Kinetic Characterization of Novel Pyrazole TGF- Receptor I Kinase Inhibitors and Their Blockade of the Epithelial-Mes
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- 作者：Sheng-Bin Peng ; Lei Yan ; Xiaoling Xia ; Scott A. Watkins ; Harold B. Brooks ; Douglas Beight ; David K. Herron ; Michael L. Jones ; John W. Lampe ; William T. McMillen ; Nicholas Mort ; J. Scott Sawyer ; and Jo
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：February 22, 2005
- 年：2005
- 卷：44
- 期：7
- 页码：2293 - 2304
- 全文大小：557K
- 年卷期：v.44,no.7(February 22, 2005)
- ISSN：1520-4995
文摘
Transforming growth factor 
(TGF-) signaling pathways regulate a wide variety of cellularprocesses including cell proliferation, differentiation, extracellular matrix deposition, development, andapoptosis. TGF- type-I receptor (TRI) is the major receptor that triggers several signaling events byactivating downstream targets such as the Smad proteins. The intracellular kinase domain of TRI isessential for its function. In this study, we have identified a short phospho-Smad peptide, pSmad3(-3),KVLTQMGSPSIRCSS(PO4)VS as a substrate of TRI kinase for in vitro kinase assays. This peptide isuniquely phosphorylated by TRI kinase at the C-terminal serine residue, the phosphorylation site of itsparent Smad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylated by onlyTRI and not TGF- type-II receptor kinase, indicating that the peptide is a physiologically relevantsubstrate suitable for kinetic analysis and screening of TRI kinase inhibitors. Utilizing pSmad3(-3) asa substrate, we have shown that novel pyrazole compounds are potent inhibitors of TRI kinase with Kivalue as low as 15 nM. Kinetic analysis revealed that these pyrazoles act through the ATP-binding siteand are typical ATP competitive inhibitors with tight binding kinetics. More importantly, these compoundswere shown to inhibit TGF--induced Smad2 phosphorylation in vivo in NMuMg mammary epithelialcells with potency equivalent to the inhibitory activity in the in vitro kinase assay. Cellular selectivityanalysis demonstrated that these pyrazoles are capable of inhibiting activin signaling but not bonemorphogenic protein or platelet-derived growth factor signal transduction pathways. Further functionalanalysis revealed that pyrazoles are capable of blocking the TGF--induced epithelial-mesenchymaltransition in NMuMg cells, a process involved in the progression of cancer, fibrosis, and other humandiseases. These pyrazoles provide a foundation for future development of potent and selective TRI kinaseinhibitors to treat human disease.
(TGF-) signaling pathways regulate a wide variety of cellularprocesses including cell proliferation, differentiation, extracellular matrix deposition, development, andapoptosis. TGF- type-I receptor (TRI) is the major receptor that triggers several signaling events byactivating downstream targets such as the Smad proteins. The intracellular kinase domain of TRI isessential for its function. In this study, we have identified a short phospho-Smad peptide, pSmad3(-3),KVLTQMGSPSIRCSS(PO4)VS as a substrate of TRI kinase for in vitro kinase assays. This peptide isuniquely phosphorylated by TRI kinase at the C-terminal serine residue, the phosphorylation site of itsparent Smad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylated by onlyTRI and not TGF- type-II receptor kinase, indicating that the peptide is a physiologically relevantsubstrate suitable for kinetic analysis and screening of TRI kinase inhibitors. Utilizing pSmad3(-3) asa substrate, we have shown that novel pyrazole compounds are potent inhibitors of TRI kinase with Kivalue as low as 15 nM. Kinetic analysis revealed that these pyrazoles act through the ATP-binding siteand are typical ATP competitive inhibitors with tight binding kinetics. More importantly, these compoundswere shown to inhibit TGF--induced Smad2 phosphorylation in vivo in NMuMg mammary epithelialcells with potency equivalent to the inhibitory activity in the in vitro kinase assay. Cellular selectivityanalysis demonstrated that these pyrazoles are capable of inhibiting activin signaling but not bonemorphogenic protein or platelet-derived growth factor signal transduction pathways. Further functionalanalysis revealed that pyrazoles are capable of blocking the TGF--induced epithelial-mesenchymaltransition in NMuMg cells, a process involved in the progression of cancer, fibrosis, and other humandiseases. These pyrazoles provide a foundation for future development of potent and selective TRI kinaseinhibitors to treat human disease.