| Cannabis sativa (marijuana) have been used for centuries for medicinal and recreational purposes but it was not until the 1960s that the structure of the psychoactive cannabinoid component, Delta 9-tetrahydrocannabinol (THC), was elucidated. THC is know to exert its effect by mimicking endogenous molecules -- the endocannabinoids (eCBs) anandamide (AEA, N-arachidonoylethanolamine and 2-arachidonoyl- sn-glycerol (2-AG) which are lipid messengers that bind two subtypes of cannabinoid (CB) receptors; CB1, which are mainly found in the central nervous system, and CB2, which are localized within the immune system. Activation of these receptors has been shown to modulate various biological functions, including memory, inflammation, mood, appetite, pain and neuroprotection. Numerous studies have established 2-AG as a key molecule involved in CB1-mediated signaling in the brain. In addition, studies have shown that following brain injury, there is a transient accumulation of 2-AG at the site of injury that serve a neuropropective role in attenuating further brain damage. Monoacylglycerol lipase (MGL) is a cytosolic, serine hydrolase that catalyzes the cleavage of monoacylglycerols (MAGs) into fatty acid and glycerol, and is the primary enzyme responsible for the degradation of 2-AG. Recent studies have also suggested a key role for MGL in the termination of 2-AG signaling.;Excessive production of free radicals associated with neuronal injury, leads to oxidative stress, an important contributor to secondary damage after brain injury. Increased reactive oxygen species (ROS) damage important cell components like DNA, lipids and proteins and eventually lead to cell death by apoptosis or necrosis. The main focus of the present dissertation is (i) to develop novel and potent inhibitors of MGL and (ii) to understand how oxidative stress in the brain regulates MGL activity. MGL inhibition in the brain during oxidative stress is both theoretically and clinically important because oxidative stress is a final common pathway for a variety of insults, such as stroke and inflammation, which can damage and eventually kill brain cells. It is therefore crucial to understand the molecular cascade triggered by oxidative stress to identify possible points of therapeutic intervention.;First, I used a screening method to identify and characterize two potent and novel triterpenoid MGL inhibitors from a library of compounds, and hereby show evidence that these compounds inhibit MGL activity through a noncompetitive and rapidly reversible mechanism. Additionally, my results suggest that this inhibition occurs through interactions with regulatory cysteine residues in a putative lid domain of MGL. Taken together, the identification and characterization of these pharmacological tools has helped to strengthened our current understanding of the mechanisms of MGL inhibition, and will increase our understanding of the specific biological role of 2-AG in endocannabinoid signaling.;Second, I investigated and characterized the effect of oxidative stress on the in vitro activity of MGL and report that hydrogen peroxide inhibits MGL through a rapidly reversible and noncompetitive mechanism. Furthermore, I established that treating primary cortical neurons with hydrogen peroxide leads to a decreases in MGL activity while increasing 2-AG levels. Additionally, I report evidence that the increase in 2-AG levels during oxidative stress protected neuronal cells from oxidant-induced cell death.;Finally, in collaboration with Dr. Marco Mor of University of Parma, Italy, we developed a novel and potent MGL inhibitor UPR1218, and I showed that this compound inhibit MGL through interaction with the regulatory cysteines Cys201 and Cys208 in the lid domain of the enzyme. In addition, I showed that UPR1218 inhibited MGL through a reversible mechanism and protected neuronal cells from oxidant-induced cell death.;Taken together, the discovery and characterization of these pharmacological tools has invariably increased our understanding of the biochemical mechanisms involved in MGL inhibition and has serve to further elucidate the physiological role of 2-AG as an important endocannabinoid signaling molecule. Additionally, my discovery that MGL is reversibly inhibited by oxidants during oxidative stress has provided a critical insight into the mechanisms and the physiological relevance of MGL modulation by oxidants during oxidative stress. Finally, this dissertation provides evidence to support the concept of MGL as a target for the development of drugs for the treatment of oxidant-mediated neuronal injuries. |
