RESUMO
BACKGROUND: Despite treatment with effective antimalarial drugs, the mortality rate is still high in severe cases of the disease, highlighting the need to find adjunct therapies that can inhibit the adhesion of Plasmodium falciparum-infected erythrocytes (Pf-iEs). OBJECTIVES: In this context, we evaluated a new heparan sulfate (HS) from Nodipecten nodosus for antimalarial activity and inhibition of P. falciparum cytoadhesion and rosetting. METHODS: Parasite inhibition was measured by SYBR green using a cytometer. HS was assessed in rosetting and cytoadhesion assays under static and flow conditions using Chinese hamster ovary (CHO) and human lymphatic endothelial cell (HLEC) cells expressing intercellular adhesion molecule-1 (ICAM1) and chondroitin sulfate A (CSA), respectively. FINDINGS: This HS inhibited merozoite invasion similar to heparin. Moreover, mollusk HS decreased cytoadherence of P. falciparum to CSA and ICAM-1 on the surface of endothelial cells under static and flow conditions. In addition, this glycan efficiently disrupted rosettes. CONCLUSIONS: These findings support a potential use for mollusk HS as adjunct therapy for severe malaria.
Assuntos
Heparitina Sulfato/farmacologia , Merozoítos/efeitos dos fármacos , Moluscos/química , Plasmodium falciparum/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Proteínas de Protozoários/efeitos dos fármacos , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
American Trypanosomiasis, a parasitic infection commonly named Chagas disease, affects millions of people all over Latin American countries. Presently, the World Health Organization (WHO) predicts that the number of international infected individuals extends to 7 to 8 million, assuming that more than 10,000 deaths occur annually. The transmission of the etiologic agent, Trypanosoma cruzi, through people migrating to non-endemic world nations makes it an emergent disease. The best promising targets for trypanocidal drugs may be classified into three main groups: Group I includes the main molecular targets that are considered among specific enzymes involved in the essential processes for parasite survival, principally Cruzipain, the major antigenic parasite cysteine proteinase. Group II involves biological pathways and their key specific enzymes, such as Sterol biosynthesis pathway, among others, specific antioxidant defense mechanisms, and bioenergetics ones. Group III includes the atypical organelles /structures present in the parasite relevant clinical forms, which are absent or considerably different from those present in mammals and biological processes related to them. These can be considered potential targets to develop drugs with extra effectiveness and fewer secondary effects than the currently used therapeutics. An improved distinction between the host and the parasite targets will help fight against this neglected disease.
Assuntos
Doença de Chagas/tratamento farmacológico , Tripanossomicidas/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Doença de Chagas/metabolismo , Humanos , Terapia de Alvo Molecular/métodos , Proteínas de Protozoários/efeitos dos fármacos , Tripanossomicidas/classificação , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismoRESUMO
Chagas disease, caused by the kinetoplastid protozoan parasite Trypanosoma cruzi, remains a relevant cause of illness and premature death and it is estimated that 6 million to 7 million people are infected worldwide. Although chemotherapy options are limited presenting serious problems, such as low efficacy and high toxicity. T. cruzi is susceptible to thiazoles, making this class of compounds appealing for drug development. Previously, thiazoles resulted in an increase in anti-T. cruzi activity in comparison to thiosemicarbazones. Here, we report the structural planning, synthesis and anti-T. cruzi evaluation of new thiazoles derivatives (3a-m and 4a-m), designed from molecular hybridization associated with non-classical bioisosterism. By varying substituents attached to the phenyl and thiazole rings, substituents were observed to retain, enhance or greatly increase their anti-T. cruzi activity, in comparison to the corresponding thiosemicarbazones. In most cases, electron-withdrawing substituents, such as bromine, 3,4-dichloro and nitro groups, greatly increased antiparasitic activity. Specifically, new thiazoles were identified that inhibit the epimastigote proliferation and were toxic for trypomastigotes without affecting macrophages viability. These compounds were also evaluated against cruzain. However, inhibition of this enzyme was not observed, suggesting that the compounds work through another mechanism. In addition, examination of T. cruzi cell death showed that these molecules induce apoptosis. In conclusion, except for compounds 3h and 3k, all thiazoles derivatives evaluated exhibited higher cytotoxic activity against the trypomastigote forms than the reference medicament benznidazole, without affecting macrophages viability. Compounds 4d and 4k were highlights, CC50 = 1.2 e 1.6 µM, respectively. Mechanistically, these compounds do not inhibit the cruzain, but induce T. cruzi cell death by an apoptotic process, being considered a good starting point for the development of new anti-Chagas drug candidates.
Assuntos
Apoptose/efeitos dos fármacos , Tiazóis/farmacocinética , Tripanossomicidas/química , Trypanosoma cruzi/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Cisteína Endopeptidases/efeitos dos fármacos , Desenho de Fármacos , Testes de Sensibilidade Parasitária , Proteínas de Protozoários/efeitos dos fármacos , Relação Estrutura-Atividade , Tiazóis/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/citologiaRESUMO
The trypanosomatid protozoa Leishmania is endemic in ~100 countries, with infections causing ~2 million new cases of leishmaniasis annually. Disease symptoms can include severe skin and mucosal ulcers, fever, anemia, splenomegaly, and death. Unfortunately, therapeutics approved to treat leishmaniasis are associated with potentially severe side effects, including death. Furthermore, drug-resistant Leishmania parasites have developed in most endemic countries. To address an urgent need for new, safe and inexpensive anti-leishmanial drugs, we utilized the IBM World Community Grid to complete computer-based drug discovery screens (Drug Search for Leishmaniasis) using unique leishmanial proteins and a database of 600,000 drug-like small molecules. Protein structures from different Leishmania species were selected for molecular dynamics (MD) simulations, and a series of conformational "snapshots" were chosen from each MD trajectory to simulate the protein's flexibility. A Relaxed Complex Scheme methodology was used to screen ~2000 MD conformations against the small molecule database, producing >1 billion protein-ligand structures. For each protein target, a binding spectrum was calculated to identify compounds predicted to bind with highest average affinity to all protein conformations. Significantly, four different Leishmania protein targets were predicted to strongly bind small molecules, with the strongest binding interactions predicted to occur for dihydroorotate dehydrogenase (LmDHODH; PDB:3MJY). A number of predicted tight-binding LmDHODH inhibitors were tested in vitro and potent selective inhibitors of Leishmania panamensis were identified. These promising small molecules are suitable for further development using iterative structure-based optimization and in vitro/in vivo validation assays.
Assuntos
Antiprotozoários/química , Leishmaniose/tratamento farmacológico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Proteínas de Protozoários/química , Bibliotecas de Moléculas Pequenas/química , Antiprotozoários/uso terapêutico , Di-Hidro-Orotato Desidrogenase , Humanos , Leishmania/química , Leishmania/efeitos dos fármacos , Leishmaniose/parasitologia , Ligantes , Simulação de Dinâmica Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas de Protozoários/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/uso terapêutico , Interface Usuário-ComputadorRESUMO
The in vitro leishmanicidal activity and cytotoxicity of pyrazole-containing macrocyclic polyamines 1-4 was assayed on Leishmania infantum and Leishmania braziliensis species. Compounds 1-4 were more active and less toxic than glucantime and both infection rates and ultrastructural alterations confirmed that 1 and 2 were highly leishmanicidal and induced extensive parasite cell damage. Modifications in the excretion products of parasites treated with 1-3 were also consistent with substantial cytoplasm alterations. Compound 2 was highlighted as a potent inhibitor of Fe-SOD in both species, whereas its effect on human CuZn-SOD was poor. Molecular modelling suggested that 2 could deactivate Fe-SOD due to a sterically favoured enhanced ability to interact with the H-bonding net that supports the enzyme`s antioxidant features.
Assuntos
Antiprotozoários/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Pirazóis/farmacologia , Superóxido Dismutase/efeitos dos fármacos , Animais , Antiprotozoários/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Leishmania braziliensis/enzimologia , Leishmania braziliensis/ultraestrutura , Leishmania infantum/enzimologia , Leishmania infantum/ultraestrutura , Leishmaniose/parasitologia , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Poliaminas/química , Poliaminas/farmacologia , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Pirazóis/química , Superóxido Dismutase/metabolismoRESUMO
Several cysteine proteinases (CPs) participate in the virulence of Trichomonas vaginalis. One of them is a 65kDa CP, CP65, involved in cytotoxicity. The aim of this work was to investigate the effect of iron on the trichomonal CP65-dependent cytotoxicity using parasites grown under distinct iron concentrations. Cytotoxicity and cell-binding assays, and zymograms were performed. At the highest iron concentration (250 microM), parasites exhibited the lowest levels of cytotoxicity and less CP65 proteolytic activity. Other cations in the culture medium did not affect the trichomonal CP65-dependent cytotoxicity as iron did. Another four trichomonad fresh isolates presented similar iron negative effect over cytotoxicity. Western blot and RT-PCR experiments also showed reduction in the amount of protein and transcript of CP65 in trichomonads grown under iron-rich conditions, as compared with parasites grown in normal and iron-depleted media. Indirect immunofluorescence using the anti-CP65 antibody showed that parasites grown in iron-rich medium expressed less CP65 than those grown in normal and iron-depleted media. Cytotoxicity inhibition experiments with the anti-CP65 antibody confirmed the iron negative effect over the CP65-dependent cytotoxicity. In conclusion, our data show that iron specifically down-regulates proteolytic activity, expression, and transcription of CP65, negatively affecting trichomonal cytotoxicity in vitro.
Assuntos
Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/toxicidade , Regulação para Baixo , Ferro/farmacologia , Trichomonas vaginalis/patogenicidade , Animais , Meios de Cultura , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Feminino , Regulação da Expressão Gênica , Células HeLa , Humanos , Ferro/metabolismo , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/toxicidade , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/crescimento & desenvolvimentoRESUMO
Cyclophilins (CyPs) are enzymes involved in protein folding, catalyzing the isomerisation of peptidyl prolyl bonds in proteins and peptides between the cis- and trans-conformations. They are also the major cellular target for the immunosuppressive drug Cyclosporin A (CsA). In Trypanosoma cruzi, the most abundantly expressed CyP is an isoform of 19 kDa, TcCyP19, in which the enzymatic activity is inhibited by CsA. Among a reported set of CsA analogues, two non-immunosuppressive compounds, H-7-94 and F-7-62, proved to be the best inhibitors of TcCyP19 enzymatic activity as well as the most efficient trypanocidal drugs. With the objective of analysing, at the molecular level, how the structural differences between the three above-mentioned inhibitors justify their different inhibitory activity on TcCyP19, three-dimensional molecular modelling structures were generated to computationally simulate behaviours and interactions. An energy-minimized model of each binary complex in water with ions was obtained. These models were then used as starting point for molecular dynamic simulations, performed with GROMOS96 program. With the resulting set of co-ordinates and energies, a comparison of the interaction between CsA and both CsA analogues in T. cruzi and human cyclophilins were performed. Within the different magnitudes analysed, the total potential complex energy exhibited the best correlation with the experimental data. The results obtained in this study support the use of this methodology when designing new lead inhibitor compounds.
Assuntos
Ciclofilinas/química , Ciclofilinas/efeitos dos fármacos , Ciclosporina/química , Ciclosporina/farmacologia , Proteínas de Protozoários/química , Proteínas de Protozoários/efeitos dos fármacos , Trypanosoma cruzi/química , Trypanosoma cruzi/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Gráficos por Computador , Simulação por Computador , Ciclofilinas/genética , Ligação de Hidrogênio , Técnicas In Vitro , Ligantes , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Homologia de Sequência de Aminoácidos , Software , Termodinâmica , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/genéticaRESUMO
Malaria is undoubtedly the world's most devastating parasitic disease, affecting 300 to 500 million people every year. Some cases of Plasmodium falciparum infection progress to the deadly forms of the disease responsible for 1 to 3 million deaths annually. P. falciparum-infected erythrocytes adhere to host receptors in the deep microvasculature of several organs. The cytoadhesion of infected erythrocytes to placental syncytiotrophoblast receptors leads to pregnancy-associated malaria (PAM). This specific maternal-fetal syndrome causes maternal anemia, low birth weight and the death of 62,000 to 363,000 infants per year in sub-Saharan Africa, and thus has a poor outcome for both mother and fetus. However, PAM and non-PAM parasites have been shown to differ antigenically and genetically. After multiple pregnancies, women from different geographical areas develop adhesion-blocking antibodies that protect against placental parasitemia and clinical symptoms of PAM. The recent description of a new parasite ligand encoded by the var2CSA gene as the only gene up-regulated in PAM parasites renders the development of an anti-PAM vaccine more feasible. The search for a vaccine to prevent P. falciparum sequestration in the placenta by eliciting adhesion-blocking antibodies and a cellular immune response, and the development of new methods for evaluating such antibodies should be key priorities in mother-child health programs in areas of endemic malaria. This review summarizes the main molecular, immunological and physiopathological aspects of PAM, including findings related to new targets in the P. falciparum var gene family. Finally, we focus on a new methodology for mimicking cytoadhesion under blood flow conditions in human placental tissue.
Assuntos
Eritrócitos/parasitologia , Malária Falciparum/imunologia , Placenta/parasitologia , Plasmodium falciparum/imunologia , Complicações Parasitárias na Gravidez/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/sangue , Antígenos de Protozoários/efeitos dos fármacos , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Adesão Celular/fisiologia , Eritrócitos/imunologia , Feminino , Humanos , Vacinas Antimaláricas , Malária Falciparum/sangue , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologia , Gravidez , Complicações Parasitárias na Gravidez/sangue , Proteínas de Protozoários/sangue , Proteínas de Protozoários/efeitos dos fármacosRESUMO
Malaria is undoubtedly the world's most devastating parasitic disease, affecting 300 to 500 million people every year. Some cases of Plasmodium falciparum infection progress to the deadly forms of the disease responsible for 1 to 3 million deaths annually. P. falciparum-infected erythrocytes adhere to host receptors in the deep microvasculature of several organs. The cytoadhesion of infected erythrocytes to placental syncytiotrophoblast receptors leads to pregnancy-associated malaria (PAM). This specific maternal-fetal syndrome causes maternal anemia, low birth weight and the death of 62,000 to 363,000 infants per year in sub-Saharan Africa, and thus has a poor outcome for both mother and fetus. However, PAM and non-PAM parasites have been shown to differ antigenically and genetically. After multiple pregnancies, women from different geographical areas develop adhesion-blocking antibodies that protect against placental parasitemia and clinical symptoms of PAM. The recent description of a new parasite ligand encoded by the var2CSA gene as the only gene up-regulated in PAM parasites renders the development of an anti-PAM vaccine more feasible. The search for a vaccine to prevent P. falciparum sequestration in the placenta by eliciting adhesion-blocking antibodies and a cellular immune response, and the development of new methods for evaluating such antibodies should be key priorities in mother-child health programs in areas of endemic malaria. This review summarizes the main molecular, immunological and physiopathological aspects of PAM, including findings related to new targets in the P. falciparum var gene family. Finally, we focus on a new methodology for mimicking cytoadhesion under blood flow conditions in human placental tissue.
Assuntos
Humanos , Animais , Feminino , Gravidez , Eritrócitos/parasitologia , Malária Falciparum/imunologia , Placenta/parasitologia , Plasmodium falciparum/imunologia , Complicações Parasitárias na Gravidez/imunologia , Proteínas de Protozoários/imunologia , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/sangue , Antígenos de Protozoários/efeitos dos fármacos , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Adesão Celular/fisiologia , Eritrócitos/imunologia , Vacinas Antimaláricas , Malária Falciparum/sangue , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologia , Complicações Parasitárias na Gravidez/sangue , Proteínas de Protozoários/sangue , Proteínas de Protozoários/efeitos dos fármacosRESUMO
Proteins rich in sulfhydryl groups, such as metallothionein, are present in several strains of the parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Metallothionein-like protein concentrations ranged from 5.1 to 13.2 pmol/mg protein depending on the parasite strain and growth phase. Nifurtimox and benznidazole, used in the treatment of Chagas' disease, decreased metallothionein activity by approximately 70%. T. cruzi metallothionein was induced by ZnCl2. Metallothionein from T. cruzi was partially purified and its monobromobimane derivative showed a molecular weight of approximately 10,000 Da by SDS-PAGE analysis. The concentration of trypanothione, the major glutathione conjugate in T. cruzi, ranged from 3.8 to 10.8 nmol/mg protein, depending on the culture phase. The addition of buthionine sulfoximine to the protozoal culture considerably reduced the concentration of trypanothione and had no effect upon the metallothionein concentration. The possible contribution of metallothionein-like proteins to drug resistance in T. cruzi is discussed.
Assuntos
Butionina Sulfoximina/farmacologia , Glutationa/análogos & derivados , Nifurtimox/farmacologia , Nitroimidazóis/farmacologia , Proteínas de Protozoários/efeitos dos fármacos , Espermidina/análogos & derivados , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Eletroforese em Gel de Poliacrilamida , Glutationa/biossíntese , Glutationa/efeitos dos fármacos , Metalotioneína/biossíntese , Metalotioneína/efeitos dos fármacos , Proteínas de Protozoários/biossíntese , Espermidina/biossíntese , Fatores de Tempo , Trypanosoma cruzi/metabolismoRESUMO
The aim of this work was to investigate the role played by iron during interaction of Tritrichomonas foetus with cultured epithelial cells. We have observed that the growth rate of T. foetus is influenced by the amount of iron available into culture medium. When organisms maintained for 24h in iron-depleted medium were transferred to an iron-rich one, many protozoan cells exhibited a cytokinesis blockage. Parasites maintained in iron-depleted medium exhibited a significant increase in cytoadhesion when compared with both controls and parasites that had been cultured in medium in which iron was replaced. T. foetus collected from iron-depleted medium also exhibited a reduction in its ability to destroy epithelial cell monolayers and a reduction in the activity of several cysteine proteases. Taken together, the results presented here demonstrate that iron may be an extracellular signal, which seems to modulate the ability of T. foetus to interact with host epithelial cells.
Assuntos
Células Epiteliais/parasitologia , Ferro/fisiologia , Tritrichomonas foetus/crescimento & desenvolvimento , 2,2'-Dipiridil/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Meios de Cultura , Relação Dose-Resposta a Droga , Endopeptidases/biossíntese , Endopeptidases/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Compostos Ferrosos/farmacologia , Células HeLa , Humanos , Indicadores e Reagentes/farmacologia , Ferro/farmacologia , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/efeitos dos fármacos , Tritrichomonas foetus/citologia , Tritrichomonas foetus/efeitos dos fármacos , Tritrichomonas foetus/metabolismoRESUMO
Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse-chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin-cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-beta-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Lectinas/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Protozoários/metabolismo , Ribonucleoproteínas/metabolismo , Trypanosoma cruzi/química , 1-Desoxinojirimicina/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Calreticulina , Sequência de Carboidratos , Clonagem Molecular , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Glicoproteínas/química , Glicoproteínas/metabolismo , Inibidores de Glicosídeo Hidrolases , Glicosilação , Hexosaminidases/farmacologia , Soros Imunes , Lectinas/efeitos dos fármacos , Lectinas/genética , Dados de Sequência Molecular , Testes de Precipitina , Dobramento de Proteína , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/imunologia , Frações Subcelulares , alfa-GlucosidasesRESUMO
Sinefungin and its cyclic analog were evaluated in vitro for activity against the multiplication of Trypanosoma cruzi. When either drug was tested for eight days on twelve T. cruzi epimastigote isolates, an 800-fold difference in drug sensitivity was found. Both drugs were trypanostatics at a concentration range from 0.1 micrograms/ml to 300 micrograms/ml. The 50% effective concentration (EC50) of sinefungin and its cyclic analog at which the growth of a given isolate was inhibited was 0.38 micrograms/ml for sinefungin and 0.31 micrograms/ml for the cyclic analog against the Ma, Marin, OPS-86, Y, and Ya isolates, and > 300 micrograms/ml for sinefungin and 217 micrograms/ml for the cyclic analog against the A-35, Bertoldo, DS, EP, ES, OPS-58, and FL isolates. Incubation of drug-sensitive isolates for more than 10 days in glucose-saline (GS) medium, but not in minimal essential medium, in the presence of a 30-fold EC50 concentration of the drug induced an increase in the drug-resistant population, which maintained this phenotype for several passages in drug-free culture medium. Growth curves were analyzed as a function of parasite inoculum; it was observed that with sinefungin-sensitive T. cruzi epimastigote isolates grown in GS medium in the presence of 10 micrograms/ml of the drug, the inhibitory effects of the drug were dependent on the initial inoculum: 1 x 10(3)-1 x 10(4) parasites/ml were strongly inhibited even after 16 days. Significant impairment of thymidine incorporation into the DNA of parasites by both drugs was observed only in drug-sensitive epimastigote isolates.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Adenosina/análogos & derivados , Antiprotozoários/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Adenosina/farmacologia , Animais , Meios de Cultura , DNA de Protozoário/biossíntese , DNA de Protozoário/efeitos dos fármacos , Humanos , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/efeitos dos fármacos , RNA de Protozoário/biossíntese , RNA de Protozoário/efeitos dos fármacos , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Using Triton X-114, glycolipids and proteins were extracted from heart muscle cells (HMC) infected with Trypanosoma cruzi clone Dm28c and from uninfected HMC, and analysed by SDS-PAGE and high-performance thin-layer chromatography (HPTLC). Two major differences were observed: (a) two proteins with a molecular mass of 92 kDa and 69 kDa were present in the uninfected cells but absent from the infected cells and (b) a 70-90 kDa protein band was detected only in parasitized cells. These differences would seem to constitute alterations taking place during the process of cell recognition and/or parasite interiorization. No differences were observed in the respective glycolipid compositions, of control and infected cells analysed by HPTLC. A glycolipid with the same mobility as the neutral glycolipid isolated from epimastigotes of T. cruzi was detected in the uninfected cells. This finding may lend support to the previously described hypothesis that molecular mimicry is implicated in the cardioneuropathology of Chagas' disease.