Binding mechanism between entrectinib and human alpha-1-acid glycoprotein: multispectral and in silico simulations

Entrectinib (ENB) is a multi-target tyrosine kinase inhibitor currently approved for the treatment of adult and pediatric patients with NTRK fusion-positive advanced recurrent solid tumors and metastatic ROS1-mutated non-small cell lung cancer. The interaction between ENB and human α1-acid glycoprotein (HAG) was assessed by multispectral experiments and computer simulations. The results demonstrated that ENB effectively quenched HAG fluorescence via a mixed quenching mechanism. Specifically, ENB bound to the hydrophobic cavity of HAG, forming an ENB-HAG complex with 1:1 stoichiometry. The binding constant (∼10 6 M −1 ) demonstrated strong binding affinity between ENB and HAG. Non-radiative energy transfer occurred between ENB and HAG, with a binding distance of 2–4 nm. Synchronous and three-dimensional fluorescence spectroscopy indicated minimal effects on the microenvironment around the TYR and TRP residues. Circular dichroism spectroscopy demonstrated a slight alteration on the conformation of HAG, specifically a reduction in β-folded content from 43.5 % to 43.3 %. Thermodynamic calculations confirmed spontaneous binding driven primarily by hydrophobic force, which was further supported by Molecular dynamics simulationsanalyzing energy contributions of individual amino acid residues. Additionally, metal ions were found to exert varying degrees of influence on the binding process.