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  2. 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

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 Biol. 2017 Nov 9;15(1):108. doi: 10.1186/s12915-017-0446-7.
Jin Huei Wong 1 Mohammad Alfatah 1 Mei Fang Sin 2 Hong May Sim 2 Chandra S Verma 1 3 4 David P Lane 5 Prakash Arumugam 6
Affiliations

Affiliations

  • 1 Bioinformatics Institute, 30 Biopolis Street, #07-01, Matrix, Singapore, 138671, Singapore.
  • 2 Department of Pharmacy, National University of Singapore, National University of Singapore 18 Science Drive 4, Singapore, 117543, Singapore.
  • 3 Department of Biological Sciences, National University of Singapore, 14 Science Drive, Singapore, 117543, Singapore.
  • 4 Nanyang Technological University, School of Biological Sciences, 50 Nanyang Drive, Singapore, 637551, Singapore.
  • 5 The p53 Laboratory, 8A Biomedical Grove, Singapore, 138648, Singapore.
  • 6 Bioinformatics Institute, 30 Biopolis Street, #07-01, Matrix, Singapore, 138671, Singapore. [email protected].
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 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.

Keywords

Protein–protein interaction inhibitors; Yeast two-hybrid assay; p53-Mdm2 interaction.

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