MDM2/p53-based live-cell quantitative FRET imaging for apoptosis drug discovery

Targeting the interaction between P53 and MDM2 to re-activate P53 to induce apoptosis is an important strategy for cancer treatment. In this study, based on the unique advantages of in situ visualization, dynamic imaging, and quantitative analysis of living cell FRET imaging, a method for screening apoptotic drugs targeting p53-MDM2 interaction was developed. A stable model of Nutlin-3-induced apoptosis was established in MCF-7 cells, which was verified by reducing mitochondrial membrane potential and increasing the proportion of nuclear chromatin condensation (from 9.16 % to 50.55 %). Biochemical methods such as WB analysis found that after activating P53, BAX expression was up-regulated through a Puma-independent pathway, which promoted BAX oligomerization. Live-cell quantitative FRET imaging found that the maximum donor center FRET efficiency (E_Dmax) of CFP-p53 and YFP-MDM2 decreased from 0.50 to 0.22 after Nutlin-3 treatment, and the co-localization coefficient decreased significantly from 83 % to 22 %, confirmed that Nutlin-3 directly disrupted the interaction between P53/MDM2, promoting P53 nuclear translocation and apoptosis. This indicated that Nutlin-3 was a direct inhibitor of the P53/MDM2 interaction. Apoptosis drug screening was performed in MCF-7 cells, and we found that the E_Dmax was 0.29 and 0.31 for the cells treated with DOX and RSV, respectively, and 0.48 for the IKE-treated cells and 0.43 for the SOR-treated cells, indicating that DOX and RSV, but not IKE and SOR, were potential P53/MDM2-dependent apoptotic drugs. In addition, Nutlin-3 treatment decreased the E_Dmax value in p53 wild-type U2OS cells from 0.43 to 0.20. In summary, our method can identify p53-MDM2 interaction inhibitors in living cells, providing a quantitative in vivo supplement for traditional target-based drug discovery.