Table 3.
Impact of biophysical evaluation of hits identified during HTS development.
| Protein target | Assay methodology | Number of compounds tested | Biophysical methods employed | Observations | Actions taken |
|---|---|---|---|---|---|
| KEAP1 | HTRF | 180 | NMR, SPR | No genuine hits identified | HTS was stopped and FBLG approach used instead |
| MALT1 | Fluorescence intensity following proteolytic cleavage | 60 | NMR | 17% of hits showed specific binding. 38% showed redox cycling behaviour. 27% were not soluble | Incorporation of a redox-artefact assay in the cascade reduced the number of redox-active compounds reaching the NMR assay from 38% to 5% |
| ERRγ | FRET | 180 | NMR | FRET assay suggested that hit rate would be low. NMR suggested that 90% were false positives | HTS in this format was not run |
| TTBK1 | ADP-glo | 250 | SPR | 69 verified hits, then profiled versus phosphorylated and non-phosphorylated protein Large number of reactive compounds identified | ADP-glo assay was not run |
| ACPER | Fluorescence intensity following substrate turnover | Tool compounds and 630 fragments | SPR, ITC | Characterisation demonstrated that compounds showing several different mechanisms of inhibition could be found | Project view on needing a cofactor competitive inhibitor was changed and assays configured to find all mechanisms |
| LTC4S | HTRF, RapidFire | Total of 50 selected from actives in one or both assays | NMR, SPR | 77% of the total hits shown to bind and also to displace tool ligand. Confirmation rate was 90% for RapidFire hits, 40% for HTRF hits | RapidFire assay prioritised for full HTS |
| aPC | 4 different assays:
1) Chromogenic cleavage 2) Peptide cleavage coupled assay 3) Peptide cleavage RapidFire 4) Fibrin clot assay |
Total of 250
identified from the assays as follows: 1) 90 2) 50 3) 10 4) 100 190 selected for biophysical testing (90 from assays 1-3 and total output from 4 |
SPR, NMR | Numbers of confirmed hits originating from each assay approach:
1) 8 2) 16 3) 9 4) 13 Fibrin clot assay was shown to identify compounds binding at a site distal from the active site |
Fibrin clot assay was selected for HTS, based on the ability to identify novel, exo site binders. |
Where KEAP1 is Kelch Like ECH Associated Protein 1, MALT1 is Mucosa-associated lymphoid tissue lymphoma translocation protein 1, ERRγ is Estrogen-related receptor gamma, TTBK1 is Tau tubulin kinase 1, ACPER is acyl carrier protein enoyl reductase, LTC4S is Leukotriene C4 synthase and aPC is activated protein c.