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

Methodology Article

Mohammad Alfatah¹  Prakash Arumugam¹  Jin Huei Wong¹  Chandra S. Verma²  Mei Fang Sin³  Hong May Sim³  David P. Lane⁴ 

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

关键词: Yeast two-hybrid assay;    Protein–protein interaction inhibitors;    p53-Mdm2 interaction;   

DOI  :  10.1186/s12915-017-0446-7

 received in 2017-06-05, accepted in 2017-10-19,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundProtein–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.MethodsWe 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.ResultsWe 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.ConclusionThe Y2H assay can be utilized for high-throughput screening of small-molecule inhibitors of PPIs and to identify domains that stabilize PPIs.

【 授权许可】

CC BY   
© Arumugam et al. 2017