Structural biochemistry studies on MAP kinase allosteric binding sites

描述(申请人提供)：MAP蛋白家族在细胞信号传递中起关键作用，MAP蛋白P381与控制包括关节炎在内的风湿性疾病的事件有关。P381MAPK是一个重要的药物发现靶点，药物设计工作主要集中在ATP催化位点的小分子抑制剂上。然而，由于p381的活性部位与其他激酶相似，这些抑制剂通常表现出剂量限制的不良反应。P381的活性形式是通过一系列辅助激酶和磷酸酶提供的磷酸化事件以及底物结合来调节的。因此，鉴定p381中可以结合底物、抑制剂或变构效应物的其他潜在位点将是非常有意义的。该提案中的初步数据确定了结合在p381的活性中心和C叶口袋中的先导化合物。这个口袋是由激酶折叠的延伸而形成的，只出现在一小群激酶中，即MAP、CDK和GSK家族。值得注意的是，初步的结构和计算分析表明，p381中的这个位置很可能适合于设计小分子来以预定的方式结合并潜在地调节该激酶的形状和相互作用。因此，拟议的研究将建立在这些研究的基础上，通过定义和比较p381的特定芦荟酸结合位点。这项提议将检验这样一个假设，即MAP激酶的活性和功能可以通过使用短肽或小分子来特定地调节，这些短肽或小分子旨在紧密结合变构相互作用部位。在具体目标1中，已知的D基序变构位点的关键功能相互作用将在p381中定义，它与某些底物和调节酶结合。其目标是产生效应器分子，调节酶的活性，并具有比自然底物更强的对该位点的结合亲和力。在具体目标2中，将定义小分子和多肽与特征较少的C叶口袋的相互作用和潜在的调节功能。总体而言，这些研究将把尖端的蛋白质结晶学和小角X射线散射分析与电子虚拟对接方法、多肽阵列研究以及体外和体内的激酶活性分析相结合。预期的结果将提供对激酶变构的关键机制见解，提供新的靶向分子调节酶活性，并揭示新的治疗策略，在没有活性位点连接毒性的情况下治疗MAP激酶相关疾病。此外，从这些关于p381的先导研究得出的结果和概念可能会导致进行研究，测试进一步优化的变构调节剂在风湿病模型中的有效性。

DESCRIPTION (provided by applicant): Abstract The MAP kinase protein family has a critical role in cellular signaling, with the MAP kinase p381 being linked to events controlling rheumatic disease that includes arthritis. p381 MAP kinase is an important drug discovery target, and drug design efforts have largely focused on small molecule inhibitors of the ATP catalytic site. However, these inhibitors have generally exhibited dose-limiting adverse effects due to similarities between the active site of p381 and other kinases. The active form of p381 is alostericaly regulated, through phosphorylation events provided by a set of auxiliary kinases and phosphatases and through substrate binding. Therefore, characterizing other potential sites within p381, which can bind substrates, inhibitors or allosteric effectors would be of significant interest. The preliminary data in this proposal identifies a lead compound bound in both the active site and the C-lobe pocket of p381. This pocket is formed from an extension to the kinase fold, and occurs in only a small group of kinases, the MAP, CDK and GSK families. Significantly, the preliminary structural and computational analyses suggest that this site within p381 is likely to be suitable for the design of small molecules to bind and potentially modulate the shape and interactions of this kinase in predetermined ways. Thus, the proposed research will build on these studies by defining and comparing specific alosteric binding sites of p381. This proposal will test the hypothesis that MAP kinase activity and function can be specifically modulated through the use of short peptides or small molecules, designed to tightly bind allosteric interaction sites. In Specific Aim 1, key functional interactions of the known 'D-motif' allosteric site, which is bound by certain substrates and regulatory enzymes, will be defined in p381. The goal is to produce effector molecules, modulating enzymatic activity and having a binding affinity for this site greater than natural substrates. In Specific Aim 2, the interactions and potential regulatory functions of small molecule and peptide interactions with the less characterized C-lobe pocket will be defined. Overall, these studies will integrate cutting-edge protein crystallography and small-angle x-ray scattering analyses, with in silico virtual docking methods, peptide array studies and in vitro and in vivo kinase activity assays. The expected results will provide key mechanistic insights into kinase allostery, provide new, targeted molecules regulating enzymatic activity, and reveal new therapeutic strategies for MAP kinase-linked diseases without the active site-linked toxicity. Moreover, the results and concepts developed from these pilot studies on p381 are likely to lead to studies that will test the efficacy of further optimized allosteric regulators, in models of rheumatic disease.