Chemical probes to potently and selectively inhibit endocannabinoid cellular reuptake

Significance

Suitable chemical tools have been instrumental in the discovery and characterization of the endocannabinoid system. However, the lack of potent and selective inhibitors for endocannabinoid transport has prevented the molecular characterization of this process. Current uptake inhibitors are poorly bioavailable to the central nervous system (CNS) and weakly selective because they also inhibit fatty acid amide hydrolase (FAAH), the major anandamide-degrading enzyme. Few studies have addressed the uptake inhibition of 2-arachidonoyl glycerol (2-AG), which is the major endocannabinoid. Here, we report a highly potent and selective endocannabinoid reuptake inhibitor. Our data indicate that endocannabinoid transport across the membrane can be targeted, leading to general antiinflammatory and anxiolytic effects in mice.

Abstract

The extracellular effects of the endocannabinoids anandamide and 2-arachidonoyl glycerol are terminated by enzymatic hydrolysis after crossing cellular membranes by facilitated diffusion. The lack of potent and selective inhibitors for endocannabinoid transport has prevented the molecular characterization of this process, thus hindering its biochemical investigation and pharmacological exploitation. Here, we report the design, chemical synthesis, and biological profiling of natural product-derived N-substituted 2,4-dodecadienamides as a selective endocannabinoid uptake inhibitor. The highly potent (IC ₅₀ = 10 nM) inhibitor N-(3,4-dimethoxyphenyl)ethyl amide (WOBE437) exerted pronounced cannabinoid receptor-dependent anxiolytic, antiinflammatory, and analgesic effects in mice by increasing endocannabinoid levels. A tailored WOBE437-derived diazirine-containing photoaffinity probe (RX-055) irreversibly blocked membrane transport of both endocannabinoids, providing mechanistic insights into this complex process. Moreover, RX-055 exerted site-specific anxiolytic effects on in situ photoactivation in the brain. This study describes suitable inhibitors to target endocannabinoid membrane trafficking and uncovers an alternative endocannabinoid pharmacology.