Quantifying the Selectivity of Protein–Protein and Small Molecule Interactions with Fluorinated Tandem Bromodomain Reader Proteins

Abstract

Multidomain bromodomain-containing proteins regulate gene expression via chromatin binding, interactions with the transcriptional machinery, and by recruiting enzymatic activity. Selective inhibition of members of the bromodomain and extra-terminal (BET) family is important to understand their role in disease and gene regulation, although due to the similar binding sites of BET bromodomains, selective inhibitor discovery has been challenging. To support the bromodomain inhibitor discovery process, here we report the first application of protein-observed fluorine (PrOF) NMR to the tandem bromodomains of BRD4 and BRDT to quantify the selectivity of their interactions with acetylated histones as well as small molecules. We further determine the selectivity profile of a new class of ligands, 1,4-acylthiazepanes, and find them to have ≥3–10-fold selectivity for the C-terminal bromodomain of both BRD4 and BRDT. Given the speed and lower protein concentration required over traditional protein-observed NMR methods, we envision that these fluorinated tandem proteins may find use in fragment screening and evaluating nucleosome and transcription factor interactions.

19F NMR viewed through two different lenses: ligand-observed and protein-observed19F NMR applications for fragment-based drug discovery

“…10a and b). 35,[127][128][129][130][131][132] In a cross-validation study of PrOF NMR, a 930-member fragment library was screened against the first bromodomain of 5FW-labeled BRD4 (BRD4 D1) in mixtures of five using both PrOF NMR and ligand-observed 1 H CPMG NMR (with and without competitor validation). 41 Comparing the individual fragments, there was 85% overlap in hit detection when using competitors.…”

“…1,4-Acylthiazepanes were later shown to have B10-fold higher affinity for the second bromodomain of BET proteins, using fluorine-labeled constructs containing the two tandem bromodomains of BRD4 and BRDT. 130 Bromodomain selectivity is challenging because of their relatively conserved acetyl lysine binding sites. Screening of protein mixtures is one method to enable selectivity information upfront.…”

“…135 The selectivity for BRD4-D1 was further confirmed using the fluorine-labeled BRD4 tandem domain construct. 130 Of the small molecule hits for BPTF from the dual screen with BRD4 D1, the tightest binders were molecules with arylurea motifs. 129 AU1 was selected for follow-up, which in its racemic form had a K d of 2.8 mM by ITC, and by PrOF NMR titrations it was determined that (S)-AU1 was the active enantiomer perturbing the reporter resonance, W2950, in the acetyl lysine binding pocket.…”

“…1,4-Acylthiazepanes were later shown to have ∼10-fold higher affinity for the second bromodomain of BET proteins, using fluorine-labeled constructs containing the two tandem bromodomains of BRD4 and BRDT. 130 …”

“… 135 The selectivity for BRD4-D1 was further confirmed using the fluorine-labeled BRD4 tandem domain construct. 130 …”