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Hit Validation: Orthogonal NMR Methods


Fragment Hit validation via Protein Observed NMR (HSQC). When the target can be expressed in bacteria, stable NMR visible isotopes can be incorporated. This allows efficient acquisition of NMR spectra (HSQC, see figure upper left) of the protein that are extremely sensitive to ligand binding.

This orthogonal technique can be used to validate and quantify binding (upper right) as well as to define a low resolution binding site on the target. At bottom left, we noted a good correlation between binding detected by TINS (T/R ratio, y axis) and the HSQC experiment (average CSP, x axis) for 86 fragment hits against this target.


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