Elsevier

Analytical Biochemistry

Volume 340, Issue 2, 15 May 2005, Pages 259-271
Analytical Biochemistry

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

https://doi.org/10.1016/j.ab.2005.02.012Get rights and content

Abstract

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.

Section snippets

Proteins

The majority of the B-ZIP domain proteins used in this study have been described elsewhere: VBP (vitellogenin gene binding protein) [17], C/EBP [18], CREB [19], c-FOS [20], and SREBP-1 (sterol regulatory element binding protein) [21]. All proteins used in this study have 13 amino acid Phi 10 epitopes at their N terminal. The c-JUND protein has the following amino acid sequence: SPIDMESQERIKAERKRMRNRIAASKCRKR KLER IARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLM LTQQLQTF. The sequence of C/EBPβ

High-throughput screen

Fluorescein-labeled DNA containing a consensus-binding site for each B-ZIP protein was added at 5 nM to a black 384-well polypropylene plate with a Biomek 2000 automated pipetting instrument. The corresponding B-ZIP protein was added at 300 nM for FOS|JUND, 400 nM for CREB, and 1000 nM for both C/EBPβ and VBP by the Biomek 2000. The test compounds were diluted to the required concentrations and transferred from the 96-well storage plates to the test plate by the Biomek 2000. After incubation for 60 

Results

Fig. 1A presents the X-ray structure of a B-ZIP homodimer bound to DNA [24]. Fig. 1B presents the increase in fluorescence polarization after a 28-bp double-stranded DNA, labeled with fluorescein at the 5′ ends of both DNA strands, binds to a FOS|JUND heterodimer. This indicates a slowing of the tumbling rate of the DNA on protein binding. Normally, the binding between two entities of similar mass does not produce an easily observable change in fluorescent anisotropy. However, in the case of

Discussion

We have developed a high-throughput fluorescence polarization screen to identify small molecules that disrupt the DNA binding of B-ZIP proteins to DNA. We screened the NCI diversity set of 1990 compounds using four B-ZIP dimers (CREB, C/EBPβ, VBP, and FOS|JUND) and identified 39 compounds that were active against at least one protein|DNA complex. Confirmation titrations identified 21 compounds that disrupted DNA binding in the micromolar range. We used three secondary screens to evaluate 12

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