Development of Assays for Inhibitors of LysRS in Mast Cell Activation

描述（由申请人提供）：拟定研究的目标是开发高通量筛选，以鉴定对肥大细胞激活中赖氨酰-tRNA合成酶（LysRS）的转录功能具有特异性的抑制剂。肥大细胞通过释放强有力的促炎介质，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器。肥大细胞的抗原诱导触发Ser 207上LysRS的磷酸化。在没有磷酸化的情况下，LysRS以“封闭”形式与细胞质多tRNA合成酶复合物MSC强烈相关，其催化Lys-tRNALys氨酰化反应以进行蛋白质合成。然而，Ser 207的磷酸化触发LysRS成为专门用于转录的“开放”构象异构体。我们的研究结果表明，通过打开结构，磷酸化的LysRS从MSC释放，从细胞质易位到细胞核，并产生Ap 4A，以激活MITF靶向基因的转录，从而激活肥大细胞。这条途径是第一个 一种参与调节肥大细胞活化的管家机制。我们的中心目标是开发新的筛选方法，以允许高通量筛选LysRS开放构象异构体的特异性抑制剂。如目标1所述，我们将开发一种新的HTS兼容的基于LysRS细胞的检测方法，并利用MLP计划提供的资源，与MLPCN中心合作，开展HTS活动，以鉴定主导LysRS药效团。一旦确认源自MLSMR（分子库小分子库）收集的初始“命中”，将进行一系列HTS兼容的直接筛选，以进一步筛选目标2中确认的“命中”。这些二次筛选包括基于新型蛋白质-蛋白质相互作用的α筛选测定、经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，将测定EC 50，并评价所选“命中物”的化学易处理性。在目标3中，我们将开发基于功能细胞的高含量成像，以量化LysRS易位的细胞效力，以确保化合物的作用机制确实是由于LysRS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免疫激活（例如，MCP 6、TPH的表达），我们将根据它们调节肥大细胞中转录激活的能力对顶级抑制剂进行排序。总的来说，我们的基于细胞的和生物化学测定，沿着针对一系列机制筛选分析先导化合物，将推动发现LysRS新型和选择性分子探针，用于其在肥大细胞活化中的非典型功能，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.