RESUMO
We have investigated the binding characteristics of a potent member of the bis-ortho-substituted five-membered nitrogen heterocycle class of ALK-5 kinase inhibitors with lysates of cultured HEK-293 cells to identify protein binding partners of potential biological significance. An affinity chromatographic resin containing an immobilized ALK-5 kinase inhibitor, 2-phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine, was used to capture specific proteins from the cell lysate. The soluble inhibitor was then used to specifically elute the proteins which selectively bound to the pharmacophore ligand structure. Application of 2-D SDS-PAGE analysis with positive and negative controls demonstrated the inhibitor bound several different proteins via selective molecular recognition processes. The structural features of the specifically eluted proteins were identified by peptide mass fingerprinting (PMF) methods and included proteins with structural, metabolic and chaperone functions. Furthermore, these PMF results identified the therapeutic target in various cancer treatment studies, HSP-70, as a potential high-affinity binding partner. These observations warrant examination of bis-ortho-substituted five-membered nitrogen heterocycles as dual ALK-5/HSP-70 inhibitors for anti-cancer drug development.
Assuntos
Antineoplásicos/química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/síntese química , Piridinas/química , Piridinas/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismoRESUMO
Novel inhibitors of TGF-ß1 and activin A signalling based on a 2-aryl-4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridine pharmacophore have been synthesised. Compounds containing phenyl or aromatic nitrogen heterocycle substituents inhibited both types of signalling with HEK-293T cells in culture, with a selectivity preference for TGF-ß1. Synthetic compounds containing pyridin-3-yl, pyrazol-4-yl, pyrazol-1-yl or 1H-imidazoyl-1-yl substituents exhibited structural and functional attributes suitable for further investigation related to the development of more potent TGF-ß inhibitors.