SAR by (Protein-Observed) ¹⁹F NMR

Abstract

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 1 H− 15 N-HSQC NMR which detects chemical shift perturbations of 15 N-labeled amides, we have applied a complementary protein-observed 19 F NMR approach using 19 F-labeled side-chains that are enriched at protein−proteininteraction interfaces. This protein-observed 19 F NMR assay is abbreviated PrOF NMR to distinguish the experiment from the more commonly employed ligand-observed 19 F 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 19 F nucleus due to its high signal sensitivity and the hyperresponsiveness of 19 F 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.

Solution NMR readily reveals distinct structural folds and interactions in doubly 13 C- and 19 F-labeled RNAs

“…Ligand-based ( 35 ) and protein-observed ( 36 ) 19 F NMR screening methods are important for identifying small drug-like molecules that act as protein inhibitors. Although most work to date has focused on proteins, recent work suggests that RNAs also contain specific binding pockets that could be easily distinguished and targeted with small molecules ( 1 , 2 ).…”

What does fluorine do to a protein? Thermodynamic, and highly-resolved structural insights into fluorine-labelled variants of the cold shock protein

“…An elegant combination of X-ray crystallography, NMR spectroscopy, and MD simulations has been recently applied to unravel the distribution of conformational states along the reaction pathway of a homodimeric enzyme 22 . Pomerantz and co-workers have successfully utilized fluoroaromatic amino acids for the implementation into proteins to report on the structure-activity relationship 23 focusing on screening of small molecule-protein interactions based on protein-observed fluorine NMR spectroscopy 24 . Moreover, incorporation of difluorotyrosine has been successfully employed enabling to report on tyrosine phosphorylation 25 or to probe distinct conformational states of a protein which are related to signalling by applying 19 F NMR spectroscopy 26 .…”

Perfluorocarbons in Chemical Biology

“…Protein fluorination can also be used to study protein-small molecule interactions with 19 F NMR. [81,82] Changes to protein conformation or hydration upon the introduction of a small molecule can be detected, which provides a unique assay for drug discovery. This technique, deemed protein-observed 19 F NMR (PrOF), is an alternative to the more commonly performed ligand-observed 19 F NMR methods, which rely on monitoring changes in fluorinated small-molecule fragments in the presence of a non-fluorinated protein target.…”

“…Protein fluorination can also be used to study protein‐small molecule interactions with 19 F NMR [81,82] . Changes to protein conformation or hydration upon the introduction of a small molecule can be detected, which provides a unique assay for drug discovery.…”