Imaging Saturation Transfer Difference (STD) NMR: Affinity and Specificity of Protein-Ligand Interactions from a Single NMR Sample

We have combined saturation transfer difference NMR (STDNMR) withchemical shift imaging (CSI) and controlled concentration gradientsof small molecule ligands to develop imaging STD NMR, a new tool forthe assessment of protein-ligand interactions. Our methodologyallows the determination of protein-ligand dissociation constants(K (D)) and assessment of the binding specificityin a single NMR tube, avoiding time-consuming titrations. We demonstratethe formation of suitable and reproducible concentration gradientsof ligand along the vertical axis of the tube, against homogeneousprotein concentration, and present a CSI pulse sequence for the acquisitionof STD NMR experiments at different positions along the sample tube.Compared to the conventional methodology in which the [ligand]/[protein]ratio is increased manually, we can perform STD NMR experiments ata greater number of ratios and construct binding epitopes in a fraction(& SIM;20%) of the experimental time. Second, imaging STD NMR alsoallows us to screen for non-specific binders, by monitoring any variationof the binding epitope map at increasing [ligand]/[protein] ratios.Hence, the proposed method does carry the potential to speed up andsmooth out the drug discovery process.

Automated summary

We developed a new tool called imaging STD NMR by combining saturation transfer difference NMR (STDNMR) with chemical shift imaging (CSI) and controlled concentration gradients of small molecule ligands. This method allows for the assessment of protein-ligand interactions, determination of dissociation constants (K(D)), and evaluation of binding specificity in a single NMR tube, saving time compared to traditional titration methods. Imaging STD NMR also allows for screening of non-specific binders, making it a potential tool to speed up and optimize the drug discovery process.