NMR methods in fragment screening: theory and a comparison with other biophysical techniques

Nuclear magnetic resonance, surface plasmon resonance and fluorescence spectroscopy (particularly fluorescence anisotropy and fluorescence lifetime) are techniques often applied to fragment screening. These methodologies are analyzed on the basis of their performance, strengths, limitations and pitfalls. NMR-based screening, despite its low intrinsic sensitivity, offers the largest dynamic range and is capable of capturing very weak interactions. Theoretical simulation demonstrates the power of some NMR experiments, in particular those with fluorine observation, in detecting protein interactions for fragments tested at concentrations orders of magnitude lower than their dissociation binding constants. This apparently counterintuitive finding enables the identification of hits that are insoluble at the concentrations required for detection by other biophysical techniques. However it is evident that several techniques should be applied and the data analyzed on the basis of their complementarities.