Label-free cell phenotypic assessment of the molecular mechanism of action of epidermal growth factor receptor inhibitors

Abstract

Epidermal growth factor receptor (EGFR) is the target of several clinically approved tyrosine kinase inhibitor (TKI) drugs including gefitinib and erlotinib in the treatment of cancer. Multiple mechanisms have been implicated in the clinical features of these drugs. However, little is known about the molecular mechanism of action of these drugs at the whole cell level. Here we applied a label-free biosensor-enabled dynamic mass redistribution (DMR) assay to assess the molecular mechanism of action of three EGFR inhibitors, gefitinib, erlotinib and AG1478, to alter the EGFR signaling in A431 and HT-29, two native cancer cell lines expressing the EGFR. The whole-cell DMR assays with the persistent inhibitor treatment showed that all inhibitors dose-dependently inhibited the EGFR signaling in both cell lines, but generally displayed higher potency in A431 than HT-29 cells. The DMR assays with the inhibitor washout showed that the washout unexpectedly increased the potency of gefitinib and AG-1478 to inhibit the EGFR signaling in A431, but slightly decreased the potency of all three inhibitors in HT29. The DMR assays under microfluidics showed that the removal of the inhibitors using buffer perfusion resulted in a time-dependent recovery of EGF signaling that is slower in A431 than HT-29 cells. In contrast, DMR assays under microfluidics showed that the removal of reversible competitive antagonists led to the full recovery of the signalling of two distinct G protein-coupled receptors (GPCRs), the β₂-adrenergic receptor in A431 and the GPR35 in HT-29 cells. Together, our results suggest that for EGFR inhibitors their uptake and retention, rather than binding kinetics, dominate their label-free cell phenotypic efficacy; however, for GPCR antagonists the binding characteristics are critical to the inhibitory effects. This study also implicates the potential of DMR assays under different simulation conditions for elucidating the cell phenotypic pharmacology, in particular transporter-related drug resistance, of kinase inhibitor drugs.