High-throughput screen for specific small-molecule inhibitors of HIF-2A activity

HIF-2A 活性特异性小分子抑制剂的高通量筛选

基本信息

批准号：
8262506
负责人：
Mei Yee Koh
金额：
$ 3.95万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-02-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8262506
关键词：
Address Aerobic Antineoplastic Agents Apoptotic Attenuated BNIP3L gene Binding Binding Sites Biological Biological Assay Cell Line Cell model Cells Chemicals Collection Complement Data Development Dimethyl Sulfoxide Epidermal Growth Factor Receptor Epithelial Gene Targeting Genes Genomics Glioblastoma Goals Growth Hour Hypoxia Hypoxia Inducible Factor Hypoxia-Responsive Elements In Vitro Laboratories Lead Libraries Link Luciferases Maintenance Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of brain Mediator of activation protein Messenger RNA Minority Modeling Morbidity - disease rate Neoplasm Metastasis Neuroblastoma Non-Small-Cell Lung Carcinoma Oncogene Proteins Operative Surgical Procedures Pathway interactions Patients Pharmaceutical Preparations Population Process Prognostic Factor Protein Isoforms Radiation therapy Reading Recurrence Regulation Renal Cell Carcinoma Renal carcinoma Reporter Role Screening procedure Signal Transduction Small Interfering RNA Solid Neoplasm Stem cells Tandem Repeat Sequences Testing Therapeutic Time United States National Institutes of Health Validation Vascular Endothelial Growth Factors Work base c-myc Genes cancer stem cell cancer therapy computerized data processing counterscreen epithelial to mesenchymal transition experience functional outcomes genome-wide high throughput screening hypoxia inducible factor 1 in vivo inhibitor/antagonist insight neoplastic neoplastic cell novel novel strategies outcome forecast promoter response self-renewal small molecule stem cell differentiation stem cell population therapy resistant tissue culture transcription factor tumor tumor initiation tumor progression

项目摘要

DESCRIPTION (provided by applicant): Most solid tumors and their metastases experience regions of hypoxia, which promotes both tumor progression and resistance to therapy. Hypoxia is also important for the proliferation and maintenance of cancer stem cells (CSC), a minority population within the heterogenous tumor mass capable of generating the diverse tumor cell population and implicated in tumor recurrence following therapy. Critical mediators of the hypoxic response are the hypoxia-inducible factors HIF-1¿ and HIF-2¿, non-redundant transcription factors regulating both overlapping and unique downstream target genes. HIF-2¿ is an important driver of tumor morbidity in renal cell carcinoma (RCC), neuroblastoma, glioblastoma (GBM) and non small cell lung cancer (NSCLC). This has been linked with the ability of HIF-2¿ (but not HIF-1¿) to drive proliferation, invasion and to promote the maintenance and growth of cancer stem cells within these tumor types. In this setting, high HIF-1¿ predicts for better patient prognosis. Hence, we propose that the specific inhibition of HIF-2¿ will be a useful strategy for treating HIF-2¿ driven tumors and for targeting the CSC population. Our goal is to identify small molecule specific inhibitors of HIF-2¿ that will lead to the development of ne anti-cancer therapies. We have generated 786-0 RCC cells stably expressing a hypoxia responsive element (HRE)-luciferase construct. The 786-0 HRE cells do not express HIF-1¿ and we will use these cells to screen the NIH MLSMSR collection for inhibitors of HIF-2¿. We have performed the necessary optimization assays for the 786-0 HRE cells in 384-well plates and find that these cells are suitable for HTS using our pan-HIF inhibitor PX478 as a positive control. Signal to background ratios were ~20, Z' factor for optimized assay conditions was 0.67, maximum DMSO concentration tolerated was 0.75% (v/v) and total assay time from cell seeding to luciferase reading was 48 hours. As a counter screen, we have generated the MIAPACA-2 HRE cells that only express HIF-1¿ and we will use these cells to eliminate hits that also inhibit HIF-1¿. As a validation screen, we will use the PANC HRE cells in which HRE luciferase activity is HIF-2¿ dependent to confirm HIF-2¿ inhibition by hits from the primary screen. As an indicator of potential anti-tumor activity, hit compounds will be evaluated using secondary assays to determine their ability to inhibit HIF-2¿ downstream target genes and functional outcomes such as epithelial to meseenchymal transition and 3D colony formation in relevant cell models such has RCC and GBM. The proposed screen will lead to the identification of novel HIF-2¿ specific inhibitors that will provide structural leads for new anti-cancer therapies. Additionally, this screen will also facilitate the identification pan HIF-1¿/ HIF-2¿ inhibitor that may have therapeutic application and provide new insights into novel mechanisms of HIF-1/2¿ is form specific regulation.

描述（由适用提供）：大多数实体瘤及其转移经历了缺氧的区域，这促进了肿瘤的进展和抗治疗的耐药性。缺氧对于癌症干细胞的增殖和维持（CSC）也很重要，癌症干细胞（CSC）是能够产生不同肿瘤细胞群体的异质肿瘤质量中的少数群体，并且在治疗后隐含肿瘤复发。低氧反应的关键介质是缺氧诱导的因素HIF-1和HIF-2，是调节重叠和独特下游靶基因的非冗余转录因子。 HIF-2？是肾细胞癌（RCC），神经母细胞瘤，胶质母细胞瘤（GBM）和非小细胞肺癌（NSCLC）中肿瘤发病率的重要驱动力。这与HIF-2¿（但不是HIF-1）的能力相关联，促进这些肿瘤类型中癌症干细胞的维持和生长。在这种情况下，高HIF-1预测患者的预后更好。因此，我们建议对HIF-2？的特定抑制作用将是治疗HIF-2驱动肿瘤和靶向CSC种群的有用策略。我们的目标是确定将导致NE抗癌疗法发展的小分子特异性抑制剂。我们已经生成了786-0 RCC细胞，稳定地表达了缺氧响应元件（HRE） - 卢糖苷酶构建体。 786-0 HRE细胞不表达HIF-1，我们将使用这些细胞来筛选NIH MLSMSR收集，以用于HIF-2的抑制剂。我们已经在384孔板中对786-0 HRE细胞进行了必要的优化测定，发现这些细胞适用于使用我们的Pan-HIF抑制剂PX478作为阳性对照的HTS。信号与背景比为〜20，优化测定条件的z'因子为0.67，最大DMSO浓度耐受性为0.75％（v/v），从细胞播种到荧光素酶读数的总测定时间为48小时。作为计数器屏幕，我们生成了仅表达HIF-1缺的Miapaca-2 HRE细胞，我们将使用这些细胞消除也抑制HIF-1。作为验证屏幕，我们将使用pancHRE细胞，其中HRE荧光素酶活性依赖于HIF-2，以确认主屏幕上的命中抑制HIF-2。作为潜在抗肿瘤活性的指标，将使用次要测定法对HIT化合物进行评估，以确定其抑制HIF-2¿下游靶基因的能力，以及在相关细胞模型中具有RCC和GBM的相关细胞模型中上皮上皮到上皮过渡和3D菌落形成等功能结果。提出的屏幕将导致对新型HIF-2？特定抑制剂的鉴定，这些抑制剂将为新的抗癌疗法提供结构铅。此外，此屏幕还将促进识别pan hif-1。 / hif-2抑制剂可能具有治疗性应用，并对HIF-1/2的新机制提供新的见解是特定的调节。