A new kinetic method for measuring agonist efficacy and ligand bias using high resolution biosensors and a kinetic data analysis framework

ABSTRACTThe kinetics/dynamics of signaling are of increasing value for G-protein-coupled receptor therapeutic development, including spatiotemporal signaling and the kinetic context of biased agonism. Effective application of signaling kinetics to developing new therapeutics requires reliable kinetic assays and an analysis framework to extract kinetic pharmacological parameters. Here we describe a platform for measuring arrestin recruitment kinetics to GPCRs using a high quantum yield, genetically encoded fluorescent biosensor, and a new analysis framework to quantify the recruitment kinetics. The sensor enabled high temporal resolution measurement of arrestin recruitment to the angiotensin AT1 and vasopressin V2 receptors. The analysis quantified the initial rate of arrestin signaling (kτ), a biologically-meaningful kinetic drug efficacy parameter, by fitting time course data using routine curve-fitting methods. Biased agonism was assessed by comparing kτ values for arrestin recruitment with those for Gq signaling via the AT1 receptor. The kτ ratio values were in good agreement with bias estimates from existing methods. This platform potentially improves and simplifies assessment of biased agonism because the same assay modality is used to compare pathways (potentially in the same cells), the analysis method is parsimonious and intuitive, and kinetic context is factored into the bias measurement.

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Novel Insights into Biased Agonism at G Protein-Coupled Receptors and their Potential for Drug Design

Current Pharmaceutical Design ◽

10.2174/1381612811319280014 ◽

2013 ◽

Vol 19 (28) ◽

pp. 5156-5166 ◽

Cited By ~ 21

Author(s):

Maria Marti-Solano ◽

Ramon Guixa-Gonzalez ◽

Ferran Sanz ◽

Manuel Pastor ◽

Jana Selent

Keyword(s):

Drug Design ◽

G Protein ◽

G Protein Coupled Receptors ◽

Biased Agonism ◽

G Protein Coupled

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Revealing the time course of laser evoked potential habituation by high temporal resolution analysis

European Journal of Pain ◽

10.1002/ejp.1823 ◽

2021 ◽

Author(s):

D. Kersebaum ◽

S.‐C. Fabig ◽

M. Sendel ◽

A. C. Muntean ◽

R. Baron ◽

...

Keyword(s):

Temporal Resolution ◽

Time Course ◽

Evoked Potential ◽

High Temporal Resolution ◽

Resolution Analysis

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Ras-mediated activation of the TORC2–PKB pathway is critical for chemotaxis

The Journal of Cell Biology ◽

10.1083/jcb.201001129 ◽

2010 ◽

Vol 190 (2) ◽

pp. 233-245 ◽

Cited By ~ 78

Author(s):

Huaqing Cai ◽

Satarupa Das ◽

Yoichiro Kamimura ◽

Yu Long ◽

Carole A. Parent ◽

...

Keyword(s):

Cell Migration ◽

Actin Polymerization ◽

Time Course ◽

Specific Inhibitor ◽

Ras Proteins ◽

Extracellular Signaling ◽

Upstream Regulator ◽

Pkb Phosphorylation ◽

G Protein Coupled

In chemotactic cells, G protein–coupled receptors activate Ras proteins, but it is unclear how Ras-associated pathways link extracellular signaling to cell migration. We show that, in Dictyostelium discoideum, activated forms of RasC prolong the time course of TORC2 (target of rapamycin [Tor] complex 2)-mediated activation of a myristoylated protein kinase B (PKB; PKBR1) and the phosphorylation of PKB substrates, independently of phosphatidylinositol-(3,4,5)-trisphosphate. Paralleling these changes, the kinetics of chemoattractant-induced adenylyl cyclase activation and actin polymerization are extended, pseudopodial activity is increased and mislocalized, and chemotaxis is impaired. The effects of activated RasC are suppressed by deletion of the TORC2 subunit PiaA. In vitro RasCQ62L-dependent PKB phosphorylation can be rapidly initiated by the addition of a PiaA-associated immunocomplex to membranes of TORC2-deficient cells and blocked by TOR-specific inhibitor PP242. Furthermore, TORC2 binds specifically to the activated form of RasC. These results demonstrate that RasC is an upstream regulator of TORC2 and that the TORC2–PKB signaling mediates effects of activated Ras proteins on the cytoskeleton and cell migration.

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Integrating High-Content Analysis into a Multiplexed Screening Approach to Identify and Characterize GPCR Agonists

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057114533146 ◽

2014 ◽

Vol 19 (7) ◽

pp. 1079-1089 ◽

Cited By ~ 6

Author(s):

Yingjie Zhu ◽

John Watson ◽

Mengjie Chen ◽

Ding Ren Shen ◽

Melissa Yarde ◽

...

Keyword(s):

Drug Discovery ◽

G Protein Coupled Receptors ◽

High Content Imaging ◽

Biased Agonism ◽

Receptor Oligomerization ◽

Sphingosine 1 Phosphate ◽

High Content Analysis ◽

Screening Assays ◽

Hit Identification ◽

G Protein Coupled

G protein–coupled receptors (GPCRs) are one of the most popular and proven target classes for therapeutic intervention. The increased appreciation for allosteric modulation, receptor oligomerization, and biased agonism has led to the development of new assay platforms that seek to capitalize on these aspects of GPCR biology. High-content screening is particularly well suited for GPCR drug discovery given the ability to image and quantify changes in multiple cellular parameters, to resolve subcellular structures, and to monitor events within a physiologically relevant environment. Focusing on the sphingosine-1-phosphate (S1P1) receptor, we evaluated the utility of high-content approaches in hit identification efforts by developing and applying assays to monitor β-arrestin translocation, GPCR internalization, and GPCR recycling kinetics. Using these approaches in combination with more traditional GPCR screening assays, we identified compounds whose unique pharmacological profiles would have gone unnoticed if using a single platform. In addition, we identified a compound that induces an atypical pattern of β-arrestin translocation and GPCR recycling kinetics. Our results highlight the value of high-content imaging in GPCR drug discovery efforts and emphasize the value of a multiassay approach to study pharmacological properties of compounds of interest.

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Biased Agonism at G Protein-Coupled Receptors: The Promise and the Challenges-A Medicinal Chemistry Perspective

Medicinal Research Reviews ◽

10.1002/med.21318 ◽

2014 ◽

Vol 34 (6) ◽

pp. 1286-1330 ◽

Cited By ~ 73

Author(s):

Jeremy Shonberg ◽

Laura Lopez ◽

Peter J. Scammells ◽

Arthur Christopoulos ◽

Ben Capuano ◽

...

Keyword(s):

G Protein ◽

Medicinal Chemistry ◽

G Protein Coupled Receptors ◽

Biased Agonism ◽

G Protein Coupled

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Mechanisms underlying agonist efficacy

Biochemical Society Transactions ◽

10.1042/bst0350733 ◽

2007 ◽

Vol 35 (4) ◽

pp. 733-736 ◽

Cited By ~ 8

Author(s):

P.G. Strange

Keyword(s):

Time Course ◽

Binding Assay ◽

Full Range ◽

Receptor Activation ◽

D2 Dopamine Receptor ◽

Pseudo First Order Reaction ◽

Rate Determining Step ◽

Binding Event ◽

Set Up ◽

G Protein Coupled

Agonist efficacy is a measure of how well an agonist can stimulate a response system linked to a receptor. Efficacy can be assessed in functional assays and various parameters (Emax, KA/EC50, Emax·KA/EC50) determined. The Emax·KA/EC50 parameter provides a good estimate of efficacy across the full range of efficacy. A convenient assay for the efficacy of agonists for some receptors is provided by the [35S]GTP[S] (guanosine 5′-[γ-[35S]thio]triphosphate)-binding assay. In this assay, the normal GTP-binding event in GPCR (G-protein-coupled receptor) activation is replaced by the binding of the non-hydrolysable analogue [35S]GTP[S]. This assay may be used to profile ligands for their efficacy, and an example here is the D2 dopamine receptor where an efficacy scale has been set up using this assay. The mechanisms underlying the assay have been probed. The time course of [35S]GTP[S] binding follows a pseudo-first-order reaction with [35S]GTP[S] binding reaching equilibrium after approx. 3 h. The [35S]GTP[S]-binding event is the rate-determining step in the assay. Agonists regulate the maximal level of [35S]GTP[S] bound, rather than the rate constant for binding. The [35S]GTP[S]-binding assay therefore determines agonist efficacy on the basis of the amount of [35S]GTP[S] bound rather than the rate of binding.

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