WaterLOGSY represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. Several relay pathways are used constructively in the experiment for transferring bulk water magnetization to the ligand. The method is particularly useful for the identification of novel scaffolds of micromolar affinity that can be then optimized using directed screening, combinatorial chemistry, medicinal chemistry and structure-based drug design. The practical aspects and range of applicability of the WaterLOGSY experiment are analyzed in detail here. Competition binding and titration WaterLOGSY permit, after proper correction, the evaluation of the dissociation binding constant. The high sensitivity of the technique in combination with the easy deconvolution of the mixtures for the identification of the active components, significantly reduces the amount of material and time needed for the NMR screening process.

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.

Site-specific titration curves for 12 histidine residues in carbon monoxy sperm whale myoglobin (MbCO) have been determined from two-dimensional (2D) double quantum NMR experiments. Eight of these histidine residues are observed to titrate over the accessible pH range, and pK(a) values have been determined; bounds on the titration midpoints of the remaining four histidines are also reported. Results for residues 48, 81, and 119 differ significantly from those estimated from earlier, one-dimensional studies, but they are in good agreement with values recently determined for metaquomyoglobin. These experimental values (plus those determined earlier for tyrosine titrations) are compared to predictions from crystal structures of myoglobin using a numerical Poisson-Boltzmann model and a Monte Carlo treatment of the multiple-site titration. An extension of existing models is described that accounts for alternate tautomers for histidines. Calculations are reported using several choices for radii and charges, and for five crystal structures, in order to assess the sensitivity of the results to details of the calculations. In general, the agreement between calculated and observed titration behavior suggests that this theoretical model captures much of the electrostatic behavior in this system, even though it ignores conformational fluctuations and the differences in mean structures that may exist between crystal and solution. Interactions among titrating groups are often important; in general, these interactions lead to more gradual individual site titrations (the mean Hill coefficient is about 0.8), and in several cases the interactions are so strong that two side chains need to be considered as a unit and single residues may participate in two-step titrations. It is suggested that histidines involved in such two-step titrations and carboxylic acid residues with abnormally low pK(a) values in the native conformation may be involved in the acid-induced partial unfolding of MbCO.

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A powerful screening by NMR methodology (WaterLOGSY), based on transfer of magnetization from bulk water, for the identification of compounds that interact with target biomolecules (proteins, RNA and DNA fragments) is described. The method exploits efficiently the large reservoir of H2O magnetization. The high sensitivity of the technique reduces the amount of biomolecule and ligands needed for the screening, which constitutes an important requirement for high throughput screening by NMR of large libraries of compounds. Application of the method to a compound mixture against the cyclin-dependent kinase 2 (cdk2) protein is presented.