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.