Publications (16)75.08 Total impact
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Article: Exploration of the Orthosteric/Allosteric Interface in Human M1 Muscarinic Receptors by Bitopic Fluorescent Ligands.
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ABSTRACT: Bitopic binding properties apply to a variety of muscarinic compounds that span and simultaneously bind to both the orthosteric and allosteric receptor sites. We provide evidence that fluorescent pirenzepine derivatives, with the M1 antagonist fused to the Bodipy [558/568] fluorophore via spacers of varying lengths, exhibit orthosteric/allosteric binding properties at muscarinic M1 receptors. This behaviour was inferred from a combination of functional, radioligand and FRET binding experiments, performed under equilibrium and kinetic conditions on EGFP-fused M1 receptors. Though displaying a common orthosteric component, the fluorescent compounds inherit bitopic properties from a linker-guided positioning of their Bodipy moiety within the M1 allosteric vestibule. Depending on linker length, the fluorophore is allowed to reach neighbouring allosteric domains, overlapping or not with the classical gallamine site, but distinct from the allosteric indocarbazolole 'WIN' site. Site-directed mutagenesis, as well as molecular modeling and ligand docking studies based on recently solved muscarinic receptor structures, further support the definition of two groups of Bodipy-pirenzepine derivatives exhibiting distinct allosteric binding poses. Thus, the linker may dictate pharmacological outcomes for bitopic molecules that are hardly predictable from the properties of individual orthosteric and allosteric building blocks. Our findings also demonstrate that the fusion of a fluorophore to an orthosteric ligand is not neutral as it may confer, unless carefully controlled, unexpected properties to the resultant fluorescent tracer. Altogether, this study illustrates the importance of a 'multifacette' experimental approach to unravel and validate bitopic ligand binding mechanisms.Molecular pharmacology 04/2013; · 4.53 Impact Factor -
Article: An antedrug of the CXCL12 neutraligand blocks experimental allergic asthma without systemic effect in the mouse.
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ABSTRACT: The chemokine receptor CXCR4 and its chemokine CXCL12 are involved in normal tissue patterning, but also in tumor cell growth and survival as well as in the recruitment of immune and inflammatory cells, as successfully demonstrated using agents that block either CXCL12 or CXCR4. In order to achieve selectivity in drug action on the CXCR4/CXCL12 pair in particular in the airways, drugs should be delivered as selectively as possible in the treated tissue, and should not diffuse in the systemic circulation where it may reach undesired organs. To this end, we used a previously unexploited Knoevenagel reaction to create a short-lived drug, or soft drug, based on the CXCL12-neutralizing small molecule, chalcone 4, which blocks binding of CXCL12 to CXCR4. We show that the compound, carbonitrile-chalcone 4, blocks the recruitment of eosinophils to the airways in ovalbumin-sensitized and challenged mice in vivo when administered directly to the airways by intranasal route, but not when administered systemically by intraperitoneal route. We show that the lack of effect at a distant site is due to the rapid degradation of the molecule to inactive fragments. This approach allows selective action of the CXCL12 neutraligands even though the target protein is widely distributed in the organism.Journal of Biological Chemistry 02/2013; · 4.77 Impact Factor -
Article: Selective Fluorescent Nonpeptidic Antagonists For Vasopressin V(2) GPCR: Application To Ligand Screening and Oligomerization Assays.
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ABSTRACT: A series of fluorescent benzazepine ligands for the arginine-vasopressin V(2) receptor (AVP V(2)R) was synthesized using "Click" chemistry. Their in vitro pharmacological profile at AVP V(2)R, 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 V(2)R (4.0 nM), an excellent selectivity toward V(2)R and antagonist properties. By changing the nature of the dye, DY647 and Lumi4-Tb probes 44 and 47 still display a high affinity for V(2)R (5.6 and 5.8 nM, respectively). These antagonists constitute the first high-affinity selective nonpeptidic fluorescent ligands for V(2)R. They enabled the development of V(2)R 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-V(2)R dimerization on cell surface.Journal of Medicinal Chemistry 09/2012; 55(20):8588-602. · 4.80 Impact Factor -
Article: Combinatorial Aid for Underprivileged Scaffolds: Solution and Solid-phase Strategies for a Rapid and Efficient Access To Novel Aza-diketopiperazines (Aza-DKP).
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ABSTRACT: An efficient solution-phase synthesis of aza-diketopiperazines (aza-DKP, triazinediones) is reported. A structurally diverse collection of c-[aza-alkylGly-Pro] derivatives and yet unreported 2,4,5-trisubstituted-1,2,4-triazine-3,6-diones has been synthesized starting from Fmoc-l-Pro-OH and various Fmoc-l-amino acids. To extend the practical value of this class of dipeptidomimetics, a general solid-phase synthesis approach amenable to library production was developed on both Wang-PS and HMBA-PS resins. The final acidic treatment of the resins in TFA/water mixture at room temperature enabled the rapid and quantitative cyclization/release highly pure triazinediones. The conformational preferences and the spatial organization of the three substituents of a representative 2,4,5-trisubstituted-1,2,4-triazine-3,6-dione were investigated by X-ray diffraction and (1)H NMR spectroscopy.ACS combinatorial science. 04/2012; -
Article: Fluorescent derivatives of AC-42 to probe bitopic orthosteric/allosteric binding mechanisms on muscarinic M1 receptors.
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ABSTRACT: Two fluorescent derivatives of the M1 muscarinic selective agonist AC-42 were synthesized by coupling the lissamine rhodamine B fluorophore (in ortho and para positions) to AC42-NH(2). This precursor, prepared according to an original seven-step procedure, was included in the study together with the LRB fluorophore (alone or linked to an alkyl chain). All these compounds are antagonists, but examination of their ability to inhibit or modulate orthosteric [(3)H]NMS binding revealed that para-LRB-AC42 shared several properties with AC-42. Carefully designed experiments allowed para-LRB-AC42 to be used as a FRET tracer on EGFP-fused M1 receptors. Under equilibrium binding conditions, orthosteric ligands, AC-42, and the allosteric modulator gallamine behaved as competitors of para-LRB-AC42 binding whereas other allosteric compounds such as WIN 51,708 and N-desmethylclozapine were noncompetitive inhibitors. Finally, molecular modeling studies focused on putative orthosteric/allosteric bitopic poses for AC-42 and para-LRB-AC42 in a 3D model of the human M1 receptor.Journal of Medicinal Chemistry 03/2012; 55(5):2125-43. · 4.80 Impact Factor -
Article: Prodrugs of a CXC Chemokine-12 (CXCL12) Neutraligand Prevent Inflammatory Reactions in an Asthma Model in Vivo
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ABSTRACT: Chalcone 4 (compound 1) is a small molecule that neutralizes the CXC chemokine CXCL12 and prevents it from acting on the CXCR4 and CXCR7 receptors. To overcome its poor solubility in aqueous buffers, we designed highly soluble analogues of compound 1, phosphate, l-seryl, and sulfate, all inactive by themselves on CXCL12 but when cleaved in vivo into 1, highly active locally at a low dose in a mouse airway hypereosinophilia model.Keywords: prodrug; solubility; CXCL12 chemokine; CXCR4 receptor; GPCR; asthma12/2011; -
Article: Identification and pharmacological properties of E339-3D6, the first nonpeptidic apelin receptor agonist.
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ABSTRACT: Apelin plays a prominent role in body fluid and cardiovascular homeostasis. To explore further upstream the role played by this peptide, nonpeptidic agonists and antagonists of the apelin receptor are required. To identify such compounds that do not exist to date, we used an original fluorescence resonance energy transfer-based assay to screen a G-protein-coupled receptor-focused library of fluorescent compounds on the human EGFP-tagged apelin receptor. This led to isolated E339-3D6 that displayed a 90 nM affinity and behaved as a partial agonist with regard to cAMP production and as a full agonist with regard to apelin receptor internalization. Finally, E339-3D6 induced vasorelaxation of rat aorta precontracted with noradrenaline and potently inhibited systemic vasopressin release in water-deprived mice when intracerebroventricularly injected. This compound represents the first nonpeptidic agonist of the apelin receptor, the optimization of which will allow development of a new generation of vasodilator and aquaretic agents.The FASEB Journal 05/2010; 24(5):1506-17. · 5.71 Impact Factor -
Article: Neutralizing endogenous chemokines with small molecules. Principles and potential therapeutic applications.
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ABSTRACT: Regulation of cellular responses to external stimuli such as hormones, neurotransmitters, or cytokines is achieved through the control of all steps of the complex cascade starting with synthesis, going through maturation steps, release, distribution, degradation and/or uptake of the signalling molecule interacting with the target protein. One possible way of regulation, referred to as scavenging or neutralization of the ligand, has been increasingly studied, especially for small protein ligands. It shows innovative potential in chemical biology approaches as well as in disease treatment. Neutralization of protein ligands, as for example cytokines or chemokines can lead to the validation of signalling pathways under physiological or pathophysiological conditions, and in certain cases, to the development of therapeutic molecules now used in autoimmune diseases, chronic inflammation and cancer treatment. This review explores the field of ligand neutralization and tries to determine to what extent small chemical molecules could substitute for neutralizing antibodies in therapeutic approaches.Pharmacology [?] Therapeutics 04/2010; 126(1):39-55. · 8.56 Impact Factor -
Article: Homodimerization of the death-associated protein kinase catalytic domain: development of a new small molecule fluorescent reporter.
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ABSTRACT: Death-Associated Protein Kinase (DAPK) is a member of the Ca2+/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases. In the in vitro study presented here, we describe the homodimerization of DAPK catalytic domain and the crucial role played by its basic loop structure that is part of the molecular fingerprint of death protein kinases. Nanoelectrospray ionization mass spectrometry of DAPK catalytic domain and a basic loop mutant DAPK protein performed under a variety of conditions was used to detect the monomer-dimer interchange. A chemical biological approach was used to find a fluorescent probe that allowed us to follow the oligomerization state of the protein in solution. The use of this combined biophysical and chemical biology approach facilitated the elucidation of a monomer-dimer equilibrium in which the basic loop plays a key role, as well as an apparent allosteric conformational change reported by the fluorescent probe that is independent of the basic loop structure.PLoS ONE 01/2010; 5(11):e14120. · 4.09 Impact Factor -
Article: Biased agonist pharmacochaperones of the AVP V2 receptor may treat congenital nephrogenic diabetes insipidus.
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ABSTRACT: 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.Journal of the American Society of Nephrology 10/2009; 20(10):2190-203. · 9.66 Impact Factor -
Article: Small neutralizing molecules to inhibit actions of the chemokine CXCL12.
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ABSTRACT: The chemokine CXCL12 and the receptor CXCR4 play pivotal roles in normal vascular and neuronal development, in inflammatory responses, and in infectious diseases and cancer. For instance, CXCL12 has been shown to mediate human immunodeficiency virus-induced neurotoxicity, proliferative retinopathy and chronic inflammation, whereas its receptor CXCR4 is involved in human immunodeficiency virus infection, cancer metastasis and in the rare disease known as the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome. As we screened chemical libraries to find inhibitors of the interaction between CXCL12 and the receptor CXCR4, we identified synthetic compounds from the family of chalcones that reduce binding of CXCL12 to CXCR4, inhibit calcium responses mediated by the receptor, and prevent CXCR4 internalization in response to CXCL12. We found that the chemical compounds display an original mechanism of action as they bind to the chemokine but not to CXCR4. The highest affinity molecule blocked chemotaxis of human peripheral blood lymphocytes ex vivo. It was also active in vivo in a mouse model of allergic eosinophilic airway inflammation in which we detected inhibition of the inflammatory infiltrate. The compound showed selectivity for CXCL12 and not for CCL5 and CXCL8 chemokines and blocked CXCL12 binding to its second receptor, CXCR7. By analogy to the effect of neutralizing antibodies, this molecule behaves as a small organic neutralizing compound that may prove to have valuable pharmacological and therapeutic potential.Journal of Biological Chemistry 07/2008; 283(34):23189-99. · 4.77 Impact Factor -
Article: 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.
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ABSTRACT: 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.Chemistry 02/2008; 14(20):6247-54. · 5.93 Impact Factor -
Article: Convenient method to access new 4,4-dialkoxy- and 4,4-diaryloxy-diaza-s-indacene dyes: Synthesis and spectroscopic evaluation.
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ABSTRACT: A straightforward method for the synthesis of original 4,4-dialkoxy- or 4,4-diaryloxy-diaza-s-indacenes (BODIPY) derivatives obtained by treatment of BODIPY 1 with various alcohols in the presence of AlCl3 is described. The novel compounds are characterized by spectroscopic properties similar to those of the parent BODIPY 1, absorption and emission spectra with similar band shapes, high molar absorption coefficients (epsilon lambda max approximately 80,000 M(-1) cm(-1)), and for most of them high fluorescence quantum yields (Phi exp from 0.52 to 0.71). Among all of the new compounds synthesized, the dye 2 h exhibits higher fluorescence quantum yield (0.71) and lifetime (4.09 ns) than compound 1 and a good chemical stability toward conditions compatible with biological cell-based assays.The Journal of Organic Chemistry 02/2007; 72(1):269-72. · 4.45 Impact Factor -
Article: Use of a fluorescent polarization based high throughput assay to identify new calmodulin ligands.
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ABSTRACT: In order to develop a fluorescence polarization (FP) assay for calcium binding proteins, a fluorescent peptides based library of 1328 compounds has been synthesized. The use of this library has been validated by setting up a FP-high-throughput screening (FP-HTS) assay for calmodulin using the synthetic gene product (synCaM). With this assay, a set of 880 FDA approved compounds was screened. Besides the promazine class, we discovered two new classes of compounds that interact with calmodulin in a calcium dependent manner. One class has compounds with anti-histaminic/spasmolytic activities, and the other one are detergents with antibacterial activities.Biochimica et Biophysica Acta 12/2006; 1763(11):1250-5. · 4.66 Impact Factor -
Article: A rapid and versatile method to label receptor ligands using "click" chemistry: Validation with the muscarinic M1 antagonist pirenzepine.
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ABSTRACT: Tagged biologically active molecules represent powerful pharmacological tools to study and characterize ligand-receptor interactions. However, the labeling of such molecules is not trivial, especially when poorly soluble tags have to be incorporated. The classical method of coupling usually necessitates a tedious final purification step to remove the excess of reagents and to isolate tagged molecules. To overcome this limitation, Cu(I)-catalyzed 1,3-dipolar cycloaddition, referred to as "click" chemistry, was evaluated as a tool to facilitate the access to labeled molecules. In order to validate the approach, we focused our attention on the incorporation of a fluorophore (Lissamine Rhodamine B), a nonfluorescent dye (Patent Blue VF), or biotin into a muscarinic antagonist scaffold derived from pirenzepine. The reaction performed in acetonitrile/water, in the presence of CuSO4 and Cu wire, allowed us to obtain three novel pirenzepine derivatives with high purity and in good yield. No coupling reagents were needed, and the quasi-stoichiometric conditions of the reaction enabled the straightforward isolation of the final product by simple precipitation and its use in bioassays. The affinity of the compounds for the human M1 muscarinic receptor fused to EGFP was checked under classical radioligand and FRET binding conditions. The three pirenzepine constructs display a nanomolar affinity for the M1 receptor. In addition, both dye-labeled derivatives behave as potent acceptors of energy from excited EGFP with a very high quenching efficiency.Bioconjugate Chemistry 17(6):1618-23. · 4.93 Impact Factor -
Article: Solid-phase preparation of a pilot library derived from the 2,3,4,5-tetrahydro-1H-benzo[b]azepin-5-amine scaffold.
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ABSTRACT: A convenient and reliable solid-phase strategy for the synthesis of di- and trisubstituted benzazepine derivatives was developed. 5-Amino-1-tert-butoxycarbonyl-2,3,4,5-tetrahydro-1H-benzo[b]azepine and 5-amino-1-tert-butoxycarbonyl-7-bromo-2,3,4,5-tetrahydro-1H-benzo[b]azepine G-protein coupled receptor-targeted (GPCR-targeted) scaffolds were efficiently synthesized in a six-step solution-phase process, immobilized on the acid-labile FMPB-AM resin, and further functionalized through acylation, sulfonation, reductive amination, alkylation, and Suzuki or Buchwald-Hartwig cross-coupling reactions. The efficacy of this strategy was exemplified by the preparation of an original pilot library of di- and trisubstituted benzazepines obtained in high purity as assessed by both 1H NMR and liquid chromatography/mass spectrometry (LC/MS) analysis.Journal of Combinatorial Chemistry 9(3):487-500. · 3.41 Impact Factor
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Institutions
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2011
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French National Centre for Scientific Research
Lyon, Rhone-Alpes, France
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2008
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Université de Strasbourg
- Faculté de pharmacie
Strasbourg, Alsace, France
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