Investigation of the binding characteristics between ligands and epidermal growth factor receptor by cell membrane chromatography.

The binding property between a ligand and its receptor is very important for numerous biological processes. In this study, we developed a high epidermal growth factor receptor (EGFR)-expression cell membrane chromatography (CMC) method to investigate the binding characteristics between EGFR and the ligands gefitinib, erlotinib, canertinib, afatinib, and vandetanib. Competitive binding analysis using gefitinib as the marker was used to investigate the interactions that occurred at specific binding sites on EGFR. The ability of displacement was measured from the HEK293-EGFR/CMC column on the binding sites occupied by gefitinib for these ligands, which revealed the following order: gefitinib (KD, 8.49 ± 0.11 × 10-7  M) > erlotinib (KD, 1.07 ± 0.02 × 10-6  M) > canertinib (KD, 1.41 ± 0.07 × 10-6  M) > afatinib (KD, 1.80 ± 0.12 × 10-6  M) > vandetanib (KD, 1.99 ± 0.03 × 10-6  M). This order corresponded with the values estimated by frontal displacement analysis and the scores obtained with molecular docking. Furthermore, thermodynamic analysis indicated that the hydrogen bond or Van der Waals force was the main interaction force in the process of EGFR binding to all 5 ligands. Overall, these results demonstrate that a CMC method could be an effective tool to investigate the binding characteristics between ligands and receptors.