Fig. 6: High-throughput drug screening platform using saFRET biosensor.

a Schematics of the high-throughput drug screening platform. First, the cells cultured in 96-well glass-bottom plates were treated either with DMSO or inhibitors from the kinase inhibitor library. After 40 min of incubation, the cells were imaged, and the FRET ratio change compared to the control cell was calculated. This platform can also allow dynamic tracking of the FRET ratio change after inhibitor treatment in single cells. b Representative FRET-Ratio images of the cells with different inhibitors corresponding to (d). Scale bars, 10 µm. c Summary of screening results. Some of the inhibitors have shown high efficiency in inhibiting ZAP70 kinase. The highlighted inhibitors represent the inhibitors targeting ZAP70 upstream signaling molecules. Dasatinib, Src kinase inhibitor; PP2, Lck/Fyn kinase inhibitor; R406, Syk inhibitor. d Top 10 selected inhibitors (From left to right, n = 24, 12, 31, 37, 26, 50, 49, 51, 46, and 46, respectively). Error bars, mean ± SD. e Counter screening using a mutant biosensor with a kinase-dead domain to subtract the noise engendered from non-specific fluorescence. The Scatter plot illustrates the FRET ratio changes in the positive and negative screenings using the saFRET biosensor fused with an active kinase or a kinase-dead domain, respectively. f–g Time-lapse FRET ratio images (f), and the normalized FRET ratio (g) of HEK293 cells before and after inhibitor treatment. The TAK-659 (10 μM) was used as the negative control, which cannot sufficiently inhibit the ZAP70 kinase. (n is shown in the figure). Error bars, mean ± SEM. Scale bars, 10 µm. See also supplementary video 7. Source data are provided as a Source Data file.