terminus with a glycosylphosphatidylinositol anchor that is important for its localization in intracellular tachyzoites. The protein localizes throughout the rhoptry bulbs of mature tachyzoites and to the outer membrane of nascent rhoptries in dividing tachyzoites, as demonstrated by immunofluorescence and immunoelectron microscopy using specific antibodies. T. gondii mutant tachyzoites lacking TgCA_RP display a growth and invasion phenotype in vitro and have atypical rhoptry morphology. The mutants also exhibit reduced virulence in a mouse model. Our results show that TgCA_RP plays an important role in the biogenesis of rhoptries

Unsupervised Discovery of Toxoplasma gondii Motility Phenotypes

Toxoplasma gondii is a parasitic protozoan that causes dis- seminated toxoplasmosis, a disease that afflicts roughly a third of the worlds population. Its virulence is predicated on its motility and ability to enter and exit nucleated cells; therefore, studies elucidating its mechanism of motility and in particular, its motility patterns in the context of its lytic cycle, are critical to the eventual development of therapeutic strate- gies. Here, we present an end-to-end computational pipeline for identifying T. gondii motility phenotypes in a completely unsupervised, data-driven way. We track the parasites before and after addition of extracellular Ca2+ to study its effects on the parasite motility patterns and use this information to parameterize the motion and group it according to similarity of spatiotemporal dynamics.Comment: 4 pages, Accepted to 2018 IEEE International Symposium on Biomedical Imagin

Proteomic Analysis of the Acidocalcisome, an Organelle Conserved from Bacteria to Human Cells

Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate

Aryloxyethyl thiocyanates are potent growth inhibitors of Trypanosoma cruzi and Toxoplasma gondii

As a part of our project aimed at searching new safe chemotherapeutic agents against parasitic diseases, several compounds structurally related to the antiparasitic agent WC-9 (4-phenoxyphenoxyethyl thiocyanate), which were modified at the terminal phenyl ring, were designed, synthesized and evaluated as growth inhibitors against Trypanosoma cruzi, the etiological agent of Chagas disease and Toxoplasma gondii, the parasite responsible of toxoplasmosis. Most of the synthetic analogues exhibited similar antiparasitic activity being slightly more potent than our lead WC-9. For example, the trifluoromethyl derivatives 15 and 16 exhibited ED50 values of 10.0 uM and 9.2 uM, respectively, against intracellular T. cruzi, whereas they showed potent action against tachyzoites of T. gondii (ED50 values 1.6 uM and 1.9 uM against T. gondii, respectively). In addition, the WC-9 analogues 48 and 61, in which the terminal aryl group was meta with respect to the alkyl chain bearing the thiocyanate group, showed potent inhibitory action against both T. cruzi and T. gondii at the very low micromolar range suggesting that para-phenyl substitution pattern is not necessarily required for biological activity.Fil: Chao, Maria Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; ArgentinaFil: Exeni Matiuzzi, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; ArgentinaFil: Storey, Melissa. University of Georgia; Estados UnidosFil: Li, Catherine. University of Georgia; Estados UnidosFil: Szajnman, Sergio Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; ArgentinaFil: Docampo, Roberto. University of Georgia; Estados UnidosFil: Moreno, Silvia N. J.. University of Georgia; Estados UnidosFil: Rodriguez, Juan Bautista. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentin

Magic-angle spinning 31 P NMR spectroscopy of condensed phosphates in parasitic protozoa: visualizing the invisible

Abstract We report the results of a solid-state 31 P nuclear magnetic resonance (NMR) spectroscopic investigation of the acidocalcisome organelles from Trypanosoma brucei (bloodstream form), Trypanosoma cruzi and Leishmania major (insect forms). The spectra are characterized by a broad envelope of spinning sidebands having isotropic chemical shifts at V V0, 3 37 and 3 321 ppm. These resonances are assigned to orthophosphate, terminal (K K) phosphates of polyphosphates and bridging (L L) phosphates of polyphosphates, respectively. The average polyphosphate chain length is V V3.3 phosphates. Similar results were obtained with whole L. major promastigotes. 31 P NMR spectra of living L. major promastigotes recorded under conventional solution NMR conditions had spectral intensities reduced with respect to solution-state NMR spectra of acid extracts, consistent with the invisibility of the solid-state phosphates. These results show that all three parasites contain large stores of condensed phosphates which can be visualized by using magic-angle spinning NMR techniques. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies

Design, synthesis and biological evaluation of sulfur-containing 1,1-bisphosphonic acids as antiparasitic agents

As part of our efforts aimed at searching for new antiparasitic agents, 2-alkylmercaptoethyl-1,1-bisphosphonate derivatives were synthesized and evaluated against Trypanosoma cruzi, the etiologic agent of Chagas disease, and Toxoplasma gondii, the responsible agent for toxoplasmosis. Many of these sulfur-containing bisphosphonates were potent inhibitors against the intracellular form of T. cruzi, the clinically more relevant replicative form of this parasite, and tachyzoites of T. gondii targeting T. cruzi or T. gondii farnesyl diphosphate synthases (FPPSs), which constitute valid targets for the chemotherapy of these parasitic diseases. Interestingly, long chain length sulfur-containing bisphosphonates emerged as relevant antiparasitic agents. Taking compounds 37, 38, and 39 as representative members of this class of drugs, they exhibited ED50 values of 15.8 μM, 12.8 μM, and 22.4 μM, respectively, against amastigotes of T. cruzi. These cellular activities matched the inhibition of the enzymatic activity of the target enzyme (TcFPPS) having IC50 values of 6.4 μM, 1.7 μM, and 0.097 μM, respectively. In addition, these compounds were potent anti-Toxoplasma agents. They had ED50 values of 2.6 μM, 1.2 μM, and 1.8 μM, respectively, against T. gondii tachyzoites, while they exhibited a very potent inhibitory action against the target enzyme (TgFPPS) showing IC50 values of 0.024 μM, 0.025 μM, and 0.021 μM, respectively. Bisphosphonates bearing a sulfoxide unit at C-3 were also potent anti-Toxoplasma agents, particularly those bearing long aliphatic chains such as 43-45, which were also potent antiproliferative drugs against tachyzoites of T. gondii. These compounds inhibited the enzymatic activity of the target enzyme (TgFPPS) at the very low nanomolar range. These bisphosphonic acids have very good prospective not only as lead drugs but also as potential chemotherapeutic agents.Fil: Recher, Marion. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; ArgentinaFil: Barboza, Alejandro Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; ArgentinaFil: Li, Zhu Hong. University of Georgia; Estados UnidosFil: Galizzi, Melina. University of Georgia; Estados UnidosFil: Ferrer, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; ArgentinaFil: Szajnman, Sergio Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; ArgentinaFil: Docampo, Roberto. University of Georgia; Estados UnidosFil: Moreno, Silvia N. J.. University of Georgia; Estados UnidosFil: Rodriguez, Juan Bautista. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentin

Using a genetically encoded sensor to identify inhibitors of Toxoplasma gondii Ca2+ Signalling

This work was supported in part by National Institutes of Health Grants AI-110027 and AI-096836 (to S. N. J. M.) and 1DP5OD017892 (to S. L.).The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca2+. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca2+ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca2+ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca2+. We define the pool of Ca2+ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca2+ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca2+. The enhancers identified are capable of releasing intracellular Ca2+ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca2+-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca2+ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca2+, underscoring the importance of these pathways and the therapeutic potential of their inhibition.Publisher PDFPeer reviewe