Oxytocic plant cyclotides as templates for G -coupled ligand design

Johannes Koehbacha, Margaret O’Brienb, Markus Muttenthalerc, Marion Miazzoa, Muharrem Akcand, Alysha G. Elliottd, Norelle L. Dalyd,e, Peta J. Harveyd, Sarah Arrowsmithf, Sunithi Gunasekerag, Terry J. Smithb,h, Susan Wrayf, Ulf Göranssong, Philip E. Dawsonc, David J. Craikd, Michael Freissmutha, and Christian W. Grubera,1

aCenter for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria; bNational Centre for Biomedical Engineering Science and hThe School of Natural Sciences, National University of Ireland, Galway, Ireland; cDepartments of Chemistry and Cell Biology, The Scripps Research Institute, La Jolla, CA 92037; dInstitute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia; eSchool of Pharmacy and Molecular Sciences, Centre for Biodiscovery and Molecular Development of Therapeutics, Queensland Tropical Health Alliance, James Cook University, Cairns, QLD 4878, Australia; fDepartment of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool L69 3BX, United Kingdom; and gDivision of Pharmacognosy, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University, 751 23 Uppsala, Sweden

Edited by Robert J. Lefkowitz, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, and approved October 23, 2013 (received for review June 24, 2013) Cyclotides are plant comprising a circular backbone and make them interesting templates for the development of novel three conserved disulfide bonds that confer them with exceptional pharmaceuticals (14). stability. They were originally discovered in Oldenlandia affinis However, five decades after the discovery of cyclotides, there based on their use in traditional African medicine to accelerate still is not any information about specific molecular targets and/ labor. Recently, cyclotides have been identified in numerous plant or mechanisms underlying their biological activities. It is known species of the coffee, violet, cucurbit, pea, potato, and grass fam- that cyclotides can, at higher concentrations, disrupt phospho- ilies. Their unique structural topology, high stability, and tolerance lipid bilayers (15, 16), because they expose hydrophobic residues to sequence variation make them promising templates for the on their surface. This endows them with physicochemical prop- development of peptide-based pharmaceuticals. However, the erties allowing for insertion into membranes and pore formation mechanisms underlying their biological activities remain largely (17, 18). Although no cyclotide target receptor has been identi- fi fied hitherto, the observed biological activities (e.g., their ute- unknown; speci cally, a receptor for a native cyclotide has not fi been reported hitherto. Using bioactivity-guided fractionation of rotonic effects) may be explained by speci c receptor-mediated an herbal peptide extract known to indigenous healers as “kalata- mechanisms. In mammals, including humans, uterine muscle kalata,” the cyclotide kalata B7 was found to induce strong contrac- contractility can be elicited by activation of various signaling pathways. One physiological regulator of uterine contraction is tility on human uterine smooth muscle cells. Radioligand displace- the . In uterine tissue, this peptide activates ment and second messenger-based reporter assays confirmed the oxytocin and V1a receptors (19–21), two members oxytocin and vasopressin V1a receptors, members of the - of the G protein-coupled receptor (GPCR) family. GPCRs are coupled receptor family, as molecular targets for this cyclotide. Fur- thermore, we show that cyclotides can serve as templates for the fi design of selective G protein-coupled receptor ligands by generating Signi cance an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. G protein-coupled receptors (GPCRs) are promising drug tar- These observations provide a proof of concept for the develop- gets: >30% of the currently marketed drugs elicit their actions ment of cyclotide-based peptide ligands. by binding to these transmembrane receptors. However, only ∼10% of all GPCRs are targeted by approved drugs. Resorting to circular plant peptide | peptide ligand design | | plant-derived compounds catalogued by ethnopharmacological chemical pharmacology | peptide drugs analyses may increase this repertoire. We provide a proof of concept by analyzing the uterotonic action of an herbal remedy used in traditional African medicine. We identified cyclic yclotides are head-to-tail cyclized plant peptides containing peptides, investigated the molecular mechanisms underlying three conserved disulfide bonds in a knotted arrangement C their uterotonic activity, and report an oxytocic plant peptide known as a cyclic cystine-knot motif (1). This confers them high that modulates the human oxytocin/vasopressin receptors. This stability (2) and presumably improves their oral bioactivity rel- naturally occurring peptide served as a template for the design fi ative to their linear counterparts (3). They were rst discovered of an oxytocin-like nonapeptide with enhanced receptor selec- Oldenlandia affinis in a decoction of DC. (Rubiaceae) leaves, an tivity, highlighting the potential of cyclotides for the discovery herbal remedy used in traditional African medicine during of peptide-based GPCR ligands. childbirth (4). The observed induction of labor and shortened delivery time were later studied on isolated rat and rabbit uteri Author contributions: C.W.G. designed research; J.K., M.O., M. Muttenthaler, M. Miazzo, and on human uterine strips (4, 5). The peptides responsible for M.A., A.G.E., N.L.D., P.J.H., S.A., S.G., and C.W.G. performed research; M. Muttenthaler, M.A., A.G.E., N.L.D., S.G., T.J.S., U.G., P.E.D., D.J.C., M.F., and C.W.G. contributed new the contractility effects (5) raised interest because they survived reagents/analytic tools; J.K., M.O., M. Muttenthaler, M. Miazzo, N.L.D., P.J.H., S.A., T.J.S., boiling, presumably as a result of their unique 3D structure, S.W., D.J.C., M.F., and C.W.G. analyzed data; and J.K., M. Muttenthaler, S.W., U.G., P.E.D., which was elucidated in 1995 (6). Since then, several plant spe- D.J.C., M.F., and C.W.G. wrote the paper. cies of the coffee (Rubiaceae) (7), violet (Violaceae) (8), legume The authors declare no conflict of interest. (Fabaceae) (9), potato (Solanaceae) (10) and grass (Poaceae) This article is a PNAS Direct Submission. families (11) have been identified to produce cyclotides. Cur- Freely available online through the PNAS open access option. rently, ∼300 sequences have been reported (12), and the pre- Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2M9O, 2MGO, and 2RU2). dicted number of >50,000 cyclotides in Rubiaceae alone (7) 1To whom correspondence should be addressed. E-mail: christian.w.gruber@meduniwien. suggests them to be one of the largest peptide classes within the ac.at. plant kingdom. Their high intercysteine sequence variability and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. PHARMACOLOGY structural plasticity (13), together with intrinsic bioactivities, 1073/pnas.1311183110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1311183110 PNAS | December 24, 2013 | vol. 110 | no. 52 | 21183–21188 Downloaded by guest on September 25, 2021 prominent drug targets, with ∼30% of all marketed drugs acting cyclotide (Fig. S3) was analyzed (i) for its ability to stimulate via modulation of these receptors (21). contractions of uterine smooth muscle cells and (ii) for its af- We used a bioactivity-guided fractionation approach com- finity to human or V1a receptor. Kalata B7 bined with pharmacological and structural analysis to elucidate displaced tritiated oxytocin or vasopressin in a dose-dependent the mechanism underlying the oxytocic activity of cyclotides and manner from the binding site of the oxytocin receptor or V1a identified a molecular target for native cyclotides. In addition, we receptor with a Ki of 50 μM and 12 μM, respectively (Fig. 2A and used cyclotides as a template to explore substitutions that en- Table 1). It also provoked significant contraction of uterine cells hanced receptor binding and agonistic activity. Our observations (i.e., 8.4% increased contraction compared with unstimulated provide a proof of concept that (i) naturally occurring peptide control cells) (Fig. 2C). We verified that the cyclotide kalata B7 libraries cover a chemical space that intersects with the sampled acted via the oxytocin receptor and/or V1a receptor on GPCRs and (ii) cyclotides can serve as a template for the design cells by applying kalata B7 together with the receptor antagonist of new classes of GPCR ligands, thus opening new avenues for (23); this coapplication of both compounds resulted in cyclotide-based drug development. a significant loss of contractility. If kalata B7 is an agonist at the oxytocin receptor and/or the V1a Results receptor, it ought to trigger signaling via a Gq-dependent pathway. Bioactivity-Guided Fractionation of Uterotonic Plant Cyclotides. An We verified this prediction by measuring the generation of inositol- herbal extract that has been used for many generations by tra- 1-phosphate (IP1) in response to the cyclotide in HEK293 cells ditional healers for its uterotonic properties was analyzed by heterologously expressing either receptor. The analysis of the con- bioactivity-guided fractionation. Dried aerial parts of O. affinis centration–response curve showed that kalata B7 was a partial ag- B were extracted by grinding, solvent partitioning, and solid C - onist at both the oxytocin receptor and the V1a receptor (Fig. 2 ) 18 μ μ phase extraction of the aqueous filtrate to yield a crude cyclotide with an EC50 of 12 Mand4 M, respectively. Agonistic activity extract. The analysis by RP-HPLC and MALDI-TOF MS was more pronounced at the oxytocin receptor (about 80% of the showed that this extract contained a number of cyclotides iden- response elicited by oxytocin) than at the V1a receptor (about 40% tified based on their mass, cysteine content, and hydrophobicity of the response elicited by vasopressin). (7) (Fig. 1A and Fig. S1). Four subfractions eluting in the range – of 18–54% acetonitrile were collected by preparative RP-HPLC Structural Characterization of Kalata B7. To understand the ligand receptor interaction, the structure of kalata B7 was determined and tested for their ability to induce contractions of human A fi uterine smooth muscle cells using a collagen gel contractility by NMR (Fig. 3 and Table S2). This revealed a well-de ned backbone around the cyclic cystine-knot motif typical for Möbius assay (22) (Fig. S2). Compared with unstimulated cells, in- β cubation with cyclotide-containing extracts showed a significant cyclotides, a type I -turn between residues 9 and 12, a type II fl β-turn between residues 16 and 19, and a type VIa1 β-turn be- decrease in the collagen gel area, which re ected an increased β contraction of the smooth muscle cells. Further RP-HPLC tween residues 22 and 25, as well as a -hairpin between residues fractionation generated 15 subfractions, of which six induced 20 and 28. As shown in Fig. 3, loop 3 of kalata B7 (-CYTQGC-) significant contraction, ranging from 6.8–18.7% increased con- is homologous to the six-residue ring sequence of human oxy- tractility over unstimulated cells (Fig. 1B). tocin (CYIQNC-). In particular, the tyrosine (Y15) and gluta- mine (Q17) residues of kalata B7 are in positions analogous to Molecular and Pharmacological Characterization of the Oxytocic those (Y2 and Q4) in oxytocin. NMR structural analysis of hu- man oxytocin confirmed the presence of a type II β-turn (24) Cyclotide Kalata B7. The 15 HPLC fractions were analyzed by B MS and tandem MS peptide sequencing, and 17 cyclotides were (Fig. 3 ). Therefore, loop 3 of kalata B7 and human oxytocin identified, with each of the fractions containing one to five share similarities in sequence and 3D structure. Furthermore, the structure of kalata B7 indicated that the side chains of Tyr peptides. Cyclotide sequences within biologically active fractions A display to human oxytocin, whereas cyclo- and Gln in loop 3 protrude from the backbone (Fig. 3 ). Hence, tides from inactive fractions lack any appreciable similarity they might be capable of interacting with the oxytocin receptor. The crucial role of the tyrosine and glutamine residues (loop 3) (Table S1). In particular, loop 3 of the cyclotide kalata B7 fi (-CYTQGC-) found in the most active fraction (Fig. 1B) and the of the cyclotide was con rmed by generating mutated variants six-residue ring of oxytocin (CYIQNC-) have related sequences. (Y replaced by A, S, or F; Q was replaced by A or E). These were all inactive or did not bind to the receptor (Fig. S4). Therefore, kalata B7 was isolated by RP-HPLC, and the purified Cyclotides as Peptide Templates for Oxytocin and Vasopressin GPCR Ligand Design. Cyclotides typically comprise 28–37 amino acids. Therefore, they are larger and more bulky than the nonapeptide ligands oxytocin and vasopressin. Thus, we used the sequence of kalata B7 as a template for the synthesis of oxytocin-like non- apeptides. Based on the sequence of loop 3 of kalata B7, four peptides were synthesized (Table 1). NMR analysis revealed negligible differences in structure relative to oxytocin, as de- termined by 1H chemical shifts (Fig. S5) and structural calcu- lations (Fig. 3 and Table S3). A comparison of the structural ensembles of the solution structure of oxytocin (Fig. 3B) with the nonapeptide kalata B7-oxytocin 1 (OT1) (Fig. 3C) revealed a similarly dynamic exocyclic tail and a defined region compris- – D fi ing residues 1 6 that overlay well (rmsd of 0.65 Å, Fig. 3 ). The Fig. 1. Bioactivity-guided fractionation of O.af nis peptide extracts. (A)An- synthetic oxytocin-like peptides were tested for binding and re- alytical RP-HPLC chromatogram of a peptide extract from O. affinis leaves after fi ceptor activation (Fig. 4 and Fig. S4). [G5, T7, S9]-oxytocin solvent extraction and in-batch C18 puri cation shows multiple peptide peaks, fi K = as determined by MALDI MS (Fig. S1). Fractions that were tested for biological (kalata B7-OT1) had improved binding af nity ( i 218 nM; Fig. 4A). This increased affinity was also evident, when assessing activity are labeled with dashed lines. Fraction OA44–45, showing the highest contractility (see B), is highlighted in gray. mAU, milli-absorption units. (B) its ability to promote luciferase transcription in cells express- = Collagen gel contractility data of partially purified cyclotide fractions (1.25 ing the human oxyocin receptor (EC50 IP1 formation 145 nM, −1 mg·mL ) on isolated human uterus smooth muscle cells. Data represent the EC50 luciferase induction = 356 nM; Table 1), where it acted as mean ± SEM. Statistical differences were analyzed using one-way ANOVA (**P a full agonist (compare with the maximum response of oxyto- < 0.01; ***P < 0.001). cin and kalata B7-OT1 in Fig. 4B). Similar to the mutated

21184 | www.pnas.org/cgi/doi/10.1073/pnas.1311183110 Koehbach et al. Downloaded by guest on September 25, 2021 Fig. 2. Receptor pharmacology and bioactivity of isolated kalata B7. (A) Binding data were obtained by measuring the displacement of radioactive [3H]oxytocin (2 nM) or [3H]arginine-vasopressin (0.75 nM) by kalata B7 (10 nM to 100 μM) and control peptide oxytocin (OT) or vasopressin (AVP) (0.1 nM to 10

μM) from 30 to 100 μg of human oxytocin receptor (solid lines) or V1a receptor (dashed lines) membranes. (B) Receptor activation was measured by recording intracellular IP1 accumulation upon stimulation with kalata B7 (1–100 μM) or control peptide (0.1 nM to 10 μM) (same labels as in A). Data were fitted by nonlinear regression (sigmoidal, variable slope) and are shown as the mean ± SEM of two to three independent experiments. Binding data were normalized to the percentage (%) of maximal binding; the 100% value refers to an average of 1.57 pmol of ligand bound per milligram of membrane for the oxytocin

receptor and 0.97 pmol bound per milligram of membrane for the V1a receptor. Fold induction of intracellullar IP1 accumulation above baseline was nor- malized to the number of cells. Ki and EC50 values are listed in Table 1. (C) Collagen gel contractility of pure kalata B7 (dark gray) at 10 μM could be abolished by cotreatment with the oxytocin- and V1a receptor antagonist atosiban (AT, light gray) at 800 nM. Statistical differences were analyzed using one-way ANOVA (*P < 0.05; **P < 0.01). n.s., not significant.

cyclotide, the kB7-OT1 mutants (Y2A) and (Q4A) had lost their pretreatment with atosiban (for amplitude, 1 nM: −0.8 ± affinity and did not activate the oxytocin receptor (Table 1 and 2.7%; 10 nM, −13.6 ± 7%; 100 nM: −2.8 ± 5.8%; for area under Fig. S4). Interestingly, kB7-OT1 proved to be selective for the the curve, 1 nM: −14.5 ± 6.9%; 10 nM, −25.5 ± 4.8%; 100 nM: oxytocin receptor because it did not compete for binding of −11.1 ± 13.7%; n = 3) (Fig. 5 B and C). radiolabeled vasopressin on any of the three human vasopressin (V1a,V1b, and V2) receptors at concentrations up to 10 μM (Fig. Discussion 4C) in contrast to native oxytocin (25). A decoction of O. affinis induces strong uterine contractions after either oral administration as a tea or intravaginal in- O. affinis Uterostimulant Effects of Extract and Kalata B7-OT1 on stillation (4, 5). In line with this contractile activity, we identified Human Myometrium. The crude “kalata-kalata” plant extract and an active principle in peptide-containing extracts of O. affinis and synthetic kB7-OT1 ([G5, T7, S9]-oxytocin) were applied directly to HPLC-purified fractions containing various cyclotides based on organ baths containing strips of human myometrium superfused the following criteria: (i) The peptide mixtures and purified − with physiological saline solution. Application of 1 mg·mL 1 crude kalata B7 elicited contractions of uterine muscle cells that were extract resulted in stimulation of contraction amplitude by +8.1 ± antagonized by the oxytocin receptor blocker atosiban; (ii) they 3.5%, whereas the area under the curve increased by 313.5 ± 96% displaced radiolabeled oxytocin from its heterologously expressed ± n = A (mean SEM; 6) (Fig. 5 ). Pretreatment with atosiban cognate receptor and vasopressin from the V1a receptor, the significantly reduced but did not abolish the stimulatory effect of closest relative of the oxytocin receptor; (iii) consistent with the the extract (Fig. S6). Application of kB7-OT1 resulted in a dose- oxytocin receptor being a Gq-coupled receptor, kalata B7 also dependent increase in both contraction amplitude (1 nM: +6.0 ± triggered the formation of the canonical signaling downstream 3.2%; 10 nM: +41.4 ± 6.4%; 100 nM: +73.2 ± 5.4%) and area cascade, resulting in the increased accumulation of IP1; and (iv) under the curve (1 nM: +2.7 ± 2.3%; 10 nM, +48.8 ± 20.3%; in organ bath experiments, both the O. affinis extract and the 100 nM; +218.3 ± 143.6%; n = 3), which was inhibited by nonapeptide kB7-OT1 ([G5, T7, S9]-oxytocin) augmented

Table 1. Pharmacology of kalata B7 and oxytocin-like peptides Binding affinity IP1 formation

Ki,M* EC50,M Peptide Sequence

OT receptor V1a receptor OT receptor V1a receptor

− − − − Kalata B7 Cyclo-GLPVCGETCTLGTCYTQGCTCSWPICKRN 5.0 ± 1.1 × 10 5 1.2 ± 0.1 × 10 5 1.2 ± 0.4 × 10 5 4.8 ± 1.0 × 10 6 −7 −5 −7 [G5,T7,S9]-OT (kB7-OT1) CYIQGCTLS-NH2 2.2 ± 0.2 × 10 >1.0 × 10 1.5 ± 0.1 × 10 n.d. 3.6 ± 0.4 × 10−7† −5 −5 −5 [T3,G5,T7,S8,S9]-OT CYTQGCTSS-NH2 >1.0 × 10 >1.0 × 10 >5.0 × 10 n.d. −5 −5 −5 [T3,P4,G5,S7,S8,T9]-OT CYTPGCSST-NH2 >1.0 × 10 >1.0 × 10 >5.0 × 10 n.d. −5 −5 −5 [P4,G5,S7,T9]-OT CYIPGCSLT-NH2 >1.0 × 10 >1.0 × 10 >5.0 × 10 n.d. −5 −5 kB7-OT1 [Y2A] CAIQGCTLS-NH2 >1.0 × 10 n.d. >5.0 × 10 n.d. −5 −5 kB7-OT1 [Q4A] CYIAGCTLS-NH2 >1.0 × 10 n.d. >5.0 × 10 n.d. −9 −9 OT CYIQNCPLG-NH2 1.8 ± 0.1 × 10 n.d. 3.4 ± 0.5 × 10 n.d. 4.2 ± 0.5 × 10−9† −10 −9 Vasopressin CYFQNCPRG-NH2 n.d. 8.0 ± 0.2 × 10 n.d. 1.0 ± 0.1 × 10

*Binding affinity (Ki) and functional receptor activation (EC50) data are the mean ± SEM of two to four independent experiments. Ki values were calculated using IC50 values according to Cheng and Prusoff (44), with a Kd value of 1.5 nM for oxytocin (OT) on the oxytocin receptor and 0.6 nM for vasopressin on the

V1a receptor. If no IC50 value has been determined, the given values represent the highest concentration tested. n.d., not determined. PHARMACOLOGY † Measurement of luciferase-coupled nuclear factor of activated T cells induction.

Koehbach et al. PNAS | December 24, 2013 | vol. 110 | no. 52 | 21185 Downloaded by guest on September 25, 2021 both receptors is highly conserved, and it is therefore not sur- prising that many drugs engage both receptors (26, 27). In fact, the oxytocin receptor antagonist atosiban, which is used clinically to delay preterm , is also a potent antagonist at the V1a receptor (23). Adverse events reported after administration of oxytocin and the original remedy kalata-kalata include a de- crease in blood pressure and cardiotoxic effects (4, 28); these might be related to the observed cross-activity, particularly if the partial agonistic action of the effect of kalata B7 on V1a receptor is taken into account (Fig. 2B). Based on the observed pharmacological properties of kalata B7, we performed a structural analysis to define candidate in- teraction residues with the oxytocin receptor. Cyclotides are three times larger than oxytocin and presumably cannot enter deep into the binding pocket of receptors. However, the NMR structure showed that the side chains of the Tyr and Gln residues in loop 3 protrude from the backbone, and hence are capable of interacting with the oxytocin receptor. To our knowledge, kalata B7 is the only cyclotide containing a tyrosine residue and a glu- tamine residue in this loop. Both residues are also present in native oxytocin. In fact, the tyrosine at position 2 in oxytocin has been shown to be important for receptor–ligand interaction with residues Y209 and F284 of the oxytocin receptor (29–31). In addition, loop 3 of kalata B7 contains a type II β-turn, which is also important for the activity of oxytocin (24). Nonapeptide analogs of oxytocin are flexible, and therefore can adopt several conformations that may allow for accommo- dating differences in the ligand binding pocket of their target receptors. In fact, of the several peptides that were designed using the kalata B7 intercysteine loop 3 as a template, kB7-OT1 ([G5, T7, S9]-oxytocin) was found to be a selective agonist at the oxytocin receptor because it stimulated the receptor in the sub- micromolar range but did not bind to any of the other related receptors (i.e., V1a,V1b,V2 receptor) up to concentrations of 10 μM(Fig.4).Weconfirmed that this agonist effectively stimulated intact human myometrium (Fig. 5). The NMR data suggest that kB7-OT1 and authentic oxytocin are very similar in their overall structure (compare 1H chemical shifts in Fig. S5). A comparison of the structural ensembles from the solution structures of oxy- tocin and kB7-OT1 reveals similarly dynamic exocyclic tails, with Fig. 3. NMR solution structures of kalata B7 (A), oxytocin (B, red), and the more defined regions of the structures overlaying well (Fig. kalata B7-OT1 ([G5, T7, S9]-oxytocin) (C, blue). Superimposition of the 20 α 3). The sequence of kB7-OT1 (CYIQGCTLS) is a combination lowest energy structures shows the backbone chain (N, C, and C atoms) and of the kalata B7-loop 3 (-CYTQGCTCS-) and oxytocin cysteine connectivity (yellow). The sequence of each peptide is shown above (CYIQNCPLG). Tyr2, Ile3, and Leu8 are known to be important the structures. The structural ensemble of the side-chain orientations of for receptor recognition of oxytocin-like peptides (30). This is in residues Y15 and Q17 of kalata B7 is illustrated. (D) Representative ribbon line with our data (Table 1). A change of Asn to Gly in position 5 diagrams of oxytocin (red) and kB7-OT1 (blue) overlaid across the backbone atoms of residues 1–6 (rmsd of 0.65 Å). of oxytocin does not impede the ability to bind and activate the receptor but contributes to receptor selectivity. This feature has been previously appreciated for oxytocin and V1a receptor contractions in human myometrium strips. Unlike the selective antagonists (27). Based on our observations, we also consider this nonapeptide, the extract also significantly stimulated frequency of relevance in the future development of selective agonists for the of contractions and atosiban did not completely abolish stimula- human oxytocin and vasopressin receptors. There has only been tory effects, suggesting the presence of other uterotonic substances modest progress made over the past two decades in identifying in the plant extract. Taken together, these observations provide selective agonists for the four receptor subtypes (23). Hence, it is, per se, of interest that a selective agonist for the oxytocin re- formal proof that the oxytocin receptor is a target of kalata fi ceptor was discovered by extracting sequence information derived cyclotides, particularly kalata B7 peptide, which was identi ed from a cyclotide, revealing the intriguing possibility that ligands by MS and peptide sequencing as the major compound in the for other human receptors could be discovered in this way. fraction (OA44–45) displaying the greatest contractility (Fig. 1 At a more general level, our work provides a proof of concept and Fig. S1). Sequence analysis of kalata B7 showed high ho- that naturally occurring peptides serve as templates for GPCR mology between its loop 3 (-CYTQGC-) and human oxytocin ligand design (25, 32). Cyclotides represent a natural combina- (CYIQNCPLG) (Table S1). In humans, oxytocin acts on the torial peptide library, and they probe a chemical space that is oxytocin and vasopressin receptors and is one of the key players difficult to target by using small organic molecules that have in the induction of labor and uterine contraction (20). Oxytocin been created by various synthetic strategies. Thus, at the very and V1a receptors are both expressed in the pregnant human uterus least, they can be anticipated to complement the existing col- and are up-regulated during parturition, making the tissue more lections of compounds that are used in drug discovery by high- sensitive for stimulation (26). The endogenous ligand oxytocin throughput screening and related approaches. In fact, cyclotides is used clinically to induce labor and to prevent life-threatening have recently been used as scaffolds to improve the stability of postpartum bleeding (20). Kalata B7 is a partial agonist on peptides that have interesting biological activities. This grafting uterine smooth muscle cells and cells expressing the human introduced peptide sequences into cyclotide loops and resulted in oxytocin and V1a receptors (Fig. 2B). The extracellular face of chimeric molecules, which bound to G protein-coupled receptors

21186 | www.pnas.org/cgi/doi/10.1073/pnas.1311183110 Koehbach et al. Downloaded by guest on September 25, 2021 3 Fig. 4. Pharmacological selectivity of synthetic kalata B7-OT1 ([G5, T7, S9]-oxytocin) on oxytocin/vasopressin receptors. (A) Binding of [ H]oxytocin (2 nM) to membranes from HEK293 cells (30–100 μg per assay) expressing the human oxytocin receptor was measured in an excess of OT peptide (0.1 nM to 1 μM) and kB7-OT1 (0.3 nM to 3 μM). (B) Ability of the peptides (0.03 nM to 10 μMOTand3nMto30μMkB7-OT1) to signal through Gq and activate downstream DNA binding elements of GPCR activation in HEK293 cells stably transfected with the human oxytocin receptor was measured with a luciferase reporter assay. Data were fitted with nonlinear regression (sigmoidal, variable slope) and are shown as the mean ±SEM of three independent experiments. Binding data are normalized to the percentage (%) of maximal binding; the 100% value refers to an average of 1.57 pmol of ligand bound per milligram of membrane. Activation data are normalized to the number of cells and fold induction

above baseline. Ki and EC50 values are listed in Table 1. (C) Selectivity of kB7-OT1 was tested on all four human receptors [i.e., the oxytocin receptor (OTR) and vasopressin V1a,V1b,andV2 receptors]. Maximal binding (100%) refers to values of 1.57 pmol/mg for the OTR, 0.97 pmol/mg for the V1a receptor, 0.78 pmol/mg for the V1b receptor, and 0.30 pmol/mg for the V2 receptor, respectively. Statistical differences were analyzed using an unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001).

(33, 34), inactivated VEGF (35), stimulated angiogenesis (36), the active segment of the cyclotide to create a selective ligand) and blocked entry of HIV via CXCR4 (37), and inhibited serine pro- documented a plant peptide sharing similarity in sequence and teases (38). Here, we used the reverse approach (i.e., we extracted activity with oxytocin. The discovery of the active ergot ingredients produced by the fungus Claviceps purpurea was instrumental to the development of modern pharmacology. Accordingly, C. purpurea has been referred to as the treasure trove of pharmacology (39). Incidentally, ergot also contains (methyl)ergometrine/(methyl)er- gonovine (i.e., the first selective uterotonic compounds introduced into clinical medicine). We are aware of the limitations of his- torical comparisons. Nevertheless, we believe that the rich diversity of cyclotides justifies that they also be considered as a potential treasure trove for drug discovery. Materials and Methods Detailed materials and methods are given in SI Materials and Methods.

Plant Extraction, RP-HPLC Fractionation, and Peptide Isolation. Aerial parts of O. affinis DC. were extracted and purified as described previously (40), yielding a starting extract of peptides. Fractionation and isolation of cyclo- tides were carried out using RP-HPLC on a Dionex Ultimate 3000 unit (Thermo-Scientific Dionex).

MS and Peptide Identification. Analysis of peptides was performed on a MALDI-TOF/TOF 4800 Analyzer (AB Sciex). MS and tandem MS experiments were carried out as described previously (8). Before MALDI analysis, samples were

desalted using C18 ZipTips (Millipore). Spectra were processed using DataExplorer software (AB Sciex), and cyclotides were characterized by manual peptide sequencing.

Cloning, Cell Culture, Transfection, and Membrane Preparation. Oxytocin re-

ceptor and V1a,V1b, and V2 receptor cDNA sequences were inserted into pEGFP-N1 plasmids to yield C-terminal GFP fusion . Preparation of stably transfected HEK293 cell lines, propagation, and membrane isolation were similar to previously described methods (41).

Radioligand Displacement Assays. Isolated membranes were incubated with radioactive agonists [3H]oxytocin (2 nM) or [3H]arginine-vasopressin (0.75 nM) and various concentrations of competing peptide. The reaction was stopped by filtration over glass fiber filters using a cell harvester. Nonspecific binding was determined in the presence of 1 μM oxytocin or vasopressin, respectively.

Functional Receptor Activation Assays. Luciferase-based reporter assays were performed as described previously (41). Briefly, HEK cells were transfected fi fl Fig. 5. Uterostimulant effects of O. affinis extract and kalata B7-OT1 ([G5, T7, S9]- with re y luciferase containing plasmid pGL4.30 luc2P. After transfection, oxytocin) on human myometrium. Spontaneous contractions of term and non- cells were seeded into 96-well plates and incubated with logarithmically laboring human myometrium superfused with physiological saline solution at 37 °C. spaced concentrations of peptides. Following incubation, medium was re- (A) Application of 1 mg·mL−1 extract of O. affinis followed by 0.5 nM oxytocin. (B) moved and cells were frozen at −80 °C. Following cell lysis, luciferase activity – Dose responses from 1 nM to 100 nM kB7-OT1.(C) Effects of kB7-OT1 in the was measured using a Promega luciferase reagent kit. IP1 accumulation PHARMACOLOGY presence of the oxytocin- and vasopressin V1a receptor antagonist, atosiban (1 μM). measurements were carried out using the Cisbio IP1 homogeneous time-

Koehbach et al. PNAS | December 24, 2013 | vol. 110 | no. 52 | 21187 Downloaded by guest on September 25, 2021 resolved fluorescence assay. Cells were incubated with peptides for 1 h prior codes for kalata B7, human OT, and [G5, T7, S9]-OT (kB7-OT1) are 2M9O, to fluorescence measurements on a SynergyH4 microplate reader (Biotek) 2MGO, and 2RU2, respectively. according to the manufacturer’s recommendations. Organ Bath Myometrial Contractility Assays. Isometric force recordings were Collagen Gel Contractility Assays. Human uterine myometrial smooth muscle made on strips of human myometrium obtained from the lower uterine cells (hTERT-HM) were cultured, and collagen gels were prepared as described incision site at caesarean section. All women gave informed written consent previously (42). Gel images were taken using a Fluorchem 8900 imager for participation. The study was approved by the North West (Liverpool East) (Alpha Innotech Corporation), and the gel area was measured using AlphaEaseFC Research Ethics Committee (LREC 10/H1002/49) and by the Research and software (Alpha Innotech Corporation). Collagen contraction, correlating to Development Director of Liverpool Women’s Hospital National Health Service · −1 decrease in gel area, was determined in quadruplicate. Data were statisti- Foundation Trust, Liverpool, United Kingdom. Crude extract (1 mg mL )or – cally analyzed using one-way ANOVA. the selective nonapeptide kB7-OT1 (1 100 nM) was added directly to the organ bath. In some experiments, strips were pretreated with atosiban (1 μM). Cyclotide and Peptide Synthesis. Peptides were synthesized using Fmoc solid- Contraction amplitude and area under the contraction curve were measured and compared with spontaneous control activity (100%) using OriginPro 8.5 phase peptide synthesis. After cleavage from resin, peptides were oxidized in software (OriginLab Corporation) as previously described (43). 0.1 M ammonium bicarbonate at pH 8.2 for 24 h and purified on RP-HPLC to yield >95% purity. ACKNOWLEDGMENTS. This work was supported by Austrian Science Fund Grant P22889-B11 (to C.W.G.). M. Muttenthaler was supported by the European NMR Spectroscopy. Samples were dissolved in 90% (vol/vol) H2O/10% (vol/vol) Union Seventh Framework Programme (FP7/2007-2013) under Grant Agree- D2O or 100% D2O and spectra recorded on a Bruker 600-MHz spectrometer ment 254897. N.L.D. is an Australian Research Council Future Fellow. D.J.C. is a at temperatures of 290K and 298K. The Protein Data Bank (www.pdb.org)ID National Health and Medical Research Council Professorial Fellow.

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