RESUMO
Cardiac fibrosis is a severe outcome of Chagas disease (CD), caused by the protozoan Trypanosoma cruzi. Clinical evidence revealed a correlation between fibrosis levels with impaired cardiac performance in CD patients. Therefore, we sought to analyze the effect of inhibitors of TGF-ß (pirfenidone), p38-MAPK (losmapimod) and c-Jun (SP600125) on the modulation of collagen deposition in cardiac fibroblasts (CF) and in vivo models of T. cruzi chronic infection. Sirius Red/Fast Green dye was used to quantify both collagen expression and total protein amount, assessing cytotoxicity. The compounds were also used to treat C57/Bl6 mice chronically infected with T. cruzi, Brazil strain. We identified an anti-fibrotic effect in vitro for pirfenidone (TGF-ß inhibitor, IC50 114.3 µM), losmapimod (p38 inhibitor, IC50 17.6 µM) and SP600125 (c-Jun inhibitor, IC50 3.9 µM). This effect was independent of CF proliferation since these compounds do not affect T. cruzi-induced host cell multiplication as measured by BrdU incorporation. Assays of chronic infection of mice with T. cruzi have shown a reduction in heart collagen by pirfenidone. These results propose a novel approach to fibrosis therapy in CD, with the prospect of repurposing pirfenidone to prevent the onset of ECM accumulation in the hearts of the patients.
Assuntos
Cardiomiopatia Chagásica , Fibrose , Camundongos Endogâmicos C57BL , Piridonas , Animais , Piridonas/farmacologia , Piridonas/uso terapêutico , Cardiomiopatia Chagásica/tratamento farmacológico , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/patologia , Camundongos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Miocárdio/patologia , Miocárdio/metabolismo , Colágeno/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Humanos , Doença Crônica , Fator de Crescimento Transformador beta/metabolismo , Modelos Animais de Doenças , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Masculino , AntracenosRESUMO
Transforming growth factor beta (TGF-ß) is a determinant for inflammation and fibrosis in cardiac and skeletal muscle in Chagas disease. To determine its regulatory mechanisms, we investigated the response of Trypanosoma cruzi-infected cardiomyocytes (CM), cardiac fibroblasts (CF), and L6E9 skeletal myoblasts to TGF-ß. Cultures of CM, CF, and L6E9 were infected with T. cruzi (Y strain) and treated with TGF-ß (1-10 ng/mL, 1 h or 48 h). Fibronectin (FN) distribution was analyzed by immunofluorescence and Western blot (WB). Phosphorylated SMAD2 (PS2), phospho-p38 (p-p38), and phospho-c-Jun (p-c-Jun) signaling were evaluated by WB. CF and L6E9 showed an increase in FN from 1 ng/mL of TGF-ß, while CM displayed FN modulation only after 10 ng/mL treatment. CF and L6E9 showed higher PS2 levels than CM, while p38 was less stimulated in CF than CM and L6E9. T. cruzi infection resulted in localized FN disorganization in CF and L6E9. T. cruzi induced an increase in FN in CF cultures, mainly in uninfected cells. Infected CF cultures treated with TGF-ß showed a reduction in PS2 and an increase in p-p38 and p-c-Jun levels. Our data suggest that p38 and c-Jun pathways may be participating in the fibrosis regulatory process mediated by TGF-ß after T. cruzi infection.
Assuntos
Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Matriz Extracelular/metabolismo , Interações Hospedeiro-Patógeno , Fator de Crescimento Transformador beta/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Biomarcadores , Linhagem Celular , Células Cultivadas , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Miócitos Cardíacos/metabolismo , Transdução de SinaisRESUMO
Transforming growth factor beta (TGF-ß) cytokine is involved in Chagas disease establishment and progression. Since Trypanosoma cruzi can modulate host cell receptors, we analysed the TGF-ß receptor type II (TßRII) expression and distribution during T. cruzi - cardiomyocyte interaction. TßRII immunofluorescent staining revealed a striated organization in cardiomyocytes, which was co-localized with vinculin costameres and enhanced (38%) after TGF-ß treatment. Cytochalasin D induced a decrease of 45·3% in the ratio of cardiomyocytes presenting TßRII striations, demonstrating an association of TßRII with the cytoskeleton. Western blot analysis showed that cytochalasin D significantly inhibited Smad 2 phosphorylation and fibronectin stimulation after TGF-ß treatment in cardiomyocytes. Trypanosoma cruzi infection elicited a decrease of 79·8% in the frequency of cardiomyocytes presenting TßRII striations, but did not interfere significantly in its expression. In addition, T. cruzi-infected cardiomyocytes present a lower response to exogenous TGF-ß, showing no enhancement of TßRII striations and a reduction of phosphorylated Smad 2, with no significant difference in TßRII expression when compared to uninfected cells. Together, these results suggest that the co-localization of TßRII with costameres is important in activating the TGF-ß signalling cascade, and that T. cruzi-derived cytoskeleton disorganization could result in altered or low TGF-ß response in infected cardiomyocytes.
Assuntos
Doença de Chagas/fisiopatologia , Costâmeros/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Miócitos Cardíacos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Parasita/efeitos dos fármacos , Camundongos , Miócitos Cardíacos/parasitologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Trypanosoma cruzi/fisiologiaRESUMO
This work describes the antitrypanocidal activity of two hydroxamic acid derivatives containing o-ethoxy (HAD1) and p-ethoxy (HAD2) as substituent in the aromatic ring linked to the isoxazoline ring. HAD1 and HAD2 induced a significant reduction in the number of intracellular parasites and consequently showed activity on the multiplication of the parasite. Treatment of cardiomyocytes and macrophages with the compounds revealed no significant loss in cell viability. Ultrastructural alterations after treatment of cardiomyocytes or macrophages infected by Trypanosoma cruzi with the IC50 value of HAD1 revealed alterations to amastigotes, showing initial damage seen as swelling of the kinetoplast. This gave a good indication of the ability of the drug to permeate through the host cell membrane as well as its selectivity to the parasite target. Both compounds HAD1 and 2 were able to reduce the cysteine peptidases and decrease the activity of metallopeptidases.
Assuntos
Doença de Chagas/tratamento farmacológico , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Células Cultivadas , Doença de Chagas/microbiologia , Relação Dose-Resposta a Droga , Ácidos Hidroxâmicos/síntese química , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Estrutura Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/microbiologia , Relação Estrutura-Atividade , Tripanossomicidas/síntese químicaRESUMO
Chagas' Disease, caused by the protozoan parasite Trypanosoma cruzi, is responsible for up to 41% of the heart failures in endemic areas in South America and is an emerging infection in regions of North America, Europe, and Asia. Treatment is suboptimal due to two factors. First, the lack of an adequate biomarker to predict disease severity and response to therapy; and second, up to 120-days treatment course coupled with a significant incidence of adverse effects from the drug currently used. Because the disease can manifest itself clinically a few years to decades after infection, controversy remains concerning the suitability of current drug treatment (benznidazole), and the efficacy of alternative drugs (e.g. posaconazole). We therefore followed the clinical course, and PCR detection of parasite burden, in a mouse model of infection for a full year following treatment with benznidazole or posaconazole. Efficacy of the two drugs depended on whether the treatment was performed during the acute model or the chronic model of infection. Posaconazole was clearly superior in treatment of acute disease whereas only benznidazole had efficacy in the chronic model. These results have important implications for the design and analysis of human clinical trials, and the use of specific drugs in specific clinical settings.
Assuntos
Doença de Chagas/tratamento farmacológico , Nitroimidazóis/farmacologia , Triazóis/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Administração Oral , Animais , Doença de Chagas/parasitologia , Modelos Animais de Doenças , Seguimentos , Masculino , Camundongos Endogâmicos C57BL , Nitroimidazóis/administração & dosagem , Reação em Cadeia da Polimerase , Triazóis/administração & dosagem , Tripanossomicidas/farmacologia , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
We discovered a series of azole antifungal compounds as effective antiprotozoal agents. They displayed promising inhibitory activities within the micromolar-submicromolar range against P. falciparum, L. donovani, and T. b. rhodesiense. Moreover, most of such compounds showed excellent nanomolar IC50 against T. cruzi, showing also very low cytotoxicity. Discussion of structure-activity relationships and biological data for these compounds are provided against the different parasites. To assess the mechanism of action against T. cruzi we proved that the most potent compounds (3b, 3j-l) inhibited the T. cruzi CYP51. Moreover, the most active derivative 3j dramatically reduced parasitemia in T. cruzi mouse model without acute toxicity.
Assuntos
Antiprotozoários/química , Antiprotozoários/farmacologia , Doença de Chagas/tratamento farmacológico , Imidazóis/química , Imidazóis/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antiprotozoários/síntese química , Antiprotozoários/uso terapêutico , Linhagem Celular , Feminino , Humanos , Imidazóis/síntese química , Imidazóis/uso terapêutico , Concentração Inibidora 50 , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Ratos , Relação Estrutura-Atividade , Trypanosoma brucei rhodesiense/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológicoRESUMO
Recent advances in cell-based, high-throughput phenotypic screening have identified new chemical compounds that are active against eukaryotic pathogens. A challenge to their future development lies in identifying these compounds' molecular targets and binding modes. In particular, subsequent structure-based chemical optimization and target-based screening require a detailed understanding of the binding event. Here, we use directed evolution and whole-genome sequencing of a drug-sensitive S. cerevisiae strain to identify the yeast ortholog of TcCyp51, lanosterol-14-alpha-demethylase (TcCyp51), as the target of MMV001239, a benzamide compound with activity against Trypanosoma cruzi, the etiological agent of Chagas disease. We show that parasites treated with MMV0001239 phenocopy parasites treated with another TcCyp51 inhibitor, posaconazole, accumulating both lanosterol and eburicol. Direct drug-protein binding of MMV0001239 was confirmed through spectrophotometric binding assays and X-ray crystallography, revealing a binding site shared with other antitrypanosomal compounds that target Cyp51. These studies provide a new probe chemotype for TcCyp51 inhibition.
Assuntos
Inibidores de 14-alfa Desmetilase/uso terapêutico , Doença de Chagas/tratamento farmacológico , Evolução Molecular Direcionada , Tripanossomicidas/uso terapêutico , Inibidores de 14-alfa Desmetilase/química , Inibidores de 14-alfa Desmetilase/farmacologia , Sequência de Aminoácidos , Doença de Chagas/parasitologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Descoberta de Drogas , Cromatografia Gasosa-Espectrometria de Massas , Simulação de Acoplamento Molecular , Plasmodium falciparum/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Espectrofotometria Ultravioleta , Esterol 14-Desmetilase/efeitos dos fármacos , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologiaRESUMO
BACKGROUND: Chagas disease, caused by the protozoan Trypanosoma cruzi, is the leading cause of heart failure in Latin America. The clinical treatment of Chagas disease is limited to two 60 year-old drugs, nifurtimox and benznidazole, that have variable efficacy against different strains of the parasite and may lead to severe side effects. CYP51 is an enzyme in the sterol biosynthesis pathway that has been exploited for the development of therapeutics for fungal and parasitic infections. In a target-based drug discovery program guided by x-ray crystallography, we identified the 4-aminopyridyl-based series of CYP51 inhibitors as being efficacious versus T.cruzi in vitro; two of the most potent leads, 9 and 12, have now been evaluated for toxicity and efficacy in mice. METHODOLOGY/PRINCIPAL FINDINGS: Both acute and chronic animal models infected with wild type or transgenic T. cruzi strains were evaluated. There was no evidence of toxicity in the 28-day dosing study of uninfected animals, as judged by the monitoring of multiple serum and histological parameters. In two acute models of Chagas disease, 9 and 12 drastically reduced parasitemia, increased survival of mice, and prevented liver and heart injury. None of the compounds produced long term sterile cure. In the less severe acute model using the transgenic CL-Brenner strain of T.cruzi, parasitemia relapsed upon drug withdrawal. In the chronic model, parasitemia fell to a background level and, as evidenced by the bioluminescence detection of T. cruzi expressing the red-shifted luciferase marker, mice remained negative for 4 weeks after drug withdrawal. Two immunosuppression cycles with cyclophosphamide were required to re-activate the parasites. Although no sterile cure was achieved, the suppression of parasitemia in acutely infected mice resulted in drastically reduced inflammation in the heart. CONCLUSIONS/SIGNIFICANCE: The positive outcomes achieved in the absence of sterile cure suggest that the target product profile in anti-Chagasic drug discovery should be revised in favor of safe re-administration of the medication during the lifespan of a Chagas disease patient. A medication that reduces parasite burden may halt or slow progression of cardiomyopathy and therefore improve both life expectancy and quality of life.
Assuntos
Inibidores de 14-alfa Desmetilase/uso terapêutico , Doença de Chagas/tratamento farmacológico , Parasitemia/tratamento farmacológico , Pirimidinas/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacos , Inibidores de 14-alfa Desmetilase/efeitos adversos , Animais , Doença de Chagas/parasitologia , Modelos Animais de Doenças , Descoberta de Drogas , Feminino , Coração/efeitos dos fármacos , Chumbo/química , Chumbo/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/patologia , Parasitemia/parasitologia , Pirimidinas/efeitos adversos , Esterol 14-Desmetilase/metabolismo , Esteróis/biossíntese , Tripanossomicidas/efeitos adversosRESUMO
Chagas disease is a neglected tropical disease caused by the flagellated protozoan Trypanosoma cruzi. The current drugs used to treat this disease have limited efficacy and produce severe side effects. Quinolines, nitrogen heterocycle compounds that form complexes with heme, have a broad spectrum of antiprotozoal activity and are a promising class of new compounds for Chagas disease chemotherapy. In this study, we evaluated the activity of a series of 4-arylaminoquinoline-3-carbonitrile derivatives against all forms of Trypanosoma cruzi in vitro. Compound 1g showed promising activity against epimastigote forms when combined with hemin (IC50<1 µM), with better performance than benznidazole, the reference drug. This compound also inhibited the viability of trypomastigotes and intracellular amastigotes. The potency of 1g in combination with heme was enhanced against epimastigotes and trypomastigotes, suggesting a similar mechanism of action that occurs in Plasmodium spp. The addition of hemin to the culture medium increased trypanocidal activity of analog 1g without changing the cytotoxicity of the host cell, reaching an IC50 of 11.7 µM for trypomastigotes. The mechanism of action was demonstrated by the interaction of compound 1g with hemin in solution and prevention of heme peroxidation. Compound 1g and heme treatment induced alterations of the mitochondrion-kinetoplast complex in epimastigotes and trypomastigotes and also, accumulation of electron-dense deposits in amastigotes as visualized by transmission electron microscopy. The trypanocidal activity of 4-aminoquinolines and the elucidation of the mechanism involving interaction with heme is a neglected field of research, given the parasite's lack of heme biosynthetic pathway and the importance of this cofactor for parasite survival and growth. The results of this study can improve and guide rational drug development and combination treatment strategies.
Assuntos
Aminoquinolinas/farmacologia , Heme/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Concentração Inibidora 50 , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Trypanosoma cruzi/fisiologia , Trypanosoma cruzi/ultraestruturaRESUMO
ABSTRACT Various extracts obtained from the red alga Plocamium brasiliense (Greville Howe & Taylor), including a fraction containing crude 5-chloro-1-(E)-chlorovinyl-2,4-dibromo-1,5-dimethylcyclohexane (1) and another containing a mixture of halogenated monoterpenes (F), as well as atomaric acid meroditerpene (2) isolated from brown alga Stypopodium zonale (J. V. Lamouroux) Papenfuss, were evaluated for their activity against Trypanosoma cruzi. The cytotoxic and trypanosomicidal effects of these extracts were evaluated in Vero cells and clinically relevant forms of T. cruzi (amastigotes and trypomastigotes). All extracts from P. brasiliense presented low cytotoxicity and moderate trypanosomicidal effects, except for the hydroalcoholic extract. The crude 1 and F fractions had enhanced trypanocidal activity but showed low selectivity. Moreover, atomaric acid (2) was identified as a hit, demonstrating a potent trypanocidal effect reaching an IC50 <10 µM against two different DTU (Yand high selectivity index (<10). These results identify marine natural products as promising candidates against Chagas disease.
RESUMO
Trypanosoma cruzi invasion is mediated by receptor-ligand recognition between the surfaces of both parasite and target cell. We have previously demonstrated the role of heparan sulfate proteoglycan in the attachment and invasion of T. cruzi in cardiomyocytes. Herein, we have isolated the T. cruzi heparin-binding proteins (HBP-Tc) and investigated the nature of cardiomyocyte heparan sulfate (HS)-binding site to the parasite surface ligand. Two major heparin-binding proteins with molecular masses of 65.8 and 59 kDa were observed in total extract of amastigote and trypomastigote forms of T. cruzi. Hydrophobic [S(35)]methionine labeled proteins eluted from heparin-sepharose affinity chromatography also revealed both proteins in trypomastigotes but only the 59 kDa is strongly recognized by biotin-conjugated glycosaminoglycans. Competition assays were performed to analyze the role of sulfated proteoglycans, including heparin, keratan sulfate and both acetylated and highly sulfated domains of heparan sulfate, in the recognition and invasion process of T. cruzi. Significant inhibitions of 84% and 35% in the percentage of infection were revealed after treatment of the parasites with heparin and the N-acetylated/ N-sulfated heparan sulfate domain, respectively, suggesting the important role of the glycuronic acid and NS glucosamine domain of the HS chain in the recognition of the HBP-Tc during the T. cruzi-cardiomyocyte interaction.
Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Células Cultivadas , Cloratos/farmacologia , Chlorocebus aethiops , Cromatografia de Afinidade , Heparina/farmacologia , Camundongos , Peso Molecular , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/parasitologia , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Trypanosoma cruzi/efeitos dos fármacos , Células VeroRESUMO
Componentes de matriz extracelular têm importante papel na patologia da doença de Chagas, podendo participar do processo de invasão do Trypanosoma cruzi ou acumular-se progressivamente no tecido cardíaco, levando à fibrose que ocorre em concomitância com a miocardite chagásica. Citocinas secretadas durante a inflamação podem funcionar como mediadoras do processo fibrótico. No entanto, os mecanismos reguladores da fibrose chagásica ainda precisam ser bem esclarecidos. Ensaios de interação T. cruzi - cardiomiócito in vitro revelaram a participação da seqüência RGD de fibronectina (FN) no processo de invasão. A análise histopatológica do miocárdio de camundongos Suíços infectados pelo T. cruzi, cepa Y, revelou a presença de intensos infiltrados inflamatórios na fase aguda da infecção experimental, enquanto animais infectados com T. cruzi, clone Dm28c, não apresentaram miocardite. Um aumento nos depósitos de FN, laminina (LM) e colágeno IV detectados por imunofluorescência indireta foi revelado apenas no miocárdio de animais infectados com T. cruzi cepa Y, mas não com Dm28c. Por outro lado, a análise da interação T. cruzi-cardiomiócito in vitro demonstrou redução na expressão de FN em cardiomiócitos altamente infectados, enquanto a LM apresentou somente alterações em sua distribuição. O tratamento das culturas de cardiomiócitos com TGF-beta e TNF-alfa provocou aumento generalizado na expressão de matriz de FN, mas não alterou a expressão de LM. A análise das imagens de culturas de cardiomiócitos infectados pelo T. cruzi (72h) com o programa KS400 revelou baixa superposição de FN com células altamente infectadas, mesmo após o tratamento com citocinas. Este evento parece estar relacionado com a quebra de miofibrilas decorrente da infecção, uma vez que o tratamento de cardiomiócitos com citocalasina D, droga que despolimeriza filamentos de actina, resultou em uma desestruturação similar das fibrilas de FN independente da presença de citocinas. A adição de...