Use of cellular impedance to characterize ligand functional selectivity at G protein-coupled receptors

Les récepteurs couplés aux protéines G (RCPGs) représentent la plus grande famille de cibles thérapeutiques pour le traitement d’une panoplie de pathologies humaines. Bien que plusieurs décennies de recherche aient permis de façonner nos connaissances sur ces protéines membranaires, notre compréhension des déterminants moléculaires de leur activité signalétique reste encore limitée. De ces domaines de recherche, une avancée récente a mis à jour un nouveau phénomène, appelé sélectivité fonctionnelle des ligands, qui a bouleversé les paradigmes décrivant leu fonctionnement de ces récepteurs. Ce concept émane d’observations montrant que l’activité pharmacologique de certains ligands n’est pas nécessairement conservée sur tout le répertoire signalétiques connu du récepteur et peu se restreindre à l'activation sélective d’un sous-groupe de voies de signalisation.Ce nouveau modèle pharmacologique de l'activation des RCPG ouvre de nouvelles possibilités pour la découverte de médicaments plus efficace et sûr, ciblant les RCPGs. En effet, il permet la conception de molécules modulant spécifiquement les voies signalétiques d’intérêt thérapeutique, sans engager les autres voies qui pourraient mener à des effets secondaires indésirables ou de la tolérance. Cette thèse décrit l'utilisation d'une nouvelle approche sans marquage, basée sur la mesure du changement l'impédance cellulaire. Par la mesure des changements cellulaires, comme la morphologie, l’adhésion et/ou la redistribution des macromolécules, cette approche permet de mesurer de façon simultanée l'activité de plusieurs voies de signalisation impliqués dans ces réponses. Utilisant le récepteur β2-adrénergique (β2AR) comme modèle, nous avons démontré que les variations dans l’impédance cellulaire étaient directement liées à l’activation de multiples voies de signalisation suite à la stimulation du récepteur par son ligand. L’agoniste type du β2AR, l’isoprotérénol, s’est avéré induire une réponse d’impédance dose-dépendante constituée, dans le temps, de plusieurs caractéristiques distinctes pouvant être bloquées de façon compétitive par l’antagoniste ICI118,551 Par l’utilisation d’inhibiteurs sélectifs, nous avons été en mesure de déterminer la contribution de plusieurs voies signalétiques canoniques, comme les voies dépendantes de Gs et Gi, la production d’AMPc et l’activation de ERK1/2, sur ces changements. De plus, la dissection de la réponse d’impédance a permis d’identifier une nouvelle voie de mobilisation du Ca2+ contribuant à la réponse globale des changements initiés par la stimulation du β2AR. Dans une autre étude, nous avons rapporté que la réponse calcique induite par le β2AR serait attribuable à une transactivation Gs-dépendant du récepteur purinergique P2Y11, lui-même couplé à la protéine Gq. La mesure d’impédance permettant de distinguer et de décrire une pléiade d’activités signalétiques, nous avons émis l’hypothèse que des ligands arborant des profils signalétiques différents généreraient des réponses d’impédance distinctes. Le criblage d’une librairie de ligands spécifiques au β2AR a révélé une grande variété de signatures d’impédance. Grâce au développement d’une approche computationnelle innovatrice, nous avons été en mesure de regrouper ces signatures en cinq classes de composés, un regroupement qui s’est avéré hautement corrélé avec le profil signalétique des différents ligands. Nous avons ensuite combiné le criblage de composés par impédance avec l’utilisation d’inhibiteurs sélectifs de voies signalétiques afin d’augmenter la résolution du regroupement. En évaluant l’impact d’une voie signalétique donnée sur la signature d’impédance, nous avons été en mesure de révéler une plus grande variété de textures parmi les ligands. De plus, cette méthode s’est avérée efficace pour prédire le profil signalétique d’une librairie de composés non caractérisés, ciblant le β2AR. Ces travaux ont mené à l’élaboration d’une méthode permettant d’exprimer visuellement la sélectivité fonctionnelle de ligands et ont révélé de nouvelles classes de composés pour ce récepteur. Ces nouvelles classes de composés ont ensuite été testées sur des cardiomyocytes humains, confirmant que les composés regroupés dans différentes classes produisent des effets distincts sur la contractilité de ces cellules. Globalement, ces travaux démontrent la pertinence de l’utilisation de l’impédance cellulaire pour une évaluation précise des différences fonctionnelles parmi les composés ciblant les RCPGs. En fournissant une représentation pluridimensionnelle de la signalisation émanant des RCPGs à l’aide d’un seul essai ne requérant pas de marquage, les signatures d’impédance représentent une stratégie simple et innovante pour l’évaluation de la fonctionnalité sélective des ligands. Cette méthode pourrait être d’une grande utilité dans le processus de découverte de nouveaux médicaments.G protein-coupled receptors (GPCRs) represent the largest family of therapeutic targets for the treatment of a wide variety of human pathologies. Decades of research have provided an extensive base of knowledge about these fascinating membrane proteins, yet significant advancements in the understanding of the structural and functional details of these important drug targets continue to accumulate to this day. One such area of research in particular that has caused a paradigm shift in the way we conceptualize receptor function is a recently identified phenomenon known as ligand functional selectivity. This concept refers to the numerous observations that the pharmacological activity of a ligand at a given receptor is not always conserved over all possible signalling events engaged by the receptor, often resulting in the selectivity of a ligand to modulate only a subset of the receptor’s signalling repertoire. This model of receptor activity reveals exciting new possibilities for the discovery of safer and more efficacious drugs targeting GPCRs; through the design of drugs specifically targeting the pathway of therapeutic interest without modulating other, uninvolved pathways which could lead to tolerance or adverse effects. This thesis will describe the use of a novel, label-free technique based on cellular impedance to further characterize ligand functional selectivity at GPCRs. By measuring changes in higher-order cellular responses, such as changes in morphology, adhesion and redistribution of macromolecules, this approach provides a means to simultaneously measure the activity of multiple signalling pathways converging on these responses. Using the β2-adrenergic receptor (β2AR) as a model system, we have demonstrated that changes in cellular impedance reflect the activity of multiple signalling events elicited following ligand stimulation of the receptor. Isoproterenol, the prototypical agonist of the β2AR, was found to elicit a dose-dependent impedance response consisting of multiple, discrete features over time, which could be blocked in a competitive manner by the antagonist ICI118,551. Using pathway-selective inhibitors, we were able to dissect the contribution of many of the canonical pathways activated by the β2AR, including Gs- and Gi-dependent signalling, as well as cAMP production and ERK1/2 activation. Furthermore, through the pharmacological dissection of this impedance response, we identified a novel Ca2+ mobilization pathway that contributes to the overall cellular response to β2AR stimulation. In a separate study of the mechanism generating this β2AR-promoted Ca2+ response, we revealed a Gs-dependent transactivation mechanism of the Gq-coupled P2Y11 purinergic receptor. Given the ability of impedance measurements to capture this pleiotropic signalling activity, we then reasoned that ligands exhibiting different signalling profiles should generate distinct impedance signatures. In screening a library of functionally selective compounds targeting the β2AR, we obtained a wide variety of impedance signatures. Through the development of a novel computational approach, we were able to cluster these signatures into five distinct compounds classes, which were highly correlated with signalling profiles of the ligands. In an extension of this approach, we then combined impedance screening with the use of pathway-selective inhibitors to determine if this would provide greater resolution in distinguishing among functionally distinct compounds. By assessing if and how a given signalling pathway contributes to a ligand’s impedance signature, we were able to reveal even more texture among ligands targeting the β2AR. Furthermore, this approach was found to be predictive of the signalling profiles of a library of uncharacterized compounds for the β2AR. This work led to the development of a visualization method to express ligand functional selectivity and revealed potentially novel classes of compounds for the receptor. These compound classes were then validated in human cardiomyocytes, confirming that compounds clustering into different classes produced distinct effects on cardiomyocyte contractility. Altogether, this work demonstrates the ability of cellular impedance to accurately measure functional differences among compounds targeting GPCRs. In providing a representation of the pluridimensionality of GPCR signalling using a single, label-free assay, impedance profiling represents an innovative strategy to assess ligand functional selectivity and may be a valuable addition to future drug discovery campaigns

Impedance Responses Reveal β2-Adrenergic Receptor Signaling Pluridimensionality and Allow Classification of Ligands with Distinct Signaling Profiles

The discovery that drugs targeting a single G protein-coupled receptor (GPCR) can differentially modulate distinct subsets of the receptor signaling repertoire has created a challenge for drug discovery at these important therapeutic targets. Here, we demonstrate that a single label-free assay based on cellular impedance provides a real-time integration of multiple signaling events engaged upon GPCR activation. Stimulation of the β2-adrenergic receptor (β2AR) in living cells with the prototypical agonist isoproterenol generated a complex, multi-featured impedance response over time. Selective pharmacological inhibition of specific arms of the β2AR signaling network revealed the differential contribution of Gs-, Gi- and Gβγ-dependent signaling events, including activation of the canonical cAMP and ERK1/2 pathways, to specific components of the impedance response. Further dissection revealed the essential role of intracellular Ca2+ in the impedance response and led to the discovery of a novel β2AR-promoted Ca2+ mobilization event. Recognizing that impedance responses provide an integrative assessment of ligand activity, we screened a collection of β-adrenergic ligands to determine if differences in the signaling repertoire engaged by compounds would lead to distinct impedance signatures. An unsupervised clustering analysis of the impedance responses revealed the existence of 5 distinct compound classes, revealing a richer signaling texture than previously recognized for this receptor. Taken together, these data indicate that the pluridimensionality of GPCR signaling can be captured using integrative approaches to provide a comprehensive readout of drug activity

Feature selection for preserving biological trajectories in single-cell data

<p>Contains preprocessed single-cell data and metadata for feature selection. Preprocessed adata objects can be accessed using the <i>read_h5ad</i> function in anndata.</p><ul><li>The RPE iterative indirect immunofluorescence imaging dataset (adata_RPE.h5ad) from Ref. (<a href="https://doi.org/10.1016/j.cels.2021.10.007">https://doi.org/10.1016/j.cels.2021.10.007</a>) was originally downloaded from the Zenodo repository (<a href="https://doi.org/10.5281/zenodo.4525425">https://doi.org/10.5281/zenodo.4525425</a>).</li><li>The PDAC iterative indirect immunofluorescence imaging datasets (listed below) were originally downloaded from the Zenodo repository (<a href="https://doi.org/10.5281/zenodo.7860332">https://doi.org/10.5281/zenodo.7860332</a>).<ul><li>adata_PDAC_BxPC3_control.h5ad</li><li>adata_PDAC_CFPAC_control.h5ad</li><li>adata_PDAC_HPAC_control.h5ad</li><li>adata_PDAC_MiaPaCa_control.h5ad</li><li>adata_PDAC_Pa01C_control.h5ad</li><li>adata_PDAC_Pa02C_control.h5ad</li><li>adata_PDAC_Pa16C_control.h5ad</li><li>adata_PDAC_PANC1_control.h5ad</li><li>adata_PDAC_UM53_control.h5ad</li></ul></li><li>The CD8+ T cell differentiation dataset (adata_CD8.h5ad) from Ref. (<a href="https://doi.org/10.1016/j.cels.2021.10.007">https://doi.org/</a><a href="https://doi.org/10.1126/sciimmunol.aaz6894">10.1126/sciimmunol.aaz6894</a>) was originally downloaded from the Gene Expression Omnibus under the accession code GSE131847 (<a href="https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE138266">https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131847</a>).</li><li>The definitive endoderm differentiation dataset (adata_DE.h5ad) contains multiplexed single-cell RNA sequencing data profiling the differentiation of human embryonic stem cells into the definitive endoderm. </li></ul&gt

Impedance signatures reveal 5 distinct compound classes among β-adrenergic ligands.

<p>(<b>A–E</b>) Impedance responses were obtained following stimulation with saturating concentrations of the following ligands: ISO (50 µM), epinephrine (EPI, 75 µM), salbutamol (SALB, 100 µM), salmeterol (SALM, 1 µM), alprenolol (ALP, 1 µM), bucindolol (BUC, 1 µM), labetalol (LAB, 1 µM), pindolol (PIN, 1 µM), propranolol (PRO, 1 µM), carvedilol (CARV, 1 µM), metoprolol (MET, 20 µM), timolol (TIM, 1 µM), ICI118,551 (ICI, 1 µM) and atenolol (ATEN, 100 µM). Impedance responses, as represented by the Cell Index, were normalized and baseline-corrected. The first 10 minutes of the impedance responses are enlarged in panels <b>F–I</b>. (<b>J</b>) Ligands were categorized according to complete linkage hierarchical clustering (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029420#s4" target="_blank"><i>Materials and Methods</i></a>) determined by comparing the area between individual curves. The dashed line represents the calculated threshold value defining compound dissimilarity. (<b>K,L</b>) Efficacies of compounds for cAMP (K) and ERK1/2 (L) pathways performed in conditions identical to those used for impedance measurements in (A–E). Data represent means of at least three independent experiments (± SEM for K and L).</p

The role of intracellular Ca²⁺ in the impedance response to β₂AR stimulation.

<p>(<b>A</b>) Peak Ca²⁺ responses generated upon stimulation with the indicated ligand (1 µM each). The intracellular Ca²⁺ concentration is represented in relative luminescence units (RLU) emitted by the obelin biosensor (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029420#s4" target="_blank"><i>Materials and Methods</i></a>). (<b>B</b>) Pre-treatment for 1 hour with the IP₃ receptor antagonist 2-aminoethoxydiphenyl borate (2-APB, 200 µM) or the intracellular Ca²⁺ chelator BAPTA-AM (20 µM) completely abolishes the ISO-promoted Ca²⁺ response. (<b>C–F</b>) Pre-treatment with 2-APB or BAPTA-AM also leads to an inhibition of the rapid ascending phase and a decrease in the maximum impedance responses of ligands from Groups I to IV: ISO (C), salbutamol (SALB, D), alprenolol (ALP, E) and propranolol (PRO, F), respectively (1 µM each). (<b>G</b>) Pre-treatment with 2-APB or BAPTA-AM significantly decreases basal intracellular [Ca²⁺]. Data represent means of at least three independent experiments (± SEM for A, B and G). For statistical analysis, individual conditions were compared in <b>G</b> using a one-way ANOVA and a Bonferroni post-hoc test. *** P<0.001 and * P<0.05.</p

β-adrenergic ligand impedance responses in rat aortic vascular smooth muscle cells (VSMCs).

<p>(<b>A</b>) Pre-treatment with the β₂-selective antagonist ICI118,551 (100 nM) for 1 hour completely abolishes the impedance response obtained following stimulation with 1 µM ISO in VSMCs. (<b>B</b>) Impedance signatures for β-adrenergic ligands representing each of the 5 compound classes defined in 6HisHA-β₂AR-HEKS cells. (<b>C</b>) Complete linkage hierarchical clustering of ligand impedance responses determined by comparing the area between individual curves (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029420#s4" target="_blank"><i>Materials and Methods</i></a>). The dashed line represents the calculated threshold value defining compound dissimilarity. Data represent the means from at least three independent experiments.</p

Analysis of the transient negative phase of the impedance response reveals differences between Group I and II vs Group III and IV ligands.

<p>The duration of the negative phase was plotted as a function of the minimum value for all ligands promoting a transient negative response (Group I–IV). Compound classes are indicated by color: Group I – blue, Group II – purple, Group III – green, and Group IV – pink. Lines of best fit were determined using weighted total least squares regression for all ligands (black dotted line, r² = 0.06, P = 0.79) and for Group III and IV compounds only (red dotted line, r² = 0.97, P = 9.0×10⁻⁵). Inset: The comparison of transient negative phases promoted by the prototypical Group I and Group III ligands EPI (dark blue) and LAB (green), respectively. Data represent means of at least three independent experiments (± SEM).</p

Increasing intracellular [Ca²⁺] accelerates the rapid ascending phase and maximum impedance response.

<p>(<b>A</b>) Impedance responses obtained following treatment with the Ca²⁺ ionophore A23187 (1 µM), the adenylyl cyclase activator forskolin (10 µM) or the combined stimulation with both. (<b>B–E</b>) Impedance responses obtained following stimulation with ISO (B), SALB (C), ALP (D) and PRO (E) (1 µM each) in the presence or absence of A23187 (1 µM). Data represent means of at least three independent experiments.</p

Ligand-induced changes in impedance are multi-featured and concentration-dependent.

<p>(<b>A</b>) Impedance measurements were obtained in 6HisHA-β₂AR-HEK293S cells following treatment with isoproterenol (ISO) at the concentrations indicated. Impedance responses (represented as changes in Cell Index) were normalized and baseline-corrected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029420#s4" target="_blank"><i>Materials and Methods</i></a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029420#pone.0029420.s009" target="_blank">Figure S9</a>. Inset: Enlargement of the transient negative phase of the response. (<b>B</b>) Features of the ISO impedance signature with the inset showing an enlargement of the first 10 minutes post-stimulation. (<b>C,D</b>) Concentration-response curves describing the maximum impedance response generated (<b>C</b>) and the slope of the rapid ascending phase (<b>D</b>) reveal distinct EC₅₀ values. Data represent means of at least three independent experiments (± SEM for C and D). For clarity, error bars are not shown for impedance responses. Typical experimental variability in the impedance responses is demonstrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029420#pone.0029420.s005" target="_blank">Figure S5</a>.</p

Cyclin F drives proliferation through SCF-dependent degradation of the retinoblastoma-like tumor suppressor p130/RBL2.

Cell cycle gene expression programs fuel proliferation and are universally dysregulated in cancer. The retinoblastoma (RB)-family of proteins, RB1, RBL1/p107, and RBL2/p130, coordinately represses cell cycle gene expression, inhibiting proliferation, and suppressing tumorigenesis. Phosphorylation of RB-family proteins by cyclin-dependent kinases is firmly established. Like phosphorylation, ubiquitination is essential to cell cycle control, and numerous proliferative regulators, tumor suppressors, and oncoproteins are ubiquitinated. However, little is known about the role of ubiquitin signaling in controlling RB-family proteins. A systems genetics analysis of CRISPR/Cas9 screens suggested the potential regulation of the RB-network by cyclin F, a substrate recognition receptor for the SCF family of E3 ligases. We demonstrate that RBL2/p130 is a direct substrate of SCFcyclin F. We map a cyclin F regulatory site to a flexible linker in the p130 pocket domain, and show that this site mediates binding, stability, and ubiquitination. Expression of a mutant version of p130, which cannot be ubiquitinated, severely impaired proliferative capacity and cell cycle progression. Consistently, we observed reduced expression of cell cycle gene transcripts, as well a reduced abundance of cell cycle proteins, analyzed by quantitative, iterative immunofluorescent imaging. These data suggest a key role for SCFcyclin F in the CDK-RB network and raise the possibility that aberrant p130 degradation could dysregulate the cell cycle in human cancers