Boosting the sensitivity of ligand-protein screening by NMR of long-lived states.
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.
Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
Nicola
N
Salvi
Roberto
R
Buratto
Aurélien
A
Bornet
Simone
S
Ulzega
Inmaculada
I
Rentero Rebollo
Alessandro
A
Angelini
Christian
C
Heinis
Geoffrey
G
Bodenhausen
D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
D004791:Enzyme Inhibitors; D008024:Ligands; D009842:Oligopeptides; C031918:glycyl-glycyl-argininal; D014568:Urokinase-Type Plasminogen Activator