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 ( K i low micromolar) μ-opioid agonists, which led to the design of bilorphin, a potent and selective μ-opioid receptor (MOPr) agonist ( K i 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit β-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting β-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.
biased agonist; glycosylation; opioid analgesic; peptide drug; μ-opioid receptor.
Copyright © 2019 the Author(s). Published by PNAS.
Conflict of interest statement
Competing interest statement: Patent Application(s) corresponding to Australian Patent Application 2018901944 The University of Sydney and The University of Queensland has been filed concerning this peptide. Title: Analgesics and Methods of Use Thereof.
Structures of bilaids, bilorphin, and bilactorphin.
A) Competition for binding of [ 3H]DAMGO to hMOPr (human recombinant MOPr) by the native bilaid YvVf-OH ( 3a), YvVf-NH 2 ( 3b) and bilorphin ([Dmt]vVf-NH 2) ( 3c), as well as bilorphin to hDOPr ([ 3H]DADLE binding to human recombinant DOPr) and hKOPr ([ 3H] U69593 binding to human recombinant KOPr). ( B) Exemplar GIRK current recorded from a rat LC neuron in response to Met-enk (Met-enk; 1 µM), bilorphin (1 µM) applied for duration of bars shown, and its reversal by coapplication of the MOPr selective antagonist, CTAP ( d-Phe-Cys-Tyr- d-Trp-Arg-Thr-Pen-Thr-NH 2, 1 µM). (Scale bars, 50 pA, 5 min.) ( C) Exemplar record of bilorphin, morphine, and Met-enk–induced G GIRK in mMOPr-expressing AtT20 cells in response to SST and opioids after alkylation of a fraction of receptors by the irreversible MOPr antagonist β-chlornaltrexamine (β-CNA). (Scale bar, 0.2 nS, 1 min.) ( D) Agonist concentration–response relationships of opioids for activation of GIRK current in LC neurons normalized to 1 µM Met-enk applied first as a reference in each cell ( n = 4–13 cells per data point; endo-2, endomorphin-2). ( E) Concentration–response curves of G GIRK induced by opioids in AtT20 cells after reducing the receptor reserve.
A) Representative images of S375 phosphorylation in AtT20 cells induced by saturating concentrations (30 µM) of Met-enk, endomorphin-2, morphine, and bilorphin after 5-min incubation. Colors enhanced uniformly for all images for presentation purposes. ( B) Time course of ligand-induced BRET signal (ratio of emission of 535 nm/475 nm, baseline subtracted) indicating β-arrestin 2 recruitment after incubation with saturating concentrations of agonists (shown by the arrow). The band represents the SE of experiments repeated independently 6 times (each experiment in triplicate). ( C) Example images of MOPr internalization 30 min after incubation with saturating concentrations of agonists. Dual staining was employed for quantification (membrane receptor in green and internalized receptor in red, colors enhanced uniformly for presentation purposes). ( D) Maximal efficacy values of endomorphin-2, morphine and bilorphin relative to Met-enk for GIRK channel activation, Ser375 phosphorylation, β-arrestin 2 recruitment, and internalization (all normalized to Met-enk; nonnormalized data in ). SI Appendix, Fig. S2
Predicted binding poses of bilorphin (purple) (
A) and endomorphin-2 (orange) ( B), and the positions of the surrounding binding pocket residues (gray) obtained after molecular docking and 1 μs of MD simulations. The salt bridge between the protonated amine of the ligands and Asp147 3.32 is marked as a dashed black line. TM7 has been removed for clarity. ( C) Ligand–residue interaction fingerprints for the bilorphin–MOPr complex (purple) and endomorphin2–MOPr complex (orange). Data are expressed as the percentage of simulation time each residue is within 4.5 Å of the ligand, with points radiating outwards from 0 to 100% in 20% increments. ( D) Principal component analysis was performed on the alpha carbons of the receptor transmembrane domains, before projecting the receptor conformations at each simulation time point onto PC1 and PC2. The bilorphin–MOPr complex is in purple, the endomorphin-2–MOPr complex in orange, and the black point indicates the conformation of the inactive MOPr model to which the peptides were docked.
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