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

Anandamide or N-arachidonoylethanolamine (AEA)
is a signaling lipid that modulates neurotransmitter release via activation
of the type 1 cannabinoid receptor (CB1R) 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.