Chemical Proteomics Reveals Off-Targets of the Anandamide Reuptake Inhibitor WOBE437

[Image: see text] Anandamide or N-arachidonoylethanolamine (AEA) is a signaling lipid that modulates neurotransmitter release via activation of the type 1 cannabinoid receptor (CB(1)R) in the brain. Termination of anandamide signaling is thought to be mediated via a facilitated cellular reuptake process that utilizes a purported transporter protein. Recently, WOBE437 has been reported as a novel, natural product-based inhibitor of AEA reuptake that is active in cellular and in vivo models. To profile its target interaction landscape, we synthesized pac-WOBE, a photoactivatable probe derivative of WOBE437, and performed chemical proteomics in mouse neuroblastoma Neuro-2a cells. Surprisingly WOBE437, unlike the widely used selective inhibitor of AEA uptake OMDM-1, was found to increase AEA uptake in Neuro-2a cells. In line with this, WOBE437 reduced the cellular levels of AEA and related N-acylethanolamines (NAEs). Using pac-WOBE, we identified saccharopine dehydrogenase-like oxidoreductase (SCCPDH), vesicle amine transport 1 (VAT1), and ferrochelatase (FECH) as WOBE437-interacting proteins in Neuro-2a cells. Further genetic studies indicated that SCCPDH and VAT1 were not responsible for the WOBE437-induced reduction in NAE levels. Regardless of the precise mechanism of action of WOB437 in AEA transport, we have identified SSCPHD, VAT1, and FECH as unprecedented off-targets of this molecule which should be taken into account when interpreting its cellular and in vivo effects.