Characterization of Cell-Surface Interactions of Ligands Using ¹⁹F NMR and DNP Hyperpolarization

The binding of small molecule ligands to membrane lipids and membrane proteins in live cells is characterized by their effect on nuclear spin relaxation. The hyperpolarization of ¹⁹F spins provides signal enhancements for the measurement of R₂ relaxation in single-scan NMR experiments. The relaxation rates of two prototype drug molecules, 2-methyl-3-(5-methylsulfanyl-[1,3,4]oxadiazol-2-yl)-6-trifluoromethyl-pyridine (ICT5040) and 4-(trifluoromethyl)benzene-1-carboximidamide (TFBC), are found to increase linearly with cell density in the presence of several different cell types. The linear increase is interpreted as the initial slope of a binding saturation curve. A stronger relaxivity of cells toward the hyperpolarized ITC5040 is observed compared to small unilamellar vesicles, when normalized to lipid concentration in each case. The difference in relaxation rates is not fully accounted for by the size difference between cells and vesicles. A larger effect of the suspension-grown DU4475 human breast cancer cells compared to adherent 4T1 mouse breast cancer cells and HEK293T human embryonic kidney cells is observed. This difference is reduced when DU4475 cells are subjected to the same trypsin treatment that is required for adherent cells, supporting the conclusion that the interaction with proteins increases the relaxivity of the cells toward the ligand molecules. The influence of these parameters suggests that model membranes are not always sufficient for binding studies, and that hyperpolarized R₂ relaxometry may be used for determining the interactions of ligand molecules with cells in biophysical studies or for drug discovery.