A tetrapeptide class of biased analgesics from an Australian fungus targets the μ-opioid receptor Zoltan Dekana,1, Setareh Sianatib,1, Arsalan Yousufb,1, Katy J. Sutcliffec,1, Alexander Gillisb, Christophe Malletb,2, Paramjit Singha, Aihua H. Jina, Anna M. Wangb, Sarasa A. Mohammadib, Michael Stewarta, Ranjala Ratnayakea, Frank Fontainea, Ernest Laceyd, Andrew M. Piggotta, Yan P. Dub, Meritxell Canalse,3, Richard B. Sessionsc, Eamonn Kellyc, Robert J. Capona,4, Paul F. Alewooda,4, and MacDonald J. Christieb,4 aInstitute for Molecular Bioscience, The University of Queensland, 4072 Brisbane, Australia; bDiscipline of Pharmacology, School of Medical Sciences, University of Sydney, NSW 2006, Australia; cSchools of Physiology, Pharmacology and Neuroscience, and Biochemistry, Biomedical Sciences Building, University of Bristol, BS8 1TD Bristol, United Kingdom; dMicrobial Screening Technologies Pty. Ltd., Smithfield, NSW, 2164, Australia; and eDrug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, 3052 Parkville, Australia Edited by Robert J. Lefkowitz, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, and approved September 18, 2019 (received for review May 22, 2019) An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded in the second position formed by a posttranslational modification 3 tetrapeptides named the bilaids with an unusual alternating LDLD isolated from frog skin, dermorphin and deltorphin II, selectively chirality. Given their resemblance to known short peptide opioid target MOPr and DOPr, respectively (14). Similar synthetic modi- K agonists, we elucidated that they were weak ( i low micromolar) fications have yielded enhanced biological stability and receptor μ-opioid agonists, which led to the design of bilorphin, a potent and selectivity. For example, introduction of D-Ala stabilizes the en- μ K selective -opioid receptor (MOPr) agonist ( i 1.1 nM). In sharp con- kephalins to proteolysis, and further substitutions yield highly trast to all-natural product opioid peptides that efficaciously recruit stable, selective MOPr agonists such as DAMGO ([D-Ala2, N- β-arrestin, bilorphin is G protein biased, weakly phosphorylating MePhe4, Gly5-ol]-enkephalin) (15). However, to our knowledge, all the MOPr and marginally recruiting β-arrestin, with no receptor endogenous opioid peptides acting on MOPr robustly recruit internalization. Importantly, bilorphin exhibits a similar G protein arrestins and produce MOPr internalization (16–19). Here, we re- bias to oliceridine, a small nonpeptide with improved overdose port the discovery of 3 tetrapeptides, bilaids A–C(Fig.1,[1a-3a]), PHARMACOLOGY safety. Molecular dynamics simulations of bilorphin and the from an Australian estuarine isolate of Penicillium sp. MST-MF667 strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that Significance could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is μ orally active with similar in vivo potency to morphine. Bilorphin Agonists of the -opioid receptor (MOPr) are currently the gold is both a unique molecular tool that enhances understanding of standard for pain treatment. However, their therapeutic usage is MOPr biased signaling and a promising lead in the development of greatly limited by side effects including respiratory depression, next generation analgesics. constipation, tolerance, and dependence. Functionally selective MOPr agonists that mediate their effects preferentially through β biased agonist | μ-opioid receptor | peptide drug | opioid analgesic | G proteins rather than -arrestin signaling are believed to pro- glycosylation duce fewer side effects. Here, we present the discovery of 3 unusual tetrapeptides with a unique stereochemical arrange- ment of hydrophobic amino acids from an Australian estuarine eveloping ligands that target G protein-coupled receptors isolate of Penicillium species. Building on these natural tem- D(GPCRs) in multiple functional states has attracted great plates we developed bilorphin, a potent and selective highly G interest, particularly with increasing knowledge of GPCR struc- – protein-biased agonist of the MOPr. Further, through the addi- ture (1 3). These novel ligands are expected to underpin the tion of a simple sugar moiety, we generated bilactorphin that is development of agonists with superior pharmaceutically relevant an effective analgesic in vivo. properties, including biased receptor signaling (4, 5), whereby one downstream signaling pathway is favored over another. For 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., example, biased agonists that signal by differentially recruiting G 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., proteins over β-arrestin to the μ-opioid receptor (MOPr) could 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., deliver better analgesics, based on the view that down-regulating 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., β-arrestin recruitment diminishes adverse side effects (4, 6–8). 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. Exploiting this concept, the G protein-biased MOPr agonist Competing interest statement: Patent Application(s) corresponding to Australian Patent oliceridine (TRV130) is a potent analgesic in rodents, with lower Application 2018901944 The University of Sydney and The University of Queensland has respiratory depression and gastrointestinal dysfunction compared been filed concerning this peptide. Title: Analgesics and Methods of Use Thereof. to morphine (7). Indeed, human clinical trials of oliceridine show This article is a PNAS Direct Submission. reduced respiratory impairment compared to morphine adminis- This open access article is distributed under Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND). tered at equi-analgesic doses (9). Reduced respiratory depression 1 delivers improved safety, potentially reducing the burden of opioid Z.D., S.S., A.Y., and K.J.S. contributed equally to this work. 2 overdoses, now at epidemic proportions in many jurisdictions (10). Present address: Neuro-Dol Basics & Clinical Pharmacology of Pain, INSERM, Université Clermont Auvergne, F-63000 Clermont-Ferrand, France. Bioactive peptides display great promise for their novel phar- 3Present address: Centre of Membrane Proteins and Receptors, University of Nottingham, macological properties (11). Since the discovery of the relatively NG7 2UH Nottingham, United Kingdom. nonselective mammalian opioid peptides, the enkephalins, other 4To whom correspondence may be addressed. Email: [email protected], endogenous mammalian ligands, including the tetrapeptide [email protected], or [email protected]. endomorphins that target the MOPr with high selectivity over This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the related κ-opioid (KOPr) and δ-opioid (DOPr) subtypes, have 1073/pnas.1908662116/-/DCSupplemental. been found (12, 13). Natural peptide agonists containing a D-Ala www.pnas.org/cgi/doi/10.1073/pnas.1908662116 PNAS Latest Articles | 1of6 Downloaded by guest on September 29, 2021 [initially reported as P. bilaii (20); maximum likelihood tree pre- A B sented in SI Appendix,Fig.S1]. Discovery of the bilaids, which re- YvVf-OH (3a) met-enk bilorphin YvYf-NH2 (3b) CTAP semble known opioid peptides but featuring an unusual alternating 100 [Dmt]-vVf-NH2 (3c, MOPr) sequence of antipodal amino acids (LDLD), led to our design of [Dmt]-vVf-NH2 (DOPr) [Dmt]-vVf-NH2 (KOPr) bilorphin (3c), a MOPr agonist, with G protein signaling bias similar 75 to oliceridine. Bilorphin adopts a distinct conformational shape and C 50 intermolecular interactions in molecular dynamics (MD) simula- (nS) – GIRK tions of the bilorphin MOPr complex, consistent with predicted G 25 G Specific binding (%) protein bias at related GPCRs. Together with its in vitro and in vivo 0 1 μM 10030 300 1010 activity, we demonstrate that bilorphin provides a scaffold for the -10-9-8-7-6-5-4 SST bilorphin morph Met-enk development of stable, orally active opioid peptides that are biased log [agonist] (M) (nM) (μM) (μM) toward G protein signaling (3g). D E 125 100 Results 100 75 To date, the major opioid peptide families have only been de- tected in vertebrates (21). Through a broader screening program, 75 50 we now report the discovery of 3 closely related tetrapeptides % of 1μM SST) GIRK current 50 (Fig. 1), bilaids A (1a, FvVf-OH), B (2a, FvVy-OH), and C (3a, ( (% of 1 μM Met-enk ) GIRK 25 25 YvVf-OH), from the Australian estuarine-derived Penicillium sp. G MST-MF667. Solvent extracts of cultivations of MST-MF667 0 0 -8 -7 -6 -5 -9 -8 -7 -6 -5 were subjected to solvent partition followed by reversed-phase log [agonist] (M) log [agonist] (M) high-performance liquid chromatography (HPLC) to yield 1a met-enk (0.15%), as well as traces of 2a (0.0018%) and 3a (0.0008%) morphine endo-2 bilorphin oliceridine (Fig. 1). The chemical structures for 1a–3a, which resemble Fig. 2. (A) Competition for binding of [3H]DAMGO to hMOPr (human opioid peptides albeit with a unique, alternating LDLD amino recombinant MOPr) by the native bilaid YvVf-OH (3a), YvVf-NH2 (3b)and 3 acid configuration, were identified by spectroscopic analyses, bilorphin ([Dmt]vVf-NH2)(3c), as well as bilorphin to hDOPr ([ H]DADLE 3 chemical derivatization and degradation, and Marfey’sanalysis binding to human recombinant DOPr) and hKOPr ([ H] U69593 binding to and total synthesis. In addition to synthesizing 1a–3a,wepre- human recombinant KOPr).