High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications

被引:167
作者
Stockwell, BR
Haggarty, SJ
Schreiber, SL
机构
[1] Harvard Univ, Howard Hughes Med Inst, Harvard Inst Chem & Cell Biol, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
[2] Harvard Univ, Howard Hughes Med Inst, Harvard Inst Chem & Cell Biol, Dept Mol & Cellular Biol, Cambridge, MA 02138 USA
来源
CHEMISTRY & BIOLOGY | 1999年 / 6卷 / 02期
关键词
cell-based assay; chemical genetics; high-throughput screening; post-translational; profiling;
D O I
10.1016/S1074-5521(99)80004-0
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Background: Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. Results: We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. Conclusions: We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.
引用
收藏
页码:71 / 83
页数:13
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