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：
8644634
负责人：
Mei Yee Koh
金额：
$ 4.24万
依托单位：
SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-06-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8644634
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 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 screening 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（由申请人提供）：大多数实体瘤及其转移灶都会经历缺氧区域，这会促进肿瘤进展和对治疗的抵抗力，缺氧对于癌症干细胞（CSC）的增殖和维持也很重要，癌症干细胞是肿瘤干细胞中的少数群体。能够产生不同肿瘤细胞群并参与治疗后肿瘤复发的异质性肿瘤块是缺氧诱导因子 HIF-1¿和 HIF-2¿ ，非冗余转录因子调节重叠和独特的下游靶基因。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) 肿瘤发病率的重要驱动因素，这与 HIF-2 的能力有关。（但不是 HIF-1¿）驱动增殖、侵袭并促进这些肿瘤类型内癌症干细胞的维持和生长。在这种情况下，高 HIF-1¿因此，我们建议特异性抑制 HIF-2¿将是治疗 HIF-2 的有用策略？我们的目标是识别 HIF-2 的小分子特异性抑制剂。这将导致新抗癌疗法的发展，我们已经产生了稳定表达缺氧反应元件（HRE）-荧光素酶结构的 786-0 RCC 细胞，786-0 HRE 细胞不表达 HIF-1¿我们将使用这些细胞来筛选 NIH MLSMSR 集合中的 HIF-2 抑制剂¿我们对 384 孔板中的 786-0 HRE 细胞进行了必要的优化测定，发现这些细胞适合使用我们的泛 HIF 抑制剂 PX478 作为阳性对照，信号与背景比率约为 20，Z。优化测定条件的因子为 0.67，最大耐受 DMSO 浓度为 0.75% (v/v)，从细胞接种到荧光素酶读数的总测定时间为作为计数器筛选，我们生成了仅表达 HIF-1 的 MIAPACA-2 HRE 细胞。我们将使用这些细胞来消除也抑制 HIF-1 的攻击？作为验证筛选，我们将使用 PANC HRE 细胞，其中 HRE 荧光素酶活性为 HIF-2¿依赖于确认 HIF-2¿作为潜在抗肿瘤活性的指标，将使用二次测定来评估命中化合物以确定其抑制 HIF-2 的能力。下游靶基因和功能结果，例如 RCC 和 GBM 等相关细胞模型中的上皮到间充质转化和 3D 集落形成。拟议的筛选将导致新型 HIF-2 的鉴定。此外，该筛选还将促进泛 HIF-1 的鉴定。 / HIF-2¿抑制剂可能具有治疗应用并为 HIF-1/2 的新机制提供新的见解¿是形式具体的规定。