6′-Guanidinonaltrindole (6′-GNTI) Is a G Protein-biased κ-Opioid Receptor Agonist That Inhibits Arrestin Recruitment

Abstract

Background: Arrestin recruitment to the -opioid receptor (KOR) has been linked to several adverse effects of analgesics, such as dysphoria and tolerance. Results: We identified 6Ј-guanidinonaltrindole (6Ј-GNTI) as a potent KOR agonist for G protein activation that fails to recruit arrestin. Conclusion: 6Ј-GNTI is an extreme G protein-biased KOR ligand. Significance: 6Ј-GNTI is a lead toward analgesics with fewer arrestin-mediated adverse effects.-Opioid receptor (KOR) agonists do not activate the reward pathway stimulated by morphine-like -opioid receptor (MOR) agonists and thus have been considered to be promising nonaddictive analgesics. However, KOR agonists produce other adverse effects, including dysphoria, diuresis, and constipation. The therapeutic promise of KOR agonists has nonetheless recently been revived by studies showing that their dysphoric effects require arrestin recruitment, whereas their analgesic effects do not. Moreover, KOR agonist-induced antinociceptive tolerance observed in vivo has also been proposed to be correlated to the ability to induce arrestin-dependent phosphorylation, desensitization, and internalization of the receptor. The discovery of functionally selective drugs that are therapeutically effective without the adverse effects triggered by the arrestin pathway is thus an important goal. We have identified such an extreme G protein-biased KOR compound, 6-guanidinonaltrindole (6-GNTI), a potent partial agonist at the KOR receptor for the G protein activation pathway that does not recruit arrestin. Indeed, 6-GNTI functions as an antagonist to block the arrestin recruitment and KOR internalization induced by other nonbiased agonists. As an extremely G protein-biased KOR agonist, 6-GNTI represents a promising lead compound in the search for nonaddictive opioid analgesic as its signaling profile suggests that it will be without the dysphoria and other adverse effects promoted by arrestin recruitment and its downstream signaling.-Opioid receptors (KOR) 2 are widely expressed in the periphery, the dorsal root ganglia, the spinal cord, and the supraspinal regions associated with pain modulation. KOR agonists have been shown to activate pain inhibitory pathways in the central nervous system, and peripherally restricted KOR agonists have been developed to target KOR located on visceral and somatic afferent nerves for relief of inflammatory, visceral, and neuropathic chronic pain (1, 2). The analgesic properties of KOR agonists are attributed to their ability to activate G proteins in the G i/o family (3, 4) as the subsequent inhibition of cAMP production (5), as well as the activation of inward rectifier potassium channels (6) and blockade of calcium channels (7), has an inhibitory effect in neurons. In contrast to MOR agonists, KOR agonists are unable to activate the reward pathway and have therefore attracted considerable attention for their ability to exert potent analgesic effects without high abuse potential (1,8).Unfortunately, KOR agonists have been found to produce other signif...

Identification of Novel Functionally Selective κ-Opioid Receptor Scaffolds

“…Furthermore, the tested morphinans (Table 3) and benzomorphans (Table 4) tested displayed very little bias. Only 69-GNTI displayed a slight G protein bias (bias factor of 6), consistent with previous studies (Rives et al, 2012;Schmid et al, 2013). Also, we found that the antagonist JDTic has no agonistic activity in either G protein or arrestin assays (Table 4).…”

“…To confirm our results from the Tango arrestin recruitment assay, we used a BRET-based arrestin-recruitment assay (Rives et al, 2012) to further analyze the compounds displaying the highest degree of bias. Salvinorin A displayed very similar potency values for the Tango and BRET assays (5.56 and 5.63 nM, respectively) ( Tables 1 and 6).…”

“…Two assays were used to assess b-arrestin translocation: the Tango assay as described previously (Barnea et al, 2008;Wu et al, 2012), and a bioluminescence resonance energy transfer (BRET)-based assay as an orthologous confirmatory assay as described previously (Rives et al, 2012). The Tango assay requires the fusion of a transcription factor to the C terminus of KOR via linker that contains a tobacco etch virus (TEV) protease cleavage site.…”

A Novel Method for Analyzing Extremely Biased Agonism at G Protein–Coupled Receptors

“…This can be seen using 69-GNTI, which produces a clear and distinct activation of response in both assays that can easily be resolved from the baseline. Although the competitive model does not confer a distinct benefit over the standard model for 69-GNTI, both the standard and competitive methods provide estimates of 69-GNTI bias toward G protein signaling that are in agreement with previous studies (Rives et al, 2012;Schmid et al, 2013). In this case, the data obtained by calculating relative affinity using the competitive analysis does not further resolve the estimation of bias; moreover, this approach may actually introduce error into the analysis, as evidenced by an increase in the width of the 95% CIs.…”

“…For example, 69-guanidinonaltrindole (69-GNTI) was initially characterized as a potent partial KOR agonist (Sharma et al, 2001). It was subsequently shown to stimulate G protein coupling yet induced very little detectable barrestin2 recruitment when compared with U69,593 [(1)-(5a,7a,8b)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide], a KOR agonist that displays high efficacy in both assays (Rives et al, 2012;Schmid et al, 2013). Using the method of bias analysis described by Ehlert (2008) and Kenakin et al (2012), referred to herein as the "standard" model, 69-GNTI was determined to be a biased agonist for G protein signaling over barrestin2 recruitment .…”