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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 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 via 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.

SUBMITTER: Urick AK

PROVIDER: S-EPMC8325173 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Inhibitor discovery for protein-protein interactions has proven difficult due to the large protein surface areas and dynamic interfaces involved. This is particularly the case when targeting transcription-factor-protein interactions. To address this challenge, structural biology approaches for ligand discovery using X-ray crystallography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy have had a significant impact on advancing small molecule inhibitors into the clinic, including the U.S. Food and Drug Administration approved drug, Venetoclax. Inspired by the protein-observed NMR approach using 1H-15N-HSQC NMR which detects chemical shift perturbations of 15N-labeled amides, we have applied a complementary protein-observed 19F NMR approach using 19F-labeled side-chains that are enriched at protein-protein-interaction interfaces. This protein-observed 19F NMR assay is abbreviated PrOF NMR to distinguish the experiment from the more commonly employed ligand-observed 19F NMR methods. In this Account, we describe our efforts using PrOF NMR as a ligand discovery tool, particularly for fragment-based ligand discovery (FBLD). We metabolically label the aromatic amino acids on proteins due to the enrichment of aromatic residues at protein interfaces. We choose the 19F nucleus due to its high signal sensitivity and the hyperresponsiveness of 19F to changes in chemical environment. Simultaneous labeling with two different types of fluorinated aromatic amino acids for PrOF NMR has also been achieved. We first describe the technical aspects of considering the application of PrOF NMR for characterizing native protein-protein interactions and for ligand screening. Several test cases are further described with a focus on a transcription factor coactivator interaction with the KIX domain of CBP/p300 and two epigenetic regulatory domains, the bromodomains of BRD4 and BPTF. Through these case studies, we highlight medicinal chemistry applications in FBLD, selectivity screens, structure-activity relationship (SAR) studies, and ligand deconstruction approaches. These studies have led to the discovery of some of the first inhibitors for BPTF and a novel inhibitor class for the N-terminal bromodomain of BRD4. The speed, ease of interpretation, and relatively low concentration of protein needed for NMR-based binding experiments affords a rapid, structural biology-based method to discover and characterize both native and new ligands for bromodomains, and it may find utility in the study of additional epigenetic proteins and transcription-factor-protein interactions.

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

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

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OmicsDI is part of the ELIXIR infrastructure

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