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Positive allosteric modulator (PAM) JNJ-46281222 and negative allosteric modulator (NAM) RO4491533-induced responses at low concentration (1.6 ± 0.09 µM) of endogenous glutamate. A) Cells were stimulated by increasing concentrations of JNJ-46281222 or RO4491533. B) Concentration-response curves were obtained from maximum (JNJ-46281222) or minimum (RO4491533) ΔCI values and normalized from vehicle-induced ΔCI values (0%) to maximum (100%) or minimum (−100%) ΔCI values. C) Responses induced by an EC 20 LY354740 concentration in the absence and presence of 1 µM JNJ-46281222. D) Responses induced by an EC 80 LY354740 concentration in the absence and presence of 1 µM RO4491533. xCELLigence traces are from representative experiments performed in duplicate. Bar graphs represent mean ± SEM of at least three experiments performed in duplicate, where data was normalized to the response of PAM or NAM on itself (1.0). Statistical analysis was performed using a two-tailed unpaired Student's t-test. * p < 0.01, ** p < 0.001.
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Label-free cellular assays using a biosensor provide new opportunities for studying G protein-coupled receptor (GPCR) signaling. As opposed to conventional in vitro assays, integrated receptor-mediated cellular responses are determined in real-time rather than a single downstream signaling pathway. In this study, we examined the potential of a labe...
Citations
... The class C GPCRs that sense amino-acids and their derivatives 31 usually exhibit significant constitutive activity 21,[32][33][34] . However, that of the calcium-sensing receptor (CaSR) 35 has not been well studied. ...
... It was recently reported that ambient L-AAs in the cell culture medium were constitutively bound in the CaSR VFT (Fig. 5a), and act as pure-PAMs during activation by extracellular Ca 2+ 45,47-53 . However, the extracellular concentrations of these L-AAs in cellular assays were difficult to control 32,45 , even in the absence of serum. To study the importance of the L-AAs in the basal activity of the CSCS mutant, we constructed several single mutations that are known to prevent L-AAs binding in the VFT binding pocket of CaSR 45,47,49 . ...
G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences. Among the class C G protein-coupled receptors that typically sense amino-acids and their derivatives, the calcium sensing receptor (CaSR) tightly controls blood calcium levels. Its constitutive activity has not yet been studied. Here, we demonstrate the importance of the inter-subunit disulfide bridges in maintaining the inactive state of CaSR, resulting in undetectable constitutive activity, unlike the other class C receptors. Deletion of these disulfide bridges results in strong constitutive activity that is abolished by mutations preventing amino acid binding. It shows that this inter-subunit disulfide link is necessary to limit the agonist effect of amino acids on CaSR. Furthermore, human genetic mutations deleting these bridges and associated with hypocalcemia result in elevated CaSR constitutive activity. These results highlight the physiological importance of fine tuning the constitutive activity of G protein-coupled receptors.
... In the past few years, the application of cellular impedance biosensor has become prevalent in tackling issues related to receptor pharmacology and the development of new therapeutic ligands. [97][98][99][100][101][102][103] The combined use of electrical impedance and pharmacological inhibitors may shed light on cellular processes, from receptors to signalling molecules. By way of illustration, Meshki et al. 104 used the electrical cellular biosensor to show that pre-treatment of HEK293 cells with the ROCK inhibitor (Y27632) eliminated the blebbing and morphological changes associated with neurokinin 1 receptor (NK1R) activation, leading to their interpretation shown in Fig. 9. ...
Herein the development of cellular impedance biosensors, electrochemical impedance spectroscopy, and the general principles and terms associated with the cell-electrode interface is reviewed. This family of techniques provides quantitative and sensitive information into cell responses to stimuli in real-time with high temporal resolution. The applications of cell-based impedance biosensors as a readout in cell biology is illustrated with a diverse range of examples. The current state of the field, its limitations, the possible available solutions, and the potential benefits of developing biosensors are discussed.
... ; effects of Y262C may rely on extended agonist exposure. Indeed, the lack of GPT in buffers in prior studies suggest ambient glutamate was likely chronically activating mGlu1, as levels of glutamate produced by cells can reach micromolar concentrations under non-GPT conditions, and incubation with an orthosteric agonist reduced Y262C activity to WT levels 14,54 . ...
Metabotropic glutamate receptor 1 (mGlu1) is a promising therapeutic target for neurodegenerative CNS disorders including spinocerebellar ataxias (SCAs). Clinical reports have identified naturally-occurring mGlu1 mutations in rare SCA subtypes and clinical symptoms of mGlu1 mutations have been described. However, how mutations alter mGlu1 function remains unknown. We explored SCA-associated mutation effects on mGlu1 cell surface expression and canonical signal transduction. Orthosteric agonists and positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs) were assessed at two functional endpoints (iCa2+ mobilisation and IP1 accumulation). mGlu1 mutants exhibited differential impacts on receptor expression, with a truncating C-terminus mutation significantly reducing mGlu1 expression. Mutations differentially influenced orthosteric ligand affinity, efficacy, and functional cooperativity between allosteric and orthosteric ligands. Loss-of-function mutations L454F and N885del reduced orthosteric affinity and efficacy, respectively. Gain-of-function Y792C mutant mGlu1 displayed enhanced constitutive activity in IP1 assays, which manifested as reduced orthosteric agonist activity. mGlu1 PAMs restored glutamate potency in iCa2+ mobilisation for loss-of-function mutations, and mGlu1 NAMs displayed enhanced inverse agonist activity at Y792C relative to wild-type mGlu1. Collectively, these data highlight distinct mechanisms by which mGlu1 mutations affect receptor function and show allosteric modulators may present a means to restore aberrant mGlu1 function in rare SCA subtypes.
... Impedancebased assays, e.g. xCELLigence (Doornbos and Heitman, 2019), have been extensively used for functional characterization of GPCR agonists, antagonists, and allosteric modulators (Doornbos et al., 2018). Since SLC substrates can act as GPCR ligands, this technology is suitable for assaying SLCs. ...
... Examples of these are SLCs for monoamine neurotransmitters (e.g. SLC6A2, SLC6A4), glutamate (SLC1) (Doornbos et al., 2018), carboxylic acids (SLC16), and fatty acids (SLC27). To further widen the scope, any SLC that is involved in or influences a process that leads to detectable changes in cell morphology could potentially be assessed (Wang W. W. et al., 2019). ...
The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.
... adenylyl cyclases (AC) activated and inhibited by Gs and Gi/o, respectively), intracellular second-messenger levels (e.g. cyclic adenosine monophosphate, cAMP, downstream of Gαs and Gαi/o and inositol phosphates, IP1/2/3, downstream of Gαq/11 signaling) and other integrated cellular responses even further downstream of receptor/G protein interactions (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). However, assessment of constitutive activity of GPCR/G protein complexes based on intracellular second messenger levels is limited to subtypes of the Gs and Gq/11 families, does not reveal functional selectivity within the same G protein family (e.g. ...
Heterotrimeric G proteins constitute the primary transducers of G protein-coupled receptor (GPCR) signaling. Besides mediating ligand-induced GPCR activation, G proteins transduce basal levels of activity in various physiological and pathophysiological settings evoked by constitutively active, native GPCRs or disease-related receptor mutants. Several generations of optical biosensors were developed and optimized to monitor GPCR ligand-induced G protein activation, however, quantitative approaches to detect constitutively active GPCRs are not available. Here, we designed and validated a set of eight bioluminescence-resonance-energy-transfer (BRET)-based G protein sensors, covering all four major families of G proteins, and established a protocol to identify constitutive GPCR/G protein signaling in living cells. These sensors rely on the encoding of all three G protein subunits on a single plasmid, enabling their cellular expression at desired relative levels and resulting in reduced signal variability in mammalian cells. Based on this sensor platform, we further present here an experimental protocol to quantify constitutive signaling of native and mutated GPCRs through these heterotrimeric transducers. This approach will aid in the characterization of constitutively active GPCRs and the exploration of their role in health and disease.
One Sentence Summary
This Resource article describes the validation of a biophysical approach to directly assess the constitutive signaling activity of G protein-coupled receptors through heterotrimeric G proteins in living cells using optical biosensors.
... Various reports demonstrate that CEI allows identification and characterization of different types of GPCR-targeting compounds (i.e., agonists, antagonists, inverse agonists, allosteric modulators) in one and the same assay (Chen et al., 2015;Doornbos et al., 2018a;Meguro et al., 2018;Moreno Delgado et al., 2017;Peters et al., 2007;Scott and Peters, 2010;Tagami et al., 2016;Zweemer et al., 2013) and this in a more relevant setting (at 37°C on live cells and in culture medium) than many of the traditional assays. ...
... Doornbos et al. reported opposite changes in the impedance for ligands with opposite pharmacological effects on the metabotropic glutamate receptor 2 (mGLUR2). Both the agonist LY354740 and the PAM JNJ-46281222 increased the impedance while antagonist LY341495 and a negative allosteric modulator (NAM) RO4491533 decreased the impedance of a consitutively active mGLUR2 in CHO-k1 cells (Doornbos et al., 2018a). The increase in impedance induced by LY354740, typically seen for G i -coupled receptors, as well as the decrease in impedance elicited by LY341495, could be blocked by PTX, suggesting that both processes are G i-mediated. ...
... As preincubation with LY341495 resulted in more pronounced positive LY354740-induced CEI profiles while preincubation with the NAM did decrease the LY354740 response, they speculated that LY341495 in fact behaves as an inverse agonist instead of an antagonist. This inverse agonism was further confirmed using PAM and NAM pretreatment experiments (Doornbos et al., 2018a). The ability of CEI to discern ligands with various activities is a big advantage of the technology (Doornbos and Heitman, 2019). ...
G Protein-Coupled Receptors (GPCRs) transduce extracellular signals and activate intracellular pathways, usually through activating associated G proteins. Due to their involvement in many human diseases, they are recognized worldwide as valuable drug targets.
Many experimental approaches help identify small molecules that target GPCRs, including in vitro cell-based reporter assays and binding studies. Most cell-based assays use one signaling pathway or reporter as an assay readout. Moreover, they often require cell labeling or the integration of reporter systems.
Over the last decades, cell-based electrical impedance biosensors have been explored for drug discovery. This label-free method holds many advantages over other cellular assays in GPCR research. The technology requires no cell manipulation and offers real-time kinetic measurements of receptor-mediated cellular changes. Instead of measuring the activity of a single reporter, the impedance readout includes information on multiple signaling events. This is beneficial when screening for ligands targeting orphan GPCRs since the signaling cascade(s) of the majority of these receptors are unknown. Due to its sensitivity, the method also applies to cellular models more relevant to disease, including patient-derived cell cultures.
Despite its advantages, remaining issues regarding data comparability and interpretability has limited implementation of cell-based electrical impedance (CEI) in drug discovery. Future optimization must include both full exploitation of CEI response data using various ways of analysis as well as further exploration of its potential to detect biased activities early on in drug discovery.
Here, we review the contribution of CEI technology to GPCR research, discuss its comparative benefits, and provide recommendations.
... They modeled the VFT domain and went on to show that Cl − ions stabilize the glutamate-induced active state of the extracellular domain of the mGlu 3 receptor and the large basal activity that had for many years been seen was due to chloride mediated positive allosteric modulation effect on low levels of residual glutamate concentrations. This is consistent with other recent reports of mGlu receptor activation being sensitive to residual levels of glutamate [99]. ...
Metabotropic glutamate (mGlu) receptors are a family of eight GPCRs that are attractive drug discovery targets to modulate glutamate action and response. Here we review the application of computational methods to the study of this family of receptors. X-ray structures of the extracellular and 7-transmembrane domains have played an important role to enable structure-based modeling approaches, whilst we also discuss the successful application of ligand-based methods. We summarize the literature and highlight the areas where modeling and experiment have delivered important understanding for mGlu receptor drug discovery. Finally, we offer suggestions of future areas of opportunity for computational work.
... To overcome these limitations label-free biosensing detection methods have been proposed [4]. The increasing number of publications on label-free methods confirms the possibility to utilize cost-effective biosensors for various cells detection [5][6][7][8]. Generally, a biosensor is constituted by a reactive layer able to recognize a specific biomolecule, and a transducer transforming the biochemical input interaction in a physical output signal as was shown in Figure 1. ...
In designing a bacteria biosensor, various issues must be addressed: the specificity of bacteria recognition, the immobilization of biomolecules that act as the bacteria receptor, and the selectivity of sensor surface. The aim of this paper was to examine how the biofunctionalized surface of Ti, Au, and Ru metals reacts in contact with strains of Escherichia coli (E. coli). The focus on metal surfaces results from their future use as electrodes in high frequency biosensors, e.g., resonant circuits or transmission-line sections. First, the surfaces of different metals were chemically functionalized with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde or with 3-glycidylooxypropyltrimethoxysilane (GPTMS) followed by N-(5-amino-1-carboxypentyl) iminodiacetic acid (AB-NTA) and NiCl 2. Secondly, the lipopolysaccharide binding protein (LBP), polyclonal anti-Escherichia coli antibody and bacteriophage protein gp37 were tested as bacteria receptors. The selectivity and specificity have been confirmed by the Enzyme-Linked Immunosorbent Assay (ELISA) and visualized by scanning electron microscopy at low landing energies. We noticed that LBP, polyclonal antibody, and gp37 were successfully immobilized on all studied metals and recognized the E. coli bacteria selectively. However, for the antibody, the highest reactivity was observed when Ti surface was modified, whereas the bacteria binding was comparable between LBP and gp37 on the functionalized Ru surfaces, independent from modification. Thus, all surfaces were biocompatible within the scope of biosensor functionality, with titanium functionalization showing the best performance.
... We have recorded concentrations ranging from 0.6 to more than 5 μM. These glutamate concentrations are in line with those measured in another recent publication (Doornbos et al., 2018). Thus, Cl − ions are pure positive allosteric modulators of the mGlu 3 receptor. ...
Due to the essential roles of glutamate, detection and response to a large range of extracellular concentrations of this excitatory amino acid are necessary for the fine-tuning of brain functions. Metabotropic glutamate receptors (mGluRs) are implicated in shaping the activity of many synapses in the central nervous system. Among the eight mGluR subtypes, there is increasing interest in studying the mGlu3 receptor which has recently been linked to various diseases, including psychiatric disorders. This receptor displays striking functional properties, with a high and, often, full basal activity, making its study elusive in heterologous systems. Here, we demonstrate that Cl- ions exert strong positive allosteric modulation of glutamate on the mGlu3 receptor. We have also identified the molecular and structural determinants lying behind this allostery: a unique interactive "chloride-lock" network. Indeed, Cl- ions dramatically stabilize the glutamate-induced active state of the extracellular domain of the mGlu3 receptor. Thus, the mGlu3 receptors' large basal activity does not correspond to a constitutive activity in absence of agonist. Instead, it results mostly from a Cl-mediated amplified response to low ambient glutamate concentrations, such as those measured in cell media. This strong interaction between glutamate and Cl- ions allows the mGlu3 receptor to sense and efficiently react to sub-micromolar concentrations of glutamate, making it the most sensitive member of mGluR family.
... Previous studies have also used this assay to study the link between receptor binding kinetics, functional activation kinetics and duration of signalling for agonist-induced responses on the dopamine D 2 and neurokinin 1 receptors [45,46]. Of note, we have previously shown that culture medium contains 100 µM endogenous glutamate [47], and therefore the enzyme glutamate-pyruvate transaminase (GPT) is often used to reduce this level. In the current study we were interested in the effect of a PAM on glutamate-responses, as this is the endogenous ligand and thus most relevant for PAM drug discovery. ...
While many orthosteric ligands have been developed for the mGlu2 receptor, little is known about their target binding kinetics and how these relate to those of the endogenous agonist glutamate. Here, the kinetic rate constants, i.e. kon and koff, of glutamate were determined for the first time followed by those of the synthetic agonist LY354740 and antagonist LY341495. To increase the understanding of the binding mechanism and impact of allosteric modulation thereon, kinetic experiments were repeated in the presence of allosteric modulators. Functional assays were performed to further study the interplay between the orthosteric and allosteric binding sites, including an impedance-based morphology assay. We found that dissociation rate constants of orthosteric mGlu2 ligands were all within a small 6-fold range, whereas association rate constants were ranging over more than three orders of magnitude and correlated to both affinity and potency. The latter showed that target engagement of orthosteric mGlu2 ligands is kon-driven in vitro. Moreover, only the off-rates of the two agonists were decreased by a positive allosteric modulator (PAM), thereby increasing their affinity. Interestingly, a PAM increased the duration of a glutamate-induced cellular response. A negative allosteric modulator (NAM) increased both on- and off-rate of glutamate without changing its affinity, while it did not affect these parameters for LY354740, indicating probe-dependency. In conclusion, we found that affinity- or potency-based orthosteric ligand optimization primarily results in ligands with high kon values. Moreover, positive allosteric modulators alter the binding kinetics of orthosteric agonists mainly by decreasing koff, which we were able to correlate to a lengthened cellular response. Together, this study shows the importance of studying binding kinetics in early drug discovery, as this may provide important insights towards improved efficacy in vivo.
