Development of Protein-Observed Fluorine Nuclear Magnetic Resonance Spectroscopy as a Ligand Discovery Technique

Abstract

Fragment-based drug design (FBDD) has been rapidly gaining traction in the drug discovery process. A central tenant of fragment-based molecular screening is to use less sophisticated small molecules to sample chemical space more efficiently. With Vemurafenib and Venetoclax as FDA approved therapeutics from FBDD and several others in Phase III clinical trials, FBDD is becoming a validated technique for drug discovery. However, because of their small size these fragments are likely to bind to their target with a low affinity, necessitating more sensitive methods to detect protein-ligand interactions during a screen. Nuclear magnetic resonance spectroscopy has emerged as one of several powerful biophysical techniques for conducting fragment screens. In this thesis, a 19F protein-observed NMR method for detecting bromodomain−ligand interactions using fluorine-labeled aromatic amino acids due to the conservation of aromatic residues in the bromodomain binding site is described. Therein, we test the sensitivity, accuracy, and speed of this method with small molecule ligands. Experiment times on the order of a few minutes and the simplicity of the NMR spectra obtained make this approach well-suited to the investigation of small- to medium-sized proteins, as well as the screening of multiple proteins in the same experiment. Simplified 19F NMR spectra allowed for simultaneous testing of multiple bromodomains to assess selectivity and identification of a new BPTF ligand. Fluorine labeling only modestly affected the Brd4 structure and function assessed by isothermal titration calorimetry, circular dichroism, and X-ray crystallography. To benchmark its potential as a ligand discovery tool, we compare the protein-observed 19F NMR screening method with the well-characterized ligand-observed 1H CPMG NMR screen. We selected the first bromodomain of Brd4 as a model system because of the high ligandability of Brd4 and the need for small molecule inhibitors of related epigenetic regulatory proteins. We conclude that for the protein class understudy here, protein-observed 19F NMR and 1H CPMG have similar sensitivity, with both being effective tools for ligand discovery. The speed, ease of interpretation, and low concentration of protein needed for binding experiments affords a new method to discover and characterize both native and new ligands.