Design And Synthesis Of Allosteric Modulators Of Cb1 Cannabinoid Receptor

Author : Derek M. Shore
Publisher :
ISBN 13 :
Total Pages : 202 pages
Book Rating : 4.:/5 (969 download)

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Download or read book Allosteric Modulation of the Human Cannabinoid-1 Receptor written by Derek M. Shore and published by . This book was released on 2014 with total page 202 pages. Available in PDF, EPUB and Kindle. Book excerpt: "The human cannabinoid-1 (CB1) receptor is a Class A, rhodopsin-like G protein-coupled receptor (GPCR). CB1 is found primarily in the central nervous system (CNS) where it participates in the regulation of neuronal activity; consequently, it is not surprising that this receptor has been implicated in numerous pathologies, including Alzheimer's disease, cancer, obesity, and pain. Unfortunately, many attempts at therapeutically targeting CB1 have failed, due to unacceptable CNS-related side effects; specifically, attempts to target CB1's orthosteric binding site (i.e. the primary binding site of endogenous, non-allosteric ligands) have been unsuccessful. These failures may be due to problems involving receptor subtype selectivity, a lack of functional selectivity, as well as a pathological interference with basal signaling. The ultimate goal of this research is to expand our understanding of CB1 signal transduction, at a molecular level, and to employ this knowledge in the development of CB1-based drug therapies. In pursuing this goal, we have used computational methods together with mutagenesis, synthesis, and pharmacological studies. The results of this work are presented here in four chapters, with each chapter acting as a foundation for subsequent investigation. In Chapter 1, we present results involving the importance of CB1's extracellular (EC) loops to its G protein-mediated signaling. Specifically, these results suggest that an ionic interaction between Lys-373 (of the EC-3 loop) and D2.63176 is important for G protein-mediated signaling. Our computational results suggest this salt bridge is important because it promotes an active conformation of the EC-3 loop, such that the EC-3 loop is pulled across the top of the receptor, `tethering' the EC-3 loop and transmembrane helix (TMH) 2. In addition, we report results that suggest that the EC-2 loop moves down (into the transmembrane core) upon activation. In Chapter 2, we report the binding site and mechanism of action of the negative CB1 allosteric modulator ORG27569. This compound has the paradoxical effects of increasing the equilibrium binding of CP55,940 (an orthosteric agonist), while at the same time antagonizing its G protein-mediated signaling. When applied alone, ORG27569 acts as an inverse agonist of G protein-mediated signaling, as well as an agonist of the ERK signaling pathway. Our results suggest that ORG27569 binds in the TMH3/6/7 region of CB1 (extending extracellularly), and promotes an intermediate conformation of CB1. In addition, ORG27569 may antagonize the G protein-mediated signaling of CP55,940 by sterically blocking conformational changes of the EC-2 and EC-3 loops, as well as by packing tightly against TMH6. We also reported that ORG27569's inverse agonism is dependent upon the formation of a hydrogen bond between its piperidine nitrogen and K3.28192. In Chapter 3, we use our model of ORG27569 docked at CB1 (in the presence of CP55,940) to design, synthesize, and characterize four analogs of ORG27569. These compounds were designed using three different strategies: 1) to form a new hydrogen bond between the analog(s) and D6.58366; 2) to form a new aromatic stack between the analog(s) an F3.25189; and 3) to test steric packing between the analog(s) and TMH6/7. The experimental results revealed that these four compounds have a unique and rich pharmacological profile. The analog PHR018 is a more efficacious negative allosteric modulator than ORG27569 (whereas PHR017 is a less efficacious modulator). The analog PHR016 is a `classical' allosteric modulator (i.e. an allosteric modulator that only affects the binding/signaling of an orthosteric ligand, with no functional effects when applied alone); PHR016's sole functional effect is to antagonize the G protein-mediated signaling of CP55,940. Finally, the analog PHR019 was observed to be a completely biased agonist for CB1 ERK signaling. To our knowledge, PHR019 is the only completely biased agonist for the ERK signaling pathway that targets a GPCR. In addition, none of these analogs acted as inverse agonists of G protein-mediated signaling. Altogether, these results suggest the remarkable therapeutic potential of CB1 allosteric-based therapies, due to the analogs' unprecedented level of functional control, as well the analogs' noninterference with basal G protein-mediated signaling. In Chapter 4, we report the binding site and mechanism of action of lipoxin A4, a positive allosteric modulator of CB1. Specifically, we used the Forced-Biased Metropolis Monte Carlo simulated annealing method (MMC), Glide docking studies, as well as molecular dynamics to identify lipoxin A4's binding site at CB1. These results suggest that lipoxin A4 binds in the TMH3/6/7 region of CB1, extending extracellularly. Unlike ORG27569 (which sterically blocks conformational changes of the EC loops), lipoxin A4 forms several electrostatic interactions with the EC loops. By forming these interactions, lipoxin A4 promotes an active conformation of the EC-3 (and EC-2) loops, thereby stabilizing an active conformation of CB1. Together, these results describe important conformational changes in the extracellular region of CB1, the binding site and mechanism of action of ORG27569, the development of unique ORG27569 analogs (including a biased agonist of the ERK pathway), and finally the binding site and mechanism of action of the positive allosteric modulator, lipoxin A4. Hopefully, this work (and future studies) will aid in the development of new therapies that target CB1."--Abstract from author supplied metadata.