Rational design, molecular characterization and dissociation mechanisms of PROTACs targeting hFAAH

Fatty acid amide hydrolase (FAAH) is a key enzyme in the endocannabinoid system and a promising therapeutic target for pain and depression. However, traditional FAAH inhibitors have faced clinical setbacks due to their limited efficacy and off-target effects. Here, we present the rational design and molecular characterization of C13p, a novel PROTAC molecule targeting hFAAH, composed of a 13-carbon linker connecting the FAAH ligand Carb4d and the CRBN ligand pomalidomide. Our results demonstrate that C13p significantly enhances the protein-protein interactions between hFAAH and CRBN, with a binding free energy of -42.2 kcal mol^-1 in the ternary complex compared with -30.5 kcal mol^-1 in the binary system. Notably, C13p exhibits stronger binding to CRBN (-13.7 kcal mol^-1) than to hFAAH (-7.9 kcal mol^-1), suggesting a stepwise assembly mechanism where C13p first binds to CRBN and subsequently recruits hFAAH. Dissociation studies reveal that hFAAH dissociates independently from the ternary complex in 81% of trajectories, with an average residence time of 0.58 s, highlighting a cyclic recruitment mechanism. We further identified K508, K505, and K501 as the potential ubiquitination sites on hFAAH, with significantly enhanced solvent exposure upon C13p binding. This work provides the first detailed mechanistic insight into PROTAC-mediated FAAH degradation, offering a novel strategy for developing next-generation therapeutics for depression-pain comorbidity.