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.
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