Distinct signatures of molecular recognition of AXL kinase by varied classes of inhibitors examined through biomolecular simulations.

Designing new anticancer agents requires understanding how ligands affect target protein dynamics. AXL kinase has recently emerged as a promising therapeutic target in cancer therapy. In this study, we performed comprehensive comparative analyses of two ligand-bound AXL kinase complexes (PDB IDs: 5U6B and 7DXL) using molecular dynamics simulations, contact mapping, residue-level flexibility analysis, binding free energy calculations, free energy surface (FES) exploration, and ligand unbinding studies. Results showed that both ligands stabilized the core region via hydrophobic and van der Waals interactions, while increasing flexibility in the peripheral regions. The macrocyclic ligand in 5U6B exhibited dynamic flexibility patterns indicating potential allosteric regulation. Conversely, the indazole-based ligand from 7DXL induced a more rigid and pre-organised binding site, stabilized by strong electrostatic and hydrophobic forces. Per-residue MMPBSA analysis revealed critical energetic hotspots collectively reinforcing the binding affinity. The ligand from 7DXL displayed favourable gas-phase energies along with cooperative electrostatic contributions, aligning with deeper FES minima and compact structural clustering. Unbinding simulations revealed significant kinetic stability: the indazole ligand had a single high-affinity egress routes, while the macrocyclic ligand had two egress routes with higher unbinding rates. Overall, our findings highlight distinct spatial and temporal mechanisms in ligand recognition and stability. The ligand from 7DXL formed a high-affinity, structurally rigid complex, while the other (5U6B) showed more flexibility and adaptability-offering unique avenues for optimization. This comprehensive analysis provides a mechanistic understanding and strategic direction for designing selective, functionally tunable AXL kinase inhibitors.