Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase
Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase
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DOI：
10.1186/1471-2407-10-7
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发表时间：
2010-01-07
期刊：
影响因子：
3.8
通讯作者：
Ivaska, Johanna
中科院分区：
文献类型：
--
作者：
Mattila, Elina;Marttila, Heidi;Ivaska, Johanna
背景：T细胞蛋白酪氨酸磷酸酶（TCPTP/TC45）是一种广泛表达的细胞内非受体蛋白酪氨酸磷酸酶，参与多种癌症相关细胞信号通路的负调控。我们先前已表明，α1β1整合素的α - 细胞质尾与TCPTP之间的相互作用通过破坏TCPTP中的抑制性分子内键来激活TCPTP。因此，抑制TCPTP中的调节性相互作用是激活TCPTP和减弱致癌性受体酪氨酸激酶（RTK）信号传导的理想策略。然而，使用小分子化合物实现这一点具有挑战性。
方法：我们开发了一种高通量兼容的检测方法，用于在体外分析重组TCPTP的活性。利用该检测方法，我们筛选了64280种小分子以鉴定TCPTP的新型激动剂。使用体外检测方法进行对TCPTP激动剂的剂量依赖性反应分析。使用表达TCPTP和无TCPTP的小鼠胚胎成纤维细胞评估TCPTP激动剂的抑制效果和特异性。蛋白质印迹分析用于评估血小板衍生生长因子受体β（PDGFRβ）和表皮生长因子受体（EGFR）磷酸化的减弱情况。通过血管内皮生长因子（VEGF）诱导的内皮细胞出芽试验分析对VEGF信号传导的抑制作用。
结果：从筛选中我们鉴定出六种TCPTP激动剂。两种化合物与α1 - 细胞质结构域竞争结合TCPTP，这表明它们通过破坏TCPTP中的分子内键，以类似于α1 - 细胞质尾的方式激活TCPTP。重要的是，其中一种化合物（亚精胺）在细胞中对TCPTP表现出特异性，因为TCPTP - / - 细胞对该化合物的抗性比表达TCPTP的细胞高43倍。这种化合物以TCPTP依赖的方式减弱细胞中PDGFRβ和血管内皮生长因子受体2（VEGFR2）的信号传导，并作为癌细胞中EGFR磷酸化的负调节剂发挥作用。
结论：在本研究中，我们表明模拟TCPTP - α1相互作用的小分子可用作TCPTP激动剂。这些数据首次提供了概念验证描述，即使用高通量筛选来鉴定可在细胞中作为RTK拮抗剂发挥作用的小分子蛋白酪氨酸磷酸酶（PTP）激活剂。
Background: T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular nonreceptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the alpha-cytoplasmic tail of alpha 1 beta 1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds.Methods: We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFR beta and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays.Results: From the screen we identified six TCPTP agonists. Two compounds competed with alpha 1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to alpha 1-cyt by disrupting the intramolecular bond in TCPTP. Importantly, one of the compounds (spermidine) displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFR beta and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells.Conclusions: In this study we showed that small molecules mimicking TCPTP-alpha 1 interaction can be used as TCPTP agonists. These data provide the first proof-of-concept description of the use of high-throughput screening to identify small molecule PTP activators that could function as RTK antagonists in cells.