title: Kinetic characterization of novel pyrazole TGF-beta receptor I kinase inhibitors and their blockade of the epithelial-mesenchymal transition.
abstract: 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.
authors.affiliation: Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285, USA.
authors.forename: Sheng-Bin
authors.initials: SB
authors.lastname: Peng
authors.forename: Lei
authors.initials: L
authors.lastname: Yan
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Xia
authors.forename: Scott A
authors.initials: SA
authors.lastname: Watkins
authors.forename: Harold B
authors.initials: HB
authors.lastname: Brooks
authors.forename: Douglas
authors.initials: D
authors.lastname: Beight
authors.forename: David K
authors.initials: DK
authors.lastname: Herron
authors.forename: Michael L
authors.initials: ML
authors.lastname: Jones
authors.forename: John W
authors.initials: JW
authors.lastname: Lampe
authors.forename: William T
authors.initials: WT
authors.lastname: McMillen
authors.forename: Nicholas
authors.initials: N
authors.lastname: Mort
authors.forename: J Scott
authors.initials: JS
authors.lastname: Sawyer
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Yingling
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D006131:Growth Inhibitors / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013058:Mass Spectrometry; D008648:Mesoderm / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000378:metabolism; D011720:Pyrazoles / Q000737:chemistry* / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D051899:Smad2 Protein; D051900:Smad3 Protein; D013379:Substrate Specificity / Q000187:drug effects; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000502:physiology; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D000255:Adenosine Triphosphate; D004268:DNA-Binding Proteins; D006131:Growth Inhibitors; D020930:MAP Kinase Kinase Kinases; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D012694:Serine; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D000099151:MAP Kinase Kinase Kinase 7; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497783:Smad2 protein, mouse; C497791:Smad3 protein, mouse; C031280:pyrazole