Jecklin Matthias Conradin, Touboul David, Jain Rishi, Toole Estee Naggar, Tallarico John, Drueckes Peter, Ramage Paul, Zenobi Renato
Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093 Zürich, Switzerland.
Anal Chem. 2009 Jan 1;81(1):408-19. doi: 10.1021/ac801782c.
Protein kinases have emerged as a major drug target in the last years. Since more than 500 kinases are encoded in the human genome, cross-reactivity of a majority of kinase inhibitors causes problems. Tools are required for a rapid classification of inhibitors according to their affinity for a certain target to refine the search for new, more specific lead compounds. Mass spectrometry (MS) is increasingly used in pharmaceutical research and drug discovery to investigate protein-ligand interactions and determination of binding affinities. We present a comparison of different existing nanoelectrospray-MS based methods to quantify binding affinities and qualitatively rank, by competitive experiments, the affinity of several clinical inhibitors. We also present a new competitive method which is derived from our previous work for quantitative assessment of binding strengths (Wortmann et al., J. Mass Spectrom. 2008, 43(5), 600-608). The human kinases studied for this purpose were p38alpha (MAPK14) and LCK (lymphocyte specific kinase), and their interaction with 17 known small molecule kinase inhibitors was probed. Moreover, we present a new method to differentiate type I from type II inhibitors (Liu, Y.; Gray, N. S. Nat. Chem. Biol. 2006, 2(7), 358-364) based on a kinetic experiment with direct MS read-out of the noncovalent complex between the human kinase and the inhibitor. This method was successfully applied to p38alpha binding to BIRB796, as well as to a BIRB796 analogue. Quantitative determination of the binding strength is also described. The results of our competitive experiments for the affinity classification of different inhibitors, as well as the results for the kinetic study, are in good agreement with IC(50) measurements and data found in the literature.
近年来，蛋白激酶已成为主要的药物靶点。由于人类基因组中编码了500多种激酶，大多数激酶抑制剂的交叉反应性引发了问题。需要根据抑制剂对特定靶点的亲和力对其进行快速分类的工具，以优化对新型、更具特异性先导化合物的搜索。质谱（MS）在药物研究和药物发现中越来越多地用于研究蛋白质-配体相互作用以及结合亲和力的测定。我们比较了现有的基于纳米电喷雾质谱的不同方法，以量化结合亲和力，并通过竞争实验对几种临床抑制剂的亲和力进行定性排序。我们还提出了一种新的竞争方法，该方法源自我们之前用于定量评估结合强度的工作（沃特曼等人，《质谱学报》，2008年，43(5)，600-608）。为此研究的人类激酶是p38α（丝裂原活化蛋白激酶14）和LCK（淋巴细胞特异性激酶），并探究了它们与17种已知小分子激酶抑制剂的相互作用。此外，我们提出了一种基于动力学实验的新方法，可通过直接质谱读出人类激酶与抑制剂之间的非共价复合物，来区分I型和II型抑制剂（刘，Y.；格雷，N.S.《自然化学生物学》，2006年，2(7)，358-364）。该方法已成功应用于p38α与BIRB796以及BIRB796类似物的结合。还描述了结合强度的定量测定。我们对不同抑制剂亲和力分类的竞争实验结果以及动力学研究结果，与IC(50)测量值和文献中的数据高度一致。
