Protein-Observed Fluorine NMR: A Bioorthogonal Approach for Small Molecule Discovery

The ¹⁹F isotope is 100% naturally abundant and is the second most sensitive and stable NMR-active nucleus. Unlike the ubiquitous hydrogen atom, fluorine is nearly absent in biological systems, making it a unique bioorthogonal atom for probing molecular interactions in biology. Over 73 fluorinated proteins have been studied by ¹⁹F NMR since the seminal studies of Hull and Sykes in 1974. With advances in cryoprobe production and fluorinated amino acid incorporation strategies, protein-based ¹⁹F NMR offers opportunities to the medicinal chemist for characterizing and ultimately discovering new small molecule protein ligands. This review will highlight new advances using ¹⁹F NMR for characterizing small molecule interactions with both small and large proteins as well as detailing NMR resonance assignment challenges and amino acid incorporation approaches

Prediction of ¹⁹F NMR Chemical Shifts in Labeled Proteins: Computational Protocol and Case Study

The structural analysis of ligand complexation in biomolecular systems is important in the design of new medicinal therapeutic agents; however, monitoring subtle structural changes in a protein’s microenvironment is a challenging and complex problem. In this regard, the use of protein-based ¹⁹F NMR for screening low-molecular-weight molecules (i.e., fragments) can be an especially powerful tool to aid in drug design. Resonance assignment of the protein’s ¹⁹F NMR spectrum is necessary for structural analysis. Here, a quantum chemical method has been developed as an initial approach to facilitate the assignment of a fluorinated protein’s ¹⁹F NMR spectrum. The epigenetic “reader” domain of protein Brd4 was taken as a case study to assess the strengths and limitations of the method. The overall modeling protocol predicts chemical shifts for residues in rigid proteins with good accuracy; proper accounting for explicit solvation of fluorinated residues by water is critical

Protein-Observed Fluorine NMR Is a Complementary Ligand Discovery Method to ¹H CPMG Ligand-Observed NMR

To evaluate its potential as a ligand discovery tool, we compare a newly developed 1D protein-observed fluorine NMR (PrOF NMR) screening method with the well-characterized ligand-observed ¹H CPMG NMR screen. We selected the first bromodomain of Brd4 as a model system to benchmark PrOF NMR because of the high ligandability of Brd4 and the need for small molecule inhibitors of related epigenetic regulatory proteins. We compare the two methods’ hit sensitivity, triaging ability, experiment speed, material consumption, and the potential for false positives and negatives. To this end, we screened 930 fragment molecules against Brd4 in mixtures of five and followed up these studies with mixture deconvolution and affinity characterization of the top hits. In selected examples, we also compare the environmental responsiveness of the ¹⁹F chemical shift to ¹H in 1D-protein observed ¹H NMR experiments. To address concerns of perturbations from fluorine incorporation, ligand binding trends and affinities were verified <i>via</i> thermal shift assays and isothermal titration calorimetry. We conclude that for the protein understudy here, PrOF NMR and ¹H CPMG have similar sensitivity, with both being effective tools for ligand discovery. In cases where an unlabeled protein can be used, 1D protein-observed ¹H NMR may also be effective; however, the ¹⁹F chemical shift remains significantly more responsive

Specific Acetylation Patterns of H2A.Z Form Transient Interactions with the BPTF Bromodomain

Post-translational lysine acetylation of histone tails affects both chromatin accessibility and recruitment of multifunctional bromodomain-containing proteins for modulating transcription. The bromodomain- and PHD finger-containing transcription factor (BPTF) regulates transcription but has also been implicated in high gene expression levels in a variety of cancers. In this report, the histone variant H2A.Z, which replaces H2A in chromatin, is evaluated for its affinity for BPTF with a specific recognition pattern of acetylated lysine residues of the N-terminal tail region. Although BPTF immunoprecipitates H2A.Z-containing nucleosomes, a direct interaction with its bromodomain has not been reported. Using protein-observed fluorine nuclear magnetic resonance (PrOF NMR) spectroscopy, we identified a diacetylation of H2A.Z on lysine residues 4 and 11, with the highest affinity for BPTF with a <i>K</i>_d of 780 μM. A combination of subsequent ¹H NMR Carr–Purcell–Meiboom–Gill experiments and photo-cross-linking further confirmed the specificity of the diacetylation pattern at lysines 4 and 11. Because of an adjacent PHD domain, this transient interaction may contribute to a higher-affinity bivalent interaction. Further evaluation of specificity toward a set of bromodomains, including two BET bromodomains (Brd4 and BrdT) and two <i>Plasmodium falciparum</i> bromodomains, resulted in one midmicromolar affinity binder, <i>Pf</i>GCN5 (<i>K</i>_d = 650 μM). With these biochemical experiments, we have identified a direct interaction of histone H2A.Z with bromodomains with a specific acetylation pattern that further supports the role of H2A.Z in epigenetic regulation

Design, Synthesis, and Characterization of a Fluorescence Polarization Pan-BET Bromodomain Probe

Several chemical probes have been developed for use in fluorescence polarization screening assays to aid in drug discovery for the bromodomain and extra-terminal domain (BET) proteins. However, few of those have been characterized in the literature. We have designed, synthesized, and thoroughly characterized a novel fluorescence polarization pan-BET chemical probe suitable for high-throughput screening, structure–activity relationships, and hit-to-lead potency and selectivity assays to identify and characterize BET bromodomain inhibitors

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

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 <i>K</i>_d = 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 multiprotein format offers a new method to discover and characterize selective ligands for bromodomain-containing proteins

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

Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease. We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay. Dihydropyrido­pyimidine <b>3</b> was subjected to further characterization and is highly selective for the BET family of bromodomains. Structure–activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity. Compound <b>3</b> was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure–activity data. An additional series of dihydropyrido­pyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement. Given these findings, novel and easily synthesized inhibitors are being introduced to the growing field of bromodomain inhibitor development