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

• 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
• 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

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瘤经历缺氧区域，这促进了肿瘤进展和对治疗的抵抗。缺氧对于癌症干细胞（CSC）的增殖和维持也是重要的，CSC是异质性肿瘤块内的少数群体，能够产生多样化的肿瘤细胞群体，并与治疗后的肿瘤复发有关。缺氧反应的关键介质是缺氧诱导因子HIF-1和HIF-2，它们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因素。这与HIF-2 <$（而不是HIF-1 <$）驱动增殖，侵袭以及促进这些肿瘤类型中癌症干细胞的维持和生长的能力有关。在这种情况下，高HIF-1预测患者预后更好。因此，我们认为特异性抑制HIF-2将是治疗HIF-2驱动的肿瘤和靶向CSC群体的有用策略。我们的目标是确定HIF-2的小分子特异性抑制剂，这将导致新的抗癌疗法的发展。我们已经产生了稳定表达缺氧反应元件（HRE）-荧光素酶构建体的786-0 RCC细胞。786-0 HRE细胞不表达HIF-1，我们将使用这些细胞筛选NIH MLSMSR收集的HIF-2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，并发现这些细胞适用于HTS，使用我们的泛HIF抑制剂PX 478作为阳性对照。信号与背景比约为20，优化测定条件的Z'因子为0.67，耐受的最大DMSO浓度为0.75%（v/v），从细胞接种到荧光素酶阅读的总测定时间为48小时。作为一个反筛选，我们已经产生了MIAPACA-2 HRE细胞，只表达HIF-1，我们将使用这些细胞来消除也抑制HIF-1的命中。作为验证筛选，我们将使用其中HRE荧光素酶活性是HIF-2依赖性的PANC HRE细胞来确认通过来自初步筛选的命中物的HIF-2抑制。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，以确定它们抑制HIF-2下游靶基因和功能结果的能力，例如在相关细胞模型（例如RCC和GBM）中的上皮向间充质转化和3D集落形成。拟议的筛选将导致识别新的HIF-2特异性抑制剂，这将为新的抗癌疗法提供结构线索。此外，该屏幕还将有助于识别pan HIF-1/ 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。