HTS for Caspase-8 selective TRAIL Sensitizers

描述(申请人提供)：TRAIL是一种潜在的非常重要的治疗方法，因为它显示了诱导癌细胞凋亡的选择性，而正常细胞是难治性的。TRAIL与死亡受体-4和-5的结合导致caspase-8的募集和典型的下游效应物caspase的激活。与许多药物一样，TRAIL的有效性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的1536孔高通量筛选(HTS)策略，用于识别仅以caspase-8依赖的方式发挥作用的TRAIL敏感剂，而不是通过一般毒性。这将通过利用缺乏caspase-8(NB7)的TRAIL抗性细胞系来实现，而不是使用由该蛋白重组的相同细胞。重要的是，由于假阳性在缺乏caspase-8诱导活性的细胞中的活性，因此在这种检测中，假阳性很容易被“剔除”。我们的Screenig策略的真正力量是基于它识别特定作用于caspase-8凋亡轴的致敏剂的能力，而不是通过普遍的毒性。我们进一步注意到，我们的系统可以识别作用于TRAIL-caspase-8外源性死亡途径中任何分子或过程的试剂，而不仅仅是caspase-8本身。在我们的初步研究中，我们建立了在384孔系统中具有很高统计严谨性(NB7+Caspase-8细胞的Z‘=0.85)的ATP消耗CellTiterGlo细胞存活分析。筛选药物库(LOPAC)已经作为一种概念验证来发现其机制是caspase-8介导的TRAIL增敏剂。使用蛋白酶体抑制剂MG-132作为TRAIL敏化阳性对照试剂，可以监测所有使用的平台的敏感性。在这里，我们建议进一步开发这一检测方法，并优化高度自动化的HTS系统，随后筛选~330,600个MLSMR收集，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，并潜在地识别新的治疗干预点。在目标2中，将通过CaspaseGlo分析系统对获得的任何“HITS”进行二次分析，以测量下游效应物Caspase的活性。同样，这项测试将被开发、优化和小型化到1536孔板格式，并将通过计算Z‘值来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%。所有检测系统都将通过高Z‘值的信号灵敏度进行评估。评估化合物对TRAIL诱导的caspase-8活性的敏化能力应该提供关于探针作用机制的初步线索，然后可以在第三次分析中使用标准分子生物学技术进一步评估这些线索。免疫沉淀和免疫印迹等生化分析与蛋白质组学研究、基因表达分析和功能基因组学筛选相结合，有望提供有价值的作用机制数据。总之，我们提出了一个扩展的~330,600个MLSMR化合物集合的HTS，具有一个独特的筛选策略，优化了对caspase-8途径特异性TRAIL敏感剂的检测。预计这类药物缺乏普遍的细胞毒性，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，这些药物可以用作工具化合物，进一步表征和研究在正常细胞和癌细胞中差异调控的细胞生存和死亡途径。

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.