Covalent-Fragment Screening of BRD4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites

BRD4,
a member of the bromodomain and extraterminal domain (BET)
family, has emerged as a promising epigenetic target in cancer and
inflammatory disorders. All reported BET family ligands bind within
the bromodomain acetyl-lysine binding sites and competitively inhibit
BET protein interaction with acetylated chromatin. Alternative chemical
probes that act orthogonally to the highly conserved acetyl-lysine
binding sites may exhibit selectivity within the BET family and avoid
recently reported toxicity in clinical trials of BET bromodomain inhibitors.
Here, we report the first identification of a ligandable site on a
bromodomain outside the acetyl-lysine binding site. Inspired by our
computational prediction of hotspots adjacent to nonhomologous cysteine
residues within the C-terminal BRD4 bromodomain (BRD4-BD2),
we performed a midthroughput mass spectrometry screen to identify
cysteine-reactive fragments that covalently and selectively modify
BRD4. Subsequent mass spectrometry, NMR, and computational docking
analyses of electrophilic fragment hits revealed a novel ligandable
site near Cys356 that is unique to BRD4 among human bromodomains.
This site is orthogonal to the BRD4-BD2 acetyl-lysine binding site
as Cys356 modification did not impact binding of the pan-BET bromodomain
inhibitor JQ1 in fluorescence polarization assays nor an acetylated
histone peptide in AlphaScreen assays. Finally, we tethered our top-performing
covalent fragment to JQ1 and performed NanoBRET assays to provide
proof of principle that this orthogonal site can be covalently targeted
in intact human cells. Overall, we demonstrate the potential of targeting
sites orthogonal to bromodomain acetyl-lysine binding sites to develop
bivalent and covalent inhibitors that displace BRD4 from chromatin.