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药物，因为它在诱导癌细胞凋亡方面表现出选择性，而正常细胞则是难治性的。TRAIL与死亡受体-4和-5结合导致caspase-8的募集和下游效应caspase的经典激活。与许多药物一样，TRAIL的效用受到先天或后天耐药性的限制。在目标1中，我们提出开发一种新的1536孔高通量筛选（HTS）策略，用于识别仅以caspase-8依赖方式起作用的trail增敏剂，而不是通过一般毒性作用。这将通过利用缺乏caspase-8 （NB7）的TRAIL抗性细胞系与用该蛋白重组的相同细胞进行比较来实现。重要的是，由于假阳性在缺乏caspase-8诱导活性的细胞中具有活性，因此在该试验中可以很容易地“清除”假阳性。我们的筛选策略的真正力量是基于其识别通过caspase-8凋亡轴特异性作用的致敏剂的能力，而不是通过普遍毒性作用。我们进一步指出，我们的系统可以识别作用于TRAIL-caspase-8外源性死亡途径中涉及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CellTiterGlo细胞存活试验，具有高统计严严性（NB7+Caspase-8细胞的Z′= 0.85）。筛选药理学药物库（LOPAC）已经进行了概念验证，以发现机制是caspase-8介导的TRAIL致敏剂。使用蛋白酶体抑制剂MG-132作为TRAIL致敏阳性对照剂，可以监测所有平台的敏感性。在这里，我们建议进一步发展这一分析，并优化系统，以实现高度自动化的HTS，随后筛选约330,600个MLSMR集合，以确定可能有助于询问与人类疾病相关的信号通路的分子探针，并潜在地确定新的治疗干预点。在Aim 2中，获得的任何“命中”将通过CaspaseGlo检测系统进行二次分析，以测量下游效应物caspase活性。同样，该分析将被开发、优化和小型化到1536孔板格式，并通过计算Z值重新确认统计有效性。更改为1536格式将导致试剂使用减少75%。所有的分析系统将被评估为具有高Z值的信号灵敏度。评估化合物对TRAIL诱导的caspase-8活性的增敏能力应该提供初步的线索，以说明探针的作用机制，然后可以在三级分析期间使用标准分子生物学技术进一步评估。生化分析，如免疫沉淀和免疫印迹对已知信号成分的询问，结合蛋白质组学研究，基因表达分析和功能基因组学筛选，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到330,600个MLSMR化合物集合的HTS，该集合具有独特的筛选策略，用于检测caspase-8途径特异性TRAIL增敏剂。这类药物预计缺乏普遍的细胞毒性，因此可以作为进一步开发的有吸引力的先导化合物。此外，这些药物可以用作工具化合物，进一步表征和研究正常细胞和癌细胞中受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.