Electrochemical biosensors based on peptide-kinase interactions at the kinase docking site

Abstract

Kinases are important cancer biomarkers and are conventionally detected based on their catalytic activity. Kinases regulate cellular activities by phosphorylation of motif-specific multiple substrate proteins, resulting in lack of selectivity of activity-based kinase biosensors. We present an alternative approach of sensing kinases based on the interactions of their allosteric docking sites with a specific partner protein. The new approach was demonstrated for the ERK2 kinase and its substrate ELK-1. A peptide derived from ELK-1 was bound to a gold electrode and ERK2 sensing was performed by electrochemical impedance spectroscopy. The sensors showed high level of target selectivity for ERK2 when compared with p38γ kinase and BSA. ERK2 was detected in its cellular concentration range, 0.2-8.0 μM. Using the flexibility of peptide design, our method is generic for developing sensitive and substrate-specific biosensors and other disease-related enzymes based on their interactions.Abstract Figure

Peptide‐Based Inhibitors that Target the Docking Site of ERK2

“…To determine whether the D domain peptides that bound ERK2 can also inhibit its interactions mediated via its DRS, we used an electrochemical impedance spectroscopy (EIS)‐based ERK2 detection assay developed in our labs [29] . The assay was comprised of the ELK1 D domain peptide, ELK1 312–321, adsorbed onto gold electrodes.…”

“…To conclude, we showed that inhibition of ERK2 can be done by using peptides that target the PPI of its DRS docking site [29] . This approach is highly versatile and can easily be utilized for different kinases and other types of enzymes, whose activities are crucial in disease.…”

“…We have recently used the interaction of ERK2 at its DRS docking site for sensing the kinase [29] . The ELK1 D domain peptide, ELK1 312–321, was adsorbed onto gold electrodes to form a peptide layer.…”

“…We first designed a library of D domain peptides derived from binding partners of ERK2 and quantified their binding with ERK2. Using the previously developed electrochemical assay, we evaluated the ability of the peptides to inhibit the interactions of ERK2 with the surface‐bound ELK1 D domain peptide, which are mediated by the DRS docking site [29] . The most active peptides penetrated HT29 colon cancer cells and decreased their viability.…”