Comparison of the biochemical and kinetic properties of the type 1 receptor tyrosine kinase intracellular domains - Demonstration of differential sensitivity to kinase inhibitors
Comparison of the biochemical and kinetic properties of the type 1 receptor tyrosine kinase intracellular domains - Demonstration of differential sensitivity to kinase inhibitors
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DOI：
10.1074/jbc.m105907200
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发表时间：
2002-01-11
影响因子：
4.8
通讯作者：
Wood, ER
中科院分区：
文献类型：
--
作者：
Brignola, PS;Lackey, K;Wood, ER
表皮生长因子受体(EGFR)、ErbB-2和ErbB-4是I型受体酪氨酸激酶家族的成员。这些受体的过度表达，特别是ErbB-2和EGFR，已被认为与多种形式的癌症有关。EGFR酪氨酸激酶活性的抑制剂正在用于癌症治疗的临床评估。这些抑制剂的效力和选择性可能会影响治疗的疗效和毒性。在这里，我们描述了EGFR、ErbB-2和ErbB-4胞内结构域的表达、纯化和生化比较。尽管这三种酶的序列同源性很高，但它们的催化性质和底物动力学却有显著的不同。例如，ErbB-2的催化活性不如EGFR稳定。与EGFR和ErbB-4相比，ErbB-2利用ATP-mg作为底物的效率较低。这三种酶对三种优化的多肽底物具有非常相似的底物偏好，但在底物协同作用方面存在差异。我们使用从这些研究中确定的生化和动力学参数来开发一种测试系统，该系统可以准确地测量I型受体家族之间的抑制剂效力和选择性。我们报道了4-苯胺基喹唑啉系列分子的选择性可以通过特定的苯胺基取代来改变。此外，这些化合物在整个细胞中都具有活性，反映了用该检测系统确定的靶向抑制的效力和选择性。
Epidermal growth factor receptor (EGFR), ErbB-2, and ErbB-4 are members of the type 1 receptor tyrosine kinase family. Overexpression of these receptors, especially ErbB-2 and EGFR, has been implicated in multiple forms of cancer. Inhibitors of EGFR tyrosine kinase activity are being evaluated clinically for cancer therapy. The potency and selectivity of these inhibitors may affect the efficacy and toxicity of therapy. Here we describe the expression, purification, and biochemical comparison of EGFR, ErbB-2, and ErbB-4 intracellular domains. Despite their high degree of sequence homology, the three enzymes have significantly different catalytic properties and substrate kinetics. For example, the catalytic activity of ErbB-2 is less stable than that of EGFR. ErbB-2 uses ATP-Mg as a substrate inefficiently compared with EGFR and ErbB-4. The three enzymes have very similar substrate preferences for three optimized peptide substrates, but differences in substrate synergies were observed. We have used the biochemical and kinetic parameters determined from these studies to develop an assay system that accurately measures inhibitor potency and selectivity between the type I receptor family. We report that the selectivity profile of molecules in the 4-anilinoquinazoline series can be modified through specific aniline substitutions. Moreover, these compounds have activity in whole cells that reflect the potency and selectivity of target inhibition determined with this assay system.