Molecular insights into the biased signaling mechanism of the µ-opioid receptor.

GPCR functional selectivity opens new opportunities for the design of safer drugs. Ligands orchestrate GPCR signaling cascades by modulating the receptor conformational landscape. Our study provides insights into the dynamic mechanism enabling opioid ligands to preferentially activate the G protein over the ß-arrestin pathways through the µ-opioid receptor (µOR). We combine functional assays in living cells, solution NMR spectroscopy, and enhanced-sampling molecular dynamic simulations to identify the specific µOR conformations induced by G protein-biased agonists. In particular, we describe the dynamic and allosteric communications between the ligand-binding pocket and the receptor intracellular domains, through conserved motifs in class A GPCRs. Most strikingly, the biased agonists trigger µOR conformational changes in the intracellular loop 1 and helix 8 domains, which may impair ß-arrestin binding or signaling. The findings may apply to other GPCR families and provide key molecular information that could facilitate the design of biased ligands.

Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases.

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts.

Selective and Wash-Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single-Molecule Imaging of µ-Opioid Receptor Dimerization.

µ-Opioid receptors (µ-ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how µ-ORs produce specific effects in living cells. We developed new fluorescent ligands based on the µ-OR antagonist E-p-nitrocinnamoylamino-dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single-molecule imaging of µ-ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of µ-ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that µ-ORs interact with each other to form short-lived homodimers on the plasma membrane. This approach provides a new strategy to investigate µ-OR pharmacology at single-molecule level.

The oligomeric state sets GABA(B) receptor signalling efficacy.

G protein-coupled receptors (GPCRs) have key roles in cell-cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA(B) receptor is one of the most abundant, being distributed in most brain regions, on either pre- or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers.

The EBI2 receptor is coexpressed with CCR5 in CD4 + T cells and boosts HIV-1 R5 replication.

OBJECTIVE: CCR5, a G protein-coupled receptor (GPCR), is used by most HIV strains as a coreceptor. In this study, we looked for other GPCR able to modify HIV-1 infection. DESIGN: We analyzed the effects of one GPCR coexpressed with CCR5, EBI2, on HIV-1 replicative cycle. METHODS: We identified GPCR expressed in primary CD4 + CCR5 + T cells by multi-RT-qPCR. We studied GPCR dimerization by FRET technology. Cell lines expressing EBI2 were established by transduction with HIV vectors. HIV-1 entry was quantified with virions harboring ß-lactamase fused to the viral protein vpr, early and late HIV-1 transcriptions by qPCR, NFkB nuclear activation by immunofluorescence and transfection, and viral production by measuring p24 concentration in culture supernatant by ELISA. RESULTS: We showed that EBI2 is naturally expressed in primary CD4 + CCR5 + T cells, and that CCR5 and EBI2 heterodimerize. We observed that this coexpression reduced viral entry by 50%. The amount of HIV reverse transcripts was similar in cells expressing or not EBI2. Finally, the presence of EBI2 induced the translocation of NFkB and activated HIV-1 genome expression. Globally, the result was a drastic HIV-1 R5, but not X4, overproduction in EBI2 -transduced cells. CONCLUSION: EBI2 expression in CD4 + CCR5 + cells boosts HIV-1 R5 productive infection. As the natural ligand for EBI2 is present in blood and lymphoid tissues, the constant EBI2 activation might increase HIV replication in CD4 + T cells. It might be of interest to test the effect of EBI2 antagonists on the residual viral production persisting in patients aviremic under treatment.

Optical Control of Adenosine A2A Receptor Using Istradefylline Photosensitivity.

In recent years, there has been growing interest in the potential therapeutic use of inhibitors of adenosine A2A receptors (A2AR) for the treatment of neurodegenerative diseases and cancer. Nevertheless, the widespread expression of A2AR throughout the body emphasizes the importance of temporally and spatially selective ligands. Photopharmacology is an emerging strategy that utilizes photosensitive ligands to attain high spatiotemporal precision and regulate the function of biomolecules using light. In this study, we combined photochemistry and cellular and in vivo photopharmacology to investigate the light sensitivity of the FDA-approved antagonist istradefylline and its potential use as an A2AR photopharmacological tool. Our findings reveal that istradefylline exhibits rapid trans-to-cis isomerization under near-UV light, and prolonged exposure results in the formation of photocycloaddition products. We demonstrate that exposure to UV light triggers a time-dependent decrease in the antagonistic activity of istradefylline in A2AR-expressing cells and enables real-time optical control of A2AR signaling in living cells and zebrafish. Together, these data demonstrate that istradefylline is a photoinactivatable A2AR antagonist and that this property can be utilized to perform photopharmacological experiments in living cells and animals.

Design, Synthesis, and Characterization of New δ Opioid Receptor-Selective Fluorescent Probes and Applications in Single-Molecule Microscopy of Wild-Type Receptors.

The delta opioid receptor (δOR or DOR) is a G protein-coupled receptor (GPCR) showing a promising profile as a drug target for nociception and analgesia. Herein, we design and synthesize new fluorescent antagonist probes with high δOR selectivity that are ideally suited for single-molecule microscopy (SMM) applications in unmodified, untagged receptors. Using our new probes, we investigated wild-type δOR localization and mobility at low physiological receptor densities for the first time. Furthermore, we investigate the potential formation of δOR homodimers, as such a receptor organization might exhibit distinct pharmacological activity, potentially paving the way for innovative pharmacological therapies. Our findings indicate that the majority of δORs labeled with these probes exist as freely diffusing monomers on the cell surface in a simple cell model. This discovery advances our understanding of OR behavior and offers potential implications for future therapeutic research.

A fluorescent sensor for GABA and synthetic GABA(B) receptor ligands.

While γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter, suitable tools to measure its concentration in living cells with high spatiotemporal resolution are missing. Herein, we describe the first ratiometric fluorescent sensor for GABA, dubbed GABA-Snifit, which senses GABA with high specificity and spatiotemporal resolution on the surface of living mammalian cells. GABA-Snifit is a semisynthetic fusion protein containing the GABA(B) receptor, SNAP- and CLIP-tag, a synthetic fluorophore and a fluorescent GABA(B) receptor antagonist. When assembled on cell surfaces, GABA-Snifit displays a GABA-dependent fluorescence emission spectrum in the range of 500-700 nm that permits sensing micromolar to millimolar GABA concentrations. The ratiometric change of the sensor on living cells is 1.8. Furthermore, GABA-Snifit can be utilized to quantify the relative binding affinities of GABA(B) receptor agonists, antagonists and the effect of allosteric modulators. These properties make GABA-Snifit a valuable tool to investigate the role of GABA and GABA(B) in biological systems.

Structure-Based Discovery of Negative Allosteric Modulators of the Metabotropic Glutamate Receptor 5.

Recently determined structures of class C G protein-coupled receptors (GPCRs) revealed the location of allosteric binding sites and opened new opportunities for the discovery of novel modulators. In this work, molecular docking screens for allosteric modulators targeting the metabotropic glutamate receptor 5 (mGlu5) were performed. The mGlu5 receptor is activated by the main excitatory neurotransmitter of the nervous central system, L-glutamate, and mGlu5 receptor activity can be allosterically modulated by negative or positive allosteric modulators. The mGlu5 receptor is a promising target for the treatment of psychiatric and neurodegenerative diseases, and several allosteric modulators of this GPCR have been evaluated in clinical trials. Chemical libraries containing fragment- (1.6 million molecules) and lead-like (4.6 million molecules) compounds were docked to an allosteric binding site of mGlu5 identified in X-ray crystal structures. Among the top-ranked compounds, 59 fragments and 59 lead-like compounds were selected for experimental evaluation. Of these, four fragment- and seven lead-like compounds were confirmed to bind to the allosteric site with affinities ranging from 0.43 to 8.6 µM, corresponding to a hit rate of 9%. The four compounds with the highest affinities were demonstrated to be negative allosteric modulators of mGlu5 signaling in functional assays. The results demonstrate that virtual screens of fragment- and lead-like chemical libraries have complementary advantages and illustrate how access to high-resolution structures of GPCRs in complex with allosteric modulators can accelerate lead discovery.

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging.

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged ß-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.

Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput-compatible format. Using this approach, we examined whether G protein-coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers--a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.

Visualizing biochemical activities in living cells through chemistry.

The development of molecular probes to visualize cellular processes is an important challenge in chemical biology. One possibility to create such cellular indicators is based on the selective labeling of proteins with synthetic probes in living cells. Over the last years, our laboratory has developed different labeling approaches for monitoring protein activity and for localizing synthetic probes inside living cells. In this article, we review two of these labeling approaches, the SNAP-tag and CLIP-tag technologies, and their use for studying cellular processes.

Closed state of both binding domains of homodimeric mGlu receptors is required for full activity.

Membrane receptors, key components in signal transduction, often function as dimers. These include some G protein-coupled receptors such as metabotropic glutamate (mGlu) receptors that have large extracellular domains (ECDs) where agonists bind. How agonist binding in dimeric ECDs activates the effector domains remains largely unknown. The structure of the dimeric ECDs of mGlu(1) solved in the presence of agonist revealed two specific conformations in which either one or both protomers are in an agonist-stabilized closed form. Here we examined whether both conformations correspond to an active form of the full-length receptor. Using a system that allows the formation of dimers made of a wild-type and a mutant subunit, we show that the closure of one ECD per dimer is sufficient to activate the receptor, but the closure of both ECDs is required for full activity.

Assembly-dependent surface targeting of the heterodimeric GABAB Receptor is controlled by COPI but not 14-3-3.

Cell surface expression of transmembrane proteins is strictly regulated. Mutually exclusive interaction with COPI or 14-3-3 proteins has been proposed as a mechanism underlying such trafficking control of various proteins. In particular, 14-3-3 dimers have been proposed to "sense" correctly assembled oligomers, allowing their surface targeting by preventing COPI-mediated intracellular retention. Here we examined whether such a mechanism is involved in the quality control of the heterodimeric G protein-coupled GABAB receptor. Its GB1 subunit, carrying the retention signal RSR, only reaches the cell surface when associated with the GB2 subunit. We show that COPI and 14-3-3 specifically bind to the GB1 RSR sequence and that COPI is involved in its intracellular retention. However, we demonstrate that the interaction with 14-3-3 is not required for proper function of the GABAB receptor quality control. Accordingly, competition between 14-3-3 and COPI cannot be considered as a general trafficking control mechanism. A possible other role for competition between COPI and 14-3-3 binding is discussed.

Pharmacological evidence for a metabotropic glutamate receptor heterodimer in neuronal cells.

Metabotropic glutamate receptors (mGluRs) are mandatory dimers playing important roles in regulating CNS function. Although assumed to form exclusive homodimers, 16 possible heterodimeric mGluRs have been proposed but their existence in native cells remains elusive. Here, we set up two assays to specifically identify the pharmacological properties of rat mGlu heterodimers composed of mGlu2 and 4 subunits. We used either a heterodimer-specific conformational LRET-based biosensor or a system that guarantees the cell surface targeting of the heterodimer only. We identified mGlu2-4 specific pharmacological fingerprints that were also observed in a neuronal cell line and in lateral perforant path terminals naturally expressing mGlu2 and mGlu4. These results bring strong evidence for the existence of mGlu2-4 heterodimers in native cells. In addition to reporting a general approach to characterize heterodimeric mGluRs, our study opens new avenues to understanding the pathophysiological roles of mGlu heterodimers.

Reversing HIV latency via sphingosine-1-phosphate receptor 1 signaling.

OBJECTIVE: In this study, we looked for a new family of latency reversing agents. DESIGN: We searched for G-protein-coupled receptors (GPCR) coexpressed with the C-C chemokine receptor type 5 (CCR5) in primary CD4 T cells that activate infected cells and boost HIV production. METHODS: GPCR coexpression was unveiled by reverse transcriptase-PCR. We used fluorescence resonance energy transfer to analyze the dimerization with CCR5 of the expressed GPCR. Viral entry was measured by flow cytometry, reverse transcription by quantitative PCR, nuclear factor-kappa B translocation by immunofluorescence, long terminal repeat activation using a gene reporter assay and viral production by p24 quantification. RESULTS: Gαi-coupled sphingosine-1-phophate receptor 1 (S1P1) is highly coexpressed with CCR5 on primary CD4 T cells and dimerizes with it. The presence of S1P1 had major effects neither on viral entry nor on reverse transcription. Yet, S1P1 signaling induced NFκB activation, boosting the expression of the HIV LTR. Consequently, in culture medium containing sphingosine-1-phophate, the presence of S1P1 enhanced the replication of a CCR5-, but also of a CXCR4-using HIV-1 strain. The S1P1 ligand FTY720, a drug used in multiple sclerosis treatment, inhibited HIV-1 productive infection of monocyte-derived dendritic cells and of severe combined immunodeficiency mice engrafted with human peripheral blood mononuclear cells. Conversely, S1P1 agonists were able to force latently infected peripheral blood mononuclear cells and lymph node cells to produce virions in vitro. CONCLUSION: Altogether these data indicate that the presence of S1P1 facilitates HIV-1 replicative cycle by boosting viral genome transcription, S1P1 antagonists have anti-HIV effects and S1P1 agonists are HIV latency reversing agents.

Human native kappa opioid receptor functions not predicted by recombinant receptors: Implications for drug design.

If activation of recombinant G protein-coupled receptors in host cells (by drugs or other ligands) has predictive value, similar data must be obtained with native receptors naturally expressed in tissues. Using mouse and human recombinant κ opioid receptors transfected into a host cell, two selectively-acting compounds (ICI204448, asimadoline) equi-effectively activated both receptors, assessed by measuring two different cell signalling pathways which were equally affected without evidence of bias. In mouse intestine, naturally expressing κ receptors within its nervous system, both compounds also equi-effectively activated the receptor, inhibiting nerve-mediated muscle contraction. However, whereas ICI204448 acted similarly in human intestine, where κ receptors are again expressed within its nervous system, asimadoline was inhibitory only at very high concentrations; instead, low concentrations of asimadoline reduced the activity of ICI204448. This demonstration of species-dependence in activation of native, not recombinant κ receptors may be explained by different mouse/human receptor structures affecting receptor expression and/or interactions with intracellular signalling pathways in native environments, to reveal differences in intrinsic efficacy between receptor agonists. These results have profound implications in drug design for κ and perhaps other receptors, in terms of recombinant-to-native receptor translation, species-dependency and possibly, a need to use human, therapeutically-relevant, not surrogate tissues.

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

Activation mechanism of the heterodimeric GABA(B) receptor.

The GABA(B) receptor was the first heteromeric G-protein coupled receptor (GPCR) identified. Indeed, both GABA(B1) and GABA(B2) subunits appear necessary to get a functional GABA(B) receptor. Soon after the cloning of both subunits, it was demonstrated that GABA(B2) was required for GABA(B1) to reach the cell surface. However, even a mutated GABA(B1) able to reach the cell surface is not functional alone despite its ability to bind GABA(B) ligands. This clearly demonstrated that GABA(B2) is not only required for the correct trafficking of GABA(B1) but also for the correct functioning of the receptor. In the present review article, we will summarize our actual knowledge of the specific role of each subunit in ligand recognition, intramolecular transduction, G-protein activation and allosteric modulation. We will show that the GABA(B) receptor is an heterodimer (not an hetero-oligomer), that agonists bind in GABA(B1), whereas GABA(B2) controls agonist affinity and is responsible for G-protein coupling. Finally, we will show that the recently identified positive allosteric modulator CGP7930 acts as a direct activator of the heptahelical domain of GABA(B2), being therefore the first GABA(B2) ligand identified so far.