Aditi Das, Ph.D. - US grants

PROJECT SUMMARY Dietary consumption of ?-3 and ?-6 fatty acids have been linked to cardiovascular health benefits in humans. The central hypothesis is that the cardiovascular physiological effects of ?-3 and ?-6 fatty acids are partly mediated by the synthesis of eicosanoids via the epoxygenase (EPOX) pathway. Herein we perform biochemical studies of some of the key enzymes in these pathways. CYP2J2 is an enzyme in the EPOX pathway that is highly expressed in the cardiovascular system in the aortic epithelium and cardiomyocytes. CYP2J2's primary effects are facilitated via epoxidation of ?-3 and ?-6 fatty acids into epoxides that exert potent anti- inflammatory, vasodilatory and pro-angiogenic effects. CYP2J2 is also implicated in cardiotoxicity of drugs. Additionally, CYP2J2 is also a membrane bound protein and exhibit unique biochemical mechanisms that are poorly characterized and are the primary focus of the current proposal. Our first goal is to understand allosteric modulation of CYP2J2 epoxygenase activity by ?-3 and ?-6 fatty acids and selected cardiotoxic drugs (doxorubicin, ebastine and terfenadine). Our second goal is to examine the metabolism of ?-3 and ?-6 fatty acid derived endocannabinoids by CYP2J2. It is predicted that similar to ?-6 endocannabinoids, the ?-3 endocannabinoids are substrates for the EPOX enzymes producing novel bioactive epoxide mediators. The third goal is to examine how the composition of membranes effect CYP2J2 activity. We use several novel approaches that includes detection of lipid mediators with mass spectrometry, innovative methodologies such as Nanodiscs to solubilize CYP2J2 and provide membrane bilayer environment. We also introduce novel concepts of lipid-drug heterotropic interactions influencing the formation of the products of these enzymes. The long-term goal of this work is to understand the interplay of the formation of the eicosanoids from dietary fatty acids.

PROJECT SUMMARY Neurodegenerative disorders, stroke, and chronic pain are associated with ongoing neuroinflammation which can be partially ameliorated by endocannabinoids. Endocannabinoids are ligands for cannabinoid receptor 1 and 2 (CB1 and CB2). CB1 receptor agonists exhibit psychotropic properties while CB2 receptor agonists have anti-neuroinflammatory effects. Consequently, there is a strong interest in the discovery of CB2 selective agonists to mitigate inflammatory pathologies while being devoid of the psychotropic activity exhibited by CB1 receptor agonists. Endocannabinoids are derived from dietary fatty acids. Anandamide (AEA) is derived from the ?-6 fatty acid arachidonic acid (AA), while docosahexaenoic ethanolamide (DHEA) is derived from the ?-3 fatty acid, docosahexaenoic acid (DHA). The ?-3 and ?-6 endocannabinoids AEA and DHEA are further metabolized by eicosanoid synthesizing enzymes such as CYP epoxygenases to form oxidized endocannabinoid products that have new biological activity. Previous studies have shown that CYP epoxygenases convert AEA into AEA-epoxide that is a CB2 selective agonist. Herein we will test our central hypothesis that, DHEA epoxide (EDP- EA) will display anti-neuroinflammatory action in microglial cells by binding to CB2 receptor and other putative receptors. In order to execute the specific aims of this proposal, we will use a combination of several innovative methods including novel targeted lipidomics (LC-MS/MS) approaches to detect lipid epoxide metabolites, receptor binding and activation studies and neuroinflammation studies using microglial cells. The overall outcome of the project will be the discovery of a new set of lipid signaling endogenous molecules, DHEA epoxides that exhibit anti-neuroinflammatory properties.  

PROJECT SUMMARY Remedies derived from the cannabis plants have been used for management of pain for centuries. Recent understanding of the clinical effects of cannabis and the corresponding cannabinoids in the treatment of pain has been focused on two major phytocannabinoids, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Though effective, these compounds cause sedation and drowsiness. On the other hand, there are more than 110 known minor phytocannabinoids; however, only a few limited studies related to their analgesic properties exists to date. The major obstacle in studying rare cannabinoids is their limited availability. Numerous purifications of plant extracts mixtures usually provide only milligram quantities of pure compounds from kilograms of plant material, making such endeavors highly nonpractical as well as unsustainable. In fact, most of minor phytocannabinoids are not commercially available or listed in NIDA?s Drug Supply Program (DSP). As synthetic organic chemists and biologists, uniquely situated at the interface of chemistry and biology, our research programs are devoted to providing solutions to the supply problem in the form of sustainable and practical syntheses as well as performing fundamental biological studies. By providing an access to rare phytochemicals by synthetic means, we expect to remove the barrier of supply as a prerequisite for studying their analgesic properties. Specifically, this proposal describes synthetic approaches to several classes of rare phytocannabinoids and systematic evaluation of their anti-inflammatory potential in microglial cells. Minor cannabinoids with strong anti-inflammatory will undergo further evaluation towards their agonism/antagonism of major endogenous pain circuitry systems including cannabinoid receptors (CB1, CB2, GPR55) and vanilloid/transient receptor potential cation channel subfamily V (TRPV), subfamily A (TRPA), subfamily M (TRPM). It is expected that these studies will establish well-defined pharmacological properties of rare phytocannabinoids with respect to the major receptors involved in pain sensation.