Baker Zachary D, Thompson Andrew R, Thomas David D, Levinson Nicholas M
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, United States.
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, Minnesota 55455, United States.
ACS Chem Biol. 2025 Jul 18;20(7):1683-1695. doi: 10.1021/acschembio.5c00224. Epub 2025 Jun 19.
Conformational changes triggered by kinase inhibitors are a major factor driving specificity and efficacy, but few scalable methods exist for differentiating induced conformations and binding modes. Using the receptor tyrosine kinase MET, we show that three classes of inhibitors can be distinguished by their contrasting effects on static and dynamic quenching of a fluorescent dye attached to the activation loop. Quenching is mediated by tyrosine residues on the flexible activation loop, and inhibitor binding induces order in the loop, sequestering the tyrosines and differentially suppressing static and dynamic quenching in a manner that is dependent on the induced structural state. Type I MET inhibitors have a large static and moderate dynamic component, type II inhibitors have only a static component, and active-state-selective inhibitors relieve both components to similar extents. These distinct dequenching signatures allow the straightforward detection of each binding mode by using parallel steady-state and time-resolved fluorescence measurements. We show that this technique can be applied to rapidly assess the effects of resistance mutations on inhibitor binding and can report on the chemical interactions and conformational changes that drive these effects. Conservation of the three activation loop tyrosine residues across many receptor tyrosine kinases suggests that this approach has broad utility.
激酶抑制剂引发的构象变化是决定特异性和疗效的主要因素，但用于区分诱导构象和结合模式的可扩展方法却很少。利用受体酪氨酸激酶MET，我们发现三类抑制剂可通过其对附着于激活环的荧光染料静态和动态猝灭的不同影响加以区分。猝灭由柔性激活环上的酪氨酸残基介导，抑制剂结合会使环产生有序性，隔离酪氨酸，并以依赖于诱导结构状态的方式差异性地抑制静态和动态猝灭。I型MET抑制剂具有较大的静态成分和中等程度的动态成分，II型抑制剂只有静态成分，而活性状态选择性抑制剂会在相似程度上减轻这两种成分。这些独特的去猝灭特征使得通过平行稳态和时间分辨荧光测量能够直接检测每种结合模式。我们表明，该技术可用于快速评估耐药性突变对抑制剂结合的影响，并可报告驱动这些影响的化学相互作用和构象变化。许多受体酪氨酸激酶中激活环上三个酪氨酸残基的保守性表明该方法具有广泛的实用性。
