Quantitative Analysis of Ligand-EGFR Interactions: A Platform for Screening Targeting Molecules

Abstract

Epidermal growth factor receptor (EGFR) is often constitutively stimulated in many cancers owing to the binding of ligands such as epidermal growth factor (EGF). Therefore, it is necessary to investigate the interaction between EGFR and its targeting biomolecules. The main aim of this study was to estimate the binding affinity and adhesion force of two targeting molecules, anti-EGFR monoclonal antibody (mAb LA1) and the peptide GE11 (YHWYGYTPQNVI), with respect to EGFR and to compare these values with those obtained for the ligand, EGF. Surface plasmon resonance (SPR) was used to determine the equilibrium dissociation constant (KD) for evaluating the binding affinity. Atomic force microscopy (AFM) was performed to estimate the adhesion force. In the case of EGFR, the KD of EGF, GE11, and mAb LA1 were 1.77 × 10−7, 4.59 × 10−4 and 2.07 × 10−9, respectively, indicating that the binding affinity of mAb LA1 to EGFR was higher than that of EGF, while the binding affinity of GE11 to EGFR was the lowest among the three molecules. The adhesion force between EGFR and mAb LA1 was 210.99 pN, which is higher than that observed for EGF (209.41 pN), while the adhesion force between GE11 and EGFR was the lowest (59.51 pN). These results suggest that mAb LA1 binds to EGFR with higher binding affinity than EGF and GE11. Moreover, the adhesion force between mAb LA1 and EGFR was greater than that observed for EGF and GE11. The SPR and AFM experiments confirmed the interaction between the receptor and targeting molecules. The results of this study might aid the screening of ligands for receptor targeting and drug delivery.

Characterization of EGF-guided MDA-MB-231 cell chemotaxis in vitro using a physiological and highly sensitive assay system

“…EGF binds to its receptor in MDA-MB-231 cells with a K d value of approximately 0.49 nM over a channel width of 1 mm. The EGFR binding affinity has been reported several times in the literature and resulted in values ranging from 0.0177 nM ( in vitro measurements) to 5 nM (binding assay in living cells) [ 36 , 37 ]. Interestingly, Björkelund et al investigated, that the affinity and kinetics of EGF binding to EGFR is greatly dependent on the cell line, with a K d value of approximately 0.2 nM for breast cancer cells [ 38 ].…”

Overview of the detection methods for equilibrium dissociation constant KD of drug-receptor interaction

“…SPR is utilized to detect whether biological molecules interact with each other, and further explore the specificity of the interaction, kinetic parameters and affinity of the interaction [41] , [42] , [43] . SPR technique provides a powerful and nondestructive tool for cell sorting, cell surface characterization, protein-protein interaction, protein-small molecule interaction, and drug discovery [41] , [43] , [44] , [45] .…”

“…SPR is a label-free and real-time detection method for monitoring biomolecular interactions [40] . In recent years, SPR has become a rapid developmental technology for studying the interaction between membrane protein receptors and ligands [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] , [56] , which is shown in Table 2 . Because of the high-throughput screening characteristic, SPR has been widely used in the identification of drug targets and the optimization of lead compounds [44] , [45] , [46] , [47] , [48] .…”

“… No. Receptor Drug Receptor material K D values References 1 EGFR EGF EGFR protein 0.177×10 −6 M [44] GE11 0.459×10 −3 M mAb LA1 2.07×10 −9 M 2 Subendothelial collagens vWf Purified protein 2.03(±0.04)×10 −9 M [45] 3 Pr55(Gag) 1,4,5-IP3 Purified protein 2170×10 −6 M [46] di-C(8)-PI 186×10 −6 M di-C(8)-PI(4,5)P2 47.4×10 −6 M 4 VEGFR2 D3 Nanobody against NTV(1–4) HUVEC cell 49(±1.8)×10 −9 M [47] 5 CD56 Monoclonal antibodies m900 Cancer cell 2.9×10 −9 M [48] Monoclonal antibodies m906 4.5×10 −9 M 6 Grp1 PH domain Biotinylated Ins(1,3,4,5)P4 Rat brain membranes 0.14×10 −6 M [49] 7 Angiotensin converting enzyme Lisinopril Angiotensin converting enzyme 1.78×10 −9 M [50] 8 rKDR1–3 VEGF165 rKDR1–3 protein 57.4×10 −9 M [51] 9 Lipoprotein lipase Bis-ANS Purified protein (0.10−0.26)×10 −6 ...…”

A bispecific enediyne-energized fusion protein targeting both epidermal growth factor receptor and insulin-like growth factor 1 receptor showing enhanced antitumor efficacy against non-small cell lung cancer

“…Firstly, EGF and IGF-1 has high affinity to their receptors with the Kd values of 1.77×10 −7 mol/L and 4.45×10 −9 mol/L, respectively [44, 45]. Secondly, the EGF and IGF-1 are both small proteins with molecular weights of 6.2 kDa and 7.6 kDa, respectively.…”