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Rapid Quantification of Protein-Ligand Binding via ¹⁹F NMR Lineshape Analysis.

ABSTRACT: Fluorine incorporation is ideally suited to many NMR techniques, and incorporation of fluorine into proteins and fragment libraries for drug discovery has become increasingly common. Here, we use one-dimensional ¹⁹F NMR lineshape analysis to quantify the kinetics and equilibrium thermodynamics for the binding of a fluorine-labeled Src homology 3 (SH3) protein domain to four proline-rich peptides. SH3 domains are one of the largest and most well-characterized families of protein recognition domains and have a multitude of functions in eukaryotic cell signaling. First, we showe that fluorine incorporation into SH3 causes only minor structural changes to both the free and bound states using amide proton temperature coefficients. We then compare the results from lineshape analysis of one-dimensional ¹⁹F spectra to those from two-dimensional ¹H-¹⁵N heteronuclear single quantum coherence spectra. Their agreement demonstrates that one-dimensional ¹⁹F lineshape analysis is a robust, low-cost, and fast alternative to traditional heteronuclear single quantum coherence-based experiments. The data show that binding is diffusion limited and indicate that the transition state is highly similar to the free state. We also measured binding as a function of temperature. At equilibrium, binding is enthalpically driven and arises from a highly positive activation enthalpy for association with small entropic contributions. Our results agree with those from studies using different techniques, providing additional evidence for the utility of ¹⁹F NMR lineshape analysis, and we anticipate that this analysis will be an effective tool for rapidly characterizing the energetics of protein interactions.

SUBMITTER: Stadmiller SS

PROVIDER: S-EPMC7231920 | 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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Rapid Quantification of Protein-Ligand Binding via ¹⁹F NMR Lineshape Analysis.

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Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis.

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OmicsDI is part of the ELIXIR infrastructure

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Rapid Quantification of Protein-Ligand Binding via ¹⁹F NMR Lineshape Analysis.