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
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 challen...
| Main Authors: | , , , , , , |
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| Format: | Journal Article |
| Language: | English |
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2018
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| Online Access: | https://hdl.handle.net/10356/88055 http://hdl.handle.net/10220/44532 |
| _version_ | 1824454731359059968 |
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| author | Wong, Jin Huei Alfatah, Mohammad Sin, Mei Fang Sim, Hong May Verma, Chandra Shekhar Lane, David P. Arumugam, Prakash |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Wong, Jin Huei Alfatah, Mohammad Sin, Mei Fang Sim, Hong May Verma, Chandra Shekhar Lane, David P. Arumugam, Prakash |
| author_sort | Wong, Jin Huei |
| collection | NTU |
| description | 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 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. |
| first_indexed | 2025-02-19T03:26:58Z |
| format | Journal Article |
| id | ntu-10356/88055 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2025-02-19T03:26:58Z |
| publishDate | 2018 |
| record_format | dspace |
| spelling | ntu-10356/880552023-02-28T17:00:21Z 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 Wong, Jin Huei Alfatah, Mohammad Sin, Mei Fang Sim, Hong May Verma, Chandra Shekhar Lane, David P. Arumugam, Prakash School of Biological Sciences Yeast Two-hybrid Assay Protein–protein Interaction Inhibitors 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 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. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version 2018-03-09T05:01:18Z 2019-12-06T16:55:04Z 2018-03-09T05:01:18Z 2019-12-06T16:55:04Z 2017 Journal Article Wong, 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-. https://hdl.handle.net/10356/88055 http://hdl.handle.net/10220/44532 10.1186/s12915-017-0446-7 en BMC Biology © 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. 17 p. application/pdf |
| spellingShingle | Yeast Two-hybrid Assay Protein–protein Interaction Inhibitors Wong, Jin Huei Alfatah, Mohammad Sin, Mei Fang Sim, Hong May Verma, Chandra Shekhar Lane, David P. Arumugam, Prakash 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 |
| 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | Yeast Two-hybrid Assay Protein–protein Interaction Inhibitors |
| url | https://hdl.handle.net/10356/88055 http://hdl.handle.net/10220/44532 |
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