Boosting Sensitivity of Ligand-Protein Screening by NMR of Long-Lived States.
Résumé
A new NMR method for the study of ligand-protein interactions by NMR exploits the unusual lifetimes of long-lived states (LLS). The new method provides better contrast between bound and free ligands, and requires a protein-ligand ratio about 25 times lower than established T1ρ methods, thus saving on costly proteins. The new LLS method was applied to the screening of inhibitors of urokinase-type plasminogen activator (uPA), which is a prototypical target of cancer research. Using only 10 μM protein, a dissociation constant KD = 180 +/- 20 nM has been determined for the strong ligand (inhibitor) UK-18, which can be compared with KD = 157 +/- 39 nM determined by the established SPR method.