Functional group-dependent stabilization and dissociation mechanisms of EGFR allosteric inhibitors.

Allosteric inhibitors targeting the αC-helix-adjacent pocket provide an alternative strategy to overcome resistance associated with ATP-site mutations. However, the structural and thermodynamic determinants governing their stability across resistant EGFR variants remain incompletely understood. Here, we comparatively investigated two fourth-generation allosteric inhibitors, JBJ-04-125-02 (JBJ) and its quinazolinone analogue C34, in EGFR™ and EGFRLR/™/CS mutants using multi-replica molecular dynamics simulations and umbrella sampling. Both ligands suppress activation-compatible motions, maintain the Lys745-Glu762 pair in a separated inactive-state geometry, and remodel ATP-site dynamics while occupying the αC-helix-adjacent allosteric pocket. The calculated binding energetics are dominated by hydrophobic interactions, whereas polar contacts involving Asp855 and Phe856 primarily govern ligand orientation and conformational control. JBJ forms a high-occupancy (∼82%) hydrogen bond with Phe856, providing a strong directional anchoring interaction enabled by its hydroxyl-bearing phenyl-acetamide moiety. In contrast, the quinazolinone scaffold of C34 lacks this functionality and relies on a broader interaction network within the allosteric pocket. Despite similar binding modes, C34 exhibits a larger and more continuously increasing PMF than JBJ along the sampled unbinding coordinate in EGFRLR/™/CS, together with greater stabilization of intermediate configurations. Together, these findings reveal distinct functional-group-dependent stabilization strategies and establish a mechanistic framework demonstrating how local chemical features encode interaction topology, ATP-site regulation, conformational sampling, and simulated unbinding behavior in EGFR allosteric inhibitors.