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 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.

A direct-to-biology high-throughput chemistry approach to reactive fragment screening

“…7 Covalent fragment-based technologies have recently emerged as a complementary approach for identification of tool molecules. [8][9][10][11][12][13] Covalent fragments leverage the ability of relatively small compounds (<300 Da) to efficiently explore chemical space. [14][15][16][17] Such fragments are typically functionalised with an electrophile to enable the capture of transient fragment-protein interactions.…”

“…α,β-unsaturated carboxylic esters and amides). [8][9][10][11][12][13]18 These platforms have enabled the development of numerous chemical probes, including for previously unliganded proteins (Fig. 1a).…”

Combined Protein- and Ligand-Observed NMR Workflow to Screen Fragment Cocktails against Multiple Proteins: A Case Study Using Bromodomains

“…The response of both resonances may indicate a conformational change in the protein upon binding or ligands accessing a second binding site near Trp1454. Although rare for bromodomains, recently an additional binding site has been discovered for the second bromodomain of BRD4 [ 52 ]. Titration of 9 with Pf GCN5 showed significant responses of both 5FW resonances ( Figure 2 ) and warranted additional exploration of a possible second binding site on Pf GCN5.…”

“…Finally, we used the in silico solvent docking program, FTMap, to identify any additional hot spot residues outside the native histone binding site [ 56 ]. This program has been used previously by Olp et al to identify a new binding site on BRD4 D2 [ 52 ]. In this case, whereas several large solvent clusters were found in the histone binding site, only a single cluster of 12 solvent molecules was found near Trp1454.…”

Therapeutic targeting of chromatin: status and opportunities

“…PROTACs are dependent on a ligand to the protein of interest, but this ligand does not necessarily need to bind the domain that is important for the protein's activity. For example, BRD4-targeting PROTACs have not only been designed from the well-studied ligands of the acetyllysine binding site [189][190][191], but also towards a ligandable allosteric site that was detected by screening of covalent fragments [192].…”