Mechanisms of Kinase Function and Drug Resistance in Cancer

描述(由申请人提供)：激酶是第二大药物靶标家族，有10种已获批准的激酶抑制剂药物和50种化合物正在进行临床试验。蛋白激酶结构域最常由癌症基因编码。在它们的ATP结合G-环中发生了几个致癌突变。Abl抑制剂Imatinib是治疗慢性粒细胞白血病的突破性药物，但约35%的患者会因Imatinib耐药的Abl激酶区域突变积累而复发，特别是在G环。因此，随着越来越多的患者接受激酶抑制剂药物的治疗，耐药性可能成为一个主要的临床问题。使用Src家族蛋白酪氨酸激酶Lyn作为一个实验上非常容易处理的例子，我们建议实施和验证一种多学科方法，该方法首先使用分子动力学(MD)模拟来相对快速地识别影响催化和抑制剂相互作用并可能导致耐药性的突变(目标1)。我们的方法接下来分析已鉴定的Lyn突变体在体内外BA/F3细胞(Aim 2)或Lyn/骨髓(Aim 3)中的活性、抑制剂相互作用和抗药性，以确定那些与生理上最相关的突变。在每个步骤中排除没有信息的突变体可以最大限度地减少实验工作，并最大限度地提高相关性和成功的可能性。我们认为这种发现耐药导致激酶突变的综合方法具有很高的创新性，因为它提供了通常只有在更长的时间段内才能通过几个实验室的努力才能获得的重要见解。这些研究是我们最近发表的发现的后续，58个真核细胞激酶在其G-环上包含一个保守的静电盐桥，这对于G-环的稳定、催化和ATP-或ATP-竞争性抑制物结合是必不可少的。Bcr-Abl的盐桥破坏导致对伊马替尼的耐药。我们的初步数据表明，在31个激酶中，包括在癌症中起重要作用的Src、Ab1、CK1和CK2家族，酸性盐桥锚还与嵌入在疏水核心中的保守的极性-芳香族或碱性氨基酸侧链静电相互作用。为了验证这种“三联体相互作用-网络结构”对G-环功能和抑制物-相互作用是必不可少的假设，以及它的破坏可能导致耐药性，我们将分析突变调节典型蛋白激酶LYn(目标1-3)、Ab1、CK1(2)和CK2a1(目标4)中变异G-环-三联体配置的不同成分的影响。为了使目标4在5年资助期内能够实现，我们将重点放在MD分析上。未来的研究将在体外和体内分析预测的高优先级突变体。我们认为这一建议非常重要，因为它实现并验证了一种有效的方法来了解分子机制，通过这些机制，在治疗上非常重要的靶类发挥作用，与小分子抑制剂相互作用，并可能产生耐药性。如果成功，我们的方法可以应用于其他目标，在药物发现项目开始时识别耐药突变，使基于结构的合理设计分子有效地抑制野生型和突变的激酶。这将有助于开发更具选择性、更少副作用和更少耐药性的倾向于治疗的药物。

DESCRIPTION (provided by applicant): Kinases are the second-largest drug-target family with 10 approved kinase inhibitor drugs and 50 compounds in clinical trials. Protein-kinase-domains are most frequently encoded by cancer-genes. Several cancer-driving mutations occur in their ATP-binding G-loops. The Abl-inhibitor Imatinib is a breakthrough-therapeutic for chronic-myelogenous-leukemia, but ~35% of the patients relapse due to accumulation of Imatinib-resistant Abl kinase-domain-mutations, particularly in the G-loop. Drug-resistance could thus become a major clincial problem as increasing patient populations are treated with kinase-inhibitor drugs. Using the Src-family protein tyrosine kinase Lyn as an experimentally very tractable example, we propose to implement and validate a multidisciplinary approach that first uses molecular dynamics (MD) simulations to relatively quickly identify mutations that affect catalysis and inhibitor interactions and can cause drug-resistance (Aim 1). Our approach next analyzes the activities, inhibitor-interactions and -resistance of the identified Lyn mutants in vitro and in vivo in Ba/F3 cells (Aim 2) or in Lyn-/- bone-marrow (Aim 3) to identify those mutations that are most relevant physiologically. Exclusion of uninformative mutants at each step minimizes experimental effort and maximizes relevance and likelihood of success. We consider this integrated approach to discover drug-resistance causing kinase mutations highly innovative, because it provides important insight that is usually only gained over much longer time periods and through the efforts of several labs. These studies follow up on our recently published finding that 58 eukaryotic kinases contain a conserved electrostatic salt-bridge across their G-loops that is essential for G-loop-stabilization, catalysis and ATP- or ATP-competitive inhibitor-binding. Salt-bridge- disruption in Bcr-Abl causes Imatinib-resistance. Our preliminary data suggest that in 31 kinases, including the Src, Abl, CK1 and CK2-families which all have important roles in cancer, the acidic salt-bridge-anchor also interacts electrostatically with a conserved polar-aromatic or basic amino-acid-side-chain embedded in a hydrophobic core. To test the hypothesis that this "triad interaction-network" architecture is essential for G-loop function and inhibitor-interactions, and that its disruption can cause drug resistance, we will analyze the effects of mutationally modulating the different components of the variant G-loop-triad-configurations in the exemplary kinases Lyn (Aims 1-3), Abl, CK1(2 and CK2a1 (Aim 4). To keep Aim 4 achievable within the 5 year funding period, we will focus on MD analyses. Future research will analyze the predicted high-priority mutants in vitro and in vivo. We consider this proposal highly significant, because it implements and validates an efficient approach to understand the molecular mechanisms through which a therapeutically very important target class functions, interacts with small-molecule inhibitors and can become drug-resistant. If successful, our approach can be applied to other targets to identify drug-resistant mutants at the onset of a drug discovery project, enabling the structure-based rational design of molecules that inhibit wildtype and mutant kinases potently. This will aid the development of more selective, less side-effect and less drug-resistance prone therapeutics.