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International audience; 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.

International audience; 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 T1/2β 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.

International audience; Biased agonists and pharmacological chaperones have demonstrated their potential to harness G protein-coupled receptor signaling and trafficking, and have collectively opened new possibilities in G protein-coupled receptor drug discovery. Combining pharmacological chaperoning and biased agonism properties into a unique given molecule would be of high therapeutic interest in many human diseases resulting from G protein-coupled receptor mutation and misfolding. This strategy perfectly fits to congenital Nephrogenic Diabetes Insipidus which is a typical conformational disease. In most of the cases, it is associated to 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 the receptor mutants and to rescue their function. Interestingly, different classes of pharmacological chaperones of the V2 receptor have proven their usefulness and efficacy, such as antagonists, agonists as well as biased agonists. These compounds, particularly small-molecule biased agonists which elicit the V2-induced Gs protein-dependent signaling pathway, but not V2-related arrestin-dependent cell responses, represent a potential therapeutic treatment of this X-linked genetic pathology.

International audience; Mambaquaretins are a group of toxins from mamba venoms, containing seven members until now, which target the vasopressin type 2 receptor (V2R). V2R, primarily expressed in the kidney, belongs to the G-protein coupled receptor family and regulates fluid osmotic pressure and diuresis through the vasopressin hormone. The Mambaquaretin-1, the very first identified member of the Mambaquaretin group, antagonizes V2R and this property gives it potential therapeutic applications to treat disorders such as hyponatremia, syndrome of inappropriate antidiuretic hormone secretion (SIADH) or polycystic kidney diseases (PKD). PKD are severe genetic diseases which affect 1 in 1,000 people in the world. However, therapeutic strategies are very limited. An animal trial was conducted in a rodent model of PKD and demonstrated the efficacy of Mambaquaretin-1 to slow down the disease progression after a 100 days treatment, validating the toxin as a novel therapeutic strategy. The pharmacodynamics of the toxin was further investigated in healthy rats to estimate the best dose to inject regarding duration of action and side effects occurrence. To define the molecular mode of interaction between V2R and Mambaquaretin-1, a structure activity relationship study was completed. X-ray diffraction approach confirmed that the toxins adopt a Kunitz fold reticulated by three disulphide bridges. Comparing the natural SAR of the seven active Mambaquaretins and exploring the V2R activity of six toxins from databases very close in sequence, interesting positions were mutated in order to determine the pharmacophore of the Mambaquaretin-1. Consequently, we could advance a receptor-toxin complex model in which the toxin interacts both with the upper part of the orthosteric site and the extracellular loops. Thanks to these results, engineered toxins are being designed to enhance its in vivo characteristics, such as blood stability or speed of elimination in the aim to propose the best therapeutic candidate.

International audience; The signaling pathway of G protein-coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V1b subtype (V1b R) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of β-arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V1b R-mediated MAP kinase pathway. Using MEF cells Knocked-out for β-arrestins 1 and 2, we demonstrated that both β-arrestins 1 and 2 play a fundamental role in internalization and recycling of V1b R with a rapid and transient V1b R-β-arrestin interaction in contrast to a slow and long-lasting β-arrestin recruitment of the V2 vasopressin receptor subtype (V2 R). Using V1b R-V2 R chimeras and V1b R C-terminus truncations, we demonstrated the critical role of the V1b R C-terminus in its interaction with β-arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation-independent manner. In parallel, V1b R MAP kinase activation was dependent on arrestins and Src-kinase but independent on G proteins. Interestingly, Src interacted with hV1b R at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V1b R involving both arrestins and Src kinase family.

International audience; 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.

G protein-coupled receptors are key proteins in the regulation of most of the physiological responses. Their conformations are generally oscillating between inactive and active forms leading to the activation of no, a few, or many signaling pathways. Although receptors can spontaneously adopt these various conformations, their interactions with ligands, other G protein-coupled receptors, or intracellular proteins (G proteins, arrestins, etc.) can stabilize one of these conformations, leading to specific cellular responses. The identification of the partners interacting with the G protein-coupled receptors and the dynamics of these interactions is therefore crucial to fully understand receptor functioning. Although it is crucial, it remains nevertheless ambitious and difficult to achieve this goal. In the last two decades, various technical strategies have been developed to investigate molecular complexes and their dynamics. In this review, we will focus on recent technological breakthroughs in fluorescent-based techniques and their impact on the understanding of G protein-coupled receptor functioning. We will give particular attention to resonance energy transfer-based strategies, their advantages, and drawbacks and to other microscopy based techniques which are efficient to investigate stability, mobility, and dynamics of molecular complexes at the cell surface.

Heterodimerization of G protein-coupled receptors has an impact on their signaling properties, but the molecular mechanisms underlying heteromer-directed selectivity remain elusive. Using purified monomers and dimers reconstituted into lipid discs, we explored how dimerization impacts the functional and structural behavior of the ghrelin receptor. In particular, we investigated how a naturally occurring truncated splice variant of the ghrelin receptor exerts a dominant negative effect on ghrelin signaling upon dimerization with the full-length receptor. We provide direct evidence that this dominant negative effect is due to the ability of the non-signaling truncated receptor to restrict the conformational landscape of the full-length protein. Indeed, associating both proteins within the same disc blocks all agonist- and signaling protein-induced changes in ghrelin receptor conformation, thus preventing it from activating its cognate G protein and triggering arrestin 2 recruitment. This is an unambiguous demonstration that allosteric conformational events within dimeric assemblies can be directly responsible for modulation of signaling mediated by G protein-coupled receptors.

International audience; 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.

International audience; G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

International audience; 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.

International audience; Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B(4) receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G(i2) protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human alpha(5) integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB(4) binding in the presence of the purified G protein G alpha(i2). The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5'-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified G alpha(i2) beta(1) gamma(2) protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.

International audience; 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.

International audience; The V2 vasopressin receptor is a G-protein-coupled receptor that regulates the renal antidiuretic response. Its third intracellular loop is involved in the coupling not only with the GαS protein but also with gC1qR, a potential chaperone of G-protein-coupled receptors. In this report, we describe the NMR solution structure of the V2 i3 loop under a cyclized form (i3_cyc) and characterize its interaction with gC1qR. i3_cyc formed a left-twisted α-helical hairpin structure. The building of a model of the entire V2 receptor including the i3_cyc NMR structure clarified the side-chain orientation of charged residues, in agreement with literature mutagenesis reports. In the model, the i3 loop formed a rigid helical column, protruding deep inside the cytoplasm, as does the i3 loop in the recently elucidated structure of squid rhodopsin. However, its higher packing angle resulted in a different structural motif at the intracellular interface, which may be important for the specific recognition of GαS. Moreover, we could estimate the apparent Kd of the i3_cyc/gC1qR complex by anisotropy fluorescence. Using a shorter and more soluble version of i3_cyc, which encompassed the putative site of gC1qR binding, we showed by NMR saturation transfer difference spectroscopy that the binding surface corresponded to the central arginine cluster. Binding to gC1qR induced the folding of the otherwise disordered short peptide into a spiral-like path formed by a succession of I and IV turns. Our simulations suggested that this folding would rigidify the arginine cluster in the entire i3 loop and would alter the conformation of the cytosolic extensions of TM V and TM VI helices. In agreement with this conformational rearrangement, we observed that binding of gC1qR to the full-length receptor modifies the intrinsic tryptophan fluorescence binding curves of V2 to an antagonist.