1Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queens-land 4072, Australia. 2Faculty of Chemistry, Institute of Biological Chemistry, Universityof Vienna, 1090 Vienna, Austria. 3School of Pharmacy, The University of Queensland,Brisbane, Queensland 4104, Australia. 4Department of Behavioral and MolecularNeurobiology, Regensburg Center of Neuroscience, University of Regensburg,93053 Regensburg, Germany. 5CNR–Institute of Neuroscience, 20129 Milan, Italy.6Department of Biotechnology and Translational Medicine, University of Milan,20129 Milan, Italy. 7Center for Physiology and Pharmacology, Medical University ofVienna, 1090 Vienna, Austria. 8Department of Cellular andMolecular Physiology, Harris-Wellbeing Preterm Birth Centre, Institute of Translational Medicine, University ofLiverpool, Liverpool L69 3BX, UK. 9National Centre for Biomedical Engineering Science,NationalUniversity of Ireland,GalwayH91CF50, Ireland. 10School of Biomedical Sciences,Faculty ofMedicine, TheUniversity ofQueensland, Brisbane, Queensland 4072, Australia.*Corresponding author. Email: [email protected], [email protected](M.M.); [email protected] (P.F.A.)
Muttenthaler et al., Sci. Signal. 10, eaan3398 (2017) 5 December 2017
Subtle modifications to oxytocin produce ligands thatretain potency and improved selectivity across speciesMarkus Muttenthaler,1,2* Åsa Andersson,1 Irina Vetter,1,3 Rohit Menon,4 Marta Busnelli,5,6
Lotten Ragnarsson,1 Christian Bergmayr,7 Sarah Arrowsmith,8 Jennifer R. Deuis,1
Han Sheng Chiu,1 Nathan J. Palpant,1 Margaret O’Brien,9 Terry J. Smith,9 Susan Wray,8
Inga D. Neumann,4 Christian W. Gruber,7,10 Richard J. Lewis,1 Paul F. Alewood1*
Oxytocin and vasopressin mediate various physiological functions that are important for osmoregulation, reproduc-tion, cardiovascular function, social behavior,memory, and learning through fourGprotein–coupled receptors that arealso implicated in high-profile disorders. Targeting these receptors is challenging because of the difficulty in obtainingligands that retain selectivity across rodents and humans for translational studies. We identified a selective and morestable oxytocin receptor (OTR) agonist by subtly modifying the pharmacophore framework of human oxytocin andvasopressin. [Se-Se]-oxytocin-OHdisplayed similar potency to oxytocin but improved selectivity for OTR, an effect thatwas retained in mice. Centrally infused [Se-Se]-oxytocin-OH potently reversed social fear in mice, confirming that thisactionwasmediatedbyOTR andnot byV1aor V1b vasopressin receptors. In addition, [Se-Se]-oxytocin-OHproduced amore regular contraction pattern than did oxytocin in a preclinical labor induction and augmentation model usingmyometrial strips from cesarean sections. [Se-Se]-oxytocin-OH had no activity in human cardiomyocytes, indicatinga potentially improved safety profile and therapeutic window compared to those of clinically used oxytocin. In con-clusion, [Se-Se]-oxytocin-OH is a novel probe for validating OTR as a therapeutic target in various biological systemsand is apromisingnew lead for therapeutic development.Ourmedicinal chemistry approachmayalsobe applicable toother peptidergic signaling systems with similar selectivity issues.
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INTRODUCTIONOxytocin and vasopressin [also known as arginine vasopressin (AVP)]are closely related neurohypophysial neuropeptides that are mainlysynthesized in the magnocellular and parvocellular neurons of thehypothalamus, transported in association with neurophysins to theposterior pituitary, and released into the systemic circulation after en-zymatic cleavage in response to relevant physiological stimuli (1, 2). Inthe periphery, oxytocin is involved in uterine smooth muscle contrac-tion during parturition, milk ejection during lactation, ejaculation, andpain (3–5), whereas centrally released oxytocin functions as a neuro-transmitter or neuromodulator that promotes multiple behaviors (6–8)such as maternal care (9, 10), partnership bonding (8, 11, 12), socialinteractions (12), and stress and anxiety responses (13–15), as largelydetermined in rodents. Intranasal administration of oxytocin elicitsbroad behavioral effects in humans, and its therapeutic potential forpsychopathologies characterized by social or emotional dysfunctions isunder clinical investigation (14, 16–21). Vasopressin increases and de-creases fluid balance and blood pressure in the periphery (22–24). Cen-trally, vasopressin is implicated in learning and memory (25–27), invarious social behaviors including pair bonding and aggression in ro-
dents (6, 8, 28, 29), and in stress- and anxiety-related behaviors (28–30).The ubiquitous involvement of the oxytocin and vasopressin signal-ing system in diverse physiological functions reflects its ancient origindating back at least 600 million years (8, 31). The oxytocin receptor(OTR) and vasopressin receptor are members of the G protein–coupledreceptor (GPCR) family (5, 32) and are attractive targets in the treat-ment 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 and vasopressin act through theoxytocin receptor (OTR) and the three AVP receptors (AVPRs; vaso-pressor V1aR, pituitary V1bR, and antidiuretic V2R). Oxytocin andvasopressin are structurally similar nonapeptides that differ only bytwo amino acids at positions 3 and 8 (Fig. 1A). Two cysteine residuesin positions 1 and 6 form the cyclic part of the molecules followed by athree-residue amidated C-terminal tail. Their chemical similarity andthe high sequence homology of the extracellular binding domains ofOTR and AVPRs (~80%) lead to substantial cross-talk, with oxytocinable to activate the AVPRs and vasopressin the OTR (34, 35). Specificreceptor functionality is thus not controlled by ligand selectivity but bycell-specific variations in receptor abundance, controlled release, receptoroligomerization, rapid clearance, and specific enzymatic degradation(36). High OTR and AVPR homology and overlapping distributionconstitute a major hurdle in the development of selective receptor ago-nists, antagonists, and therapeutic candidates (37, 38). The identificationof novel drug leads is further complicated by substantial species differ-ences, such that rodent and human selectivity typically do not overlap,thereby restricting clinical translation (37–39). For example, clinicallyused oxytocin and vasopressin analogs including desmopressin (40, 41),carbetocin (42), and atosiban (37, 43, 44) are receptor subtype–selectivein rats but not in humans. This difference is mainly due to rapid bio-degradation, renal clearance, and a limited administration window duringwhich they can be used clinically without side effects (40, 44, 45). Despite
these limitations, oxytocin remains the ligand of choice in the clinic to in-duce and progress labor (46). The lack of a complete set of selective recep-tor agonists and antagonists further limits our ability to characterize thephysiological responses for each subtype receptor and their relevance indisease. We therefore commenced a program to overcome these limita-tions and to producemore selective ligands for this fundamental signalingsystem (31, 47–51).Here, we demonstrated that smallmodifications to thestructural framework of the pharmacophore of the endogenous and phar-macologically unselective neuropeptides could be used to tune selectivityand generate analogs with an improved selectivity profile that was con-served across mouse and human, thereby facilitating translational studies.
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RESULTSRationale for subtle modificationsTraditional medicinal chemistry strategies that modify single or mul-tiple functional groups of oxytocin and vasopressin commonly yieldselectivity improvements that are not retained across species (37, 38).On the basis of structural data for oxytocin (4, 48, 52–54) and earlierstudies (37, 50, 55–57), we knew that oxytocin and vasopressin bind-ing and receptor activation are sensitive to even minor modifications.For example, ring reduction by one sulfur atom or disulfide bond re-placement by a dicarba bridge (−CH2−CH2−) causes complete loss ofactivity (56). N-terminal deamination of oxytocin analogs often leads
Fig. 1. Overviewof the pharmacophore frameworkmodifications to oxytocin and vasopressin. (A) NMR structure of oxytocin (OT), with framework residuesmarked in redthat 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); D,deletion of residue; bold, modification; P, position; term, terminus; AVP, arginine vasopressin. (B) Synthesis of selenocysteine building blocks for Boc-SPPS to enable sulfur toselenium replacements. (C andD) Functional screenof oxytocin analogs 1 to 10 (C) andvasopressin analogs 11 to 16 (D) at thehumanOT receptor (hOTR), hV1aR, andhV1bRusingthe fluorescent imaging plate reader (FLIPR) Ca2+ signaling assay and at the hV2R by measuring cyclic adenosine monophosphate (cAMP) accumulation. Taller bars in graphsindicate loss of function at that particular receptor. The last row of beige bars illustrates howmodifications affected potency compared to oxytocin at the hOTR and vasopressin atthe hV2R (black). (E) Functional screen of oxytocin analogs 1 to 7, vasopressin, and dAVP at the hOTR, hV1aR, and hV1bR as assessed bymeasuring second-messenger inositol1-phosphate (IP1) accumulation and at the hV2R as assessed bymeasuring second-messenger cAMP accumulation. CNS (central nervous system) and PNS (peripheral nervoussystem) indicate where these receptors can be found in humans. Exact EC50 values are shown in tables S2 and S3.
to improved OTR activation, which has led to the generation of theslightly more stable and hydrophobic oxytocin superagonist deamino-oxytocin (dOT) and the clinically used ligands carbetocin, desmopressin,and atosiban (37, 55, 57). Furthermore, changing Gly9 to Val9 in ox-ytocin and vasopressin switches these agonists to antagonists. In ad-dition, [Gly9,Val]-oxytocin not only acts as an antagonist at the humanV1aR (hV1aR) but also no longer binds to the hV1bR and the hV2R atconcentrations up to 10 mM (50). For vasopressin, replacement of Gly9
by a carboxylic acid markedly decreases its vasopressor (V1aR) andantidiuretic (V2R) activity in rats (58). We also included replacementof the disulfide bond with a diselenide bond, which has been describedas a structurally isosteric disulfide bond mimic that can increase po-tency and selectivity due to the slightly more hydrophobic character ofthe diselenide bond (56, 59). Moreover, the lower redox potential ofthe diselenide bond correlates to higher stability in a reducing environ-ment and can therefore be exploited to improve peptide half-life(56, 59–61). We therefore introduced subtle modifications to thepharmacophore framework of oxytocin and vasopressin (Fig. 1A andtable S1) to identify those that yield selectivity gains that are retainedacross species.
Peptide synthesisTwo building blocks [Boc-L-Sec(Meb)-OH and dSec(Meb)-OH] weresynthesized to generate the desired sulfur or selenium modifications(Fig. 1, A and B, and table S1). To acquire Boc-L-Sec(Meb)-OH insufficient quantities, we optimized various protocols (62–64) to obtain28.7 g of pure Boc-L-Sec(Meb)-OH in only three steps (62%overall yieldin a 4-day procedure; Fig. 1B). We also devised a new synthetic strategyfor dSec(Meb)-OH, yielding the building block in a two-step synthesiswith an overall yield of 12% (Fig. 1B). With these building blocks inhand, all of the oxytocin and vasopressin analogs 1 to 16 (Fig. 1A andtable S1)were synthesized by Boc-SPPS (tert-butyloxycarbonyl solid-phasepeptide synthesis), folded in 0.1 M NH4HCO3, and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) (59, 65).
Functional response of novel oxytocin andvasopressin ligandsA fluorescent imaging plate reader (FLIPR) Ca2+ mobilization assay(for OTR, V1aR, and V1bR) and a second-messenger cyclic adenosinemonophosphate (cAMP) assay (for V2R) showed that oxytocin acti-vated all four human receptors. Half-maximal effective concentration(EC50) at the hV1aR and hV1bR was 10- to 300-fold higher than thatat the hOTR (EC50: hOTR ≈ hV2R < hV1bR < hV1aR) (Fig. 1C andtable S2). Deletion of the N-terminal amino group (dOT) increasedthe ability of the ligand to activate all of the AVPRs, suggesting an im-proved fit of dOT in the hydrophobic transmembrane binding pocket(47) across all four receptors. Replacement of the disulfide bond withthe isosteric yet slightly more hydrophobic diselenide bond ([Se-Se]-OT and d[Se-Se]-OT) was well tolerated and did not lead to markedchanges in potency. In particular, d[Se-Se]-OT showed the least changein potency and selectivity compared to oxytocin. Exchange of theC-terminal amide to acid in oxytocin (OT-OH) resulted in weakerpotency at all receptors, and truncations of the C terminus of oxytocin(compounds 8, 9, and 10) reduced the potency at all receptors withcomplete loss of activity at the hV2R (up to 10 mM). Compounds 6,7, 9, and 10 all showed substantial drops in interaction with AVPRswhile retaining good potency at the hOTR, thereby indicating thatthe C terminus of oxytocin is a good target for improving selectivityfor the hOTR against the three AVPRs.
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In particular, the combination of diselenide replacement andC-terminal amide to acid change yielded [Se-Se]-OT-OH (compound6) with an improved selectivity profile for OTR compared to oxytocin(Fig. 1C and table S2). [Se-Se]-OT-OH displayed low nanomolarpotency (7.3 nM) and partial agonism (Emax = 52%) at the hOTR,no activation of the hV1aR (>10 mM), a 600-fold higher EC50 at thehV1bR, and a 15-fold higher EC50 at the hV2R (109 nM). N-terminaldeamination of [Se-Se]-OT-OH yielded d[Se-Se]-OT-OH, which didnot have improved potency at the OTR but at all three AVPRs, thushaving a less interesting selectivity profile than [Se-Se]-OT-OH (Fig. 1Cand table S2). d[Se-Se]-OT-OH was also a partial agonist at the hOTR(Emax = 59%) and a full agonist at the AVPRs.
Subtle modifications to vasopressin also modulated potency andselectivity without, however, yielding a clear lead compound (Fig. 1Dand table S2). Vasopressin activated all four receptors, including thehOTR (EC50: hV2R > hV1bR > hV1aR > hOTR). Deamination of theN terminus [deamino-AVP (dAVP)] did not affect receptor activa-tion, and the resulting ligand had potencies similar to that of vaso-pressin. The disulfide-to-diselenide exchange ([Se-Se]-AVP andd[Se-Se]-AVP) was well tolerated and did not substantially change thepotency or selectivity profile. Deletion of Gly9 in dAVP and dAVP-OHresulted in substantial drops in potency at all four receptors.
Oxytocin analogs 1 to 7, vasopressin, and dAVP were further testedusing the homogeneous time-resolved fluorescence (HTRF) inositol1-phosphate (IP1) assay (Fig. 1E and table S3) to allow comparisonwith the downstream intracellular Ca2+ changes measured using theFLIPR assay. The HTRF-IP1 assay correlated well with the FLIPR assay(table S4), and the potency and selectivity trends, particularly withthe 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 oxy-tocin and no activity at the hV1aR and hV1bR at concentrations up to10 mM (table S3).
The Ca2+ concentration–response curves of [Se-Se]-OT-OH at thehOTR, hV1aR, and hV1bR (representative curves in Fig. 2A; rawFLIPRdata in fig. S1, A and B) highlight its partial agonism at the hOTR, itsreceptor selectivity, and its weak antagonism at the hV1aR [17% in-hibition; half-maximal inhibitory concentration (IC50), 132 nM](Fig. 2A; fig. S1, C and D; and table S2). The representative cAMPconcentration–response curves of [Se-Se]-OT-OH at the hV2R showedthat [Se-Se]-OT-OHwas a full agonist at this receptor but not as potentas oxytocin and vasopressin (Fig. 2B). The IP1 concentration–responsecurves of [Se-Se]-OT-OH at the hOTR, hV1aR, and hV1bR showedthat the same selectivity, potency, and partial agonism trends were alsoobserved using second-messenger IP1 measurements (Fig. 2C).
Binding analysis at the hOTR, hV1aR, hV1bR, and hV2RBinding data for the oxytocin analogs 1 to 7, vasopressin, and dAVPwere obtained in radioligand displacement assays at all four human re-ceptors (table S5). The experimental inhibition constants (Ki) correlatedwell with the functional data (tables S2 and S3). [Se-Se]-OT-OH dis-placed the 125I-V1a antagonist ([125I]phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2) at the hV1aR with a Ki of 57 nM,thereby confirming the weak antagonistic effects observed in the FLIPRassay (Fig. 2D and table S5).
Schild regression analysis for binding modeTo confirm competitive binding of [Se-Se]-OT-OH at the hOTR, weperformed a series of Schild regression experiments with oxytocinand [Se-Se]-OT-OH against the hOTR/Gq antagonist atosiban using
the HTRF-IP1 assay. Increasing concentrations of atosiban shiftedthe concentration-response curves of oxytocin (fig. S2A) and [Se-Se]-OT-OH (fig. S2B) to the right, resulting in a linear Schild plot that isrepresentative of competitive binding at the hOTR.
Binding and functional analysis of [Se-Se]-OT-OH andd[Se-Se]-OT-OH at murine OTR, V1aR, V1bR, and V2R[Se-Se]-OT-OH retained its functional selectivity for OTR in mice andwas inactive at murine V1aR (mV1aR) and mV1bR concentrations upto 10 mM (Fig. 3A, fig. S3A, and table S3). [Se-Se]-OT-OH activatedmOTR with a potency similar to that of oxytocin (table S3) (66).[Se-Se]-OT-OH activated mV2R with an EC50 that was ~300-foldlarger than that of vasopressin (67). d[Se-Se]-OT-OH also retainedits selectivity profile in mice and was again less selective than [Se-Se]-OT-OH because it activated all four murine receptor subtypes (fig. S3Band table S3). Both compounds were partial agonists at the mOTR andfull agonists at the mV2R. The binding data of both compounds (tableS5) correlated well with the functional data (table S3), including low-affinity binding of [Se-Se]-OT-OH at the mV1bR fitting with the ab-sence of activation at the mV1bR and binding to the mV1aR confirmingmV1aR antagonism. [Se-Se]-OT-OH displayed biphasic binding tomV1aR and mV2R (fig. S4A and table S5), and d[Se-Se]-OT-OHdisplayed biphasic binding to mV2R (fig. S4B and table S5).
Human serum stability assay[Se-Se]-OT-OH had a ~2-fold longer half-life of 25 hours compared tooxytocin, which had a half-life of 12 hours (Fig. 3B).
Human myometrial cell and strip contractility assays asproxies for induction and augmentation of laborTo determine whether the partial agonism of [Se-Se]-OT-OH andd[Se-Se]-OT-OH in the pharmacological studies would affect theirphysiological activity compared to the full agonist oxytocin, we tested
Muttenthaler et al., Sci. Signal. 10, eaan3398 (2017) 5 December 2017
the ability of compounds 1, 6, and 7 to induce contraction by activatingthe hOTR in myometrial cells immortalized with the human telomerasereverse transcriptase (hTERT-HM) (68) in a collagen gel contractilityassay (69, 70). Both [Se-Se]-OT-OH and d[Se-Se]-OT-OH induceda contractile response comparable to oxytocin (fig. S5). [Se-Se]-OT-OHand d[Se-Se]-OT-OH increased contractility by 7.4 and 9% respectively,compared to 9% for oxytocin. We then confirmed these findings incontractility studies that used strips of human myometrium from cesar-ean sections. [Se-Se]-OT-OH and oxytocin increased contraction ampli-tude in a concentration-dependent manner (Fig. 3, C and D, and fig. S6)and had similar potencies (fig. S6). The contraction profile inducedby [Se-Se]-OT-OH was, however, more phasic and frequent comparedto a more tonic-like activity induced by oxytocin at the concentrationsused (Fig. 3, C and D).
Behavioral efficacy in a mouse model of social fear[Se-Se]-OT-OH was tested in the social fear conditioning (SFC) para-digm (71), which specifically generates social fear (as measured byreduced social investigation 24 hours after fear conditioning) in micewithout any confounding behavioral alterations. [Se-Se]-OT-OH re-duced social fear and facilitated social fear extinction 10 min after itsintracerebroventricular infusion compared with vehicle-treated socialfear–conditioned mice (Fig. 4A). This effect was similar to that of syn-thetic oxytocin and [Thr4,Gly7]-OT, a highly selective mOTR agonist(66) that also potently reduced social fear (Fig. 4A). All unconditionedcontrol mice treated with either vehicle, [Se-Se]-OT-OH, oxytocin, or[Thr4,Gly7]-OT showed similar extents of social preference behav-ior as reflected by active exploration of conspecifics. Independently ofsubsequent treatment, all social fear–conditioned and unconditioned
Fig. 2. Pharmacological characterization of [Se-Se]-OT-OH at all four humanoxytocin and vasopressin receptors. (A) Representative Ca2+ concentration–response curves of [Se-Se]-OT-OH at the hOTR, hV1aR, and hV1bR. (B) RepresentativecAMP concentration–response curves of oxytocin, vasopressin, and [Se-Se]-OT-OH atthe hV2R. (C) Representative IP1 concentration–response curves of [Se-Se]-OT-OH atthe hOTR, hV1aR, and hV1bR. (D) Representative radioligand concentration-displacement curves for [Se-Se]-OT-OH at the hOTR, hV1aR, hV1bR, and hV2R. Allcurves were normalized to percentage of response or displacement of the control lig-and (oxytocin for OTR and vasopressin for AVPRs). Data in (A) to (D) aremeans ± SEMofresults obtained from at least n = 3 separate experiments, each performed in triplicate.
Fig. 3. Comparison of selectivity, stability, and ability to augment myometrialstrip contractions between [Se-Se]-OT-OH and oxytocin. (A) Functional selectivityprofile of [Se-Se]-OT-OH and OT over all four human (h) and murine (m) oxytocinand vasopressin receptors. OTR, V1aR, and V1bR activity was measured by IP1accumulation. V2R activity was measured by cAMP accumulation. (B) Metabolicstability of [Se-Se]-OT-OH (t1/2 = 25 hours) compared to oxytocin (t1/2 = 12 hours)in human serum. Data in (A) and (B) are means ± SEM of results obtained from atleast n = 3 separate experiments, each performed in triplicate. (C and D) Repre-sentative contraction pattern for human myometrial strips exposed to increasingdoses of oxytocin (C) or [Se-Se]-OT-OH (D). n = 5 women for [Se-Se]-OT-OH, and n = 8women for oxytocin.
groups showed similar investigation of a nonsocial stimulus (small emp-ty cage), indicating similar amounts of nonsocial anxiety. Duringthe social fear recall (Fig. 4B), all groups of mice, irrespective of theirtreatment, showed high social investigation, which indicated reducedsocial fear and successful social fear extinction.
Maximal Ca2+ concentrations in human cardiomyocytes as aproxy for assessing cardiovascular safetyV1aR is highly abundant in the vascular smooth muscle and heart(cardiomyocytes), and V1aR activation has been linked to cardiovascularrisks (72–76). As a proxy to assess cardiovascular risks, we measuredmaximal intracellular Ca2+ concentrations in human cardiomyocytestreated with the various ligands (Fig. 5). Vasopressin and oxytocinconcentration-dependently caused an increase in maximal intracellularCa2+ in human cardiomyocytes with EC50 values of 121 and 174 nM,respectively, which aligned well with our pharmacological results ofhV1aR activation by oxytocin (tables S2 and S3). Consistent with itsimproved selectivity (OTR activation without V1aR activation), [Se-Se]-OT-OH did not affect the maximal intracellular Ca2+ concentration(up to 10 mM) in cardiomyocytes (Fig. 5).
DISCUSSIONAgonists and antagonists with good selectivity for the four oxytocin andvasopressin receptor subtypes are in short supply (37, 38, 77). Physio-logical function and dysfunction of the receptors are therefore studiedby time- and cost-intensive knockout models, which have linked thesubtypes to various disorders including autism, schizophrenia, epilepsy,stress, aggression, depression, anxiety, and pain (5, 8, 16, 33). Therapeuticdevelopment targeting these disorders has so far failed because of thechallenge of developing subtype-selective ligands that retain their se-lectivity across species, thereby limiting translational studies withclinically relevant animal models (37, 38, 78).
Subtle pharmacophore framework modifications—A newstrategy to retain selectivity across murine andhuman receptorsThis structure-activity relationship study demonstrated that subtlemodifications to the pharmacophore framework of the endogenous
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ligands oxytocin and vasopressin could tune potency and selectivityand yield ligands ([Se-Se]-OT-OH and d[Se-Se]-OT-OH) with an im-proved selectivity profile that was conserved across mouse and human(Fig. 3A and table S3). In particular, the replacement of the disulfidebond with the diselenide bond in combination with the C-terminalamide to acid change in oxytocin yielded [Se-Se]-OT-OH, the com-pound with the most pronounced selectivity gain for the OTR (Fig.3A). Although an amide-to-acid modification can generally beconsidered a subtle modification, it seems that either the free lonepair of the nitrogen atom is involved in receptor recognition and ac-tivation or the negative charge of the acid contributes to activity lossat the AVPRs. Substitution of the disulfide bond by the diselenide bondslightly increases the bond length (+0.3 Å), torsion angle (D +11°),and hydrophobicity (59, 60). In particular, the change in torsionangle could play a part in the selectivity differences observed by push-ing the ligand into a left-handed conformer, as observed in the crystalstructure of dOT (54). Both modifications (diselenide bond and C-terminal acid) had to be present to improve selectivity.
The increased selectivity of [Se-Se]-OT-OH makes it an excellentprobe to delineate OTR function in the central nervous system (CNS)in behavioral animal models (where V2R is not present) and a prom-ising lead molecule for the clinic, which we further explored in thisstudy. We recommend that an effective concentration of 50 nM ora 10-fold higher concentration than oxytocin (in comparative stu-dies with oxytocin) is used to ensure OTR-mediated action whileretaining OTR preference. The weak antagonism (17% inhibition ofvasopressin at the hV1aR) does not affect the investigation of OTRfunction but needs to be taken into account either when bindingstudies are carried out ([Se-Se]-OT-OH binds to the hV1aR with aKi of 56.8 nM) or when [Se-Se]-OT-OH is administered in the pres-ence of V1aR ligands. [Se-Se]-OT-OH is a partial agonist for OTR;however, this did not affect biological function as suggested by ourmyometrial contractility study, which showed that OTR activationresulted in a full response similar to that induced by oxytocin (Fig.3, C and D, and fig. S5). Partial agonists can be beneficial in treatingchronic disorders such as pain and autism because they often do nottrigger the development of adverse effects such as overstimulation, de-sensitization, adaptation, tolerance, and dependence as seen with fullagonists.
Fig. 4. Social fear conditioning (SFC)mouse study. (A and B) Unconditioned (SFC−) and conditioned (SFC+) mice were intracerebroventricularly infused with either vehicle(2 ml of Ringer’s solution; n = 9 SFC− mice; n = 10 SFC+ mice) or [Se-Se]-OT-OH (250 mM/2 ml; 500 pmol; n = 7 SFC− mice; n = 7 SFC+ mice). Oxytocin (250 mM/2 ml;500 pmol; n = 10 mice) and [Thr4,Gly7]-OT (250 mM/2 ml; 500 pmol; n = 4 mice) were only infused into SFC+ mice 10 min before extinction training. Percentage ofmice that investigated three nonsocial stimuli (empty cage) and six social stimuli (cage with a conspecific) during social fear extinction (day 2; A) and six socialstimuli during social fear extinction recall (day 3; B) is shown. Data are means ± SEM and were analyzed using two-way analysis of variance (ANOVA) for repeatedmeasures. (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 < 0.05SFC+/vehicle compared to SFC+/OT and SFC−/vehicle.
General conclusions and guidelines for future oxytocin andvasopressin ligand designN-terminal deamination of oxytocin ligands improves binding andpotency at all four receptors, renders oxytocin slightly more hydro-phobic, and improves its stability against proteases (55). AlthoughN-terminal deamination of vasopressin did not affect potency andselectivity, we still recommend deamination because it offers enhancedproteolytic stability. Considering the advancements in the area of di-sulfide mimetics, we recommend the replacement of the disulfidebond by a nonreducible and therefore metabolically more stable thio-or selenoether bond (4, 56). Modification of the C terminus of oxyto-cin and vasopressin could yield novel antagonists, considering thisstudy and the replacement of Gly9 in oxytocin and vasopressin by Val,which results in an agonist-to-antagonist switch at the hV1aR for bothligands (50). The FLIPR is a valuable instrument for primary screeningof compounds that target oxytocin and vasopressin receptors becauseit rapidly provides agonist and antagonist information with high-throughput capabilities. Follow-up characterization of selected leadsby ligand binding, second-messenger quantification, and arrestin re-cruitment can then be used to better understand ligand kinetics andsecondary messenger signaling pathways. The structure-activity rela-tionship study presented here assessed affinity and functional data(FLIPR and IP1) of the most commonly used control compounds ox-ytocin, vasopressin, dOT, and dAVP over all four receptor subtypesin a single study and could be a reference for future oxytocin andvasopressin ligand development.
Comparison of the FLIPR and IP1 assaysOnly three discrepancies with more than 50-fold difference were de-tected between the FLIPR and the IP1 assays (oxytocin and dOT at thehV1bR and d[Se-Se]-OT at the hV1aR; table S4), suggesting signalingbias for these ligands at these receptors. The characteristic Ca2+ tran-sients elicited by activation of Gaq-coupled GPCRs such as OTR,V1aR, and V1bR originate from the production of IP3 downstreamof phospholipase C activation, which induces the release of Ca2+ fromintracellular stores through IP3 receptors, termination of receptor sig-
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naling, and subsequent uptake of increased intracellular Ca2+ intostores and the extracellular compartment through Ca2+ adenosine tri-phosphatases (79). In addition, Ca2+ signals can be magnified or in-hibited through intracellular feedback mechanisms such as Ca2+-inducedCa2+ release, activation of downstream effectors that modulate intra-cellular Ca2+ amounts, or modification of Ca2+ signals through alter-nate G proteins such as Gai, which is involved in OTR and vasopressinreceptor signaling (5, 80). Thus, whereas IP3 production is a requisitestep for initiation of a Ca2+ signal, other factors contribute to the mag-nitude and kinetics of the observed Ca2+ transients elicited by activationof GPCRs. In contrast, the IP1 assay indirectly quantifies the productionof IP3 through accumulation of its breakdown product IP1. It isplausible that the observed differences arise because of mechanisticdifferences that contribute to increases in intracellular Ca2+ and accu-mulation of IP1. The cellular consequences of such differences remainunclear to date, although Ca2+ signaling is a physiologically relevantconsequence of GPCR activation. The difference in signaling producedby these ligands indicates a potential signaling bias, a phenomenon thathas been previously studied for this receptor class (43, 80–82).
[Se-Se]-OT-OH modulating central actions: Reversal ofsocial fearOxytocin is linked to multiple CNS disorders such as autism, bipolardisorders, schizophrenia, pain, anxiety, and depression (5, 8, 16, 33).Studies showing that intranasal oxytocin administration can modulatesocial behavior in humans have resulted in increased interest and clin-ical trials exploring the therapeutic potential of intranasal oxytocin forthese disorders. The exact target pharmacology and underlyingmechanisms of action are, however, still under debate because oxyto-cin can also signal through V1aR and V1bR (tables S2 to S5). Hence,receptor subtype–specific probes are critical because of the partlyopposing behavioral effects of oxytocin and vasopressin, especiallyin the context of anxiety and stress regulation (14). [Se-Se]-OT-OHallows for a clear and simple distinction between the involvementof OTR compared to V1aR and V1bR in the CNS without the use oftime- and cost-intensive knockout models or coapplication of species-selective V1a and V1bR antagonists. This is particularly valuablefor therapeutic target validation and future drug development. Here,we chose to behaviorally test our novel probe in the SFC mousemodel, which has clinical implication to treat social anxiety disorders(15, 71, 83), and to compare it to the commonly used mOTR-selectiveagonist [Thr4,Gly7]-OT, which does not retain its selectivity profilefor human receptors. The use of [Se-Se]-OT-OH confirmed that thisaction is mediated through OTR and not through V1aR or V1bR,and [Se-Se]-OT-OH displayed similar effects to [Thr4,Gly7]-OT (Fig.4, A and B).
[Se-Se]-OT-OH modulating peripheral action: Laborinduction and augmentationSelectivity against V1aR to reduce cardiovascular side effects is of par-ticular importance for peripheral applications because V1aR is the pri-mary receptor subtype in the vascular smooth muscle and heart(72–76). Clinically, oxytocin is administered intravenously to inducelabor, augment weak contractions in labor, and treat postpartum hem-orrhage and intranasally to elicit lactation. Because of its activationof AVPRs, oxytocin administration has only a limited therapeuticwindow of use, and adverse effects reported in the mother includeanaphylactic reaction, postpartum hemorrhage, cardiac arrhythmia,fatal afibrinogenemia, pelvic hematoma, subarachnoid hemorrhage,
Fig. 5. Effects of vasopressin, oxytocin, and [Se-Se]-OT-OH on maximal intra-cellular Ca2+ inhumancardiomyocytes. FLIPR assays showing the effect of vasopres-sin, oxytocin, and [Se-Se]-OT-OHon themaximal intracellular Ca2+ concentrations. Dataare means ± SEM, n = 4 wells per data point.
hypertensive episodes, rupture of the uterus, convulsions, coma, anddeath due to water intoxication. In the neonate, adverse effects reportedinclude bradycardia, cardiac arrhythmia, jaundice, retinal hemorrhage,permanent CNS or brain damage, seizures, and death (46, 84, 85).Hence, the lack of AVPR activation in OTR agonists is needed todiminish side effects.
[Se-Se]-OT-OH may be such a candidate because it only minimallyraised 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 mediatesmany of its cardiovascular side effects. Furthermore, [Se-Se]-OT-OHand oxytocin were equipotent in augmenting contractility in humanmyometrial preparations, although [Se-Se]-OT-OH displayed a moreregular contraction pattern (Fig. 3D). By contrast, oxytocin reducedcontraction frequency in a concentration-dependent fashion but in-creased the contraction duration, resulting in much greater total con-tractile activity (Fig. 3C), which can lead to uterine hyperstimulationand rupture, which are life-threatening events for mother and fetus(86, 87). [Se-Se]-OT-OH’s inactivity at V1aR, a receptor that is alsoabundant in the myometrium during pregnancy and is involved incontraction (88, 89), in combination with its partial agonism at theOTR, resulted in an improved contractility profile, suggesting that itwould be a more controlled and safer alternative to oxytocin to induceand augment human labor. Thus, [Se-Se]-OT-OH not only reducedAVPR-related side effects but also offered additional advantages toaid labor induction and progression through a more regular increaseof contraction strength without the risk of uterine hyperstimulationor rupture.
Human serum stability of [Se-Se]-OT-OHEnzymatic stability is a key characteristic for many peptide drug can-didates, and hence, it was important to characterize the stability of[Se-Se]-OT-OH compared to oxytocin. Diselenide bonds exhibit alower redox potential than disulfide bonds, rendering such mimeticsmore difficult to reduce, a feature that can be exploited in improvingthe half-life of peptide drug candidates (4, 56, 59–61). On the otherhand, peptides with C-terminal amides are more stable than peptideswith C-terminal acids. The improved stability of [Se-Se]-OT-OH inhuman serum (Fig. 3B) suggests that the impact of the more redox-stable diselenide bond outweighs the disadvantage of the C-terminalacid modification. However, the experimentally determined in vitrohuman serum half-life (which was in hours) of oxytocin and [Se-Se]-OT-OH does not reflect physiologically relevant in vivo half-life (whichis in minutes) because it does not take renal clearance into account.Nonetheless, it can assess even small stability improvements, whichcould be important for treating CNS disorders through delivery methods[such as treatment of autism in children or treatment of migraines inadults (21)] that are not affected by renal clearance.
In conclusion, we showed that subtle modifications to the pharma-cophore framework of oxytocin can lead to enhanced selectivity pro-files that are conserved across species (mouse and human), therebyovercoming a substantial hurdle in ligand development. This proof-of-concept study provides a framework that could be applied to similarcomplex peptidergic signaling systems. The lead ligand derived fromthis study, [Se-Se]-OT-OH, is a valuable probe that can effectivelydelineate the physiological responses of OTR and is particularly suitedfor use in animal behavior studies looking at the central roles of OTR.Its functional selectivity and close structural similarity to oxytocin areimportant features that will help to clarify the contributions of the
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oxytocin signaling system in health and disease. The improved selec-tivity, stability, and safety profile of [Se-Se]-OT-OH could lead to su-perior alternatives to oxytocin in the clinic for peripheral indicationssuch as labor induction and progression and postpartum hemorrhage,as well as for central indications such as autism, migraine, schizo-phrenia, anxiety, and stress, where clinical trials have shown that in-tranasally administered oxytocin is safe and therapeutically effective(20, 21, 90–93). [Se-Se]-OT-OH has the potential to advance ourunderstanding of the OTR as a therapeutic target in different animalmodels and represents a promising lead molecule for various high-profile OTR-related indications.
MATERIALS AND METHODSSelenocysteine building block synthesisAn optimized protocol with yield-improving modifications was devel-oped on the basis of 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 scaleof 20 g with an overall yield of 62%. A new two-step strategy was de-vised for the synthesis of dSec(Meb)-OH with an overall yield of 12%(Fig. 1B).
Peptide synthesisPeptides 1 to 16 were assembled manually by Boc-SPPS using theHBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hex-afluorophosphate)–mediated in situ neutralization protocol (59, 65).The analogs were cleaved with hydrogen fluoride (94), purified byRP-HPLC, folded over 24 hours in 0.1 M NH4HCO3 buffer at pH8.2 (1 mg/10 ml; ~100 mM), and purified by RP-HPLC to >95% pu-rity. Peptide concentrations were determined on the basis of peakarea detected at 214 nm by analytical RP-HPLC against oxytocinand vasopressin as standards with known peptide content establishedby amino acid analysis. Using the Beer-Lambert law, the peptide con-centrations were calculated on the basis of absorptions of standardsand samples using calculated extinction coefficients (95–97).
Transfection and membrane preparation for FLIPR,HTRF-IP1, and radioligand displacement assays for thehuman receptorshOTR, hV1aR, hV1bR, and hV2R complementary DNAs (cDNAs)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 (2200 Ci/mmol), FlashBlue GPCR Scintillating Beads, TopSeal-A 96-wellsealing film, and 384-well white OptiPlates were from PerkinElmer LifeSciences. The HTRF-IP1 assay kit was from Cisbio International.
COS-1 cells grown in Dulbecco’s modified Eagle’s medium (DMEM)and 5% fetal bovine serum (FBS) in 150-mm plates were transientlytransfected with plasmid DNA (14.5 mg) encoding the hOTR, hV1aR,hV1bR, or hV2R using Lipofectamine 2000 (29 ml; Invitrogen). The cellswere harvested 48 hours after transfection and homogenized using anUltra-Turrax homogenizer (22,000 rpm) in assay buffer [50 mM tris-HCl, 10 mM MgCl2 (5 mM MgCl2 for oxytocin), and 0.1% bovineserum albumin (BSA) (pH 7.4)] with cOmplete protease inhibitorcocktail (Roche Diagnostics). The homogenate was centrifuged at 484g(2000 rpm) for 10 min, and the resulting supernatant was centrifugedat 23,665g (14,000 rpm) for 30 min. The pellet was resuspended in ap-propriate buffer without protease inhibitor containing 10% glycerol andstored at −80°C until assayed.
FLIPR assay for the hOTR, hV1aR, and hV1bRCOS-1 cells transfected with the hOTR, hV1aR, or hV1bR were plated24 hours before the experiment at a density of 35,000 to 50,000 cellsper well on black-walled 96-well imaging plates (Corning). Cells wereloaded for 30 min at 37°C with Fluo-4-AM [4-(6-acetoxymethoxy-2,7-difluoro-3-oxo-9-xanthenyl)-4′-methyl-2,2′-(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester] (4.8 mM) inphysiological salt solution (PSS; 140 mM NaCl, 11.5 mM glucose,5.9 mM KCl, 1.4 mM MgCl2, 1.2 mM NaH2PO4, 5 mM NaHCO3,1.8 mM CaCl2, and 10 mM Hepes) containing 0.3% fatty acid–freeBSA. To allow for complete dye de-esterification, cells were washed withPSS for 5 to 10 min before transferring them to a FLIPRTETRA (Molec-ular Devices) fluorescent plate reader. Ca2+ responses were measuredusing a cooled charge-coupled device (CCD) camera with excitationat 470 to 495 nM and emission at 515 to 575 nM. The baseline fluo-rescence was set to a minimum of 1000 arbitrary fluorescence units byadjusting camera gain and excitation intensity. Compounds were addedas 3× concentrated stock solutions in PSS, with 10 baseline fluorescencereadings before compound addition followed by fluorescence readingevery second for 180 s.
Raw fluorescence data were converted to DF/F values by subtract-ing baseline fluorescence readings from subsequent time points anddividing the difference by baseline fluorescence values, as previously de-scribed (98). For the concentration-response curves, maximum DF/Fvalues after the addition of compounds were plotted against agonist con-centration and normalized to the response elicited by the native ligand(oxytocin for the hOTR and vasopressin for the hV1aR and hV1bR).A four-parameter Hill equation with a Hill coefficient of 1 was fittedto the data using GraphPad Prism (version 4.00).
cAMP assay for hV2RThe cDNA plasmid clones for hV2R were a gift from R. Schülein(FMP, Berlin). The hV2R sequence was inserted into pKaede-MN1(MBL Life Science) using Eco RI and Hind III restriction sites to yieldthe wild-type receptor. The conditions for the propagation of humanembryonic kidney (HEK) 293 cells and the creation of stably trans-fected cell lines were similar to those described previously (48, 99).Briefly, cells were transfected with CaPO4 transfection, and 1.8 × 106 ofHEK293 cells per 10-cm dish were prepared. Twenty microliters of DNA,with a concentration of 1 mg/ml, was mixed with 480 ml of H2O + CaCl2(430 ml of H2O + 50 ml of CaCl2) and added to 500 ml of Hepes-bufferedsaline solution. After 6 min, the solution was added to the DMEM high-glucose medium (PAA Laboratories), supplemented with L-glutamineand gentamicin, and the cells were incubated for 4 hours at 37°C. After aglycerol shock, DMEM was added again and the cells were put back at37°C. In the following days, selection through the antibiotic Geneticin[geneticin G418-BC liquid (50 mg/ml; Biochrom)] took place.
Cells were grown in six-well plates. The adenine nucleotide pool wasmetabolically labeled by incubating confluent monolayers with 3H-adenine (1 mCi per well) ([2,8-3H]-adenine; 27.2 Ci/mmol, PerkinElmerLife Sciences) for 16 hours as described previously (100). After pre-incubation, fresh medium that contained 100 mM RO201724 (a cell-permeable, selective inhibitor of cAMP-specific phosphodiesterase;Calbiochem)was added. After 4 hours, cAMP formationwas stimulatedby the hV2R agonist vasopressin (50 pM to 1 mM), oxytocin (60 pMto 1 mM), synthetic oxytocin analogs (700 pM to 10 mM), or forskolin(30 mM; a cell-permeable diterpenoid that has antihypertensive, positiveinotropic, and adenylyl cyclase–activating properties; Sigma-Aldrich)for 20 min at 37°C. Assays were performed in at least three separate
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experiments in triplicate. The formation of 3H-cAMP was determinedaccording 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 valueswere presented as means ± SEM.
HTRF-IP1 assay for the human receptorsCOS-1 cells were transiently transfected with plasmid DNA encodingthe hOTR, hV1aR, or hV1bR using a Lipofectamine 2000/DNA ratioof 2 in DMEM. Assays measuring IP1 accumulation were performed48 hours after transfection according to the manufacturer’s protocol.Briefly, cells were incubated with increasing concentrations of oxyto-cin and vasopressin analogs (10 pM to 10 mM) in stimulation buffercontaining LiCl for 1 hour in 37°C and 5% CO2 in white 384-wellOptiPlates. Cells were lysed by the addition of the HTRF reagents,the europium cryptate–labeled anti-IP1 antibody, and the d2-labeledIP1 analog and diluted in lysis buffer. The assays were incubated for1 hour at room temperature. The emission signals at 590 and 665 nmwere measured after excitation at 340 nm using the EnVision multi-label plate reader (PerkinElmer Life Sciences). Signal was presented asthe HTRF ratio: F = [(F665nm/F590nm) × 104]. For concentration-responsecurves, the HTRF ratio values after the addition of compounds wereplotted against the ligand concentration and normalized to the responseelicited without ligand (negative control). Sigmoidal curves for the cal-culation of the EC50 values were fitted to individual data points by non-linear regression with a Hill coefficient of 1, using the software packagePrism (GraphPad Software).
Radioligand displacement assay for the human receptorsCompetitive binding assays were performed using FlashBlue GPCRScintillating Beads (PerkinElmer Life Sciences) as previously described(56). Reactions containing increasing concentrations of competingoxytocin and vasopressin analogs (10 pM to 10 mM); FlashBlue GPCRScintillating Beads (100 mg for the hOTR and hV1aR; 200 mg for thehV1bR and hV2R); hOTR, hV1aR, hV1bR, and hV2R membranepreparations (5 mg of protein); and radioligand [3H-OT (2 nM) forthe hOTR, 125I-V1a antagonist ([125I]phenylacetyl-d-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2) (0.5 nM) for the hV1aR, and 3H-AVP (3 nM)for the hV1bR and hV2R] in assay buffer [50 mM tris-HCl, 10 mMMgCl2(5 mM MgCl2 for oxytocin), and 0.1% BSA (pH 7.4)] were established in96-well white polystyrene plates with clear flat bottoms in a total reactionvolume of 80 ml. Radioligand binding was detected using a Wallac 1450MicroBeta scintillation counter (PerkinElmer Life Sciences). Sigmoidalcurves for the calculation of the IC50 values were fitted to individualdata points by nonlinear regression with a Hill coefficient of −1, usingthe software package Prism (GraphPad Software).
Radioligand displacement assay for the murine receptorsCompetitive binding assays were performed using 3H-OT and 3H-AVP (PerkinElmer Life Sciences) and increasing concentrations of theindicated peptides at 30°C onmembranes prepared fromHEK293 cellstransfected with the mOTR, mV1aR, mV1bR, or mV2R as describedpreviously (66). Compound affinities (Ki) were determined by meansof competition experiments in which the unlabeled compound con-centrations varied from 10 pM to 10 mM to displace 4 nM 3H-OT forthe mOTR and 3H-AVP for themV1aR, mV1bR, andmV2R. Ligandbinding data and Ki were analyzed by means of nonlinear regressionand binding-competitive fitting using Prism version 5 (GraphPadSoftware).
HTRF-IP1 assay and cAMP determination for themurine receptorsIP1 and cAMP accumulation were determined in HEK293 cells tran-siently transfected with DNA plasmids encoding the mOTR, mV1aR,mV1bR, or mV2R using HTRF assays (IP1 and cAMP assays, CisbioInternational) as previously described (66). Sigmoidal curves for the calcu-lation of the EC50 values were fitted by nonlinear regression log(agonist)versus response fitting, using Prism version 5 (GraphPad Software).
Stability assaysPeptides were added to human serum, and aliquots were taken and ana-lyzed by RP-HPLC and liquid chromatography–mass spectrometry at 1-,2-, 3-, 4-, 12-, 24-, and 48-hour time points as previously described (56).
SFC mouse modelSFCwas performed as previously described (71). Sample sizes (>4mice)were determined on the basis of previous studies (71, 83, 101). All ex-perimental procedures were performed in accordance with the Guidefor the Care and Use of Laboratory Animals of the Government ofOberpfalz and the guidelines of the National Institutes of Health. Allmale CD1mice (8 to 10weeks of age at the start of experiments; CharlesRiver Laboratories) were group-housed under standard laboratoryconditions (12-hour light/12-hour dark cycle, lights on at 06:00, 22°C,60% humidity, and food and water ad libitum) in polycarbonate cages(16 cm × 22 cm × 14 cm). All experimental procedures were performedbetween 08:00 and 15:00. Surgeries were performed under isofluraneanesthesia, and extreme care was taken to minimize animal suffering.Analysis of the SFC paradigm was performed using GraphPad Prismversion 6.0 (GraphPad Software).
On day 1 of the SFC paradigm, mice were transferred from theirhome cage into the conditioning chamber; after a 30-s adaptationperiod, an empty cage was placed in the conditioning chamber as anonsocial stimulus that mice were allowed to investigate for 3 minbefore it was replaced by an identical cage containing an unfamiliarmale conspecific (which represented the social stimulus). SFC−
mice were allowed to investigate the social stimulus for 3 min with-out receiving any foot shocks, whereas SFC+ mice were given a 1-selectric foot shock (0.7 mA) each time they investigated (sniffed)the social stimulus. Mice were returned to their home cage when nofurther social contact was made for 2 min. All mice investigated thenonsocial (empty cage) stimulus to a similar extent and received asimilar number of foot shocks (table S6). On day 2, which consisted ofsocial fear extinction training, mice were exposed to three nonsocialstimuli (empty cages) in their home cage to assess nonsocial investi-gation as a parameter of nonsocial fear and general anxiety-relatedbehavior. Mice were then exposed to six unfamiliar social stimuli(six different male mice) to assess social investigation as a parameterof social fear. Mice received single acute intracerebroventricular in-fusions (35 to 45 s) of either vehicle (2 ml of sterile Ringer’s solution),oxytocin (2 ml of 250 mM; 500 pmol), [Se-Se]-OT-OH (2 ml of 250 mM;500 pmol), or [Thr4,Gly7]-OT (2 ml of 250 mM; 500 pmol) 10 min beforesocial fear extinction. On day 3, which assessed social fear extinctionrecall, mice were exposed in their home cage to six different unfamiliarsocial stimuli to investigate whether repeated exposure to socialstimuli during extinction leads to a complete reversal of social fear.
Human myometrial cell contractility assayHuman uterine myometrial smooth muscle cells (hTERT-HM) werecultured, collagen gels were prepared, and the assays were performed
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as described previously (48, 70). Briefly, cells were cultured in DMEM/F-12/10% FBS (Invitrogen). Collagen gels were prepared from rat tailtype 1 collagen (Sigma-Aldrich) to a final concentration of 1.5mg/mland seeded in 24-well culture dishes, with 150,000 hTERT-HM cellsper well. Cells in collagen gels were allowed to equilibrate overnight inserum-free DMEM. The ligands of interest (1 mMconcentration) wereadded to the serum-free medium, and the gels were released from thesides of the wells. FBS [10% (v/v)] was used as a positive control forcontraction, and unstimulated cells were used as the negative control inall experiments. Gel imageswere captured over time using a FluorChem8900 imager, and the area (in square centimeters) of the gels wasmeasured using AlphaEaseFC software (Alpha Innotech Corporation).A decrease in gel area correlated with an increase in contractility.
Tissue bath myometrial contractility assaysBiopsies of myometrium were obtained from women undergoingterm (39 to 40 weeks) prelabor elective cesarean section delivery atLiverpool Women’s Hospital, Liverpool, UK. All women gave writteninformed consent to participate, and the study was approved by theNorth West (Liverpool East) Research Ethics Committee (reference#10/H1002/49) and by the Research and Development director atLiverpool Women’s Hospital NHS Foundation Trust, Liverpool, UK.A full-thickness biopsy (~2 cm3) was cut from the upper lip of thelower uterine incision site and placed into Hanks’ balanced salt so-lution at 4°C (102). In the laboratory, strips of myometrium (1 mm ×2 mm × 5 mm) were dissected and placed between a force transducerand a fixed hook using aluminum clips. Strips were continually super-fused with PSS [154 mMNaCl, 5.6 mM KCl, 1.2 mMMgSO4, 7.8 mMglucose, 10.9 mM Hepes, and 2.0 mM CaCl2 (pH 7.4)] maintained at36°C. After stable spontaneous contractions developed, the strips wereexposed to rising concentrations of oxytocin or [Se-Se]-OT-OH. Datawere analyzed by measuring the amplitude of contraction using OriginPro 9.0 software (OriginLab Corporation) as described previously(48, 103, 104). The effect of [Se-Se]-OT-OH or oxytocin was com-pared to the activity preceding the application of the first dose, andthe data represent the change in activity compared to spontaneouscontrol activity (100%). Concentration-response curves were fittedusing nonlinear regression to calculate EC50 values.
Human cardiomyocyte assayWild-type WTC11 human-induced pluripotent stem cells (hiPSCs)were used in this study. Undifferentiated cells were maintained inmTeSR1 medium (STEMCELL Technologies). Standard cardiomyocyte-directed differentiation using a monolayer platform was performedwith a modified protocol based on previous reports (105–109). Thedifferentiation setup was initiated by plating undifferentiated hiPSCsas single cells. The cultures were treated with 1 mMCHIR-99021 (CaymanChemical) for 24 hours before reaching confluence. Cells were in-duced to differentiate (designated day 0) by replacing the culturingmedium with RPMI 1640 medium (catalog number 11875-119, ThermoFisher Scientific) containing 3 mM CHIR-99021, BSA (500 mg/ml), andascorbic acid (213 mg/ml). On day 3, the medium was changed to RPMI1640 medium with BSA and ascorbic acid containing 1 mM XAV-939(Tocris Bioscience). On day 5, the medium was changed to RPMI1640 containing ascorbic acid and BSA. On day 7, the medium wasreplaced with RPMI 1640 containing B-27 supplement with insulin(catalog number 17504-044, Thermo Fisher Scientific). Cells wereharvested using trypsin, replated into a black-walled 384-well imagingplate coated with CellBIND (Sigma-Aldrich) at a density of 4 × 104 cells
per well and cultured for 8 days before the experiments. Functionalactivity was assessed using a high-throughput FLIPRTETRA (Molec-ular Devices) FLIPR assay. The growth medium was removed and re-placed with Calcium 4 (no-wash) dye (Molecular Devices) diluted inPSS [140 mM NaCl, 11.5 mM glucose, 5.9 mM KCl, 1.4 mM MgCl2,1.2 mMNaH2PO4, 5 mMNaHCO3, 1.8 mM CaCl2, and 10 mMHepes(pH 7.4)] according to the manufacturer’s instructions and incubatedat 37°C for 30 min. Changes in intracellular Ca2+ amounts were mea-sured using a cooled CCD camera (excitation, 470 to 495 nm; emission,515 to 575 nm) with reads taken every 0.4 s for 240 s after the additionof compounds. The maximum intracellular Ca2+ amount was deter-mined using ScreenWorks (version 2.2.0.14, Molecular Devices).
Statistical analysisAll 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 statisticalsignificance for the humanmyometrial cell contractility and organ bathassays. Two-way ANOVA was used to analyze the SFC data. P values<0.05 were considered significant.
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SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/10/508/eaan3398/DC1Materials and MethodsFig. 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, andmV2R.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.Table S2. Functional potencies for oxytocin and vasopressin analogs at the hOTR, hV1aR,hV1bR, and hV2R.Table S3. Functional potencies for oxytocin and vasopressin analogs at the human and murineOTR, 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 humanand murine OTR, V1aR, V1bR, and V2R.Table S6. Overview of the number of electric foot shocks per mouse.Reference (110)
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Acknowledgments: We acknowledge A. Jones for his help with mass spectrometry,A. Mulligan for his contribution to some of the synthetic work, L. Rash for conductingthe qualitative control and concentration determination of tested analogs, andM. Freissmuth and B. Chini for their continuous support. Funding: This work was supportedby National Health and Medical Research Council (NHMRC) Project (1063803) andProgram grants (1072113 and 1063803). I.V. was supported by an Australian ResearchCouncil (ARC) Future Fellowship grant (FT130101215) and R.J.L. was supported byan NHMRC Fellowship. M.B. is an Umberto Veronesi Foundation Postdoctoral Fellow(FUV 2017). M.M. received funding from the European Union Seventh FrameworkProgramme (FP7/2007-2013) under Marie Curie Actions grant agreement no. 254897 and2013-BP-B-00109, from the Secretary of Universities and Research of the Economy andKnowledge Department of the Government of Catalonia, from the ARC (DE150100784),and from the European Research Council under the European Union’s Horizon 2020Research and Innovation Programme grant agreement no. 714366. C.W.G. is an ARC FutureFellow (FT140100730), and his research was supported by the Vienna Science andTechnology Fund (WWTF) through project grant LS13-017. S.W. and S.A. were supportedby a Harris-Wellbeing Preterm Birth Research Centre grant administered by Wellbeingof Women, UK. I.D.N. was supported by the DFG (Ne 465/27-1), German Ministryof Education and Research (BMBF; OptiMD; 01EE1401A), and European Union SeventhFramework (FemNAT CD; Health-F2-2013-602407). Author contributions: M.M. designedand performed the synthesis of the peptide analogs and developed the selenocysteinechemistry. A.A., I.V., M.B., L.R., C.B., R.J.L., and C.W.G. performed the pharmacologicalanalysis of the analogs. J.R.D., H.S.C., and N.J.P. performed the cardiomyocyte experiments.S.A., S.W., M.O., and T.J.S. performed the uterine strip contraction studies. R.M. andI.D.N. performed the behavioral in vivo experiments. M.M. and P.F.A. directed the projectand 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 ona U.S. patent (2013/0130,985) for oxytocin peptide analogs. The other authors declare thatthey have no competing interests. Data and materials availability: The hiPSC line(WTC11 hiPSCs) used to generate the cardiomyocytes in this study requires a materialtransfer agreement from Bruce Conklin’s laboratory at University of California, SanFrancisco (Gladstone Institute).
Submitted 30 March 2017Accepted 7 November 2017Published 5 December 201710.1126/scisignal.aan3398
Citation: M. Muttenthaler, Å. Andersson, I. Vetter, R. Menon, M. Busnelli, L. Ragnarsson, C. Bergmayr,S. Arrowsmith, J. R. Deuis, H. S. Chiu, N. J. Palpant, M. O’Brien, T. J. Smith, S. Wray, I. D. Neumann,C. W. Gruber, R. J. Lewis, P. F. Alewood, Subtle modifications to oxytocin produce ligands thatretain potency and improved selectivity across species. Sci. Signal. 10, eaan3398 (2017).
across speciesSubtle modifications to oxytocin produce ligands that retain potency and improved selectivity
D. Neumann, Christian W. Gruber, Richard J. Lewis and Paul F. AlewoodIngaSarah Arrowsmith, Jennifer R. Deuis, Han Sheng Chiu, Nathan J. Palpant, Margaret O'Brien, Terry J. Smith, Susan Wray,
Markus Muttenthaler, Åsa Andersson, Irina Vetter, Rohit Menon, Marta Busnelli, Lotten Ragnarsson, Christian Bergmayr,
are solely mediated by the oxytocin receptor.thatcross-talk between oxytocin, vasopressin, and their receptors, this compound will also be helpful in identifying effects
mice and induced contractile activity in human myometrial strips without affecting cultured cardiomyocytes. Given thecompound that was more selective for the oxytocin receptor than for the vasopressin receptors. It reduced social fear in
. generated ligands based on oxytocin with subtle modifications, yielding a leadet aloxytocin receptor. Muttenthaler However, oxytocin triggers adverse cardiovascular side effects because it activates the vasopressin receptor and the
Oxytocin is clinically used to induce labor, and there is interest in using this peptide to treat social disorders.A more selective oxytocin receptor agonist
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