PROJECT SUMMARY
The endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) is released from postsynaptic spines and activates
CB1 receptors on axon terminals to regulate ion-channel activity and neurotransmitter release. Monoglyceride
lipase (MGL) – a presynaptic hydrolase that hydrolyzes 2-AG into arachidonic acid and glycerol – stops this
retrograde signaling process. Small-molecule inhibitors that target MGL have provided insights into the
functions of 2-AG and have been recently advanced to clinical development. Current drug development efforts
in this area are focused on agents that interact irreversibly or reversibly with the catalytic site of MGL. There
are, however, serious disadvantages to either of these approaches: irreversible inhibitors cause excessive 2-
AG accumulation and consequent CB1 desensitization, while reversible orthosteric inhibitors must compete for
active-site binding with 2-AG and other monoacylglycerols, which may reach high micromolar concentrations in
the relevant biophase (e.g., presynaptic membranes). We have shown that the reversible peroxide-dependent
sulfenylation of C201 and C208 in MGL stabilizes a catalytically inactive conformation of the enzyme, and thus
enhances 2-AG-mediated signaling at CB1 receptors. In this revised R21 application, we propose to develop
allosteric MGL inhibitors that target this regulatory region and, by doing so, may be able to bypass substrate
competition. In particular, we identified a class of benzisothiazolin-3(2H)-one derivatives that inhibit MGL
activity with high potency by interacting in a reversible manner with the regulatory cysteines C201 and C208.
We propose to use a combination of experimental and computational approaches – molecular modeling,
structure-activity relationship studies, site-directed mutagenesis, mass spectrometry, and in vivo pharmacology
– to modify these lead molecules and generate potent and systemically active allosteric MGL inhibitors. These
compounds will advance our understanding of 2-AG-mediated signaling by allowing us to test whether 2-AG
deactivation – which lies at the very core of endocannabinoid signaling – might be modulated by physiological
and pathological factors that affect such status, such as ischemia. They might also serve as starting point for
the discovery of novel therapeutics for pain, substance use disorders and other human diseases.