Molecular mechanisms of mutant mu-opioid receptors where naloxone acts as an agonist

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

Abstract: Pain management is often one of the most difficult aspects of treatment for patients suffering from acute or chronic pain. The mu-opioid receptor (MOR) agonist, morphine, and its derivatives are highly used in pain management strategies. However, these medications have many side effects including respiratory depression, gastrointestinal problems, as well as dependence and addiction liabilities. For these reasons, innovative new modalities for pain management continue to be needed. One new approach to the design of opioid therapies for chronic pain with reduced liabilities is a targeted-gene therapy strategy developed by the lab of Dr. Ping-Yee Law at the University of Minnesota. This strategy makes novel use of a MOR S4.54A mutant at which the classical opioid antagonist, naloxone, acts as a partial agonist. Targeted gene therapy studies using this mutant have shown that naloxone becomes an antinociceptive agent at the S4.54A mutant both in vitro and in vivo. Because expression of the mutant MOR is targeted to the spinal cord injection site region, systemic administration of naloxone results in antagonism of all other (native) MOR's. The reduced number of receptors activated in this paradigm results in no measurable dependence/addiction as seen with traditional mu agonists like morphine. Despite the clear success of basing this strategy on the S4.54A MOR mutant, the origins of this unusual phenotype are not yet understood. It was therefore the overall goal of this dissertation to identify the molecular basis for the agonism of naloxone at this novel S4.54A mutant. To this end, a model of the wild-type and S4.54A mu opioid receptor was developed and ligand docking studies were used to probe this model. The opioid receptors, delta, kappa and mu, belong to the Class A subfamily of G-Protein Coupled Receptors (GPCRs). These are integral membrane proteins that possess seven transmembrane helices (TMHs) arranged to form a closed bundle with loops that extend both extracellularly and intracellularly. The N-terminus is extracellular and the C-terminus is intracellular. In recent years, X-ray crystallography studies have yielded structures of numerous GPCRs. In 2012, the nociception/orphanin FQ receptor and the mu, delta and kappa opioid receptor crystal structures were published. Prior to the release of the MOR crystal structure, we developed a homology model of WT MOR using the β2-AR crystal structure2,3 as a template with substitutions for TMH 1, 2, 4 and 7 based on sequence divergences, as described in methods. This model was then used for studies of the MOR including analyzing the receptor for cholesterol and palmitoylaiton interactions as well as modeling a homodimer interface for the MOR based on experimental data. In 2012, new models of WT MOR and the S4.54A/L mutants were developed using the MOR crystal structure. The conformational change in TMH4 that would be created upon the S4.54A mutation was examined using the simulated annealing/Monte Carlo method, Conformational Memories, and the result was incorporated into the model. The S4.54A mutant model was then used for naloxone docking studies using Glide. These studies revealed that in the crystal structure, Y3.34 forms a hydrogen bond with the sidechain of S4.54; however, in the S4.54A MT MOR, this interaction is broken as there is no polar partner for Y3.34. The breaking of this interaction allows the extracellular end of TMH4 to kink away from TMH3 and towards TMH5, which leads to changes in the packing of the receptor binding pocket. In the wild type MOR, naloxone interacts with D3.32 and sits in close proximity to the binding pocket "toggle switch" residue, W6.48, restricting its movement. However, in the S4.54A MT MOR, naloxone sits higher in the binding pocket, away from W6.48 and interacts with D3.32 and E5.35. In this higher location, naloxone exerts no effect on W6.48, permitting W6.48 to assume an active state conformation. This shift in binding pocket location for naloxone may be the origin of naloxone's partial agonism in the S4.54A MOR mutant. We also explored additional experimental data generated in Dr. Ping Law's lab for other mutations at the 4.54 locus. Mutating S4.54 to Phe or Gly results in the same phenotype as the S4.54A mutation. On the other hand, for Ile or Val mutants, naloxone behaves as in WT MOR. We propose that in the case of the S4.54 I / V, an increase in hydrophobic interactions between W4.50 and I/V4.54 allow TMH4 to maintain its wild type conformation. However, while the S4.54F is also able to increase hydrophobic interactions, its size prevents the helix from maintaining the wild type shape. In the S4.54L mutant, there is no increase in hydrophobic interactions and the orientation of the leucine gives rise to a straighter TMH4, as seen in the S4.54A MT MOR. The S4.54G mutant offers additional flexibility and a higher turn ratio, with 5 residues per turn in that region such that the extracellular end of TMH4 moves away from TMH3 and towards TMH5. Additionally, Law and coworkers have published studies using a S4.54L/T7.44A/C7.47S triple mutant MOR that gives rise to naloxone acting as a full agonist.4 While this gene therapy has been shown in cells and in spinal cord, the underlying mechanism is unknown. A triple mutant MOR model was developed and analyzed to determine the molecular mechanism for which naloxone acts as an agonist. The binding pocket for mu opioid ligands is formed by TMHs 3, 5 and 6 in the wild type receptor, as seen in the crystal structure with β-FNA5 and in our glide dock of naloxone (see Chapter 3). As studied in the single mutant MOR, S4.54 is a lipid facing residue. Interestingly, both of the mutated residues on TMH7 (T7.44 and C7.47) in the triple mutant MOR also face lipid. We report here that the combination of the S4.54L mutation on TMH4 along with TMH7 face shift changes occur upon mutation of T7.44 and C7.47 produce overall packing changes that give rise to a different binding pocket than seen in the wild type or single mutant MORs. These changes result in naloxone's ability to fully activate the S4.54L/T7.44A/C7.47S MOR.

Additional Information

Language: English
Date: 2013
Gene therapy, Opioid, Pain
Chronic pain $x Alternative treatment
Opioids $x Receptors
Opioids $x Therapeutic use

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