A new Kunitz-type snake toxin family associated with an original mode of interaction with the vasopressin 2 receptor.

BACKGROUND AND PURPOSE: Venomous animals express numerous Kunitz-type peptides. The mambaquaretin-1 (MQ1) peptide identified from the Dendroaspis angusticeps venom is the most selective antagonist of the arginine-vasopressin V2 receptor (V2R) and the only unique Kunitz-type peptide active on a GPCR. We aimed to exploit other mamba venoms to enlarge the V2R-Kunitz peptide family and gain insight into the MQ1 molecular mode of action. EXPERIMENTAL APPROACH: We used a bio-guided screening assay to identify novel MQs and placed them phylogenetically. MQs were produced by solid-phase peptide synthesis and characterized in vitro by binding and functional tests and in vivo by diuresis measurement in rats. KEY RESULTS: Eight additional MQs were identified with nanomolar affinities for the V2R, all antagonists. MQs form a new subgroup in the Kunitz family, close to the V2R non-active dendrotoxins and to two V2R-active cobra toxins. Sequence comparison between active and non-active V2R Kunitz peptides highlighted five positions, among which four are involved in V2R interaction and belong to the two large MQ1 loops. We finally determined that eight positions, part of these two loops, interact with the V2R. The variant MQ1-K39A showed a higher affinity for the hV2R, but not for the rat V2R. CONCLUSIONS AND IMPLICATIONS: A new function and mode of action is associated with the Kunitz peptides. The number of MQ1 residues involved in V2R binding is large and may explain its absolute selectivity. MQ1-K39A represents the first step in the improvement of the MQ1 design from a medicinal perspective.

Carbohydrate-Based NK1R Antagonists with Broad-Spectrum Anticancer Activity.

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 d-galactose or l-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 (kinact 0.209 ± 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.

A snake toxin as a theranostic agent for the type 2 vasopressin receptor.

Rationale: MQ1, a snake toxin which targets with high nanomolar affinity and absolute selectivity for the type 2 vasopressin receptor (V2R), is a drug candidate for renal diseases and a molecular probe for imaging cells or organs expressing V2R. Methods: MQ1's pharmacological properties were characterized and applied to a rat model of hyponatremia. Its PK/PD parameters were determined as well as its therapeutic index. Fluorescently and radioactively labeled MQ1 were chemically synthesized and associated with moderate loss of affinity. MQ1's dynamic biodistribution was monitored by positron emission tomography. Confocal imaging was used to observe the labeling of three cancer cell lines. Results: The inverse agonist property of MQ1 very efficiently prevented dDAVP-induced hyponatremia in rats with low nanomolar/kg doses and with a very large therapeutic index. PK (plasma MQ1 concentrations) and PD (diuresis) exhibited a parallel biphasic decrease. The dynamic biodistribution showed that MQ1 targets the kidneys and then exhibits a blood and kidney biphasic decrease. Whatever the approach used, we found a T1/2α between 0.9 and 3.8 h and a T1/2ß between 25 and 46 h and demonstrated that the kidneys were able to retain MQ1. Finally, the presence of functional V2R expressed at the membrane of cancer cells was, for the first time, demonstrated with a specific fluorescent ligand. Conclusion: As the most selective V2 binder, MQ1 is a new promising drug for aquaresis-related diseases and a molecular probe to visualize in vitro and in vivo V2R expressed physiologically or under pathological conditions.

Pharmacological Chaperones as Potential Therapeutic Strategies for Misfolded Mutant Vasopressin Receptors.

Pharmacological chaperones recently opened new possibilities in G protein-coupled receptor drug discovery. Even more interestingly, some unique ligands combine pharmacological chaperoning and biased agonism properties, boosting their therapeutic interest in many human diseases resulting from G protein-coupled receptor mutation and misfolding. These compounds displaying dual characteristics would constitute a perfect treatment for congenital Nephrogenic Diabetes Insipidus, a typical conformational disease. This X-linked genetic pathology is mostly associated with inactivating mutations of the renal arginine-vasopressin V2 receptor leading to misfolding and intracellular retention of the receptor, causing the inability of patients to concentrate their urine in response to the antidiuretic hormone. Cell-permeable pharmacological chaperones have been successfully challenged to restore plasma membrane localization of many V2 receptor mutants. In addition, different classes of specific ligands such as antagonists, agonists as well as biased agonists of the V2 receptor have proven their usefulness in rescuing mutant receptor function. This is particularly relevant for small-molecule biased agonists which only trigger Gs protein activation and cyclic adenosine monophosphate production, the V2-induced signaling pathway responsible for water reabsorption. In parallel, high-throughput screening assays based on receptor trafficking rescue approaches have been developed to discover novel V2 pharmacological chaperone molecules from different chemical libraries. These new hit compounds, which still need to be pharmacologically characterized and functionally tested in vivo, represent promising candidates for the treatment of congenital Nephrogenic Diabetes Insipidus.

Design, synthesis and biological studies of a library of NK1-Receptor Ligands Based on a 5-arylthiosubstituted 2-amino-4,6-diaryl-3-cyano-4H-pyran core: Switch from antagonist to agonist effect by chemical modification.

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 (SS,RC) 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.

Green mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease.

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs.

Quantitative MALDI-MS Binding Assays: An Alternative to Radiolabeling.

Radiolabeling of ligands is still the gold standard in the study of high-affinity receptor-ligand interactions. In an effort toward safer and simpler alternatives to the use of radioisotopes, we developed a quantitative and highly sensitive matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) method that relies on the use of chemically tagged ligands designed to be specifically detectable when present as traces in complex biological mixtures such as cellular lysates. This innovative technology allows easy, sensitive detection and accurate quantification of analytes at the sub-nanomolar level. After statistical validation, we were able to perform pharmacological evaluations of G protein-coupled receptor (V1A-R)-ligand interactions. Both saturation and competitive binding assays were successfully processed.

Expression, purification and NMR characterization of the cyclic recombinant form of the third intracellular loop of the vasopressin type 2 receptor.

The vasopressin type 2 (V2R) receptor belongs to the class of G-protein coupled receptors. It is mainly expressed in the membrane of kidney tubules, where it is activated by the extracellular arginine vasopressin. In men, inactivating and activating mutations cause nephrogenic diabetes insipidus and the nephrogenic syndrome of inappropriate antidiuresis respectively. Like most GPCRs, V2R's third intracellular loop (V2R-i3) is involved in the binding and activation of its major effector, the GαS protein. We overexpressed the V2R224₋274 fragment corresponding to V2R-i3 as a fusion protein with thioredoxin A at the N-terminus and a hexahistidine tag between the two proteins. Recombinant V2R-i3 was designed to harbor N- and C-terminal cysteines, in order to introduce a disulfide bond between N- and C-terminal extremities and hence reproduce the hairpin fold presumably present in the full-length receptor. The fusion protein was produced as inclusion bodies in Escherichia coli and purified by nickel affinity chromatography under denaturing conditions. After a refolding step, thioredoxin and hexahistidine tags were specifically cleaved with the tobacco etch virus protease. The hydrolysis yield, initially very low, increased up to 80% thanks to optimization of buffers and refolding methods. The cleaved fragment, V2224₋274, devoid of any tag, was then eluted with low imidazole concentrations in a second nickel affinity chromatography in denaturing conditions. The final yield was sufficient to prepare a ¹5N-¹³C labeled NMR sample suitable for triple resonance experiments. We assigned all NMR resonances and confirmed the correct peptide sequence. As expected, the peptide forms a hairpin stabilized by a disulfide bond between its N- and C-terminal parts, thus mimicking its native structure in the full-length receptor. This study may provide a strategy for producing and studying the structure/function relationship of GPCR fragments.

[Pharmacological chaperones: a potential therapeutic treatment for conformational diseases]. / Chaperons pharmacologiques : un espoir thérapeutique pour les pathologies conformationnelles.

Many genetic and neurodegenerative diseases in humans result from protein misfolding and/or aggregation. These diseases are named conformational diseases. As a result, the misfolded non functional proteins are rejected and misrouted by the cellular quality control system, and cannot play their endogenous physiological roles. Specific compounds (ligands, substrates or inhibitors) known as pharmacological chaperones are able to bind and stabilize these misfolded proteins. Their interaction allows the target proteins to escape the quality control system and to be functionally rescued. These pharmacochaperones may possess different intrinsic activity: they can be antagonists (inhibitors), agonists (activators) or allosteric modulators of the target receptors, ionic channels or enzymes. Pharmacological chaperones have obviously a therapeutic potential to treat rare diseases like cystic fibrosis, retinitis pigmentosa, nephrogenic diabetes insipidus, Fabry disease, Gaucher disease, but also for cancers and more frequent and highly invalidant neurodegenerative disorders such as Alzheimer's disease or Parkinson's disease.

Biased agonist pharmacochaperones of the AVP V2 receptor may treat congenital nephrogenic diabetes insipidus.

X-linked congenital nephrogenic diabetes insipidus (cNDI) results from inactivating mutations of the human arginine vasopressin (AVP) V2 receptor (hV(2)R). Most of these mutations lead to intracellular retention of the hV(2)R, preventing its interaction with AVP and thereby limiting water reabsorption and concentration of urine. Because the majority of cNDI-hV(2)Rs exhibit protein misfolding, molecular chaperones hold promise as therapeutic agents; therefore, we sought to identify pharmacochaperones for hV(2)R that also acted as agonists. Here, we describe high-affinity nonpeptide compounds that promoted maturation and membrane rescue of L44P, A294P, and R337X cNDI mutants and restored a functional AVP-dependent cAMP signal. Contrary to pharmacochaperone antagonists, these compounds directly activated a cAMP signal upon binding to several cNDI mutants. In addition, these molecules displayed original functionally selective properties (biased agonism) toward the hV(2)R, being unable to recruit arrestin, trigger receptor internalization, or stimulate mitogen-activated protein kinases. These characteristics make these hV(2)R agonist pharmacochaperones promising therapeutic candidates for cNDI.

Differential coupling of the vasopressin V1b receptor through compartmentalization within the plasma membrane.

We show here that the rat vasopressin V(1b) receptor simultaneously activates both the G(q/11)-inositol phosphate (IP) and G(s)-cAMP pathways when transiently expressed in Chinese hamster ovary, human embryonic kidney (HEK) 293, and COS-7 cells and stimulated with arginine-vasopressin. Higher concentrations of the hormone, however, were needed to trigger the cAMP pathway. The nonmammalian analog arginine-vasotocin and the selective V(1b) agonist d[Cha(4)]vasopressin also activated the cAMP and IP pathways, although d[Cha(4)]-vasopressin elicited the two responses with equivalent potencies. We determined that the V(1b) receptor is present as a homodimer at the plasma membrane. Treatment of V(1b)-transfected HEK-293 cells with methyl-beta-cyclodextrin, a drug known to dissociate cholesterol-rich domains of the plasma membrane, shifted the EC(50) of the vasopressin-induced cAMP accumulation to lower concentrations and, remarkably, increased the hormone efficacy related to the activation of this second messenger system. In parallel, the vasopressin-mediated activation of the IP pathway was slightly reduced without modification of its EC(50). These results suggest that, as with many other G protein-coupled receptors, when transfected in heterologous cell systems, the V(1b) receptor forms dimers that signal differentially through the G(q/11) and G(s) proteins depending on the nature of the ligand as well as on its localization within specialized compartments of the plasma membrane. The present study thus illustrates how signal transduction associated with the activation of a G protein-coupled receptor can be versatile and highly dependent on both the cell context and the chemical nature of the extracellular signaling messenger.

Peptide and non-peptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and OT receptors: research tools and potential therapeutic agents.

Oxytocin (OT) and vasopressin (AVP) mediate their biological actions by acting on four known receptors: The OT (uterine) and the AVP V(1a) (vasopressor), V(1b) (pituitary), V(2) (renal) receptors and a fifth putative AVP V(1c)? (vasodilating) receptor. This presentation will summarize some highlights of the recent progress, in the design and synthesis of selective peptide agonists, antagonists, radioiodinated ligands, fluorescent ligands and bivalent ligands for these receptors. Here we present published and unpublished pharmacological data on the most widely used agonists, antagonists and labelled ligands. The pharmacological properties of promising new selective OT antagonists and V(1b) agonists are also presented. This review should serve as a useful guide for the selection of the most appropriate ligand for a given study. The current status of non-peptide OT and AVP antagonists and agonists is also summarized. The relative merits of peptide and non-peptide AVP and OT agonists and antagonists as: (1) research tools and (2) therapeutic agents will be evaluated. Many of the receptor selective peptide agonists and antagonists from this and other laboratories are far more widely used as pharmacological tools for studies on the peripheral and central effects of OT and AVP than their non-peptide counterparts. In addition to OT and to a lesser extent AVP (pitressin), a number of OT and AVP analogues; such as carbetocin (OT agonist) dDAVP (desmopressin, V(2) agonist), terlipressin (V(1a) agonist), felypressin (V(1a) agonist) and atosiban (Tractocile OT antagonist) are also in clinical use. Despite much early promise, no non-peptide V(1a) or OT antagonists are currently in clinical trials. While a number of orally active non-peptide V(2) antagonists (Vaptans); notably, Tolvaptan, Lixivaptan and Satavaptan, are currently in Phase III clinical trials; to date, only the mixed V(2)/V(1a), antagonist Conivaptan (Vaprisol), has been approved by the US FDA for clinical use (by i.v. administration), for the treatment of euvolemic and hypervolemic hyponatremia in hospitalized patients. Promising new non-peptide V(1b) and OT antagonists, as well as non-peptide V(2) and OT agonists are now in pre-clinical development.

Solid-phase organic tagging resins for labeling biomolecules by 1,3-dipolar cycloaddition: application to the synthesis of a fluorescent non-peptidic vasopressin receptor ligand.

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.

Toward efficient drug screening by homogeneous assays based on the development of new fluorescent vasopressin and oxytocin receptor ligands.

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.

Crosslinking photosensitized by a ruthenium chelate as a tool for labeling and topographical studies of G-protein-coupled receptors.

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.

Identification of the binding sites of the SR49059 nonpeptide antagonist into the V1a vasopressin receptor using sulfydryl-reactive ligands and cysteine mutants as chemical sensors.

To identify the binding site of the human V1a vasopressin receptor for the selective nonpeptide antagonist SR49059, we have developed a site-directed irreversible labeling strategy that combines mutagenesis of the receptor and use of sulfydryl-reactive ligands. Based on a three-dimensional model of the antagonist docked into the receptor, hypothetical ligand-receptor interactions were investigated by replacing the residues potentially involved in the binding of the antagonist into cysteines and designing analogues of SR49059 derivatized with isothiocyanate or alpha-chloroacetamide moieties. The F225C, F308C, and K128C mutants of the V1a receptor were expressed in COS-7 or Chinese hamster ovary cells, and their pharmacological properties toward SR49059 and its sulfydryl-reactive analogues were analyzed. We demonstrated that treatment of the F225C mutant with the isothiocyanate-derivative compound led to dose-dependent inhibition of the residual binding of the radio-labeled antagonist [125I]HO-LVA. This inhibition is probably the consequence of a covalent irreversible chemical modification, which is only possible when close contacts and optimal orientations exist between reactive groups created both on the ligand and the receptor. This result validated the three-dimensional model hypothesis. Thus, we propose that residue Phe225, located in transmembrane domain V, directly participates in the binding of the V1a-selective nonpeptide antagonist SR49059. This conclusion is in complete agreement with all our previous data on the definition of the agonist/antagonist binding to members of the oxytocin/vasopressin receptor family.