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The tropomyosin receptor kinase (TRK) family regulates key oncogenic signaling pathways, and genetic alterations in NTRK genes are implicated in a broad spectrum of malignancies. Although TRK inhibitors such as entrectinib effectively demonstrate robust clinical efficacy in NTRK fusion-positive tumors, their long-term therapeutic utility is frequently limited by the emergence of acquired resistance mutations, including the G595R substitution.

In this study, molecular dynamics simulations and MM-GBSA binding free energy calculations were employed to investigate the mechanistic impact of the G595R mutation on entrectinib binding. Our analyses reveal that substitution of glycine with the sterically bulky, positively charged arginine residue at position 595 induces severe steric clash within the ATP-binding pocket, disrupts conserved hydrophobic packing interactions, and displaces the N-methylpiperazinyl moiety of entrectinib. Moreover, the G595R mutant exhibits elevated root mean square deviation of the bound ligand and enhanced conformational flexibility in the glycine-rich loop (G-loop). Quantitative MM-GBSA decomposition identifies a substantial increase in binding free energy, attributable predominantly to attenuated van der Waals contributions and loss of key hydrogen bonds with Tyr591, Met592, His594, and Leu657. Domain cross-correlation analysis demonstrates weakened dynamic coupling between the G-loop and the hinge region, critical for allosteric control of kinase activity, thereby compromising the structural integrity required for high-affinity inhibitor binding. These findings provide a mechanistic explanation for entrectinib resistance at atomic resolution and illustrate how a single-point mutation can trigger long-range perturbations in protein dynamics and interdomain communication.