Please use this identifier to cite or link to this item: https://doi.org/10.1186/s12915-017-0446-7
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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 p53
dc.contributor.authorWong, J.H
dc.contributor.authorAlfatah, M
dc.contributor.authorSin, M.F
dc.contributor.authorSim, H.M
dc.contributor.authorVerma, C.S
dc.contributor.authorLane, D.P
dc.contributor.authorArumugam, P
dc.date.accessioned2020-09-01T00:49:08Z
dc.date.available2020-09-01T00:49:08Z
dc.date.issued2017
dc.identifier.citationWong, J.H, Alfatah, M, Sin, M.F, Sim, H.M, Verma, C.S, Lane, D.P, Arumugam, P (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. ScholarBank@NUS Repository. https://doi.org/10.1186/s12915-017-0446-7
dc.identifier.issn17417007
dc.identifier.urihttps://scholarbank.nus.edu.sg/handle/10635/173762
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 protein-protein 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 ABC9? yeast strain that is highly permeable to small molecules. In the ABC9? 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. © 2017 Arumugam et al.
dc.sourceUnpaywall 20200831
dc.subjectprotein binding
dc.subjectprotein MDM2
dc.subjectprotein p53
dc.subjectSaccharomyces cerevisiae protein
dc.subjectbinding site
dc.subjectmetabolism
dc.subjectmolecular library
dc.subjectprotein domain
dc.subjectSaccharomyces cerevisiae
dc.subjecttwo hybrid system
dc.subjectBinding Sites
dc.subjectProtein Binding
dc.subjectProtein Interaction Domains and Motifs
dc.subjectProto-Oncogene Proteins c-mdm2
dc.subjectSaccharomyces cerevisiae
dc.subjectSaccharomyces cerevisiae Proteins
dc.subjectSmall Molecule Libraries
dc.subjectTumor Suppressor Protein p53
dc.subjectTwo-Hybrid System Techniques
dc.typeArticle
dc.contributor.departmentDEPT OF PHARMACY
dc.contributor.departmentBIOLOGY (NU)
dc.contributor.departmentDEPT OF MEDICINE
dc.contributor.departmentDEPT OF BIOLOGICAL SCIENCES
dc.description.doi10.1186/s12915-017-0446-7
dc.description.sourcetitleBMC Biology
dc.description.volume15
dc.description.issue1
dc.description.page108
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