RESUMO
This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A 'proof of concept' molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.
Assuntos
Doença de Chagas/tratamento farmacológico , Inibidores de Cisteína Proteinase/uso terapêutico , Dipeptídeos/uso terapêutico , Desenho de Fármacos , Tripanossomicidas/uso terapêutico , Compostos de Vinila/uso terapêutico , Animais , Cisteína Endopeptidases , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450 , Humanos , Fenilalanina/análogos & derivados , Piperazinas , Proteínas de Protozoários/antagonistas & inibidores , Compostos de Tosil , Estados Unidos , United States Food and Drug AdministrationRESUMO
This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A "proof of concept" molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.
Assuntos
Animais , Humanos , Doença de Chagas/tratamento farmacológico , Inibidores de Cisteína Proteinase/uso terapêutico , Desenho de Fármacos , Dipeptídeos/uso terapêutico , Tripanossomicidas/uso terapêutico , Compostos de Vinila/uso terapêutico , Cisteína Endopeptidases , /antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Estados Unidos , United States Food and Drug AdministrationRESUMO
Several Cdc2p-related protein kinases (CRKs) have been described in trypanosomatids but their role in the control of the cell cycle nor their biological functions have been addressed. In Trypanosoma cruzi two CRKs have been identified, TzCRK1 and TzCRK3. In this work we further characterize T. cruzi CRK1 and report the identification of three novel associating cyclins. We demonstrate that CRK1 levels and localization do not vary during the cell cycle, and show that it is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplast), suggesting a putative control function in this organelle. Using purified anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cruzi epimastigote forms a protein kinase activity which is not inhibited by CDK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomoucine. Lastly, using the yeast two-hybrid system we identified three novel cyclin-like proteins able to associate with TzCRK1, and demonstrate that two of these cyclins also bind the T. cruzi CRK3 protein, indicating that these two CRKs are cyclin-dependent kinases.
Assuntos
Ciclinas/isolamento & purificação , Proteínas Quinases/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2 , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/isolamento & purificação , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Citoplasma/enzimologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Histonas/metabolismo , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Cinetina , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Piperidinas/farmacologia , Testes de Precipitina , Proteínas Quinases/isolamento & purificação , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Purinas/farmacologia , Proteína do Retinoblastoma/metabolismo , Alinhamento de Sequência , Trypanosoma cruzi/metabolismoRESUMO
Endemic in most American countries, Chagas' disease causes high morbidity and mortality. Recent experimental and clinical evidence shows the importance of chemotherapy in both the acute and chronic phases of this disease. However, treatment is yet limited by the toxicity associated to available drugs. This review describes the design, evolution, and selection of dipeptides that interrupt the intracellular cycle of T. cruzi and cure acute experimental infections in laboratory animals. Peptido-mimetic inhibitors specifically bind cruzain, a T. cruzi cystein protease. The inhibitors cause alterations in the Golgi complex and ER, accumulation of unprocessed enzyme within Golgi cisternae, and decrease of mature cruzain within lysosomes. The most effective compound, N-Pip-F-hF-VS phi, cured an acute lethal infection in experimental animals. Myocardial lesions, lymphocyte infiltration and intracellular amastigote clusers were absent in treated animals. Preliminary toxicology and pharmacokinetic analyses suggest the lack of toxicity associated to high doses and prolonged treatment regimes. Protease inhibitors may soon become good chemotherapeutic alternatives for acute and chronic Chagas' disease.
Assuntos
Antiprotozoários/uso terapêutico , Doença de Chagas/tratamento farmacológico , Cisteína Endopeptidases/uso terapêutico , Inibidores de Cisteína Proteinase/uso terapêutico , Proteínas de Protozoários/uso terapêutico , Doença Aguda , Animais , Doença de Chagas/patologia , Doença Crônica , Cisteína Endopeptidases/química , Cisteína Endopeptidases/farmacologia , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C3H , Proteínas de Protozoários/química , Proteínas de Protozoários/farmacologia , RatosRESUMO
The complete intracellular cycle of the Leishmania mexicana mexicana G. S. strain was quantified in human macrophages and in the mouse IC-21 macrophage line utilizing a culture system that allows the direct observation of individual intracellular parasites. A wide range of pre-replicative lag periods exists, implying that promastigotes may be in any phase of their DNA synthetic cycle when phagocytosed by the macrophage. Amastigotes replicated 2-3 times, after which the host cell died and liberated amastigotes that were taken up by other macrophages and continued to replicate. The mean amastigote population-doubling time in human macrophages (17.5 h) was not statistically different from promastigotes growing in axenic culture (16.4 h), but was nearly 2-fold less than amastigotes growing in mouse-derived IC-21 macrophages (33.7 h). These observations are markedly different from cover-glass culture assays of Leishmania-macrophage interactions and provide an unambiguous description of the intracellular cycle of Leishmania mexicana mexicana.
Assuntos
Leishmania mexicana/crescimento & desenvolvimento , Animais , DNA/biossíntese , Humanos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Axenic culture amastigote-like forms of Trypanosoma cruzi, grown at 28 degrees C, reach a stationary phase after two generations, and differentiate to epimastigotes, which then resume growth. Axenic culture amastigotes readily ferment glucose to succinate and acetate, and do not excrete NH3; they have high activities of hexokinase and phosphoenolpyruvate carboxykinase, and very low citrate synthase activity; cytochrome o is absent, and cytochrome b-like is present at a very low level. Epimastigotes catabolize glucose and produce succinate and acetate at a considerably lower rate; they exhibit lower levels of hexokinase and carboxykinase, and much higher levels of citrate synthase and cytochromes o and b-like. They catabolize amino acids, as shown by excretion of NH3 to the medium. The results suggest that axenic culture amastigotes have an essentially glycolytic metabolism, and they acquire the ability to oxidize substrates such as amino acids only after differentiation to epimastigotes.
Assuntos
Glucose/metabolismo , Trypanosoma cruzi/metabolismo , Aminoácidos/metabolismo , Animais , Metabolismo dos Carboidratos , Fermentação , Concentração de Íons de Hidrogênio , Oxirredução , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
The oligosaccharide transferred from a dolichol-P-P derivative to proteins in the assembly of N-linked glycoproteins in Leishmania mexicana appeared to be Man6GlcNAc2. It was found that this compound underwent transient glucosylation once bound to protein but that Man6GlcNAc2 was the oligosaccharide present in mature glycoproteins. No complex type saccharides were detected. The structure of the oligosaccharide appeared to be similar to that of the core of compounds transferred from dolichol-P-P derivatives in protein glycosylation in Trypanosoma cruzi or animal cells.