Discovery of New E-Selectin Inhibitors by Virtual Screening, Fluorescence Binding Assays, and STD NMR Experiments

E‐selectin is an endothelial protein that participates in the adhesion of metastatic cancer cells, and is therefore a relevant target for antitumor therapeutic intervention. In this work, virtual screening was used to identify new E‐selectin inhibitors from a subset of drug‐like molecules retrieved from the ZINC database, including the physiological ligand sLex as reference structure (PDB ID: 1G1T). Four hits were chosen and subjected to molecular dynamics simulations and fluorescence binding assays, which led to the determination of experimental dissociation constants between 333 and 1012 μm. The candidate with the highest affinity was studied by saturation transfer difference (STD) NMR experiments and complete relaxation and conformational exchange matrix analysis of saturation transfer (CORCEMA‐ST), aimed at identifying the preferable binding mode with E‐selectin. Our results revealed that this new inhibitor binds more strongly than sLex in the E‐selectin binding site, in good agreement with simulation predictions. These properties will prove valuable for the future design of drugs that target E‐selectin. Cell adhesion blocked: The structure of a new non‐carbohydrate E‐selectin inhibitor was identified by virtual screening modeling and validated through fluorescence binding assays and STD NMR spectroscopy. The measured binding free energy was −4.7 kcal mol−1, which corresponds to a tighter association than the physiological ligand sLex.