Chemical competition
NMR provides a sensitive, spatially resolved approach for observing a ligand binding to a protein. However, because of the timescales involved and protein concentrations required, NMR is limited to measuring affinities of relatively weakly binding compounds, typically in the micromolar to millimolar range. Thus, NMR has been useful for the early stages of drug discovery, but SPR or ITC are typically used to determine compound affinities during lead optimization. Zhang et al. now report an NMR method for determining the relative binding affinities of low nanomolar to subnanomolar ligands. In one approach, the two-dimensional spectrum is determined for each ligand bound separately to an isotopically labeled protein. A 1:1 mixture of the ligands is then incubated with the protein and allowed to equilibrate. The relative binding constants of the two compounds can be directly related to their relative peak intensities. In a less accurate but faster and less protein-intensive approach, the one-dimensional spectra of two ligands are measured separately and in an equamolar mixture. When protein is added to the mixture, the NMR signal broadens for any bound ligand, enabling a calculation of the relative Kd values from the amount of each ligand that remains unbound. With this method, NMR can be used to accurately rank potent lead compounds. (Angew. Chem. Int. Ed. 48, 6691–6694, 2009) JK
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