A high-throughput fluorescence–anisotropy screen that identifies small molecule inhibitors of the DNA binding of B-ZIP transcription factors

• Published in: Analytical biochemistry 2005-05, Vol.340 (2), p.259-271
• Main Authors: Rishi, Vikas, Potter, Timothy, Laudeman, Julie, Reinhart, Russel, Silvers, Thomas, Selby, Michael, Stevenson, Timothy, Krosky, Paula, Stephen, Andrew G., Acharya, Asha, Moll, Jon, Oh, Won Jun, Scudiero, Dominic, Shoemaker, Robert H., Vinson, Charles
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; AP-1; B-ZIP protein; Binding Sites; C/EBP; CCAAT-Enhancer-Binding Protein-alpha - chemistry; Circular Dichroism; CREB; Dimerization; DNA - metabolism; DNA-Binding Proteins - antagonists & inhibitors; DNA-Binding Proteins - metabolism; Drug Evaluation, Preclinical - methods; Ethidium - chemistry; Fluorescence anisotropy/fluorescence polarization; Fluorescence Polarization - methods; High-throughput screen; Hot Temperature; Leucine Zippers; Polymerase Chain Reaction; Protein Denaturation; Protein Structure, Tertiary; Small molecules; Thermodynamics; Transcription Factors - antagonists & inhibitors; Transcription Factors - metabolism; VBP
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2005.02.012

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.