Beatriz Garat

Vanadium polypyridyl compounds as potential antiparasitic and antitumoral agents: New achievements

In the search for new therapeutic tools against diseases produced by kinetoplastid parasites five vanadyl complexes, [V(IV)O(L-2H)(phen)], including 1,10-phenanthroline (phen) and tridentate salicylaldehyde semicarbazone derivatives as ligands have been synthesized and characterized in the solid state and in solution by using different techniques. EPR suggested a distorted octahedral geometry with the tridentate semicarbazone occupying three equatorial positions and phen coordinated in an equatorial/axial mode. The compounds were evaluated in vitro on epimastigotes of Trypanosoma cruzi, causative agent of Chagas disease, Leishmania panamensis and Leishmania chagasi and on tumor cells. The complexes showed higher in vitro anti-trypanosomal activities than the reference drug Nifurtimox (IC(50) values in the range 1.6-3.8 μM) and increased activities in respect to the free semicarbazone ligands. In vitro activity on promastigote and amastigote forms of Leishmania showed interesting results. The compounds [VO(L1-2H)(phen)] and [VO(L3-2H)(phen)], where L1 = 2-hydroxybenzaldehyde semicarbazone and L3 = 2-hydroxy-3-methoxybenzaldehyde semicarbazone, resulted active (IC(50) 2.74 and 2.75 μM, respectively, on promastigotes of L. panamensis; IC(50) 19.52 and 20.75 μM, respectively, on intracellular amastigotes of L. panamensis) and showed low toxicity on THP-1 mammalian cells (IC(50) 188.55 and 88.13 μM, respectively). In addition, the complexes showed cytotoxicity on human promyelocytic leukemia HL-60 cells with IC(50) values of the same order of magnitude as cisplatin. The interaction of the complexes with DNA was demonstrated by different techniques, suggesting that this biomolecule could be a potential target either in the parasites or in tumor cells.

Potent in vitro anti-Trypanosoma cruzi activity of pyridine-2-thiol N-oxide metal complexes having an inhibitory effect on parasite-specific fumarate reductase.

In the search for new therapeutic tools against Chagas disease (American trypanosomiasis) palladium and platinum complexes of the bioactive ligand pyridine-2-thiol N-oxide were exhaustively characterized and evaluated in vitro. Both complexes showed high in vitro growth inhibition activity (IC50 values in the nanomolar range) against Trypanosoma cruzi, the causative agent of the disease. They were 39–115 times more active than the antitrypanosomal drug Nifurtimox. The palladium complex showed an approximately threefold enhancement of the activity compared with the parent compound. In addition, owing to their low unspecific cytotoxicity on mammalian cells, the complexes showed a highly selective antiparasite activity. To get an insight into the mechanism of action of these compounds, DNA, redox metabolism (intraparasite free-radical production) and two parasite-specific enzymes absent in the host, namely, trypanothione reductase and NADH-fumarate reductase, were evaluated as potential parasite targets. Additionally, the effect of metal coordination on the free radical scavenger capacity previously reported for the free ligand was studied. All the data strongly suggest that trypanocidal action of the complexes could mainly rely on the inhibition of the parasite-specific enzyme NADH-fumarate reductase.

Synthesis and characterization of a pyridine-2-thiol N-oxide gold(I) complex with potent antiproliferative effect against Trypanosoma cruzi and Leishmania sp. insight into its mechanism of action ☆

In the search for new therapeutic tools against parasitic diseases caused by the Kinetoplastids Leishmania spp. and Trypanosoma cruzi, a novel gold(I) triphenylphosphine complex with the bioactive coligand pyridine-2-thiol N-oxide (mpo) was synthesized and characterized by using analytical and conductometric measurements, electrospray ionization-mass spectrometry (ESI) and electronic, FTIR and (1)H and (31)P NMR spectroscopies. A dinuclear structure is suggested for the complex. At a 1 microM concentration the complex induced in vitro after 30 min a potent leishmanicidal effect (LD(50)) against promastigotes of Leishmania (L.) mexicana while on Leishmania (V.) braziliensis with the same concentration only a leishmanistatic effect (IC(75)) was observed 48 h after treatment. Similar differential susceptibilities were also found when testing the ligand mpo, but at a higher dose (5 microM). In addition, the compound showed growth inhibitory effect on Dm28c T. cruzi epimastigotes in culture (IC(50) 0.09 microM), being even more active than the anti-trypanosomal reference drug Nifurtimox (IC(50) 6 microM). DNA interaction studies showed that this biomolecule does not constitute a main target for the mpo complex currently tested. Instead, the significant potentiation of the antiproliferative effect against both Leishmania species and T. cruzi could be associated to the inhibition of NADH fumarate reductase, a kinetoplastid parasite-specific enzyme absent in the host. Furthermore, due to its low unspecific cytotoxicity on mammalian cells (J774 macrophages), the new gold complex showed a selective anti-parasite activity. It constitutes a promising new potent chemotherapeutic alternative to be evaluated in vivo in experimental models of leishmaniasis and Chagas disease.

Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi

BackgroundDue to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms.ResultsHere we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect’s life stages using RNA-seq and ribosome profiling. The observed steady state transcript levels support constitutive transcription and maturation implying the existence of distinctive posttranscriptional regulatory mechanisms controlling gene expression levels at those parasite stages. Meanwhile, the downregulation of a large proportion of the translatome indicates a key role of translation control in differentiation into the infective form. The previously described proteomic data correlate better with the translatomes than with the transcriptomes and translational efficiency analysis shows a wide dynamic range, reinforcing the importance of translatability as a regulatory step. Translation efficiencies for protein families like ribosomal components are diminished while translation of the transialidase virulence factors is upregulated in the quiescent infective metacyclic trypomastigote stage.ConclusionsA large subset of genes is modulated at the translation level in two different stages of Trypanosoma cruzi life cycle. Translation upregulation of virulence factors and downregulation of ribosomal proteins indicates different degrees of control operating to prepare the parasite for an infective life form. Taking together our results show that translational regulation, in addition to regulation of steady state level of mRNA, is a major factor playing a role during the parasite differentiation.

Functional Genomic Characterization of mRNAs Associated with TcPUF6, a Pumilio-like Protein from Trypanosoma cruzi

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle.

DNA as molecular target of analogous palladium and platinum anti-Trypanosoma cruzi compounds: A comparative study

In the search for drugs with anti-trypanosome activity, we had previously synthesized two series of platinum and palladium analogous compounds of the formula [M(II)Cl(2)(HL)], where HL were bioactive 5-nitrofuryl or 5-nitroacroleine thiosemicarbazone derivatives. In this work, we thoroughly characterized [M(II)Cl(2)(HL)] complexes interaction with DNA by using different techniques: gel electrophoresis, DNA viscosity measurements, circular dichroism (CD) and atomic force microscopy (AFM). Electrophoresis results showed that all complexes induced a withdrawal of DNA superhelicity demonstrated by a decrease in electrophoretic mobility of supercoiled DNA form. This effect on migration was dependent on dose but also on the nature of both the metal and the ligand. In general, the effect produced by palladium complexes was significantly more intense than that observed for the corresponding platinum analogs. Differences between palladium and platinum complexes were also observed in CD experiments. While palladium complexes induce evident calf thymus (CT)-DNA profile changes compatible with B-DNA to Z-DNA conformational transition, no clear effect was observed for platinum ones. Additionally, AFM studies showed that changes in the shape of plasmid DNA, like supercoiling, kinks and thickness increase resulted more intense for the former. In addition, either Pd or Pt complexes increased the viscosity of CT DNA solutions in a concentration dependent manner. Although the nature of DNA interaction of both series of analogous palladium and platinum complexes seemed to be similar, an explanation for the observed differential intensity of the effect could be related to the known kinetic stability differences between palladium and platinum compounds.

Cytotoxic palladium complexes of bioreductive quinoxaline N1,N4-dioxide prodrugs

Four new palladium(II) complexes with the formula Pd(L)(2), where L are quinoxaline-2-carbonitrile N(1),N(4)-dioxide derivatives, were synthesized as a contribution to the chemistry and pharmacology of metal compounds with this class of pharmacologically interesting bioreductive prodrugs. Compounds were characterized by elemental, conductometric and thermogravimetric analyses, fast atom bombardment mass spectrometry (FAB-MS) and electronic, Fourier transform infrared (FTIR) and (1)H-nuclear magnetic resonance spectroscopies. The complexes were subjected to cytotoxic evaluation on V79 cells in hypoxic and aerobic conditions. In addition, a preliminary study on interaction with plasmid DNA in normoxia was performed. Complexes showed different in vitro biological behavior depending on the nature of the substituent on the quinoxaline ring. Pd(L1)(2) and Pd(L2)(2), where L1 is 3-aminoquinoxaline-2-carbonitrile N(1),N(4)-dioxide and L2 is 3-amino-6(7)-methylquinoxaline-2-carbonitrile N(1),N(4)-dioxide, showed non selective cytotoxicity, being cytotoxic either in hypoxic or in aerobic conditions. On the other hand, Pd(L3)(2), where L3 is 3-amino-6(7)-chloroquinoxaline-2-carbonitrile N(1),N(4)-dioxide, resulted in vitro more potent cytotoxin in hypoxia (P=5.0 microM) than the corresponding free ligand (P=9.0 microM) and tirapazamine (P=30.0 microM), the first bioreductive cytotoxic drug introduced into clinical trials. In addition, it showed a very good selective cytotoxicity in hypoxic conditions, being non-cytotoxic in normoxia. Its hypoxic cytotoxicity relationship value, HCR, was of the same order than those of other hypoxia selective cytotoxins (i.e., Mitomycine C, Misonidazole and the N-oxide RB90740). Interaction of the complexes with plasmid DNA in normoxia showed dose dependent ability to relax the negative supercoiled forms via different mechanisms. Pd(L2)(2) introduced a scission event in supercoiled DNA yielding the circular relaxed form. Meanwhile, both Pd(L1)(2) and Pd(L3)(2) produced the loss of negative supercoils rendering a family of topoisomers with reduced electrophoretic mobility. Pd(L3)(2) showed a more marked effect than Pd(L1)(2). Indeed, for the highest doses assayed, Pd(L3)(2) was even able to introduce positive supercoils on the plasmid DNA.

Analysis of the DNA Interaction of Copper Compounds Belonging to the Casiopeínas® Antitumoral Series

Casiopeínas® are mixed-chelate copper complexes with antitumor tested potential. Their activity, both in vitro and in vivo, as antiproliferative, cytotoxic, and genotoxic drugs has been assessed. Biological results of these copper compounds have deserved some of them entering clinical trials. Significant efforts have been devoted to the in-depth identification of their mechanism of action. Using gel electrophoresis analysis, we have previously shown that the interaction of the Casiopeínas® Cas II-gly, [Cu(4,7-dimethyl-1,10-phenanthroline)(glycinate)]NO3 with DNA, triggers the cleavage of the biomolecule by a free radical mechanism. In this work, we further study the behavior of different complexes of the same Casiopeínas® series also including glycinate as co-ligand {Cas VI-gly (5,6 dimethyl-1,10-phenanthroline glycinato copper(II) nitrate), Cas VII-gly (1,10-phenanthroline glycinato copper(II) nitrate), and Cas IX-gly (2,2′-bipyridine glycinato copper(II) nitrate)} and of a Casiopeínas® with a different co-ligand (Cas III-Cs; 4,7-dimethyl-1,10-phenanthroline salicylaldehydato-copper(II) nitrate). While all of them produce DNA degradation, the performance in the presence of a radical scavenger suggests the existence of differences in their mechanism of interaction with DNA.

New chemotypes as Trypanosoma cruzi triosephosphate isomerase inhibitors: a deeper insight into the mechanism of inhibition

Abstract Context: Triosephosphate isomerase (TIM) is a ubiquitous enzyme that has been targeted for the discovery of new small molecular weight compounds used against Trypanosoma cruzi, the causative agent of Chagas disease. We have identified phenazine and 1,2,6-thiadiazine chemotypes as novel inhibitors of TIM from T. cruzi (TcTIM). Objective: Study the mechanism of TcTIM inhibition by a phenazine derivative and by a 1,2,6-thiadiazine derivative. Methods: We performed biochemical and theoretical molecular docking studies to characterize the interaction of the derivatives with wild-type and mutant TcTIM. Results and conclusion: At low micromolar concentrations, the compounds induce highly selective irreversible inactivation of parasitic TIM. The molecular docking simulations indicate that the phenazine derivative likely interferes with the association of the two monomers of the dimeric enzyme by locating at the dimer interface, while 1,2,6-thiadiazine could act as an inhibitor binding to a region surrounding Cys-118.

A novel type of single-stranded nucleic acid binding protein recognizing a highly frequent motif in the intergenic regions of Trypanosoma cruzi.

Regulation of gene expression in trypanosomatids is not yet well understood. Genes are organized in long polycistronic transcriptional units separated by intergenic regions that may contain the signaling information for nucleic acid processing. Poly-dinucleotides are frequent in these regions and have been proposed to be involved in regulation of gene expression. Previously, we have reported that [dT-dG] are highly frequent, asymmetrically strand distributed, and constitute targets for specific protein binding [Biochem. Biophys. Res. Commun. 287 (2001) 98]. Here, we present the purification and characterization of a new type of single stranded nucleic acid binding protein (Tc38) that recognizes specifically the motif poly[dT-dG] in this parasite. The protein has a deduced molecular weight of 38kDa and its salient characteristics include an isoelectric point of 9.34, a high frequency of Ser, Leu, and di-amino acids. Neither compositional nor architectural conserved domains could be detected in database searches. Recombinant Tc38 was expressed as a GST fusion protein, purified, and used to analyze target specificity by electrophoretic mobility shift assays. The unusual characteristics of the protein together with the peculiar features of the specific nucleic acid target suggest the existence of a novel event that may be involved in the mechanisms of gene expression in trypanosomatids.