ABSTRACT
An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.
Subject(s)
Analgesics, Opioid/pharmacology , Fungal Proteins/pharmacology , Oligopeptides/pharmacology , Penicillium/chemistry , Receptors, Opioid, mu/agonists , Analgesics, Opioid/chemistry , Animals , Binding Sites , Cell Line, Tumor , Fungal Proteins/chemistry , HEK293 Cells , Humans , Mice , Molecular Docking Simulation , Oligopeptides/chemistry , Protein Binding , Receptors, Opioid, mu/chemistry , Receptors, Opioid, mu/metabolismABSTRACT
Opioid efficacy on mu-receptor may be influenced by various Gi/o-G-protein subunits interacting with intracellular face of receptor. Pertussis toxin-insensitive Galphai1 and Galphai2 subunits tethered with mu-receptor were stably transfected into AtT20 cells to (i) determine coupling of different alpha-subunits on opioid efficacy, and (ii) determine coupling to downstream effectors, for example, calcium and potassium channels. After pertussis toxin, stimulation of [35S]GTP-gamma-S incorporation persisted. Both constructs were able to couple to native calcium and potassium channels, with endomorphins 1 and 2 equally effective. However, pertussis toxin abolished opioid actions on calcium and potassium channels suggesting strong coupling to endogenous G-proteins, and that differences in coupling efficacy to Galphai1 and Galphai2 previously observed are restricted to initial step of signaling cascade.