High Affinity Immobilization of Proteins Using the CrAsH/TC Tag.

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.

Site-specific, reversible and fluorescent immobilization of proteins on CrAsH-modified surfaces for microarray analytics.

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.

Discovery of N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline (EW-7197): a highly potent, selective, and orally bioavailable inhibitor of TGF-β type I receptor kinase as cancer immunotherapeutic/antifibrotic agent.

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-β 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.

4-([1,2,4]Triazolo[1,5-a]pyridin-6-yl)-5(3)-(6-methylpyridin-2-yl)imidazole and -pyrazole derivatives as potent and selective inhibitors of transforming growth factor-β type I receptor kinase.

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.

Synthesis and biological evaluation of 2-benzylamino-4(5)-(6-methylpyridin-2-yl)-5(4)-([1,2,4]triazolo[1,5-a]-pyridin-6-yl)thiazoles as transforming growth factor-β type 1 receptor kinase inhibitors.

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.

Synthesis and biological evaluation of 1-substituted-3-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrazoles as transforming growth factor-β type 1 receptor kinase inhibitors.

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.

Synthesis and biological evaluation of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)pyrazoles as transforming growth factor-β type 1 receptor kinase inhibitors.

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.

Synthesis and biological evaluation of 1-substituted-3(5)-(6-methylpyridin-2-yl)-4-(quinolin-6-yl)pyrazoles as transforming growth factor-β type 1 receptor kinase inhibitors.

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.