NanoBRET Approaches to Study Ligand Binding to GPCRs and RTKs

Stoddart, Leigh A.; Kilpatrick, Laura E.; Hill, Stephen J.

doi:10.1016/j.tips.2017.10.006

Cited by 86 publications

(120 citation statements)

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“…The availability of fluorescent ligands in combination with the recently engineered Nluc luciferase allows the development of NanoBRET-based ligand-receptor-binding assays (Stoddart et al, 2015(Stoddart et al, , 2018aSoave et al, 2016). Since NanoBRET only occurs if the fluorescent ligand is bound to the Nluc-tagged GPCR, this assay can be performed in a homogeneous format without additional washing steps to separate unbound from bound fluorescent ligand and is, therefore, well suited to detect real-time ligand binding to GPCRs that are expressed on living cells.…”

Section: Discussionmentioning

confidence: 99%

Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H₃ and H₄ Receptors on Living Cells

et al. 2018

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Receptor-binding affinity and ligand-receptor residence time are key parameters for the selection of drug candidates and are routinely determined using radioligand competition-binding assays. Recently, a novel bioluminescence resonance energy transfer (BRET) method utilizing a NanoLuc-fused receptor was introduced to detect fluorescent ligand binding. Moreover, this NanoBRET method gives the opportunity to follow fluorescent ligand binding on intact cells in real time, and therefore, results might better reflect in vivo conditions as compared with the routinely used cell homogenates or purified membrane fractions. In this study, a real-time NanoBRET-based binding assay was established and validated to detect binding of unlabeled ligands to the histamine H 3 receptor (H 3 R) and histamine H 4 receptor on intact cells. Obtained residence times of clinically tested H 3 R antagonists were reflected by their duration of H 3 R antagonism in a functional receptor recovery assay.

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Section: Discussionmentioning

confidence: 99%

Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H₃ and H₄ Receptors on Living Cells

et al. 2018

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“…BRET techniques have been successfully used to determine the real time kinetics of ligand binding to GPCRs (Stoddart et al, 2018, Sykes et al, 2019. In BRET ligand-binding experiments, we investigated the ability of the selective A3R antagonist MRS 1220, K5, K17 or K18 to inhibit specific binding of the fluorescent A3R antagonist CA200645 to Nluc-A3R.…”

Section: Kinetics Of A3r Antagonists Determined Through Bretmentioning

confidence: 99%

Pharmacological Characterisation of Novel Adenosine Receptor A₃R Antagonists

Barkan

Lagarias

Vrontaki

et al. 2019

Preprint

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Author Contributions:KB, AK and GL conceived and designed the research; KB performed the mammalian assays; KK conducted radioligand binding experiments, PL, DS, EV and AK performed the molecular dynamic simulations; SH derived the equations for the 'Rapid competitor dissociation kinetics' model; KB, GL and AK analysed data; KB and GL wrote manuscript, AK revised and edited the manuscript. Summary Background and PurposeThe adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between ARs orthosteric binding sites, obtaining highly selective receptor antagonists is a challenging but critical task.Experimental approach 39 potential A3R, antagonists were screened using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Further, a likely binding pose of the most potent antagonist (K18) was determined through molecular dynamics (MD) simulations and consistent calculated binding 2 free energy differences between K18 and congeners, using a homology model of A3R, combined with mutagenesis studies. Key ResultsWe demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (<1 µM) competitive antagonist. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazole group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by MD simulations identified the residues important for binding in the A3R orthosteric site. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. Conclusions and ImplicationsThese results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Word count: 241

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“…The development of fluorescence-based technologies has advanced our pharmacological understanding of GPCRs, RTKs, and other classes of membrane proteins (Stoddart, Kilpatrick, & Hill, 2018;Stoddart, White, Nguyen, Hill, & Pfleger, 2016). For example, real-time ligand binding can be quantified in living cells using BRET (Stoddart et al, 2015;Stoddart et al, 2016).…”

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confidence: 99%

Comparison of the ligand‐binding properties of fluorescent VEGF‐A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET

Peach

Kilpatrick

Woolard

et al. 2019

British J Pharmacology

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Background and Purpose Vascular endothelial growth factor A (VEGF‐A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF 165 a to nanoluciferase‐tagged VEGF receptor 2 (VEGFR2) in living cells is that the BRET signal is not sustained and declines over time. This may be secondary to receptor internalisation. Here, we have compared the binding of three fluorescent VEGF‐A isoforms to VEGFR2 in cells and isolated membrane preparations. Experimental Approach Ligand‐binding kinetics were monitored in both intact HEK293T cells and membranes (expressing nanoluciferase‐tagged VEGFR2) using BRET between tagged receptor and fluorescent analogues of VEGF 165 a, VEGF 165 b, and VEGF 121 a. VEGFR2 endocytosis in intact cells expressing VEGFR2 was monitored by following the appearance of fluorescent ligand‐associated receptors in intracellular endosomes using automated quantitative imaging. Key Results Quantitative analysis of the effect of fluorescent VEGF‐A isoforms on VEGFR2 endocytosis in cells demonstrated that they produce a rapid and potent translocation of ligand‐bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF‐A isoforms to VEGFR2. In isolated membrane preparations, ligand‐binding association curves were maintained for the duration of the 90‐min experiment. Measurement of the k off at pH 6.0 in membrane preparations indicated shorter ligand residence times than those obtained at pH 7.4. Conclusions and Implications These studies suggest that rapid VEGF‐A isoform‐induced receptor endocytosis shortens agonist residence times on the receptor (1/ k off ) as VEGFR2 moves from the plasma membrane to the intracellular endosomes.

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NanoBRET Approaches to Study Ligand Binding to GPCRs and RTKs

Cited by 86 publications

References 67 publications

Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H₃ and H₄ Receptors on Living Cells

Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H₃ and H₄ Receptors on Living Cells

Pharmacological Characterisation of Novel Adenosine Receptor A₃R Antagonists

Comparison of the ligand‐binding properties of fluorescent VEGF‐A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET

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NanoBRET Approaches to Study Ligand Binding to GPCRs and RTKs

Cited by 86 publications

References 67 publications

Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H3 and H4 Receptors on Living Cells

Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H3 and H4 Receptors on Living Cells

Pharmacological Characterisation of Novel Adenosine Receptor A3R Antagonists

Comparison of the ligand‐binding properties of fluorescent VEGF‐A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET

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Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H₃ and H₄ Receptors on Living Cells

Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H₃ and H₄ Receptors on Living Cells

Pharmacological Characterisation of Novel Adenosine Receptor A₃R Antagonists