Selective Nonpeptidic Fluorescent Ligands for Oxytocin Receptor

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Brief Article

Selective Non-Peptidic Fluorescent Ligands for Oxytocin Receptor: Design, Synthesis and Application to Time-Resolved FRET Binding Assay Iuliia Karpenko, Jean-François Margathe, Thieric Rodriguez, Elsa Pflimlin, Elodie Dupuis, Marcel Hibert, Thierry Durroux, and Dominique Bonnet J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Feb 2015 Downloaded from http://pubs.acs.org on February 2, 2015

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Journal of Medicinal Chemistry

Selective Non-Peptidic Fluorescent Ligands for Oxytocin Receptor: Design, Synthesis and Application to Time-Resolved FRET Binding Assay. Iuliia A. Karpenko,† Jean-François Margathe,† Thiéric Rodriguez,‡ Elsa Pflimlin,‡ Elodie Dupuis,§ Marcel Hibert,† Thierry Durroux,*,‡ and Dominique Bonnet*,† †

Laboratoire d’Innovation Thérapeutique, UMR7200 CNRS/Université de Strasbourg, Labex MEDALIS, Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France. ‡

Institut de Génomique Fonctionnelle, Département de Pharmacologie Moléculaire, CNRS UMR 5203, INSERM U1191, Université de Montpellier, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France.

§

Cisbio Bioassays, Parc Marcel Boiteux, 30200 Codolet, France.

KEYWORDS. Oxytocin receptor, Time-Resolved-FRET, Tag-lite, GPCR, fluorescent probes.

ABSTRACT: The design and the synthesis of the first high-affinity fluorescent ligands for oxytocin receptor (OTR) are described. These compounds enabled the development of a TR-FRET based assay for OTR, readily amenable to high throughput screening. The validation of the assay was achieved by competition experiments with both peptide and nonpeptide OTR ligands as competitors. These probes represent the first selective fluorescent ligands for the oxytocin G protein-coupled receptor.

INTRODUCTION There is growing evidence that the neuropeptide oxytocin (OT) modulates complex social behaviors and social cognition. OT has been reported in human to improve prosocial behavior and trust,1 social memory,2 and to decrease fear associated with social phobia.3 It is considered as a promising target for therapeutic interventions in a variety of mental diseases characterized by anxiety and alteration in social function.4 Of particular interest, some recent studies tend to demonstrate the role of OT and its receptors (OTR) in autism spectrum disorders (ASD).5,6 Thereby, the intranasal administration of OT to ASD patients resulted in significant reduction of repetitive behavior,7 improvements in affective speech comprehension,8 increase of brain activity during social judgments9 and promotion of social interactions.10 However, these studies are generally hampered by the lack of potent, specific and centrally bioavailable ligands. Therefore, we decided to develop efficient chemical tools to study the pharmacology of OTR both in vitro and in vivo. Time-resolved Förster resonance energy transfer (TRFRET), utilizing rare-earth lanthanides with long emission half-lives as donor fluorophores, combines standard FRET with the time-resolved fluorescence detection. The high sensitivity of this approach associated with efficient fluorescent chemical probes provides an excellent opportunity to investigate ligand-protein and protein-protein interactions. Thus, TR-FRET has been successfully applied

to the development of high throughput screening (HTS) assays11 and to detect GPCR oligomerization not only at the surface of living cells12 but also in native tissue.13

Figure 1. Chemical structure of oxytocin and OTR antagonists 1, 2 and 3.

However, the prerequisite for the development of such approaches is to design and to synthesize efficient fluorescent ligands that retain the pharmacological profiles of

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non-labeled probes. In addition to be highly potent and selective, these ligands should be also non-peptidic to allow the in vivo imaging and/or oligomerization studies as recently reported for vasopressin V2 receptor.14 To date, the only fluorescent probes to investigate OTR functions and distribution in native tissues are peptide OT analogues that have a poor bioavailability (poor blood-brain barrier permeability, short half-life, rapid clearance).15 Moreover, few reported peptide fluorescent ligands showed a lack of selectivity for OTR versus the vasopressin V1a and V1b receptors.16 In the present work, we report the design and the synthesis of the first high-affinity selective fluorescent nonpeptidic ligands for OTR. With these probes in hands, a new TR-FRET based assay for OTR was developed and validated by competition experiments with known agonist/antagonist ligands. This assay will be used to accelerate the discovery of small-molecule ligands for OT receptors. In addition, the fluorescent probes described herein could represent useful tools to investigate the in vivo structure, functioning and oligomerization of OTR.

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et al.,17 was alkylated with tert-butyl bromoacetate in the presence of potassium carbonate to lead to ester 5 (85% yield). Hydrolysis of the tert-butyl ester moiety under acidic conditions followed by the coupling of the resulting carboxylic acid with tert-butyl carbazate gave a clean access to the hydrazide 6 (83% yield, two steps). A subsequent treatment of 6 under basic conditions was followed by the coupling of the resulting acid with 3azidopropylamine providing 7 (73% yield, two steps). Next, the Boc protecting group was removed under acidic conditions and the resulting hydrazide was involved into the acid-catalyzed condensation with isatin to yield hydrazone 8 (75% yield, two steps). Its final treatment with triphenylphosphine afforded the target amine 9 in 35% yield, ready to be acylated with activated esters of fluorescein and DY647.

Scheme 2. Synthesis of the conjugatable amino derivatives 13 and 14.a

RESULTS AND DISCUSSION Our strategy to develop fluorescent ligands for OTR was to introduce fluorescent dyes (fluorescein and DY647, which are commonly used in TR-FRET applications) onto two selective high-affinity non-peptide OTR antagonists, 217 and 3 (PF-3274167)18 (Figure 1). The attachment of dyes on 2 was envisaged via the lateral amino-group whereas for 3 it was decided to elongate its methoxymethyl chain. Dyes were incorporated through spacers that differ in terms of length and nature. Scheme 1. Synthesis of the conjugatable amino derivative 9.a a

Reagents and conditions: (i) tBuOK, MeI, THF; (ii) Benzoyloxyacetic acid hydrazide, TFA, THF; (iii) BCl3, CH2Cl2; (iv) KOH, TsO-(CH2-CH2-O)3-CH2-CH2-NBoc2, DMF; (v) TFA, CH2Cl2; (vi) KOH, TsO-(CH2-CH2-O)7-CH2-CH2-NBoc2, DMF.

The synthesis of fluorescent derivatives of triazole 3 (Scheme 2) started from the thiourea 10, which was obtained in excellent yield as previously reported.18 Its subsequent conversion to the corresponding Smethylisothiourea by treatment with methyl iodide in the presence of potassium carbonate was followed by a condensation with benzoyloxyacetic acid hydrazide19 to afford the substituted triazole 11 (41% yield, two steps).

a

Reagents and conditions: (i) tert-Butyl bromoacetate, K2CO3, DMF; (ii) TFA, CH2Cl2, then BocHNNH2, EDCI, HOBt, DIEA, THF; (iii) LiOH, THF-H2O, then 3azidopropylamine, PyBOP, NMM, CH2Cl2; (iv) HCl, dioxane then isatin, EtOH-AcOH : 95-5; (v) Ph3P, THF, H2O.

Amino precursors of fluorescent ligands derived from 2 were synthesized following the route depicted in Scheme 1. Sulfonamide 4, obtained as described by Quattropani,

The benzoyl protecting group was readily removed by treatment with boron trichloride in dichloromethane to afford 12 in 90% yield. The latter was alkylated with NdiBoc amino protected PEG4 or PEG8 chains synthesized as previously described.20,21 A subsequent treatment in acidic conditions to remove the terminal Boc protecting groups enabled the access to amines 13 and 14 in 60% and 30% yield, respectively (two steps).


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Table 1. Physicochemical and spectroscopic properties of fluorescent compounds 15-20. HPLC, cpd

molecular

M/Z

formula

calculated e

15

4.68

C55H50ClN7O12S

534.6542

16

3.96

C60H65ClN8O12S3

611.1864

17 18 19 20 5-SFX DY647-NHS a

tR a (min)

4.38 4.39 3.94 3.97 4.00 3.17

C53H55ClFN7O13

e

526.6855

e

e

f

1205.4255

C66H86ClFN8O17S2

e

691.2691

761.85

e

614.7391

emission

c

(%)

d

h

Fluorescein

501

522

nd

DY647

652

667

nd

Fluorescein

497

525

79

Fluorescein

497

525

77

f

DY647

650

667

39

e

DY647

650

667

40

1205.4251 691.2696

absorption

g

-

Fluorescein

496

522

51

g

-

DY647

648

664

34

586.55

C36H40N3O10 S2Na

e

534.6530

e

C58H70ClFN8O13S2 C31H26N2O10

(HR-MS) e

614.7368

quantum yield

b

λmax (nm)

fluorophore

611.1852

526.6843

C61H71ClFN7O17

M/Z found

b

The detection was performed at 220 nm. The absorption and the emission parameters were measured in HEPES/0.1% BSA, c d pH 7.4. The excitation wavelength was set to 470 nm for fluorescein derivatives and to 600 nm for DY647 derivatives. Fluorescence quantum yields were measured using fluorescein in 0.1 M NaOH (QY = 91%) or DID in MeOH as a reference (QY = 33%). e 2+ f + g h For [M+2H] /2. For [M+H] . Molecular weight. not determined.

Then, the amino derivatives of 2 and 3 were readily conjugated with commercially available Nhydroxysuccinimide esters of 6-(fluorescein-5carboxamido)hexanoic acid (5-SFX) or of DY647 (DY647NHS) in DMSO in the presence of the Hünig’s base (Scheme 3). The resulting fluorescent compounds 15-20 were isolated by semi-preparative reverse-phase HPLC. Their identity was confirmed by HRMS and their purity was determined by analytical RP-HPLC. Scheme 3. Conjugation of amines 9, 13 and 14 with the fluorescent dyesa.

a

Reagents: (i) 5-SFX or DY647-NHS, DIEA, DMSO.

The spectroscopic properties of fluorescent derivatives 1520 were studied in HEPES/0.1% BSA buffer, which is

commonly used for in vitro cell experiments. The absorption and fluorescence maxima of all the six compounds are very close to those of the parent fluorescein and DY647 dyes, whereas the fluorescence quantum yields (QY) for probes 17-20 are more sensitive to the chemical derivatization of the dyes especially for fluorescein (Table 1). In contrast, the length of the spacer (n=3 or n=7) does not impact the QY value. The affinities of the fluorescent derivatives of isatin 2 and triazole 3 for OTR were evaluated by TR-FRET binding assay with the Tag-lite technology which combines the homogeneous time-resolved fluorescence (HTRF) detection with a covalent labeling technology called SNAPtag.11 Saturation TR-FRET experiments were performed using fluorescent ligands as acceptors of the resonance energy transfer and SNAP-tagged receptors labeled with terbium cryptate Lumi4-Tb as FRET donor. To ensure that the N-terminal labeling of OTR did not modify the functional activity of the receptor, EC50 of oxytocin was evaluated and was found to be very close to that determined on wild type receptor (8 nM vs 5 nM, respectively; see SI for detailed experiments). The TR-FRET efficiency was measured as a ratio of the acceptor fluorescence at 665 nm (DY647) or 520 nm (fluorescein) and the donor luminescence (Lumi4-Tb) at 620 nm. According to obtained results, all fluorescent ligands displayed a nanomolar affinity for OTR (Table 2), close to those of parent OTR ligands 2 and 3, indicating that the introduction of the rather bulky fluorophores did not influence the interaction of ligands with the receptor, regardless of the length of the spacer and the nature of the fluorophore. Indeed, no difference in the dissociation constants was observed between the triazole derivatives with PEG chains of 4 and 8 units, suggesting that the PEG4 spacer was already long enough to hold away the fluorophore from the receptor binding pocket



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Table 2. Determination of the dissociation constants of compounds 15-20 by TR-FRET saturation experiments. a

cpd

binding, TR-FRET (Kd, nM) OTR

V1aR

V1bR

0.65

42

nd

c

3

9.5

>1000

>1000

>1000

15

1.73 ± 0.09

61.0 ± 8.9

>1000

135 ± 5

16

1.37 ± 0.17

19.7 ± 1.8

>1000

30.5 ± 2.5

2

d

V2R

b

nd

17

1.59 ± 0.38

>1000

>1000

>1000

18

2.05 ± 0.58

>1000

>1000

>1000

19

1.59 ± 0.39

>1000

>1000

509 ± 122

20

1.86 ± 0.51

>1000

>1000

353 ± 82

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and DY647- derived tracers were in good accordance with those previously reported by the classical radioactive binding assay (Table 3). By contrast, inhibition constant estimated for agonist OT was found higher than the dissociation constant estimated by radioactive binding assays, whatever the tracers used. Such differences in the estimation of the affinity of a ligand has already been reported and can be explained by the existence of a positive or negative binding cooperativity.25

a

The dissociation constants (Kd) of fluorescent ligands were directly determined by time-resolved FRET saturation experiments. Results are expressed as mean ± SEM of three (compounds 17-20) or two (compounds 15-16) independent b separate experiments performed in triplicate. Ki values obtained from radioactive binding competition assays from 17 c 18 d Quattropani, et al. and from Brown, et al. Not determined.

The selectivity of the fluorescent OTR ligands was also investigated towards the three subtypes of the vasopressin receptor family known to have the highest sequence homology (≈ 25%) for OTR22 (Table 2). The incorporation of the fluorescein onto the isatin 2 decreased its selectivity against the vasopressin V1a receptor (Kd(V1aR)/Kd(OTR) = 65 for 2 and 35 for 15), while the labeling of 2 with DY647 (16) turned it almost non-selective versus V1aR (Kd(V1aR)/Kd(OTR) = 14). Moreover, 16 displayed also high affinity (30 nM) for the human V2 receptor. In contrast to isatin derivatives, all fluorescent probes derived from triazole 3 showed an excellent selectivity versus the vasopressin receptors with almost no affinity for the V1b and V1a subtypes (Table 2 and Figure 2A). Surprisingly, introduction of fluorophores onto triazole 3 not only maintained the ability of the resulting fluorescent probes 17-20 to bind to OTR, but also increased their affinity by one order of magnitude and drastically improved their selectivity versus V1aR. The nature of the dye plays also an important role on the binding affinities for V2R. Indeed, unlike probes 17 and 18 labeled with fluorescein, both triazoles 19 and 20 labeled with DY647 displayed submicromolar affinities for V2R (509 and 353 nM, respectively), suggesting that the red fluorophore could participate in V2R binding. To evaluate the relevance of the fluorescent ligands in screening assay, TR-FRET competition experiments were performed on OTR expressing cells (Figure 2B), using 17 (green probe) and 19 (red probe) as tracers and the agonist peptide OT, the antagonist peptide OTA (d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr-NH29]VT)23 and the non-peptidic antagonist 124 as competitors (Figure 1). Thus, the affinities of the antagonists determined by TRFRET competition experiments with both fluorescein-

Figure 2. TR-FRET based assays with ligand 17. (A) Saturation experiments with 17 on the cells expressing OTR, V1aR, V1bR or V2R. (B) Competition experiments with unlabeled OT, OTA and 1, and probe 17 as a tracer on the cells expressing OTR. (C) Binding kinetics of 17 at 0.316 nM (blue), 1 nM (red), 3.16 nM (black) and 10 nM (violet) to OTR. (D) Effects of 17 on inositol phosphate (IP) accumulation: Inhibition of OT-induced IP accumulation by increasing concentrations of 17 (black closed circle). IP accumulation in the presence of 17 (1 µM) (open red circle). IP accumulation in basal condition (open black triangle). Values are mean ± SEM of the three independent experiments performed in triplicate.

To further characterize the binding properties of antagonists 17 and 19 at OTR, ligand binding kinetics were monitored by FRET on cells expressing SNAP-OTR. The binding of fluorescent probes at various concentrations was measured at different times (Figure 2C and Figure S3) giving access to their association and dissociation kinetics (Kon = (3.57 ± 0.20) × 106 and (4.03 ± 0.09) × 106 M-1. min-1 and Koff = (18.9 ± 1.5) × 10-3 and (10.9 ± 1.1) × 10-3 min-1, for 17 and 19, respectively), the plateau being reached after 1 hour. To complement the characterization of 17 and 19, their functional activity was determined by the measurement of inositol phosphate (IP) accumulation following cell incubation with 1 µM of the fluorescent ligands (Figure 2D and Figure S3, red open circle). None of the probes


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increases the intracellular concentration of second messengers but both fully inhibited OT-induced IP accumulation in a dose-dependent manner (Kinact = 6.4 ± 1.4 nM and 5.5 ± 0.3 nM, respectively), highlighting the antagonistic character of 17 and 19 (Figure 2D and S3). Table 3. Determination of the inhibition constants of OT, OTA and 1 on the OTR when using 17 and 19 as tracers. binding, Ki (nM) ligand

tracer

a

reference

17

19

value

OT

9.53 ± 1.57

6.60 ± 1.70

0.79

OTA

0.67 ± 0.07

0.45 ± 0.01

0.21

1

6.22 ± 1.63

6.60 ± 1.27

27.8

a

b

have found applications for the development of a TRFRET based assay readily amenable to HTS. This assay represents a powerful alternative to the classical radioactive binding assay and should facilitate the discovery of novel small-molecule ligands for OTR to further investigate the role of OTR in autism. These probes have also found application to specifically visualize OTR at the cells surface. Combined with their high selectivity towards V1aR, V1bR and V2R, we anticipate that these probes could facilitate the in vivo localization of OTR and the detection of receptor oligomers in native tissues.13

26

EXPERIMENTAL SECTION

23

General procedure for the coupling of fluorescein and DY647 onto amines 9, 13 and 14. A solution of 5SFX (1 equiv) or DY647-NHS (1 equiv) in anhydrous DMSO was added to a solution of 9, 13 or 14 (0.9 equiv) in anhydrous DMSO. Following addition of DIEA (10 equiv), the mixture was stirred at r.t. under argon atmosphere for 2 hours (15, 17, 18) or for 1 hour (16, 19, 20). The completion of the reaction was monitored by analytical RPHPLC. The expected labeled compounds were isolated by semi-preparative RP-HPLC using a linear gradient of solvent B in solvent A. Fractions containing the products of interest were lyophilized and further checked by analytical RP-HPLC.

24

b

Ki were determined with Cheng-Prusoff equation. Ki values from radioligand binding competition assays.

Finally, to highlight the potential of probes 17 and 19 to visualize OTR at the cell surface, confocal laser scanning microscopy experiments were achieved on HEK293 cells overexpressing native OTR. As shown in Figures 3A and 3C, both probes enabled the labeling of OTR at low concentration (10 nM). Following the addition of an excess of OTR peptide ligand carbetocin (2 µM), fluorescent ligand binding was fully reversed, clearly demonstrating the specificity of 17 and 19 for OTR (Figure 3B and 3D). Noteworthy, the fluorescence excitation of probe 19 at 635 nm allowed overcoming the autofluorescence of cells detected with probe 17 excited at 488 nm (Figure 3B and 3D).

Fluorescein-labeled compound 15. Yellow powder (458 nmol, 36%). RP-HPLC purity: >95%; tR = 4.68 min. HRMS (ESI): calcd for C55H52ClN7O12S ([M+2H]2+/2) 534.6542, found 534.6530. DY647-labeled compound 16. Blue powder (517 nmol, 37%). RP-HPLC purity: >95%; tR = 3.96 min. HRMS (ESI): calcd for C60H67ClN8O12S3 ([M+2H]2+/2) 611.1864, found 611.1852. Fluorescein-labeled compound 17. Yellow powder (350 nmol, 37%). RP-HPLC purity: >95%; tR = 4.38 min. HRMS (ESI): calcd for C53H57ClFN7O13 ([M+2H]2+/2) 526.6843, found 526.6855. Fluorescein-labeled compound 18. Yellow powder (574 nmol, 57%). RP-HPLC purity: >95%; tR = 4.39 min. HRMS (ESI): calcd for C61H73ClFN7O17 ([M+2H]2+/2) 614.7368, found 614.7391. DY647-labeled compound 19. Blue powder (150 nmol, 15%). RP-HPLC purity: >95%; tR = 3.94 min. HRMS (ESI): calcd for C58H71ClFN8O13S2 ([M+H]+) 1205.4255, found 1205.4251. DY647-labeled compound 20. Blue powder (480 nmol, 48%). RP-HPLC purity: >95%; tR = 3.97 min. HRMS (ESI): calcd for C66H88ClFN8O17S2 ([M+2H]2+/2) 691.2691, found 691.2696.

Figure 3. Confocal microscopy studies. Confocal images of HEK293 cells expressing wild-type OTR with 10 nM of 17 (A, B) or 19 (C, D) without (A, C) or with (B, D) carbetocin (2 µM).

CONCLUSION In summary, we have designed and synthesized the first selective fluorescent antagonists for OTR. These probes

TR-FRET ligand-binding assays with Tag-lite technology. These assays were performed as previously described.27 Briefly, HEK293 cells were cultivated in DMEM medium supplemented with 10% FCS and 1% penicillin/streptomycin antibiotics. As described in Cottet et al.,28 confluent cells were dissociated and plated in black 96-well plates previously coated with poly-ornithine (diluted at 0.1 mg/mL in sterile water, incubated 30 min at 37



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°C, washed with sterile PBS). The cells were treated with Lipofectamine 2000 complex of plasmid DNA coding for the OT, V1a, V1b or V2 receptors (Cisbio Bioassays) fused to the SNAP-tag suicide enzyme in Opti-MEM medium. Plates were incubated for 48 hours (OTR) or 24 hours (V1aR, V1bR or V2R) at 37 °C and 5% CO2. Expressed receptors fused to SNAP-tag were then labeled with luminescent donor (BG-Lumi4-Tb) at 100 nM in 100 µL/well of the Tag-lite labelling medium (Cisbio Biossays), for 1 hour at 37 °C. Cells were then washed 3 times with the Tag-lite medium and incubated with the fluorescent ligands. For saturation assays, increasing concentrations of fluorescent ligands were diluted in 100 µL of the Tag-lite medium. Noteworthy, the spectroscopic properties of the fluorescent probes (λabs, λem and quantum yields) were found very close in Tag-lite buffer than those determined in HEPES/BSA (Table S1, supporting information). For competition assays, a fixed concentration of fluorescent ligand (equal to its Kd) was mixed with an increasing concentration of cold competitor ligand (OT, OTA or 1) in the Taglite medium. Plates were read in an HTRF-compatible multi-well plate reader (excitation at 340 nM, donor emission measured at 620 nm and acceptor emission at 665 nm (DY647) or 520 nm (Fluorescein), 150 µs delay, 500 µs integration). To determine the incubation time necessary to reach the equilibrium, TR-FRET and fluorescence signals were measured after various time of incubation. Dissociation (saturation) and inhibition (competition) constants were determined when equilibrium was reached by fitting experimental data with Graphpad Prism 6 (GraphPad Software). For competition experiments, 1 hour incubation at r.t. or 4 hour long incubation at 4 °C to prevent receptor internalization were used.

ASD, autism spectrum disorders; Cpd, compound; DID, 1,1'dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine; DIEA, N,N-diisopropylethylamine; DMEM, dulbecco modified Eagle's minimal essential medium; EDCI, 1-ethyl-3-(3dimethylaminopropyl)carbodiimide; FCS, foetal calf serum; HOBt, hydroxybenzotriazole; Kd, dissociation constant; NMM, N-methylmorpholine OT, oxytocin; OTR, oxytocin receptor; PyBOP, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate; RPHPLC, reverse phase high performance liquid chromatography; r.t., room temperature; TR-FRET, time-resolved Förster resonance energy transfer; V1aR, arginine-vasopressin V1a receptor; V1bR, arginine-vasopressin V1b receptor; V2R, arginine-vasopressin V2 receptor

REFERENCES (1)

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ASSOCIATED CONTENT Supporting Information. The synthesis and full characterization of compounds 5-9 and 11-14; The functional characterization of the SNAP-tag labeled OTR; This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION (9)

Corresponding Author * (D.B.) Phone : +33 368 85 42 36; Email: [email protected]. * (T.D.) Phone : +33 434 35 92 64; Email: thierry.durroux@ igf.cnrs.fr

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the French “Ministère de la Recherche”, the Fonds Unique Interministériel and OSEO (Cell2lead program n° F1005035J/ATFUAA00LB/AAP9), the Centre National de la Recherche Scientifique, the Université de Strasbourg. We warmly thank Pascale Buisine and Patrick Wehrung for MS analyses.

ABBREVIATIONS

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Figure 2. TR-FRET based assays with ligand 17. (A) Satu-ration experiments with 17 on the cells expressing OTR, V1aR, V1bR or V2R. (B) Competition experiments with unlabeled OT, OTA and 1, and probe 17 as a tracer on the cells expressing OTR. (C) Binding kinetics of 17 at 0.316 nM (blue), 1 nM (red), 3.16 nM (black) and 10 nM (violet) to OTR. (D) Effects of of 17 on IP accumulation: Inhibi-tion of OT-induced IP accumulation by increasing con-centration of 17 (black closed circle). IP accumulation in the presence of 17 (1µM) (open red circle). IP accumula-tion in basal condition (open black triangle). Values are mean ± SEM of the three independent experiments per-formed in triplicate. 176x181mm (144 x 144 DPI)



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Figure 3. Confocal microscopy studies. Confocal images of HEK293 cells expressing wild-type OTR with 10 nM of 17 (A, B) or 19 (C, D) with (A, C) or without (B, D) carbetocin (2 µM). 83x82mm (150 x 150 DPI)


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595x157mm (144 x 144 DPI)


Selective Nonpeptidic Fluorescent Ligands for Oxytocin Receptor

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