Abstract
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.
Highlights
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
Nutlin inhibits the interaction of p53 (1–52) with Mdm2 in pdr1Δ pdr3Δ pdr5Δ cells We considered the nature of the p53-Mdm2 interaction
To identify residues involved in the additional p53Mdm2 interaction, we examined the conserved residues around the region (116–143) of p53 that is minimally required for nutlin resistance in the yeast two-hybrid assay (Y2H) assay and stable interaction with Mdm2 in the in vitro binding assay (Fig. 8a)
Summary
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. While several in vitro assays to screen for PPI inhibitors are available, they are often expensive, cumbersome, and require large amounts of purified protein. Limited in vivo assays are available to screen for small-molecule inhibitors of PPI. It has been estimated that approximately 350,000 types of PPIs occur in a human cell [1]. This offers innumerable opportunities to develop PPI inhibitors and to control cellular processes. Obtaining a small-molecule inhibitor of PPIs is not trivial. There has been some success over the last couple of decades in obtaining small-molecule PPI inhibitors [3]. It has been observed that a few key residues in the PPI interface contribute to the bulk of the binding energy in a PPI [4] and, could be targeted for inhibition by a small molecule
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