A Fluorescence Anisotropy Assay for the Muscarinic M1 G-protein-Coupled Receptor

Huwiler, Kristin G.; Rosier, Therese De; Hanson, Bonnie J.; Vogel, Kurt W.

doi:10.1089/adt.2009.0257

Cited by 20 publications

(13 citation statements)

References 32 publications

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“…Although FP assays have been applied to the study of several members of GPCRs, such as melanocortin-4 receptor [20], 5-HT receptors [21], muscarinic receptors [22], the application of FP to ARs has not yet been successful mainly due to the difficulty in the design of an appropriate FP ligand. Earlier studies were also limited by the lack of sensitive fluorescence detectors in addition to the suitable fluorescent probes.…”

Section: Discussionmentioning

confidence: 99%

Novel Alexa Fluor-488 labeled antagonist of the A2A adenosine receptor: Application to a fluorescence polarization-based receptor binding assay

Kecskés

Kumar

Yoo

et al. 2010

Biochemical Pharmacology

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Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A2A adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A2AAR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a Ki value of 111±16 nM in radioligand binding using [3H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A2AAR. In a cyclic AMP functional assay, MRS5346 was shown to be an A2AAR antagonist. MRS5346 did not show any effect on A1 and A3 ARs in binding or the A2BAR in a cyclic AMP assay at 10 μM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A2AAR binding. The FP signal was optimal with 20 nM MRS5346 and 150 μg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The Kdvalue of MRS5346 calculated from kinetic parameters was 16.5 ± 4.7 nM. In FP competition binding experiments using MRS5346 as a tracer, Ki values of known AR agonists and antagonists consistently agreed with Ki values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs.

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Section: Discussionmentioning

confidence: 99%

Novel Alexa Fluor-488 labeled antagonist of the A2A adenosine receptor: Application to a fluorescence polarization-based receptor binding assay

Kecskés

Kumar

Yoo

et al. 2010

Biochemical Pharmacology

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“…This method has been used to characterize ligand binding to receptors of hormones like endothelin (Junge et al, 2010) and melanocortin (Veiksina et al, 2010). However, it has also been demonstrated for GPCRs of small molecules like acetylcholine for mACh receptors (Huwiler et al, 2010) and serotonin (Tõntson et al, 2014). However, the ratiometric nature of this assay format generates certain limitations for itself-the changes in anisotropy can only be detected if the ratio of bound to free fluorescent ligand has been significantly altered (Nosjean et al, 2006).…”

Section: Binding Determined By Fluorescence Anisotropymentioning

confidence: 99%

Ligand Residence Time at G-protein–Coupled Receptors—Why We Should Take Our Time To Study It

et al. 2015

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Over the past decade the kinetics of ligand binding to a receptor have received increasing interest. The concept of drug-target residence time is becoming an invaluable parameter for drug optimization. It holds great promise for drug development, and its optimization is thought to reduce off-target effects. The success of long-acting drugs like tiotropium support this hypothesis. Nonetheless, we know surprisingly little about the dynamics and the molecular detail of the drug binding process. Because protein dynamics and adaptation during the binding event will change the conformation of the protein, ligand binding will not be the static process that is often described. This can cause problems because simple mathematical models often fail to adequately describe the dynamics of the binding process. In this minireview we will discuss the current situation with an emphasis on G-protein-coupled receptors. These are important membrane protein drug targets that undergo conformational changes upon agonist binding to communicate signaling information across the plasma membrane of cells.

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“…These results suggest that lipoparticle-based FP assays can be developed for other GPCRs incorporated into lipoparticles. An important caveat of this approach is worth noting: while the target protein may be expressed and isolated, key accessory proteins, such as binding partners that modulate the GPCR’s affinity towards its ligands, may be absent from the preparation, which in turn can lead to skewed assay results [27, 28]. …”

Section: Fp Applicationsmentioning

confidence: 99%

Fluorescence polarization assays in small molecule screening

Lea

Simeonov²

2010

Expert Opinion on Drug Discovery

316

225

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Importance of the field Fluorescence polarization (FP) is a homogeneous method that allows rapid and quantitative analysis of diverse molecular interactions and enzyme activities. This technique has been widely utilized in clinical and biomedical settings, including the diagnosis of certain diseases and monitoring therapeutic drug levels in body fluids. Recent developments in the field has been symbolized by the facile adoption of FP in high-throughput screening (HTS) and small molecule drug discovery of an increasing range of target classes. Areas covered in this review The article provides a brief overview on the theoretical foundation of FP, followed by updates on recent advancements in its application for various drug target classes, including G-protein coupled receptors (GPCRs), enzymes and protein-protein interactions (PPIs). The strengths and weaknesses of this method, practical considerations in assay design, novel applications, and future directions are also discussed. What the reader will gain The reader will be informed of the most recent advancements and future directions of FP application to small molecule screening. Take home message In addition to its continued utilization in high-throughput screening, FP has expanded into new disease and target areas and has been marked by increased use of labeled small molecule ligands for receptor binding studies.

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A Fluorescence Anisotropy Assay for the Muscarinic M1 G-protein-Coupled Receptor

Cited by 20 publications

References 32 publications

Novel Alexa Fluor-488 labeled antagonist of the A2A adenosine receptor: Application to a fluorescence polarization-based receptor binding assay

Novel Alexa Fluor-488 labeled antagonist of the A2A adenosine receptor: Application to a fluorescence polarization-based receptor binding assay

Ligand Residence Time at G-protein–Coupled Receptors—Why We Should Take Our Time To Study It

Fluorescence polarization assays in small molecule screening

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