In the past decade, the potential of harnessing the ability of nuclear magnetic resonance (NMR) spectroscopy to monitor intermolecular interactions as a tool for drug discovery has been increasingly appreciated in academia and industry. In this Perspective, we highlight some of the major applications of NMR in drug discovery, focusing on hit and lead generation, and provide a critical analysis of its current and potential utility.

The Na,K-ATPase hydrolyzes ATP to drive the coupled extrusion and uptake of Na+ and K+ ions across the plasma membrane. Here, we report two high-resolution NMR structures of the 213-residue nucleotide-binding domain of rat alpha1 Na,K-ATPase, determined in the absence and the presence of ATP. The nucleotide binds in the anti conformation and shows a relative paucity of interactions with the protein, reflecting the low-affinity ATP-binding state. Binding of ATP induces substantial conformational changes in the binding pocket and in residues located in the hinge region connecting the N- and P-domains. Structural comparison with the Ca-ATPase stabilized by the inhibitor thapsigargin, E2(TG), and the model of the H-ATPase in the E1 form suggests that the observed changes may trigger the series of events necessary for the release of the K+ ions and/or disengagement of the A-domain, leading to the eventual transfer of the gamma-phosphate group to the invariant Asp369.

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Determining the three-dimensional structure of a small molecule-protein complex with weak affinity can be a significant challenge. We present a paramagnetic NMR method to determine intermolecular structure restraints based on pseudocontact shifts (PCSs). Since the ligand must be in fast exchange between free and bound states and the fraction bound can be as low as a few percent, the method is ideal for ligands with high micromolar to millimolar dissociation constants. Paramagnetic tags are attached, one at a time, in a well-defined way via two arms at several sites on the protein surface. The ligand PCSs were measured from simple 1D (1)H spectra and used as docking restraints. An independent confirmation of the complex structure was carried out using intermolecular NOEs. The results show that structures derived from these two approaches are similar. The best results are obtained if the magnetic susceptibility tensors of the tags are known, but it is demonstrated that with two-armed probes, the magnetic susceptibility tensor can be predicted with sufficient accuracy to provide a low-resolution model of the ligand orientation and the location of the binding site in the absence of isotope-labeled protein. This approach can facilitate fragment-based drug discovery in obtaining structural information on the initial fragment hits.