Development of Assays for Inhibitors of LysRS in Mast Cell Activation

肥大细胞激活中 LysRS 抑制剂检测方法的开发

基本信息

批准号：
8729500
负责人：
Min Guo
金额：
$ 35.91万
依托单位：
SCRIPPS FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-05 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8729500
关键词：
Affect Allergic Amino Acyl-tRNA Synthetases Aminoacylation Animal Model Antigens Area Binding Biochemical Biological Assay Cell Line Cell Nucleus Cells Cellular biology Chemicals Collaborations Collection Complex Coupled Cytoplasm Cytoplasmic Granules Disease Ensure Enzymes Fluorescence Resonance Energy Transfer Foundations Future Gene Targeting Genetic Transcription Goals Histamine Housekeeping Human Hypersensitivity Image Immune In Vitro Inflammation Mediators Inhibitory Concentration 50 Lead Libraries Luc Gene Luciferases Lysine-Specific tRNA Lysine-tRNA Ligase MAPK14 gene Measurement Mediator of activation protein Molecular Bank Molecular Probes Monitor Pathway interactions Peptide Hydrolases Phase Phosphorylation Production Protein Biosynthesis Reaction Regulation Regulator Genes Reporter Research Resources Series Signaling Protein Specificity Structure Testing Therapeutic Time Toxic effect Transcriptional Activation Triage Tryptase Two-Hybrid System Techniques Validation allergic response assay development base conformer cytokine design diadenosine tetraphosphate high throughput screening immune activation inhibitor/antagonist mast cell miniaturize novel pharmacophore protein protein interaction repository response screening small molecule tool

项目摘要

DESCRIPTION (provided by applicant): The goals of the proposed research are to develop high throughput screens to identify inhibitors specific for the transcriptional function of lysyl-tRNA synthetase (LysRS) in mast activation. By releasing potent pro-inflammatory mediators, mast cell is an essential effectors in the elicitation of allergic responses. Our recent studies indicate that an essential component of the protein synthesis machinery-LysRS --is a key regulator for the production of these mediators. Antigen induction of mast cells triggers the phosphorylation of LysRS on Ser207. In the absence of phosphorylation, LysRS is strongly associated with the cytoplasmic multi-tRNA synthetase complex MSC in a "closed" form, which catalyzes Lys-tRNALys aminoacylation reaction for protein synthesis. However, phosphorylation of Ser207 triggers LysRS into an "open" conformer that functions exclusively for transcription. Our results show that, by opening up the structure, phosphorylated LysRS is released from MSC, translocates from cytoplasm to the nucleus, and generates Ap4A to activate the transcription of MITF-targeted genes for mast cell activation. This pathway is the first example of a housekeeping machinery involved in the regulation of mast cell activation. Our central goal is to develop novel screening assays to allow a high throughput screen for specific inhibitors of the LysRS open conformer. As outlined in Aim 1, we will generate a novel HTS-compatible LysRS cell-based assay and, using the resources provided by the MLP initiative and in collaboration with a MLPCN center, will perform a HTS campaign to identify lead LysRS pharmacophores. Once initial 'hits' derived from the MLSMR (molecular libraries small molecule repository) collection are confirmed, a series of HTS-compatible direct screen will be performed to further triage confirmed 'hits' in Aim 2. These secondary screens include a novel protein-protein interaction-based alpha screen assay, a validated cell-based transcription activation assay, and a biochemical activity assay. To rank order compound activity, EC50's will be determined and the chemical tractability of the chose 'hits' will be evaluated. In Aim 3, we will develop a functional cell-based high-content imaging, to quantify cellular potency on the translocation of LysRS, to ensure that the mechanism of action of compounds is indeed due to LysRS inhibition. Finally, using a mast cell line, we will test if our inhibitors affect the mast cell immune activaton (e.g., the expression of MCP6, TPH) and we will rank order of top inhibitors for their ability to modulate transcriptional activation in mast cells. Collectively, our cell-based and biochemical assays, along with profiling lead compounds against a series of mechanistic screens will drive the discovery of LysRS novel and selective molecular probes for its non-canonical function in mast cell activation, with an ultimate goal of developing unique anti-allergy compound.

描述（由申请人提供）：拟议的研究的目标是开发高吞吐量筛选，以识别针对桅杆激活中赖氨酸-TRNA合成酶（LYSR）转录功能的抑制剂。通过释放有效的促炎性介质，肥大细胞是引起过敏反应的重要效应子。我们最近的研究表明，蛋白质合成机械 - lysrs的重要组成部分 - 是生产这些介体的关键调节剂。肥大细胞的抗原诱导会触发Ser207上LYSR的磷酸化。在没有磷酸化的情况下，LYSR与“封闭”形式中的细胞质多TRNA合成酶复合MSC密切相关，该形式催化了lys-trnalys氨基酰基化反应以促进蛋白质合成。然而，Ser207的磷酸化触发了LYSR，进入了专门用于转录的“开放”构象体。我们的结果表明，通过打开结构，磷酸化的LYSR从MSC释放，从细胞质转移到核，并生成AP4A以激活MITF靶向基因的转录以进行肥大细胞激活。该途径是第一个示例涉及肥大细胞激活调节的管家机械。我们的中心目标是开发新颖的筛选测定法，以允许对LYSRS开放构象体的特定抑制剂进行高吞吐量屏幕。如AIM 1中概述的那样，我们将生成一种新颖的HTS兼容LYSRS基于细胞的测定法，并使用MLP计划提供的资源并与MLPCN中心合作，将执行HTS运动，以识别Lysr Lysrs Pharmacophores。 Once initial 'hits' derived from the MLSMR (molecular libraries small molecule repository) collection are confirmed, a series of HTS-compatible direct screen will be performed to further triage confirmed 'hits' in Aim 2. These secondary screens include a novel protein-protein interaction-based alpha screen assay, a validated cell-based transcription activation assay, and a biochemical activity assay.为了对顺序复合活动进行排名，将确定EC50，并评估选择“命中”的化学障碍。在AIM 3中，我们将开发一种基于功能性细胞的高含量成像，以量化LYSR易位的细胞效力，以确保化合物的作用机理确实是由于LYSR抑制作用所致。最后，使用肥大细胞系，我们将测试我们的抑制剂是否影响肥大细胞免疫激活（例如，MCP6，TPH的表达），我们将对它们调节肥大细胞中转录激活的能力对顶部抑制剂进行排序。总的来说，我们的基于细胞的和生化测定，以及针对一系列机械筛选的铅化合物的分析，将推动LYSRS新颖和选择性分子探针在肥大细胞激活中的非基本功能，并最终的目标是开发独特的抗抗校准化合物。