HTS for Caspase-8 selective TRAIL Sensitizers

描述（由申请人提供）：TRAIL是一种潜在的非常重要的治疗剂，因为它显示出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的结合导致半胱天冬酶-8的募集和下游效应物半胱天冬酶的经典活化。与许多药物一样，TRAIL的有效性受到先天或后天抗性的限制。在目标1中，我们提出了一种新的1536孔高通量筛选（HTS）策略的发展，用于识别TRAIL敏化剂，其仅以caspase-8依赖的方式起作用，而不是通过一般毒性。这将通过利用与用蛋白质重构的相同细胞相比缺乏半胱天冬酶-8（NB 7）的TRAIL抗性细胞系来实现。重要的是，由于假阳性命中在缺乏半胱天冬酶-8诱导活性的细胞中的活性，在该测定中可以容易地“清除”假阳性命中。我们的筛选策略的真实的力量是基于它能够识别特异性通过caspase-8凋亡轴而不是通过全身毒性起作用的致敏剂。我们进一步注意到，我们的系统可以识别作用于TRAIL-胱天蛋白酶-8外源性死亡途径中涉及的任何分子或过程的试剂，而不仅仅是胱天蛋白酶-8本身。在我们的初步研究中，我们已经在384孔系统中开发了ATP消耗CellTiterGlo细胞存活测定，具有高统计学严谨性（对于NB 7+胱天蛋白酶-8细胞，Z' = 0.85）。药理学试剂库（LOPAC）的筛选已经作为概念验证进行，以发现其机制是caspase-8介导的TRAIL敏化剂。使用蛋白酶体抑制剂MG-132作为TRAIL致敏阳性对照剂允许监测所有使用的平台的灵敏度。在这里，我们建议进一步开发这种检测方法，并优化高度自动化HTS的系统，随后筛选约330，600 MLSMR集合，以确定可能有助于询问与人类疾病相关的信号传导途径的分子探针，并可能确定新的治疗干预点。在目的2中，将通过CaspaseGlo测定系统对获得的任何“命中”进行二次分析，以测量下游效应物胱天蛋白酶活性。将再次开发、优化并将该测定小型化至1536孔板格式，并通过计算Z'值重新确认统计学有效性。变更为1536格式将导致试剂使用量减少75%。将评估所有测定系统的高Z'值信号灵敏度。化合物敏化TRAIL诱导的半胱天冬酶-8活性的能力的评估应提供关于所述探针作用机制的初步线索，其然后可在三级分析期间使用标准分子生物学技术进一步评估。生物化学分析，如免疫沉淀和免疫印迹询问已知的信号成分，加上蛋白质组学研究，基因表达分析和功能基因组学筛选，预计将提供有价值的作用机制的数据。 总之，我们提出了一个HTS的扩大~ 330，600 MLSMR收集的化合物与独特的筛选策略优化的检测caspase-8途径特异性TRAIL敏化剂。预期此类试剂缺乏全身细胞毒性，因此可作为有吸引力的先导化合物用于进一步开发。此外，这些试剂可以用作工具化合物，以进一步表征和研究在正常细胞和癌细胞中差异调节的细胞存活和死亡途径。

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.