Markus Muttenthaler

University of Vienna

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Paul F. Alewood

University of Queensland

Christian W. Gruber

Medical University of Vienna

Richard J. Lewis

University of Queensland

Nayara Braga Emídio

National Institute of Diabetes and Digestive and Kidney Diseases

David J. Craik

University of Queensland

Sarah Arrowsmith

University of Liverpool

Philip E. Dawson

Scripps Research Institute

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Åsa Andersson

University of Queensland

Athanasios Makristathis

Medical University of Vienna

Christoph Gasché

Medical University of Vienna

Cooperative Institutions

University of Queensland

Medical University of Vienna

University of Vienna

Australian Research Council

Brisbane School of Theology

Research Institute for Bioscience and Biotechnology

Centre National de la Recherche Scientifique

ARC Centre of Excellence for Innovations in Peptide and Protein Science

United States Naval Research Laboratory

University of Washington

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Cyclotides as templates for peptide GPCR ligand design - discovery of the target receptor of the oxytocic plant peptide kalata B7

Johannes Koehbach Margaret O’Brien Markus Muttenthaler Marion Miazzo Muharrem Akcan

Publication Information Koehbach, J,O'Brien, M,Muttenthaler, M,Miazzo, M,Akcan, M,Elliott, AG,Daly, NL,Harvey, PJ,Arrowsmith, S,Gunasekera, S,Smith, TJ,Wray, S,Goransson, U,Dawson, PE,Craik, DJ,Freissmuth, M,Gruber, CW (2013) 'Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design'. Proceedings Of The National Academy Of Sciences Of The United States Of America, 110 (52):21183-21188.

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10.2210/pdb2m9o/pdb

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Neuropeptide signalling systems – An underexplored target for venom drug discovery

Biochemical Pharmacology (2020)

Helen C. Mendel Quentin Kaas Markus Muttenthaler

Signalling

10.1016/j.bcp.2020.114129

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Peptide engineering - controlling the folding of disulfide-rich peptides

Markus Muttenthaler

Disulfide bonds play a decisive role in the folding and stability in most classes of extra-cellular bioactive peptides including hormones, neurotransmitters, growth factors, enzyme inhibitors, antimicrobial peptides and venom toxins. Controlling the folding of disulfide-rich peptides is not trivial and often determines the rate and the success of their synthesis. This work introduces an alternative strategy to direct disulfide formation by substituting cysteine by the nearly isosteric selenocysteine. It describes a novel methodology that allows efficient access to conopeptide libraries via an on-resin oxidation approach and furthermore analyzes disulfide bridge mimetics of increased stability of oxytocin, involving new synthetic routes and revealing an interesting functional selectivity switch for the human oxytocin receptor. Chapter 1 & 2 serve as an overall introduction with the main focus on the chemical aspects of the 21st proteinogenic amino acid selenocysteine in peptides and proteins. It describes the physicochemical properties of selenium/sulfur and selenocysteine/cysteine based on comprehensive structural (X-ray, NMR, CD) and biological data, and illustrates why selenocysteine is considered the most conservative substitution for cysteine. The synthetic methods on selenocysteine incorporation into peptides and proteins were reviewed, including an overview of the selenocysteine building block syntheses for Boc- and Fmoc- solid phase peptide synthesis. Selenocysteine-mediated reactions are addressed such as native chemical ligation and dehydroalanine formation towards peptide conjugation. Selenopeptides have very interesting and distinct properties, which led to a diverse range of applications such as structural, functional and mechanistic probes, robust scaffolds, enzyme engineering, peptide conjugation and folding tools, all discussed in this review. Chapter 3 contains the core work of the thesis and describes how selenocysteine was used to control regioselective folding of an important class of disulfide -rich peptides, the α−conotoxins. Driven by the physicochemical differences of cysteine and selenocysteine such as lower pKa, lower redox potential and higher nucleophilicity, it was possible to direct the peptides into desired folds by replacing pairs of cysteine residues with isosteric selenocysteine residues. Selective access to the globular and ribbon isoforms of α-AuIB, α-MI, α-[A10L]-PnIA, α-RgIA and α-ImI was achieved through oxidative folding in situ in less than ten minutes. CD and NMR analysis confirmed the isosteric character of the α-selenoconotoxins and comparison of the crystal structures of α-PnIA and Sec[2,4]-[A10L]-PnIA (1.4 A) showed an increase of bond length of only 0.3 A and Se-Se torsion angles within 4-6o of the corresponding Cys analogue. The α-selenoconotoxins exhibited comparable or more potent inhibition of acetylcholine-evoked currents mediated by nAChR subtypes (α3β2, α7, α1β1δγ) expressed in Xenopus oocytes and on rat hemi-diaphragm contraction assays. Studies in rat plasma and in equimolar glutathione solutions showed increased stability to scrambling of the selenocysteine isoforms. Chapter 4 describes the development of resin-supported chemistry using Boc-SPPS that leads directly to correctly folded α-conotoxins on resin. Drug lead optimization, high-throughput screening and structure-activity relationship studies often require easy synthetic access to a large number of analogues. This has been hampered by tedious and time-consuming folding and purification processes. The use of a safety-catch acid labile linker on a water compatible, amphiphilic polyethyleneglycol support (ChemMatrix) allows side-chain deprotection and subsequent oxidation on-resin in aqueous phosphate buffer solution at pH 8.4. A selection of small to medium sized peptides, including the α-conotoxins MI, Vc1.1 and PnIA, were synthesized and oxidized on resin. The solid support had an effect on the isomer formation of α-MI and α-Vc1.1, yielding all three possible isomers. Control over the isomer formation in Sec[3,8]-MI was obtained by replacing a pair of cysteine residues with a pair of isosteric selenocysteine residues, showing that seleno-chemistry is compatible with on-resin chemistry. Peptide cleavage was achieved by reductive acidolysis and peptides were obtained in >90% purity either by individual cleavage and RP-HPLC purification using labeled solvent-permeable resin bags, or by an efficient ‘one-pot’ cleavage and RP-HPLC-purification step. Chapter 5 investigates structure-activity relationships of more stable disulfide bond mimetics of oxytocin (lanthionine, cystathionine and selenium analogues) for the human oxytocin and vasopressin V1a receptor. A new on-resin thioether formation and two novel selenocysteine building blocks (Nα−tert-butyloxycarbonyl-acetamidomethyl-L-seleno-cysteine (Boc-L-Sec(Acm)-OH) and Nα−tert-butyloxycarbonyl-4-nitrobenzyl-L-selenocysteine (Boc-L-Sec(pNB)-OH)) were developed for analogue synthesis. The cystathionine and selenylsulfur analogues retained full binding and functional activity, while the lanthionine analogues surprisingly abolished activity. Metabolic stability in rat and human plasma was improved over oxytocin (12h) in the selenium analogues (20-25h), and in the all-D-, N-to C-terminal-cyclized- and all-D-retroinverse-oxytocin (> 48h). Substitution of the C-terminal carboxylic amide to a carboxylic acid in the selenium analogue [C1,6U]-Oxytocin-OH revealed an interesting 2600-fold functional selectivity switch for the human oxytocin receptor. The chemistry introduced in this study facilitates access to analogues with enhanced metabolic stability that retained full activity and has the potential to be applied to other important classes of disulfide-rich peptides.

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The Tumor Suppressor TFF1 Occurs in Different Forms and Interacts with Multiple Partners in the Human Gastric Mucus Barrier: Indications for Diverse Protective Functions

International Journal of Molecular Sciences (2020)

Jörn Heuer Franziska Heuer René Stürmer Sönke Harder Hartmut Schlüter

TFF1 is a protective peptide of the Trefoil Factor Family (TFF), which is co-secreted with the mucin MUC5AC, gastrokine 2 (GKN2), and IgG Fc binding protein (FCGBP) from gastric surface mucous cells. Tff1-deficient mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas, indicating that Tff1 is a tumor suppressor. As a hallmark, TFF1 contains seven cysteine residues with three disulfide bonds stabilizing the conserved TFF domain. Here, we systematically investigated the molecular forms of TFF1 in the human gastric mucosa. TFF1 mainly occurs in an unusual monomeric form, but also as a homodimer. Furthermore, minor amounts of TFF1 form heterodimers with GKN2, FCGBP, and an unknown partner protein, respectively. TFF1 also binds to the mucin MUC6 in vitro, as shown by overlay assays with synthetic 125I-labeled TFF1 homodimer. The dominant presence of a monomeric form with a free thiol group at Cys-58 is in agreement with previous studies in Xenopus laevis and mouse. Cys-58 is likely highly reactive due to flanking acid residues (PPEEEC58EF) and might act as a scavenger for extracellular reactive oxygen/nitrogen species protecting the gastric mucosa from damage by oxidative stress, e.g., H2O2 generated by dual oxidase (DUOX).

10.3390/ijms21072508

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The Structural Integrity of Disulfide Bonds in Tff1 Under Reducing Conditions

Dilsah Nur Elmaci Gene Hopping Werner Hoffmann Markus Muttenthaler Matthias Stein

Structural integrity

10.2139/ssrn.4878122

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Sequences, gene- and peptide structures of ant inotocins.

Gruber Christian W.Markus Muttenthaler

10.1371/journal.pone.0032559.g004

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Design and structural validation of peptide–drug conjugate ligands of the kappa-opioid receptor

Nature Communications (2023)

Edin Muratspahić Kristine Deibler Jianming Han Nataša Tomašević Kirtikumar B. Jadhav

Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-β-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-β-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-β-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.

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κ-opioid receptor

10.1038/s41467-023-43718-w

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Ligand-Based Peptide Design and Combinatorial Peptide Libraries to Target G Protein-Coupled Receptors

Current Pharmaceutical Design (2010)

Christian W. Gruber Markus Muttenthaler Michael Freissmuth

G protein-coupled receptors (GPCRs) are considered to represent the most promising drug targets; it has been repeatedly said that a large fraction of the currently marketed drugs elicit their actions by binding to GPCRs (with cited numbers varying from 30-50%). Closer scrutiny, however, shows that only a modest fraction of (∼60) GPCRs are, in fact, exploited as drug targets, only ∼20 of which are peptide-binding receptors. The vast majority of receptors in the humane genome have not yet been explored as sites of action for drugs. Given the drugability of this receptor class, it appears that opportunities for drug discovery abound. In addition, GPCRs provide for binding sites other than the ligand binding sites (referred to as the “orthosteric site”). These additional sites include (i) binding sites for ligands (referred to as “allosteric ligands”) that modulate the affinity and efficacy of orthosteric ligands, (ii) the interaction surface that recruits G proteins and arrestins, (iii) the interaction sites of additional proteins (GIPs, GPCR interacting proteins that regulate G protein signaling or give rise to G protein-independent signals). These sites can also be targeted by peptides. Combinatorial and natural peptide libraries are therefore likely to play a major role in identifying new GPCR ligands at each of these sites. In particular the diverse natural peptide libraries such as the venom peptides from marine cone-snails and plant cyclotides have been established as a rich source of drug leads. High-throughput screening and combinatorial chemistry approaches allow for progressing from these starting points to potential drug candidates. This will be illustrated by focusing on the ligand-based drug design of oxytocin (OT) and vasopressin (AVP) receptor ligands using natural peptide leads as starting points. Keywords: GPCR, drug, ligand-based design, peptide, cyclotide, conotoxin, oxytocin, vasopressin

10.2174/138161210793292474

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Conopressin-T from Conus tulipa Reveals an Antagonist Switch in Vasopressin-like Peptides

Journal of Biological Chemistry (2008)

Sébastien Dutertre Daniel E. Croker Norelle L. Daly Åsa Andersson Markus Muttenthaler

We report the discovery of conopressin-T, a novel bioactive peptide isolated from Conus tulipa venom. Conopressin-T belongs to the vasopressin-like peptide family and displays high sequence homology to the mammalian hormone oxytocin (OT) and to vasotocin, the endogenous vasopressin analogue found in teleost fish, the cone snail's prey. Conopressin-T was found to act as a selective antagonist at the human V1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro7 and Gly9), which are replaced with Leu7 and Val9 in conopressin-T. Whereas conopressin-T binds only to OT and V1a receptors, an L7P analogue had increased affinity for the V1a receptor and weak V2 receptor binding. Surprisingly, replacing Gly9 with Val9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V1a receptor. NMR structures of both conopressin-T and L7P analogue revealed a marked difference in the orientation of the exocyclic tripeptide that may serve as templates for the design of novel ligands with enhanced affinity for the V1a receptor. We report the discovery of conopressin-T, a novel bioactive peptide isolated from Conus tulipa venom. Conopressin-T belongs to the vasopressin-like peptide family and displays high sequence homology to the mammalian hormone oxytocin (OT) and to vasotocin, the endogenous vasopressin analogue found in teleost fish, the cone snail's prey. Conopressin-T was found to act as a selective antagonist at the human V1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro7 and Gly9), which are replaced with Leu7 and Val9 in conopressin-T. Whereas conopressin-T binds only to OT and V1a receptors, an L7P analogue had increased affinity for the V1a receptor and weak V2 receptor binding. Surprisingly, replacing Gly9 with Val9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V1a receptor. NMR structures of both conopressin-T and L7P analogue revealed a marked difference in the orientation of the exocyclic tripeptide that may serve as templates for the design of novel ligands with enhanced affinity for the V1a receptor. The vasopressin (AVP) 3The abbreviations used are:AVPvasopressinAVTvasotocinCon-Sconopressin-SCon-Tconopressin-TIPtotal inositol phosphateOToxytocinOTROT receptorHPLChigh pressure liquid chromatographyRPreversed phaseCHOChinese hamster ovaryGPCRG protein-coupled receptorMSmass spectrometry. and oxytocin (OT) peptides were originally discovered and identified as neurohypophysial hormones in mammals (1Birnbaumer M. Trends Endocrinol. Metab. 2000; 11: 406-410Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). In humans, AVP acts via three vasopressin receptors (vascular V1aR, pituitary V1bR, and renal V2R), whereas OT acts via one OT receptor (OTR). All targets are members of the G protein-coupled receptor family (2Barberis C. Mouillac B. Durroux T. J. Endocrinol. 1998; 156: 223-229Crossref PubMed Scopus (238) Google Scholar). Peripherally, they regulate water balance, the control of blood pressure, and contraction of uterine smooth muscle and mammary myoepithelium (3Treschan T.A. Peters J. Anesthesiology. 2006; 105 (quiz 639-540): 599-612Crossref PubMed Scopus (255) Google Scholar). Centrally, these peptides affect levels of aggression, depression, and young parent bonding (4Febo M. Numan M. Ferris C.F. J. Neurosci. 2005; 25: 11637-11644Crossref PubMed Scopus (163) Google Scholar, 5Ferris C.F. Lu S.F. Messenger T. Guillon C.D. Heindel N. Miller M. Koppel G. Bruns Robert F. Simon N.G. Pharmacol Biochem. Behav. 2006; 83: 169-174Crossref PubMed Scopus (87) Google Scholar, 6Kozorovitskiy Y. Hughes M. Lee K. Gould E. Nat. Neurosci. 2006; 9: 1094-1095Crossref PubMed Scopus (123) Google Scholar). Endogenous analogues of OT and AVP have been reported in nonmammalian vertebrates, annelids, molluscs, and insects, suggesting an old lineage for these peptides (7Hoyle C.H. Brain Res. 1999; 848: 1-25Crossref PubMed Scopus (154) Google Scholar). Surprisingly, two variants were also found in the venom of predatory cone snails. The original discovery of these two AVP analogues, named conopressins, was based on the characteristic “scratching” effect observed upon intracerebral injection into mice (8Cruz L.J. Santos de V. Zafaralla G.C. Ramilo C.A. Zeikus R. Gray W.R. Olivera B.M. J. Biol. Chem. 1987; 262: 15821-15824Abstract Full Text PDF PubMed Google Scholar). Although the sequences of conopressins are similar to vasopressin itself, they have an additional positive charge in position 4, which is only found in two other endogenous vasopressin analogues, cephalotocin (Octopus vulgaris) and annetocin (Eisenia foetida). Conopressin-S was isolated from Conus striatus, whereas conopressin-G was first isolated from Conus geographus venom but later found in Conus imperialis venom as well as in tissue extracts of the nonvenomous snails Lymnea stagnalis and Aplysia californica and the leech Erpobdella octoculata (9McMaster D. Kobayashi Y. Lederis K. Peptides. 1992; 13: 413-421Crossref PubMed Scopus (26) Google Scholar, 10Salzet M. Bulet P. Dorsselaer Van A. Malecha J. Eur. J. Biochem. 1993; 217: 897-903Crossref PubMed Scopus (79) Google Scholar, 11Kesteren Van R.E. Smit A.B. Lange De R.P. Kits K.S. Golen Van F.A. Der Schors Van R.C. With De N.D. Burke J.F. Geraerts W.P. J. Neurosci. 1995; 15: 5989-5998Crossref PubMed Google Scholar). vasopressin vasotocin conopressin-S conopressin-T total inositol phosphate oxytocin OT receptor high pressure liquid chromatography reversed phase Chinese hamster ovary G protein-coupled receptor mass spectrometry. Molluscs of the genus Conus produce bioactive peptides in a combinatorial fashion. As demonstrated for the snake toxins (12Fry B.G. Genome Res. 2005; 15: 403-420Crossref PubMed Scopus (354) Google Scholar), most conotoxins or conopeptides are believed to be derived from an endogenous structural template (13Dutertre S. Lumsden N.G. Alewood P.F. Lewis R.J. FEBS Lett. 2006; 580: 3860-3866Crossref PubMed Scopus (34) Google Scholar). Because conopressin-G is widely distributed, it may represent the endogenous hormone in Gastropods and Annelids. However, a role in prey capture has also been proposed (14Nielsen D.B. Dykert J. Rivier J.E. McIntosh J.M. Toxicon. 1994; 32: 845-848Crossref PubMed Scopus (42) Google Scholar). In this study, we report the discovery of conopressin-T isolated from Conus tulipa venom. Pharmacological characterizations of Con-T across human receptors revealed that it is a selective V1a antagonist, with partial agonist activity at the OT receptor and no detectable activity at V1b and V2 receptors. The exocyclic tripeptide segment of conopressin-T shows unusual sequence divergence. L7P-Con-T had increased affinity at the V1a receptor but minimal effect on the selectivity profile across all human receptors compared with Con-T. Interestingly, replacing Gly9 with Val9 in OT and AVP converted these peptides from full agonist to full antagonist at the V1a receptor, demonstrating the role of position 9 as an antagonist switch in these peptides. Finally, the NMR structures of conopressin-T and its L7P analogue provide new templates for the design of novel pharmacological agents with enhanced activity at the V1a receptor. Materials—t-Butoxycarbonyl-protected amino acids and reagents used during chain assembly and HPLC purification (dimethylformamide, dichloromethane, acetonitrile, and trifluoroacetic acid) were peptide synthesis grade purchased from Auspep (Melbourne, Australia) and Novabiochem (San Diego, CA). 4-methylbenzhydrylamine-NH2 resin was obtained from Applied Biosystems (Foster City, CA). Most standard chemicals were purchased from Sigma, Roche Applied Science, or Merck, unless otherwise indicated. AVP and OT came from Bachem (Bubendorf, Switzerland), and fetal calf serum was from Sigma. Myo-[2-3H]inositol was from PerkinElmer Life Sciences. Minimal essential medium and Dulbecco's modified Eagle's medium were purchased from Invitrogen, and inositol-free Dulbecco's modified Eagle's medium came from ICN Biochemicals (Orsay, France). Dowex AG1-X8 formate form 200-400 mesh was purchased from Bio-Rad. Human V1a and V1b MTS membranes from HEK293 cells, human V2 MTS membranes from CHO-K1 cells, [3H]OT ([tyrosyl-2,6-3H]oxytocin; 40 Ci/mmol), 125I-linear vasopressin V1a receptor antagonist ([125I]phenylacetyl-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2; 2,200 Ci/mmol), [3H]AVP ([Arg8,tyrosyl-3,5-3H]vasopressin; 19.4 Ci/mmol), FlashBlue™ GPCR scintillation beads, Betaplate Scintillant, GF/B filtermats, sample bags, and TopSeal-A 96-well plate sealing film were from PerkinElmer Life Sciences, [Arg8]vasopressin from Auspep (Melbourne, Australia), Costar 96-well white polystyrene plates with clear flat bottoms from Corning Glass, pcDNA3.1/V5-His©TOPO®TA expression kit, ThermalAce™ DNA polymerase, SuperScript™ III RNase H-reverse transcriptase, Red hot TaqDNA polymerase, F-12 nutrient mixture (HAM) from Invitrogen, QuikChange™ XL site-directed mutagenesis kit from Stratagene (La Jolla, CA), human placenta total RNA from Ambion (Austin, TX), trypsin-Versene (EDTA), and Serum Supreme from Cambrex Biosciences (Walkersville, MD), all primers and oligonucleotides from Sigma, TransIT®-CHO transfection kit from Mirus (Madison, WI), and CHO-K1 cells from the American Tissue Culture Collection (Manassas, VA). Isolation and Sequencing—Specimens of Conus tulipa were collected from the Great Barrier Reef, Australia. Venom ducts were dissected, and crude venom prepared as previously described (15Lewis R.J. Nielsen K.J. Craik D.J. Loughnan M.L. Adams D.A. Sharpe I.A. Luchian T. Adams D.J. Bond T. Thomas L. Jones A. Matheson J.L. Drinkwater R. Andrews P.R. Alewood P.F. J. Biol. Chem. 2000; 275: 35335-35344Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Initial fractionation of the venom was carried out by RP-HPLC, and fractions were stored at 4 °C until further use. Conopressin-T was purified from fraction 30 using a 1% linear gradient of 0-60% solvent B in A over 60 min (A = 0.05% trifluoroacetic acid (aqueous); B = 0.045% trifluoroacetic acid, 90% acetonitrile) on an analytical C18 Phenomenex column. The flow rate was 1 ml/min, and the absorbance was monitored at 214 nm. N-terminal sequencing was carried out on an Applied Biosystems Procise HT Protein Sequencer (BRF, Newcastle, Australia) using ∼20 pmol of purified peptide. Peptide Synthesis—Conopressin-T and analogues were synthesized using the method described in Ref. 13Dutertre S. Lumsden N.G. Alewood P.F. Lewis R.J. FEBS Lett. 2006; 580: 3860-3866Crossref PubMed Scopus (34) Google Scholar, with minor modifications. Briefly, conopressin-T and L7P-conopressin-T were synthesized manually using t-butoxycarbonyl chemistry with in situ neutralization protocols (16Schnolzer M. Alewood P. Jones A. Alewood D. Kent S.B. Int. J. Pept. Protein Res. 1992; 40: 180-193Crossref PubMed Scopus (938) Google Scholar) on a 0.5-mmol scale for residues 9 and 8. The peptide was then nitrogen-dried and split into two syntheses of 0.25 mmol for the remaining 7 residues of conopressin-T and L7P-conopressin-T. After HF cleavage, the crude peptides were purified by semipreparative RP-HPLC using a linear gradient of 0-60% B at 3 ml/min while monitoring UV absorbance at 214 nm. Air oxidation was carried out by dissolving 10 mg of the crude peptides in 45 ml of 0.1 m NH4HCO3 (pH 8.25) with vigorous stirring at room temperature for 1 h. Whereas L7P-conopressin-T dissolved readily at concentrations up to 0.5 mg/ml, conopressin-T was found to aggregate instantly. To overcome this, 1 mg of reduced conopressin-T was first dissolved in a 2:1 ratio of solution A and B and then slowly added to the oxidation buffer. Prior to purification, the solution was acidified to pH 3 with neat trifluoroacetic acid and analyzed by analytical C18 HPLC and electrospray-MS. Oxidized conopressin-T and L7P-conopressin-T were then purified by semipreparative RP-HPLC using the same chromatographic conditions as above. Pure fractions, as determined by analytical RP-HPLC and mass spectrometry, were pooled and lyophilized. Stock solutions of conopressin-T and L7P-conopressin-T were made in MilliQ water and quantified by analytical HPLC using a commercial AVP standard. Cloning of Human OT Receptor—A 1.3-kb fragment of OT cDNA, containing the full coding region of the human OT receptor, was isolated from human placental cDNA constructed from total placental RNA using SuperScript™ III reverse transcriptase. The following forward (5′-GGTAGAGGATTCCCGCTCATTTG-3′) and reverse (5′-GGGGAGGGATACAAACTGATAGG-3′) primers were used to isolate the DNA fragment. This fragment was subcloned into the mammalian expression vector pcDNA3.1/V5-his-TOPO. Following sequence analysis, 8 nucleotide changes were found over the published OT sequence (NM_000916) encoding 7 amino acid changes with one change being conserved. The nucleotide changes were T47C, A281G, A256C, G532C, T646C, and A1121G, which correspond to amino acid changes of V16A, H73R, M86L, G178R, F216L, and N374S. These changes were mutated to the published sequence using a QuikChange™ XL site-directed mutagenesis kit. OT Receptor Expression and Membrane Preparation—DNA was prepared and used for transfection in CHO-K1 cells. CHO-K1 cells were propagated in 150-mm plates with F-12 medium containing 10% Serum Supreme at 37 °C in a humidified atmosphere with 5% CO2. Transient transfection was performed with an appropriate plasmid using the TransIT®-CHO transfection reagent method. Briefly, the method involved combining TransIT®-CHO reagent (72 μl) in serum-free F-12 medium incubated for 5 min at room temperature. Following the addition of receptor plasmid (24 μg), this mixture was incubated for 10 min at room temperature. CHO Mojo reagent (16 μl) was added to the mixture and incubated for 15 min at room temperature prior to the addition to the cells. The transfected cells were harvested within 24 h by the following method. Confluent 150-mm plates were washed with phosphate-buffered saline (10 mm phosphate buffer, pH 7.4, 2.7 mm KCl, 137 mm NaCl), harvested by scraping into ice-cold harvest buffer (50 mm Tris-HCl, pH 7.4, 5 mm MgCl2), followed by homogenization with a Polytron homogenizer and centrifugation at 100 × g for 10 min at 4 °C. Supernatants were recovered and centrifuged at 22,000 × g for 1 h at 4 °C. Membrane pellets were resuspended in 0.5 ml of ice-cold assay buffer A (50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 0.1% bovine serum albumin, containing 10% glycerol and aliquots stored at -80 °C until use. Receptor Binding Studies—Receptor binding assays were performed using FlashBlue™ GPCR scintillation beads. Flash-Blue™ GPCR beads are 3-μm polystyrene scintillating beads with wheat germ agglutinin covalently attached on the surface. These beads allow development of a homogeneous GPCR radioligand binding assay using cellular membranes. Briefly, FlashBlue™ GPCR SPA binding assays were performed in 96-well white polystyrene plates with clear flat bottoms (Costar). Radioligand (OTR [3H]OT (2 nm), V1a 125I-labeled linear V1a antagonist (21 pm), V1b [3H]AVP (0.5 nm), V2 [3H]AVP (0.85 nm)) was added to each membrane preparation, followed by the addition of various concentrations of competing compounds (1 pm to 10 μm) in a total volume of 80 μl containing assay buffer A or B (50 mm Tris-HCl, pH 7.4, 0.1% bovine serum albumin, and either 5 mm MgCl2 (A) or 10 mm MgCl2 (B)) (OT and V2 assay, buffer A; V1a and V1b assay, buffer B). The final reaction volume per well comprised 20 μl of compound/buffer, 20 μl of FlashBlue™ GPCR beads (OTR, V1a, and V2, 100 μg; V1b, 200 μg), and 20 μl of membrane, and the assay was initiated by the addition of 20 μl of radioligand. The plate was then sealed with TopSeal-A sealing film and incubated with shaking for 1 h at room temperature. Radioligand binding was then assessed for 30 s/well on a 1450 Microbeta scintillation counter (Wallac). All binding data were analyzed using GraphPad PRISM (GraphPAD Software, Inc., San Diego, CA). Each data point was performed in triplicate and derived from at least three separate experiments. The inhibitory dissociation constant (Ki) was calculated using the following formula: Ki = IC50/(1 + [L]/Kd), where [L] is the concentration of radioligand present, and Kd is the dissociation constant of radioligand (17Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12293) Google Scholar). Inositol Phosphate Assays—Inositol phosphate accumulation was determined as previously described (18Derick S. Cheng L.L. Voirol M.J. Stoev S. Giacomini M. Wo N.C. Szeto H.H. Mimoun Ben M. Andres M. Gaillard R.C. Guillon G. Manning M. Endocrinology. 2002; 143: 4655-4664Crossref PubMed Scopus (61) Google Scholar). Briefly, CHO cells stably transfected with AVP/OT receptors were plated at 100,000 cells/well. Cells were grown for 24 h in their respective culture medium (see above) and further incubated for another 24-h period in a serum- and inositol-free medium supplemented with 1 μCi·ml-1 myo-[2-3H]inositol. Cells were then washed twice with Hanks' buffered saline medium, incubated for 15 min in this medium supplemented with 20 mm LiCl, and further stimulated for 15 min with increasing concentrations of analogues to be tested. The reaction was stopped by adding perchloric acid (5%, v/v). Total inositol phosphates (IPs) accumulated were extracted and purified on a Dowex AGI-X8 anion exchange chromatography column and counted. NMR Spectroscopy—Samples for 1H NMR measurements contained ∼1 mm peptide in 95% H2O, 5% D2O (v/v) at pH ∼3. Spectra were recorded at 290 K on a Bruker AVANCE-600 spectrometer. Two-dimensional NMR spectra were recorded in phase-sensitive mode using time-proportional phase incrementation for quadrature detection in the t1 dimension (19Daly N.L. Ekberg J.A. Thomas L. Adams D.J. Lewis R.J. Craik D.J. J. Biol. Chem. 2004; 279: 25774-25782Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 20Rosengren K.J. Daly N.L. Plan M.R. Waine C. Craik D.J. J. Biol. Chem. 2003; 278: 8606-8616Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). The two-dimensional experiments consisted of a TOCSY using a MLEV-17 spin lock sequence with a mixing time of 80 ms, DQF-COSY, and NOESY with mixing times of 200-300 ms. Solvent suppression was achieved using a modified WATERGATE sequence. Spectra were acquired over 6024 Hz with 4096 complex data points in F2 and 512 increments in the F1 dimension. 3JHN-Hα coupling constants were measured from a one-dimensional spectrum or from the DQF-COSY spectrum. Spectra were processed on a Silicon Graphics Indigo work station using XWINNMR (Bruker) software. The t1 dimension was zero-filled to 1024 real data points, and 90° phase-shifted sine bell window functions were applied prior to Fourier transformation. Structure Calculations—Preliminary structures were calculated using a torsion angle simulated annealing protocol within the program DYANA (21Guntert P. Mumenthaler C. Wuthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2555) Google Scholar). Final structures were calculated using simulated annealing and energy minimization protocols within CNS version 1.1 (22Brunger A.T. Adams P.D. Rice L.M. Structure. 1997; 5: 325-336Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The starting structures were generated using random (φ,ψ) dihedral angles and energy-minimized to produce structures with the correct local geometry. A set of 50 structures was generated by a torsion angle simulated annealing protocol (19Daly N.L. Ekberg J.A. Thomas L. Adams D.J. Lewis R.J. Craik D.J. J. Biol. Chem. 2004; 279: 25774-25782Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 20Rosengren K.J. Daly N.L. Plan M.R. Waine C. Craik D.J. J. Biol. Chem. 2003; 278: 8606-8616Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). This protocol involved a high temperature phase comprising 4000 steps of 0.015 ps of torsion angle dynamics, a cooling phase with 4000 steps of 0.015 ps of torsion angle dynamics during which the temperature was lowered to 0 K, and finally an energy minimization phase comprising 500 steps of Powell minimization. Structures consistent with restraints were subjected to further molecular dynamics and energy minimization in a water shell, as described by Linge and Nilges (23Linge J.P. Nilges M. J. Biomol. NMR. 1999; 13: 51-59Crossref PubMed Scopus (239) Google Scholar). The refinement in explicit water involved the following steps. First, heating to 500 K via steps of 100 K, each comprising 50 steps of 0.005 ps of Cartesian dynamics. Second, 2500 steps of 0.005 ps of Cartesian dynamics at 500 K before a cooling phase where the temperature was lowered in steps of 100 K, each comprising 2500 steps of 0.005 ps of Cartesian dynamics. Finally, the structures were minimized with 2000 steps of Powell minimization. Structures were analyzed using PROMOTIF (24Hutchinson E.G. Thornton J.M. Protein Sci. 1996; 5: 212-220Crossref PubMed Scopus (997) Google Scholar) and PROCHECK-NMR (25Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4428) Google Scholar). Isolation and Sequencing of Conopressin-T—A peptide with a monoisotopic mass of 1107.54 Da was purified from C. tulipa venom (Fig. 1, A-C). The Tris(2-carboxyethyl)phosphine-reduced peptide displayed a mass of 1109.5 Da, suggesting the presence of one disulfide bond (data not shown). N-terminal sequencing produced the sequence CYIQNCLRV with a calculated mass of 1110.53 Da, consistent with one disulfide bond (-2 Da) and C-terminal amidation (-1 Da). A BLAST search revealed that this sequence belonged to the vasopressin peptide family characterized by a disulfide-containing ring (residues 1-6) and a short exocyclic C-terminal tripeptide (residues 7-9). Conopressin-T had 7 of 9 residues identical to vasotocin (AVT), including the first six residues also found in oxytocin and mesotocin, and Arg8 known to be essential for the pressor activity in vasopressin. However, two highly conserved residues, Pro7 and Gly9, found in other vasopressin-like peptides as well as conopressin-G and conopressin-S are modified to Leu7 and Val9 in conopressin-T. De novo mass spectrometry sequencing (data not shown) unambiguously confirmed the sequence of Con-T as CYIQNCLRV-NH2. Synthesis of Conopressin-T and L7P-Conopressin-T—Con-T and its analogues were synthesized on 4-methylbenzhydrylamine resin using solid-phase peptide synthesis t-butoxycarbonyl chemistry to further validate the primary sequence and allow characterization of its biological activity. Lyophilized crude peptides from HF cleavage were dissolved in 0.5% trifluoroacetic acid and analyzed on analytical C18 Phenomenex RP-HPLC column. The chromatograms and electrospray-MS revealed that the syntheses contained >90% of the expected products; consequently, the oxidation was carried out directly on the crude peptides following the conditions described under “Experimental Procedures.” During synthesis, we noticed that the replacement of Leu7 by Pro7 had a marked effect on solubility. L7P-Con-T dissolved readily in oxidation buffer to 0.5 mg/ml, whereas Con-T aggregated instantly in this solvent. This difference suggested that a Leu or a Pro at position 7 significantly modified the physical properties of the peptides. Oxidized synthetic and native Con-T were indistinguishable when co-injected in a C18 Phenomenex analytical RP-HPLC column (Fig. 1D). Conopressin-T and L7P-Conopressin-T Are Selective V1a Receptor Antagonists—Con-T and L7P-Con-T were tested for their ability to displace radioligand from human AVP and OT receptors expressed in HEK and CHO cells (Fig. 2A and Table 1). Con-T showed the highest affinity for human OTR (Ki = 108 nm) and V1aR (Ki = 319 nm) and no detectable activity at V1b and V2 receptors at up to 10 μm peptide. In contrast, L7P-Con-T was 8-fold more potent than Con-T at the V1aR (Ki = 37 nm) but had similar affinity for OTR (Ki = 132 nm). L7P-Con-T was also found to have a weak effect at V2R (Ki = 1.8 μm), whereas no displacement of radioligand was detected at the V1bR at up to 10 μm peptide. For comparison, we also tested the activity of conopressin-S (Con-S) across these receptors. Con-S does not bind to V2R (up to 10 μm peptide), has similar affinity for OTR (Ki = 175 nm) but is less potent at V1a (Ki = 827 nm). In contrast to Con-T and L7P-Con-T, Con-S binds with high affinity to V1bR (Ki = 8.3 nm), although the displacement of specific [3H]AVP binding was incomplete (a 30-40% resistant component remained; see Fig. 2A). Therefore, conopressin-T, -S, and -L7P have distinct pharmacological profiles on human receptors, with the lack of effect of Con-T on radioligand binding to both V1b and V2 receptors being unique (supplemental Fig. 1).TABLE 1Binding affinities (Ki, nm) on human vasopressin and oxytocin receptorsCompoundV1aRV1bRV2ROTRCon-T319 ± 15>10,000>10,000108 ± 9L7P-Con-T37 ± 2>10,0001836 ± 392132 ± 10Con-S827 ± 768.3 ± 1.6>10,000175 ± 13AVP0.6 ± 0.020.085 ± 0.014.9 ± 0.32110 ± 25G9V-AVP25 ± 2170 ± 2224 ± 3114 ± 35OT37 ± 3222 ± 22823 ± 1221.5 ± 0.42G9V-OT466 ± 43>10,000>10,000464 ± 171Values are mean ± S.E. obtained from at least three separate experiments, each performed in triplicate. Open table in a new tab Values are mean ± S.E. obtained from at least three separate experiments, each performed in triplicate. The functional properties of Con-T and L7P-Con-T were investigated on CHO cells expressing human receptors (Fig. 2B and Table 2). Con-T did not stimulate phospholipase C activity in cells expressing V1aR at ≤10 μm peptide. However, Con-T at 10 μm showed partial agonist activity at the V1bR and OTR, producing 9 and 22% of AVP and OT maximal activity, respectively. L7P-Con-T displayed a similar profile, with no agonist activity at the V1aR at ≤10 μm and partial agonist activity at the V1bR and OTR (4 and 28% of AVP and OT maximal activity, respectively) at 10 μm peptide. In contrast, both Con-T and L7P-Con-T induced a potent and concentration-dependent inhibition of AVP-stimulated IP production in CHO cells expressing V1aR, with Kinact values of 329 and 90 nm, respectively (Table 2). These values correlated well with the binding affinities. Thus, both peptides are full antagonists at the human V1aR subtype and weak partial agonists for the V1bR and OTR (Fig. 2B). Due to the absence of significant binding to V2R, the functional activity of Con-T and L7P-Con-T was not investigated on this subtype.TABLE 2Antagonist and agonist actions of Con-T, L7P-Con-T, G9V-AVP, and G9V-OT on IP signaling through human vasopressin and oxytocin receptorsCompoundV1aRV1bROTRCon-TNo agonist activity at 10 μm (1 ± 7% of AVP maximum activity)Weak agonist activity (9 ± 3% of AVP max activity at 10 μm)Partial agonist (22 ± 3% of OT maximum activity; Kact 37 ± 30 nm)Antagonist, Kinact 329 ± 58 nmNo antagonist activity at 10 μmNo antagonist activity at 10 μmL7P-Con-TNo agonist activity at 10 μmWeak agonist activity at 10 μm (4 ± 2% of AVP max activity)Partial agonist (28 ± 3% of OT max activity; Kact = 16 nm)Antagonist, Kinact 90 ± 12 nmNo antagonist activity at 10 μmNo antagonist activity at 10 μmG9V-AVPNo agonist activity at 10 μmPartial agonist (5-10% of OT maximum activity; Kact 320 ± 145 nm)Antagonist, Kinact 40 ± 10 nmNDaND, not determined.No antagonist activity at 10 μmG9V-OTNo agonist activity at 10 μmPartial agonist (25-30% of OT maximum activity; Kact 124 ± 37 nm)Antagonist, Kinact 391 ± 85 nmNDNo antagonist activity at 10 μma ND, not determined. Open table in a new tab Gly9 → Val Replacement in OT and AVP Acts as an Antagonist Switch at the V1a Receptor—L7P-conopressin-T and vasotocin differs only at position 9, yet L7P-conopressin-T acts as a full antagonist at the V1a receptor (Fig. 2B), whereas the closely related vasotocin is known to function as an agonist (29Mahlmann S. Meyerhof W. Hausmann H. Heierhorst J. Schonrock C. Zwiers H. Lederis K. Richter D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1342-1345Crossref PubMed Scopus (145) Google Scholar). To investigate if the Val9 modification in conopressin-T could switch agonist to antagonist activity in related peptides, we tested this modification in OT and AVP. First, the binding properties of these analogues were assessed on human receptors (Fig. 3A and Table 1). G9V-AVP was equipotent at V1aR and V2R (∼25 nm) and 5-7-fold less potent at the OTR and V1bR (114 and 170 nm, respectively). Compared with AVP, G9V-AVP affinity ranged from 5-fold less at V2R to 2000-fold less at V1bR. In contrast, G9V-OT was equipotent at the V1a and OT receptors (∼465 nm), whereas it was unable to fully displace radioligands at the V1b and V2 subtypes at concentrations up to 10 μm. Compared with OT, G9V-OT affinity ranged from 13-fold less at V1aR to 309-fold less at OTR. The functional properties of G9V-AVP and G9V-OT were investigated on CHO cells expressing human receptors (Fig. 3B and Table 2). Both peptides failed to stimulate phospholipase C activity in cells expressing V1aR at ≤10 μm peptide. However, G9V-AVP and G9V-OT both acted as partial agonists at the OTR, eliciting ∼5-10% and ∼25-30% of OT maximal activity, respectively. In contrast, both peptides induced a potent and concentration-dependent inhibition of AVP-stimulated IP production in CHO cells expressing V1aR. The affinities (Kinact) of G9V-AVP (40 nm) and G9V-OT (391 nm) correlate well with the binding data at the V1a receptor (25 and 466 nm, respectively). Clearly, the G9V replacemen

Vasotocin

Tripeptide

10.1074/jbc.m706477200

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Oxytocin and vasopressin signaling in health and disease

Trends in Biochemical Sciences (2024)

Monika Perisic Katrina Woolcock Anke Hering Helen C. Mendel Markus Muttenthaler

Neurohypophysial peptides are ancient and evolutionarily highly conserved neuropeptides that regulate many crucial physiological functions in vertebrates and invertebrates. The human neurohypophysial oxytocin/vasopressin (OT/VP) signaling system with its four receptors has become an attractive drug target for a variety of diseases, including cancer, pain, cardiovascular indications, and neurological disorders. Despite its promise, drug development faces hurdles, including signaling complexity, selectivity and off-target concerns, translational interspecies differences, and inefficient drug delivery. In this review we dive into the complexity of the OT/VP signaling system in health and disease, provide an overview of relevant pharmacological probes, and discuss the latest trends in therapeutic lead discovery and drug development.

10.1016/j.tibs.2024.01.010

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