Allosteric modulation of the human cannabinoid-1 receptor: negative modulators, positive modulators, and biased agonists

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)

Derek M. Shore (Creator)

Institution

The University of North Carolina at Greensboro (UNCG )

Web Site: http://library.uncg.edu/

Advisor

Patricia Reggio

Abstract: 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 CB<sub>1</sub> 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 CB<sub>1</sub> (extending extracellularly), and promotes an intermediate conformation of CB<sub>1</sub>. 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 CB<sub>1</sub> (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.

Allosteric modulation of the human cannabinoid-1 receptor: negative modulators, positive modulators, and biased agonists
PDF (Portable Document Format)
227946 KB
Created on 12/1/2014
Views: 1511

Additional Information

Publication

Dissertation

Language: English

Date: 2014

Keywords

Allosteric modulation, Biased agonist, Cannabinoid receptor, Drug design, G protein-coupled receptor, Molecular modeling

Subjects

Cannabinoids $x Receptors

Cannabinoids $x Physiological effect

Cannabinoids $x Therapeutic use

Molecules $x Models

Drugs $x Design

Email this document to