Covalent-fragment screening of BRD4 identifies a ligandable site orthogonal to the acetyl-lysine binding sites
Abstract
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…
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