Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules

Abstract

Bromodomain-containing protein dysregulation is linked to cancer, diabetes, and inflammation. Selective inhibition of bromodomain function is a newly proposed therapeutic strategy. We describe a 19F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of 19F resonances within fluorine-labeled proteins enables the simultaneous analysis of two fluorinated bromodomains by NMR. A library of 229 small molecules was screened against the first bromodomain of Brd4 and the BPTF bromodomain. We report the first small molecule selective for BPTF over Brd4, termed AU1. The Kd = 2.8 μM for AU1 which is active in a cell-based reporter assay. No binding is detected with Brd4. Three new Brd4 inhibitors with submicromolar affinity were also discovered. Brd4 hits were validated in a thermal stability assay and potency determined via fluorescence anisotropy. The speed, ease of interpretation, and low protein concentration needed for protein-observed 19F NMR experiments in a multi-protein format, offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.

Molecular Basis for the N-Terminal Bromodomain-and-Extra-Terminal-Family Selectivity of a Dual Kinase–Bromodomain Inhibitor

“…The observed affinity for BRD4(1) was 3-fold weaker than the previously reported data of the compound used directly from the library stock, indicating a possible discrepancy in concentration or purity. Consistent with our prior report, 26 the para-substituted analogue, II , bound substantially more weakly. However, a 3,4-dimethyl-substituted analogue with an O-substituted pyrimidine, compound III , bound with increased affinity (IC 50 = 2.9 ± 0.2 μ M).…”

BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen

“…21,28–31 PrOF NMR utilizes 19 F NMR spectra of fluorine-labeled proteins, in this case 5-fluorotryptophan (5FW) labeled Brd4(1) and BrdT(1). Because of the high environmental sensitivity of the 19 F protein resonances, perturbations to the 19 F NMR spectrum of the protein, such as resonance broadening or shifting in the presence of ligand, correlate to ligand binding.…”

Paramagnetic relaxation enhancement for protein-observed 19F NMR as an enabling approach for efficient fragment screening

“…19 However, binding is non-specific and the change in chemical shift does not reach a saturation point up to 200 mM Ni-DTPA (Figure S5). Similarly, the chemical shift of fluorine resonances can change substantially upon titration of deuterated solvent, underscoring the large environmental sensitivity of the fluorine nucleus.…”

“…Changes in melting temperature were ≤1.2 °C by this method (Figure S6). To assess the functional effect of Ni-DTPA on binding, fluorescence anisotropy was used to quantitate the binding affinity between a fluorescently labelled BET bromodomain ligand BI-BODIPY 19,33 and Brd4 (Figure S7). Binding was minimally perturbed, with only minor perturbations to the dissociation constant upon increasing concentrations of Ni-DTPA (Table S1).…”