High-throughput strategy to identify inhibitors of histone-binding domains.
Many epigenetic proteins recognize the posttranslational modification state of chromatin through their histone-binding domains and thereby recruit nuclear complexes to specific loci within the genome. A number of these domains have been implicated in cancer and other diseases through aberrant binding of chromatin; therefore, identifying small molecules that disrupt histone binding could be a powerful mechanism for disease therapy. We have developed a high-throughput assay for the detection of histone peptide-domain interactions utilizing AlphaScreen technology. Here, we describe how the assay can be first optimized and then performed for high-throughput screening of small molecule-binding inhibitors. We also describe strategies for biochemical validation of small molecules identified.
Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, USA.
Elise K
EK
Wagner
Brittany N
BN
Albaugh
John M
JM
Denu
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D003627:Data Interpretation, Statistical; D057166:High-Throughput Screening Assays; D006657:Histones / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D059629:Signal-To-Noise Ratio; D054852:Small Molecule Libraries; D014584:User-Computer Interface
D004268:DNA-Binding Proteins; D006657:Histones; D010446:Peptide Fragments; D054852:Small Molecule Libraries