p300/CREB binding protein associated factor (PCAF/KAT2B) and general control non-derepressible 5 (GCN5/KAT2A) are multidomain proteins that have been implicated in retroviral infection, inflammation pathways and cancer development. However, outside of viral replication, little is known about the dependence of these effects on the C-terminal bromodomain. GSK4027 is as a chemical probe for the PCAF/GCN5 bromodomain and GSK4028 is the enantiomeric negative control. The probe was optimized from a weakly potent, non-selective pyridazinone hit to deliver high potency for the PCAF/GCN5 bromodomain, high solubility, cellular target engagement and ≥18000-fold selectivity over the BET family, together with ≥70 fold selectivity over the wider bromodomain families.
PCAF IC50 40 nM in a TR-FRET binding competition assay using truncated PCAF bromodomain and a fluorescently tagged bromodomain ligand.
PCAF Ki 1.4 nM in a BROMOscan assay run at DiscoverX.
GCN5 Ki 1.4 nM in a BROMOscan assay run at DiscoverX.
Co-crystal structure with GCN5: PDB ID 5MLJ
>70 fold selectivity over other bromodomain targets including BRPF3 (100 nM), BRD1 (110 nM), FALZ (130 nM) and BRPF1 (140 nM) In BROMOscan assay run at DiscoverX.
GSK internal enhanced, cross-screening panel (eXP); a full curve against 53 biochemical and phenotypic assays did not reveal any off-target binding <3 µM.
IC50 60 nM in Promega NanoBRET assay, measuring displacement of NanoLuc-tagged full length PCAF from Halo-tagged histone H3.3 in HEK293 cells.
Cellular health assay looking at mitochondrial integrity, nuclear size and membrane permeability found no changes up to 200 µM.