Identification of upregulators of human ATP-binding cassette transporter A1 via high-throughput screening of a synthetic and natural compound library
Identification of upregulators of human ATP-binding cassette transporter A1 via high-throughput screening of a synthetic and natural compound library
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DOI：
10.1177/1087057108320545
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发表时间：
2008-08-01
影响因子：
--
通讯作者：
Si, Shuyi
中科院分区：
文献类型：
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作者：
Gao, Jie;Xu, Yanni;Si, Shuyi
ATP结合盒转运体A1（ABCA 1）是一种膜转运体，通过介导胆固醇和磷脂向贫脂载脂蛋白A-I的细胞外排，直接参与高密度脂蛋白（HDL）的生物合成。因此，由于ABCA 1在胆固醇稳态和HDL代谢中的关键作用，鉴定新的ABCA 1上调剂将有益于动脉粥样硬化的预防和/或治疗。本研究建立了ABCA 1上调产物的高通量检测方法，并用于筛选合成和天然化合物库。使用ABCA 1启动子-荧光素酶构建体稳定转染并在96孔参考ABCA 1上调物（氧固醇、9-顺式-视黄酸、噻唑烷二酮、环腺苷一磷酸、维拉帕米、非诺贝特和制瘤素M）中校准的人肝癌HepG 2细胞，在96孔格式中进行基于细胞的高通量筛选。在2600种化合物中，4种微生物化合物（吡咯霉素、阿克拉霉素、大豆苷元和pratensin）通过高通量筛选试验被挑选为命中物，并且这些化合物通过实时定量逆转录聚合酶链反应和Western印迹分析被进一步鉴定为ABCA 1表达的上调剂。
The ATP-binding cassette trasporter A1 (ABCA1) is a membrane transporter that directly contributes tohigh-density lipoprotein (HDL) biogenesis by mediating the cellular efflux of cholesterol and phospholipids to lipid-poor apolipoprotein A-I. Therefore, identification of a novel upregulator of ABCA1 would be beneficial for atherosclerosis prevention and/or therapy because of ots pivotal role in cholesterol homeostasis and HDL metabolism. In this study, a high-throughput assay mthod for ABCA1 upregulates was developed and used for screening a synthetic and natural compound library. The cell-based high-throughput screen is conducted in a 96-well format using the human hepatoma HepG2 cells stably transfected with ABCA1 promoter-luciferase construct and calibrated in a 96-well reference ABCA1 upregulates (oxysterols, 9-cis-retinoic acid, thiazolidinediones, cyclic adenosine monophosphate, verapamil, fenofibrate, and oncostatin M). Among 2600 compounds, 4 microbial compounds (pyrromycin, aclarubin, daidzein, and pratensein) were picled up as hits by the high-throughput screening assay, and those compounds were further identified as upregulators of ABCA1 expression by real-time quantative reverse transcription-polymerase chain reaction and Western blot analysis.