PDB ID for probe-target interaction (3D structure):
5AD3
Structure-activity relationship:
Yes, briefly described in paper Nature Chem. Biol. 2016, 12, 1097.
Potency assay (off target):
Bromoscan (DiscoveRx); biBET shows selectivity for the BET family proteins compared with other BRDs.
Probe Selectivity in Vitro:
In selectivity panels of 123 kinases (ThermoFisher) and 147 secondary pharmacology assays (Cerep), biBET showed no detectable kinase activity at 1 μM. Only ten of the other secondary pharmacology assays registered potencies below 10 μM, with the most potent being the 5HT1B receptor (EC50=0.93 μM; selectivity ratio of 1,400-fold relative to BRD4).
In cell validation
Potency in cells:
IC50
100 pM
Potency assay (cells):
BiBET disrupted BRD4-mediator complex foci with pIC50=10 in U2OS cells.
Other cellular endpoints are characterized extensively including nanoBRET target engagement and growth inhibition assays with consistent potency values. Potencies in cells against all BET proteins are 100 pM.
Mutations in BRD4(2) confirmed bivalent mechanism of action in BRET assay (pIC50 for full-length BRD4=9.6, BRD4(1)=5.6 and pIC50 mutant full-length BRD4=6.6, BRD4(1)=6.8).
Target engagement assay (cells):
Yes, using Nano-BRET tracer and histone-displacement assays in HCT116 and HEK293 cells, biBET is active against BRD4, BRD2, BRD3 and BRDT.
Toxicity
Cytoxicity assay:
Yes
Notes on cytotoxicity:
Cytotoxicity is consistent with the mechanism of action. In Alamar Blue assays, biBET is cytotoxic for BRD4-dependent hematalogical cell lines (e.g., MV4.11, MM.1S, RS4-11).