Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway.
Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway.
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发表时间：
2002-08
期刊：
影响因子：
11.2
通讯作者：
A. Rapisarda;B. Uranchimeg;D. Scudiero;M. Selby;E. Sausville;R. Shoemaker;G. Melillo
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文献类型：
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作者：
A. Rapisarda;B. Uranchimeg;D. Scudiero;M. Selby;E. Sausville;R. Shoemaker;G. Melillo
缺氧诱导因子1 （HIF-1）是缺氧转录反应的主要调控因子。HIF-1参与了血管生成（如血管内皮生长因子（VEGF）和诱导型一氧化氮合酶）和无氧代谢（如糖酵解酶）相关基因的调控。HIF-1对血管生成至关重要，并与肿瘤进展有关。此外，HIF-1 α的过表达已在许多常见的人类癌症中得到证实。因此，HIF-1是开发新型癌症治疗药物的一个有吸引力的分子靶点。我们开发了一种基于细胞的高通量筛选方法，用于鉴定HIF-1途径的小分子抑制剂。我们对人胶质瘤细胞U251进行了基因工程改造，使其稳定表达一种重组载体，其中荧光素酶报告基因受三份典型缺氧反应元件（U251- hre）的控制。U251-HRE细胞以缺氧和hif -1依赖的方式一致表达荧光素酶。我们现在报告国家癌症研究所“多样性集”试点筛选的结果，这是一个大约2000种化合物的集合，被选中代表国家癌症研究所化学储存库中更大的化学多样性。我们发现四种化合物特异性抑制hif -1依赖性诱导的荧光素酶，但不抑制由组成型启动子驱动的荧光素酶表达。此外，这些化合物抑制缺氧诱导的U251细胞中VEGF mRNA和蛋白的表达。有趣的是，有三种化合物与喜树碱类似物和拓扑异构酶(Topo)- 1抑制剂密切相关。我们发现，在抑制HIF-1和VEGF的同时，所测试的Topo-I抑制剂的活性与诱导环氧合酶2mrna表达有关。基于荧光素酶的高通量筛选是鉴定HIF-1转录激活小分子抑制剂的可行工具。此外，我们的研究结果表明，Topo-I功能的改变可能与hif -1依赖性诱导基因表达的抑制有关。
Hypoxia-inducible factor 1 (HIF-1) is a master regulator of the transcriptional response to oxygen deprivation. HIF-1 has been implicated in the regulation of genes involved in angiogenesis [e.g., vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase] and anaerobic metabolism (e.g., glycolytic enzymes). HIF-1 is essential for angiogenesis and is associated with tumor progression. In addition, overexpression of HIF-1 alpha has been demonstrated in many common human cancers. Therefore, HIF-1 is an attractive molecular target for development of novel cancer therapeutics. We have developed a cell-based high-throughput screen for the identification of small molecule inhibitors of the HIF-1 pathway. We have genetically engineered U251 human glioma cells to stably express a recombinant vector in which the luciferase reporter gene is under control of three copies of a canonical hypoxia-responsive element (U251-HRE). U251-HRE cells consistently expressed luciferase in a hypoxia- and HIF-1-dependent fashion. We now report the results of a pilot screen of the National Cancer Institute "Diversity Set," a collection of approximately 2000 compounds selected to represent the greater chemical diversity of the National Cancer Institute chemical repository. We found four compounds that specifically inhibited HIF-1-dependent induction of luciferase but not luciferase expression driven by a constitutive promoter. In addition, these compounds inhibited hypoxic induction of VEGF mRNA and protein expression in U251 cells. Interestingly, three compounds are closely related camptothecin analogues and topoisomerase (Topo)-I inhibitors. We show that concomitant with HIF-1 and VEGF inhibition, the activity of the Topo-I inhibitors tested is associated with induction of cyclooxygenase 2 mRNA expression. The luciferase-based high-throughput screen is a feasible tool for the identification of small molecule inhibitors of HIF-1 transcriptional activation. In addition, our results suggest that altered Topo-I function may be associated with repression of HIF-1-dependent induction of gene expression.