A facile method to screen inhibitors of protein-protein interactions including MDM2-p53 displayed on T7 phage |
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Authors: | Ishi Kazutomo Sugawara Fumio |
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Affiliation: | Genome and Drug Research Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. |
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Abstract: | 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'. |
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Keywords: | PBS, phosphate-buffered saline DMSO, dimethyl sulfoxide SDS, sodium dodecyl sulfate GST, glutathione S-transferase pNPP, p-nitrophenyl phosphate CBB, Coomassie brilliant blue R250 IPTG, isopropyl-beta- smallcaps" >d-galactoside PFU, plaque forming unit |
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