RESUMEN
SAR about the B-ring of a series of N(2)-aroyl anthranilamide factor Xa (fXa) inhibitors is described. B-ring o-aminoalkylether and B-ring p-amine probes of the S1' and S4 sites, respectively, afforded picomolar fXa inhibitors that performed well in in vitro anticoagulation assays.
Asunto(s)
Antitrombina III/síntesis química , Inhibidores del Factor Xa , Colorantes Fluorescentes/farmacología , ortoaminobenzoatos/farmacología , Anticoagulantes/química , Antitrombina III/química , Sitios de Unión , Química Farmacéutica/métodos , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Colorantes Fluorescentes/síntesis química , Humanos , Cinética , Modelos Químicos , Conformación Molecular , Relación Estructura-Actividad , ortoaminobenzoatos/síntesis químicaRESUMEN
Transforming growth factor beta (TGF-beta) signaling pathways regulate a wide variety of cellular processes including cell proliferation, differentiation, extracellular matrix deposition, development, and apoptosis. TGF-beta type-I receptor (TbetaRI) is the major receptor that triggers several signaling events by activating downstream targets such as the Smad proteins. The intracellular kinase domain of TbetaRI is essential for its function. In this study, we have identified a short phospho-Smad peptide, pSmad3(-3), KVLTQMGSPSIRCSS(PO4)VS as a substrate of TbetaRI kinase for in vitro kinase assays. This peptide is uniquely phosphorylated by TbetaRI kinase at the C-terminal serine residue, the phosphorylation site of its parent Smad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylated by only TbetaRI and not TGF-beta type-II receptor kinase, indicating that the peptide is a physiologically relevant substrate suitable for kinetic analysis and screening of TbetaRI kinase inhibitors. Utilizing pSmad3(-3) as a substrate, we have shown that novel pyrazole compounds are potent inhibitors of TbetaRI kinase with K(i) value as low as 15 nM. Kinetic analysis revealed that these pyrazoles act through the ATP-binding site and are typical ATP competitive inhibitors with tight binding kinetics. More importantly, these compounds were shown to inhibit TGF-beta-induced Smad2 phosphorylation in vivo in NMuMg mammary epithelial cells with potency equivalent to the inhibitory activity in the in vitro kinase assay. Cellular selectivity analysis demonstrated that these pyrazoles are capable of inhibiting activin signaling but not bone morphogenic protein or platelet-derived growth factor signal transduction pathways. Further functional analysis revealed that pyrazoles are capable of blocking the TGF-beta-induced epithelial-mesenchymal transition in NMuMg cells, a process involved in the progression of cancer, fibrosis, and other human diseases. These pyrazoles provide a foundation for future development of potent and selective TbetaRI kinase inhibitors to treat human disease.
Asunto(s)
Células Epiteliales/citología , Inhibidores de Crecimiento/química , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Mesodermo/citología , Inhibidores de Proteínas Quinasas/química , Pirazoles/química , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Línea Celular , Cromatografía Líquida de Alta Presión , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/química , Células Epiteliales/efectos de los fármacos , Inhibidores de Crecimiento/metabolismo , Células HeLa , Humanos , Cinética , Quinasas Quinasa Quinasa PAM/metabolismo , Espectrometría de Masas , Mesodermo/química , Mesodermo/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/metabolismo , Pirazoles/metabolismo , Serina/metabolismo , Proteína Smad2 , Proteína smad3 , Especificidad por Sustrato/efectos de los fármacos , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
We have expanded our previously reported series of pyrazole-based inhibitors of the TGF-beta type I receptor kinase domain (TbetaR-I) to now include new 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole analogues. Limited examination of the SAR of this new series in both enzyme and cell based in vitro assays has revealed selectivity differences with respect to p38 MAP kinase (p38 MAPK) depending on the nature of the 'warhead' group on the dihydropyrrolopyrazole ring. As with our original pyrazole series, phenyl substituents tended to show greater selectivity against p38 MAPK than those comprised of the quinoline-4-yl moiety. We have also achieved co-crystallization and X-ray analysis of compounds 3 and 15, two potent examples of this new series, with the TbetaR-I receptor kinase domain.
Asunto(s)
Receptores de Activinas Tipo I/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Pirazoles/síntesis química , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Activinas Tipo I/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Cristalografía por Rayos X , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Concentración 50 Inhibidora , Proteínas Serina-Treonina Quinasas , Pirazoles/metabolismo , Pirazoles/farmacología , Quinolinas/química , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Pyrazole-based inhibitors of the transforming growth factor-beta type I receptor kinase domain (TbetaR-I) are described. Examination of the SAR in both enzyme- and cell-based in vitro assays resulted in the emergence of two subseries featuring differing selectivity versus p38 MAP kinase. A common binding mode at the active site has been established by successful cocrystallization and X-ray analysis of potent inhibitors with the TbetaR-I receptor kinase domain.