The endocannabinoid system has been historically targeted for medicinal purposes through the use of cannabis. Two cannabinoid receptors, CB1 and CB2, have been identified as the mediators of the biological effects of the main psychoactive component in Cannabis sativa; delta-9-tetrahydrocannabinol. The CB1 receptor is a class A G-protein coupled receptor (GPCR), and is the most abundant neuro-modulatory receptor in the CNS. With its location at presynaptic termini, it regulates the function of other receptors, including the dopamine, serotonin, GABA, opioid and glutamate receptors.
Many potential therapeutic applications targeting the CB1 receptor are under investigation. However, the unformed knowledge in drug-receptor interactions at the molecular level, and in the CB1 receptor signaling properties, has hampered the transition of many synthesized CB1 receptor agonists and antagonists to clinical use. In the studies to be presented, computational and mutational methods are employed to improve our understanding of the CB1 receptor structure, its conformational changes during activation, and the dynamics of drug-receptor interaction.
In the first chapter, background information on the CB1 receptor is provided; including its structure, drugs that act at the CB1 receptor, G-protein and β-arrestin mediated signaling pathways, as well as, CB1 receptor regulation by other proteins.
The second chapter includes a discussion of the biased signaling through class A GPCRs in general and through the CB1 receptor specifically. A set of hypothesis-driven mutations on the CB1 receptor yielded a CB1 receptor that exhibits biased signaling through the β-arrestin pathway. Results from this study provide better understanding of the molecular mechanisms of biased signaling at the CB1 receptor specifically, and in Class A GPCRs in general. In addition, biased mutants developed here should assist structure-based drug design of CB1 receptor ligands with β-arrestin functional selectivity.
In the third chapter, molecular dynamics simulations are presented that were used to investigate the docking of 5-(4-chlorophenyl)-N-[(1R,2R)-2-hydroxycyclohexyl]-6-(2- methoxyethoxy)-3-pyridinecarboxamide (14h) inside the CB1 receptor. 14h has been described previously as a peripherally selective, high affinity CB1 receptor antagonist. However, the compound exhibits higher affinity for the human CB1 receptor compared to rodent CB1. Recent inactive state crystal structures (PDB ID: 5TGZ, 5U09) of the CB1 receptor have been published showing the membrane proximal region of the N-terminus invading the receptor binding pocket, steering an important interaction site (K3.28) away from binding pocket. Thus, results from the docking study will help in the remodeling of the CB1 receptor’s N-terminus based on a mutational study that identified an N-terminal residue (M106 in rodent CB1 compared to I105 in human CB1) as the determinant of the species differential affinity of 14h at the CB1 receptor.
The fourth chapter is focused on the biarylpyrazole derivative; 5-(4-Chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (SR-141716A (SR)). SR is a potent and a selective CB1 receptor antagonist and inverse agonist. The lack of force field parameters for SR has hampered the study of its interaction dynamics with the receptor in a lipid bilayer. This work describes the development of missing CHARMM force field parameters for SR based on a previously published parameterization method for drug-like molecules, as well as an all atom MD simulation study of SR in a fully hydrated bilayer. Generated parameters were successful in reproducing target data from SR crystal structures equilibrated at the MP2/6-31G* level of theory. MD simulations show that SR can adopt multiple orientations in the lipid bilayer, it can rotate in all directions and move freely between leaflets.
Advances in crystallization and expression techniques made the crystallization of the CB1 receptor possible. However, those techniques, as well as, the static nature of the crystal structures, compromise the structural information for the receptor and undermine the dynamics of drug-receptor interactions. Computational, as well as, mutational studies would provide ancillary information that enhance our understanding of the CB1 receptor. Current studies increase our molecular level understanding of the conformational changes that accompany CB1 receptor activation. They also highlight the dynamics of drug-receptor interactions in a bilayer. Results from these studies will aid the development of new ligands that target the CB1 receptor.
|Advisor:||Reggio, Patricia H.|
|Commitee:||McFarland, Sherri, Peterson, Kimberly, Taylor, Ethan|
|School:||The University of North Carolina at Greensboro|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- North Carolina|
|Source:||DAI-B 79/11(E), Dissertation Abstracts International|
|Subjects:||Computational chemistry, Medicine, Pharmaceutical sciences|
|Keywords:||Biased signaling, Cannabinoids, Drug design, GPCR, MD simulation, Modelling|
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