Workflow of high-throughput screening using a focused library
| Step | Test compound concentration | Number of compounds | % of picked compounds | Inhibition cut-off |
| Library compound | - | 2,092 | - | - |
| AlphaLISA (AL) assay hits | 5, 10, and 20 µM | 327 | 15.6 | Inhibition >35% at 5 µM and >50% at 10 µM |
| Filtering using quenching (AL) | 10 µM | 58 | 2.77 | Quenching <27% at 10 µM and inhibition >55% at 10 µM |
| Lipinski’s rule | 50 | 2.39 | ≤1 violation | |
| Validation/dose response hits (FP and AL) | 0.01–20 µM | 5 | 0.24 | IC50 < 3 µM (FP) and <10 µM (AL) |
| Step | Test compound concentration | Number of compounds | % of picked compounds | Inhibition cut-off |
| Library compound | - | 2,092 | - | - |
| AlphaLISA (AL) assay hits | 5, 10, and 20 µM | 327 | 15.6 | Inhibition >35% at 5 µM and >50% at 10 µM |
| Filtering using quenching (AL) | 10 µM | 58 | 2.77 | Quenching <27% at 10 µM and inhibition >55% at 10 µM |
| Lipinski’s rule | 50 | 2.39 | ≤1 violation | |
| Validation/dose response hits (FP and AL) | 0.01–20 µM | 5 | 0.24 | IC50 < 3 µM (FP) and <10 µM (AL) |
Selection criteria and number of compounds selected during each step of screening.