HTS for inhibitors of RBPJ co-repressor complex

描述（由申请人提供）：本申请是针对PAR-10-182开发HTS测定。目标是确定小分子探针，破坏转录因子RBPJ和SHARP之间的相互作用，SHARP是一种支架蛋白，将其与共抑制因子和组蛋白去乙酰化酶连接起来，以抑制靶基因。在体外细胞培养和小鼠体内，需要小分子抑制剂来探测RBPJ:SHARP相互作用的功能。我们的遗传数据显示，RBPJ [J重组信号序列结合蛋白，又名CSL-1和无毛素抑制因子]的衰减增加了心脏毛细血管的数量，并改善心肌功能和心肌梗死（MI）后的生存。从机制上讲，RBPJ的衰减激活了编码血管生成和心脏保护因子的基因，这些基因可能解释了有益的影响。尽管减弱RBPJ对心脏有深远的影响，但目前仍有许多问题无法解决，如作用时间与心脏病自然进展的关系、剂量效应、以及药物抑制是否能有效维持心脏功能。因此，小分子探针将用于细胞培养和心肌损伤动物模型。先前的研究表明，SHARP:RBPJ界面很小，可能是可药物化的。我们的主要HTS分析将识别破坏RBPJ与SHARP的RBPJ结合域之间结合的分子。在这个项目中，我们已经生成了一个稳定的细胞系用于筛选，该细胞系含有荧光素酶报告基因构建体，并表达RBPJ-VP16和SHARP-GAL4 DNA结合域融合蛋白。这些蛋白之间的相互作用通过转录激活荧光素酶报告基因。初步数据显示一个信号到背景bbbb40和Z' = 0.81。目标1将把它发展成384孔或1536孔格式的筛选试验，并采用合适的阳性和阴性板对照。目标2将使该分析适用于HTS，开发二级TR-FRET分析，以及选择性计数筛选。目的包括优化HTS的分析参数和开发探针开发的关键路径。

DESCRIPTION (provided by applicant): This application is to develop a HTS assay in response to PAR-10-182. The goal is to identify small molecule probes that disrupt the interaction between the transcription factor RBPJ and SHARP, a scaffold protein that links it to co-repressors and histone deacetylases to repress target genes. Small molecule inhibitors are needed to probe the function of the RBPJ:SHARP interaction in vitro cell culture and in vivo in mice. We present genetic data showing that attenuation of RBPJ [J Recombination signal sequence Binding Protein, aka CSL-1 and Suppressor of Hairless, Su(H)] increases the number of capillaries in the heart, and improves heart muscle function and survival after myocardial infarction (MI). Mechanistically, attenuation RBPJ activates genes that encode angiogenic and cardioprotective factors that might account for the beneficial effects. Although attenuating RBPJ profoundly affects the heart, a number of questions remain unapproachable with current tools, such as timing of action relative to the natural progression of heart disease, dose effects, and whether or not pharmacological inhibition would be an effective therapy to maintain cardiac function. Hence, the small molecule probes will be used in cell culture and animal models of myocardial injury. Prior studies have shown that the SHARP:RBPJ interface is small and likely to be druggable. Our primary HTS assay will identify molecules that disrupt binding between RBPJ and the RBPJ-binding domain of SHARP. For this project, we have already generated a stable cell line for screening that contains a luciferase reporter construct and expresses RBPJ-VP16 and SHARP-GAL4 DNA binding domain fusion proteins. Interaction between these proteins transcriptionally activates the luciferase reporter gene. Preliminary data show a signal to background >40 and the Z' = 0.81. Aim 1 will develop this into an assay for screening in 384- or 1536 well format with suitable positive and negative plate controls. Aim 2 will adapt the assay for HTS, develop a secondary TR-FRET assay, as well as counter screens for selectivity. The Aims include optimizing assay parameters for HTS and development of a critical path for probe development. PUBLIC HEALTH RELEVANCE: There is a large unmet need for novel drug targets for heart disease. Myocardial angiogenesis is considered such a target, but the proteins that coordinate production of the multiple factors needed for normal vessel formation remain unclear. The preliminary data are significant because they indicate that RBPJ is critical for this process; hence, this project is to develop tool compounds to test the physiological effects of pharmacological inhibition of RBPJ.