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

描述(申请人提供)：成人脑瘤占所有癌症的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。这些肿瘤起源于神经胶质细胞，似乎是多灶性的。手术是不成功的，最初的切除总是伴随着大脑中不同解剖位置的复发。这些肿瘤对放化疗有抵抗力。多年来，死亡率很高，治疗选择也没有明显改善。显然，需要更多的研究来增加这种癌症的治疗选择，即使在今天，这种癌症的诊断相当于死刑。我们的实验室已经产生了大量的初步数据，表明胶质瘤细胞对触发内质网应激反应(ERSR)而导致的细胞死亡很敏感。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抵抗力。在ERSR过程中，人和大鼠胶质瘤细胞产生更高水平的GRP78，GRP78是ERSR的关键设计师，也是参与ER中蛋白质折叠的伴侣，而不是星形胶质细胞。后一项发现表明，未能控制胶质瘤细胞中蛋白质的展开。ERSR修复机制的失败依次导致ER相关的caspase(4/12)激活，从而导致细胞凋亡。基于这些证据，我们构建了U87-MG，在GRP78启动子的控制下表达荧光素酶，并建立了稳定的细胞系，为寻找新的ERSR诱导剂奠定了基础。我们已经用两个已知ERSR激活剂的阳性对照，如衣霉素和thapsigargin，验证了该方法。这两个积极的控制措施是有效的，而且成本低廉。我们已经设置了成功运行检测所需的所有参数，如生长介质、细胞数量、孵育时间、给药方案。Z因子平均在0.67以上，变异系数显著低于10%，阴性对照和阳性对照暴露细胞的S/B均大于100。动态范围宽，刺激倍数达8~10倍，阳性对照浓度低于最大值。我们使用自动化合物电镀、手动细胞分配和自动Bright Glow添加和读取，对照Prestwick II文库(1120个FDA批准的化合物+一些天然分子)进行筛选，鉴定出12个匹配项。我们的后续测试表明，大多数化合物都能够上调GRP78-Luc。我们将重点放在3部热门影片的筛选后验证上。螺环酮确实激活了ERSR，上调了天然的GRP78，激活了ER相关的caspase，并导致了胶质瘤细胞的显著死亡。布洛芬是从第三个热门药物布洛芬开发出来的第二类化合物。奎纳克林调节GRP78，导致胶质瘤细胞死亡。我们还在小鼠皮肤U87-MG移植瘤中测试了喹卡林和一种源自初始SAR的化合物，发现在两周的单一药物治疗中，肿瘤生长显著减少。在动物肿瘤标本中也观察到靶向调节。我们建议使用这种方法对较大的小分子文库进行自动化的高通量筛选，如MLSPCN(200,000)。我们将筛选这些文库以1)鉴定能够诱导ERSR的小分子；为了支持该中心进行HIT聚类分析、商业类比和初步的基本构效关系分析，寻找有趣的化学支架来提炼我们的探针2)使用三级实验来验证选定的化合物的有效性和作用机制。到本项目结束时，我们将表征HIT化合物，将它们组织为感兴趣的化学类别，测试原型支架对神经胶质毒性的影响，并收集对其作用机制的见解。我们工作的附加值可以从以下可能性中确定出来：一些受攻击的化合物可能会启动一项更大的努力，旨在开发和测试活性和安全的分子，作为可能的治疗方法，用于对抗成人原发胶质细胞肿瘤。

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.