Kinetic characterization of novel pyrazole TGF-β receptor I kinase inhibitors and their blockade of the epithelial-mesenchymal transition
Kinetic characterization of novel pyrazole TGF-β receptor I kinase inhibitors and their blockade of the epithelial-mesenchymal transition
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DOI：
10.1021/bi048851x
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发表时间：
2005-02-22
期刊：
影响因子：
2.9
通讯作者：
Yingling, JM
中科院分区：
文献类型：
--
作者：
Peng, SB;Yan, L;Yingling, JM
转化生长因子β (tgf - β)信号通路调节多种细胞过程，包括细胞增殖、分化、细胞外基质沉积、发育和凋亡。tgf - β - 1型受体（TbetaRI）是通过激活下游靶标（如Smad蛋白）触发几种信号事件的主要受体。TbetaRI的胞内激酶结构域对其功能至关重要。在这项研究中，我们已经确定了短磷酸化smad肽pSmad3(-3)， KVLTQMGSPSIRCSS(PO4)VS作为TbetaRI激酶的底物，用于体外激酶检测。该肽在体内被TbetaRI激酶在其亲本Smad蛋白的c端丝氨酸残基（磷酸化位点）唯一磷酸化。特异性分析表明，该肽仅被TbetaRI磷酸化，而不被tgf - β ii型受体激酶磷酸化，这表明该肽是一种生理相关的底物，适合用于TbetaRI激酶抑制剂的动力学分析和筛选。利用pSmad3（-3）作为底物，我们发现新的吡唑类化合物是有效的TbetaRI激酶抑制剂，其K-i值低至15 nM。动力学分析表明，这些吡唑通过ATP结合位点起作用，是典型的ATP竞争性抑制剂，具有紧密的结合动力学。更重要的是，在NMuMg乳腺上皮细胞中，这些化合物被证明可以抑制tgf - β诱导的Smad2磷酸化，其效力相当于体外激酶试验中的抑制活性。细胞选择性分析表明，这些吡唑能够抑制激活素信号通路，但不能抑制骨形态发生蛋白或血小板衍生生长因子信号转导通路。进一步的功能分析表明，吡唑能够阻断tgf - β诱导的NMuMg细胞的上皮-间质转化，这一过程涉及癌症、纤维化和其他人类疾病的进展。这些吡唑为未来开发有效的选择性TbetaRI激酶抑制剂治疗人类疾病提供了基础。
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.