Flexibility-driven design of etoposide derivatives targeting Daam1: a thermodynamics-guided strategy against breast cancer metastasis.

Balancing molecular flexibility and rigidity remains a key challenge in rational drug design. Here, the Daam1 formin homology 2 (FH2) domain, a regulator of tumor metastasis, was used as a model to investigate the role of flexible side chains in targeted inhibition. Molecular docking, micro-scale thermophoresis, and molecular dynamics simulations showed that the flexible peptide thymopentin binds Daam1 more stably than etoposide through sustained interactions with Arg692 and Asn695. Guided by these insights, the 4' position of etoposide was optimized using free energy perturbation combined with enhanced sampling MD. Two derivatives, V2 and V3, exhibited improved binding free energies. In vitro assays demonstrated that V2 and V3 enhanced inhibition of breast cancer cell migration and reduced Daam1 expression to approximately half of the level observrd with etoposide. This study establishes a flexibility-driven optimization strategy and highlights FEP-enhanced MD as a robust framework for rational drug design.