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种已批准的激酶抑制剂药物和50种处于临床试验中的化合物。蛋白激酶结构域最常由癌症基因编码。一些癌症驱动突变发生在它们的atp结合g环中。Abl抑制剂伊马替尼是治疗慢性骨髓性白血病的突破性药物，但约35%的患者由于耐伊马替尼的Abl激酶结构域突变积累而复发，特别是在g环。随着越来越多的患者接受激酶抑制剂药物治疗，耐药性可能成为一个主要的临床问题。以src家族蛋白酪氨酸激酶Lyn为例，我们建议实施并验证一种多学科方法，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（Aim 1）。接下来，我们的方法分析了鉴定出的Lyn突变体在体外和体内Ba/F3细胞（Aim 2）或Lyn-/-骨髓（Aim 3）中的活性、抑制剂相互作用和耐药性，以确定那些在生理上最相关的突变。在每个步骤中排除无信息的突变体可以使实验努力最小化，并使相关性和成功的可能性最大化。我们认为这种发现引起激酶突变的耐药性的综合方法具有高度创新性，因为它提供了通常只有在更长的时间内通过几个实验室的努力才能获得的重要见解。这些研究跟进了我们最近发表的发现，58种真核激酶在其g环上含有保守的静电盐桥，这对于g环稳定、催化和ATP或ATP竞争性抑制剂结合至关重要。Bcr-Abl的盐桥破坏导致伊马替尼耐药。我们的初步数据表明，在31种激酶中，包括Src、Abl、CK1和ck2家族，它们都在癌症中发挥重要作用，酸性盐桥锚也与嵌入在疏水核心中的保守极性芳香或碱性氨基酸侧链发生静电相互作用。为了验证这种“三元相互作用网络”结构对G-loop功能和抑制剂相互作用至关重要的假设，以及它的破坏可能导致耐药性，我们将分析突变调节典型激酶Lyn （Aims 1-3）、Abl、CK1(2)和CK2a1 （Aim 4）中变异G-loop三元结构的不同组分的影响。为确保目标4能在5年资助期内实现，我们将重点进行医学发展分析。未来的研究将在体外和体内分析预测的高优先突变体。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理解治疗上非常重要的靶标类的分子机制，通过这种机制，靶标类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靶标，在药物发现项目开始时识别耐药突变体，从而实现基于结构的合理设计分子，有效抑制野生型和突变型激酶。这将有助于开发更具选择性、副作用更小、耐药倾向更低的治疗方法。

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. PUBLIC HEALTH RELEVANCE: While kinases have become the second-largest family of drug targets due to their paramount roles in causing diseases such as cancer, the accumulation of drug-resistant mutant kinases in patients treated with kinase inhibitor drugs remains a major therapeutic problem. To promote our understanding of the molecular mechanisms mediating kinase function, small-molecule inhibition and in particular the development of drug- resistance, we propose an innovative, multidisciplinary approach that, if successful, can be applied to any potential drug-target protein whose structure is known. The results will advance our understanding of kinase- function and aid the development of new and improved kinase-inhibitor drugs that are less prone to undesired side-effects, and less susceptible to drug-resistance. .