Structural biochemistry studies on MAP kinase allosteric binding sites

描述（由申请人提供）：摘要MAP激酶蛋白家族在细胞信号传导中具有关键作用，MAP激酶p381与控制包括关节炎在内的风湿性疾病的事件有关。p381 MAP激酶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和其他激酶的活性位点之间的相似性，这些抑制剂通常表现出剂量限制性的不良反应。p381的活性形式是通过一组辅助激酶和磷酸酶提供的磷酸化事件以及通过底物结合进行变构调节的。因此，表征p381内可以结合底物、抑制剂或变构效应物的其他潜在位点将具有重大意义。该提案中的初步数据确定了在p381的活性位点和C叶口袋中结合的先导化合物。该口袋由激酶折叠的延伸形成，并且仅在一小组激酶（MAP、CDK和GSK家族）中出现。值得注意的是，初步的结构和计算分析表明，p381内的这个位点可能适合于设计小分子，以预定的方式结合并潜在地调节这种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的特定变构结合位点。该提案将测试MAP激酶活性和功能可以通过使用短肽或小分子进行特异性调节的假设，所述短肽或小分子被设计为紧密结合变构相互作用位点。在特定目标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，该变构位点与某些底物和调节酶结合。目标是产生效应分子，调节酶活性并对该位点具有比天然底物更大的结合亲和力。在具体目标2中，将定义小分子和肽与特征较少的C端半段口袋的相互作用和潜在调节功能。总的来说，这些研究将整合尖端的蛋白质晶体学和小角度X射线散射分析，与计算机虚拟对接方法，肽阵列研究以及体外和体内激酶活性测定。预期的结果将为激酶变构提供关键的机制见解，提供新的靶向分子调节酶活性，并揭示MAP激酶相关疾病的新治疗策略，而没有活性位点相关的毒性。此外，从这些p381的初步研究中开发的结果和概念可能会导致在风湿性疾病模型中测试进一步优化的变构调节剂的功效的研究。

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.