HTS for Caspase-8 selective TRAIL Sensitizers

用于 Caspase-8 选择性 TRAIL 敏化剂的 HTS

• 批准号: 8262536
• 负责人: KRISTIINA VUORI
• 金额: $ 4.88万
• 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/8262536
• 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; 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; Screening procedure; 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; 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 是一种潜在的非常重要的治疗方法，因为它显示出诱导癌细胞凋亡的选择性，而正常细胞则难以治愈。 TRAIL 与死亡受体 4 和 -5 结合导致 caspase-8 的募集和下游效应器 caspase 的经典激活。与许多药物一样，TRAIL 的用途受到先天或后天耐药性的限制。在目标 1 中，我们建议开发一种新型 1536 孔高通量筛选 (HTS) 策略，用于识别仅以 caspase-8 依赖方式而不是通过一般毒性发挥作用的 TRAIL 敏化剂。与用蛋白质重建的相同细胞相比，这将通过利用缺乏 caspase-8 (NB7) 的 TRAIL 抗性细胞系来实现。重要的是，假阳性命中在该测定中很容易被“清除”，因为它们在缺乏 caspase-8 诱导活性的细胞中具有活性。我们的筛选策略的真正威力是基于其识别敏化剂的能力，这些敏化剂通过 caspase-8 细胞凋亡轴而不是通过普遍毒性发挥作用。我们进一步注意到，我们的系统可以识别作用于 TRAIL-caspase-8 外在死亡途径中涉及的任何分子或过程的药物，而不仅仅是作用于 caspase-8 本身。在我们的初步研究中，我们在 384 孔系统中开发了具有高统计严谨性的 ATP 消耗 CellTiterGlo 细胞存活测定（对于 NB7+Caspase-8 细胞，Z' = 0.85）。药理学药物库 (LOPAC) 的筛选已作为概念验证进行，以发现其机制由 caspase-8 介导的 TRAIL 敏化剂。使用蛋白酶体抑制剂 MG-132 作为 TRAIL 敏化阳性对照剂可以监测所有使用平台的敏感性。在这里，我们建议进一步开发这种测定并优化高度自动化的 HTS 系统，随后筛选约 330,600 个 MLSMR 集合，以期识别可能有助于询问与人类疾病相关的信号通路的分子探针，并可能识别新的治疗干预点。在目标 2 中，将通过 CaspaseGlo 测定系统对获得的任何“命中”进行二次分析，以测量下游效应器 caspase 活性。同样，该测定将被开发、优化并小型化为 1536 孔板格式，并且将通过计算 Z' 值重新确认统计有效性。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高 Z' 值的信号灵敏度。对化合物敏化 TRAIL 诱导的 caspase-8 活性的能力的评估应该提供关于探针作用机制的初步线索，然后可以在三级分析过程中使用标准分子生物学技术进一步评估。生化分析（例如已知信号成分的免疫沉淀和免疫印迹询问）与蛋白质组学研究、基因表达分析和功能基因组学筛选相结合，预计将提供有价值的作用机制数据。 总之，我们提出了对扩展的约 330,600 个 MLSMR 化合物集合进行 HTS，并采用针对 caspase-8 通路特异性 TRAIL 敏化剂的检测进行优化的独特筛选策略。预计此类药物没有普遍的细胞毒性，因此可以作为有吸引力的先导化合物进行进一步开发。此外，此类试剂可用作工具化合物，以进一步表征和研究正常细胞和癌细胞中差异调节的细胞存活和死亡途径。