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NK1R antagonists, investigated for the treatment of several pathologies, have shown encouraging results in the treatment of several cancers. In the present study, we report on the synthesis of carbohydrate-based NK1R antagonists and their evaluation as anticancer agents against a wide range of cancer cells. All of the prepared compounds, derived from either-arabinose, have shown high affinity and NK1R antagonistic activity with a broad-spectrum anticancer activity and an important selectivity, comparable to Cisplatin. This strategy has allowed us to identify the galactosyl derivative 14α , as an interesting hit exhibiting significant NK1R antagonist effect (kinact0.209 ± 0.103 μM) and high binding affinity for NK1R (IC50= 50.4 nM,Ki= 22.4 nM by measuring the displacement of [125I] SP from NK1R). Interestingly, this galactosyl derivative has shown marked selective cytotoxic activity against 12 different types of cancer cell lines. Ministerio de Economía y Competitividad CTQ2016-78580-C2- 2-R, CTQ2016-78580-C2-1-R, CTQ2017-86655-R, PID2019-104767RB-I00 Ministerio de Ciencia e Innovación PID2019-104767RB-I00 Junta de Andalucía P06-FQM-01852, CV20-04221, FQM-313, FQM-102

International audience; Oxytocin (OT) and its receptor (OT-R) are implicated in the etiology of autism spectrum disorders (ASD), and OT-R is a potential target for therapeutic intervention. Very few nonpeptide oxytocin agonists have currently been reported. Their molecular and in vivo pharmacology remain to be clarified, and none of them has been shown to be efficient in improving social interaction in animal models relevant to ASD. In an attempt to rationalize the design of centrally active nonpeptide full agonists, we studied in a systematic way the structural determinants of the affinity and efficacy of representative ligands of the V-1a and V-2 vasopressin receptor subtypes (V-1a-R and V-2-R) and of the oxytocin receptor. Our results confirm the subtlety of the structure affinity and structure efficacy relationships around vasopressin/oxytocin receptor ligands and lead however to the first nonpeptide OT receptor agonist active in a mouse model of ASD after peripheral ip administration.

A library of 5-arylthiosubstituted 2-amino-4,6-diaryl-3-cyano-4H-pyrans has been synthesized as a new family of non-peptide NK1 receptor ligands by a one-pot cascade process. Their biological effects via interaction with the NK1 receptor were experimentally determined as percentage of inhibition (for antagonists) and percentage of activation (for agonists), compared to the substance P (SP) effect, in IPone assay. A set of these amino compounds was found to inhibit the action of SP, and therefore can be considered as a new family of SP-antagonists. Interestingly, the acylation of the 2-amino position causes a switch from antagonist to agonist activity. The 5-phenylsulfonyl-2-amino derivative 17 showed the highest antagonist activity, while the 5-p-tolylsulfenyl-2-trifluoroacetamide derivative 20R showed the highest agonist effect. As expected, in the case of the 5-sulfinylderivatives, there was an enantiomeric discrimination in favor of one of the two enantiomers, specifically those with (S,R) configuration. The anticancer activity studies assessed by using human A-549 lung cancer cells and MRC-5 non-malignant lung fibroblasts, revealed a statistically significant selective cytotoxic effect of some of these 2-amino-4H-pyran derivatives toward the lung cancer cells. These studies demonstrated that the newly synthesized 4H-pyran derivatives can be used as a starting point for the synthesis of novel SP-antagonists with higher anticancer activity in the future., We are grateful to the Spanish Ministerio de Economía y Competitividad (MEC) (grant number CTQ2013-49066-C2-2-R and CTQ2016-78580-C2-2-R) and the Junta de Andalucía (grant number P11-FQM-08046) for financial support. We gratefully thank CITIUS for NMR facilities.

Classical fluorescence-based approaches to monitor ligand-protein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn-on probes for a G protein-coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor-specific turn-on response. The new ligand was successfully applied for background-free imaging and quantification of oxytocin receptors in living cells.

Fluorescent ligands for GPCRs (G-protein-coupled receptors) have been synthesized for a long time but their use was usually restricted to receptor localization in the cell by fluorescent imaging microscopy. During the last two decades, the emergence of new fluorescence-based strategies and the concomitant development of fluorescent measurement apparatus have dramatically widened the use of fluorescent ligands. Among the various strategies, TR (time-resolved)-FRET (fluorescence resonance energy transfer) approaches exhibit an interesting potential to study GPCR interactions with various partners. We have derived various sets of ligands that target different GPCRs with fluorophores, which are compatible with TR-FRET strategies. Fluorescent ligands labelled either with a fluorescent donor (such as europium or terbium cryptate) or with a fluorescent acceptor (such as fluorescein, dy647 or Alexa Fluor® 647), for example, kept high affinities for their cognate receptors. These ligands turn out to be interesting tools to develop FRET-based binding assays. We also used these fluorescent ligands to analyse GPCR oligomerization by measuring FRET between ligands bound to receptor dimers. In contrast with FRET strategies, on the basis of receptor labelling, the ligand-based approach we developed is fully compatible with the study of wild-type receptors and therefore with receptors expressed in native tissues. Therefore, by using fluorescent analogues of oxytocin, we demonstrated the existence of oxytocin receptor dimers in the mammary gland of lactating rats.

Among the different classes of integral membrane proteins, G protein-coupled receptors (GPCR) constitute the largest family. They are involved in most essential physiological functions and particularly play a key role in cell-to-cell communication and sensory signal transduction. They represent targets for approximately 30% of currently marketed drugs. In order to better understand their functioning, define their tridimensional structure and develop novel selective and efficient therapeutic compounds, it is crucial to purify these proteins for a full characterization. However, this biochemical step is not trivial since GPCR are present in membranes at very low levels and they require detergents to be extracted from their natural lipid environment and be handled as functional proteins. No universal strategy for GPCR production, purification and stabilization is currently available; each single GPCR possesses a unique set of physicochemical characteristics, preference for some detergents upon solubilization and specific conditions for purification. During the last decade, major breakthroughs regarding overexpression, purification and above all GPCR stabilization, thanks to amphipols and nanodiscs, opened very exciting perspectives for structural and dynamic investigations of these membrane proteins. The aim of this chapter is to provide an overview of the different aspects of GPCR handling.

The eight metabotropic glutamate receptors (mGluRs) are key modulators of synaptic transmission and are considered promising targets for the treatment of various brain disorders. Whereas glutamate acts at a large extracellular domain, allosteric modulators have been identified that bind to the seven transmembrane domain (7TM) of these dimeric G-protein-coupled receptors (GPCRs). We show here that the dimeric organization of mGluRs is required for the modulation of active and inactive states of the 7TM by agonists, but is not necessary for G-protein activation. Monomeric mGlu2, either as an isolated 7TM or in full-length, purified and reconstituted into nanodiscs, couples to G proteins upon direct activation by a positive allosteric modulator. However, only a reconstituted full-length dimeric mGlu2 activates G protein upon glutamate binding, suggesting that dimerization is required for glutamate induced activation. These data show that, even for such well characterized GPCR dimers like mGluR2, a single 7TM is sufficient for G-protein coupling. Despite this observation, the necessity of dimeric architecture for signaling induced by the endogenous ligand glutamate confirms that the central core of signaling complex is dimeric.

International audience; G-protein-coupled receptors (GPCRs), the largest family of integral membrane proteins, participate in the regulation of many physiological functions and are the targets of approximately 30% of currently marketed drugs. However, knowledge of the structural and molecular bases of GPCR functions remains limited owing to difficulties related to their overexpression, purification and stabilization. The development of new strategies aimed at obtaining large amounts of functional GPCRs is therefore crucial. Here, we review the most recent advances in the production and functional folding of GPCRs from Escherichia coli inclusion bodies. Major breakthroughs open exciting perspectives for structural and dynamic investigations of GPCRs. In particular, combining targeting to bacterial inclusion bodies with amphipol-assisted folding is emerging as a highly powerful strategy.

Very few nonpeptide oxytocin agonists have currently been reported, and none of them seem suitable for the in vivo investigation of the oxytocin mediated functions. In an attempt to rationalize the design of better tools, we have systematically studied the structural determinants of the affinity and efficacy of representative ligands of the V(1a), V(2), and OT receptor subtypes. Despite apparently obvious similarity between the ligand structures on one hand, and between the receptor subtypes on the other hand, the binding affinity and the functional activity profiles of truncated and hybrid ligands highlight the subtlety of ligand-receptor interactions for obtaining nonpeptide OT receptor agonists.

Different series of fluorescent agonists and antagonists have been developed and characterized for arginine-vasopressin and oxytocin G protein-coupled receptors. Both cyclic and linear peptide analogs of the neurohypophysial hormones are useful tools for investigating receptor localization and trafficking, analysing receptor structural organization, and developing new receptor-selective high-throughput ligand screening assays.

A series of fluorescent ligands designed for vasopressin and oxytocin G protein-coupled receptors was synthesized and characterized to develop fluorescence polarization or homogeneous time-resolved fluorescence (HTRF) binding assays. These ligands, labeled with europium pyridine-bis-bipyridine cryptate or with Alexa 488,546,647 selectively bound to the vasopressin V1a and oxytocin receptors with high affinities and exhibited antagonistic properties. The affinities of several unlabeled ligands determined by our homogeneous assays on membrane preparations or on intact cells into 96- and 384-well plate formats were similar to those determined by usual radioligand binding methods. Compared to other binding assays, the polarization and HTRF binding assays are nonradiaoactive, therefore safer to perform, yet very sensitive and homogeneous, therefore easier and faster to automate. These methods are thus suitable for efficient drug high-throughput screening procedures and can easily be applied to other G protein-coupled receptor models.

Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand–receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O2)11]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (Kd1 = 0.14 ± 0.02 nM and Kd2 = 1.4 ± 0.18 nM, and Kd1 = 5.5 ± 0.7 nM and Kd2 = 620 ± 117 nM, respectively), whereas only one binding affinity (Kd = 5.5 ± 0.4 nM) could be observed for propionyl[Met(O2)11]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).

The purpose was to apply oxidative crosslinking reactions to the study of recognition and signaling mechanisms associated to G-protein-coupled receptors. Using a ruthenium chelate, Ru(bipy)(3)(2+), as photosensitizer and visible light irradiation, in the presence of ammonium persulfate, we performed fast and efficient covalent labeling of the B(2) bradykinin receptor by agonist or antagonist ligands possessing a radio-iodinated phenol moiety. The chemical and topographical specificities of these crosslinking experiments were investigated. The strategy could also be applied to the covalent labeling of the B(1) bradykinin receptor, the AT(1) angiotensin II receptor, the V(1a) vasopressin receptor and the oxytocin receptor. Interestingly, we demonstrated the possibility to covalently label the AT(1) and B(2) receptors with functionalized ligands. The potential applications of metal-chelate chemistry to receptor structural and signaling studies through intramolecular or intermolecular crosslinking are presented.

Three-dimensional models of G protein-coupled receptors (GPCR) have been defined using most experimental data available and protein modeling techniques. The endogenous ligand binding sites have been qualitatively described and putative receptor activation mechanisms have been proposed. The model has been recently refined to take into account recent crystallographic data. Most experimental results published are in excellent qualitative agreement with the initial model. We have undertaken to study more systematically by site directed mutagenesis the vasopressin/oxytocin receptor binding domain as a prototype of neuropeptide receptors. The experimental results are in very good agreement with the models. The residues responsible for the neuropeptide binding have been identified and confirm the predicted localization of the neuromediator in the transmembrane domain of the receptors. The side chain of the 8th residue of vasopressin interacts with a non-conserved receptor residue located in the first extracellular loop. As predicted from the model, this interaction is completely responsible for the selectivity of the ligand-receptor interaction. Finally, aromatic residues which allow the modulation of the efficacy of agonists have been identified.

The steroid hormone progesterone (P4) is essential for establishing and maintaining pregnancy in mammals. One of its functions includes maintenance of uterine quiescence by decreasing uterine sensitivity to the uterotonic peptide hormone oxytocin. Although it is generally held that steroid hormones such as P4 act at a genomic level by binding to nuclear receptors and modulating the expression of specific target genes, we show here that the effect of P4 on uterine sensitivity to oxytocin involves direct, non-genomic action of P4 on the uterine oxytocin receptor (OTR), a member of the G-protein-coupled receptor family. P4 inhibits oxytocin binding to OTR-containing membranes in vitro, binds with high affinity to recombinant rat OTR expressed in CHO cells, and suppresses oxytocin-induced inositol phosphate production and calcium mobilization. These effects are highly steroid- and receptor-specific, because binding and signalling functions of the closely related human OTR are not affected by P4 itself but by the P4 metabolite 5beta-dihydroprogesterone. Our findings provide the first evidence for a direct interaction between a steroid hormone and a G-protein-coupled receptor and define a new level of crosstalk between the peptide- and steroid-hormone signalling pathways.

The neurohypophyseal hormones vasopressin and oxytocin are two closely related nonapeptides, synthesized mainly by the magnocellular neurons of the hypothalamus. Their peptide sequences differ only in the amino acids at positions 3 and 8, but, for both hormones, the formation of a disulfide bond between Cys residues at the 1 and 6 positions results in a peptide constituted of a 6 amino acid cyclic part and a 3 amino acid C-terminal part. Both peptides exert various hormonal effects. Circulating oxytocin is mostly known for its ability to elicit the contraction of uterine smooth muscle at term and that of myoepithelial cells that surround the alveoli of the mammary gland during lactation. The main endocrine functions of arginine vasopressin (AVP) are the facilitation of water reabsorption by the kidney and the contraction of smooth muscle cells in arteries. AVP released in the portal blood in the median eminence acts as a potent secretagogue of adrenocorticotropin. In addition, it has become clear that vasopressin and oxytocin, besides mediating well-documented functions at the periphery, are also critically involved in numerous central processes including higher cognitive functions such as memory and learning (see Barberis & Tribollet 1996 for review). Vasopressin and oxytocin were the first biologically active peptides to be synthesized. This achievement, by Du Vigneaud and coworkers, 40 years ago, ushered in the modern era of peptide chemistry. During the subsequent decades, many structural analogues of the neurohypophyseal hormones have been synthesized and pharmacologically characterized (Manning & Sawyer 1993). Peripheral vasopressin and oxytocin receptors have been classified on the basis of both the second messenger system coupled to the receptors and the affinity of a series of vasopressin and oxytocin analogues with enhanced selectivity for a certain receptor type. These classification criteria have led to the distinction of V1a vasopressin (liver, smooth muscle cells from blood vessels, and most peripheral tissues expressing vasopressin receptors), V1b vasopressin (adenohypophysis), V2 vasopressin (kidney) and oxytocin (uterus, mammary gland) receptors ( Jard et al. 1988). To date central receptors have been identified as being of the vasopressin V1a and oxytocin subtypes. Moreover, a great number of molecular probes have been developed, including agonists and antagonists, and radiolabelled, fluorescent or photosensitive ligands. These make this receptor family a good model with which to study structure–function relationships. Today, the four different receptor subtypes have been cloned in mammals, lower vertebrates and invertebrates. Molecular cloning studies have renewed interest in these neurohormone receptors. Vasopressin V1b receptor mRNA has been detected in peripheral tissues (kidney, thymus, heart, lung, spleen, uterus and breast) and numerous areas of the brain in the rat (Lolait et al. 1995), and this receptor subtype has also been characterized in rat adrenal (Grazzini et al. 1996). These studies suggest that the vasopressin V1b receptor may serve additional and unknown functions in the brain and at the periphery. The primary focus of this review is to summarize recent studies that have led to novel insights into the molecular bases of vasopressin and oxytocin receptor functions.

A series of fluorescent benzazepine ligands for the arginine-vasopressin V₂ receptor (AVP V₂R) was synthesized using "Click" chemistry. Their in vitro pharmacological profile at AVP V₂R, V(1a)R, V(1b)R, and oxytocin receptor was measured by binding assay and functional studies. Compound 9p, labeled with Lissamine Rhodamine B using novel solid-phase organic tagging (SPOrT) resin, exhibited a high affinity for V₂R (4.0 nM), an excellent selectivity toward V₂R and antagonist properties. By changing the nature of the dye, DY647 and Lumi4-Tb probes 44 and 47 still display a high affinity for V₂R (5.6 and 5.8 nM, respectively). These antagonists constitute the first high-affinity selective nonpeptidic fluorescent ligands for V₂R. They enabled the development of V₂R time-resolved FRET-based assay readily amenable to high-throughput screening. Taking advantage of their selectivity, these compounds were also successfully involved in the study of V(1a)R-V₂R dimerization on cell surface.

Two novel solid-phase organic tagging (SPOrT) resins were synthesized to facilitate the labeling of peptides and small organic compounds with a fluorescent probe. Both resins were obtained from the commercially available backbone amide linker (BAL) resin. Following the solid-phase synthesis of model compounds, a tripeptide and benzazepine, the fluorescent probe derived from Lissamine Rhodamine B was incorporated through CuI-catalyzed 1,3-dipolar cycloaddition. Final cleavage in acidic media enabled access to both types of molecules in good yield with high purity. The SPOrT resin was successfully applied to the preparation of the first non-peptidic fluorescent compound with a nanomolar affinity for the human vasopressin V2 receptor (V2R) subtype. This molecule will find application in binding assays that use polarization or fluorescence resonance energy-transfer (FRET) techniques. The SPOrT resins are also well suited for other tags and the parallel synthesis of a fluorescently tagged library for protein screening.