RESUMO
Human African trypanosomiasis (HAT, sleeping sickness) and American trypanosomiasis (Chagas disease) are endemic zoonotic diseases caused by genomically related trypanosomatid protozoan parasites (Trypanosoma brucei and Trypanosoma cruzi, respectively). Just a few old drugs are available for their treatment, with most of them sharing poor safety, efficacy, and pharmacokinetic profiles. Only fexinidazole has been recently incorporated into the arsenal for the treatment of HAT. In this work, new multifunctional Ru(II) ferrocenyl compounds were rationally designed as potential agents against these pathogens by including in a single molecule 1,1'-bis(diphenylphosphino)ferrocene (dppf) and two bioactive bidentate ligands: pyridine-2-thiolato-1-oxide ligand (mpo) and polypyridyl ligands (NN). Three [Ru(mpo)(dppf)(NN)](PF6) compounds and their derivatives with chloride as a counterion were synthesized and fully characterized in solid state and solution. They showed in vitro activity on bloodstream T. brucei (EC50 = 31-160 nM) and on T. cruzi trypomastigotes (EC50 = 190-410 nM). Compounds showed the lowest EC50 values on T. brucei when compared to the whole set of metal-based compounds previously developed by us. In addition, several of the Ru compounds showed good selectivity toward the parasites, particularly against the highly proliferative bloodstream form of T. brucei. Interaction with DNA and generation of reactive oxygen species (ROS) were ruled out as potential targets and modes of action of the Ru compounds. Biochemical assays and in silico analysis led to the insight that they are able to inhibit the NADH-dependent fumarate reductase from T. cruzi. One representative hit induced a mild oxidation of low molecular weight thiols in T. brucei. The compounds were stable for at least 72 h in two different media and more lipophilic than both bioactive ligands, mpo and NN. An initial assessment of the therapeutic efficacy of one of the most potent and selective candidates, [Ru(mpo)(dppf)(bipy)]Cl, was performed using a murine infection model of acute African trypanosomiasis. This hit compound lacks acute toxicity when applied to animals in the dose/regimen described, but was unable to control parasite proliferation in vivo, probably because of its rapid clearance or low biodistribution in the extracellular fluids. Future studies should investigate the pharmacokinetics of this compound in vivo and involve further research to gain deeper insight into the mechanism of action of the compounds.
Assuntos
Compostos Ferrosos , Rutênio , Tripanossomicidas , Trypanosoma cruzi , Compostos Ferrosos/química , Compostos Ferrosos/farmacologia , Compostos Ferrosos/síntese química , Trypanosoma cruzi/efeitos dos fármacos , Ligantes , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Tripanossomicidas/síntese química , Animais , Rutênio/química , Rutênio/farmacologia , Camundongos , Metalocenos/química , Metalocenos/farmacologia , Metalocenos/síntese química , Trypanosoma brucei brucei/efeitos dos fármacos , Testes de Sensibilidade Parasitária , Estrutura Molecular , Compostos Organometálicos/farmacologia , Compostos Organometálicos/química , Compostos Organometálicos/síntese química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/síntese químicaRESUMO
Current treatment for Chagas' disease is based on two drugs, Nifurtimox and Benznidazol, which have limitations that reduce the effectiveness and continuity of treatment. Thus, there is an urgent need to develop new, safe and effective drugs. In previous work, two new metal-based compounds with trypanocidal activity, Pd-dppf-mpo and Pt-dppf-mpo, were fully characterized. To unravel the mechanism of action of these two analogous metal-based drugs, high-throughput omics studies were performed. A multimodal mechanism of action was postulated with several candidates as molecular targets. In this work, we validated the ergosterol biosynthesis pathway as a target for these compounds through the determination of sterol levels by HPLC in treated parasites. To understand the molecular level at which these compounds participate, two enzymes that met eligibility criteria at different levels were selected for further studies: phosphomevalonate kinase (PMK) and lanosterol 14-α demethylase (CYP51). Molecular docking processes were carried out to search for potential sites of interaction for both enzymes. To validate these candidates, a gain-of-function strategy was used through the generation of overexpressing PMK and CYP51 parasites. Results here presented confirm that the mechanism of action of Pd-dppf-mpo and Pt-dppf-mpo compounds involves the inhibition of both enzymes.
RESUMO
Trypanosoma cruzi is a digenetic unicellular parasite that alternates between a blood-sucking insect and a mammalian, host causing Chagas disease or American trypanosomiasis. In the insect gut, the parasite differentiates from the non-replicative trypomastigote forms that arrive upon blood ingestion to the non-infective replicative epimastigote forms. Epimastigotes develop into infective non-replicative metacyclic trypomastigotes in the rectum and are delivered via the feces. In addition to these parasite stages, transitional forms have been reported. The insect-feeding behavior, characterized by few meals of large blood amounts followed by long periods of starvation, impacts the parasite population density and differentiation, increasing the transitional forms while diminishing both epimastigotes and metacyclic trypomastigotes. To understand the molecular changes caused by nutritional restrictions in the insect host, mid-exponentially growing axenic epimastigotes were cultured for more than 30 days without nutrient supplementation (prolonged starvation). We found that the parasite population in the stationary phase maintains a long period characterized by a total RNA content three times smaller than that of exponentially growing epimastigotes and a distinctive transcriptomic profile. Among the transcriptomic changes induced by nutrient restriction, we found differentially expressed genes related to managing protein quality or content, the reported switch from glucose to amino acid consumption, redox challenge, and surface proteins. The contractile vacuole and reservosomes appeared as cellular components enriched when ontology term overrepresentation analysis was carried out, highlighting the roles of these organelles in starving conditions possibly related to their functions in regulating cell volume and osmoregulation as well as metabolic homeostasis. Consistent with the quiescent status derived from nutrient restriction, genes related to DNA metabolism are regulated during the stationary phase. In addition, we observed differentially expressed genes related to the unique parasite mitochondria. Finally, our study identifies gene expression changes that characterize transitional parasite forms enriched by nutrient restriction. The analysis of the here-disclosed regulated genes and metabolic pathways aims to contribute to the understanding of the molecular changes that this unicellular parasite undergoes in the insect vector.
Assuntos
Adaptação Fisiológica , Doença de Chagas , Insetos , Estágios do Ciclo de Vida , Inanição , Trypanosoma cruzi , Animais , Diferenciação Celular , Doença de Chagas/genética , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Insetos/metabolismo , Insetos/parasitologia , Insetos/fisiologia , Mamíferos/parasitologia , Transcriptoma/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/isolamento & purificação , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/fisiologia , Inanição/genética , Inanição/parasitologia , Inanição/fisiopatologia , Adaptação Fisiológica/genética , Adaptação Fisiológica/fisiologia , Estágios do Ciclo de Vida/genética , Estágios do Ciclo de Vida/fisiologiaRESUMO
Chagas' disease (American Trypanosomiasis) is an ancient and endemic illness in Latin America caused by the protozoan parasite Trypanosoma cruzi. Although there is an urgent need for more efficient and less toxic chemotherapeutics, no new drugs to treat this disease have entered the clinic in the last decades. Searching for metal-based prospective antichagasic drugs, in this work, multifunctional Re(I) tricarbonyl compounds bearing two different bioactive ligands were designed: a polypyridyl NN derivative of 1,10-phenanthroline and a monodentate azole (Clotrimazole CTZ or Ketoconazol KTZ). Five fac-[Re(CO)3(NN)(CTZ)](PF6) compounds and a fac-[Re(CO)3(NN)(KTZ)](PF6) were synthesized and fully characterized. They showed activity against epimastigotes (IC50 3.48-9.42 µM) and trypomastigotes of T. cruzi (IC50 0.61-2.79 µM) and moderate to good selectivity towards the parasite compared to the VERO mammalian cell model. In order to unravel the mechanism of action of our compounds, two potential targets were experimentally and theoretically studied, namely DNA and one of the enzymes involved in the parasite ergosterol biosynthetic pathway, CYP51 (lanosterol 14-α-demethylase). As hypothesized, the multifunctional compounds shared in vitro a similar mode of action as that disclosed for the single bioactive moieties included in the new chemical entities. Additionally, two relevant physicochemical properties of biological interest in prospective drug development, namely lipophilicity and stability in solution in different media, were determined. The whole set of results demonstrates the potentiality of these Re(I) tricarbonyls as promising candidates for further antitrypanosomal drug development.
Assuntos
Antiprotozoários , Doença de Chagas , Compostos Organometálicos , Trypanosoma cruzi , Humanos , Doença de Chagas/tratamento farmacológico , Compostos Organometálicos/química , Antiprotozoários/química , Cetoconazol/químicaRESUMO
Human African trypanosomiasis (sleeping sickness) and leishmaniasis are prevalent zoonotic diseases caused by genomically related trypanosomatid protozoan parasites (Trypanosoma brucei and Leishmania spp). Additionally, both are co-endemic in certain regions of the world. Only a small number of old drugs exist for their treatment, with most of them sharing poor safety, efficacy, and pharmacokinetic profiles. In this work, new multifunctional Ru(II) ferrocenyl compounds were rationally designed as potential agents against these trypanosomatid parasites by including in a single molecule 1,1'-bis(diphenylphosphino)ferrocene (dppf) and two bioactive bidentate ligands: 8-hydroxyquinoline derivatives (8HQs) and polypyridyl ligands (NN). Three [Ru(8HQs)(dppf)(NN)](PF6) compounds were synthesized and fully characterized. They showed in vitro activity on bloodstream Trypanosoma brucei (IC50 140-310 nM) and on Leishmania infantum promastigotes (IC50 3.0-4.8 µM). The compounds showed good selectivity towards T. brucei in respect to J774 murine macrophages as mammalian cell model (SI 15-38). Changing hexafluorophosphate counterion by chloride led to a three-fold increase in activity on both parasites and to a two to three-fold increase in selectivity towards the pathogens. The compounds affect in vitro at least the targets of the individual bioactive moieties included in the new chemical entities: DNA and generation of ROS. The compounds are stable in solution and are more lipophilic than the free bioactive ligands. No clear correlation between lipophilicity, interaction with DNA or generation of ROS and activity was detected, which agrees with their overall similar anti-trypanosoma potency and selectivity. These compounds are promising candidates for further drug development.
Assuntos
Leishmania infantum , Compostos Organometálicos , Trypanosoma brucei brucei , Trypanosoma cruzi , Humanos , Camundongos , Animais , Espécies Reativas de Oxigênio , Linhagem Celular , Compostos Organometálicos/química , Ligantes , DNA , MamíferosRESUMO
Five newly synthetized fac-Re(I) tricarbonyl compounds were explored as prospective antitrypanosomal agents. The biological activity of the whole series was evaluated preliminarily against the epimastigote form of Trypanosoma cruzi. All compounds showed activity against epimastigotes with IC50 values in the low micromolar range. The most active compound [fac-Re(I)(CO)3(tmp)(CTZ)](PF6), with CTZ = clotrimazole and tmp = 3,4,7,8-tetramethyl-1,10-phenantroline, showed good selectivity towards the parasites and thus was selected to carry out further metallomic studies. For this task, a newly bioanalytical method based on microwave plasma atomic emission spectrometry (MP-AES) was developed and validated. The accuracy of the method was ensured by testing a certified reference material. Results of rhenium elemental analysis by MP-AES agreed with the proposed formula of the studied compounds, contributing to the overall validation of the method, which was then applied to evaluate the percentage of rhenium uptaken by the parasites and the association of the compounds with parasite biomacromolecules. Metallomics results showed low total rhenium percentage uptaken by parasites (â¼1.2%) and preferential accumulation in the soluble proteins fraction (â¼82.8%). Thus, the method based on MP-AES turned out to be an economical and green alternative for metallomics studies involving potential rhenium metallodrugs. Moreover, a comparison against rhenium determination by electrothermal atomic absorption spectrometry (ET-AAS) was included.
Assuntos
Rênio , Micro-Ondas , Plasma , Estudos Prospectivos , Rênio/química , Espectrofotometria Atômica/métodosRESUMO
Chagas disease is caused by the protozoan Trypanosoma cruzi (T. cruzi). It remains the major parasitic disease in Latin America and is spreading worldwide, affecting over 10 million people. Hundreds of new compounds with trypanosomicidal action have been identified from different sources such as synthetic or natural molecules, but they have been deficient in several stages of drug development (toxicology, scaling-up, and pharmacokinetics). Previously, we described a series of compounds with simple structures, low cost, and environmentally friendly production with potent trypanosomicidal activity in vitro and in vivo. These molecules are from three different families: thiazolidenehydrazines, diarylideneketones, and steroids. From this collection, we explored their capacity to inhibit the triosephosphate isomerase and cruzipain of T. cruzi. Then, the mechanism of action was explored using NMR metabolomics and computational molecular dynamics. Moreover, the mechanism of death was studied by flow cytometry. Consequently, five compounds, 314, 793, 1018, 1019, and 1260, were pre-clinically studied and their pharmacologic profiles indicated low unspecific toxicity. Interestingly, synergetic effects of diarylideneketones 793 plus 1018 and 793 plus 1019 were evidenced in vitro and in vivo. In vivo, the combination of compounds 793 plus 1018 induced a reduction of more than 90% of the peak of parasitemia in the acute murine model of Chagas disease.
RESUMO
Five heteroleptic compounds, [VVO(IN-2H)(L-H)], where L are 8-hydroxyquinoline derivatives and IN is a Schiff base ligand, were synthesized and characterized in both the solid and solution state. The compounds were evaluated on epimastigotes and trypomastigotes of Trypanosoma cruzi as well as on VERO cells, as a mammalian cell model. Compounds showed activity against trypomastigotes with IC50 values of 0.29-3.02 µM. IN ligand and the new [VVO2(IN-H)] complex showed negligible activity. The most active compound [VVO(IN-2H)(L2-H)], with L2 = 5-chloro-7-iodo-8-hydroxyquinoline, showed good selectivity towards the parasite and was selected to carry out further biological studies. Stability studies suggested a partial decomposition in solution. [VVO(IN-2H)(L2-H)] affects the infection potential of cell-derived trypomastigotes. Low total vanadium uptake by parasites and preferential accumulation in the soluble proteins fraction were determined. A trypanocide effect was observed when incubating epimastigotes with 10 × IC50 values of [VVO(IN-2H)(L2-H)] and the generation of ROS after treatments was suggested. Fluorescence competition measurements with DNA:ethidium bromide adduct showed a moderate DNA interaction of the complexes. In vivo toxicity study on C. elegans model showed no toxicity up to a 100 µM concentration of [VVO(IN-2H)(L2-H)]. This compound could be considered a prospective anti-T. cruzi agent that deserves further research.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Oxiquinolina/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Vanádio/farmacologia , Animais , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Estrutura Molecular , Oxiquinolina/química , Testes de Sensibilidade Parasitária , Tripanossomicidas/síntese química , Tripanossomicidas/química , Vanádio/químicaRESUMO
Treatment for Chagas disease, a parasitosis caused by Trypanosoma cruzi, has always been based on two drugs, nifurtimox and benznidazole, despite the toxic side effects described after prolonged prescription. In this work, we study a new prospective antitrypanosomal drug based on vanadium, here named VIVO(5Brsal)(aminophen). We found a good IC50 value, (3.76 ± 0.08) µM, on CL Brener epimastigotes. The analysis of cell death mechanism allowed us to rule out the implication of a mechanism based on early apoptosis or necrosis. Recovery assays revealed a trypanostatic effect, accompanied by cell shape and motility alterations. An uptake mostly associated with the insoluble fraction of the parasites was deduced through vanadium determinations. Concordantly, no drastic changes of the parasite transcriptome were detected after 6 h of treatment. Instead, proteomic analysis uncovered the modulation of proteins involved in different processes such as energy and redox metabolism, transport systems, detoxifying pathways, ribosomal protein synthesis, and proteasome protein degradation. Overall, the results here presented lead us to propose that VIVO(5Brsal)(aminophen) exerts a trypanostatic effect on T. cruzi affecting parasite insoluble proteins.
RESUMO
OBJECTIVES: The propose was to compare this new implant macrogeometry with a control implant with a conventional macrogeometry. MATERIALS AND METHODS: Eighty-six conical implants were divided in two groups (n = 43 per group): group control (group CON) that were used conical implants with a conventional macrogeometry and, group test (group TEST) that were used implants with the new macrogeometry. The new implant macrogeometry show several circular healing cambers between the threads, distributed in the implant body. Three implants of each group were used to scanning electronic microscopy (SEM) analysis and, other eighty samples (n = 40 per group) were inserted the tibia of ten rabbit (n = 2 per tibia), determined by randomization. The animals were sacrificed (n = 5 per time) at 3-weeks (Time 1) and at 4-weeks after the implantations (Time 2). The biomechanical evaluation proposed was the measurement of the implant stability quotient (ISQ) and the removal torque values (RTv). The microscopical analysis was a histomorphometric measurement of the bone to implant contact (%BIC) and the SEM evaluation of the bone adhered on the removed implants. RESULTS: The results showed that the implants of the group TEST produced a significant enhancement in the osseointegration in comparison with the group CON. The ISQ and RTv tests showed superior values for the group TEST in the both measured times (3- and 4-weeks), with significant differences (p < 0.05). More residual bone in quantity and quality was observed in the samples of the group TEST on the surface of the removed implants. Moreover, the %BIC demonstrated an important increasing for the group TEST in both times, with statistical differences (in Time 1 p = 0.0103 and in Time 2 p < 0.0003). CONCLUSIONS: Then, we can conclude that the alterations in the implant macrogeometry promote several benefits on the osseointegration process.