Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design

Significance

G protein-coupled receptors (GPCRs) are promising drug targets: >30% of the currently marketed drugs elicit their actions by binding to these transmembrane receptors. However, only ∼10% of all GPCRs are targeted by approved drugs. Resorting to plant-derived compounds catalogued by ethnopharmacological 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 peptides, investigated the molecular mechanisms underlying their uterotonic activity, and report an oxytocic plant peptide that modulates the human oxytocin/vasopressin receptors. This naturally occurring peptide served as a template for the design of an oxytocin-like nonapeptide with enhanced receptor selectivity, highlighting the potential of cyclotides for the discovery of peptide-based GPCR ligands.

Abstract

Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as “kalata-kalata,” the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V_1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands.

Cyclotides are head-to-tail cyclized plant peptides containing three conserved disulfide bonds in a knotted arrangement known as a cyclic cystine-knot motif (1). This confers them high stability (2) and presumably improves their oral bioactivity relative to their linear counterparts (3). They were first discovered in a decoction of Oldenlandia affinis DC. (Rubiaceae) leaves, an herbal remedy used in traditional African medicine during childbirth (4). The observed induction of labor and shortened delivery time were later studied on isolated rat and rabbit uteri and on human uterine strips (4, 5). The peptides responsible for the contractility effects (5) raised interest because they survived boiling, presumably as a result of their unique 3D structure, which was elucidated in 1995 (6). Since then, several plant species of the coffee (Rubiaceae) (7), violet (Violaceae) (8), legume (Fabaceae) (9), potato (Solanaceae) (10) and grass (Poaceae) families (11) have been identified to produce cyclotides. Currently, ∼300 sequences have been reported (12), and the predicted number of >50,000 cyclotides in Rubiaceae alone (7) suggests them to be one of the largest peptide classes within the plant kingdom. Their high intercysteine sequence variability and structural plasticity (13), together with intrinsic bioactivities, make them interesting templates for the development of novel pharmaceuticals (14).

However, five decades after the discovery of cyclotides, there still is not any information about specific molecular targets and/or mechanisms underlying their biological activities. It is known that cyclotides can, at higher concentrations, disrupt phospholipid bilayers (15, 16), because they expose hydrophobic residues on their surface. This endows them with physicochemical properties allowing for insertion into membranes and pore formation (17, 18). Although no cyclotide target receptor has been identified hitherto, the observed biological activities (e.g., their uterotonic effects) may be explained by specific receptor-mediated mechanisms. In mammals, including humans, uterine muscle contractility can be elicited by activation of various signaling pathways. One physiological regulator of uterine contraction is the neuropeptide oxytocin. In uterine tissue, this peptide activates oxytocin and vasopressin V_1a receptors (19–21), two members of the G protein-coupled receptor (GPCR) family. GPCRs are prominent drug targets, with ∼30% of all marketed drugs acting via modulation of these receptors (21).

We used a bioactivity-guided fractionation approach combined with pharmacological and structural analysis to elucidate the mechanism underlying the oxytocic activity of cyclotides and identified a molecular target for native cyclotides. In addition, we used cyclotides as a template to explore substitutions that enhanced receptor binding and agonistic activity. Our observations provide a proof of concept that (i) naturally occurring peptide libraries cover a chemical space that intersects with the sampled GPCRs and (ii) cyclotides can serve as a template for the design of new classes of GPCR ligands, thus opening new avenues for cyclotide-based drug development.

Results

Bioactivity-Guided Fractionation of Uterotonic Plant Cyclotides.

An herbal extract that has been used for many generations by traditional healers for its uterotonic properties was analyzed by bioactivity-guided fractionation. Dried aerial parts of O. affinis were extracted by grinding, solvent partitioning, and solid C₁₈-phase extraction of the aqueous filtrate to yield a crude cyclotide extract. The analysis by RP-HPLC and MALDI-TOF MS showed that this extract contained a number of cyclotides identified based on their mass, cysteine content, and hydrophobicity (7) (Fig. 1A and Fig. S1). Four subfractions eluting in the range of 18–54% acetonitrile were collected by preparative RP-HPLC and tested for their ability to induce contractions of human uterine smooth muscle cells using a collagen gel contractility assay (22) (Fig. S2). Compared with unstimulated cells, incubation with cyclotide-containing extracts showed a significant decrease in the collagen gel area, which reflected an increased contraction of the smooth muscle cells. Further RP-HPLC fractionation generated 15 subfractions, of which six induced significant contraction, ranging from 6.8–18.7% increased contractility over unstimulated cells (Fig. 1B).

Fig. 1.

Bioactivity-guided fractionation of O.affinis peptide extracts. (A) Analytical RP-HPLC chromatogram of a peptide extract from O. affinis leaves after solvent extraction and in-batch C₁₈ purification shows multiple peptide peaks, as determined by MALDI MS (Fig. S1). Fractions that were tested for biological activity are labeled with dashed lines. Fraction OA_44–45, showing the highest contractility (see B), is highlighted in gray. mAU, milli-absorption units. (B) Collagen gel contractility data of partially purified cyclotide fractions (1.25 mg⋅mL⁻¹) on isolated human uterus smooth muscle cells. Data represent the mean ± SEM. Statistical differences were analyzed using one-way ANOVA (**P < 0.01; ***P < 0.001).

Molecular and Pharmacological Characterization of the Oxytocic Cyclotide Kalata B7.

The 15 HPLC fractions were analyzed by MS and tandem MS peptide sequencing, and 17 cyclotides were identified, with each of the fractions containing one to five peptides. Cyclotide sequences within biologically active fractions display sequence homology to human oxytocin, whereas cyclotides from inactive fractions lack any appreciable similarity (Table S1). In particular, loop 3 of the cyclotide kalata B7 (-CYTQGC-) found in the most active fraction (Fig. 1B) and the six-residue ring of oxytocin (CYIQNC-) have related sequences. Therefore, kalata B7 was isolated by RP-HPLC, and the purified cyclotide (Fig. S3) was analyzed (i) for its ability to stimulate contractions of uterine smooth muscle cells and (ii) for its affinity to human oxytocin receptor or V_1a receptor. Kalata B7 displaced tritiated oxytocin or vasopressin in a dose-dependent manner from the binding site of the oxytocin receptor or V_1a receptor with a K_i of 50 μM and 12 μM, respectively (Fig. 2A and Table 1). It also provoked significant contraction of uterine cells (i.e., 8.4% increased contraction compared with unstimulated control cells) (Fig. 2C). We verified that the cyclotide kalata B7 acted via the oxytocin receptor and/or V_1a receptor on uterus cells by applying kalata B7 together with the receptor antagonist atosiban (23); this coapplication of both compounds resulted in a significant loss of contractility.

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 V_1a 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 V_1a receptor. Fold induction of intracellullar IP1 accumulation above baseline was normalized to the number of cells. K_i 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 V_1a 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.

Table 1.

Pharmacology of kalata B7 and oxytocin-like peptides

Peptide	Sequence	Binding affinity		IP1 formation
		Ki, M*		EC50, M
		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
[G5,T7,S9]-OT (kB7-OT1)	CYIQGCTLS-NH2	2.2 ± 0.2 × 10−7	>1.0 × 10−5	1.5 ± 0.1 × 10−7	n.d.
				3.6 ± 0.4 × 10−7†
[T3,G5,T7,S8,S9]-OT	CYTQGCTSS-NH2	>1.0 × 10−5	>1.0 × 10−5	>5.0 × 10−5	n.d.
[T3,P4,G5,S7,S8,T9]-OT	CYTPGCSST-NH2	>1.0 × 10−5	>1.0 × 10−5	>5.0 × 10−5	n.d.
[P4,G5,S7,T9]-OT	CYIPGCSLT-NH2	>1.0 × 10−5	>1.0 × 10−5	>5.0 × 10−5	n.d.
kB7-OT1 [Y2A]	CAIQGCTLS-NH2	>1.0 × 10−5	n.d.	>5.0 × 10−5	n.d.
kB7-OT1 [Q4A]	CYIAGCTLS-NH2	>1.0 × 10−5	n.d.	>5.0 × 10−5	n.d.
OT	CYIQNCPLG-NH2	1.8 ± 0.1 × 10−9	n.d.	3.4 ± 0.5 × 10−9	n.d.
				4.2 ± 0.5 × 10−9†
Vasopressin	CYFQNCPRG-NH2	n.d.	8.0 ± 0.2 × 10−10	n.d.	1.0 ± 0.1 × 10−9

^*Binding affinity (K_i) and functional receptor activation (EC₅₀) data are the mean ± SEM of two to four independent experiments. K_i values were calculated using IC₅₀ values according to Cheng and Prusoff (44), with a K_d value of 1.5 nM for oxytocin (OT) on the oxytocin receptor and 0.6 nM for vasopressin on the V_1a receptor. If no IC₅₀ value has been determined, the given values represent the highest concentration tested. n.d., not determined.

^†Measurement of luciferase-coupled nuclear factor of activated T cells induction.

If kalata B7 is an agonist at the oxytocin receptor and/or the V_1a receptor, it ought to trigger signaling via a G_q-dependent pathway. We verified this prediction by measuring the generation of inositol-1-phosphate (IP1) in response to the cyclotide in HEK293 cells heterologously expressing either receptor. The analysis of the concentration–response curve showed that kalata B7 was a partial agonist at both the oxytocin receptor and the V_1a receptor (Fig. 2B) with an EC₅₀ of 12 μM and 4 μM, respectively. Agonistic activity was more pronounced at the oxytocin receptor (about 80% of the response elicited by oxytocin) than at the V_1a receptor (about 40% of the response elicited by vasopressin).

Structural Characterization of Kalata B7.

To understand the ligand–receptor interaction, the structure of kalata B7 was determined by NMR (Fig. 3A and Table S2). This revealed a well-defined backbone around the cyclic cystine-knot motif typical for Möbius cyclotides, a type I β-turn between residues 9 and 12, a type II β-turn between residues 16 and 19, and a type VIa1 β-turn between residues 22 and 25, as well as a β-hairpin between residues 20 and 28. As shown in Fig. 3, loop 3 of kalata B7 (-CYTQGC-) is homologous to the six-residue ring sequence of human oxytocin (CYIQNC-). In particular, the tyrosine (Y15) and glutamine (Q17) residues of kalata B7 are in positions analogous to those (Y2 and Q4) in oxytocin. NMR structural analysis of human oxytocin confirmed the presence of a type II β-turn (24) (Fig. 3B). Therefore, loop 3 of kalata B7 and human oxytocin share similarities in sequence and 3D structure. Furthermore, the structure of kalata B7 indicated that the side chains of Tyr and Gln in loop 3 protrude from the backbone (Fig. 3A). Hence, they might be capable of interacting with the oxytocin receptor. The crucial role of the tyrosine and glutamine residues (loop 3) of the cyclotide was confirmed by generating mutated variants (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).

Fig. 3.

NMR solution structures of kalata B7 (A), oxytocin (B, red), and kalata B7-OT₁ ([G5, T7, S9]-oxytocin) (C, blue). Superimposition of the 20 lowest energy structures shows the backbone chain (N, C, and C^α atoms) and cysteine connectivity (yellow). The sequence of each peptide is shown above the structures. The structural ensemble of the side-chain orientations of residues Y15 and Q17 of kalata B7 is illustrated. (D) Representative ribbon diagrams of oxytocin (red) and kB7-OT₁ (blue) overlaid across the backbone atoms of residues 1–6 (rmsd of 0.65 Å).

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 nonapeptides. 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 determined by ¹H chemical shifts (Fig. S5) and structural calculations (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 comprising residues 1–6 that overlay well (rmsd of 0.65 Å, Fig. 3D). The synthetic oxytocin-like peptides were tested for binding and receptor activation (Fig. 4 and Fig. S4). [G5, T7, S9]-oxytocin (kalata B7-OT₁) had improved binding affinity (K_i = 218 nM; Fig. 4A). This increased affinity was also evident, when assessing its ability to promote luciferase transcription in cells expressing the human oxyocin receptor (EC_{50 IP1 formation} = 145 nM, EC_{50 luciferase induction} = 356 nM; Table 1), where it acted as a full agonist (compare with the maximum response of oxytocin and kalata B7-OT₁ in Fig. 4B). Similar to the mutated cyclotide, the kB7-OT₁ mutants (Y2A) and (Q4A) had lost their affinity and did not activate the oxytocin receptor (Table 1 and Fig. S4). Interestingly, kB7-OT₁ proved to be selective for the oxytocin receptor because it did not compete for binding of radiolabeled vasopressin on any of the three human vasopressin (V_1a, V_1b, and V₂) receptors at concentrations up to 10 μM (Fig. 4C) in contrast to native oxytocin (25).

Fig. 4.

Pharmacological selectivity of synthetic kalata B7-OT₁ ([G5, T7, S9]-oxytocin) on oxytocin/vasopressin receptors. (A) Binding of [³H]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 μM OT and 3 nM to 30 μM kB7-OT₁) to signal through G_q 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 gene 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. K_i 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 V_1a, V_1b, and V₂ receptors]. Maximal binding (100%) refers to values of 1.57 pmol/mg for the OTR, 0.97 pmol/mg for the V_1a receptor, 0.78 pmol/mg for the V_1b 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).

Uterostimulant Effects of O. affinis Extract and Kalata B7-OT₁ on Human Myometrium.

The crude “kalata-kalata” plant extract and synthetic kB7-OT₁ ([G5, T7, S9]-oxytocin) were applied directly to organ baths containing strips of human myometrium superfused with physiological saline solution. Application of 1 mg⋅mL⁻¹ crude extract resulted in stimulation of contraction amplitude by +8.1 ± 3.5%, whereas the area under the curve increased by 313.5 ± 96% (mean ± SEM; n = 6) (Fig. 5A). Pretreatment with atosiban significantly reduced but did not abolish the stimulatory effect of the extract (Fig. S6). Application of kB7-OT₁ resulted in a dose-dependent increase in both contraction amplitude (1 nM: +6.0 ± 3.2%; 10 nM: +41.4 ± 6.4%; 100 nM: +73.2 ± 5.4%) and area under the curve (1 nM: +2.7 ± 2.3%; 10 nM, +48.8 ± 20.3%; 100 nM; +218.3 ± 143.6%; n = 3), which was inhibited by pretreatment with atosiban (for amplitude, 1 nM: −0.8 ± 2.7%; 10 nM, −13.6 ± 7%; 100 nM: −2.8 ± 5.8%; for area under the curve, 1 nM: −14.5 ± 6.9%; 10 nM, −25.5 ± 4.8%; 100 nM: −11.1 ± 13.7%; n = 3) (Fig. 5 B and C).

Fig. 5.

Uterostimulant effects of O. affinis extract and kalata B7-OT₁ ([G5, T7, S9]-oxytocin) on human myometrium. Spontaneous contractions of term and nonlaboring human myometrium superfused with physiological saline solution at 37 °C. (A) Application of 1 mg⋅mL⁻¹ extract of O. affinis followed by 0.5 nM oxytocin. (B) Dose–responses from 1 nM to 100 nM kB7-OT₁. (C) Effects of kB7-OT₁ in the presence of the oxytocin- and vasopressin V_1a receptor antagonist, atosiban (1 μM).

Discussion

A decoction of O. affinis induces strong uterine contractions after either oral administration as a tea or intravaginal instillation (4, 5). In line with this contractile activity, we identified an active principle in peptide-containing extracts of O. affinis and HPLC-purified fractions containing various cyclotides based on the following criteria: (i) The peptide mixtures and purified kalata B7 elicited contractions of uterine muscle cells that were antagonized by the oxytocin receptor blocker atosiban; (ii) they displaced radiolabeled oxytocin from its heterologously expressed cognate receptor and vasopressin from the V_1a receptor, the closest relative of the oxytocin receptor; (iii) consistent with the oxytocin receptor being a G_q-coupled receptor, kalata B7 also triggered the formation of the canonical signaling downstream cascade, resulting in the increased accumulation of IP1; and (iv) in organ bath experiments, both the O. affinis extract and the nonapeptide kB7-OT₁ ([G5, T7, S9]-oxytocin) augmented contractions in human myometrium strips. Unlike the selective nonapeptide, the extract also significantly stimulated frequency of contractions and atosiban did not completely abolish stimulatory effects, suggesting the presence of other uterotonic substances in the plant extract. Taken together, these observations provide formal proof that the oxytocin receptor is a target of kalata cyclotides, particularly kalata B7 peptide, which was identified by MS and peptide sequencing as the major compound in the fraction (OA_44–45) displaying the greatest contractility (Fig. 1 and Fig. S1). Sequence analysis of kalata B7 showed high homology between its loop 3 (-CYTQGC-) and human oxytocin (CYIQNCPLG) (Table S1). In humans, oxytocin acts on the oxytocin and vasopressin receptors and is one of the key players in the induction of labor and uterine contraction (20). Oxytocin and V_1a receptors are both expressed in the pregnant human uterus and are up-regulated during parturition, making the tissue more sensitive for stimulation (26). The endogenous ligand oxytocin is used clinically to induce labor and to prevent life-threatening postpartum bleeding (20). Kalata B7 is a partial agonist on uterine smooth muscle cells and cells expressing the human oxytocin and V_1a receptors (Fig. 2B). The extracellular face of both receptors is highly conserved, and it is therefore not surprising that many drugs engage both receptors (26, 27). In fact, the oxytocin receptor antagonist atosiban, which is used clinically to delay preterm birth, is also a potent antagonist at the V_1a receptor (23). Adverse events reported after administration of oxytocin and the original remedy kalata-kalata include a decrease 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 V_1a receptor is taken into account (Fig. 2B).

Based on the observed pharmacological properties of kalata B7, we performed a structural analysis to define candidate interaction 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 glutamine 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 accommodating 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 submicromolar range but did not bind to any of the other related receptors (i.e., V_1a, V_1b, V₂ receptor) up to concentrations of 10 μM (Fig. 4). We confirmed 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 oxytocin and kB7-OT₁ reveals similarly dynamic exocyclic tails, with the more defined regions of the structures overlaying well (Fig. 3). The sequence of kB7-OT₁ (CYIQGCTLS) is a combination of the kalata B7-loop 3 (-CYTQGCTCS-) and oxytocin (CYIQNCPLG). Tyr2, Ile3, and Leu8 are known to be important for receptor recognition of oxytocin-like peptides (30). This is in line with our data (Table 1). A change of Asn to Gly in position 5 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 V_1a receptor antagonists (27). Based on our observations, we also consider this of relevance in the future development of selective agonists for the human oxytocin and vasopressin receptors. There has only been modest progress made over the past two decades in identifying selective agonists for the four receptor subtypes (23). Hence, it is, per se, of interest that a selective agonist for the oxytocin receptor was discovered by extracting sequence information derived from a cyclotide, revealing the intriguing possibility that ligands for other human receptors could be discovered in this way.

At a more general level, our work provides a proof of concept that naturally occurring peptides serve as templates for GPCR ligand design (25, 32). Cyclotides represent a natural combinatorial peptide library, and they probe a chemical space that is difficult to target by using small organic molecules that have been created by various synthetic strategies. Thus, at the very least, they can be anticipated to complement the existing collections of compounds that are used in drug discovery by high-throughput screening and related approaches. In fact, cyclotides have recently been used as scaffolds to improve the stability of peptides that have interesting biological activities. This grafting introduced peptide sequences into cyclotide loops and resulted in chimeric molecules, which bound to G protein-coupled receptors (33, 34), inactivated VEGF (35), stimulated angiogenesis (36), blocked entry of HIV via CXCR4 (37), and inhibited serine proteases (38). Here, we used the reverse approach (i.e., we extracted the active segment of the cyclotide to create a selective ligand) and documented a plant peptide sharing similarity in sequence and 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)ergonovine (i.e., the first selective uterotonic compounds introduced into clinical medicine). We are aware of the limitations of historical 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 cyclotides 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 receptor and V_1a, V_1b, and V₂ receptor cDNA sequences were inserted into pEGFP-N1 plasmids to yield C-terminal GFP fusion proteins. 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 with firefly luciferase containing plasmid pGL4.30 luc2P. After transfection, cells were seeded into 96-well plates and incubated with logarithmically spaced concentrations of peptides. Following incubation, medium was removed and cells were frozen at −80 °C. Following cell lysis, luciferase activity was measured using a Promega luciferase reagent kit. IP1 accumulation measurements were carried out using the Cisbio IP1 homogeneous time-resolved fluorescence assay. Cells were incubated with peptides for 1 h prior to fluorescence measurements on a SynergyH4 microplate reader (Biotek) according to the manufacturer’s recommendations.

Collagen Gel Contractility Assays.

Human uterine myometrial smooth muscle cells (hTERT-HM) were cultured, and collagen gels were prepared as described previously (42). Gel images were taken using a Fluorchem 8900 imager (Alpha Innotech Corporation), and the gel area was measured using AlphaEaseFC software (Alpha Innotech Corporation). Collagen contraction, correlating to decrease in gel area, was determined in quadruplicate. Data were statistically analyzed using one-way ANOVA.

Cyclotide and Peptide Synthesis.

Peptides were synthesized using Fmoc solid-phase peptide synthesis. After cleavage from resin, peptides were oxidized in 0.1 M ammonium bicarbonate at pH 8.2 for 24 h and purified on RP-HPLC to yield >95% purity.

NMR Spectroscopy.

Samples were dissolved in 90% (vol/vol) H₂O/10% (vol/vol) D₂O or 100% D₂O and spectra recorded on a Bruker 600-MHz spectrometer at temperatures of 290K and 298K. The Protein Data Bank (www.pdb.org) ID codes for kalata B7, human OT, and [G5, T7, S9]-OT (kB7-OT1) are 2M9O, 2MGO, and 2RU2, respectively.

Organ Bath Myometrial Contractility Assays.

Isometric force recordings were made on strips of human myometrium obtained from the lower uterine incision site at caesarean section. All women gave informed written consent for participation. The study was approved by the North West (Liverpool East) Research Ethics Committee (LREC 10/H1002/49) and by the Research and Development Director of Liverpool Women's Hospital National Health Service Foundation Trust, Liverpool, United Kingdom. Crude extract (1 mg⋅mL⁻¹) or 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). Contraction amplitude and area under the contraction curve were measured and compared with spontaneous control activity (100%) using OriginPro 8.5 software (OriginLab Corporation) as previously described (43).