Fluorine Pseudocontact Shifts Used for Characterizing the Protein-Ligand Interaction Mode in the Limit of NMR Intermediate Exchange.

The characterization of protein-ligand interaction modes becomes recalcitrant in the NMR intermediate exchange regime as the interface resonances are broadened beyond detection. Here, we determined the (19) F low-populated bound-state pseudocontact shifts (PCSs) of mono- and di-fluorinated inhibitors of the BRM bromodomain using a highly skewed protein/ligand ratio. The bound-state (19) F PCSs were retrieved from (19) F chemical exchange saturation transfer (CEST) in the presence of the lanthanide-labeled protein, which was termed the (19) F PCS-CEST approach. These PCSs enriched in spatial information enabled the identification of best-fitting poses, which agree well with the crystal structure of a more soluble analog in complex with the BRM bromodomain. This approach fills the gap of the NMR structural characterization of lead-like inhibitors with moderate affinities to target proteins, which are essential for structure-guided hit-to-lead evolution.