Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.
Anal Chem. 2013 Aug 6;85(15):6995-7002. doi: 10.1021/ac4012655. Epub 2013 Jul 23.
The discovery of activation state dependent kinase inhibitors, which bind specifically to the inactive conformation of the protein, is considered to be a promising pathway to improved cancer treatments. Identifying such inhibitors is challenging, however, because they can have Kd values similar to molecules known to inhibit kinase function by interacting with the active form. Further, while inhibitor induced changes within the kinase tertiary structure are significant, few technologies are able to correctly assign inhibitor binding modes in a high-throughput fashion based exclusively on protein-inhibitor complex formation and changes in local protein structure. We have developed a new assay, using ion mobility-mass spectrometry, capable of both rapidly detecting inhibitor binding and classifying the resultant kinase binding modes. Here, we demonstrate the ability of our approach to classify a broad set of kinase inhibitors, using micrograms of protein, without the need for protein modification or tagging.
激活状态依赖性激酶抑制剂的发现被认为是改善癌症治疗的有前途的途径，因为这些抑制剂特异性地结合蛋白质的非活性构象。然而，鉴定此类抑制剂具有挑战性，因为它们的 Kd 值可能与通过与活性形式相互作用来抑制激酶功能的已知分子相似。此外，尽管激酶三级结构内的抑制剂诱导变化很显著，但很少有技术能够仅基于蛋白质-抑制剂复合物形成和局部蛋白质结构变化以高通量方式正确分配抑制剂结合模式。我们开发了一种新的使用离子淌度-质谱法的测定法，能够快速检测抑制剂结合并对所得激酶结合模式进行分类。在这里，我们展示了我们的方法使用微克蛋白质而无需蛋白质修饰或标记即可对广泛的激酶抑制剂进行分类的能力。
