Determination of Ligand-Binding Affinity (K _d) Using Transverse Relaxation Rate (R ₂) in the Ligand-Observed ¹H NMR Experiment and Applications to Fragment-Based Drug Discovery

High hit rates from initial ligand-observed NMR screeningcan makeit challenging to prioritize which hits to follow up, especially incases where there are no available crystal structures of these hitsbound to the target proteins or other strategies to provide affinityranking. Here, we report a reproducible, accurate, and versatile quantitativeligand-observed NMR assay, which can determine K (d) values of fragments in the affinity range of low & mu;Mto low mM using transverse relaxation rate R (2) as the observable parameter. In this study, we examined thetheory and proposed a mathematical formulation to obtain K (d) values using non-linear regression analysis. We designedan assay format with automated sample preparation and simplified dataanalysis. Using tool compounds, we explored the assay reproducibility,accuracy, and detection limits. Finally, we used this assay to triagefragment hits, yielded from fragment screening against the CRBN/DDB1complex.