LAUSR

BIA 10‐2474 is a time‐dependent inhibitor of fatty acid amide hydrolase (FAAH) that was under clinical development for the treatment of neurological conditions when the program was terminated after one subject died and four were hospitalized with neurological symptoms during a first‐in‐human clinical study. The present work describes the mechanism of FAAH inhibition by BIA 10‐2474 as a target‐specific covalent inhibition, supported by quantum mechanics and molecular modelling studies. The inhibitor incorporates a weakly reactive electrophile which, upon specific binding to the enzyme‘s active site, is positioned to react readily with the catalytic residues. The reactivity is enhanced on‐site by the increased molarity at the reaction site and by specific inductive interactions with FAAH. In the second stage, the inhibitor reacts with the enzyme‘s catalytic nucleophile to form a covalent enzyme‐inhibitor adduct. The hydrolysis of this adduct is shown to be unlikely under physiological conditions, therefore leading to irreversible inactivation of FAAH. The results also reveal the important role played by FAAH Thr236 in the reaction with BIA 10‐2474, which is specific to FAAH and is not present in other serine hydrolases. It forms a hydrogen bond with the imidazole nitrogen of the inhibitor and helps lowering the activation free energy of the first step of the reaction, by pre‐orienting and stabilizing the inhibitor in a near‐reactive configuration. In the second step, Thr236 can also serve as a mechanistic alternative to protonate the leaving group.