Atomic Force Microscopy Lifetime Analysis: An Intuitive Method for Evaluating Receptor Tyrosine Kinase Dimer-Targeting Inhibitors

Abstract

Overexpression of receptor tyrosine kinases (RTKs) or binding to ligands can lead to the formation of specific unliganded and liganded RTK dimers, and these two RTK dimers are potential targets for preventing tumor metastasis. Traditional RTK dimer inhibitor analysis was mostly based on end point assays, which required cumbersome cell handling and behavior monitoring. There are still challenges in developing intuitive process-based analytical methods to study RTK dimer inhibitors, especially those used to visually distinguish between unliganded and liganded RTK dimer inhibitors. Herein, taking the mesenchymal–epithelial transition factor (MET) receptor, an intuitive method for evaluating MET inhibitors has been developed based on atomic force microscopy (AFM) lifetime analysis. The time interval between the start of the force and the bond break point was regarded as the bond lifetime, which could reflect the stability of the MET dimer. The results showed that there was a significant difference in the lifetime (τ) of unliganded MET dimers (τ₁ = 207.87 ± 4.69 ms) and liganded MET dimers (τ₂ = 330.58 ± 15.60 ms) induced by the hepatocyte growth factor, and aptamer SL1 could decrease τ₁ and τ₂, suggesting that SL1 could inhibit both unliganded and liganded MET dimers. However, heparin only decreased τ₂, suggesting that it could inhibit only the liganded MET dimer. AFM-based lifetime analysis methods could monitor RTK dimer status rather than provide overall average results, allowing for intuitive process-based analysis and evaluation of RTK dimers and related inhibitors at the single-molecule level. This study provides a novel complementary strategy for simple and intuitive RTK inhibitor research.