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Ranking of high-affinity ligands by NMR spectroscopy.

NMR spectroscopy is a powerful biophysical technique to detect and characterize molecular interactions. Its high sensitivity and robustness to detect weakly binding ligands makes it an attractive tool, particularly for the early phases of drug-discovery research that comprise hit finding and hit validation. Several methods are available to determine dissociation constants (KD) by NMR spectroscopy, [4–7] including the direct titration of protein target with increasing amounts of ligand and competition-based experiments, such as NMR reporter screening. However, these methods generally work only for weakly or moderately binding ligands, but not for tightly binding ligands. This situation precludes application of these methods to the later lead-optimization stage of drug-discovery projects. Potent compounds are generally evaluated in a functional assay, or by using different biophysical techniques, such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). In practice, however, data are sometimes conflicting, and ITC and SPR may not always be applicable. In these cases, application of an independent biophysical technique, such as NMR spectroscopy, would be desirable. Herein we describe a new NMR spectroscopic method that allows the precise determination of relative binding affinities of two tightly binding ligands. This approach is a valuable tool for the lead optimization process. Direct titration in NMR spectroscopy is not applicable for high-affinity ligands for two reasons: First, high-affinity ligands generally have slow dissociation kinetics (slow koff), so that upon titration of a ligand, a second signal set corresponding to the complexed state gradually appears while the signal set for the unbound state gradually disappears. In contrast, in the low-affinity case, fast koff rates generally lead to the shifting of signals as the fraction of complexed state increases, and the extent of chemical shift change can be conveniently plotted as a function of ligand concentration in order to determine the dissociation constant, KD. Second, it is a fundamental principle in biophysics that dissociation constants can be precisely measured only for protein concentrations in the range of KD. The high protein concentrations (typically double-digit micromolar) required for NMR spectroscopic measurements therefore allow the precise measurement of KD values in the micromolar or millimolar range, but not much lower. This is because the KD is encoded in the curvature of the titration curve, and the curvature is not precisely measurable for high affinities (Figure 1). Reporter screening extends this limit to the highnanomolar range, but not further since the dynamic range of quantification is about an order of magnitude around the reporter ligand, and the reporter ligand must show weak or intermediate binding.