Boosting the Sensitivity of Ligand–Protein Screening by NMR of Long-Lived States

• Published in: Journal of the American Chemical Society Vol. 134; no. 27; pp. 11076 - 11079
• Main Authors: Salvi, Nicola, Buratto, Roberto, Bornet, Aurélien, Ulzega, Simone, Rentero Rebollo, Inmaculada, Angelini, Alessandro, Heinis, Christian, Bodenhausen, Geoffrey
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 11.07.2012
• Subjects: Binding Sites; Chemical Sciences; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Humans; Ligands; Magnetic Resonance Spectroscopy - methods; Models, Molecular; Oligopeptides - chemistry; Oligopeptides - pharmacology; Organic chemistry; Protein Binding; Urokinase-Type Plasminogen Activator - antagonists & inhibitors; Urokinase-Type Plasminogen Activator - chemistry; Urokinase-Type Plasminogen Activator - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja303301w

A new NMR method for the study of ligand–protein interactions exploits the unusual lifetimes of long-lived states (LLSs). The new method provides better contrast between bound and free ligands and requires a protein–ligand ratio ca. 25 times lower than for established T 1ρ 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. With only 10 μM protein, a dissociation constant (K D) of 180 ± 20 nM was determined for the strong ligand (inhibitor) UK-18, which can be compared with K D = 157 ± 39 nM determined by the established surface plasmon resonance method.