Quantifying protein-drug lifetimes in human cells by ¹⁹F NMR spectroscopy

The cellular environment is a complex and crowded space, with organelles, compartments and multitudes of molecules engaged in intricate networks of communication that modulate binary protein-ligand/protein interactions. As a result, it is becoming increasingly appreciated that evaluations of protein-drug binding should be carried out in the native cellular environment. Here, we present a proof-of-concept study where we measured the lifetime (1/k_off) of a protein-drug complex in human cells by ¹⁹F NMR spectroscopy using fluorinated Cyclophilin A (CypA) bound to Cyclosporine A (CsA). Harnessing the exceptional detection sensitivity of the trifluoromethyl group attached at the para position of Phe60 in CypA, high-quality 2D ¹⁹F-¹⁹F exchange spectra were obtained in cells. Essentially identical k_off values were observed in cells and in vitro, suggesting that the overall impact of the cellular environment on the lifetime of tfmF60 CypA/CsA complex is minimal. Using similar approaches for quantifying protein-drug lifetimes in the native cellular environment paves the way for efficiently screening drug libraries in human cells by ¹⁹F NMR spectroscopy.