Please use this identifier to cite or link to this item: https://doi.org/10.1186/s12915-017-0446-7
Title: 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
Authors: Wong, J.H
Alfatah, M
Sin, M.F
Sim, H.M 
Verma, C.S 
Lane, D.P 
Arumugam, P 
Keywords: protein binding
protein MDM2
protein p53
Saccharomyces cerevisiae protein
binding site
metabolism
molecular library
protein domain
Saccharomyces cerevisiae
two hybrid system
Binding Sites
Protein Binding
Protein Interaction Domains and Motifs
Proto-Oncogene Proteins c-mdm2
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins
Small Molecule Libraries
Tumor Suppressor Protein p53
Two-Hybrid System Techniques
Issue Date: 2017
Citation: Wong, 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
Abstract: Background: 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.
Source Title: BMC Biology
URI: https://scholarbank.nus.edu.sg/handle/10635/173762
ISSN: 17417007
DOI: 10.1186/s12915-017-0446-7
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