Binding Mechanism of UAMC-1110 to Fibroblast Activation Protein

Fibroblast activation protein (FAP) is an enzyme highly expressed in cancer-associated fibroblasts. Over the past years, several FAP inhibitors (FAPIs) have been developed for tumor imaging. These approaches typically involve covalently linking a metal-loaded chelator to a small molecule ligand that targets FAP. Prolonged tumor retention of FAPIs is desirable to support theranostic applications, which integrate both diagnostic and therapeutic functions. Therefore, we sought to elucidate the binding mechanism of our in-house-designed FAPI lead compound, UAMC-1110. Initial molecular dynamics simulations yielded a noncovalent binding pose of the ligand. Subsequently, QM/MM calculations were performed to investigate the covalent binding mechanism of UAMC-1110. Our results corroborate that the compound forms a covalent complex with the receptor through a reaction pathway that begins with proton transfer from S624 to H734, followed by a concerted, asynchronous nucleophilic attack of S624 on the nitrile warhead. The resulting imidate anion is stabilized via two possible routes: (1) protonation mediated by H734 through two water molecules, and (2) direct protonation by Y541 followed by reprotonation. Covalent MD simulations reveal that H734 interacts more frequently with the imidate anion than Y541, identifying a previously unrecognized and kinetically favored protonation pathway via H734, further supported by water-density and Gibbs energy calculations. Simulations of the final product state additionally indicate that neither residue remains in proximity to the covalent adduct, making reversibility of the reaction complex unlikely and instead suggesting hydrolysis as the most plausible mechanism for complex dissociation.