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

Abstract

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 H 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 theF chemical shift to H in 1D-protein observedH NMR experiments. To address concerns of perturbations from fluorine incorporation, ligand binding trends and affinities were verified via thermal shift assays and isothermal titration calorimetry. We conclude that for the protein understudy here, PrOF NMR and H CPMG have similar sensitivity, with both being effective tools for ligand discovery. In cases where an unlabeled protein can be used, 1D protein-observedH NMR may also be effective; however, the F chemical shift remains significantly more responsive.

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

“…Recovery of the signal can be induced by addition of a high-affinity competitor. 42 An initial experiment with only H2A.Z AcK4,11 shows the two acetyl singlet resonances near 1.88 ppm. When either Pf GCN5 or the BPTF bromodomain was added, a significant decrease in resonance intensity is observed, as expected if the ligand were binding to the bromodomain.…”

The Biophysical Probes 2-fluorohistidine and 4-fluorohistidine: Spectroscopic Signatures and Molecular Properties

“…Additionally, biosynthetic incorporation of either fluorohistidine isomer introduces a valuable spectroscopic label for 19 F NMR studies. Fluorine NMR spectra provide vastly simplified information relative to traditional protein NMR spectra, with the ability to report on site-specific dynamics 62 63 and ligand-binding events 64 . While the origin of changes in fluorine chemical shifts are not systematically understood, this work provides some insight on how environment influences chemical shift.…”

Current NMR Techniques for Structure-Based Drug Discovery

“…Sequence-specific 19 F-incorporation into a target protein is accomplished by substituting the desired amino acids with fluorinated analogs [ 112 , 113 , 114 , 115 , 116 ], since the natural abundance of 19 F is 100%. 19 F-NMR is a useful alternative to 1 H-based NMR approaches for the quantitative and qualitative investigation of various structural and functional characteristics of the target protein, such as conformational fluctuation dynamics, timescale of enzymatic reaction turnover, and exchange rates of protein-ligand interactions [ 54 , 112 , 117 ]. The superior susceptibility of 19 F chemical shifts against its chemical environment is useful for a protein-based NMR approach in SBDD and for characterization of protein structure formation and thermal stability [ 54 , 118 ].…”