8CKF

Crystal Structure of the first bromodomain of human BRD4 L94C variant in complex with racemic 3,5-dimethylisoxazol ligand

Summary for 8CKF

• Entry DOI: 10.2210/pdb8ckf/pdb
• Descriptor: Bromodomain-containing protein 4, 3-(3,5-dimethyl-1,2-oxazol-4-yl)-5-[(~{R})-oxidanyl(pyridin-3-yl)methyl]phenol, 3-(3,5-dimethyl-1,2-oxazol-4-yl)-5-[(~{S})-oxidanyl(pyridin-3-yl)methyl]phenol, ... (4 entities in total)
• Functional Keywords: bromodomain, ligand, epigenetics, reader domain, transcription regulation, gene regulation
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 1
• Total formula weight: 18153.68

Authors

Thomas, A.M.,McDonough, M.A.,Schiedel, M.,Conway, S.J. (deposition date: 2023-02-15, release date: 2023-08-02, Last modification date: 2023-11-29)

Primary citation

Thomas, A.M.,Serafini, M.,Grant, E.K.,Coombs, E.A.J.,Bluck, J.P.,Schiedel, M.,McDonough, M.A.,Reynolds, J.K.,Lee, B.,Platt, M.,Sharlandjieva, V.,Biggin, P.C.,Duarte, F.,Milne, T.A.,Bush, J.T.,Conway, S.J.
Mutate and Conjugate: A Method to Enable Rapid In-Cell Target Validation.
Acs Chem.Biol., 18:2405-2417, 2023

PubMed Abstract: Target validation remains a challenge in drug discovery, which leads to a high attrition rate in the drug discovery process, particularly in Phase II clinical trials. Consequently, new approaches to enhance target validation are valuable tools to improve the drug discovery process. Here, we report the combination of site-directed mutagenesis and electrophilic fragments to enable the rapid identification of small molecules that selectively inhibit the mutant protein. Using the bromodomain-containing protein BRD4 as an example, we employed a structure-based approach to identify the L94C mutation in the first bromodomain of BRD4 [BRD4(1)] as having a minimal effect on BRD4(1) function. We then screened a focused, KAc mimic-containing fragment set and a diverse fragment library against the mutant and wild-type proteins and identified a series of fragments that showed high selectivity for the mutant protein. These compounds were elaborated to include an alkyne click tag to enable the attachment of a fluorescent dye. These clickable compounds were then assessed in HEK293T cells, transiently expressing BRD4(1) or BRD4(1), to determine their selectivity for BRD4(1) over other possible cellular targets. One compound was identified that shows very high selectivity for BRD4(1) over all other proteins. This work provides a proof-of-concept that the combination of site-directed mutagenesis and electrophilic fragments, in a mutate and conjugate approach, can enable rapid identification of small molecule inhibitors for an appropriately mutated protein of interest. This technology can be used to assess the cellular phenotype of inhibiting the protein of interest, and the electrophilic ligand provides a starting point for noncovalent ligand development.

PubMed: 37874862
DOI: 10.1021/acschembio.3c00437

Experimental method

X-RAY DIFFRACTION (1.88 Å)