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，以确定在肥大激活中赖氨酰-tRNA合成酶(LysRS)转录功能的特异性抑制物。肥大细胞通过释放强有力的促炎介质，是诱发过敏反应的重要效应者。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-是这些介体产生的关键调节因子。肥大细胞的抗原诱导触发Ser207上LysRS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的多tRNA合成酶复合体MSC结合，MSC催化Lys-tRNALys氨酰化反应合成蛋白质。然而，Ser207的磷酸化将LysRS触发为一种“开放”的构象，其功能仅限于转录。我们的结果表明，通过开放结构，磷酸化的LysRS从MSC释放，从细胞质转移到细胞核，并产生Ap4A来激活MITF靶向基因的转录，从而激活肥大细胞。这条路径是第一个例子 肥大细胞激活过程中涉及的一种内务管理机构。我们的中心目标是开发新的筛选分析方法，以便高通量筛选LysRS开放构象的特定抑制剂。如目标1所述，我们将生成一种新的HTS兼容的LysRS细胞化验方法，并将利用MLP倡议提供的资源并与MLPCN中心合作，开展HTS活动，以确定领先的LysRS药效团。一旦来自MLSMR(分子文库小分子储存库)收集的初始“HITS”得到确认，将进行一系列HTS兼容的直接筛选，以进一步对AIM 2中确认的“HITS”进行分类。这些二次筛选包括一种新的基于蛋白质相互作用的阿尔法筛选分析、一种有效的基于细胞的转录激活分析和一种生化活性分析。为了对化合物活性进行排序，将确定EC50‘S’，并将评估所选‘HITS’的化学易处理性。在目标3中，我们将开发一种基于功能细胞的高含量成像，以定量细胞对LysRS转位的效力，以确保化合物的作用机制确实是由于LysRS的抑制。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的免疫激活(例如，MCP6、TPH的表达)，并根据它们调节肥大细胞转录激活的能力对顶级抑制剂进行排序。总体而言，我们基于细胞和生化的分析，以及针对一系列机械筛查的先导化合物，将推动LysRS新型选择性分子探针的发现，以实现其在肥大细胞激活中的非规范功能，最终目标是开发独特的抗过敏化合物。

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.