Construction and validation of GPR55 active and inactive state in silico models through the use of biological assays, mutation data, and structure activity relationships

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Mary A. Lingerfelt (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Patricia Reggio

Abstract: G-protein coupled receptors (GPCRs) function as both gatekeepers and molecular messengers of the cell. They relay signals that span the cell membrane mediating nearly every significant physiological process and currently represent the target of about 30% of all drugs. The signals they transmit can arise from a remarkable variety of stimuli which includes, but is not limited to, photons, neurotransmitters and hormones. GPR55, a rhodopsin-like (Class A) GPCR, has received a great deal of attention due to its emerging involvement in a multitude of physiological processes and its putative identity as a third type of cannabinoid receptor. Characterizations of GPR55 knock-out mice reveal a role for the receptor in controlling inflammatory pain, neuropathic pain, and bone resorption.1 Myriad other studies indicate that GPR55 activation may play a part in oncogenesis and metathesis. GPR55 can be found in numerous tissue types throughout the body and is also highly expressed throughout the cerebellum and surrounding central nervous system lending credence to the idea that this receptor may play a more crucial physiological role than originally thought.2 GPR55 has an extensive physiological profile and has been shown to respond uniquely to a great number of diverse compounds. Specifically, it has been shown to recognize many cannabinoid compounds, including CB1 and CB2 endogenous ligands, phytocannabinoids and synthetic cannabinoids. Similar to the ligands of the CB1 and CB2 receptors, the endogenous ligand of GPR55, lysophosphatidylinositol (LPI), is a lipid-derived molecule.3 LPI activates ERK1/2 and increases [Ca2+] and, to date, there has been no evidence that LPI interacts with the other cannabinoid receptors. Despite innumerable prospective clinical uses hinted at by the aforementioned research no low nanomolar potency ligands of GPR55 have been identified. Nor has there been a radio-ligand developed to characterize the binding site of this receptor. Lack of such tools is a great impediment to any forward progress towards developing the GPR55 receptor as a therapeutic target for drug design. The following research details the creation of both a GPR55 active- and a GPR55 inactive- state homology model. Towards this goal, Chapter I details the background of the discovery, pharmacological relevance and ligand scope of GPR55. Its purpose is to establish a framework for the research that follows and highlight the medical importance of this elusive receptor. Chapter II describes the synthetic preparation of antagonists of GPR55 for use in preliminary SAR studies. The original high throughput screen that lead to the identification of novel GPR55 scaffold chemotypes from the screening of over 300,000 compounds gave rise to the piperidinyloxadiazolone compound CID23612552 and the synthetic diversification of what was then dubbed Scaffold 1. A detailed description of the methods used in the construction of the updated R and R* state of GPR55 models is handled in Chapter III. A combination of Conformational Memories4,5 (using the CHARMM forcefield), Ligand Conformational Analysis (performed using Spartan (Wavefunction, Inc., Irvine, CA)) and Macromodel/Maestro/Glide (from the Schrödinger suite) was used to build and refine both GPR55 model states. Chapter IV then covers model validation and refinement. Using the phenylpiperazine (ML184 CID2440433) and mutations performed in the lab of Dr. Mary Abood (Temple University) it was shown that the current iteration of the GPR55 R* model was indeed a valid representation of the activated state of this receptor. This chapter also provides information that gives rise to the “Future Directions” chapter, Chapter V. This final chapter is a look forward to the research that still remains to be done to ensure that these models will function as the accurate tools that they have the potential to be. We used the GPR55 R bundle to suggest antagonist structures that will maximize ligand/receptor interactions and hopefully give rise to nanomolar potency molecules. These ligands will need to be synthesized and tested. We also identified key residues in the active bundle (GPR55 R*) that could be mutated to enhance or verify ligand binding. Mutations that destroy receptor function, while interesting, would not have the same utility as the aforementioned kinds of mutations.

Additional Information

Language: English
Date: 2016
Agonist, Antagonist, GPR55, Homology, Mutation
G proteins $x Receptors
Cannabinoids $x Receptors
Ligands (Biochemistry)

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