RESUMO
RATIONALE: Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine. An ideal therapeutic approach would involve an effective method at achieving direct conversion of injured areas to functional tissue in situ. OBJECTIVE: The aim of this study was to develop a strategy that identified and evaluated the potential of specific micro (mi)RNAs capable of inducing reprogramming of cardiac fibroblasts directly to cardiomyocytes in vitro and in vivo. METHODS AND RESULTS: Using a combinatorial strategy, we identified a combination of miRNAs 1, 133, 208, and 499 capable of inducing direct cellular reprogramming of fibroblasts to cardiomyocyte-like cells in vitro. Detailed studies of the reprogrammed cells demonstrated that a single transient transfection of the miRNAs can direct a switch in cell fate as documented by expression of mature cardiomyocyte markers, sarcomeric organization, and exhibition of spontaneous calcium flux characteristic of a cardiomyocyte-like phenotype. Interestingly, we also found that miRNA-mediated reprogramming was enhanced 10-fold on JAK inhibitor I treatment. Importantly, administration of miRNAs into ischemic mouse myocardium resulted in evidence of direct conversion of cardiac fibroblasts to cardiomyocytes in situ. Genetic tracing analysis using Fsp1Cre-traced fibroblasts from both cardiac and noncardiac cell sources strongly suggests that induced cells are most likely of fibroblastic origin. CONCLUSIONS: The findings from this study provide proof-of-concept that miRNAs have the capability of directly converting fibroblasts to a cardiomyocyte-like phenotype in vitro. Also of significance is that this is the first report of direct cardiac reprogramming in vivo. Our approach may have broad and important implications for therapeutic tissue regeneration in general.
Assuntos
Transdiferenciação Celular , Fibroblastos/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação da Expressão Gênica , Terapia Genética/métodos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/administração & dosagem , Contração Miocárdica , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Isquemia Miocárdica/terapia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Inibidores de Proteínas Quinases/farmacologia , Recuperação de Função Fisiológica , Regeneração , Proteínas S100/genética , Proteínas S100/metabolismo , Transfecção , Proteína Vermelha FluorescenteRESUMO
Starting from potent aldehyde inhibitors with poor drug properties, derivatization to semicarbazones led to the identification of a series of semicarbazone-based cathepsin K inhibitors with greater solubility and better pharmacokinetic profiles than their parent aldehydes. Furthermore, a representative semicarbazone inhibitor attenuated bone resorption in an ex vivo rat calvarial bone resorption model. However, based on enzyme inhibition comparisons at neutral pH, semicarbazone hydrolysis rates, and 13C NMR experiments, these semicarbazones probably function as prodrugs of aldehydes.
Assuntos
Aldeídos/química , Catepsinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Semicarbazonas/farmacologia , Animais , Catepsina K , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Conformação Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Ratos , Semicarbazonas/síntese química , Semicarbazonas/química , Solubilidade , Relação Estrutura-AtividadeRESUMO
An orally bioavailable series of ketoamide-based cathepsin K inhibitors with good pharmacokinetic properties has been identified. Starting from a potent inhibitor endowed with poor drug properties, conformational constraint of the P(2)-P(3) linker and modifications to P(1') elements led to an enhancement in potency, solubility, clearance, and bioavailability. These optimized inhibitors attenuated bone resorption in a rat TPTX hypocalcemic bone resorption model.
Assuntos
Amidas/síntese química , Catepsinas/antagonistas & inibidores , Inibidores de Cisteína Proteinase/síntese química , Cetonas/síntese química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Sítios de Ligação , Disponibilidade Biológica , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Catepsina K , Catepsinas/química , Inibidores de Cisteína Proteinase/farmacocinética , Inibidores de Cisteína Proteinase/farmacologia , Modelos Animais de Doenças , Hipocalcemia/tratamento farmacológico , Hipocalcemia/metabolismo , Cetonas/farmacocinética , Cetonas/farmacologia , Ratos , Ratos Wistar , Solubilidade , Relação Estrutura-AtividadeRESUMO
Conversion of the proline-derived cyanamide lead to an acyclic cyanamide capable of forming an additional hydrogen bond with cathepsin K resulted in a large increase in inhibitory activity. An X-ray structure of a co-crystal of a cyanamide with cathepsin K confirmed the enzyme interaction. Furthermore, a representative acyclic cyanamide inhibitor 6r was able to attenuate bone resorption in the rat calvarial model.
Assuntos
Catepsinas/antagonistas & inibidores , Cianamida/química , Inibidores de Cisteína Proteinase/farmacologia , Osteogênese/efeitos dos fármacos , Animais , Sítios de Ligação , Reabsorção Óssea , Catepsina B/antagonistas & inibidores , Catepsina H , Catepsina K , Catepsina L , Cristalografia por Raios X , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/síntese química , Modelos Animais de Doenças , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
A novel series of ketoamide-based inhibitors of cathepsin K has been identified. Modifications to P(2) and P(3) elements were crucial to enhancing inhibitory activity. Although not optimized, a selected inhibitor was effective in attenuating type I collagen hydrolysis in a surrogate assay of bone resorption.