Searching for novel ligands for the cannabinoid and related receptors.

The first purpose of the present dissertation was to apply a high throughput assay to systematically screen a library of food and drug administration (FDA)-approved drugs as potential ligands for the cannabinoid receptor 2 (CB2). A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring changes in intracellular cAMP levels was validated and found to be suitable for testing ligands that may act on CB2. Among the 640 FDA-approved drugs screened, raloxifene, a drug used to treat/prevent post-menopausal osteoporosis, was identified for the first time to be a novel CB2 inverse agonist. The dissertation reporting these results demonstrated that by acting on CB2, raloxifene enhances forskolin-stimulated cAMP accumulation in a concentration-dependent manner. Furthermore, the data showed that raloxifene competes concentration-dependently for specific [3H]CP-55,940 binding to CB2. In addition, raloxifene pretreatment caused a rightward shift of the concentration-response curves of the cannabinoid agonists CP-55,940, HU-210, and WIN55,212-2. Raloxifene antagonism is vi most likely competitive in nature, as these rightward shifts were parallel and were not associated with any changes in the efficacy of cannabinoid agonists on CB2. The discovery that raloxifene is as an inverse agonist for CB2 suggests that it might be possible to repurpose this FDA-approved drug for novel therapeutic indications for which CB2 is a target. Furthermore, identifying raloxifene as a CB2 inverse agonist also provides important novel mechanisms of actions to explain the known therapeutic effects of raloxifene. The second purpose of the current study was to investigate the ability of the third-generation selective estrogen receptor modulators (SERMs) bazedoxifene and lasofoxifene to bind and act on CB2 cannabinoid receptor. We have identified, for the first time, that CB2 is a novel target for bazedoxifene and lasofoxifene. Our results showed that bazedoxifene and lasofoxifene were able to compete for specific [3H]CP-55,940 binding to CB2 in a concentration-dependent manner. Our data also demonstrated that by acting on CB2, bazedoxifene and lasofoxifene concentration-dependently enhanced forskolin-stimulated cAMP accumulation. Furthermore, bazedoxifene and lasofoxifene caused parallel, rightward shifts of the CP-55,940, HU-210, and WIN55,212-2 concentration-response curves without altering the efficacy of these cannabinoid agonists on CB2, which indicates that bazedoxifene- and lasofoxifene-induced CB2 antagonism is most likely competitive in nature. Our discovery that CB2 is a novel target for bazedoxifene and lasofoxifene suggests that these third-generation SERMs can potentially be repurposed for novel therapeutic indications for which CB2 is a target. In addition, identifying bazedoxifene and lasofoxifene as CB2 inverse agonists also provides important novel mechanisms of actions to explain the known therapeutic effects of these SERMs. The third purpose of this dissertation was to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. Using novel fatty acid amides and detailed potency and efficacy analyses, the dissertation reporting these results demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, the dissertation reporting these results demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor. The fourth purpose of this dissertation was to use a fragment-based approach, categorical-SAR (cat-SAR), to model ligands for GPR119. Using compounds that are known GPR119 agonists and compounds that were confirmed experimentally that are not GPR119 agonists, four distinct cat-SAR models were developed. Using a leave-one out validation routine, the best GPR119 model had an overall concordance of 99 %, a sensitivity of 99 %, and a specificity of 100 %. The dissertation reporting these results from the in-depth fragment analysis of several known GPR119 agonists was consistent with previously reported GPR119 structure-activity relationship (SAR) analyses. Overall, while the dissertation reporting these results indicates the development a highly predictive cat-SAR model that can be potentially used to rapidly screen for prospective GPR119 ligands the applicability domain must be taken into consideration. Moreover, the dissertation demonstrating these results was the first report that the cat-SAR expert system can be used to model G protein-coupled receptor ligands, many of which are important therapeutic agents

Searching for novel ligands for the cannabinoid and related receptors.

A cell-based, Homogenous Time Resolved Fluorescence (HTRF) method was optimized and used to test a library of 60 putative endocannabinoids for activity towards CB1 or CB2, and to test cannabinoid ligands and fatty acid amides for GPRl19 by measuring cAMP levels in this study. The Z\u27 factors for the assay were greater than 0.5 for all three receptors and the assay was able to can tolerate up to 1% DMSO demonstrating a robust and suitable technology for screening. The known cannabinoid and GPRl19 agonists exhibited the rank order of potency expected for CB1/CB2/GPRl19. Our data demonstrate that none of the amides, N-acyl amino acids (glycine and alanine) Acyl-dopamines, and Acyl-GABAs was able to activate either CB1 or CB2. However the ethanoamides Dihomo-gamma-linolenoyl ethanolamide (DLEA) and DTEA Docosatetra-7Z,1 OZ, 13Z, 16Z-enoyl ethanolamide (DTEA) were found to activate CB1 and CB2. Our data provide direct evidence to support the hypothesis that unsaturation in the acyl chain of fatty acid ethanolamides affects the ability of these compounds to activate GPRl19. Our results suggested that GPRl19 activation requires certain structural requirements for the charged head groups of the fatty acid amides