학술논문
Determination of Ligand-Binding Affinity (Kd) Using Transverse Relaxation Rate (R2) in the Ligand-Observed 1H NMR Experiment and Applications to Fragment-Based Drug Discovery
Document Type
Article
Author
Liu, Manjuan; Mirza, Amin; McAndrew, P. Craig; Thapaliya, Arjun; Pierrat, Olivier A.; Stubbs, Mark; Hahner, Tamas; Chessum, Nicola E. A.; Innocenti, Paolo; Caldwell, John; Cheeseman, Matthew D.; Bellenie, Benjamin R.; van Montfort, Rob L. M.; Newton, Gary K.; Burke, Rosemary; Collins, Ian; Hoelder, Swen
Source
Journal of Medicinal Chemistry; 20230101, Issue: Preprints
Subject
Language
ISSN
00222623; 15204804
Abstract
High hit rates from initial ligand-observed NMR screening can make it challenging to prioritize which hits to follow up, especially in cases where there are no available crystal structures of these hits bound to the target proteins or other strategies to provide affinity ranking. Here, we report a reproducible, accurate, and versatile quantitative ligand-observed NMR assay, which can determine Kdvalues of fragments in the affinity range of low μM to low mM using transverse relaxation rate R2as the observable parameter. In this study, we examined the theory and proposed a mathematical formulation to obtain Kdvalues using non-linear regression analysis. We designed an assay format with automated sample preparation and simplified data analysis. Using tool compounds, we explored the assay reproducibility, accuracy, and detection limits. Finally, we used this assay to triage fragment hits, yielded from fragment screening against the CRBN/DDB1 complex.