A high-throughput fluorescence脙脙脙脙脙脙anisotropy screen that identifies small molecule inhibitors of the DNA binding of B-ZIP transcription factors
- 作者：Rishi ; Vikas ; Potter ; Timothy ; Laudeman ; Julie ; Reinhart ; Russel ; Silvers ; Thomas ; Selby ; Michael ; et. al.
- 关键词：High-throughput screen ; B-ZIP protein ; Fluorescence anisotropy/fluorescence polarization ; Small molecules ; CREB ; VBP ; C/EBP ; AP-1
- 刊名：Analytical Biochemistry
- 出版年：2005
- 期刊代码：93_00032697
- 类别：imm
- 出版时间：May 15, 2005
- 卷：340
- 期：2
- 页码：259-271
- 文件大小：1.09 M
摘要
We have developed a high-throughput fluorescence anisotropy screen, using a 384-well format, to identify small molecules that disrupt the DNA binding of B-ZIP proteins. Binding of a B-ZIP dimer to fluorescently labeled DNA can be monitored by fluorescence anisotropy. We screened the National Cancer Institute diversity set of 1990 compounds to identify small molecules that disrupt the B-ZIP|DNA complex of CREB, C/EBPβ, VBP, and AP-1 (FOS|JUND) bound to their cognate DNA sequence. We identified 21 compounds that inhibited the DNA binding of at least one B-ZIP protein, and 12 representative compounds were grouped depending on whether they displaced ethidium bromide from DNA. Of the 6 compounds that did not displace ethidium bromide, 2 also inhibited B-ZIP binding to DNA in a secondary electrophoretic mobility shift assay screen with some specificity. Thermal stability monitored by circular dichroism spectroscopy demonstrated that both compounds bound the basic region of the B-ZIP motif. NSC13778 preferentially binds C/EBPα 1000-fold better than it binds C/EBPβ. Chimeric proteins combining C/EBPα and C/EBPβ mapped the binding of NSC13778 to three amino acids immediately N terminal of the leucine zipper of C/EBPα. These experiments suggest that the DNA binding of B-ZIP transcription factors is a potential target for clinical intervention.