Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons

Diacylglycerol lipase
(DAGL)-α and -β are enzymes responsible
for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol
(2-AG). Selective and reversible inhibitors are required to study
the function of DAGLs in neuronal cells in an acute and temporal fashion,
but they are currently lacking. Here, we describe the identification
of a highly selective DAGL inhibitor using structure-guided and a
chemoproteomics strategy to characterize the selectivity of the inhibitor
in complex proteomes. Key to the success of this approach is the use
of comparative and competitive activity-based proteome profiling (ABPP),
in which broad-spectrum and tailor-made activity-based probes are
combined to report on the inhibition of a protein family in its native
environment. Competitive ABPP with broad-spectrum fluorophosphonate-based
probes and specific β-lactone-based probes led to the discovery
of α-ketoheterocycle LEI105 as a potent, highly selective, and
reversible dual DAGL-α/DAGL-β inhibitor. LEI105 did not
affect other enzymes involved in endocannabinoid metabolism including
abhydrolase domain-containing protein 6, abhydrolase domain-containing
protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase
and did not display affinity for the cannabinoid CB1 receptor.
Targeted lipidomics revealed that LEI105 concentration-dependently
reduced 2-AG levels, but not anandamide levels, in Neuro2A cells.
We show that cannabinoid CB1-receptor-mediated short-term
synaptic plasticity in a mouse hippocampal slice model can be reduced
by LEI105. Thus, we have developed a highly selective DAGL inhibitor
and provide new pharmacological evidence to support the hypothesis
that “on demand biosynthesis” of 2-AG is responsible
for retrograde signaling.