A-1331852

Precise targeted drug screening is the key to improve the efficiency of tumour targeted therapy. This report presents a live cell FRET two-hybrid assay-based targeted drug screening method (FRET-HBTDS). In FRET-HBTDS, the cells co-expressing donor- and acceptor-labelled targets were cultured in 96-well plates, quantitative FRET imaging was then performed on a self-built automated FRET microscope (FRETscope) well-by-well, and FRET two-hybrid assay was used to obtain the maximum donor-centre FRET efficiency (E_Dmax), maximum acceptor-centre FRET efficiency (E_Amax) and stoichiometric ratios (N_A/N_D). The FRET-HBTDS method was performed on the FRETscope with a 20 × objective for the cells co-expressing CFP-Bcl-xL and YFP-Bak to assess the action of eight compounds (A1331852, S63845, AC, DSF/Cu, Met, REGO, SOFA, ABT199) on the interaction between Bcl-xL and Bak. After 7 h of treatment with these compounds respectively, only the A1331852 group showed significantly lower E_Dmax and E_Amax compared to the control group, and a significant increase in N_A/N_D, suggesting that A1331852 unlocks the direct interaction between Bcl-xL and Bak, thus releasing Bak to induce cell death. In addition, the N_A/N_D of the DSF/Cu group was significantly higher than of the control group, suggesting that DSF/Cu altered the stoichiometry of the Bcl-xL-Bak complex. Our data firmly demonstrate that A1331852 unlocks the binding state of Bcl-xL and Bak, while DSF/Cu modifies the structure of the Bcl-xL-Bak complex. These findings demonstrate that FRET-HBTDS can be used to assess the efficacy of a drug by revealing the binding state and complex molecular structure of the target proteins using FRET technology in living cells, which may be a potential targeted drug screening method.