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DOI: 10.1177/1087057106299161 Discovery of Ligands for Nurr1 by Combined Use of NMR Screening with Different Isotopic and Spin-Labeling StrategiesMolecular Structure, Amgen, Thousand Oaks, CA, lpoppe{at}amgen.com
Molecular Structure, Amgen, Thousand Oaks, CA
Molecular Structure, Amgen, Thousand Oaks, CA
Protein Science, Amgen, Thousand Oaks, CA
Small Molecule Drug Discovery, Amgen, Thousand Oaks, CA
Protein Science, Amgen, Thousand Oaks, CA
Molecular Structure, Amgen, Thousand Oaks, CA A comprehensive approach to target screening, hit validation, and binding site determination by nuclear magnetic resonance (NMR) spectroscopy is presented. NMR 19F 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 (Kd = 20 µM) for Nurr1 and identification of the binding site location through residue-specific 15N isotope labeling and derivatization of Cys residues with 2-mercaptoethanol-1-13C. (Journal of Biomolecular Screening 2007:301-311)
Key Words: Nurr1 ligands • NMR screening • fragment-based approach • isotope labeling • spin labeling
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