Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary conservation of the 600-million-year-old oxytocin/vasopressin signalling system. We isolated the insect oxytocin/vasopressin orthologue inotocin from the black garden ant (Lasius niger), identified and cloned its cognate receptor and determined its pharmacological properties on the insect and human oxytocin/vasopressin receptors. Subsequently, we identified a functional dichotomy: inotocin activated the insect inotocin and the human vasopressin V1b receptors, but inhibited the human V1aR. Replacement of Arg8 of inotocin by D-Arg8 led to a potent, stable and competitive V1aR-antagonist ([D-Arg8]-inotocin) with a 3,000-fold binding selectivity for the human V1aR over the other three subtypes, OTR, V1bR and V2R. The Arg8/D-Arg8 ligand-pair was further investigated to gain novel insights into the oxytocin/vasopressin peptide-receptor interaction, which led to the identification of key residues of the receptors that are important for ligand functionality and selectivity. These observations could play an important role for development of oxytocin/vasopressin receptor modulators that would enable clear distinction of the physiological and pathological responses of the individual receptor subtypes.

Center for Physiology and Pharmacology Medical University of Vienna Schwarzspanierstrasse 17 1090 Vienna Austria

CUBE Division of Computational Systems Biology Department of Microbiology and Ecosystem Science University of Vienna Althanstrasse 14 1090 Vienna Austria

Department of Drug Design and Pharmacology University of Copenhagen Jagtvej 162 2100 Copenhagen Denmark

Harris Wellbeing Preterm Birth Research Centre Department of Cellular and Molecular Physiology Institute of Translational Medicine University of Liverpool L69 3BX United Kingdom

Institute for Molecular Bioscience The University of Queensland QLD 4072 Brisbane Australia

IST Austria Am Campus 1 3400 Klosterneuburg Austria

Laboratory of Radioisotopes Institute of Organic Chemistry and Biochemistry CAS Flemingovo nám 2 CZ 16610 Prague 6 Czech Republic

School of Biomedical Sciences The University of Queensland QLD 4072 Brisbane Australia

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Lander E. S. et al.. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001). PubMed

Fredriksson R., Lagerstrom M. C., Lundin L. G. & Schioth H. B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272 (2003). PubMed

Pierce K. L., Premont R. T. & Lefkowitz R. J. Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3, 639–650 (2002). PubMed

Rosenbaum D. M., Rasmussen S. G. F. & Kobilka B. K. The structure and function of G-protein-coupled receptors. Nature 459, 356–363 (2009). PubMed PMC

Stevens R. C. et al.. The GPCR Network: a large-scale collaboration to determine human GPCR structure and function. Nat. Rev. Drug Discov. 12, 25–34 (2013). PubMed PMC

Hill S. J. G-protein-coupled receptors: past, present and future. Br. J. Pharmacol. 147 Suppl 1, S27–37 (2006). PubMed PMC

Gruber C. W., Muttenthaler M. & Freissmuth M. Ligand-based peptide design and combinatorial peptide libraries to target G protein-coupled receptors. Curr. Pharm. Des. 16, 3071–3088 (2010). PubMed PMC

Palczewski K. G protein-coupled receptor rhodopsin. Annu. Rev. Biochem. 75, 743–767 (2006). PubMed PMC

Gruber C. W., Koehbach J. & Muttenthaler M. Exploring bioactive peptides from natural sources for oxytocin and vasopressin drug discovery. Future Med. Chem. 4, 1791–1798 (2012). PubMed PMC

Manning M. et al.. Oxytocin and vasopressin agonists and antagonists as research tools and potential therapeutics. J. Neuroendocrinol. 24, 609–628 (2012). PubMed PMC

Landgraf R. Neuropeptides in anxiety modulation. Handb. Exp. Pharmacol. 335–369 (2005). PubMed

Meyer-Lindenberg A., Domes G., Kirsch P. & Heinrichs M. Oxytocin and vasopressin in the human brain: social neuropeptides for translational medicine. Nat. Rev. Neurosci. 12, 524–538 (2011). PubMed

Manning M. & Sawyer W. H. Design, synthesis and some uses of receptor-specific agonists and antagonists of vasopressin and oxytocin. J Recept Res 13, 195–214 (1993). PubMed

Arrowsmith S. & Wray S. Oxytocin: its mechanism of action and receptor signalling in the myometrium. J. Neuroendocrinol. 26, 356–369 (2014). PubMed

Landgraf R. The involvement of the vasopressin system in stress-related disorders. CNS Neurol. Disord. Drug Targets 5, 167–179 (2006). PubMed

Wersinger S. R., Ginns E. I., O’Carroll A. M., Lolait S. J. & Young W. S. 3rd. Vasopressin V1b receptor knockout reduces aggressive behavior in male mice. Mol. Psychiatry 7, 975–984 (2002). PubMed

Alescio-Lautier B. & Soumireu-Mourat B. Role of vasopressin in learning and memory in the hippocampus. Prog. Brain Res. 119, 501–521 (1998). PubMed

Kirsch P. et al.. Oxytocin modulates neural circuitry for social cognition and fear in humans. J. Neurosci. 25, 11489–11493 (2005). PubMed PMC

Gruber C. W. Physiology of invertebrate oxytocin and vasopressin neuropeptides. Exp. Physiol. 99, 55–61 (2014). PubMed PMC

Hoyle C. H. Neuropeptide families and their receptors: evolutionary perspectives. Brain Res. 848, 1–25 (1999). PubMed

Gruber C. W. & Muttenthaler M. Discovery of defense- and neuropeptides in social ants by genome-mining. PLoS One 7, e32559 (2012). PubMed PMC

Proux J. P. et al.. Identification of an arginine vasopressin-like diuretic hormone from Locusta migratoria. Biochem. Biophys. Res. Commun. 149, 180–186 (1987). PubMed

Barberis C., Mouillac B. & Durroux T. Structural bases of vasopressin/oxytocin receptor function. J. Endocrinol. 156, 223–229 (1998). PubMed

Stafflinger E. et al.. Cloning and identification of an oxytocin/vasopressin-like receptor and its ligand from insects. Proc. Natl. Acad. Sci. USA. 105, 3262–3267 (2008). PubMed PMC

Chini B. et al.. Identification of a single residue responsible for agonist selectivity in the oxytocin-vasopressin receptors. Ann. N. Y. Acad. Sci. 812, 218–221 (1997). PubMed

Slusarz M. J., Gieldon A., Slusarz R. & Ciarkowski J. Analysis of interactions responsible for vasopressin binding to human neurohypophyseal hormone receptors-molecular dynamics study of the activated receptor-vasopressin-G(alpha) systems. J. Pept. Sci. 12, 180–189 (2006). PubMed

Rodrigo J. et al.. Mapping the binding site of arginine vasopressin to V1a and V1b vasopressin receptors. Mol. Endocrinol. 21, 512–523 (2007). PubMed

Hruby V. J., Chow M. S. & Smith D. D. Conformational and structural considerations in oxytocin-receptor binding and biological activity. Annu. Rev. Pharmacol. Toxicol. 30, 501–534 (1990). PubMed

Muttenthaler M. et al.. Modulating oxytocin activity and plasma stability by disulfide bond engineering. J. Med. Chem. 53, 8585–8596 (2010). PubMed

Fjellestad-Paulsen A., Soderberg-Ahlm C. & Lundin S. Metabolism of vasopressin, oxytocin, and their analogues in the human gastrointestinal tract. Peptides 16, 1141–1147 (1995). PubMed

Manglik A. et al.. Crystal structure of the micro-opioid receptor bound to a morphinan antagonist. Nature 485, 321–326 (2012). PubMed PMC

Yin J., Mobarec J. C., Kolb P. & Rosenbaum D. M. Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant. Nature 519, 247–250 (2015). PubMed

Isberg V. et al.. Generic GPCR residue numbers - aligning topology maps while minding the gaps. Trends Pharmacol. Sci. 36, 22–31 (2015). PubMed PMC

Agerso H. et al.. Pharmacokinetics and renal excretion of desmopressin after intravenous administration to healthy subjects and renally impaired patients. Br. J. Clin. Pharmacol. 58, 352–358 (2004). PubMed PMC

Koehbach J. et al.. Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design. Proc. Natl. Acad. Sci. USA. 110, 21183–21188 (2013). PubMed PMC

Liutkevičiūtė Z. & Gruber C. W. In Molecular Neuroendocrinology: From Genome to Physiology INF Masterclass in Neuroendocrinology (eds Murphy D. & Gainer H.) Ch. 1, 3–23 (John Wiley & Sons, Ltd, 2016).

Chini B. et al.. Tyr115 is the key residue for determining agonist selectivity in the V1a vasopressin receptor. EMBO J. 14, 2176–2182 (1995). PubMed PMC

Kenakin T. Analytical pharmacology and allosterism: the importance of quantifying drug parameters in drug discovery. Drug Discov Today Technol 10, e229–235 (2013). PubMed

Davie B. J., Christopoulos A. & Scammells P. J. Development of M1 mAChR allosteric and bitopic ligands: prospective therapeutics for the treatment of cognitive deficits. ACS Chem Neurosci 4, 1026–1048 (2013). PubMed PMC

Jakubik J., Bacakova L., Lisa V., el-Fakahany E. E. & Tucek S. Activation of muscarinic acetylcholine receptors via their allosteric binding sites. Proc Natl Acad Sci USA 93, 8705–8709 (1996). PubMed PMC

Satsu H. et al.. A sphingosine 1-phosphate receptor 2 selective allosteric agonist. Bioorg. Med. Chem. 21, 5373–5382 (2013). PubMed PMC

Riemer R. K. & Heymann M. A. Regulation of uterine smooth muscle function during gestation. Pediatr Res 44, 615–627 (1998). PubMed

Yuan W. & Lopez Bernal A. Cyclic AMP signalling pathways in the regulation of uterine relaxation. BMC Pregnancy Childbirth 7 Suppl 1, S10 (2007). PubMed PMC

Conner M. et al.. Systematic analysis of the entire second extracellular loop of the V(1a) vasopressin receptor: key residues, conserved throughout a G-protein-coupled receptor family, identified. J. Biol. Chem. 282, 17405–17412 (2007). PubMed

Huang W. et al.. Structural insights into micro-opioid receptor activation. Nature 524, 315–321 (2015). PubMed PMC

Isogai S. et al.. Backbone NMR reveals allosteric signal transduction networks in the beta1-adrenergic receptor. Nature 530, 237–241 (2016). PubMed

Chini B. & Manning M. Agonist selectivity in the oxytocin/vasopressin receptor family: new insights and challenges. Biochem. Soc. Trans. 35, 737–741 (2007). PubMed

Manning M. et al.. Peptide and non-peptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and OT receptors: research tools and potential therapeutic agents. Prog. Brain Res. 170, 473–512 (2008). PubMed

Manning M. et al.. Carboxy terminus of vasopressin required for activity but not binding. Nature 308, 652–653 (1984). PubMed

Schmieder R. & Edwards R. Quality control and preprocessing of metagenomic datasets. Bioinformatics 27, 863–864 (2011). PubMed PMC

Kopylova E., Noe L. & Touzet H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211–3217 (2012). PubMed

Grabherr M. G. et al.. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011). PubMed PMC

Parra G., Bradnam K. & Korf I. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 23, 1061–1067 (2007). PubMed

Altschul S. F., Gish W., Miller W., Myers E. W. & Lipman D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990). PubMed

Larkin M. A. et al.. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007). PubMed

Katoh K. & Standley D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013). PubMed PMC

Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014). PubMed PMC

Huson D. H. & Scornavacca C. Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks. Syst. Biol. 61, 1061–1067 (2012). PubMed

Park Y. et al.. Analysis of transcriptome data in the red flour beetle, Tribolium castaneum. Insect Biochem. Mol. Biol. 38, 380–386 (2008). PubMed PMC

Li B. et al.. Genomics, transcriptomics, and peptidomics of neuropeptides and protein hormones in the red flour beetle Tribolium castaneum. Genome Res. 18, 113–122 (2008). PubMed PMC

Schnolzer M., Alewood P., Jones A., Alewood D. & Kent S. B. In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. Int. J. Pept. Protein Res. 40, 180–193 (1992). PubMed

Muttenthaler M. et al.. Solving the alpha-conotoxin folding problem: efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antagonists. J. Am. Chem. Soc. 132, 3514–3522 (2010). PubMed

Muttenthaler M., Albericio F. & Dawson P. E. Methods, setup and safe handling for anhydrous hydrogen fluoride cleavage in Boc solid-phase peptide synthesis. Nat. Protoc. 10, 1067–1083 (2015). PubMed

Sarantakis D., Teichman J., Lien E. L. & Fenichel R. L. A novel cyclic undecapeptide, WY-40, 770, with prolonged growth hormone release inhibiting activity. Biochem. Biophys. Res. Commun. 73, 336–342 (1976). PubMed

Cheng Y. & Prusoff W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973). PubMed

Isberg V. et al.. GPCRdb: an information system for G protein-coupled receptors. Nucleic Acids Res. 44, D356–364 (2016). PubMed PMC

Bernstein F. C. et al.. The Protein Data Bank. A computer-based archival file for macromolecular structures. Eur. J. Biochem. 80, 319–324 (1977). PubMed

Magrane M. & Consortium U. UniProt Knowledgebase: a hub of integrated protein data. Database (Oxford) 2011, bar009 (2011). PubMed PMC

Webb B. & Sali A. Comparative Protein Structure Modeling Using MODELLER. Curr Protoc Bioinformatics 47, 561–5632 (2014). PubMed

Wang C. K. et al.. Molecular grafting onto a stable framework yields novel cyclic peptides for the treatment of multiple sclerosis. ACS Chem. Biol. 9, 156–163 (2014). PubMed PMC

Luckas M. J. & Wray S. A comparison of the contractile properties of human myometrium obtained from the upper and lower uterine segments. BJOG 107, 1309–1311 (2000). PubMed

Livingstone C. D. & Barton G. J. Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation. Comput. Appl. Biosci. 9, 745–756 (1993). PubMed