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Fluorinated bromodomains for small molecule inhibitor discovery

Protein-protein interaction (PPI) modulation offers many new opportunities for treating disease, but selective targeting remains a grand challenge. For early-stage chemical probe development, Professor William Pomerantz' lab takes advantage of the unique properties of fluorine using nuclear magnetic resonance spectroscopy (19F NMR). They recently published a report in ACS Chemical Biology, describing an application of their method for studying fluorinated transcription factors called bromodomains. Because fluorine is structurally similar to hydrogen, they can label proteins with fluorine atoms using the natural biosynthetic machinery. Due to fluorine’s absence in all naturally occurring biopolymers, they have a specific probe to study proteins by 19F NMR, a technique they call protein-observed fluorine NMR or PrOF NMR. Fluorine’s signal sensitivity and simplified spectra, make it a powerful NMR nucleus for accelerating PPI ligand discovery.

In their report, Pomerantz, along with graduate student Andrew Urick and postdoctoral research fellow Neeraj Mishra, Ph.D., describe a 19F 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. They test the sensitivity, accuracy, and speed of this method with small molecule ligands (+)-JQ1, BI2536, Dinaciclib, TG101348, and acetaminophen using three bromodomains Brd4, BrdT and BPTF. 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 Brd4 structure and function assessed by isothermal titration calorimetry and circular dichroism. In collaboration with the Moffitt Cancer Center, x-ray crystallographic analysis of their fluorinated protein also showed little structural perturbation. 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.

Figure and legend:
Aromatic amino acids are in close proximity to the bromodomain binding site. Ribbon diagram of Brd4(1) bound to (+)-JQ1 (PDB ID 3MXF). Tyrosine and tryptophan are indicated as sticks. Red and yellow indicate side-chains within 5 and 10 Å respectively. (Right) (+)-JQ1 and fluorinated amino acids used in this study.