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

Abstract

Fatty acid amide hydrolase (FAAH) is one of key regulatory enzymes of the endocannabinoid system. Inhibiting FAAH activity can significantly increase the level of endogenous anandamide (AEA), indirectly activating cannabinoid receptors and exerting multiple therapeutic effects such as analgesia and antidepressant. The clinical transformation of traditional FAAH small-molecule inhibitors has been hindered due to the insufficient efficacy, significant toxic and side effects. Seeking proteolysis targeting chimeras (PROTACs) for FAAH has become an important alternative. Based on structural biology information and distance statistical strategies, this work first successfully designed a novel PROTAC molecule named C13p targeting FAAH. By comparing molecular recognition and conformational changes in the binary (i.e., hFAAH_CRBN) and ternary (hFAAH_C13p_CRBN) complexes formed by hFAAH, the E3 ligase Cereblon (CRBN) and C13p, the regulatory mechanism of C13p on hFAAH's ubiquitination was revealed. Compared with hFAAH_CRBN, the contact of E3 ligase-target enzyme in hFAAH_C13p_CRBN becomes closer, accompanied by the enhanced H-bond interactions between E3 ligase and the ligand. According to the calculated binding free energy, C13p tends to form a relatively stable binary complex with CRBN first, and then recruit hFAAH relying on the ligand at the other end. Through statistical analysis of metadynamics simulation trajectory, the independent dissociation probability of hFAAH is higher than the co-dissociation of hFAAH_C13p. The dissociation of hFAAH presents the characteristics from spatial restriction to free diffusion, with an average residence time of 0.58 s. This work not only clarifies the intramolecular interactions for the hFAAH_CRBN and hFAAH_C13p_CRBN systems, along with the potential ubiquitination sites of the target enzyme, but also provides a feasible strategy for the design of potential high-activity PROTAC molecules targeting FAAH.