RESUMO
Protein microarrays represent important tools for biomedical analysis. We have recently described the use of the biarsenical-tetracysteine (TC) tag for the preparation of protein microarrays. The unique feature of this tag enables the site-specific immobilization of TC-containing proteins on biarsenical-modified surfaces, resulting in a fluorescence enhancement that allows the direct quantification of the immobilized proteins. Moreover, the reversibility of the binding upon incubation with large quantities of thiols permits the detachment of the proteins from the surface, thereby enabling recovery of the substrate to extend the life time of the slide. Herein, we describe our recent results that further extend the applicability of the CrAsH/TC tag to the fabrication of biochips. With this aim, the immobilization of proteins on surfaces has been investigated using two different spacers and two TC tags, the minimal TC sequence (CCPGCC) and an optimized motif (FLNCCPGCCMEP). While the minimal peptide motif enables a rapid recycling of the slide, the optimized TC sequence reveals an increased affinity due to its greater resistance to displacement by thiols. Moreover, the developed methodology was applied to the immobilization of proteins via on-chip ligation of recombinant protein thioesters.
Assuntos
Proteínas Imobilizadas/química , Compostos Organometálicos/química , Análise Serial de Proteínas/métodos , Proteínas Recombinantes/química , Cisteína/química , Peptídeos/química , Compostos de Sulfidrila/químicaRESUMO
A series of 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5(3)-(6-methylpyridin-2-yl)imidazoles and -pyrazoles 14a-c, 15a-c, 16a, 16b, 19a-d, 21a, and 21b has been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. Among them, the pyrazole derivative 21b inhibited ALK5 phosphorylation with an IC50 value of 0.018 µM and showed 95% inhibition at 0.03 µM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct. The 21b showed a high selectivity index of 284 against p38α MAP kinase. The binding pose of 21b generated by docking analysis reveals that it fits well into the ATP binding cavity of ALK5 by forming several hydrogen bond interactions.
Assuntos
Imidazóis/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/química , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Humanos , Imidazóis/síntese química , Imidazóis/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/síntese química , Pirazóis/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Relação Estrutura-AtividadeRESUMO
A series of 1-substituted-3-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrazoles 14a-ae, 16a, 16b, and 21a-c has been prepared and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-(4-methoxyphenyl)-3-(6-methylpyridin-2-yl)-1H-pyrazole-1-carbothioamide (14n) inhibited ALK5 phosphorylation with IC(50) value of 0.57 nM and showed 94% inhibition at 100 nM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/química , Pirazóis/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Animais , Linhagem Celular , Humanos , Insetos/citologia , Luciferases/análise , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/síntese química , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A series of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinolin-6-yl)pyrazoles 14a-e, 15a-e, 17a-c, and 18a-d have been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The 6-quinolinyl pyrazole analogue 14b inhibited ALK5 phosphorylation with IC(50) value of 0.022 µM and showed 84% inhibition at 0.1 µM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/síntese química , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Linhagem Celular , Ensaios Enzimáticos , Humanos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Relação Estrutura-AtividadeRESUMO
A novel technique for protein immobilization onto CrAsH-modified surfaces is presented. This approach enables an efficient, reversible and fluorogenic immobilization of proteins. Moreover, expressed proteins can also be directly immobilized from cellular lysates without prior purification. The immobilized proteins are suitable for protein-protein interaction studies and the fluorescence enhancement upon immobilization can be employed for the direct detection of the immobilized protein without the need for secondary detection methods.
Assuntos
Fluoresceínas/química , Corantes Fluorescentes/química , Proteínas/química , Motivos de Aminoácidos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Microscopia de Fluorescência , Análise Serial de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas/metabolismo , Succinimidas/química , Propriedades de SuperfícieRESUMO
A series of 2-substituted-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)imidazoles was synthesized and evaluated to optimize a prototype inhibitor of TGF-ß type I receptor kinase (ALK5), 6. Combination of replacement of a quinoxalin-6-yl moiety of 6 with a [1,2,4]triazolo[1,5-a]pyridin-6-yl moiety, insertion of a methyleneamino linker, and a o-F substituent in the phenyl ring markedly increased ALK5 inhibitory activity, kinase selectivity, and oral bioavailability. The 12b (EW-7197) inhibited ALK5 with IC50 value of 0.013 µM in a kinase assay and with IC50 values of 0.0165 and 0.0121 µM in HaCaT (3TP-luc) stable cells and 4T1 (3TP-luc) stable cells, respectively, in a luciferase assay. Selectivity profiling of 12b using a panel of 320 protein kinases revealed that it is a highly selective ALK5/ALK4 inhibitor. Pharmacokinetic study with 12b·HCl in rats showed an oral bioavailability of 51% with high systemic exposure (AUC) of 1426 ng × h/mL and maximum plasma concentration (Cmax) of 1620 ng/mL. Rational optimization of 6 has led to the identification of a highly potent, selective, and orally bioavailable ALK5 inhibitor 12b.
Assuntos
Compostos de Anilina/síntese química , Antifibrinolíticos/síntese química , Antineoplásicos/síntese química , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Triazóis/síntese química , Administração Oral , Compostos de Anilina/farmacocinética , Compostos de Anilina/farmacologia , Compostos de Anilina/toxicidade , Animais , Antifibrinolíticos/farmacocinética , Antifibrinolíticos/farmacologia , Antifibrinolíticos/toxicidade , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Disponibilidade Biológica , Descoberta de Drogas , Células HEK293 , Humanos , Imunoterapia , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/toxicidade , Ratos , Receptor do Fator de Crescimento Transformador beta Tipo I , Relação Estrutura-Atividade , Triazóis/farmacocinética , Triazóis/farmacologia , Triazóis/toxicidadeRESUMO
A series of 2-benzylamino-4(5)-(6-methylpyridin-2-yl)-5(4)-([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazoles 12a-ab, 13a, 13b, and 18a-d has been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The N-(3-fluorobenzyl)-4-(6-methylpyridin-2-yl)-5-([1,2,4]triazolo[1,5-a]pyridin-6-yl)thiazol-2-amine (12b) inhibited ALK5 phosphorylation with an IC(50) value of 7.01 nM and showed 61% inhibition at 30 nM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.
Assuntos
Queratinócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Receptores de Fatores de Crescimento Transformadores beta/química , Tiazóis/síntese química , Linhagem Celular Transformada , Ensaios Enzimáticos , Genes Reporter , Humanos , Queratinócitos/enzimologia , Luciferases , Simulação de Acoplamento Molecular , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Relação Estrutura-Atividade , Tiazóis/farmacologia , TransfecçãoRESUMO
A series of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)pyrazoles 14a-d, 15a-d, 17a, 17b, 18a-d, 19a, and 19b has been synthesized and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The 2-[3-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-pyrazol-1-yl]-N-phenylethanethioamide (18a) inhibited ALK5 phosphorylation with an IC(50) value of 0.013 µM and showed 80% inhibition at 0.1 µM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.