Automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish
Automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish
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DOI：
10.1158/0008-5472.can-07-3126
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发表时间：
2007-12-01
期刊：
影响因子：
11.2
通讯作者：
Sandberg, Eric M.
中科院分区：
文献类型：
--
作者：
Tran, T. Cameron;Sneed, Blossom;Sandberg, Eric M.
病理性血管生成已成为几种主要疾病的重要治疗靶点。斑马鱼为整个脊椎动物系统的高通量药物发现提供了潜力。我们开发了第一个定量的，自动化的检测方法，利用斑马鱼胚胎进行抗血管生成化合物的鉴定。该测定使用具有荧光血管的转基因斑马鱼以促进图像分析。我们开发了在384孔板中对斑马鱼进行自动药物和成像的方法，并开发了一种自定义算法来量化斑马鱼中血管生成血管的数量。该测定用于筛选LOPAC 1280化合物文库中的抗血管生成化合物。两种已知的抗血管生成化合物SU 4312和AG 1478被鉴定为命中物。此外，还发现了一种以前未知的抗血管生成活性化合物靛玉红-3 '-单肟（IRO）。我们发现，每种命中化合物在斑马鱼中具有剂量依赖性的抗血管生成活性。在斑马鱼血管生成试验中，SU 4312、AG 1478和IRO的IC 50分别为1.8、8.5和0.31 μ mol/L。IRO具有命中化合物的最高效力。此外，IRO抑制人脐静脉内皮细胞管形成和增殖（IC 50分别为6.5和0.36 μ mol/L）。因此，它是最初在斑马鱼试验中发现的第一种抗血管生成化合物，在基于人内皮细胞的血管生成试验中也具有可证实的活性。
Pathologic angiogenesis has emerged as an important therapeutic target in several major diseases. Zebrafish offer the potential for high-throughput drug discovery in a whole vertebrate system. We developed the first quantitative, automated assay for antiangiogenic compound identification using zebrafish embryos. This assay uses transgenic zebrafish with fluorescent blood vessels to facilitate image analysis. We developed methods for automated drugging and imaging of zebrafish in 384-well plates and developed a custom algorithm to quantify the number of angiogenic blood vessels in zebrafish. The assay was used to screen the LOPAC1280 compound library for antiangiogenic compounds. Two known antiangiogenic compounds, SU4312 and AG1478, were identified as hits. Additionally, one compound with no previously known antiangiogenic activity, indirubin-3'-monoxime (IRO), was identified. We showed that each of the hit compounds had dose-dependent antiangiogenic activity in zebrafish. The IC50 of SU4312, AG1478, and IRO in the zebrafish angiogenesis assay was 1.8, 8.5, and 0.31 mu mol/L, respectively. IRO had the highest potency of the hit compounds. Moreover, IRO inhibited human umbilical vein endothelial cell tube formation and proliferation (IC50 of 6.5 and 0.36 mu mol/L, respectively). It is therefore the first antiangiogenic compound discovered initially in a zebrafish assay that also has demonstrable activity in human endothelial cell-based angiogenesis assays.