Conversion of MIG6 peptide from the nonbinder to binder of lung cancer-related EGFR by phosphorylation and cyclization

Development of chemical drugs and biologic agents to target human epidermal growth factor receptor (EGFR) has long been established as a promising therapeutic strategy for lung cancer. Previously, it was found that the tumor-suppressor protein MIG6 is a negative regulator of the EGFR kinase, which can bind at the activation interface of asymmetric dimer of EGFR kinase domains to disrupt EGFR dimerization and then inactivate the kinase. The protein adopts two separated segments, that is, MIG6s1 and MIG6s2, to directly interact with EGFR. Here, by computational modeling and analysis of the intermolecular interaction between EGFR kinase domain and MIG6s2 peptide, we demonstrated that the dephosphorylated MIG6s2 peptide can be converted from nonbinder to weak binder and then to moderate binder of EGFR by phosphorylation and cyclization, respectively; the former introduces strong electrostatic potential to EGFR–peptide complex by forming two geometrically satisfactory salt bridges across the complex interface, while the latter minimizes entropy penalty upon the binding of highly flexible peptide to EGFR. Subsequently, the linear phosphorylated and dephosphorylated peptides and phosphorylated cyclic peptide were synthesized and purified, and their binding affinities to the recombinant protein of human EGFR kinase domain were determined by fluorescence polarization titration. As expected theoretically, the linear dephosphorylated peptide has no observable binding to the kinase, and further phosphorylation and cyclization can confer, respectively, low and moderate affinities to the peptide, suggesting a good consistence between the computational analysis and experimental assay.