Development of Assays for Inhibitors of LysRS in Mast Cell Activation

描述（由申请人提供）：拟议研究的目标是开发高通量筛选，以鉴定在肥大激活中lysyl-tRNA合成酶（LysRS）转录功能特异性抑制剂。肥大细胞通过释放强效的促炎介质，是引发过敏反应的重要效应因子。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 -是这些介质生产的关键调节因子。肥大细胞的抗原诱导触发LysRS在Ser207上的磷酸化。在没有磷酸化的情况下，LysRS以“封闭”的形式与细胞质多trna合成酶复合物MSC密切相关，从而催化lyss - trnalys氨基酰化反应以合成蛋白质。然而，Ser207的磷酸化会触发LysRS变成一个“开放”的构象，仅用于转录。我们的研究结果表明，通过打开结构，磷酸化的LysRS从MSC释放，从细胞质转运到细胞核，并产生Ap4A来激活mitf靶向基因的转录，从而激活肥大细胞。这条路是第一个例子

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.