Significance Agonists of the μ-opioid receptor (MOPr) are currently the gold standard for pain treatment. However, their therapeutic usage is greatly limited by side effects including respiratory depression, constipation, tolerance, and dependence. Functionally selective MOPr agonists that mediate their effects preferentially through G proteins rather than β-arrestin signaling are believed to produce fewer side effects. Here, we present the discovery of 3 unusual tetrapeptides with a unique stereochemical arrangement of hydrophobic amino acids from an Australian estuarine isolate of Penicillium species. Building on these natural templates we developed bilorphin, a potent and selective highly G protein-biased agonist of the MOPr. Further, through the addition of a simple sugar moiety, we generated bilactorphin that is an effective analgesic in vivo.

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 (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.

Developing ligands that target G protein-coupled receptors (GPCRs) in multiple functional states has attracted great interest, particularly with increasing knowledge of GPCR structure (1⇓–3). These novel ligands are expected to underpin the development of agonists with superior pharmaceutically relevant properties, including biased receptor signaling (4, 5), whereby one downstream signaling pathway is favored over another. For example, biased agonists that signal by differentially recruiting G proteins over β-arrestin to the μ-opioid receptor (MOPr) could deliver better analgesics, based on the view that down-regulating β-arrestin recruitment diminishes adverse side effects (4, 6⇓–8). Exploiting this concept, the G protein-biased MOPr agonist oliceridine (TRV130) is a potent analgesic in rodents, with lower respiratory depression and gastrointestinal dysfunction compared to morphine (7). Indeed, human clinical trials of oliceridine show reduced respiratory impairment compared to morphine administered at equi-analgesic doses (9). Reduced respiratory depression delivers improved safety, potentially reducing the burden of opioid overdoses, now at epidemic proportions in many jurisdictions (10).

Bioactive peptides display great promise for their novel pharmacological properties (11). Since the discovery of the relatively nonselective mammalian opioid peptides, the enkephalins, other endogenous mammalian ligands, including the tetrapeptide endomorphins that target the MOPr with high selectivity over the related κ-opioid (KOPr) and δ-opioid (DOPr) subtypes, have been found (12, 13). Natural peptide agonists containing a d-Ala in the second position formed by a posttranslational modification isolated from frog skin, dermorphin and deltorphin II, selectively target MOPr and DOPr, respectively (14). Similar synthetic modifications have yielded enhanced biological stability and receptor selectivity. For example, introduction of d-Ala stabilizes the enkephalins to proteolysis, and further substitutions yield highly stable, selective MOPr agonists such as DAMGO ([d-Ala2, N-MePhe4, Gly5-ol]-enkephalin) (15). However, to our knowledge, all endogenous opioid peptides acting on MOPr robustly recruit arrestins and produce MOPr internalization (16⇓⇓–19). Here, we report the discovery of 3 tetrapeptides, bilaids A–C (Fig. 1, [1a-3a]), from an Australian estuarine isolate of Penicillium sp. MST-MF667 [initially reported as P. bilaii (20); maximum likelihood tree presented in SI Appendix, Fig. S1]. Discovery of the bilaids, which resemble known opioid peptides but featuring an unusual alternating sequence of antipodal amino acids (LDLD), led to our design of bilorphin (3c), a MOPr agonist, with G protein signaling bias similar to oliceridine. Bilorphin adopts a distinct conformational shape and intermolecular interactions in molecular dynamics (MD) simulations of the bilorphin–MOPr complex, consistent with predicted G protein bias at related GPCRs. Together with its in vitro and in vivo activity, we demonstrate that bilorphin provides a scaffold for the development of stable, orally active opioid peptides that are biased toward G protein signaling (3g).

Fig. 1. Structures of bilaids, bilorphin, and bilactorphin.

Discussion Nature has inspired many of the most well-known and widely used analgesics, from natural salicin in willow (Salix) bark to synthetic aspirin, from opioid poppy alkaloids such as morphine and codeine to synthetic hydrocodone (Vicodin), oxycodone (OxyContin), and buprenorphine (Subutex). Notwithstanding their value in alleviating pain, serious adverse side effects, combined with the challenge of addiction, abuse, and acquired tolerance, render these analgesics (particularly opioids) far from ideal. There is an urgent need to discover and develop new, safer and more efficacious analgesics, with mechanisms of actions that mitigate against risk. We therefore investigated the analgesic potential of a class of tetrapeptides, the bilaids (1a–3a), isolated from a Penicillium fungus. Taking advantage of an unprecedented natural scaffold comprising alternating LDLD configuration amino acids, which imparts inherent biostability, we designed a peptide-based G protein-biased MOPr agonist, bilorphin (3c). Furthermore, we assembled proof-of-concept data that this pharmacophore can be optimized to yield an orally active MOPr agonist analgesic, bilactorphin (3g). G protein-biased opioid agonists have been proposed as a route to improving therapeutic profile (4, 7, 8). Among known peptide opioid agonists, which typically are biased toward β-arrestin signaling relative to morphine (19, 34), the pharmacological profile of bilorphin is most unusual, although a synthetic opioid cyclopeptide with G protein bias was recently reported (44). Bilorphin enjoys an opioid signaling bias comparable to oliceridine, a G protein-biased drug candidate in phase III clinical trials. Glycosylation of bilorphin produced an analog active in vivo via s.c. and oral administration, validating the bilorphin tetrapeptide backbone as a platform for further development of druggable signaling-biased opioid agonists. Preclinical development of other G protein-biased agonists shows a favorable profile with reduced respiratory depression and constipation. The first such compound to reach clinical trials, oliceridine, was reported to have an increased therapeutic window between antinociceptive and respiratory depressive activity (7) and appears to be safer in humans than morphine for equi-analgesic doses (9). Similarly, a series of substituted fentanyl analogs was observed to produce an increased therapeutic window for respiratory depression in mice, correlating with increased G protein versus β-arrestin 2 recruitment (8). To investigate whether bias could be explained by the differential interaction of bilorphin and endomorphin-2 with MOPr, or by distinct receptor conformational changes initiated by each, we undertook MD simulations with bilorphin and compared this to the arrestin-biased opioid, endomorphin-2, bound to MOPr. Both peptides were docked to the orthosteric binding site of MOPr and displayed differences in ligand–residue interactions, which may translate to their differing bias profiles. Notably, endomorphin-2 transiently interacted with residues in ECL1 and ECL2, including the conserved residue Leu219, proposed to be important for arrestin-bias and ligand residence time at the 5-HT 2A and 5-HT 2B receptors and other aminergic GPCRs (45, 46). The cryo-electron microscopy–resolved structure of the DAMGO–MOPr–G i complex also showed DAMGO, which robustly recruits arrestin, interacting with the receptor extracellular loops (41). In contrast, bilorphin did not contact the extracellular loops and instead interacted with TM1. Intriguingly, the extracellular end of TM1 has also been identified as part of the binding pocket for the G protein-biased GLP-1 agonist, ExP5 (47) and, in addition, has been implicated in the allosteric communication between the binding site and intracellular domain for oliceridine at the MOPr (48). Moreover, the interactions between the peptides and the MOPr binding pocket appear to translate to the divergent conformational changes observed by principal component analysis, resulting in the MOPr adopting a distinct conformation with bilorphin bound compared to endomorphin-2. Specifically, with bound endomorphin-2, the extracellular portions of the transmembrane domains moved inwards so that the orthosteric binding pocket contracted relative to the bilorphin-bound MOPr. On the intracellular side of the receptor TM5, 6, and 7 adopted distinct positions depending on the bound peptide, mainly an inward shift of these helices in the presence of endormorphin-2, resulting in a more occluded intracellular cavity for this arrestin-biased ligand. Of interest, this is in line with the proposed binding pocket for arrestins at the base of the GPCR being slightly smaller than those for G proteins (49, 50). While it remains challenging at present to associate ligand-induced GPCR conformations with differential coupling to G proteins or arrestins, particularly in the absence of a large structurally diverse panel of biased MOPr agonists, the subtle differences in ligand–residue interactions and conformations of the MOPr helices that we have modeled here may represent the initial changes induced by the oppositely biased peptides, bilorphin and endomorphin-2. These different interactions and MOPr conformations may well lead to the different signaling profiles reported for these biased peptide agonists at MOPr. It remains uncertain, however, whether G protein bias per se is the sole property contributing to the improved safety of new opioid drugs such as oliceridine (9). Using receptor knockdown, we have shown here that oliceridine has very low G protein efficacy compared with morphine, similar to findings using receptor depletion with a cAMP assay system (37). Similar low efficacy results have recently been reported for another opioid, PZM21, also claimed to be safer than morphine (4, 51). Furthermore, it is difficult to evaluate maximal G protein efficacy of novel biased agonists in other studies because assays were insensitive to the relatively low G protein efficacy of morphine (7, 8). Very low G protein efficacy may indeed be a confounding factor in the preclinical and clinical studies of side effect profile, given that other agonists with very low G protein efficacy such as buprenorphine are not strongly G protein biased (19) but are well characterized to produce less respiratory depression, and overdose death than highly efficacious agonists such as morphine and methadone (52). Because bilorphin is strongly G protein biased and has nearly equivalent maximal G protein efficacy to morphine, further development of BBB penetrant analogs that can release the parent molecule will facilitate direct test of the influence of bias without being confounded by differing G protein efficacy. Finally, we have elucidated an analgesic pharmacophore based on the discovery of the bilaids and related bilorphin and bilactorphin motifs. Unusually, they derive from a microbial source, an untapped resource for analgesics and worthy of further investigation. This observation suggests that microbes may be an untapped resource for new analgesics, deserving of further investigation. Experimental Procedures. Full details on the materials and methods used are available in SI Appendix. Solvent extracts solid phase cultivations of MST-MF667 were subjected to solvent partition followed by reversed-phase HPLC, and chemical structures were identified on the basis of detailed spectroscopic analysis, chemical derivatization and degradation, Marfey’s analysis and total synthesis. All peptides were assembled manually by stepwise solid-phase peptide synthesis. MOPr activity was initially screened using competition opioid radioligand binding to membranes from cultured cells expressing hMOPr, hDOPr, or hKOPr receptors, then agonist activity screened at hMOPr using inhibition of forskolin-stimmulated cAMP formation. Agonist activation of MOPr-coupled GIRK channels was then quantified using superfusion onto LC neurons in rate brain slices using whole-cell patch clamp recording. Signaling pathway analysis was quantified in AtT20 cells stably expressing mMOPr using perforated patch recording for GIRK channels activation (G protein signal), fluorescence immunohistochemistry for Ser375 phosphorylation and MOPr internalization and arrestin recruitment with a BRET-based approach. Molecular docking was performed using the Bristol University Docking Engine (53). The selected peptide–MOPr complexes were then embedded in a lipid and cholesterol bilayer and used in all-atom MD simulations. Multiple simulations (8 × 125 ns), with different initial velocities, were performed under the Amber ff14SB and Lipid14 forcefields, to yield a total of 1 μs of trajectory data for each peptide. Behavioral assays of analgesia were performed using the hotplate latency assay in mice. Statistical analyses were performed as described in SI Appendix. All values are expressed as means ± SEM, except where noted otherwise (SI Appendix, Fig. S2).

Acknowledgments This work was supported by Program Grant APP1072113 from the National Health and Medical Research Council of Australia (to P.F.A. and M.J.C.).

Footnotes Author contributions: Z.D., A.Y., A.G., C.M., A.M.W., S.A.M., M.C., R.B.S., E.K., R.J.C., P.F.A., and M.J.C. designed research; Z.D., S.S., A.Y., K.J.S., A.G., C.M., P.S., A.H.J., A.M.W., S.A.M., M.S., R.R., F.F., E.L., A.M.P., and Y.P.D. performed research; E.L. contributed new reagents/analytic tools; Z.D., S.S., A.Y., K.J.S., A.G., C.M., P.S., A.H.J., A.M.W., S.A.M., M.S., R.R., F.F., A.M.P., Y.P.D., M.C., R.B.S., E.K., R.J.C., P.F.A., and M.J.C. analyzed data; and Z.D., S.S., A.Y., K.J.S., A.G., S.A.M., M.C., R.B.S., E.K., R.J.C., P.F.A., and M.J.C. wrote the paper.

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.

This article is a PNAS Direct Submission.

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