| The endocannabinoids arachidonoylethanolamide (anandamide) and 2-arachidonoyl- sn-glycerol (2-AG) are lipid messengers that bind two subtypes of cannabinoid (CB) receptors; CB1, which are localized primarily in the central nervous system, and CB2, which are found within the immune system. Activation of these receptors has been shown to modulate numerous biological functions, including pain, inflammation, mood, appetite, and memory. 2-AG has been established as a key molecule involved in CB1-mediated signaling in the brain.;Monoacylglycerol lipase (MGL), a cytosolic serine hydrolase that catalyzes the cleavage of monoacylglycerols (MAGs) into fatty acid and glycerol, is the primary enzyme responsible for the degradation of 2-AG. Recent work suggests a key role for MGL in the termination of 2-AG signaling. In the present dissertation, I used an experimental approach combining molecular, biochemical, pharmacological, and computational techniques to elucidate mechanisms of MGL activity and inhibition. I generated a purified recombinant rat MGL and used it to study the functional properties of this enzyme in vitro. First, I investigated the mechanism by which the MGL inhibitor URB602 interacts with the purified enzyme. The results of this study demonstrated that URB602 inhibits MGL activity by a non-competitive and partially reversible mechanism. In addition, I showed that incubation with this compound elevates 2-AG levels in hippocampal slice cultures. Next, using an approach that combined molecular modeling and site-directed mutagenesis, I established a role for cysteine residues in MGL function, and identified a novel class of isothiazolinone-based compounds that inhibit MGL activity with nanomolar potencies by interacting with specific cysteine residues within the protein. Finally, I used a compound library-screening approach to identify and characterize two novel triterpenoid-based MGL inhibitors, and report evidence that these compounds inhibit MGL activity by a non-competitive, rapidly reversible mechanism. In addition, my results suggest this inhibition occurs through interactions with cysteine residues in a regulatory region of MGL known as the lid domain. Together, the identification and characterization of these pharmacological tools has helped to broaden our current understanding of the mechanisms which underlie MGL inhibition, and will help gain further insights on the specific biological role of 2-AG in endocannabinoid-mediated signaling. |
