Applications of Fluorine Magnetic Resonance for Small-Molecule Screening, Ligand Development, and Oxygen Sensing

Abstract

Protein-protein interactions (PPIs) play a vital role in biological processes but are difficult to target therapeutically. However, targeting PPIs is an important challenge because their dysregulation is linked to many various disease states including cancers and neurological disorders. While high throughput screening (HTS) has long been the standard method for drug discovery, fragment-based screening (FBS) has emerged as a promising alternative strategy due to its greater coverage of chemical space with smaller library sizes. Successful cases like Vemurafenib and Venetoclax, continue to bolster FBS efforts. Though many techniques, including X-ray crystallography, surface plasmon resonance, and thermal shift assays, have all been used as screening tools, the central hypothesis of this dissertation is that 19F NMR is a powerful and time efficient FBS tool that is complementary to existing tools and is useful for characterizing proteins and small molecule ligands. Protein-Observed Fluorine NMR Spectroscopy (PrOF NMR) due to its high speed, lack of background signals, environmental sensitivity, is an ideal method to use for both ligand discovery and characterization of ligand-protein interactions. Herein, we describe the application of PrOF NMR to two proteins in particular, the KIX domain of CBP/p300 which is part of a larger transcriptional activation complex, and the first bromodomain of BrdT, an epigenetic “reader” protein that has been validated as a target for male contraception. We demonstrate the use of PrOF NMR as a primary screening tool for KIX, identifying key pharmacophores for KIX binding. We also demonstrate the use of PrOF NMR for characterizing ligand-protein interactions, uncovering a new binding site in KIX, distinct from its two native transcription factor binding sites. Validation of hits from other screening campaigns can also be followed via PrOF NMR, and the quantitative information obtained can be used to guide the structure-activity relationship (SAR) process for further ligand development. Beyond ligand discovery in proteins, fluorine magnetic resonance can also be applied as an imaging and oximetry tool. Given the sensitivity of fluorine and its applications in both biophysical and biomedical contexts, fluorine magnetic resonance serves as a new tool for small-molecule screening, ligand development, and oxygen sensing.