One-sentence summary: An oxytocin derivative that may not trigger adverse side effects has been generated.

Editor’s summary:

A more selective oxytocin receptor agonist

Oxytocin is clinically used to induce labor, and there is growing interest in using this peptide to treat

social disorders. However, oxytocin activates not only the oxytocin receptor but also vasopressin

receptors which can trigger adverse side effects. Muttenthaler et al. generated ligands based on

oxytocin with subtle modifications, yielding a lead compound that was more selective for the oxytocin

receptor than for the vasopressin receptors. It reduced social fear in mice and induced contractile

activity in human myometrial strips without affecting cultured cardiomyocytes. Given the crosstalk

between oxytocin, vasopressin, and their receptors, this compound is a promising therapeutic lead with

an improved safety profile and a valuable probe in identifying effects that are solely mediated by the

oxytocin receptor.

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Subtle modifications to oxytocin produce ligands that retain potency and

improved selectivity across species

Markus Muttenthaler1,2*, Åsa Andersson1, Irina Vetter1,3, Rohit Menon4, Marta Busnelli5,6, Lotten

Ragnarsson1, Christian Bergmayr7, Sarah Arrowsmith8, Jennifer R. Deuis1, Han Sheng Chiu1, Nathan

J. Palpant1, Margaret O'Brien9, Terry J. Smith9, Susan Wray8, Inga Neumann4, Christian W. Gruber7,10,

Richard J. Lewis1 and Paul F. Alewood1*

1 Institute for Molecular Bioscience, The University of Queensland, 4072, Brisbane, Australia

2 Faculty of Chemistry, Institute of Biological Chemistry, University of Vienna, 1090, Vienna,

Austria

3 School of Pharmacy, The University of Queensland, 4104, Brisbane, Australia

4 Department of Behavioral and Molecular Neurobiology, Regensburg Center of Neuroscience,

University of Regensburg, 93053, Regensburg, Germany

5 CNR - Institute of Neuroscience, 20129, Milan, Italy

6 Department of Biotechnology and Translational Medicine, University of Milan, 20129, Milan,

Italy

7 Center for Physiology and Pharmacology, Medical University of Vienna, 1090, Vienna, Austria

8 Department of Cellular and Molecular Physiology, Harris-Wellbeing Preterm Birth Centre,

Institute of Translational Medicine, University of Liverpool, L69 3BX, Liverpool, United Kingdom

9 National Centre for Biomedical Engineering Science, National University of Ireland, H91 CF50,

Galway, Ireland

10 School of Biomedical Sciences, The University of Queensland, 4072, Brisbane, Australia

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* Correspondence to:

Markus Muttenthaler Email: m.muttenthaler@uq.edu.au Tel: +61 7 3346 2985

Email: markus.muttenthaler@univie.ac.at Tel: +43 1 4277 70512

Paul F. Alewood Email: p.alewood@uq.edu.au Tel: +61 7 3346 2982

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ABSTRACT

Oxytocin and vasopressin mediate various physiological functions that are important for

osmoregulation, reproduction, cardiovascular function, social behavior, memory and learning through

four G protein-coupled receptors that are also implicated in high-profile disorders. Targeting these

receptors is challenging because of the difficulty in obtaining ligands that retain selectivity across

rodents and humans for translational studies. We identified a selective and more stable oxytocin

receptor (OTR) agonist by subtly modifying the pharmacophore framework of human oxytocin and

vasopressin. [Se-Se]-Oxytocin-OH displayed similar potency to oxytocin but improved selectivity for

OTR, an effect that was retained in mice. Centrally infused [Se-Se]-Oxytocin-OH potently reversed

social fear in mice, confirming that this action was mediated by OTR and not by V1a or V1b

vasopressin receptors. Additionally, [Se-Se]-Oxytocin-OH produced a more regular contraction pattern

than did oxytocin in a preclinical labor induction and augmentation model using myometrial strips from

cesarean sections. [Se-Se]-Oxytocin-OH had no activity in human cardiomyocytes, indicating a

potentially improved safety profile and therapeutic window compared to those of clinically used

oxytocin. In conclusion, [Se-Se]-Oxytocin-OH is a novel probe for validating OTR as a therapeutic

target in various biological systems and is a promising new lead for therapeutic development. Our

medicinal chemistry approach may also be applicable to other peptidergic signaling systems with

similar selectivity issues.

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INTRODUCTION

Oxytocin and vasopressin (also known as arginine-vasopressin) are closely related neurohypophysial

neuropeptides that are mainly synthesized in the magnocellular and parvocellular neurons of the

hypothalamus, transported in association with neurophysins to the posterior pituitary, and released into

the systemic circulation after enzymatic cleavage in response to relevant physiological stimuli (1, 2). In

the periphery, oxytocin is involved in uterine smooth muscle contraction during parturition, milk

ejection during lactation, ejaculation, and pain (3-5), while centrally released oxytocin functions as a

neurotransmitter or neuromodulator that promotes multiple behaviors (6-8) such as maternal care (9,

10), partnership bonding (8, 11, 12), social interactions (12), and stress and anxiety responses (13-15),

as largely determined in rodents. Intranasal administration of oxytocin elicits broad behavioral effects

in humans and its therapeutic potential for psychopathologies characterized by social or emotional

dysfunctions is under clinical investigation (14, 16-21). Vasopressin increases and decreases fluid

balance and blood pressure in the periphery (22-24). Centrally, vasopressin is implicated in learning

and memory (25-27), in various social behaviors including pair bonding and aggression in rodents (6,

8, 28, 29), and in stress- and anxiety-related behaviors (28-30). The ubiquitous involvement of the

oxytocin and vasopressin signaling system in diverse physiological functions reflects its ancient origin

dating back at least 600 million years (8, 31). The oxytocin and vasopressin receptors are members of

the G protein-coupled receptor (GPCR) family (5, 32) and are attractive targets in the treatment of

various high-profile disorders including cancer, pain, autism, schizophrenia, anxiety, and reproductive

and cardiovascular disorders (5, 8, 16, 33).

In humans and rodents, oxytocin acts through one receptor (OTR) and vasopressin through three

receptors (AVPRs: vasopressor V1aR, pituitary V1bR, antidiuretic V2R). Oxytocin and vasopressin are

structurally similar nonapeptides that differ only by two amino acids at positions 3 and 8 (Fig. 1A).

Two cysteine residues in positions 1 and 6 form the cyclic part of the molecules followed by a 3-

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residue amidated C-terminal tail. Their chemical similarity and the high sequence homology of the

extracellular binding domains of OTR and AVPRs (~80%) leads to substantial cross talk, with oxytocin

able to activate the AVPRs and vasopressin OTR (34, 35). Specific receptor functionality is thus not

controlled by ligand selectivity, but by cell-specific variations in receptor abundance, controlled

release, receptor oligomerization, rapid clearance and specific enzymatic degradation (36). High

oxytocin and vasopressin receptor homology and overlapping distribution constitute a major hurdle in

the development of selective receptor agonists, antagonists and therapeutic candidates (37, 38). The

identification of novel drug leads is further complicated by substantial species differences, such that

rodent and human selectivity typically do not overlap, thereby restricting clinical translation (37-39).

For example, clinically used oxytocin and vasopressin analogues including desmopressin (40, 41),

carbetocin (42) and atosiban (37, 43, 44) are receptor-subtype selective in rats but not in humans. This

difference is mainly due to rapid biodegradation, renal clearance and a limited administration window

during which they can be used clinically without side effects (40, 44, 45). Despite these limitations,

oxytocin remains the ligand of choice in the clinic to induce and progress labor (46). The lack of a

complete set of selective receptor agonists and antagonists further limits our ability to characterize the

physiological responses for each subtype receptor and their relevance in disease. We therefore

commenced a program to overcome these limitations and to produce more selective ligands for this

fundamental signaling system (31, 47-51). In this study, we demonstrated that small modifications to

the structural framework of the pharmacophore of the endogenous and pharmacologically unselective

neuropeptides could be used to tune selectivity and generate analogues with an improved selectivity

profile that was conserved across mouse and human, thereby facilitating translational studies.

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RESULTS

Rationale for subtle modifications. Traditional medicinal chemistry strategies that modify single or

multiple functional groups of oxytocin and vasopressin commonly yield selectivity improvements that

are not retained across species (37, 38). Based on structural data for oxytocin (4, 48, 52-54) and earlier

studies (37, 50, 55-57), we knew that oxytocin and vasopressin binding and receptor activation are

sensitive to even minor modifications. For example, ring reduction by one sulfur atom or disulfide

bond replacement by a dicarba bridge (-CH2-CH2-) causes complete loss of activity (56). N-terminal

deamination of oxytocin analogues often leads to improved OTR activation, which has led to the

generation of the slightly more stable and hydrophobic oxytocin super-agonist deamino-Oxytocin

(dOT) and the clinically used ligands Carbetocin, Desmopressin and Atosiban. (37, 55, 57).

Furthermore, changing Gly9 to Val9 in oxytocin and vasopressin switches these agonists to antagonists.

In addition, [Gly9Val]-oxytocin not only acts as an antagonist at the hV1aR but also no longer binds to

the hV1bR and the hV2R at concentrations up to 10 M (50). For vasopressin, replacement of Gly9 by

a carboxylic acid markedly decreases its vasopressor (V1aR) and antidiuretic (V2R) activity in rats

(58). We also included replacement of the disulfide bond with a diselenide bond, which has been

described as a structurally isosteric disulfide bond mimic that can increase potency and selectivity due

to the slightly more hydrophobic character of the diselenide bond (56, 59). Moreover, the lower redox

potential of the diselenide bond correlates to higher stability in a reducing environment and can

therefore be exploited to improve peptide half-life (56, 59-61). We therefore introduced subtle

modifications to the pharmacophore framework of oxytocin and vasopressin (Table S1 and Fig. 1A) to

identify those that yield selectivity gains that are retained across species.

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Peptide synthesis. Two building blocks [Boc-L-Sec(Meb)-OH and dSec(Meb)-OH] were synthesized

to generate the desired sulfur or selenium modifications (Fig. 1A & B, Table S1). To acquire Boc-L-

Sec(Meb)-OH in sufficient quantities, we optimized various protocols (62-64) to obtain 28.7 g of pure

Boc-L-Sec(Meb)-OH in only three steps (Fig. 1B, 62% overall yield in a four day procedure). We also

devised a new synthetic strategy for dSec(Meb)-OH, yielding the building block in a two-step synthesis

with an overall yield of 12% (Fig. 1B). With these building blocks in hand, all of the oxytocin and

vasopressin analogues 1-16 (Fig. 1A, Table S1) were synthesized by Boc-SPPS, folded in 0.1M

NH4HCO3 and purified by RP-HPLC (59, 65).

Functional response of novel oxytocin and vasopressin ligands.

A Fluorescent Imaging Plate Reader (FLIPR) Ca2+ mobilization assay (for OTR, V1aR, V1bR) and a

second messenger cyclic adenosine monophosphate (cAMP) assay (for V2R) showed that oxytocin

activated all four human (h) receptors. EC50 at the hV1aR and hV1bR were 10-300-fold higher than at

hOTR (EC50: hOTR≈hV2R<hV1bR<hV1aR) (Fig. 1C, Table S2). Deletion of the N-terminal amino

group (dOT) increased the ability of the ligand to activate all of the AVPRs, suggesting an improved fit

of dOT in the hydrophobic transmembrane binding pocket (47) across all four receptors. Replacement

of the disulfide bond with the isosteric, yet slightly more hydrophobic diselenide bond ([Se-Se]-OT,

d[Se-Se]-OT) was well tolerated and did not lead to marked changes in potency. In particular, d[Se-

Se]-OT showed the least change in potency and selectivity compared to oxytocin. Exchange of the C-

terminal amide to acid in oxytocin (OT-OH) resulted in weaker potency at all receptors and truncations

of the C-terminus of oxytocin (compounds 8, 9, 10) yielded less potent EC50 at all receptors with

complete loss of activity at hV2R (up to 10 µM). Compounds 6, 7, 9 and 10 all showed substantial

drops in interaction with AVPRs while retaining good potency at hOTR, thereby indicating that the C-

terminus of oxytocin is a good target for improving selectively for hOTR against the three AVPRs.

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In particular, the combination of diselenide replacement and C-terminal amide to acid change yielded

[Se-Se]-OT-OH (compound 6) with an improved selectivity profile for OTR compared to oxytocin

(Fig. 1C, Table S2). [Se-Se]-OT-OH displayed low nanomolar potency (7.3 nM) and partial agonism

(Emax=52%) at hOTR, no activation of hV1aR (>10 µM), a 600-fold higher EC50 at hV1bR, and a 15-

fold higher EC50 at hV2R (109 nM). N-terminal deamination of [Se-Se]-OT-OH yielded d[Se-Se]-OT-

OH, which did not have improved potency at OTR, yet at all three AVPRs, thus having a less

interesting selectivity profile than [Se-Se]-OT-OH (Fig. 1C, Table S2). d[Se-Se]-OT-OH was also a

partial agonist at hOTR (Emax=59%) and full agonist at the AVPRs.

Subtle modifications to vasopressin also modulated potency and selectivity without however yielding a

clear lead compound (Fig. 1D, Table S2). Vasopressin activated all four receptors, including hOTR

(EC50: hV2R>hV1bR>hV1aR>hOTR). Deamination of the N-terminus (dAVP) did not affect receptor

activation and the resulting ligand had potencies similar to that of vasopressin. The disulfide to

diselenide exchange ([Se-Se]-AVP, d[Se-Se]-AVP) was well tolerated and did not substantially change

the potency or selectivity profile. Deletion of Gly9 in dAVP and dAVP-OH resulted in substantial drops

in potency at all four receptors.

Oxytocin analogues 1-7, vasopressin and dAVP were further tested using the homogeneous time-

resolved fluorescence (HTRF) inositol-1-phosphate (IP1) assay (Fig. 1E, Table S3) to allow comparison

with the downstream intracellular Ca2+ changes measured using the FLIPR assay. The HTRF-IP1 assay

correlated well with the FLIPR assay (Table S4) and the potency and selectivity trends, particularly

with lead compound [Se-Se]-OT-OH were confirmed. [Se-Se]-OT-OH had an EC50 that was only 2.6-

fold less potent than that of oxytocin and no activity at hV1aR and hV1bR at concentrations up to 10

µM (Table S3).

The Ca2+-concentration-response curves of [Se-Se]-OT-OH at hOTR, hV1aR and hV1bR

(representative curves in Fig. 2A; raw FLIPR data in Fig. S1A-B) highlight its partial agonism at

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hOTR, its receptor selectivity and its weak antagonism at hV1aR (17% inhibition, IC50=132 nM) (Fig.

2A & Fig. S1C-D, Table S2). The representative cAMP-concentration-response of [Se-Se]-OT-OH at

the hV2R shows that [Se-Se]-OT-OH is a full agonist at this receptor, however not as potent as

oxytocin and vasopressin (Fig. 2B). The IP1-concentration-response curves of [Se-Se]-OT-OH at

hOTR, hV1aR and hV1bR show that the same selectivity, potency and partial agonism trend is also

observed using second messenger IP1 measurements (Fig. 2C).

Binding analysis at human OTR, V1aR, V1bR, and V2R. Binding data for the oxytocin analogues

1-7, vasopressin and dAVP were obtained in radioligand-displacement assays at all four human

receptors (Table S5). The experimental inhibition constants (Ki) correlated well with the functional data

(Table S2 & S3). [Se-Se]-OT-OH displaced the 125I-V1a antagonist at hV1aR with a Ki of 57 nM,

thereby confirming the weak antagonistic effects observed in the FLIPR assay (Fig. 2D, Table S5).

Schild regression analysis for binding mode. To confirm competitive binding of [Se-Se]-OT-OH at

hOTR, we performed a series of Schild regression experiments with oxytocin and [Se-Se]-OT-OH

against hOTR antagonist atosiban using the HTRF-IP1 assay. Increasing concentrations of atosiban

shifted the concentration-response curves of oxytocin (Fig. S2A) and [Se-Se]-OT-OH (Fig. S2B) to the

right, resulting in a linear Schild plot which is representative of competitive binding at hOTR.

Binding and functional analysis of [Se-Se]-OT-OH and d[Se-Se]-OT-OH at murine OTR, V1aR,

V1bR and V2R. [Se-Se]-OT-OH retained its functional selectivity for OTR in mice and was inactive

at mV1aR and mV1bR up to concentrations of 10 µM (Fig. 3A, Table S3, Fig. S3A). [Se-Se]-OT-OH

activated mOTR with a potency similar to that of oxytocin (Table S3) (66). [Se-Se]-OT-OH activated

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mV2R with an EC50 that was ~300-fold larger than that of vasopressin (67). d[Se-Se]-OT-OH also

retained its selectivity profile in mice, and was again less selective than [Se-Se]-OT-OH since it

activated all four murine receptors subtypes (Fig. S3B, Table S3). Both compounds were partial

agonists at mOTR and full agonists at mV2R. The binding data of both compounds (Table S5)

correlated well with the functional data (Table S3), including low affinity binding of [Se-Se]-OT-OH at

mV1bR fitting with absence of activation at mV1bR and binding to the mV1aR confirming mV1aR

antagonism. [Se-Se]-OT-OH displayed biphasic binding to mV1aR and mV2R (Table S5, Fig. S4A),

and d[Se-Se]-OT-OH biphasic binding to mV2R (Table S5, Fig. S4B).

Human serum stability assay. [Se-Se]-OT-OH had a ~2-fold longer half-life of 25 h compared to

oxytocin, which had a half-life of 12 h (Fig. 3B).

Human myometrial cell and strip contractility assays as proxies for induction and augmentation

of labor. To determine if the partial agonism of [Se-Se]-OT-OH and d[Se-Se]-OT-OH in the

pharmacological studies would affect their physiological activity compared to the full agonist oxytocin,

we tested the ability of compounds 1, 6 and 7 to induce contraction by activating hOTR in myometrial

cells immortalized with the human telomerase reverse transcriptase (hTERT-HM) (68) in a collagen gel

contractility assay (69, 70). Both [Se-Se]-OT-OH and d[Se-Se]-OT-OH induced a contractile response

comparable to oxytocin (Fig. S5). [Se-Se]-OT-OH and d[Se-Se]-OT-OH increased contractility by

7.4% and 9% respectively, compared to 9% for oxytocin. We then confirmed these findings in

contractility studies that used strips of human myometrium from cesarean sections. [Se-Se]-OT-OH

and oxytocin increased contraction amplitude in a concentration-dependent manner (Fig. 3C & D, Fig.

S6) and had similar potencies (Fig. S6). The contraction profile induced by [Se-Se]-OT-OH was

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however more phasic and frequent compared to a more tonic-like activity induced by oxytocin at the

concentrations used (Fig. 3C & D).

Behavioral efficacy in a mouse model of social fear. [Se-Se]-OT-OH was tested in the social fear

conditioning paradigm (71) which specifically generates social fear (as measured by reduced social

investigation 24 h after fear conditioning) in mice without any confounding behavioral alterations. [Se-

Se]-OT-OH reduced social fear and facilitated social fear extinction 10 min after its

intracerebroventricular infusion compared with vehicle-treated social fear conditioned mice (Fig. 4A).

This effect was similar to that of synthetic oxytocin and of [Thr4,Gly9]-OT, a highly selective mOTR

agonist (66) that also potently reduced social fear (Fig. 4A). All unconditioned control mice treated

with either vehicle, [Se-Se]-OT-OH, oxytocin or [Thr4,Gly9]-OT showed similar extents of social

preference behavior as reflected by active exploration of conspecifics. Independently of subsequent

treatment, all social fear conditioned and unconditioned groups showed similar investigation of a non-

social stimulus (small empty cage), indicating similar amounts of non-social anxiety. During the social

fear recall (Fig. 4B), all groups of mice, irrespective of their treatment, showed high social

investigation which indicated reduced social fear and successful social fear extinction.

Maximal Ca2+ concentrations in human cardiomyocytes as a proxy for assessing cardiovascular

safety. V1aR is highly abundant in the vascular smooth muscle and heart (cardiomyocytes), and V1aR

activation has been linked to cardiovascular risks (72-76). As a proxy to assess cardiovascular risks, we

measured maximal intracellular Ca2+ concentrations in human cardiomyocytes treated with the various

ligands (Fig. 5). Vasopressin and oxytocin concentration-dependently caused an increase in maximal

intracellular Ca2+ in human cardiomyocytes with EC50 values of 121 nM and 174 nM, respectively,

which aligned well with our pharmacological results of hV1aR activation by oxytocin (Table S2 & S3). 12

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Consistent with its improved selectivity (OTR activation without V1aR activation), [Se-Se]-OT-OH did

not affect the maximal intracellular Ca2+ concentration in cardiomyocytes up to 10 µM (Fig. 5).

DISCUSSION

Agonists and antagonists with good selectivity for the four oxytocin and vasopressin receptor subtypes

are in short supply (37, 38, 77). Physiological function and dysfunction of the receptors are therefore

studied by time- and cost-intensive knockout models, which have linked the subtypes to various

disorders including autism, schizophrenia, epilepsy, stress, aggression, depression, anxiety and pain (5,

8, 16, 33). Therapeutic development targeting these disorders has so far failed due to the challenge of

developing subtype-selective ligands that retain their selectivity across species, thereby limiting

translational studies with clinically relevant animal models (37, 38, 78).

Subtle pharmacophore framework modifications - a new strategy to retain selectivity across

murine and human receptors. This structure-activity relationship study demonstrated that subtle

modifications to the pharmacophore framework of the endogenous ligands oxytocin and vasopressin

could tune potency and selectivity, and yield ligands ([Se-Se]-OT-OH and d[Se-Se]-OT-OH) with an

improved selectivity profile that was conserved across mouse and human (Table S3, Fig. 3A). In

particular, the replacement of the disulfide bond with the diselenide bond in combination with the C-

terminal amide to acid change in oxytocin yielded [Se-Se]-OT-OH, the compound with the most

pronounced selectivity gain for OTR (Fig. 3A). Although an amide to acid modification can generally

be considered a subtle modification, it seems that either the free lone pair of the nitrogen atom is

involved in receptor recognition and activation or the negative charge of the acid contributes to activity

loss at the AVPRs. Substitution of the disulfide bond by the diselenide bond slightly increases the bond

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length (+ 0.3 Å), torsion angle ( +11º) and hydrophobicity (59, 60). In particular, the change in torsion

angle could play a part in the selectivity differences observed by pushing the ligand into a left-handed

conformer, as observed in the crystal structure of dOT (54). Both modifications (diselenide bond and

C-terminal acid) had to be present to introduce the selectivity improvement.

The increased selectivity of [Se-Se]-OT-OH makes it an excellent probe to delineate OTR function in

the CNS in behavioral animal models (where V2R is not present) and a promising lead molecule for the

clinic, which we further explored in this study. We recommend that an effective concentration of 50

nM or a 10-fold higher concentration than oxytocin (in comparative studies with oxytocin) is used to

ensure OTR-mediated action while retaining OTR preference. The weak antagonism (17% inhibition of

vasopressin at hV1aR) does not affect investigation of OTR function, but needs to be taken into

account when either binding studies are carried out ([Se-Se]-OT-OH binds to hV1aR with a K i=56.8

nM) or when [Se-Se]-OT-OH is administered in presence of V1aR ligands (for example, vasopressin

can still activate the hV1aR at 83% in the presence of [Se-Se]-OT-OH). [Se-Se]-OT-OH is a partial

agonist for OTR; however, this did not affect biological function as suggested by our myometrial

contractility study, which showed that OTR activation resulted in a full response similar to that induced

by oxytocin (Fig. 3C & D, Fig. S5). Partial agonists can be beneficial in treating chronic disorders such

as pain and autism since they often do not trigger the development of adverse effects such as

overstimulation, desensitization, adaptation, tolerance and dependence as seen with full agonists.

General conclusions and guidelines for future oxytocin and vasopressin ligand design. N-terminal

deamination of oxytocin ligands improves binding and potency at all four receptors, renders oxytocin

slightly more hydrophobic and improves its stability against proteases (55). Although N-terminal

deamination of vasopressin did not affect potency and selectivity, we still recommend deamination

because it offers enhanced proteolytic stability. Considering the advancements in the area of disulfide

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mimetics we recommend replacement of the disulfide bond by a non-reducible and therefore

metabolically more stable thio- or seleno-ether bond (4, 56). Modification of the C-terminus of

oxytocin and vasopressin could yield novel antagonists, considering this study and the replacement of

Gly9 in oxytocin and vasopressin by Val which resulted in an agonist to antagonist switch at the hV1aR

for both ligands (50). The FLIPR is a valuable instrument for primary screening of compounds that

target oxytocin and vasopressin receptors since it rapidly provides agonist and antagonist information

with high-throughput capabilities. Follow-up characterization of selected leads by ligand binding,

second messenger quantification and arrestin recruitment can then be used to better understand ligand

kinetics and secondary messenger signaling pathways. The structure-activity relationship study

presented here assessed affinity and functional data (FLIPR and IP1) of the most commonly used

control compounds oxytocin, vasopressin, dOT and dAVP over all four receptor subtypes in a single

study and could be a template for future oxytocin and vasopressin ligand development.

Comparison of the FLIPR and IP1 assays

Only three discrepancies with more than 50-fold difference were detected between the FLIPR and the

IP1 assays (oxytocin and dOT at hV1bR and d[Se-Se]-OT at hV1aR; Table S4) suggesting signaling

bias for these ligands at these receptors. The characteristic Ca2+ transients elicited by activation of Gq-

coupled GPCRs such as OTR, V1aR and V1bR originate from production of IP3 downstream of PLC-

activation, which induces release of Ca2+ from intracellular stores through IP3 receptors, termination of

receptor signaling and subsequent uptake of increased intracellular Ca2+ into stores and the extracellular

compartment through Ca2+ ATPases (79). In addition, Ca2+ signals can be magnified or inhibited

through intracellular feedback mechanisms such as Ca2+-induced Ca2+ release, activation of downstream

effectors that modulate intracellular Ca2+ amounts, or modification of Ca2+ signals through alternate G-

proteins such as Gi, which is involved in oxytocin and vasopressin receptor signaling (5, 80). Thus,

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whereas IP3 production is a requisite step for initiation of a Ca2+ signal, other factors contribute to the

magnitude and kinetics of the observed Ca2+ transients elicited by activation of GPCRs. In contrast, the

IP1 assay indirectly quantifies production of IP3 through accumulation of its breakdown product IP1. It

is plausible that the observed differences arise because of mechanistic differences that contribute to

increases in intracellular Ca2+ and accumulation of IP1. The cellular consequences of such differences

remain unclear to date, although Ca2+ signaling is a physiologically relevant consequence of GPCR

activation. The difference in signaling produced by these ligands indicates a potential signaling bias, a

phenomenon that has been previously studied for this receptor class (43, 80-82).

[Se-Se]-OT-OH modulating central actions: Reversal of social fear. Oxytocin is linked to multiple

CNS disorders such as autism, bipolar disorders, schizophrenia, pain, anxiety and depression (5, 8, 16,

33). Studies showing that intranasal oxytocin administration can modulate social behavior in humans

has resulted in increased interest and clinical trials exploring the therapeutic potential of intranasal

oxytocin for these disorders. The exact target pharmacology and underlying mechanisms of action are

however still under debate since oxytocin also can signal through V1aR and V1bR (Table S2-S5).

Hence, receptor subtype-specific probes are critical because of the partly opposing behavioral effects of

oxytocin and vasopressin, especially in the context of anxiety and stress regulation (14). [Se-Se]-OT-

OH allows for a clear and simple distinction between the involvement of OTR compared to V1aR and

V1bR in the CNS without the use of time and cost intensive knock-out models or co-application of

species selective V1a and V1bR antagonists. This is particularly valuable for therapeutic target

validation and future drug development. In this study, we chose to behaviorally test our novel probe in

the social fear conditioning mouse model, which has clinical implication to treat social anxiety

disorders (15, 71, 83), and to compare it to the commonly used mOTR-selective agonist [Thr4,Gly7]-OT

(TGOT), which does not retain its selectivity profile for human receptors. The use of [Se-Se]-OT-OH

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confirmed that this action is mediated through OTR and not through V1aR nor V1bR, and [Se-Se]-OT-

OH displayed similar effects as [Thr4,Gly7]-OT (Fig. 4A & B).

[Se-Se]-OT-OH modulating peripheral action: Labor induction and augmentation. Selectivity

against V1aR to reduce cardiovascular side effects is of particular importance for peripheral

applications since V1aR is the primary receptor subtype in the vascular smooth muscle and heart (72-

76). Clinically, oxytocin is administered intravenously to induce labor, augment weak contractions in

labor and treat postpartum hemorrhage, and intranasally to elicit lactation. Due to its activation of

AVPRs, oxytocin administration has only a limited therapeutic window of use and adverse effects

reported in the mother include anaphylactic reaction, postpartum hemorrhage, cardiac arrhythmia, fatal

afibrinogenemia, pelvic hematoma, subarachnoid hemorrhage, hypertensive episodes, rupture of the

uterus, convulsions, coma and death due to water intoxication. In the neonate, adverse effects reported

include bradycardia, cardiac arrhythmia, jaundice, retinal hemorrhage, permanent CNS or brain

damage, seizures and death (46, 84, 85). Hence, lack of AVPR activation in OTR agonists is needed to

diminish side effects.

[Se-Se]-OT-OH may be such a candidate because it only minimally raised intracellular Ca2+

concentrations in human cardiomyocytes, in contrast to oxytocin, which displayed nanomolar activity

(Fig. 5). V1aR is highly abundant in human cardiomyocytes where it mediates many of its

cardiovascular side effects. Furthermore, [Se-Se]-OT-OH and oxytocin were equipotent in augmenting

contractility in human myometrial preparations, although [Se-Se]-OT-OH displayed a more regular

contraction pattern (Fig. 3D). By contrast, oxytocin reduced contraction frequency in a concentration

dependent fashion but increased the contraction duration resulting in much greater total contractile

activity (Fig. 3C), which can lead to uterine hyperstimulation and rupture, which are life-threatening

events for mother and fetus (86, 87). [Se-Se]-OT-OH’s inactivity at V1aR, a receptor that is also

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abundant in the myometrium during pregnancy and is involved in contraction (88, 89), in combination

with its partial agonism at OTR, resulted in an improved contractility profile, suggesting it would be a

more controlled and safer alternative to oxytocin to induce and augment human labor. [Se-Se]-OT-OH

thus not only reduced AVPR-related side effects but also offered additional advantages to aid labor

induction and progression through a more regular increase of contraction strength without the risk of

uterine hyperstimulation or rupture.

Human serum stability of [Se-Se]-OT-OH. Enzymatic stability is a key characteristic for many

peptide drug candidates, and hence it was important to characterize the stability of [Se-Se]-OT-OH

compared to oxytocin. Diselenide bonds exhibit a lower redox potential than disulfide bonds, rendering

such mimetics more difficult to reduce, a feature that can be exploited in improving the half-life of

peptide drug candidates (4, 56, 59-61). Peptides with C-terminal amides on the other hand are more

stable than peptides with C-terminal acids. The improved stability of [Se-Se]-OT-OH in human serum

(Fig. 3B) suggests that the impact of the more redox stable diselenide bond outweighs the disadvantage

of the C-terminal acid modification. However, the experimentally determined in vitro human serum

half-life (which was in hours) of oxytocin and [Se-Se]-OT-OH does not reflect physiologically relevant

in vivo half-life (which is in minutes) as it does not take renal clearance into account. Nonetheless, it

can assess even small stability improvements, which could be important for treating CNS disorders

through delivery methods (such as treatment of autism in children or migraines in adults (21)) that are

not affected by renal clearance.

In conclusion, we showed that subtle modifications to the pharmacophore framework of oxytocin can

lead to enhanced selectivity profiles that are conserved across species (mouse and human), thereby

overcoming a substantial hurdle in ligand development. This proof-of-concept study provides a 18

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framework that could be applied to similar complex peptidergic signaling systems. The lead ligand

derived from this study, [Se-Se]-OT-OH, is a valuable probe that can effectively delineate the

physiological responses of OTR and is particularly suited for use in animal behavior studies looking at

the central roles of OTR. Its functional selectivity and close structural similarity to oxytocin are

important features that will help to clarify the contributions of the oxytocin and OTR signaling system

in health and disease. The improved selectivity, stability and safety profile of [Se-Se]-OT-OH could

lead to superior alternatives to oxytocin in the clinic for peripheral indications such as labor induction

and progression, and postpartum hemorrhage, as well as for central indications such as autism,

migraine, schizophrenia, anxiety and stress, where clinical trials have shown that intranasal

administered oxytocin is safe and therapeutically effective (20, 21, 90-93). [Se-Se]-OT-OH has the

potential to advance our understanding of the OTR as a therapeutic target in different animal models

and represents a promising lead molecule for various high-profile OTR-related indications.

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MATERIALS AND METHODS

Selenocysteine building block synthesis. An optimized protocol with yield-improving modifications

was developed based on Chocat et al. (62), Tanaka et al. (63) and Oikawa et al. (64) enabling the

synthesis of the Boc-L-Sec(Meb)-OH at a scale of 20 g with an overall yield of 62%. A new two-step

strategy was devised for the synthesis of dSec(Meb)-OH with an overall yield of 12% (Fig. 1B).

Peptide synthesis. Peptides 1-16 were assembled manually by Boc-SPPS using the HBTU-mediated in

situ neutralization protocol (59, 65). The analogues were cleaved with hydrogen fluoride (94), purified

by RP-HPLC, folded over 24 h in 0.1M NH4HCO3 buffer at pH 8.2 (1 mg/10 mL, ~100 µM) and

purified by RP-HPLC to >95% purity. Peptide concentrations were determined based on peak area

detected at 214 nm by analytical RP-HPLC against oxytocin, vasopressin as standards with known

peptide content established by amino acid analysis. Using Beer-Lambert Law, the peptide

concentrations were calculated based on absorptions of standards and samples using calculated

extinction coefficients (95-97).

Transfection and membrane preparation for FLIPR, HTRF-IP1 and radioligand displacement

assays for the human receptors. hOTR, hV1aR, hV1bR and hV2R cDNA were obtained from

Origene Technologies. [Tyrosyl-2,6-3H]-oxytocin (3H-OT); 46.3 Ci/mmol, [Phenylalanyl-3,4,5-3H(N)]-

AVP (3H-AVP); 2200 Ci/mmol, 125I-linear vasopressin hV1aR antagonist ([125I]phenylacetyl-D-

Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2); 2200 Ci/mmol, FlashBLUE GPCR scintillating beads,

TopSeal-A 96-well sealing film and 384-well white optiplates were from PerkinElmer Life Sciences.

The HTRF-IP1 assay kit was from CisBio International.

COS-1 cells grown in Dulbecco’s modified Eagle’s medium and 5% fetal bovine serum in 150 mm

plates were transiently transfected with plasmid DNA (14.5 µg) encoding the human OT, V1a, V1b or

V2 receptor using Lipofectamine-2000 (29 µL; Invitrogen). The cells were harvested 48 h post

transfection and homogenized using an Ultra turrax homogenizer (22,000 rpm) in assay buffer (50 mM 20

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Tris-HCl, 10 mM MgCl2 (5 mM MgCl2 for oxytocin), 0.1% BSA, pH 7.4) with complete protease

inhibitor cocktail (Roche Diagnostics). The homogenate was centrifuged at 484 g (2,000 rpm) for 10

min and the resulting supernatant was centrifuged at 23,665 g (14,000 rpm) for 30 min. The pellet was

re-suspended in appropriate buffer without protease inhibitor containing 10% glycerol and stored at –

80º C until assayed.

FLIPR assay for the human OTR, V1aR and V1bR. COS-1 cells transfected with hOTR, hV1aR or

hV1bR were plated 24 h prior to the experiment at a density of 35,000–50,000 cells/well on black-

walled 96-well imaging plates (Corning). Cells were loaded for 30 min at 37 °C with Fluo-4-AM (4.8

µM) in physiological salt solution (PSS; composition NaCl 140 mM, glucose 11.5 mM, KCl 5.9 mM,

MgCl2 1.4 mM, NaH2PO4 1.2 mM, NaHCO3 5 mM, CaCl2 1.8 mM, HEPES 10 mM) containing 0.3%

fatty-acid-free BSA. To allow for complete dye de-esterification, cells were washed 5-10 min with PSS

before transfer to a FLIPRTETRA (Molecular Devices) fluorescent plate reader. Ca2+ responses were

measured using a cooled CCD camera with excitation at 470–495 nM and emission at 515–575 nM.

The baseline fluorescence was set to a minimum of 1,000 arbitrary fluorescence units by adjusting

camera gain and excitation intensity. Compounds were added as 3x concentrated stock solutions in

PSS, with 10 baseline fluorescence readings prior to compound addition followed by fluorescence

reading every second for 180 seconds.

Raw fluorescence data was converted to ΔF/F values by subtracting baseline fluorescence readings

from subsequent time points and dividing by baseline fluorescence values, as previously described

(98). For concentration-response curves, maximum ΔF/F values after addition of compounds were

plotted against agonist concentration and normalized to the response elicited by the native ligand

(oxytocin for hOTR and vasopressin for hV1aR and hV1bR). A 4-parameter Hill equation with Hill

coefficient = 1 was fitted to the data using GraphPad Prism (Version 4.00).

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cAMP assay for hV2R. The cDNA plasmid clones for hV2R were a gift from Ralf Schülein (FMP,

Berlin). The hV2R sequence was inserted into pKaede-MN1 (MBL) using EcoRI and HindIII

restriction sites to yield the wild type receptor. The conditions for the propagation of HEK293 cells,

creation of stably transfected cell lines were similar as described previously (48, 99). Briefly, cells were

transfected with CaPO4 transfection and 1,8x106 of HEK293 cells per 10 cm dish were prepared. 20 µL

DNA, with a concentration of 1 µg/µL, were mixed with 480 µL H2O + CaCl2 (430 µL H2O + 50 µL

CaCl2) and added to 500 µL of HEPES-buffered saline solution. After 6 min, the solution was added to

the DMEM high glucose medium (PAA), supplemented with L-glutamine and gentamicin, and the cells

were incubated for 4 h at 37 °C. After a glycerol-shock, DMEM medium was added again and the cells

were put back at 37 °C. In the following days, selection through the antibiotic Geneticin (geneticin

G418-BC liquid – 50 mg/mL (Biochrom) took place.

Cells were grown in 6-well plates. The adenine nucleotide pool was metabolically labeled by

incubating confluent monolayers for 16 h with 3H-adenine (1 μCi/well) ([2,8-3H]-adenine; 27.2

Ci/mmol, PerkinElmer Life Sciences) as described earlier (100). After preincubation, fresh medium

was added that contained 100 μM RO201724 (a cell-permeable, selective inhibitor of cAMP-specific

phosphodiesterase; Calbiochem). After 4 h, cAMP formation was stimulated by the hV2R agonist

vasopressin (50 pM – 1 μM), oxytocin (60 pM – 1 μM), synthetic oxytocin analogues (700 pM – 10

μM) or forskolin (30 μM; a cell-permeable diterpenoid that possesses anti-hypertensive, positive

inotropic, and adenylyl cyclase activating properties; Sigma-Aldrich) for 20 min at 37 °C. Assays were

performed at least 3 separate experiments in triplicate. The formation of 3H-cAMP was determined

according to Bergmayr et al. (100). Potency (EC50) and efficacy (Emax) were calculated by fitting the

data to a three-parameter logistic equation (Hill equation) using a Levenberg-Marquardt algorithm.

EC50 values were presented as mean ± SEM.

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HTRF-IP1 assay for the human receptors. COS-1 cells were transiently transfected with plasmid

DNA encoding the human OTR, V1aR or V1bR using Lipofectamine-2000/DNA ratio of 2 in

Dulbecco’s modified Eagle’s medium. Assays measuring IP1 accumulation were performed 48 h after

transfection according to manufacturer’s protocol. Briefly, cells were incubated with increasing

concentration of oxytocin and vasopressin analogues (10 pM to 10 µM) in stimulation buffer

containing LiCl for 1 h in 37 C, 5% CO2 in white 384-well Optiplates. Cells were lysed by the addition

of the HTRF reagents, the europium cryptate-labeled anti-IP1 antibody and the d2-labeled IP1 analogue,

diluted in lysis buffer. The assays were incubated for 1 h at room temperature. The emission signals at

590 nm and 665 nm were measured after excitation at 340 nm using the Envision multilabel plate

reader (PerkinElmer Life Sciences). Signal was presented as the HTRF ratio: F=[(fluorescence-

665nm/fluorescence-590nm) x 104]. For concentration-response curves, the HTRF ratio values after

addition of compounds were plotted against the ligand concentration and normalized to the response

elicited without ligand (the negative control). Sigmoidal curves for the calculation of the half-maximal

excitatory concentration (EC50) values were fitted to individual data points by non-linear regression

with Hill coefficient = 1, using the software package Prism (GraphPad Software).

Radioligand displacement assay for the human receptors. Competitive binding assays were

performed using FlashBLUE GPCR scintillation beads (PerkinElmer) as previously described (56).

Reactions containing increasing concentrations of competing oxytocin and vasopressin analogues (10

pM to 10 µM), FlashBLUE GPCR beads (100 µg for hOTR and hV1aR; 200 µg for hV1bR and

hV2R), human OT, V1a, V1b, V2 receptor membrane preparations (5 µg protein) and radioligand (3H-

OT (2 nM) for hOTR, 125I-V1a antagonist ([125I]phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-

Tyr-NH2) (0.5 nM) for hV1aR, and 3H-AVP (3 nM) for hV1bR and hV2R) in assay buffer (50 mM

Tris-HCl, 10 mM MgCl2 (5 mM MgCl2 for oxytocin), 0.1% BSA, pH 7.4) were established in 96-well

white polystyrene plates with clear flat bottoms in a total reaction volume of 80 µL. Radioligand

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binding was detected using a Wallac 1450 MicroBeta scintillation counter (PerkinElmer Life Sciences).

Sigmoidal curves for the calculation of the half-maximal inhibitory concentration (IC50) values were

fitted to individual data points by non-linear regression with Hill coefficient = –1, using the software

package Prism (GraphPad Software).

Radioligand displacement assay for the murine receptors. Competitive binding assays were

performed using 3H-OT and 3H-AVP (Perkin Elmer) and increasing concentration of the indicated

peptides at 30 °C on membranes prepared from HEK293 cells transfected with OTR, V1aR, V1bR or

V2R murine receptors as described previously (66). Compounds affinities (Ki) were determined by

means of competition experiments in which the unlabeled compound concentrations varied from 10 pM

to 10 µM to displace 4 nM 3H-OT for mOTR and 3H-AVP for mV1aR, mV1bR and mV2R. Ligand

binding data and Ki were analyzed by means of non-linear regression and binding-competitive fitting

using Prism version 5 (GraphPad Software).

HTRF-IP1 assay and cAMP determination for the murine receptors. IP1 and cAMP accumulation

were determined in HEK293 cells transiently transfected with DNA plasmids encoding the murine OT,

V1a, V1b or V2 murine receptors using HTRF assays (IP1 and cAMP assays, CisBio International) as

previously described (66). Sigmoidal curves for the calculation of the half-maximal effective

concentration (EC50) values were fitted by non-linear regression log(agonist) vs. response fitting, using

Prism version 5 (GraphPad Software).

Stability assays. Peptides were added to human serum and aliquots were taken and analyzed by RP-

HPLC and LCMS at 1, 2, 3, 4, 12, 24 and 48 h time points as previously described (56).

Social fear conditioning mouse model. SFC was performed as previously described (71). Sample

sizes (>4 mice) were determined based on previous studies (71, 83, 101). All experimental procedures

were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the

Government of Oberpfalz and the guidelines of the NIH. All male CD1 mice (Charles River, Sulzfeld, 24

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Germany, 8-10 weeks of age at the start of experiments) were group-housed under standard laboratory

conditions (12/12 h light/dark cycle, lights on at 06:00, 22 °C, 60 humidity, food and water ad libitum)

in polycarbonate cages (16 x 22 x 14 cm). All experimental procedures were performed between 08:00

and 15:00 h. Surgeries were performed under isoflurane anaesthesia and extreme care was taken to

minimize animal suffering. Analysis of the SFC paradigm was performed using GraphPad Prism

version 6.0 (GraphPad Software).

On day 1 of the social fear conditioning paradigm, mice were transferred from their home cage into the

conditioning chamber; after a 30-s adaptation period, an empty cage was placed in the conditioning

chamber as a non-social stimulus that mice were allowed to investigate for 3 min, before it was

replaced by an identical cage containing an unfamiliar male conspecific (which represented the social

stimulus). SFC- mice were allowed to investigate the social stimulus for 3 min without receiving any

foot shocks, whereas SFC+ mice were given a 1-s electric foot shock (0.7 mA) each time they

investigated (sniffed) the social stimulus. Mice were returned to their home cage when no further social

contact was made for 2 min. All mice investigated the non-social (empty cage) stimulus to a similar

extent and received similar number of foot shocks (Table S6). On day 2, which consisted of social fear

extinction training, mice were exposed to 3 non-social stimuli (empty cages) in their home cage to

assess non-social investigation as a parameter of non-social fear and general anxiety-related behavior.

Mice were then exposed to 6 unfamiliar social stimuli (6 different male mice) to assess social

investigation as a parameter of social fear. Mice received single acute intracerebroventricular infusions

(35-45 s) of either vehicle (2 µL; sterile Ringer solution), oxytocin (2 µL of 250 µM; 500 pmol), [Se-

Se]-OT-OH (2 µL of 250 µM; 500 pmol) or [Thr4,Gly7]-OT (2 µL of 250 µM; 500 pmol) 10 min prior

to social fear extinction. On day 3, which assessed social fear extinction recall, mice were exposed in

their home cage to 6 different unfamiliar social stimuli to investigate whether repeated exposure to

social stimuli during extinction leads to a complete reversal of social fear.

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Human myometrial cell contractility assay. Human uterine myometrial smooth muscle cells

(hTERT-HM) were cultured, collagen-gels were prepared, and the assays were performed as described

previously (48, 70). Briefly, cells were cultured in DMEM-F-12/10% FBS (Invitrogen). Collagen gels

were prepared from rat tail Type 1 collagen (Sigma Aldrich), to a final concentration of 1.5 mg/mL,

and seeded in 24-well culture dishes, with 150,000 hTERT-HM cells per well. Cells in collagen gels

were allowed to equilibrate overnight in serum free DMEM medium. The ligands of interest (1 μM

concentration) were added to the serum free media and the gels released from the sides of the wells.

10% (vol/vol) FBS was used as a positive control for contraction, and unstimulated cells as the

negative control in all experiments. Gel images were captured over time, using a FluorchemTM 8900

imager, the area (cm2) of the gels were measured using AlphaEaseFC software (Alpha Innotech

Corporation). A decrease in gel area correlated with an increase in contractility.

Tissue bath myometrial contractility assays. Biopsies of myometrium were obtained from women

undergoing term (39-40 weeks) pre-labor elective cesarean section delivery at Liverpool Women’s

Hospital, Liverpool, UK. All women gave written informed consent to participate, and the study was

approved by the North West (Liverpool East) Research Ethics Committee (Ref: 10/H1002/49) and by

the Research and Development director at Liverpool Women’s Hospital NHS Foundation Trust,

Liverpool, UK. A full thickness biopsy (~2 cm3) was cut from the upper lip of the lower uterine

incision site and placed into Hanks balanced salt solution at 4 °C (102). In the laboratory, strips of

myometrium (1 mm x 2 mm x 5 mm) were dissected and placed between a force transducer and a fixed

hook using aluminum clips. Strips were continually superfused with physiological saline solution (PSS;

154 mM NaCl, 5.6 mM KCl, 1.2 mM MgSO4, 7.8 mM glucose, 10.9 mM Hepes, 2.0 mM CaCl2, pH

7.4) maintained at 36 °C. After stable spontaneous contractions developed, the strips were exposed to

rising concentrations of oxytocin or [Se-Se]-OT-OH. Data were analyzed by measuring the amplitude

of contraction using Origin Pro 9.0 software (Origin Lab Corporation) as described previously (48,

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103, 104). The effect of [Se-Se]-OT-OH or oxytocin was compared to the activity preceding the

application of the first dose and data represent the change in activity compared to spontaneous control

activity (100%). Concentration-response curves were fitted using non-linear regression to calculate

EC50 values.

Human cardiomyocyte assay. Wild-type WTC11 human induced pluripotent stem cells (hiPSCs)

were used in this study. Undifferentiated cells were maintained in mTeSR1 media (Stem Cell

Technologies). Standard cardiomyocyte directed differentiation using a monolayer platform was

performed with a modified protocol based on previous reports (105-109). The differentiation set up was

initiated by plating undifferentiated hiPSCs as single cells. The cultures were treated with 1 µM CHIR-

99021 (Cayman Chemical) for 24 h before reaching confluence. Cells were induced to differentiate

(designated day 0) by replacing the culturing media with Roswell Park Memorial Institute (RPMI)

1640 media (catalog number 11875-119, Thermo Fisher Scientific) containing 3 µM CHIR-99021, 500

µg/mL bovine serum albumin (BSA) and 213 µg/mL ascorbic acid. On day 3, media was changed to

RPMI 1640 media with BSA and ascorbic acid containing 1 µM XAV-939 (Tocris Bioscience). On day

5 media was changed to RPMI 1640 containing ascorbic acid and BSA. On day 7 the media is replaced

with RPMI 1640 containing B-27 supplement with insulin (catalog number 17504-044, Thermo Fisher

Scientific). Cells were harvested using trypsin, re-plated into a 384-well black walled imaging plate

coated with CellBIND (Sigma Aldrich) at a density of 4 × 104 cells per well and cultured for 8 days

prior to experiments. Functional activity was assessed using a high-throughput FLIPRTETRA (Molecular

Devices) fluorescent imaging plate reader assay. The growth medium was removed and replaced with

Calcium 4 No-wash dye (Molecular Devices) diluted in physiological salt solution (PSS; composition

in mM: NaCl 140, glucose 11.5, KCl 5.9, MgCl2 1.4, NaH2PO4 1.2, NaHCO3 5, CaCl2 1.8, HEPES 10;

pH 7.4) according to the manufacturer's instructions and incubated at 37 °C for 30 min. Changes in

intracellular Ca2+ amounts were measured using a cooled CCD camera (excitation 470–495 nm;

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emission 515–575 nm) with reads taken every 0.4 s for 240 s after addition of compound. The

maximum intracellular Ca2+ amount was determined using ScreenWorks (Version 2.2.0.14, Molecular

Devices).

Statistical analysis

All statistical comparisons were made using GraphPad Prism software and values were expressed as

means ± SEM. Student’s t test and one-way analysis of variance (ANOVA) were used to determine

statistical significance for the human myometrial cell contractility and organ bath assays. Two-way

ANOVA was used to analyze the SFC data. P values <0.05 were considered significant.

SUPPLEMENTARY MATERIALS

Materials and Methods

Fig. S1. Representative raw FLIPR data.

Fig. S2. Schild plot analysis.

Fig. S3. Functional study of [Se-Se]-OT-OH and d[Se-Se]-OT-OH at the mOTR, mV1aR, mV1bR, and

mV2R.

Fig. S4. Binding study of [Se-Se]-OT-OH and d[Se-Se]-OT-OH at the mOTR, mV1aR, mV1bR, and

mV2R.

Fig. S5. Human myometrial cell contractility assay.

Fig. S6. Human myometrial strip contractility assay.

Table S1. Overview of the synthesized peptides including details on their modifications.

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Table S2. Functional potencies for oxytocin and vasopressin analogs at the hOTR, hV1aR, hV1bR, and

hV2R.

Table S3. Functional potencies for oxytocin and vasopressin analogues at the human and murine OTR,

V1aR, V1bR, and V2R.

Table S4. Comparison of functional data from the FLIPR and HTRF-IP1 assays.

Table S5. Radioligand displacement data for oxytocin and vasopressin analogs at the human and

murine OTR, V1aR, V1bR, and V2R.

Table S6. Overview of the number of electric foot shocks per mouse.

Reference (110)

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REFERENCES AND NOTES

1. H. Gainer, Cell-specific gene expression in oxytocin and vasopressin magnocellular neurons.

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ACKNOWLEDGMENTS

We acknowledge Alun Jones for the help with the mass spectrometry, Andrew Mulligan for their

contribution to some of the synthetic work, Dr. Lachlan Rash for conducting the qualitative control and

concentration determination of tested analogues, and Prof. Michael Freissmuth and Dr. Bice Chini for

their continuous support.

FUNDING

This work was supported by NHMRC Project [1063803] and Program grants [1072113 & 1063803].

I.V. is supported by an ARC Future Fellowship grant [FT130101215] and R.J.L. by an NHMRC

Fellowship. M.B. is an Umberto Veronesi Foundation Postdoctoral Fellow (FUV 2017). M.M. received

funding from the European Union Seventh Framework Program (FP7/2007-2013) under Marie Curie

Actions grant agreement n°[254897] and [2013-BP-B-00109], from the Secretary of Universities and

Research of the Economy and Knowledge Department of the Government of Catalonia, from the ARC

[DE150100784], and from the European Research Council (ERC) under the European Union’s Horizon

2020 research and innovation programme grant agreement n°[714366]. C.W.G. is an ARC Future

Fellow [FT140100730] and his research is supported by the Vienna Science and Technology Fund

(WWTF) through project grant [LS13-017]. S.W. and S.A. are supported by a grant (SW) and

fellowship (SA) from the Harris-Wellbeing Centre for Preterm research.

AUTHOR CONTRIBUTIONS

M.M. designed and performed the synthesis of the peptide analogs and developed the selenocysteine

chemistry. A.A., I.V., M.B., L.R. C.B., R.J.L., C.W.G. performed the pharmacological analysis of the

analogs. J.R.D., H.S.C. and N.J.P. performed the cardiomyocyte experiments. S.A., S.W., M.O. and

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T.J.S. performed the uterine strip contraction studies. R.M. and I.N. performed the behavioral in vivo

experiments. M.M. and P.F.A. directed the project and wrote the paper. All authors contributed to the

discussion, preparation of figures, and interpretation of the results.

COMPETING INTERESTS

M. M. and P. F. A. are named on a US patent (2013/0130,985) for oxytocin peptide analogs. The other

authors declare that they have no competing interests.

DATA AND MATERIALS AVAILABILITY

The human induced pluripotent stem cell line (WTC11 hiPSCs) used to generate the cardiomyocytes in

this study came from Bruce Conklin’s laboratory at UCSF (Gladstone Institute).

FIGURE LEGENDS

Fig. 1. Overview of the pharmacophore framework modifications to oxytocin and vasopressin. A

NMR structure of oxytocin, with framework residues marked in red that were the focus of this study.

The table provides an overview of all synthesized peptides with details on their modifications. U,

selenocysteine; d, deamino (N-terminus); , deletion of residue; bold, modification; P, position; term,

terminus. B Synthesis of selenocysteine building blocks for Boc-SPPS to enable sulfur to selenium

replacements. C & D Functional screen of oxytocin analogues 1-10 (C) and vasopressin analogues 11-

16 (D) at human OTR, V1aR, and V1bR using the FLIPR Ca2+ signaling assay and at human V2R by

measuring cAMP accumulation. Taller bars in graphs indicate loss of function at that particular

receptor. The last row of beige bars illustrates how modifications affected potency compared to 44

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oxytocin at hOTR and vasopressin at the hV2R (black). E Functional screen of oxytocin analogues 1-7,

vasopressin and dAVP at the human OTR, V1aR, and V1bR as assessed by measuring second

messenger IP1 accumulation and at the hV2R as assessed by measuring second messenger cAMP

accumulation. CNS (central nervous system) and PNS (peripheral nervous system) indicate where these

receptors can be found in humans. Exact EC50 values are in Table S2 & S3.

Fig. 2. Pharmacological characterization of [Se-Se]-OT-OH at all four human oxytocin and

vasopressin receptors. A Representative concentration-Ca2+ response curves of [Se-Se]-OT-OH at the

hOTR, hV1aR and hV1bR. B Representative concentration-cAMP response curves of oxytocin,

vasopressin and [Se-Se]-OT-OH at the hV2R. C Representative concentration-IP1 response curves of

[Se-Se]-OT-OH at the hOTR, hV1aR and hV1bR. D Representative concentration-response curves for

radioligand displacement assay of [Se-Se]-OT-OH at the hOTR, hV1aR, hV1bR and hV2R. All curves

were normalized to % of response of control ligand (oxytocin for OTR and vasopressin for AVPRs).

Data in (A) to (D) are presented as means ± SEM of results obtained from at least N=3 separate

experiments, each performed in triplicate.

Fig. 3. Comparison of selectivity, stability and ability to augment myometrial strip contractions

between [Se-Se]-OT-OH and oxytocin. A Functional selectivity profile of [Se-Se]-OT-OH and OT

over all four human (h) and murine (m) oxytocin and vasopressin receptors. OTR, V1aR, and V1bR

activity was measured by IP1 accumulation. V2R activity was measured by cAMP accumulation. B

Metabolic stability of [Se-Se]-OT-OH (t1/2=25 h) compared to oxytocin (t1/2=12 h) in human serum.

Data in (A, B) is presented as means ± SEM of results obtained from at least N=3 separate experiments,

each performed in triplicate. C & D Representative contraction pattern for human myometrial strips

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exposed to increasing doses of oxytocin (C) or [Se-Se]-OT-OH (D). N=5 women for [Se-Se]-OT-OH,

N=8 women for oxytocin.

Fig. 4. Social fear conditioning (SFC) mouse study. (A and B) Unconditioned (SFC-) and

conditioned (SFC+) mice were intracerebroventricularly infused with either vehicle (Ringer solution 2

µL; N=9 SFC- mice; N=10 SFC+ mice) or [Se-Se]-OT-OH (250 µM/2 µL, 500 pmol; N=7 SFC- mice;

N=7 SFC+ mice). Oxytocin (250 µM/2 µL; 500 pmol; N=10 mice) and [Thr4,Gly7]-OT (250 µM/2 µL;

500 pmol; N=4 mice) were only infused into SFC+ mice 10 min prior to extinction training. Percentage

of mice that investigated three non-social stimuli (empty cage) and six social stimuli (cage with a

conspecific) during social fear extinction (day 2; A) and six social stimuli during social fear extinction

recall (day 3; B). Data are represented as mean ± SEM. Data were analyzed using two-way ANOVA

for repeated measures. (A) #P <0.05 SFC+/Vehicle compared to SFC+/[Se-Se]-OT-OH; *P <0.05

SFC+/Vehicle compared to SFC+/OT, SFC+/[Thr4,Gly7]-OT and SFC-/Vehicle; (B) $P <0.05

SFC+/Vehicle compared to SFC+/OT and SFC-/Vehicle.

Fig. 5. Effects of vasopressin, oxytocin and [Se-Se]-OT-OH on maximal intracellular Ca2+ in

human cardiomyocytes. FLIPR assays showing the effect of vasopressin, oxytocin and [Se-Se]-OT-

OH on the maximal intracellular Ca2+ concentrations. Data are presented as mean ± SEM, N=4 wells

per data point.

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