Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/88055
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dc.contributor.authorWong, Jin Hueien
dc.contributor.authorAlfatah, Mohammaden
dc.contributor.authorSin, Mei Fangen
dc.contributor.authorSim, Hong Mayen
dc.contributor.authorVerma, Chandra Shekharen
dc.contributor.authorLane, David P.en
dc.contributor.authorArumugam, Prakashen
dc.date.accessioned2018-03-09T05:01:18Zen
dc.date.accessioned2019-12-06T16:55:04Z-
dc.date.available2018-03-09T05:01:18Zen
dc.date.available2019-12-06T16:55:04Z-
dc.date.issued2017en
dc.identifier.citationWong, J. H., Alfatah, M., Sin, M. F., Sim, H. M., Verma, C. S., Lane, D. P., et al. (2017). A yeast two-hybrid system for the screening and characterization of small-molecule inhibitors of protein–protein interactions identifies a novel putative Mdm2-binding site in p53. BMC Biology, 15(1), 108-.en
dc.identifier.urihttps://hdl.handle.net/10356/88055-
dc.identifier.urihttp://hdl.handle.net/10220/44532en
dc.description.abstractBackground: Protein–protein interactions (PPIs) are fundamental to the growth and survival of cells and serve as excellent targets to develop inhibitors of biological processes such as host-pathogen interactions and cancer cell proliferation. However, isolation of PPI inhibitors is extremely challenging. While several in vitro assays to screen for PPI inhibitors are available, they are often expensive, cumbersome, and require large amounts of purified protein. In contrast, limited in vivo assays are available to screen for small-molecule inhibitors of PPI. Methods: We have engineered a yeast strain that is suitable for screening of small-molecule inhibitors of proteinprotein interaction using the Yeast 2-hybrid Assay. We have optimised and validated the assay using inhibitors of the p53-Mdm2 interaction and identified a hitherto unreported putative Mdm2-binding domain in p53. Results: We report a significantly improved and thoroughly validated yeast two-hybrid (Y2H) assay that can be used in a high throughput manner to screen for small-molecule PPI inhibitors. Using the p53-Mdm2 interaction to optimize the assay, we show that the p53-Mdm2 inhibitor nutlin-3 is a substrate for the yeast ATP-binding cassette (ABC) transporter Pdr5. By deleting nine ABC transporter-related genes, we generated a ABC9Δ yeast strain that is highly permeable to small molecules. In the ABC9Δ strain, p53-Mdm2 interaction inhibitors, like AMG232 and MI-773, completely inhibited the p53-Mdm2 interaction at nanomolar concentrations in the Y2H assay. In addition, we identified a conserved segment in the core DNA-binding domain of p53 that facilitates stable interaction with Mdm2 in yeast cells and in vitro. Conclusion: The Y2H assay can be utilized for high-throughput screening of small-molecule inhibitors of PPIs and to identify domains that stabilize PPIs.en
dc.description.sponsorshipASTAR (Agency for Sci., Tech. and Research, S’pore)en
dc.format.extent17 p.en
dc.language.isoenen
dc.relation.ispartofseriesBMC Biologyen
dc.rights© 2017 Arumugam et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.en
dc.subjectYeast Two-hybrid Assayen
dc.subjectProtein–protein Interaction Inhibitorsen
dc.titleA yeast two-hybrid system for the screening and characterization of small-molecule inhibitors of protein–protein interactions identifies a novel putative Mdm2-binding site in p53en
dc.typeJournal Articleen
dc.contributor.schoolSchool of Biological Sciencesen
dc.identifier.doi10.1186/s12915-017-0446-7en
dc.description.versionPublished versionen
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item.grantfulltextopen-
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