In Vitro Effects of Ligand Bias on Primate Mu Opioid Receptor Downstream Signaling.

Xiao Zhang,Eric J Vallender,Shaurita D Hutchins,Bruce E Blough

doi:10.3390/ijms21113999

Abstract

Interest has emerged in biased agonists at the mu opioid receptor (MOR) as a possible means for maintaining potent analgesis with reduced side effect profiles. While approaches measuring in vitro biased agonism are used in the development of these compounds, their therapeutic utility will ultimately be determined by in vivo functional effects. Nonhuman primates (NHPs) are the most translational model for evaluating the behavioral effects of candidate medications, but biased signaling of these drugs at NHP MOR receptors has been unstudied. The goal of the current work was to characterize MOR ligand bias in rhesus macaques, focusing on agonists that have previously been reported to show different patterns of biased agonism in rodents and humans. Downstream signaling pathways that responded to MOR activation were identified using a luciferase reporter array. Concentration-response curves for specific pathways (cAMP, NF-ĸB, MAPK/JNK) were generated using six agonists previously reported to differ in terms of signaling bias at rodent and human MORs. Using DAMGO as a reference ligand, relative cAMP, NF-ĸB and MAPK/JNK signaling by morphine, endomorphin-1, and TRV130 were found to be comparable between species. Further, the bias patterns of across ligands for NF-ĸB and MAPK/JNK were largely similar between species. There was a high degree of concordance between rhesus macaque and human MOR receptor signaling bias for all agonists tested, further demonstrating their utility for future translational behavioral studies.

Highlights

Recent advances in the study of G-protein coupled receptor (GPCR) pharmacology have demonstrated that various intracellular pathways can be differentially activated in a ligand-specific manner, a phenomenon referred to as biased agonism [1]
HEK293 cell lines containing either human or rhesus macaque mu opioid receptor (MOR) were reverse transfected into a Cignal Finder GPCR Signaling 10-Pathway Reporter Array (Qiagen; Valencia, CA, USA) that contained inducible transcriptional factor responsive luciferase reporter constructs for common GPCR downstream signaling pathways: ATF2/3/4, cAMP, MAPK/ERK, MAPK/JNK, MEF2, Hedgehog, PI3K/AFT, IL-6, PKC/Ca2+, and NF-kB
Previous work has demonstrated that activation of MAPK/JNK is driven by β-arrestin2 recruitment [45], suggesting that measuring MAPK/JNK may be an appropriate proxy for β-arrestin2 recruitment

Summary

Introduction

Recent advances in the study of G-protein coupled receptor (GPCR) pharmacology have demonstrated that various intracellular pathways can be differentially activated in a ligand-specific manner, a phenomenon referred to as biased agonism [1]. Other studies in rodents have not shown the same reductions in side effects [17,18,19,20,21,22]. This disconnect between in vitro assays and in vivo behavior may result from numerous factors, including binding kinetic effects on signaling [23], receptor availability [17], and cellular context [24]. Explicit comparisons of biased agonists between human and rodent kappa opioid receptors found significant species differences [25,26], as well as magnitude, if not in rank order, for ligand bias in the MOR [27]

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