학술논문

Kinetic Characterization of Novel Pyrazole TGF-β Receptor I Kinase Inhibitors and Their Blockade of the Epithelial--Mesenchymal Transition.
Document Type
Article
Source
Biochemistry. 2/22/2005, Vol. 44 Issue 7, p2293-2304. 10p.
Subject
*TRANSFORMING growth factors-beta
*IMMUNOMODULATORS
*PYRAZOLES
*EXTRACELLULAR matrix
*PEPTIDES
*EPITHELIAL cells
Language
ISSN
0006-2960
Abstract
Transforming growth factor β (TGF-β) signaling pathways regulate a wide variety of cellular processes including cell proliferation, differentiation, extracellular matrix deposition, development, and apoptosis. TGF-β type-I receptor (TβRI) is the major receptor that triggers several signaling events by activating downstream targets such as the Smad proteins. The intracellular kinase domain of TβR1 is essential for its function. In this study, we have identified a short phospho-Sad peptide, pSmad3(-3), KVLTQMGSPSIRCSS(PO4)VS as a substrate of TβRI kinase for in vitro kinase assays. This peptide is Uniquely phosphorylated by TβRI kinase at the C-terminal serine residue, the phosphorylation site of its parent Sad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylate by only TβRI and not TGF-β type-II receptor kinase, indicating that the peptide is a physiologically relevant substrate suitable for kinetic analysis and screening of TβRI kinase inhibitors. Utilizing pSmad3(-3) as a substrate, we have shown that novel pyrazole compounds are potent inhibitors of TβRI kinase with Ki 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-β-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-β-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 TβRI kinase inhibitors to treat human disease. [ABSTRACT FROM AUTHOR]