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Covalent-Fragment Screening of Brd4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites

submitted on 11.07.2019, 15:03 and posted on 12.07.2019, 17:41 by Michael Olp, Daniel Sprague, Stefan Kathman, Ziyang Xu, Alexandar Statsyuk, Brian Smith

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 non-homologous cysteine residues within the C-terminal Brd4 bromodomain (Brd4-BD2), we performed a mid-throughput 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 all 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. Finally, we tethered covalent fragments 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.


Discovering and Exploiting Selectivity within Tandem Bromodomains

National Institute of General Medical Sciences

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Pharmacological Inhibitors of Nedd4 ubiquitin ligase as anticancer therapeutics

National Institute of General Medical Sciences

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Chemistry of Life Processes Predoctoral Training Program

National Institute of General Medical Sciences

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American Heart Association grant 15SDG25830057

American Cancer Society institutional research grants 14-247-29-IRG, 86-004-26-IRG, and 93-037-18-IRG

American Diabetes Association grant 1-18-IBS-068

Michael Keelan Research Foundation

Advancing a Healthier Wisconsin endowment

Northwestern University

Pew Charitable Trusts

ACS Medicinal Chemistry Predoctoral Fellowship

CLP Lambert Fellowship

Medical Scientist Training Program

National Institute of General Medical Sciences

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Email Address of Submitting Author


Medical College of Wisconsin


United States

ORCID For Submitting Author


Declaration of Conflict of Interest

No conflict of interest

Version Notes

Original version submitted on 6/18/2019