Rubsicolins are naturally occurring G-protein-biased delta opioid receptor peptides

Abstract

187 Total word count: 2299 Figures: 2 Tables: 2 References: 26 2 AbstractThe impact that β-arrestin proteins have on G-protein-coupled receptor trafficking, signaling and physiological behavior has gained much appreciation over the past decade. A number of studies have attributed the side effects associated with the use of naturally occurring and synthetic opioids, such as respiratory depression and constipation, to excessive recruitment of β-arrestin. These findings have led to the development of biased opioid small molecule agonists that do not recruit β-arrestin, activating only the canonical G-protein pathway. Similar Gprotein biased small molecule opioids have been found to occur in nature, particularly within kratom, and opioids within salvia have served as a template for the synthesis of other G-proteinbiased opioids. Here, we present the first report of naturally occurring peptides that selectively activate G-protein signaling pathways with minimal β-arrestin recruitment. We find that rubiscolin peptides, which are produced as cleavage products of the plant protein rubisco, bind to and activate G-protein signaling at  opioid receptors. However, unlike the naturally occurring  opioid peptides leu-enkephalin and deltorphin II, the rubiscolin peptides only very weakly recruit β-arrestin 2 and have undectable recruitment of β-arrestin 1 at the  opioid receptor.

Isolation and Pharmacological Characterization of Six Opioidergic Picralima nitida Alkaloids

“…Binding assays were performed on membranes isolated from CHO cells stably expressing the δOR or μOR and from U2OS cells stably expressing the κOR (DiscoverX) as previously described using tritiated radioligands ([ 3 H]DAMGO, [ 3 H]U69,593, and [ 3 H]DPDPE for μOR, κOR, and δOR, respectively). 39 GloSensor cAMP Inhibition Assay. cAMP inhibition assays were performed as previously described in HEK293 cells transiently transfected with pGloSensor22F-cAMP (Promega, Madison, WI, USA) and either FLAG-mouse δOR, HA-mouse μOR, or FLAGmouse κOR.…”

Discovery of Potent Kappa Opioid Receptor Agonists Derived from Akuammicine

“…The competitive radioligand binding assay was performed on the membrane preparations as previously described with using tritiated radioligands [ 3 H]DAMGO and [ 3 H]U69,593 for the μOR and the κOR, respectfully. 75 Statistics. Cellular pharmacological data was analyzed using GraphPad 9 (GraphPad Prism software, La Jolla, CA, United States) and is presented as mean ± SEM.…”