Abstract

A new high-expression endothelial growth factor receptor (EGFR) cell membrane chromatography (CMC) method was applied to recognize the ligands acting on EGFR specifically, and investigate the affinity of gefitinib/HMQ1611 to EGFR. In the self and direct competitive assay, gefitinib/HMQ1611 was used as a competitor in the mobile phase to evaluate the effect of the competitor’s concentrations on the retention of the ligands, respectively, and the competition between gefitinib and HMQ1611 binding to EGFR was also been examined. The retention behavior indicated that gefitinib had one type of binding sites on the EGFR, and the equilibrium dissociation constant (KD) was (9.11 ± 1.89) x 10–6 M; HMQ1611 had two major binding regions on the EGFR, and the KD values obtained from the model were (2.39 ± 0.33) x 10-7 and (3.87 ± 0.93) x 10-5 M for HMQ1611 at the high- and low-affinity sites, respectively. The competition between gefitinib and HMQ1611 occurred at the low-affinity sites on the EGFR. The low-affinity sites were of higher concentrations and contributed to a much larger part of retention of HMQ1611. The results suggested that gefitinib and HMQ1611 competed for the common binding sites on the EGFR, no matter the ligand was used as an analyte or a competitor.

Keywords: Binding site, cell membrane chromatography, epidermal growth factor receptor, HMQ1611, self and direct competitive assay, zonal elution, Molecular Docking Assay, EGFR, ligand.