ERK2: Structure, Function and Inhibition

描述（由申请人提供）：BRAF致癌突变发生在大约8%的人类癌症中，并导致Ser/ thr特异性蛋白激酶ERK1和ERK2的持续活性，这是细胞进入人类细胞周期S期的关键介质。为了开始理解ERK1/2控制底物识别和催化的原理，ERK27Ets1（酶-底物）复合物的结构和动力学将在Aim 1中确定，使用一系列复杂的核磁共振技术。转录因子Elk-1 （ERK1/2的另一个底物）的半合成双底物类似物将被构建来模拟磷酸化转移并以类似的方式进行分析。这些研究将得到x射线晶体结构方法的补充。在Aim 2中，阻断ERK1/2与其蛋白质配体之间相互作用的有效且高选择性靶向分子将通过在共识结合序列中大大增加侧链多样性来获得。这些靶向分子的作用机制和特异性将通过酶动力学和结构方法来确定。在Aim 3中，靶向分子将通过蛋白质转导结构域被递送到细胞中，其功效取决于其抑制生长因子刺激的ERK1/2活性和细胞生长的能力。值得注意的是，这个多学科的研究项目将首次提供ERK1/2的有效和特异性抑制剂，并将允许ERK1/2底物招募位点被验证为人类癌症的潜在药物靶点。本文开发的方法将迅速适用于其他蛋白激酶，从而潜在地适用于许多人类疾病，并将为未来在临床前小鼠模型中的转化研究提供基础。公共卫生相关性：这项应用的重点是一种名为ERK2的酶，其活性在许多人类癌症中上调。拟议的研究将提供关于这种酶如何与其他细胞蛋白相互作用的详细结构信息。将开发一种新技术，以快速识别可以阻止ERK2结合其他细胞蛋白的细胞渗透性分子。这将在人类疾病模型中验证ERK2作为药物靶点，并为验证同一酶家族的其他成员提供实验基础。所提出的研究将具有重要意义，并将有益于人类健康。

DESCRIPTION (provided by applicant): BRAF oncogenic mutations occur in approximately 8% of human cancers and lead to sustained activity of the Ser/Thr-specific protein kinases ERK1 and ERK2, which are critical mediators of cell entry into the S- phase of the human cell cycle. To begin to understand the principles governing substrate recognition and catalysis by ERK1/2, the structure and dynamics of the ERK27Ets1 (enzyme7substrate) complex will be determined in Aim 1, using an array of sophisticated NMR techniques. A semi-synthetic bisubstrate analog of transcription factor Elk-1, another substrate of ERK1/2, will be constructed to mimic phosphoryl transfer and analyzed in a similar manner. These studies will be complimented by X-ray crystal structure approaches. In Aim 2, potent and highly selective Targeting Molecules that block interactions between ERK1/2 and its protein ligands will be acquired by greatly increasing the side chain diversity within consensus binding sequences. The mechanism of action and specificity of these targeting molecules will be determined using enzyme kinetics and structural approaches. In Aim 3, the Targeting Molecules will be delivered into cells using protein transduction domains, and their efficacy determined with respect to their ability to inhibit Growth Factor-stimulated ERK1/2 activity and cell growth. Significantly, this multi-disciplined research program will provide potent and specific inhibitors of ERK1/2 for the first time and will allow ERK1/2 substrate-recruiting sites to be validated as potential drug targets for human cancers. The methodology developed herein will be rapidly applicable to other protein kinases, thereby potentially embracing many human diseases, and will provide the basis for future translational studies in pre-clinical mouse models. PUBLIC HEALTH RELEVANCE: The focus of this application is an enzyme called ERK2 whose activity is upregulated in a large number of human cancers. The proposed studies will provide detailed structural information on how this enzyme interacts with other cellular proteins. A new technique will be developed to rapidly identify cell-permeable molecules that can prevent ERK2 from binding other cellular proteins. This will provide validation of ERK2 as a drug target, in models of human disease, and provide the experimental basis for validating other members of the same enzyme family. The studies proposed will be of high significance and will benefit human health.