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

## TL;DR

This paper introduces a method using 19F NMR and DNP hyperpolarization to study how small molecules bind to cells, showing that cell type and protein interactions affect binding behavior.

## Contribution

The study demonstrates that hyperpolarized 19F NMR can detect ligand-cell interactions in live cells, revealing differences not captured by model membranes.

## Key findings

- Relaxation rates of ligands increase linearly with cell density, indicating binding saturation.
- Hyperpolarized ITC5040 shows stronger relaxivity in cells than in vesicles, not fully explained by size differences.
- Suspension-grown cells show higher relaxivity than adherent cells, reduced after trypsin treatment, suggesting protein involvement.

## Abstract

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 R
2 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 R
2 relaxometry may be used for determining the
interactions of ligand molecules with cells in biophysical studies
or for drug discovery.