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STD NMR is a powerful ligand-based tool for screening small molecules and low molecular weight fragments for their interaction with a given macromolecule, and it has become the spectroscopic technique of choice for the study of medium/weak affinity protein-ligand interactions. In the pharmaceutical industry, there is a great interest in the accurate and fast determination of protein-fragment binding affinities, typically low. STD NMR is a uniquely suited technique to accurately determine weak proteinligand affinities. However, a drawback of the technique is that, in order to gain quantitative structural or affinity information from STD NMR experiments, long series of experiments at increasing values of the saturation time of the protein must be carried out, to get the full analysis of the so-called STD NMR build-up curve (“initial slopes approach”). To resolve this limitation, we have developed a protocol that allows to get accurate initial slopes using STD NMR data acquired at only 2 saturation times. We demonstrate that our protocol, called the Reduced Dataset STD NMR approach (rd-STD NMR), allows the very fast determination of dissociation constants of low affinity protein-ligand interactions.

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