Use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic SRP-Sec61 targeting/translocation pathway
Fig 4
Whole cell assay: biosynthesis of various target proteins in HEK 293 cells treated with compounds FMP-503533, FMP-214534, FMP-401319, FMP-208236 and FMP-214219.
To analyze activity and selectivity of the compounds, HEK 293 cells were transiently transfected with the original screening target CRF1R.GFP (red column) and various GFP-tagged integral membrane proteins. Cells were treated with the compounds (FMP-503533, 20 μM; FMP-214534, 25 μM; FMP-401319, 25 μM; FMP-208236, 20 μM; FMP-214219, 10 μM) or DMSO for 19 h and the total GFP fluorescence of the cells was analyzed using flow cytometry as a measure of biosynthesis. Note that compounds FMP-214534, FMP-401319, FMP-208236 and FMP-214219 affected the biosynthesis of all target proteins whereas compound FMP-503533 impaired only biosynthesis of the original screening target CRF1R.GFP. Columns represent mean values of biosynthesis (% of matched DMSO control, red line) calculated out of 3–10 independent experiments indicated in the individual panels ±SD. P values: p ≤ 0.001 (***), p ≤ 0.01 (**), p ≤ 0.05 (*), p ≥ 0.1 (not significant, ns).