Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy

[Image: see text] While there has been recent success in the development of KRas(G12C) inhibitors, unmet needs for selective inhibitors of KRas(G12D) and the remaining oncogenic KRas proteins remain. Here, we applied trifluoromethyl-containing ligands of KRas proteins as competitive probe ligands to assay the occupancy of the switch II pocket by (19)F NMR spectroscopy. Structure–activity-relationship studies of probe ligands increased the sensitivity of the assay and identified structures that differentially detected each nucleotide state of KRas(G12D). These differences in selectivity, combined with the high resolution of (19)F NMR spectroscopy, enabled this method to be expanded to assay both nucleotide states of the protein simultaneously.