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

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.

Keywords: biased agonism; morphine; nonhuman primate; opioid; second messenger signaling.

Figure 1

cAMP, NF-ĸB, MAPK/JNK, and MAPK/ERK respond to MOR activation by 10 µM DAMGO (solid bars) compared to saline (hatched bars) in both species, (a) human and (b) rhesus macaque. * indicates p < 0.05 compared to the negative control, # indicates p < 0.05 in the DAMGO group compared to saline.

Figure 2

The concentration-dependent inhibition/activation of signaling by MOR ligands. Inhibition of forskolin-induced cAMP activation in human (a) and rhesus macaque (d) MORs. Stimulation of NF-ĸB in human (b) and rhesus macaque (e) MORs. Stimulation of MAPK/JNK in human (c) and rhesus macaque (f) MORs. Morphine (blue), met-enkephalin (cyan), endomorphin-1 (orange), β-endorphin (purple), and TRV130 (green).

Figure 3

The ∆log($\frac{\mathsf{\tau }}{{\mathrm{K}}_A}$) values from three pathways for all ligands are shown for human (a–e) and rhesus macaque (f–j) MORs. * indicates p < 0.05 between signaling pathways.

Figure 4

Web chart reflecting the calculated bias factor from ∆∆log($\frac{\mathsf{\tau }}{{\mathrm{K}}_A}$) for all ligands in human (a) and rhesus macaque (b). Empty circles represent p < 0.05 on ∆log($\frac{\mathsf{\tau }}{{\mathrm{K}}_A}$) values compared between the test ligand and the reference ligand DAMGO. Closed circles represent p < 0.05 on ∆log($\frac{\mathsf{\tau }}{{\mathrm{K}}_A}$ ) values comparisons between human and rhesus macaque. Morphine (blue), met-enkephalin (cyan), endomorphin-1 (orange), β-endorphin (purple), TRV130 (green).