Chemobiologie des RCPG

L’objectif de cette thématique est de concevoir et de synthétiser des nouveaux ligands fluorescents qui vont permettre de développer des essais innovants afin de déterminer rapidement l’affinité d’un ligand pour un RCPG ainsi que sa sélectivité vis-à-vis d’autres RCPG. Ces sondes vont permettre également de visualiser ces récepteurs à la surface de cellules et d’étudier leur architecture fonctionnelle (homo- et hétérodimérisation).

Convenient access to fluorescent probes by chemoselective acylation of hydrazinopeptides: Application to the synthesis of the first far-red ligand for Apelin receptor imaging.

Chem. Eur. J., 2016, 22, 1399-1405.

In this article, we develop a convenient method to facilitate the solution-phase fluorescent labelling of peptides which is based on the chemoselective acylation of a-hydrazinopeptides. This approach combines the advantages to use commercially available amine-reactive dyes and very mild conditions that are fully compatible with the chemical sensitivity of the dyes. The usefulness of this approach was demonstrated by the labelling of apelin-13 peptide. Various fluorescent probes were readily synthesized enabling the rapid optimization of their affinities for Apelin receptor. Thus, the first far-red fluorescent ligand with subnanomolar affinity for Apelin receptor was characterized and enabled to track efficiently the receptor in living cells by fluorescence confocal microscopy.

Convenient access to fluorescent probes by chemoselective acylation of hydrazinopeptides: Application to the synthesis of the first far-red ligand for Apelin receptor imaging.

Selective non-peptidic fluorescent ligands for oxytocin receptor: Design, synthesis and application to time-resolved FRET binding assay.

J. Med. Chem., 2015, 58, 2547-2552.

The design and the synthesis of 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 done by competition experiments with both peptide and non-peptide OTR ligands as competitors. These probes represent the first selective fluorescent ligands for the oxytocin G protein-coupled receptor.

Selective non-peptidic fluorescent ligands for oxytocin receptor: Design, synthesis and application to time-resolved FRET binding assay.

Squaraine as bright, stable and environment-sensitive far-red label for receptor-specific cellular imaging.

Chem. Commun., 2015, 51, 2960-2963.

Herein, we show that a bright far-red arylidene-squaraine dye is stable against nucleophiles, in contrast to arene-squaraines, and exhibits fluorescence enhancement in apolar media combined with high photostability. It was successfully applied to detect the oxytocin G protein-coupled receptor at the cell surface. 

Squaraine as bright, stable and environment-sensitive far-red label for receptor-specific cellular imaging.

Fluorogenic squaraine dimers with polarity-sensitive folding as bright far-red probes for background-free bioimaging.

J. Am. Chem. Soc., 2015, 137, 405-412.

Polarity-sensitive fluorogenic dyes raised considerable attention because they can turn on their fluorescence after binding to biological targets, allowing background-free imaging. However, their brightness is limited and they do not operate in the far-red region. Here, we present a new concept of fluorogenic dye based on a squaraine dimer that unfolds on changing environment from aqueous to organic, and thus turns on its fluorescence. The obtained dimers can be considered as the brightest polarity-sensitive fluorogenic molecules reported to date, having ~660,000 M-1cm-1 extinction coefficient and up to 40% quantum yield, whereas far-red operation region enables both in vitro and in vivo applications. 

Fluorogenic squaraine dimers with polarity-sensitive folding as bright far-red probes for background-free bioimaging.

Time-Resolved FRET binding assay to investigate hetero-oligomer binding properties: proof of concept with dopamine D1/D3 heterodimer.

ACS Chem. Biol., 2014, DOI: 10.1021/cb5007568

http://www.cnrs.fr/inc/communication/direct_labos/bonnet2.htm

G protein-coupled receptors (GPCRs) have been described to form hetero-oligomers. The importance of these complexes in physiology and pathology is considered crucial, and hetero-dimers represent promising new targets to discover innovative therapeutics. However, there is a lack of binding assays to allow the evaluation of ligand affinity for GPCR hetero-oligomers. Using dopamine receptors and more specifically the D1 and D3 receptors as GPCR models, we developed a time-resolved FRET (TR-FRET) based assay to determine ligand affinity for the D1/D3 heteromer. The newly reported assay, easy to implement on other G protein-coupled receptors, constitutes an attractive strategy to screen for heteromer ligands.

Graphical abstract : Time-Resolved FRET binding assay to investigate hetero-oligomer binding properties: proof of concept with dopamine D1/D3 heterodimer

Red Fluorescent Turn-On Ligands for Imaging and Quantifying G Protein-Coupled Receptors in Living Cells

ChemBioChem, 2014, 15, 359-363.

http://www.cnrs.fr/inc/communication/direct_labos/bonnet.htm

Receptor, You Can Turn Me On: The first red fluorescent turn-on ligands capable to recognize the target G protein-coupled receptor (oxytocin) at the surface of living cells were developed. The molecular design, based on a fluorogenic dye grafted to a peptide agonist through a long polar spacer, ensures strong specific turn-on response that enables visualization and rapid quantification of the receptors in situ.

Graphical abstract : Red Fluorescent Turn-On Ligands for Imaging and Quantifying G Protein-Coupled Receptors in Living Cells

Selective Fluorescent Non-peptidic Antagonists For Vasopressin V2 GPCR: Application To Ligand Screening and Oligomerization Assays.

J. Med. Chem., 2012, 55, 8588-8602.

A series of fluorescent benzazepine ligands for the arginine-vasopressin V2 receptor (AVP V2R) was synthesized using “Click” chemistry. These antagonists constitute the first high-affinity selective non-peptidic 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 V1aR-V2R dimerization on cell surface.

Graphical abstract : Selective Fluorescent Non-peptidic Antagonists For Vasopressin V2 GPCR: Application To Ligand Screening and Oligomerization Assays.

Fluorescent derivatives of AC-42 to probe bitopic orthosteric/allosteric binding mechanisms on muscarinic M1 receptors

J. Med. Chem., 2012, 55, 2125-2143.

Two fluorescent derivatives of the M1 muscarinic selective agonist AC-42 were synthesized by coupling the lissamine rhodamine B (LRB) fluorophore to AC42-NH2. This precursor, prepared according to an original seven-step procedure, was included in the study together with the LRB fluorophore. 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.

Graphical abstract : Fluorescent derivatives of AC-42 to probe bitopic orthosteric/allosteric binding mechanisms on muscarinic M1 receptors

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.

Chem. Eur. J., 2008, 14, 6247-54.

“SPOrT” resins. Two novel “Solid Phase Organic Tagging” resins have been developed to facilitate the labelling of peptides and small organic compounds. The strategy has been successfully applied to the synthesis of the first non-peptidic fluorescent compound with nanomolar affinity for human vasopressin V2 receptor subtype.

Graphical abstract : 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