Table 2.
Primary Screening and Confirmatory Dose–Response Results
| |
|
Number of hits (PubChem AID) |
|
|||||
|---|---|---|---|---|---|---|---|---|
| Screening | Compounds tested | Bcl-2 | Bcl-B | Bcl-xL | Bcl-W | Bfl-1 | Mcl-1 | Total hitsa |
| Primary | 194,829 | 116 (950) | 142 (951) | 82 (1007) | 48 (952) | 237 (1008) | 196 (1009) | 385a |
| Confirmatory dose response | 834 | 9 (1328) | 6b (1327) | 6 (1322) | 18 (1330) | 97 (1320) | 3 (1329) | 27a |
| Powder dose response | 42 | 6 (2075) | 9/2c (2077) | 0 (2084) | 5 (2081) | 23 (2080) | 0 (2086) | 9a |
| FP (1) | 47 | 3 | Not tested | Not tested | Not tested | 3 | Not tested | 3 |
| FP (2) | 10 | 1 | 3 | 0 | Not tested | 0 | Not tested | 4d |
| ITC | 4 | Not tested | 3 | 0 | Not tested | Not tested | Not tested | 3 |
Some compounds that did not pass the threshold for being declared as active were nevertheless selected to be tested in follow-up assays.
Bfl-1 hits were not included in the number of total hits because the dose–response results from the original Bfl-1 preparation were not replicated with a new preparation in the FP assay dose–response analysis; total hits is thus the number of unique compounds affecting one or more of the other five antiapoptotic Bcl-2 family members. We hypothesized that redox-based modulation of cysteine 55 in the BH3 binding pocket could account for the discrepancy. However, the C55A mutant of Bfl-1 bound Bim normally and the compounds did not show the activity that they had against the original Bfl-1 preparation (data not shown). We then performed a multiplex analysis in which the original preparation of Bfl-1, the fresh preparation used in the FP assay, and the mutant protein were bound to individual bead set. These data recapitulated the activities of the individual preparations, and the fresh preparation set is being uploaded to PubChem to reconcile the earlier results.
Compound SID 85203040 was declared inactive in this assay, with an EC50=18.2 μM higher than the chosen threshold (10 μM).
Only two Bcl-B hits in this assay were inhibitors; seven other compounds “increased” the binding of the fluorescent peptide and were considered artifacts; compound SID 85203037 was declared inactive in this assay, with an EC50=16.3 μM higher than the chosen threshold (10 μM).
A benzoquinone reactive compound was also identified as hit on all protein targets, but was discarded.
AID, assay identification numbers; BH3, Bcl-2 homology region 3; FP, fluorescence polarization; ITC, isothermal titration calorimetry.