TY - JOUR
T1 - Time-resolved fluorescence resonance energy transfer assay for discovery of small-molecule inhibitors of methyl-CpG binding domain protein 2
AU - Wyhs, Nicolas
AU - Walker, David
AU - Giovinazzo, Hugh
AU - Yegnasubramanian, Srinivasan
AU - Nelson, William G.
N1 - Funding Information:
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by funding from the Prostate Cancer Foundation, the Patrick C. Walsh Prostate Cancer Research Fund, Department of Defense Prostate Cancer Research Program predoctoral training grant (W81XWH-11-1-0618 to N.W.), and NIH/NCI grants CA70196 and CA58236.
PY - 2014/8
Y1 - 2014/8
N2 - Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. Although both assays performed well in 96-well format, the TR-FRET assay (Z factor = 0.58) emerged as a superior screening strategy compared with FP (Z factor = 0.08) when evaluated in an HTS 384-well plate format. Using TR-FRET, we screened the Sigma LOPAC library for MBD2-MBD inhibitors and identified four compounds that also validated in a dose-response series. This included two known DNA intercalators (mitoxantrone and idarubicin) among two other inhibitory compounds (NF449 and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide, demonstrating that the activity was nonspecific. Our results provide proof of principle for using TR-FRET-based HTS to identify small-molecule inhibitors of MBD2 and other DNA-protein interactions.
AB - Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)-based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. Although both assays performed well in 96-well format, the TR-FRET assay (Z factor = 0.58) emerged as a superior screening strategy compared with FP (Z factor = 0.08) when evaluated in an HTS 384-well plate format. Using TR-FRET, we screened the Sigma LOPAC library for MBD2-MBD inhibitors and identified four compounds that also validated in a dose-response series. This included two known DNA intercalators (mitoxantrone and idarubicin) among two other inhibitory compounds (NF449 and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide, demonstrating that the activity was nonspecific. Our results provide proof of principle for using TR-FRET-based HTS to identify small-molecule inhibitors of MBD2 and other DNA-protein interactions.
KW - DNA-protein interaction inhibitor
KW - MBD2
KW - MBD2 small-molecule inhibitor
KW - TR-FRET
KW - fluorescence polarization
KW - high-throughput screening assay
KW - methylated-CpG binding domain protein 2
KW - time-resolved fluorescence resonance energy transfer
UR - http://www.scopus.com/inward/record.url?scp=84904599346&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84904599346&partnerID=8YFLogxK
U2 - 10.1177/1087057114526433
DO - 10.1177/1087057114526433
M3 - Article
C2 - 24608100
AN - SCOPUS:84904599346
SN - 1087-0571
VL - 19
SP - 1060
EP - 1069
JO - Journal of Biomolecular Screening
JF - Journal of Biomolecular Screening
IS - 7
ER -