Abstract
Abstract
Radioligands targeting fibroblast activation protein alpha (FAP) are promising tools for cancer diagnosis and therapy. However, the molecular binding mode of clinically established UAMC-1110-derived ligands such as FAPI-46 remains insufficiently characterized. To simplify synthesis while preserving key binding properties, we designed two novel derivatives—FAP-DD-101 (short aminoalkyl linker) and FAP-DD-102 (extended aminohexanoic acid spacer)—and evaluated them alongside FAPI-46 using molecular docking and all-atom molecular dynamics (MD) simulations. Across all derivatives, we identified a highly stable, energetically favorable charge-anchored binding mode, in which the 2-cyano-4,4-difluoropyrrolidine head anchors in a negatively charged subregion formed by Glu202, Glu203, and Glu204, mimicking the binding architecture observed in the FAP–linagliptin complex. MD trajectories confirmed that FAPI-46 and FAP-DD-101 maintain exceptional stability in this pose, with several non-anchored FAPI-46 trajectories spontaneously evolving toward engagement with this electrostatic subregion. For FAP-DD-102, linker elongation slightly reduced pose robustness, yet initial charge-anchored states still yielded superior average binding energies and vector stability compared to non-anchored poses. Overall, these findings elucidate the key structural determinants of FAP recognition, establishing a rational framework for designing synthetically accessible FAPI radiotracers with retained binding stability.