High throughput bioluminescence screening for glioma therapeutics

描述(由申请人提供)： 肿瘤坏死因子相关的凋亡诱导配体(TRAIL)被认为是一种潜在的抗癌药物，然而相当数量的癌细胞，特别是多形性胶质母细胞瘤(GBM)对TRAIL诱导的细胞凋亡具有抵抗力。该方案的目的是评价自然分泌的Gaussia荧光素酶(Gluc)作为高通量药物筛选报告的作用，以确定使胶质瘤细胞对TRAIL敏感的小分子。不同的胶质瘤细胞将通过基因转移来表达Gluc，通过使用96孔格式板发光仪对等量的条件培养液进行生物发光测量，可以随着时间的推移监测Gluc的表达，从而监测细胞的活力。最初，Gluc分析的不同成分，包括井与井之间的变化，以及底物随时间的稳定性和最佳底物剂量，将针对96孔板格式进行优化。接下来，将测试不同的胶质瘤细胞系，包括从患者肿瘤切片分离的原代GBM细胞对TRAIL的敏感性或抵抗力。最后，将使用小分子库筛选原代GBM细胞，以寻找使其对TRAIL敏感的药物，其中一些是FDA批准的已知可以穿越血脑屏障的药物。使原代GBM细胞对TRAIL敏感的候选药物将在体外剂量和时间依赖的实验中进一步表征，并可能在我们的实验性胶质瘤-Gluc模型中进一步表征。4秒的Gluc分析时间使其成为HTS的宝贵工具，并可轻松应用于384孔板格式，在相对较短的时间内筛选数千种化合物。

DESCRIPTION (provided by applicant): Tumor Necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anti-cancer agent, however, considerable numbers of cancer cells, especially glioblastoma multiforme (GBM), are resistant to apoptosis induction by TRAIL. The aim of this proposal is to evaluate the naturally secreted Gaussia Luciferase (Gluc) as a reporter for high throughput drug screening in order to identify small molecules that sensitize glioma cells to TRAIL. Different glioma cells will be engineered by gene transfer to express Gluc which expression, and therefore cell viability, can be monitored over time by subjecting an aliquot of the conditioned medium to bioluminescence measurements using a 96-wells format plate luminometer. Initially, different components of the Gluc assay including well-to-well variation, as well as substrate stability over time and optimal substrate dose will be optimized for a 96-well plate format. Next, different glioma cell lines, including primary GBM cells dissociated from patient tumor sections, will be tested for their sensitivity or resistance to TRAIL. Finally, primary GBM cells will be screened for drugs that will sensitize them to TRAIL using small molecule libraries some of which are FDA-approved drugs known to cross the blood-brain barrier. The drug candidates that sensitize primary GBM cells to TRAIL will be further characterized in dose- and time-dependent experiments in vitro and potentially in our experimental glioma-Gluc models, in vivo. The Gluc assay time of 4 seconds makes it a valuable tool for HTS and can be easily applied into 384-well plate format to screen thousands of compounds over a relatively short period of time.