A fully validated assay to identify activators of ERSR in human glioma cells: ide

描述（由申请人提供）：成人脑肿瘤占所有癌症的1.3%，占所有癌症相关死亡的2.2%。在美国，成人脑癌每年导致22，000人死亡。这些肿瘤来源于神经胶质细胞，似乎是多灶性的。手术是不成功的，并且总是伴随着在大脑中不同解剖位置的复发的初始切除。这些肿瘤对放疗和化疗有抵抗力。死亡率很高，多年来治疗选择没有显著改善。很明显，需要更多的研究来增加这种癌症的治疗选择，即使在今天，这种癌症的诊断也相当于死刑。 本实验室已获得大量的初步数据，表明胶质瘤细胞对触发内质网应激反应（ERSR）诱导的细胞死亡敏感。另一方面，正常的神经胶质细胞和神经元似乎对ERSR介导的细胞死亡具有抗性。在ERSR期间，人类和大鼠胶质瘤细胞产生更高水平的GRP 78，GRP 78是ERSR的关键架构师，并且是参与ER中蛋白质折叠的伴侣蛋白。后一个发现表明未能管理神经胶质瘤细胞中的蛋白质解折叠。ERSR修复机制的失败反过来又导致ER相关的caspase（4/12）激活，以及随后的细胞凋亡。基于这些证据，我们将U87-MG改造成在GRP 78启动子的控制下表达荧光素酶，并产生稳定的细胞系，以便我们可以鉴定新的ERSR诱导剂。我们已经用两种已知ERSR激活剂（如衣霉素和毒胡萝卜素）的阳性对照品验证了该测定。这两种阳性对照有效且廉价。我们已经设置了成功运行测定所需的所有参数，例如生长培养基、细胞数量、孵育时间、给药方案。Z因子平均评估值高于0.67，CV显著低于10%，对于阴性和阳性对照暴露细胞，S/B均超过100。动态范围较宽，阳性对照品的次最大浓度刺激达到8-10倍。我们使用自动化化合物铺板、手动细胞分配和自动化Bright Glow添加和阅读，针对Prestwick II文库（1120种FDA批准的化合物+一些天然分子）筛选测定，并鉴定了12个命中。我们的后续测试表明，大多数化合物能够上调GRP 78-Luc。我们将筛选后验证集中在3个命中上。螺哌隆确实激活ERSR，上调天然GRP 78，激活ER相关的半胱天冬酶，并导致神经胶质瘤细胞的显著死亡。第二类化合物是从第三种药物布洛芬中开发出来的。奎纳克林调节GRP 78导致胶质瘤细胞死亡。我们还在小鼠的皮肤U87-MG异种移植物中测试了奎纳克林和衍生自初始SAR的化合物，并发现在两周的单一药剂施用中肿瘤生长显著减少。在动物肿瘤标本中也观察到靶向调节。 我们建议使用该测定法对较大的小分子文库（如MLSPCN（200，000））进行自动化高通量筛选。我们将筛选这些文库以1）鉴定能够诱导ERSR的小分子;以支持中心进行命中聚类分析、商业类比和初始基本结构-活性关系分析，寻找感兴趣的化学支架以改进我们的探针2）使用三级测定法验证所选化合物的功效和作用机制鉴定。在这个项目结束时，我们将描述热门化合物的特征，将它们组织为感兴趣的化学类别，测试原型支架对胶质细胞毒性的影响，并收集对其作用机制的见解。我们的工作的附加价值可以确定的可能性，一些击中化合物将有可能启动一个更大的努力，旨在开发和测试活性和安全的分子作为可能的治疗方法，用于对抗成人原发性脑胶质瘤谱系。

DESCRIPTION (provided by applicant): Adult brain tumors represent 1.3% of all the cancers and 2.2% of all the cancer related deaths. Adult brain cancers cause 22,000 deaths every year in the USA. These tumors derive from glial cells and seem to be multifocal. Surgery is unsuccessful and invariably the initial resection is accompanied by relapses at different anatomic locations in the brain. These tumors are resistant to radio- and chemo-therapy. Mortality rate are high and treatment option have not significantly improved over the course of the years. It is clear that more research is needed to increase treatment options in this type of cancer whose diagnosis, even today, represent the equivalent of a death sentence. Our laboratory has produced a large amount of preliminary data indicating that glioma cells are sensible to cell death induced by triggering endoplasmic reticulum stress response (ERSR). On the other hand, normal glial cells and neurons seem to be resistant to ERSR-mediated cell death. Human and rat glioma cells during ERSR produce higher levels of GRP78, the key architect of ERSR and a chaperone involved in protein folding in the ER than astrocytes. This latter finding indicates the failure to manage protein unfolding in glioma cells. Failure of ERSR repair mechanisms in turns is responsible for ER-associated caspase (4/12) activation, and consequent apoptosis. Based on these evidences, we engineered U87-MG to express luciferase under the control of the GRP78 promoter and generated a stable cell line so that we could identify novel ERSR inducers. We have validated the assay with two positive controls known activators of ERSR such as tunicamycin and thapsigargin. These two positive controls are effective and inexpensive. We have set all the parameters needed to successfully run the assay, such as growth medium, cell number, incubation time, drugging protocols. Z factor were assessed above 0.67 on average, CV were significantly below 10%, for both negative and positive controls exposed cells, S/B was in excess of 100. The dynamic range was wide and stimulation reached 8-10 folds with submaximal concentrations of the positive controls. We screened the assay using automated compounds plating, manual cell dispensing and automated Bright Glow addition and reading, against the Prestwick II library (1120 FDA approved compounds + some natural molecules) and identified 12 hits. Our follow up testing has shown that most of the compounds were able to upregulate GRP78-Luc. We focused post screening validation on 3 of the hits. Spiperone does indeed activate ERSR, upregulate native GRP78, activate ER-associated caspases and cause marked death of glioma cells. A second class of compounds has been developed from ibuprofen the third hit. Quinacrine modulates GRP78 resulting in glioma cell death. We have also tested quinacrine and a compound derived from initial SAR in a skin U87-MG xenograft in mice and found a significant reduction of tumor growth in a two week single agent administration. Also target modulation was observed in the animal tumor specimens. We propose to use this assay to perform automated high throughput screen of larger small molecules libraries such as the MLSPCN (200,000). We will screen these libraries to 1) identify small molecules able to induce ERSR; to support the center performing hit clustering analysis, commercial analoging and initial basic structure- activity relationship analysis, looking for interesting chemical scaffolds to refine our probes 2) to validate selected compounds using tertiary assay for efficacy and mechanism of action identification. By the end of this project we will have characterized the hit compounds, organized them for chemical classes of interest, tested the effect of prototypes scaffolds on gliotoxicity, and gathered insight on their mechanism of action. The added value of our work could be identified in the possibility that some of the hit compounds will have the potential to initiate a larger effort aiming to develop and test active and safe molecules as possible therapeutics to use in the battle against adult primary brain tumors of the glial lineage.