HTS for inhibitors of RBPJ co-repressor complex

RBPJ 共阻遏物复合物抑制剂的 HTS

• 批准号: 8182854
• 负责人: MARK MERCOLA
• 金额: $ 19.1万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8182854
• 关键词: Accounting; Address; Adult; Affect; Animal Model; Attenuated; Award; Binding; Binding Proteins; Biochemical; Biological Assay; Blood capillaries; Cardiac; Cardiac Myocytes; Cell Culture Techniques; Cells; Cessation of life; Chimeric Proteins; Complex; Critical Pathways; DNA Binding Domain; Data; Development; Development Plans; Dose; Drug Delivery Systems; Embryonic Development; Equipment; Fluorescence Resonance Energy Transfer; Funding; Future; Gene Targeting; Genes; Genetic; Genetic Recombination; Genetic Transcription; Goals; Heart; Heart Diseases; Histones; In Vitro; Individual; Injury; Institutes; Knock-out; Libraries; Ligands; Link; Luciferases; Mediating; Modeling; Mus; Muscle function; Myocardial; Myocardial Infarction; Myocardium; Pathologic Processes; Peptide Signal Sequences; Performance; Physiological; Plasmids; Process; Production; Proteins; Protocols documentation; Reading; Reagent; Recruitment Activity; Relative (related person); Reporter; Reporter Genes; Repression; Reverse Transcriptase Polymerase Chain Reaction; Scaffolding Protein; Screening procedure; Signal Transduction; Testing; Therapeutic; Time; Transfection; Transgenic Organisms; United States National Institutes of Health; VP 16; angiogenesis; assay development; attenuation; base; capillary; cost; design; effective therapy; follow-up; heart function; high throughput screening; improved; in vivo; inhibitor/antagonist; notch protein; novel; prevent; research study; response; small molecule; stable cell line; therapeutic effectiveness; tool; transcription factor

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.

描述（由申请人提供）：本申请旨在开发针对 PAR-10-182 的 HTS 测定。目标是鉴定破坏转录因子 RBPJ 和 SHARP 之间相互作用的小分子探针，SHARP 是一种支架蛋白，将 RBPJ 与共阻遏物和组蛋白脱乙酰酶连接起来，从而抑制靶基因。需要小分子抑制剂来探索体外细胞培养物和小鼠体内 RBPJ:SHARP 相互作用的功能。我们提供的遗传数据显示，RBPJ [J 重组信号序列结合蛋白，又名 CSL-1 和无毛抑制因子，Su(H)] 的减弱可增加心脏中毛细血管的数量，并改善心肌梗塞 (MI) 后的心肌功能和存活率。从机制上讲，RBPJ 减弱会激活编码血管生成和心脏保护因子的基因，这可能是产生有益效果的原因。尽管减弱 RBPJ 对心脏有深远影响，但目前的工具仍然无法解决许多问题，例如相对于心脏病自然进展的作用时间、剂量效应以及药物抑制是否是维持心脏功能的有效疗法。因此，小分子探针将用于细胞培养和心肌损伤的动物模型。先前的研究表明 SHARP:RBPJ 界面很小并且可能是可药物化的。我们的主要 HTS 测定将识别破坏 RBPJ 和 SHARP 的 RBPJ 结合域之间结合的分子。对于这个项目，我们已经生成了用于筛选的稳定细胞系，其中包含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。这些蛋白质之间的相互作用在转录上激活荧光素酶报告基因。初步数据显示背景信号 >40，Z' = 0.81。目标 1 将其开发为一种用于在 384 或 1536 孔格式中进行筛选的测定方法，并具有合适的阳性和阴性板对照。目标 2 将调整 HTS 检测方法，开发二级 TR-FRET 检测方法，以及选择性计数器筛选。目标包括优化 HTS 的测定参数和开发探针开发的关键路径。 公共卫生相关性：心脏病的新药物靶点的需求尚未得到满足。心肌血管生成被认为是这样的目标，但协调正常血管形成所需的多种因子的产生的蛋白质仍不清楚。初步数据很重要，因为它们表明 RBPJ 对此过程至关重要；因此，该项目旨在开发工具化合物来测试RBPJ药理抑制的生理效应。