Fig. 5: High throughput screening (HTS) for drugs that disrupt 14-3-3:BAD interactions.

A Schematic of HTS workflow. Cells were transfected with BRET sensor or control sensors in 10 cm dishes and allowed 48 h for sensor expression. Subsequently, cells were harvested and re-plated into 384-well plates at a density of 20,000 cells per well 24 h prior to drug screening. Diluted previously approved drugs (PADs) were added to corresponding wells with final concentrations of 200 µM, 20 µM, 2 µM, and 200 nM, respectively. After a 3-hour incubation, the BRET ratio was measured Each PAD was assessed twice at each concentration, but only the highest value for each concentration was recorded. B The heatmap shows BRET reduction for 1971 PADs at concentrations of 200 µM, 20 µM, 2 µM, and 200 nM. C Subsequent screenings were conducted at 20 µM, and PADs were selected based on the median BRET reduction (26.80%) of those that demonstrated a BRET reduction greater than zero. To include the hits near this threshold, a BRET reduction of 25% was set as the selection criterion for primary screening. This led to the selection of 162 PADs for re-screening in a 96-well plate format. D, E 101 hits were further selected to assess their capacity to induce cell death in NIH-3T3 cells. Hoechst/propidium iodide incorporation assays were used to examine cell death, calculated as the ratio of propidium iodide-positive cells to Hoechst-positive cells. Cell death data were collected from three independent experiments, each with duplicate measurements. Scatter density plots are used to visualize the relationship between drug-induced BRET reduction and their capacity to induce cell death at 24 h (D) or 48 h (E) post-treatment.