HTS for Caspase-8 selective TRAIL Sensitizers

用于 Caspase-8 选择性 TRAIL 敏化剂的 HTS

• 批准号: 8400429
• 负责人: KRISTIINA VUORI
• 金额: $ 4.73万
• 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: 2011-12-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8400429
• 关键词: Adverse effects; Apical; Apoptosis; Apoptotic; Binding; Biochemical; Biological Assay; Blocking Antibodies; Caspase; Cell Death; Cell Line; Cell Survival; Cells; Cessation of life; Chemotherapy-Oncologic Procedure; Clinical Trials; Collaborations; Collection; Communities; Complex; Consumption; Coupled; DEVDase; Data; Detection; Development; Engineering; Excision; Gene Expression Profiling; Health Benefit; Immunoblotting; Immunoprecipitation; Induction of Apoptosis; Lead; Libraries; Ligands; MG132; Malignant Neoplasms; Measures; Mediating; Methodology; Molecular; Molecular Biology Techniques; Molecular Probes; Monitor; Normal Cell; Pathway interactions; Peptide Hydrolases; Pharmaceutical Preparations; Post Translational Modification Analysis; Process; Proteasome Inhibitor; Proteins; Proteomics; Protocols documentation; PubChem; Public Health; Reagent; Refractory; Research; Resistance; Signal Pathway; Signal Transduction; Stress; Sum; System; TNF-related apoptosis-inducing ligand; TNFRSF10A gene; TNFRSF10B gene; TNFSF10 gene; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Toxic effect; Tumor Necrosis Factor Ligand Superfamily Member 6; Work; base; cancer cell; caspase-8; data sharing; death receptor-4; functional genomics; high throughput screening; human TNFRSF10A protein; human disease; innovation; interest; kinase inhibitor; miniaturize; neoplastic cell; neuroblastoma cell; novel; novel therapeutic intervention; novel therapeutics; prevent; protease Re; receptor; reconstitution; response; screening; tool; tumor progression

DESCRIPTION (provided by applicant): TRAIL is a potentially very important therapeutic as it shows selectivity for inducing apoptosis in cancer cells whilst normal cells are refractory. TRAIL binding to Death Receptors-4 and -5 leads to recruitment of caspase-8 and classical activation of downstream effectors caspases. As with many drugs, TRAIL's usefulness is limited by resistance, either innate or acquired. In Aim 1, we propose the development of a novel 1536-well high-throughput screening (HTS) strategy for identifying TRAIL-sensitizing agents that act solely in a caspase-8 dependent manner, rather than through general toxicity. This will be accomplished by utilizing a TRAIL resistant cell line lacking caspase-8 (NB7) compared to the same cells reconstituted with the protein. Importantly, false positive hits can easily be "weeded out" in this assay due to their activity in cells lacking caspase-8-inducible activity. Our screenig strategy's real power is based in its ability to identify sensitizing agents that act specifically through the caspase-8 apoptotic axis rather than through generalized toxicity. We further note that our system can identify agents acting on any molecule or process involved in the TRAIL-caspase-8 extrinsic death pathway and not solely on caspase-8 itself. In our preliminary studies, we have developed the ATP consumption CellTiterGlo cell survival assay in a 384-well system with high statistical rigor (Z' = 0.85 for the NB7+Caspase-8 cells). Screening of the library of pharmacological agents (LOPAC) has already been performed as a proof-of-concept to discover TRAIL sensitizers whose mechanism is caspase-8 mediated. Use of the proteasome inhibitor MG-132 as a TRAIL sensitizing positive control agent allows for monitoring of sensitivity across all platforms used. Here, we propose to develop this assay further and optimize the system for highly automated HTS, and subsequently to screen the ~330,600 MLSMR collection with a view to identifying molecular probes that may assist in the interrogation of signaling pathways relevant to human disease and to potentially identify novel therapeutic intervention points. In Aim 2, secondary analysis of any "hits" obtained will be carried out by the CaspaseGlo assay system, in order to measure downstream effectors caspase activity. Again this assay will be developed, optimized and miniaturized to 1536-well plate format and statistical validity will be re confirmed by calculating Z' values. Change to 1536-format will result in reductions in reagent use of 75%. All assay systems will be assessed for signal sensitivity with high Z' values. Assessment of compounds ability to sensitize TRAIL induced caspase-8 activity should provide initial clues as to say probes mechanisms of action, which can then be further assessed using standard molecular biology techniques during tertiary analysis. Biochemical analysis such as immunoprecipitation and immunoblot interrogation of the known signaling components coupled with proteomic studies, gene expression analysis and functional genomics screens are expected to provide valuable mechanism of action data. In sum, we propose a HTS of the expanded ~330,600 MLSMR collection of compounds with a unique screening strategy optimized for the detection of caspase-8 pathway specific TRAIL sensitizing agents. Such agents are expected to lack generalized cellular toxicity, and could therefore function as attractive lead compounds for further development. Additionally, such agents can be used as tool compounds to further characterize and study cellular survival and death pathways that are differentially regulated in normal and cancer cells.

描述（由申请人提供）：TRAIL是一种潜在非常重要的治疗性，因为它表明了诱导癌细胞凋亡的选择性，而正常细胞是难治性的。与死亡受体-4和-5结合的跟踪导致caspase-8的募集和下游效应子胱天蛋白酶的经典激活。与许多药物一样，Trail的用途受到先天或获得的阻力的限制。在AIM 1中，我们提出了一种新型的1536孔高通量筛查（HTS）策略的开发，用于识别仅以caspase-8依赖性方式起作用的跟踪敏感剂，而不是通过一般的毒性。与与蛋白质重构的相同细胞相比，使用缺乏caspase-8（NB7）的抗轨道细胞系来实现这一点。重要的是，由于缺乏caspase-8诱导活性的细胞活性，因此可以在该测定中轻松“淘汰”假阳性命中。我们的Screenig策略的真正力量基于其识别特有通过caspase-8凋亡轴而不是通过普遍毒性起作用的敏化剂的能力。我们进一步指出，我们的系统可以识别作用于TRAIL-Caspase-8外在死亡途径的任何分子或过程的药物，而不仅仅是caspase-8本身。在我们的初步研究中，我们在384孔系统中开发了ATP消耗细胞存活测定法具有高统计严格的严格性（NB7+Caspase-8细胞的Z'= 0.85）。筛选药理学剂图书馆（LOPAC）已被作为概念验证，以发现其机制为caspase-8介导的步道灵敏度。将蛋白酶体抑制剂MG-132用作径向敏化阳性对照剂，可以监视所有使用的平台上的灵敏度。在这里，我们建议进一步开发该测定法，并优化高度自动化的HTS系统，然后筛选〜330,600 MLSMR收集，以识别可能有助于询问与人类疾病相关的信号通路并潜在地识别新的治疗干预点的分子探针。在AIM 2中，CaspaseGlo分析系统将对获得的任何“命中”进行二级分析，以测量下游效应子caspase活性。同样，该测定将开发，优化和小型化至1536孔板格式，并通过计算Z值来确认统计有效性。更改为1536格式将导致试剂使用量减少75％。所有测定系统都将评估具有高Z值的信号灵敏度。评估化合物使TRAIL诱导的caspase-8活性敏感的能力应提供的初始线索，以说探针的作用机制，然后可以在高等教育分析过程中使用标准分子生物学技术进一步评估。对已知信号传导成分与蛋白质组学研究，基因表达分析和功能基因组学筛查的已知信号传导成分的免疫沉淀和免疫印迹询问等生化分析有望提供有价值的动作数据机制。 总而言之，我们提出了扩展的〜330,600 mLSMR集合的HTS，其化合物集合，具有针对检测caspase-8途径特异性跟踪敏化剂的独特筛选策略。预计此类药物将缺乏普遍的细胞毒性，因此可以充当进一步发展的有吸引力的铅化合物。此外，此类药物可以用作工具化合物，以进一步表征和研究在正常和癌细胞中受到差异调节的细胞存活和死亡途径。