Mass Spectrometry-guided structural analysis of protein kinase inhibitor complexes

背景：蛋白磷酸化是由蛋白激酶催化的，在癌症等个性化人类疾病中，蛋白激酶已成为非常重要的药物靶点。现在有太多的物理技术可以用来测量激酶之间的配体(药物)结合倾向。蛋白激酶和假激酶(具有相似整体折叠的催化缺陷变体)是目前临床上重要的药物靶点，已有30种激酶抑制剂被批准用于治疗癌症和炎症，还有数百种正在开发中。然而，激酶抑制剂通常缺乏特异性，并且由于脱离靶点与细胞内呈现的非预期蛋白质构象结合而产生不想要的副作用。不幸的是，我们对影响药物与动态激酶相互作用的结构性因素知之甚少。在本课程中，我们将利用技术进步来研究和量化特定的激酶构象如何与不同类别的药物结合。挑战：用X射线结晶学研究激酶的一个固有问题是我们无法测量结构动力学或激酶：对结晶不妥协的配体相互作用。我们的解决方案是将最先进的结构质谱学(MS)与X射线结晶学结合起来，培养新一代跨学科科学家。结构MS是一个发展迅速的领域，它利用了大分子络合物、磷酸化、配体结合和构象信息都保存在气相中的事实。与构象相关的基于MS的亲和力(定量KD信息)可以为互补的结构方法(如X射线结晶学)提供信息，并揭示或确认新的药物结合模式和激酶中的变构网络。通过将结构洞察力与生物信息学(包括公开的药物结合数据)相结合，我们为MRC Diman DTP学生提供了一个强有力的资助理由。该项目是利物浦大学和利兹大学MRC、CR-UK和BBSRC资助的小组之间的合作，将使用定量物理方法来研究具有小分子配体的复合体中的蛋白激酶，重点放在批准的或晚期临床药物上。通过比较“开放”和“关闭”(介于“不活跃”和“活跃”之间)构象状态的配体结合和非结合群体，存在于高度动态的激酶信号复合体队列中，包括研究较少的假激酶，我们的项目代表了一个独特的多学科培训机会，解决了MRC战略研究和技能优先事项。它代表了一个由经验丰富的生化和生物物理科学家组成的监督团队与非常成功的学生培训组合之间的三方合作。我们的三个科学目标是：1)使用天然离子迁移率-质谱法评估功能性蛋白激酶和假性蛋白激酶复合体的稳定性和动力学2)开发一种基于构象的生物信息学管道，使用气相(MS)和X射线结晶学相结合的方法来报告药物结合模式(S)3)将研究结果转化为对激酶复合体和临床激酶抑制剂的药物结合参数的定量分析学生的主要成果包括：1)沉浸在研究蛋白激酶及其与Drugs2相互作用的最新研究中2)培训网络的成员，包括蛋白质组研究中心(利物浦)和阿斯特伯里大学结构分子生物学中心(利兹)3)一系列关于MRC优先技能的培训，包括对激酶：抑制剂复合体的定量结构数据的计算/生物信息学分析

Background: Protein phosphorylation is catalyzed by protein kinases, which have become hugely important drug targets in 'personalized' human diseases such as cancer. A plethora of physical techniques are now available to measure ligand (drug)-binding propensity amongst kinases. Protein kinases and pseudokinases (catalytically deficient variants with a similar overall fold) represent important current drug targets in the clinic, with >30 kinase inhibitors approved for cancer and inflammatory conditions and hundreds more in development. However, kinase inhibitors often suffer from a lack of specificity, and unwanted side-effects arise due to off-target binding to unintended protein conformations presented to them in the cell. Unfortunately, we know little about structural factors that influence dynamic kinase interactions with drugs. In this studentship, we will exploit technological advances to examine and quantify how specific kinase conformations bind different classes of drugs.The challenge: An inherent issue with studying kinases by X-ray crystallography is our inability to measure structural dynamics or kinase:ligand interactions that are intransigent to crystallisation. Our solution is to marry state-of-the-art structural Mass Spectrometry (MS) with X-ray crystallography, training a new generation of cross-disciplinary scientist. Structural MS is a rapidly developing field which takes advantage of the fact that macromolecular complexes, phosphorylation, ligand binding and conformational information are all preserved in the gas phase. MS-based affinity (quantitative Kd information) pertaining to conformation can inform complementary structural approaches (such as X-Ray crystallography), and reveal, or confirm, novel drug binding modes and allosteric networks in kinases. By marrying structural insights with bioinformatics (including publically available drug-binding data), we have created a strong case for funding for an MRC DiMeN DTP studentship.This project is a collaboration between MRC, CR-UK and BBSRC-funded groups at the Universities of Liverpool and Leeds, and will employ quantitative physical approaches to study protein kinases in complex with small molecule ligands, focusing on approved or late-phase clinical drugs. By comparing ligand-bound and unbound populations of 'open' and 'closed' (lying between 'inactive' and 'active') conformational states that exist amongst highly dynamic cohorts of kinase signaling complexes, including poorly studied pseudokinases, our project represents a unique multidisciplinary training opportunity that addresses MRC strategic research and skill priorities. It represents a three-way collaboration between an experienced supervisory team of biochemical and biophysical scientists with very successful portfolios of student training. Our three scientific goals are:1) Evaluation of stability and dynamics of functional protein kinase and pseudokinase complexes using native Ion Mobility-Mass Spectrometry2) Development of a conformation-based bioinformatic pipeline to report drug binding mode(s) using combined gas phase (MS) and X-ray crystallography approaches3) Translation of findings into a quantitative analysis of drug-binding parameters for kinase complexes and clinical kinase inhibitorsKey outcomes for the student include:1) Immersion in state-of-the art research studying protein kinases and their interaction with drugs2) Member of a training network involving the Centre for Proteome Research (Liverpool) and the Astbury Centre for Structural Molecular Biology (Leeds)3) A portfolio of training in MRC priority skill sets, including computational/bioinformatic analysis of quantitative structural data for kinase:inhibitor complexes