Mass Spectrometry-guided structural analysis of protein kinase inhibitor complexes

背景：蛋白质磷酸化是由蛋白激酶催化的，它已成为“个性化”人类疾病（如癌症）中非常重要的药物靶点。现在有大量的物理技术可用于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）是临床中重要的当前药物靶点，bbb30激酶抑制剂已被批准用于癌症和炎症，还有数百种正在开发中。然而，激酶抑制剂通常缺乏特异性，并且由于与细胞中呈现的非预期蛋白质构象脱靶结合而产生不必要的副作用。不幸的是，我们对影响动态激酶与药物相互作用的结构因素知之甚少。在本研究中，我们将利用技术进步来检查和量化特定激酶构象如何结合不同类别的药物。挑战：通过x射线晶体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：配体相互作用，这些相互作用对结晶是不妥协的。我们的解决方案是将最先进的结构质谱（MS）与x射线晶体学结合起来，培养新一代的跨学科科学家。结构质谱是一个迅速发展的领域，它利用了大分子复合物、磷酸化、配体结合和构象信息都保存在气相中这一事实。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射线晶体学）提供信息，并揭示或确认新的药物结合模式和激酶的变构网络。通过将结构见解与生物信息学（包括公开可用的药物结合数据）相结合，我们为MRC DiMeN DTP奖学金的资助创造了一个强有力的案例。该项目是MRC， CR-UK和bbsrc资助的利物浦大学和利兹大学的小组之间的合作，将采用定量物理方法研究蛋白激酶与小分子配体的复合物，重点研究已批准或后期临床药物。通过比较高度动态的激酶信号复合物（包括研究较少的假激酶）中存在的“开放”和“封闭”（介于“非活性”和“活性”之间）构象状态的配体结合和非结合种群，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)使用天然离子流动-质谱法评估功能性蛋白激酶和假激酶复合物的稳定性和动力学；2)开发基于构象的生物信息学管道，使用联合气相（MS）和x射线晶体学方法报告药物结合模式；3)将研究结果转化为激酶复合物和临床激酶抑制剂的药物结合参数的定量分析包括：1)沉浸在最先进的研究中，研究蛋白激酶及其与药物的相互作用；2)参与蛋白质组研究中心（利物浦）和Astbury结构分子生物学中心（利兹）的培训网络成员；3)MRC优先技能集的培训组合，包括激酶：抑制剂复合物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