Protein-Observed Fluorine NMR Is a Complementary Ligand Discovery Method to 1H CPMG Ligand-Observed NMR
| Autor: | William C. K. Pomerantz, Haitao Hu, Luis Pablo Calle, Juan F. Espinosa, Andrew K. Urick |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Magnetic Resonance Spectroscopy Chemistry Analytical chemistry chemistry.chemical_element Fluorine General Medicine Fluorine-19 NMR Ligands Ligand (biochemistry) Biochemistry Small molecule Affinities Combinatorial chemistry Article Bromodomain 03 medical and health sciences 030104 developmental biology Proton NMR Molecular Medicine Molecule |
| Zdroj: | ACS Chem Biol |
| ISSN: | 1554-8937 1554-8929 |
| Popis: | 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 (1)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 the (19)F chemical shift to (1)H in 1D-protein observed (1)H 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 (1)H CPMG have similar sensitivity, with both being effective tools for ligand discovery. In cases where an unlabeled protein can be used, 1D protein-observed (1)H NMR may also be effective; however, the (19)F chemical shift remains significantly more responsive. |
| Databáze: | OpenAIRE |
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