Discovery of ligands for Nurr1 by combined use of NMR screening with different isotopic and spin-labeling strategies.
A comprehensive approach to target screening, hit validation, and binding site determination by nuclear magnetic resonance (NMR) spectroscopy is presented. NMR (19)F signal perturbation was used to screen a small compound library and identify candidate ligands to the target of interest. Ligand dissociation constants were measured using a pegylated form of the protein, which resulted in a 2-fold increase in the strength of the saturation transfer difference signal. The initial small-molecule hits were further optimized by combining a residue-specific labeling strategy, to identify the specific sites of interaction with the protein, with a second site screening approach based on relaxation enhancement using a paramagnetic probe. The advantages of this combination strategy in the identification and optimization of weak binding chemical entities early in a program are illustrated with the discovery of a low micromolar ligand (K(d) = 20 microM) for Nurr1 and identification of the binding site location through residue-specific (15)N isotope labeling and derivatization of Cys residues with 2-mercaptoethanol-1-(13)C.
Poppe
Leszek
L
Molecular Structure, Amgen, Thousand Oaks, CA 91230, USA. lpoppe@amgen.com
Harvey
Timothy S
TS
Mohr
Christopher
C
Zondlo
James
J
Tegley
Christopher M
CM
Nuanmanee
Opas
O
Cheetham
Janet
J
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005461:Fluorine; D006801:Humans; D007553:Isotope Labeling; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D015203:Reproducibility of Results; D013113:Spin Labels; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D008024:Ligands; C539882:NR4A2 protein, human; D009587:Nitrogen Isotopes; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D013113:Spin Labels; D014157:Transcription Factors; D005461:Fluorine