Abstract
In drug development programmes, multiple assays are needed for the determination of protein-compound interactions and evaluation of potential use in assays with protein-protein interactions. In this protocol we describe the waterLOGSY NMR method for confirming protein-ligand binding events.
Highlights
[Background] As more altered protein forms are found in disease cells, there has been an increase in drug discovery programmes that rely on primary screening of target proteins with small molecule libraries followed by further medicinal chemistry campaigns to increase the potency of the chemicals emanating from the screens
There is a raft of orthogonal assays that contribute to verification of hit chemical matter and allow stratification of compounds for selecting the best ones to take into hit-to-lead stages and onwards to lead optimization
The direct interaction of the ubiquitous water molecules with free ligands leads to an increase in the observed ligand signal intensity due to a direct magnetic interaction between water and ligand protons known as a positive nuclear Overhauser effect
Summary
1. Norell® Select SeriesTM 3 mm NMR tubes (Sigma-Aldrich, catalog number: NORS36008) 2. Eppendorf Tube® volume (0.5 ml), PCR clean Soda lime Pasteur pipettes unplugged, capacity 2 ml, 230 mm length (VWR, catalog number: 612-1702) 4. MBL® natural rubber (SciLabware, catalog number: BIBBBPP004) 5. Pipette tips: 10 μl graduated tips (Starlab, catalog number: S1111-3800) 200 μl yellow tips (Starlab, catalog number: S1111-0806) 1,000 μl blue tips (Starlab, catalog number: S1111-6801) 6. Phosphate buffered saline tablet (Sigma-Aldrich, catalog number: P4417) 7. D2O (deuterium oxide 99.9 atom % D) (Sigma-Aldrich, catalog number: 151882) 9. DMSO-d6 (dimethyl sulfoxide-d6 "100%", 99.96 atom % D) (Sigma-Aldrich, catalog number: 156914). 2. Scientific Industries SITM Vortex-GenieTM 2 (Scientific Industries, model: SI-0266, catalog number: 15557335). PH meter (Jenway, catalog number: 351001) 5.
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