Ultrasensitive Detection of Aß42 Seeds in Cerebrospinal Fluid with a Nanopipette-Based Real-Time Fast Amyloid Seeding and Translocation Assay.

In this work, early-stage Aß42 aggregates were detected using a real-time fast amyloid seeding and translocation (RT-FAST) assay. Specifically, Aß42 monomers were incubated in buffer solution with and without preformed Aß42 seeds in a quartz nanopipette coated with L-DOPA. Then, formed Aß42 aggregates were analyzed on flyby resistive pulse sensing at various incubation time points. Aß42 aggregates were detected only in the sample with Aß42 seeds after 180 min of incubation, giving an on/off readout of the presence of preformed seeds. Moreover, this RT-FAST assay could detect preformed seeds spiked in 4% cerebrospinal fluid/buffer solution. However, in this condition, the time to detect the first aggregates was increased. Analysis of Cy3-labeled Aß42 monomer adsorption on a quartz substrate after L-DOPA coating by confocal fluorescence spectroscopy and molecular dynamics simulation showed the huge influence of Aß42 adsorption on the aggregation process.

Concerted conformational changes control metabotropic glutamate receptor activity.

Allosteric modulators bear great potential to fine-tune neurotransmitter action. Promising targets are metabotropic glutamate (mGlu) receptors, which are associated with numerous brain diseases. Orthosteric and allosteric ligands act in synergy to control the activity of these multidomain dimeric GPCRs. Here, we analyzed the effect of such molecules on the concerted conformational changes of full-length mGlu2 at the single-molecule level. We first established FRET sensors through genetic code expansion combined with click chemistry to monitor conformational changes on live cells. We then used single-molecule FRET and show that orthosteric agonist binding leads to the stabilization of most of the glutamate binding domains in their closed state, while the reorientation of the dimer into the active state remains partial. Allosteric modulators, interacting with the transmembrane domain, are required to stabilize the fully reoriented active dimer. These results illustrate how concerted conformational changes within multidomain proteins control their activity, and how these are modulated by allosteric ligands.

Leptin brain entry via a tanycytic LepR-EGFR shuttle controls lipid metabolism and pancreas function.

Metabolic health depends on the brain's ability to control food intake and nutrient use versus storage, processes that require peripheral signals such as the adipocyte-derived hormone, leptin, to cross brain barriers and mobilize regulatory circuits. We have previously shown that hypothalamic tanycytes shuttle leptin into the brain to reach target neurons. Here, using multiple complementary models, we show that tanycytes express functional leptin receptor (LepR), respond to leptin by triggering Ca2+ waves and target protein phosphorylation, and that their transcytotic transport of leptin requires the activation of a LepR-EGFR complex by leptin and EGF sequentially. Selective deletion of LepR in tanycytes blocks leptin entry into the brain, inducing not only increased food intake and lipogenesis but also glucose intolerance through attenuated insulin secretion by pancreatic ß-cells, possibly via altered sympathetic nervous tone. Tanycytic LepRb-EGFR-mediated transport of leptin could thus be crucial to the pathophysiology of diabetes in addition to obesity, with therapeutic implications.

G protein-coupled receptors can control the Hippo/YAP pathway through Gq signaling.

The Hippo pathway is an evolutionarily conserved kinase cascade involved in the control of tissue homeostasis, cellular differentiation, proliferation, and organ size, and is regulated by cell-cell contact, apical cell polarity, and mechanical signals. Miss-regulation of this pathway can lead to cancer. The Hippo pathway acts through the inhibition of the transcriptional coactivators YAP and TAZ through phosphorylation. Among the various signaling mechanisms controlling the hippo pathway, activation of G12/13 by G protein-coupled receptors (GPCR) recently emerged. Here we show that a GPCR, the ghrelin receptor, that activates several types of G proteins, including G12/13, Gi/o, and Gq, can activate YAP through Gq/11 exclusively, independently of G12/13. We revealed that a strong basal YAP activation results from the high constitutive activity of this receptor, which can be further increased upon agonist activation. Thus, acting on ghrelin receptor allowed to modulate up-and-down YAP activity, as activating the receptor increased YAP activity and blocking constitutive activity reduced YAP activity. Our results demonstrate that GPCRs can be used as molecular switches to finely up- or down-regulate YAP activity through a pure Gq pathway.

Targeted pH switched europium complexes monitoring receptor internalisation in living cells.

We report the design and evaluation of pH responsive luminescent europium(iii) probes that allow conjugation to targeting vectors to monitor receptor internalisation in cells. The approach adopted here can be used to tag proteins selectively and to monitor uptake into more acidic organelles, thereby enhancing the performance of time-resolved internalisation assays that require pH monitoring in real time.

Nanobody-Based Quantification of GTP-Bound RHO Conformation Reveals RHOA and RHOC Activation Independent from Their Total Expression in Breast Cancer.

As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 µg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.

The pharmacologic characterization of allosteric molecules: Gq protein activation.

Context: Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts in vivo. Objective: This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales. Materials and methods: Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration-response curves were fit to the functional allosteric model to yield indices of agonism (τB), effects on affinity (α cooperativity), and efficacy (ß cooperativity). Results: It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model. Conclusions: The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these in vivo where a range of tissue sensitivities is encountered.

Time-Resolved FRET-Based Assays to Characterize G Protein-Coupled Receptor Hetero-oligomer Pharmacology.

Although G protein-coupled receptor (GPCR) oligomerization is a matter of debate, it has been shown that the nature of the GPCR partners within the oligomers can influence the pharmacological properties of the receptors. Therefore, finding specific ligands for homo- or hetero-oligomers opens new perspectives for drug discovery. However, no efficient experimental strategy to screen for such ligands existed yet. Indeed, conventional binding strategies do not discriminate ligand binding on GPCR monomers, homo- or hetero-oligomers. To address this issue, we recently developed a new assay based on a time-resolved FRET method that is easy to implement and that can focus on ligand binding specifically on the hetero-oligomer.

HTRF® Total and Phospho-YAP (Ser127) Cellular Assays.

The YAP protein is a co-transcription factor increasing the expression of genes involved in cell proliferation and repressing the expression of genes important for cell differentiation and apoptosis. It is regulated by several inputs, like the Hippo pathway, through the action of kinases that phosphorylate YAP on several residues. The level of phosphorylation of the residues serine 127 (S127) of YAP is generally assessed in cellular models, native tissues, and organs, as a marker of YAP activity and location, and is regulated by numerous partners. This phosphorylation event is classically detected using a western blot technical approach. Here, we describe a novel approach to detect both the relative amount of total YAP (T-YAP assay) and the phosphorylation of the residue S127 of YAP (S127-P-YAP assay) using a HTRF®-based method. This easy-to-run method can easily be miniaturized and allows for a high-throughput analysis in 96/384-well plate format, requiring less cellular material and being more rapid than other approaches.

Gain of affinity for VEGF165 binding within the VEGFR2/NRP1 cellular complex detected by an HTRF-based binding assay.

Neuroplin 1 (NRP1), a transmembrane protein interacting with Vascular Endothelial Growth Factor VEGF-A165 (called here VEGF165) and the tyrosine kinase Receptor 2 (VEGFR2) promote angiogenesis and vascular homeostasis. In a pathophysiological context, several studies suggested that VEGFR2 and NRP1 mediate tumor development and progression. Given the involvement of the VEGF165 network in promoting tumor angiogenesis, NRP1, VEGFR2 and VEGF165 have been identified as targets for anti-angiogenic therapy. No binding assay exists to monitor specifically the binding of VEGF165 to the VEGFR2/NRP1 complex in intact cells. We established a binding assay based on the homogenous time-resolved fluorescence (HTRF®) technology. This unique binding assay enables to assess the interaction of VEGF165 with VEGFR2 or NRP1 within the VEGFR2/NRP1 complex. Ligand binding saturation experiments revealed that VEGF165 binds the VEGFR2/NRP1 complex at the cell surface with a ten to twenty-fold higher affinity compared to SNAP-VEGFR2 or SNAP-NRP1 receptors alone not engaged in the heteromeric complex. The assay allows characterizing the impact of NRP1 ligands on VEGF165 to the complex. It shows high specificity, reproducibility and robustness, making it compatible with high throughput screening (HTS) applications for identifying new VEGF165 antagonists selective for NRP1 or the VEGFR2/NRP1 complex.

Equilibrium Assays Are Required to Accurately Characterize the Activity Profiles of Drugs Modulating Gq-Protein-Coupled Receptors.

This paper discusses the process of determining the activity of candidate molecules targeting Gq-protein activation through G-protein-coupled receptors for possible therapeutic application with two functional assays; calcium release and inositol phosphate metabolism [inositol monophosphate (IP1)]. While both are suitable for detecting ligand activity (screening), differences are seen when these assays are used to quantitatively measure ligand parameters for therapeutic activity. Specifically, responses for Gq-related pathways present different and dissimulating patterns depending on the functional assay used to assess them. To investigate the impact of functional assays on the accuracy of compound pharmacological profiles, five exemplar molecules [partial agonist, antagonist, inverse agonist, positive allosteric modulator (PAM) agonist, and positive ß-PAM] targeting either muscarinic M1 or ghrelin receptors were tested using two functional assays (calcium release and IP1) and the results were compared with theoretical pharmacological models. The IP1 assay is an equilibrium assay that is able to determine the correct (i.e., internally consistent) pharmacological profiles of all tested compounds. In contrast, the nonequilibrium nature of calcium assays yields misleading classification of most of the tested compounds. Our study suggests that the use of an equilibrium assay, such as IP1, is mandatory for the optimal use of pharmacological models that can both identify mechanisms of action and also convert descriptive-to-predictive data for therapeutic systems. Such assays allow the identification of consistent and simple scales of activity that can guide medicinal chemistry in lead optimization of candidate molecules for therapeutic use.

Inhibition of neuronal FLT3 receptor tyrosine kinase alleviates peripheral neuropathic pain in mice.

Peripheral neuropathic pain (PNP) is a debilitating and intractable chronic disease, for which sensitization of somatosensory neurons present in dorsal root ganglia that project to the dorsal spinal cord is a key physiopathological process. Here, we show that hematopoietic cells present at the nerve injury site express the cytokine FL, the ligand of fms-like tyrosine kinase 3 receptor (FLT3). FLT3 activation by intra-sciatic nerve injection of FL is sufficient to produce pain hypersensitivity, activate PNP-associated gene expression and generate short-term and long-term sensitization of sensory neurons. Nerve injury-induced PNP symptoms and associated-molecular changes were strongly altered in Flt3-deficient mice or reversed after neuronal FLT3 downregulation in wild-type mice. A first-in-class FLT3 negative allosteric modulator, discovered by structure-based in silico screening, strongly reduced nerve injury-induced sensory hypersensitivity, but had no effect on nociception in non-injured animals. Collectively, our data suggest a new and specific therapeutic approach for PNP.

Allosteric nanobodies uncover a role of hippocampal mGlu2 receptor homodimers in contextual fear consolidation.

Antibodies have enormous therapeutic and biotechnology potential. G protein-coupled receptors (GPCRs), the main targets in drug development, are of major interest in antibody development programs. Metabotropic glutamate receptors are dimeric GPCRs that can control synaptic activity in a multitude of ways. Here we identify llama nanobodies that specifically recognize mGlu2 receptors, among the eight subtypes of mGluR subunits. Among these nanobodies, DN10 and 13 are positive allosteric modulators (PAM) on homodimeric mGlu2, while DN10 displays also a significant partial agonist activity. DN10 and DN13 have no effect on mGlu2-3 and mGlu2-4 heterodimers. These PAMs enhance the inhibitory action of the orthosteric mGlu2/mGlu3 agonist, DCG-IV, at mossy fiber terminals in the CA3 region of hippocampal slices. DN13 also impairs contextual fear memory when injected in the CA3 region of hippocampal region. These data highlight the potential of developing antibodies with allosteric actions on GPCRs to better define their roles in vivo.

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.

A Time-Resolved FRET Cell-Based Binding Assay for the Apelin Receptor.

Analogues of apelin-13 carrying diverse spacers and an ad hoc DY647-derived fluorophore were designed and synthesized by chemoselective acylation of α-hydrazinopeptides. The resulting probes retain very high affinity and efficacy for both the wild-type and SNAP-tagged apelin receptor (ApelinR). They give a time-resolved FRET (TR-FRET) signal with rare-earth lanthanides used as donor fluorophores grafted onto the SNAP-tagged receptor. This specific signal allowed the validation of a binding assay with a high signal-to-noise ratio. In such an assay, the most potent sub-nanomolar fluorescent probe was found to be competitively displaced by the endogenous apelin peptides with binding constants similar to those obtained in a classical radioligand assay. We have thus validated the first TR-FRET cell-based binding assay for ApelinR with potential high-throughput screening applications.

FRET-Based Sensors Unravel Activation and Allosteric Modulation of the GABAB Receptor.

The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABAB, which is a target for various therapeutic applications. It is an obligatory heterodimer made of GB1 and GB2 that can be regulated by positive allosteric modulators (PAMs). The molecular mechanism of activation of the GABAB receptor remains poorly understood. Here, we have developed FRET-based conformational GABAB sensors compatible with high-throughput screening. We identified conformational changes occurring within the extracellular and transmembrane domains upon receptor activation, which are smaller than those observed in the related metabotropic glutamate receptors. These sensors also allow discrimination between agonists of different efficacies and between PAMs that have different modes of action, which has not always been possible using conventional functional assays. Our study brings important new information on the activation mechanism of the GABAB receptor and should facilitate the screening and identification of new chemicals targeting this receptor.

HTS-compatible FRET-based conformational sensors clarify membrane receptor activation.

Cell surface receptors represent a vast majority of drug targets. Efforts have been conducted to develop biosensors reporting their conformational changes in live cells for pharmacological and functional studies. Although Förster resonance energy transfer (FRET) appears to be an ideal approach, its use is limited by the low signal-to-noise ratio. Here we report a toolbox composed of a combination of labeling technologies, specific fluorophores compatible with time-resolved FRET and a novel method to quantify signals. This approach enables the development of receptor biosensors with a large signal-to-noise ratio. We illustrate the usefulness of this toolbox through the development of biosensors for various G-protein-coupled receptors and receptor tyrosine kinases. These receptors include mGlu, GABAB, LH, PTH, EGF and insulin receptors among others. These biosensors can be used for high-throughput studies and also revealed new information on the activation process of these receptors in their cellular environment.

Protein-Protein Interaction Inhibition (2P2I)-Oriented Chemical Library Accelerates Hit Discovery.

Protein-protein interactions (PPIs) represent an enormous source of opportunity for therapeutic intervention. We and others have recently pinpointed key rules that will help in identifying the next generation of innovative drugs to tackle this challenging class of targets within the next decade. We used these rules to design an oriented chemical library corresponding to a set of diverse "PPI-like" modulators with cores identified as privileged structures in therapeutics. In this work, we purchased the resulting 1664 structurally diverse compounds and evaluated them on a series of representative protein-protein interfaces with distinct "druggability" potential using homogeneous time-resolved fluorescence (HTRF) technology. For certain PPI classes, analysis of the hit rates revealed up to 100 enrichment factors compared with nonoriented chemical libraries. This observation correlates with the predicted "druggability" of the targets. A specific focus on selectivity profiles, the three-dimensional (3D) molecular modes of action resolved by X-ray crystallography, and the biological activities of identified hits targeting the well-defined "druggable" bromodomains of the bromo and extraterminal (BET) family are presented as a proof-of-concept. Overall, our present study illustrates the potency of machine learning-based oriented chemical libraries to accelerate the identification of hits targeting PPIs. A generalization of this method to a larger set of compounds will accelerate the discovery of original and potent probes for this challenging class of targets.

Exploring Selective Inhibition of the First Bromodomain of the Human Bromodomain and Extra-terminal Domain (BET) Proteins.

A midthroughput screening follow-up program targeting the first bromodomain of the human BRD4 protein, BRD4(BD1), identified an acetylated-mimic xanthine derivative inhibitor. This compound binds with an affinity in the low micromolar range yet exerts suitable unexpected selectivity in vitro against the other members of the bromodomain and extra-terminal domain (BET) family. A structure-based program pinpointed a role of the ZA loop, paving the way for the development of potent and selective BET-BRDi probes.

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.