Fluorescent ligands to investigate GPCR binding properties and oligomerization

2013 ◽  
Vol 41 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Martin Cottet ◽  
Orestis Faklaris ◽  
Amadine Falco ◽  
Eric Trinquet ◽  
Jean-Philippe Pin ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescent Imaging ◽  
Binding Assays ◽  
Time Resolved ◽  
Receptor Localization ◽  
Long Time ◽  
Fluorescent Ligands ◽  
Receptor Dimers ◽  
G Protein Coupled

Fluorescent ligands for GPCRs (G-protein-coupled receptors) have been synthesized for a long time but their use was usually restricted to receptor localization in the cell by fluorescent imaging microscopy. During the last two decades, the emergence of new fluorescence-based strategies and the concomitant development of fluorescent measurement apparatus have dramatically widened the use of fluorescent ligands. Among the various strategies, TR (time-resolved)-FRET (fluorescence resonance energy transfer) approaches exhibit an interesting potential to study GPCR interactions with various partners. We have derived various sets of ligands that target different GPCRs with fluorophores, which are compatible with TR-FRET strategies. Fluorescent ligands labelled either with a fluorescent donor (such as europium or terbium cryptate) or with a fluorescent acceptor (such as fluorescein, dy647 or Alexa Fluor® 647), for example, kept high affinities for their cognate receptors. These ligands turn out to be interesting tools to develop FRET-based binding assays. We also used these fluorescent ligands to analyse GPCR oligomerization by measuring FRET between ligands bound to receptor dimers. In contrast with FRET strategies, on the basis of receptor labelling, the ligand-based approach we developed is fully compatible with the study of wild-type receptors and therefore with receptors expressed in native tissues. Therefore, by using fluorescent analogues of oxytocin, we demonstrated the existence of oxytocin receptor dimers in the mammary gland of lactating rats.

scholarly journals Detection of Allosteric Kinase Inhibitors by Displacement of Active Site Probes

2012 ◽  
Vol 17 (6) ◽  
pp. 813-821 ◽  
Author(s):  
Connie S. Lebakken ◽  
Laurie J. Reichling ◽  
Jason M. Ellefson ◽  
Steven M. Riddle
Keyword(s):  
Active Site ◽  
Kinase Inhibitors ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Published Data ◽  
Binding Assays ◽  
Allosteric Inhibitors ◽  
Time Resolved ◽  
Inactive Form ◽  
Competitive Inhibitors

Non–adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)–based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes (“tracers”). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC50 values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.

scholarly journals Real-Time Detection of Interactions between the Human Oxytocin Receptor and G Protein-Coupled Receptor Kinase-2

2004 ◽  
Vol 18 (5) ◽  
pp. 1277-1286 ◽  
Author(s):  
Ahmed Hasbi ◽  
Dominic Devost ◽  
Stéphane A. Laporte ◽  
Hans H. Zingg
Keyword(s):  
Real Time ◽  
G Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Oxytocin Receptor ◽  
Dominant Negative ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Time Resolved ◽  
G Protein Coupled

Abstract Although the oxytocin receptor (OTR) mediates many important functions including uterine contractions, milk ejection, and maternal behavior, the mechanisms controlling agonist-induced OTR desensitization have remained unclear, and attempts to demonstrate involvement of a G protein-coupled receptor kinase (GRK) have so far failed. Using the OTR as a model, we demonstrate here directly for the first time the dynamics of agonist-induced interactions of a GRK with a G protein-coupled receptor in real time, using time-resolved bioluminescence resonance energy transfer. GRK2/receptor interactions started within 4 sec, peaked at 10 sec, and decreased to less than 40% within 8 min. By contrast, β-arrestin/OTR interactions initiated only at 10 sec, reached plateau levels at 120 sec, but remained stable with little decrease thereafter. Physical GRK2/OTR association was further demonstrated by coimmunoprecipitation of endogenous GRK2 with activated OTR. In COS-7 cells, which express low levels of GRK2 and β-arrestin, overexpression of GRK2 and β-arrestin increased receptor phosphorylation, desensitization, and internalization to the high levels observed in human embryonic kidney 293 cells. By contrast, specific inhibition of endogenous GRK2 by dominant-negative mutants robustly inhibited OTR phosphorylation and internalization as well as arrestin/OTR interactions. These data characterize the temporal and causal relationship of GRK-2/OTR and β-arrestin/OTR interactions and establish GRK/OTR interaction as a prerequisite for β-arrestin-mediated OTR desensitization.

scholarly journals Fluorescence Lifetime–Based Competitive Binding Assays for Measuring the Binding Potency of Protease Inhibitors In Vitro

2014 ◽  
Vol 19 (6) ◽  
pp. 870-877 ◽  
Author(s):  
Andreas Boettcher ◽  
Nathalie Gradoux ◽  
Edwige Lorthiois ◽  
Trixi Brandl ◽  
David Orain ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Serine Proteases ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Competitive Binding ◽  
Binding Assays ◽  
Time Resolved ◽  
Design And Synthesis ◽  
Competitive Binding Assays

Fluorescence lifetime (FLT)–based assays have developed to become highly attractive tools in drug discovery. All recently published examples of FLT-based assays essentially describe their use for monitoring enzyme-mediated peptide modifications, such as proteolytic cleavage or phosphorylation/dephosphorylation. Here we report the development of competitive binding assays as novel, inhibitor-centric assays, principally employing the FLT of the acridone dye Puretime 14 (PT14) as the readout parameter. Exemplified with two case studies on human serine proteases, the details of the rationale for both the design and synthesis of probes (i.e., active site–directed low-molecular-weight inhibitors conjugated to PT14) are provided. Data obtained from testing inhibitors with the novel assay format match those obtained with alternative formats such as FLT-based protease activity and time-resolved fluorescence resonance energy transfer–based competitive binding assays.

scholarly journals Identification of Small-Molecule Frequent Hitters from AlphaScreen High-Throughput Screens

2013 ◽  
Vol 19 (5) ◽  
pp. 715-726 ◽  
Author(s):  
Kenji Schorpp ◽  
Ina Rothenaigner ◽  
Elena Salmina ◽  
Jeanette Reinshagen ◽  
Terence Low ◽  
...  
Keyword(s):  
Small Molecule ◽  
Detection System ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Assays ◽  
Time Resolved ◽  
Protein Protein Interaction ◽  
Nickel Chelate ◽  
High Throughput Screens

Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)–compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni2+-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies.

scholarly journals Fluorescent ligands for dopamine D2/D3 receptors

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Anni Allikalt ◽  
Nirupam Purkayastha ◽  
Khajidmaa Flad ◽  
Maximilian F. Schmidt ◽  
Alina Tabor ◽  
...  
Keyword(s):  
Total Internal Reflection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Affinities ◽  
R And D ◽  
D3 Receptors ◽  
Total Internal Reflection Microscopy ◽  
Binding Pockets ◽  
Fluorescent Ligands ◽  
G Protein Coupled

AbstractFluorescent ligands are versatile tools for the study of G protein-coupled receptors. Depending on the fluorophore, they can be used for a range of different applications, including fluorescence microscopy and bioluminescence or fluorescence resonance energy transfer (BRET or FRET) assays. Starting from phenylpiperazines and indanylamines, privileged scaffolds for dopamine D2-like receptors, we developed dansyl-labeled fluorescent ligands that are well accommodated in the binding pockets of D2 and D3 receptors. These receptors are the target proteins for the therapy for several neurologic and psychiatric disorders, including Parkinson’s disease and schizophrenia. The dansyl-labeled ligands exhibit binding affinities up to 0.44 nM and 0.29 nM at D2R and D3R, respectively. When the dansyl label was exchanged for sterically more demanding xanthene or cyanine dyes, fluorescent ligands 10a-c retained excellent binding properties and, as expected from their indanylamine pharmacophore, acted as agonists at D2R. While the Cy3B-labeled ligand 10b was used to visualize D2R and D3R on the surface of living cells by total internal reflection microscopy, ligand 10a comprising a rhodamine label showed excellent properties in a NanoBRET binding assay at D3R.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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