Structural biochemistry studies on MAP kinase allosteric binding sites

MAP 激酶变构结合位点的结构生物化学研究

• 批准号: 8454542
• 负责人: John Jefferson Perry
• 金额: $ 9.02万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8454542
• 关键词: Active Sites; Adverse effects; Affinity; Allosteric Regulation; Allosteric Site; Architecture; Arthritis; Binding; Binding Sites; Biochemical; Biological Assay; Cartoons; Catalytic Domain; Cells; Cellular biology; Characteristics; Computer Simulation; Crystallography; Data; Disease; Docking; Dose-Limiting; Drug Design; Enzymes; Event; Exhibits; Family; Goals; Gray unit of radiation dose; Human; Hydrogen Bonding; In Vitro; Individual; Inflammatory; Inflammatory Response; Lead; Link; Lobe; MAPK14 gene; Methods; Mitogen-Activated Protein Kinases; Mitogens; Modeling; Molecular Conformation; Nature; Pathway interactions; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Pilot Projects; Process; Protein Binding; Protein Conformation; Protein Family; Protein Kinase C; Protein Kinase Interaction; Proteins; Research; Rheumatism; Role; Serine; Shapes; Signal Transduction; Site; Solutions; Specificity; Structural Biochemistry; Surface; Testing; Threonine; Toxic effect; Vertebral column; Work; abstracting; base; design; drug discovery; drug use screening; efficacy testing; human MAPK14 protein; in vivo; inhibitor/antagonist; insight; interest; mitogen-activated protein kinase p38; novel; novel therapeutics; protein complex; small molecule; transcription factor; virtual

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.

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病的事件有关。 p381 MAP激酶是一个重要的药物发现靶标，药物设计工作主要集中在ATP催化位点的小分子抑制剂上。但是，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限制剂量的不良反应。 p381的活性形式是通过一组辅助激酶和磷酸酶以及底物结合提供的磷酸化事件来调节提体的。因此，表征p381中可以结合底物，抑制剂或变构效应子的其他潜在位点将具有重大关注。该提案中的初步数据确定了p381的活性位点和C-Lobe口袋中的铅化合物。该口袋是从激酶折叠的扩展形成的，仅在一小群激酶，地图，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中的该位点可能适合于设计小分子以结合并有可能以预定的方式调节该激酶的形状和相互作用。因此，拟议的研究将通过定义和比较p381的特定呈进型结合位点来基于这些研究。该建议将检验以下假设：MAP激酶活性和功能可以通过使用短肽或小分子（旨在紧密结合变构相互作用位点）进行特异性调节。在特定的目标1中，将在p381中定义已知的“ D-MOTIF”变构位点的关键功能相互作用，该位点受某些底物和调节酶的约束。目的是产生效应子分子，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。在特定的目标2中，将定义小分子和肽相互作用与较低的C-Lobe口袋的相互作用和潜在调节功能。总体而言，这些研究将与硅虚拟对接方法，肽阵列研究以及体外以及体内激酶活性测定法相结合的最先进的蛋白质晶体学和小角度X射线散射分析。预期的结果将为激酶变构提供关键的机械见解，提供调节酶活性的新的，有针对性的分子，并揭示了无主动位点连接毒性的MAP激酶连接疾病的新治疗策略。此外，这些对p381的试点研究得出的结果和概念可能会导致研究在风湿病模型中测试进一步优化的变构调节剂的功效。