A facile method to screen inhibitors of protein-protein interactions including MDM2-p53 displayed on T7 phage.
Protein-protein interactions are essential in many biological processes including cell cycle and apoptosis. It is currently of great medical interest to inhibit specific protein-protein interactions in order to treat a variety of disease states. Here, we describe a facile multiwell plate assay method using T7 phage display to screen for candidate inhibitors of protein-protein interactions. Because T7 phage display is an effective method for detecting protein-protein interactions, we aimed to utilize this technique to screen for small-molecule inhibitors that disrupt these types of interaction. We used the well-characterized interaction between p53 and MDM2 and an inhibitor of this interaction, nutlin 3, as a model system to establish a new screening method. Phage particles displaying p53 interacted with GST-MDM2 immobilized on 96-well plates, and the interaction was inhibited by nutlin 3. Multiwell plate assay was then performed using a natural product library, which identified dehydroaltenusin as a candidate inhibitor of the p53-MDM2 interaction. We discuss the potential applications of this novel T7 phage display methodology, which we propose to call 'reverse phage display'.
Ishi
Kazutomo
K
Genome and Drug Research Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Sugawara
Fumio
F
D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D019151:Peptide Library; D010879:Piperazines / Q000494:pharmacology; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D007093:Imidazoles; D019151:Peptide Library; D010879:Piperazines; D054852:Small Molecule Libraries; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3; D051736:Proto-Oncogene Proteins c-mdm2