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）：拟议研究的目标是开发高通量筛选，以鉴定肥大激活中赖氨酰-tRNA合成酶（LysRS）转录功能特异的抑制剂。通过释放有效的促炎介质，肥大细胞是引发过敏反应的重要效应器。我们最近的研究表明，蛋白质合成机制的一个重要组成部分——LysRS——是这些介质产生的关键调节因子。肥大细胞的抗原诱导触发 Ser207 上的 LysRS 磷酸化。在没有磷酸化的情况下，LysRS以“闭合”形式与细胞质多tRNA合成酶复合物MSC密切相关，其催化Lys-tRNALys氨酰化反应以进行蛋白质合成。然而，Ser207 的磷酸化会触发 LysRS 形成专门用于转录的“开放”构象异构体。我们的结果表明，通过打开结构，磷酸化的 LysRS 从 MSC 中释放，从细胞质易位到细胞核，并生成 Ap4A 来激活 MITF 靶向基因的转录，从而激活肥大细胞。该路径是第一个示例参与肥大细胞激活调节的管家机器。我们的中心目标是开发新的筛选方法，以高通量筛选 LysRS 开放构象异构体的特定抑制剂。正如目标 1 中所述，我们将生成一种新型的 HTS 兼容的 LysRS 基于细胞的检测方法，并利用 MLP 计划提供的资源并与 MLPCN 中心合作，开展 HTS 活动来识别主要的 LysRS 药效团。一旦确认来自 MLSMR（分子库小分子存储库）集合的初始“命中”，将进行一系列 HTS 兼容的直接筛选，以进一步分类目标 2 中确认的“命中”。这些二级筛选包括一种新型蛋白质-基于蛋白质相互作用的 α 筛选测定、经过验证的基于细胞的转录激活测定和生化活性测定。为了对化合物活性进行排序，将确定 EC50 并评估所选“命中”的化学易处理性。在目标 3 中，我们将开发一种基于功能性细胞的高内涵成像，以量化细胞对 LysRS 易位的效力，以确保化合物的作用机制确实是由于 LysRS 抑制。最后，我们将使用肥大细胞系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），并且我们将根据其调节肥大细胞转录激活的能力对顶级抑制剂进行排序。总的来说，我们的基于细胞和生化检测，以及针对一系列机械筛选的先导化合物分析，将推动 LysRS 新型选择性分子探针的发现，以了解其在肥大细胞激活中的非典型功能，最终目标是开发独特的抗过敏化合物。