scholarly journals Live-cell microscopy or fluorescence anisotropy with budded baculoviruses - which way to go with measuring ligand binding to M4 muscarinic receptors?

2021 ◽  
Author(s):  
Maris-Johanna Tahk ◽  
Jane Torp ◽  
Mohammed A. S. Ali ◽  
Dmytro Fishman ◽  
Leopold Parts ◽  
...  
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
Cell Segmentation ◽  
Binding Affinities ◽  
Dissociation Kinetics ◽  
Drug Candidates ◽  
Receptor Research ◽  
G Protein Coupled

M4 muscarinic receptor is a G protein-coupled receptor that has been associated with alcohol and cocaine abuse, Alzheimer's disease and schizophrenia which makes it an interesting drug target. For many G protein-coupled receptors, the development of high-affinity fluorescence ligands has expanded the options for high throughput screening of drug candidates and serve as useful tools in fundamental receptor research. So far, the lack of suitable fluorescence ligands has limited studying M4 receptor ligand binding. Here, we explored the possibilities of using fluorescence-based methods for studying binding affinity and kinetics to M4 receptor of both labeled and unlabeled ligands. We used two TAMRA-labeled fluorescence ligands, UR-MK342 and UR-CG072, for assay development. Using budded baculovirus particles as M4 receptor preparation and fluorescence anisotropy method, we determined the affinities and binding kinetics of both fluorescence ligands. The fluorescence ligands could also be used as reported probes for determining binding affinities of a set of unlabeled ligands. Based on these results, we took a step further towards a more natural signaling system and developed a method using live CHO-K1-hM4R cells and automated fluorescence microscopy suitable for routine determination of unlabeled ligand affinities. For quantitative image analysis, we developed random forest and deep learning-based pipelines for cell segmentation. The pipelines were integrated into the user-friendly open-source Aparecium software. All developed assays were suitable for measuring fluorescence ligand saturation binding, association and dissociation kinetics as well as for screening binding affinities of unlabeled ligands.

scholarly journals High Throughput Quantitation of cAMP Production Mediated by Activation of Seven Transmembrane Domain Receptors

1999 ◽  
Vol 4 (1) ◽  
pp. 27-32 ◽  
Author(s):  
Ilona Kariv ◽  
Michelle E. Stevens ◽  
Davette L. Behrens ◽  
Kevin R. Oldenburg
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Transmembrane Domain ◽  
Receptor Function ◽  
Camp Production ◽  
Ligand Interaction ◽  
G Protein Coupled ◽  
Native Ligand

Impairment of G protein—coupled seven-transmembrane (7 TM) receptor function has been implicated in a variety of different pathologic conditions, suggesting that the discovery of specific antagonists may lead to the development of successful therapeutic agents. The effect of different agents on receptor-ligand interaction is often measured directly in a receptor binding assay; however, this assay format can be time consuming and does not detect agents that interact at sites distal to the native ligand binding site. Cyclic adenosine monophospate (cAMP) represents a ubiquitous second messenger generated in response to ligand binding to many 7 TM receptors. The present study describes the practical adaptation of scintillation proximity methodology, using FlashPlate™ (NEN Life Sciences, Boston, MA) technology to evaluate cAMP production. The bioassay is based on competition between endogenously produced cAMP and exogenously added radiolabeled [125I]-cAMP. Cyclic AMP capture is mediated through an anti-cAMP antibody onto a microplate well surface. Removal of unbound radioligand is not necessary because only ligand within ≤20 μm of the plate surface is detected due to the proximity effect. The data indicate that the use of scintillation proximity technology allows accurate and specific evaluation of G protein—coupled receptor function as measured by cAMP production and is suitable for high throughput screening.

New small molecule fluorescent probes for G protein-coupled receptors: valuable tools for drug discovery

2021 ◽  
Vol 13 (1) ◽  
pp. 63-90
Author(s):  
Joshua W Conner ◽  
Daniel P Poole ◽  
Manuela Jörg ◽  
Nicholas A Veldhuis
Keyword(s):  
Drug Discovery ◽  
Ligand Binding ◽  
G Protein ◽  
Small Molecule ◽  
G Protein Coupled Receptors ◽  
Signaling Proteins ◽  
Drug Candidates ◽  
Compound Screening ◽  
G Protein Coupled ◽  
Single Approach

G protein-coupled receptors (GPCRs) are essential signaling proteins and tractable therapeutic targets. To develop new drug candidates, GPCR drug discovery programs require versatile, sensitive pharmacological tools for ligand binding and compound screening. With the availability of new imaging modalities and proximity-based ligand binding technologies, fluorescent ligands offer many advantages and are increasingly being used, yet labeling small molecules remains considerably more challenging relative to peptides. Focusing on recent fluorescent small molecule studies for family A GPCRs, this review addresses some of the key challenges, synthesis approaches and structure–activity relationship considerations, and discusses advantages of using high-resolution GPCR structures to inform conjugation strategies. While no single approach guarantees successful labeling without loss of affinity or selectivity, the choice of fluorophore, linker type and site of attachment have proved to be critical factors that can significantly affect their utility in drug discovery programs, and as discussed, can sometimes lead to very unexpected results.

Mechanochemical Activation of Class-B G-Protein-Coupled Receptor upon Peptide–Ligand Binding

Nano Letters ◽  
2020 ◽  
Vol 20 (7) ◽  
pp. 5575-5582 ◽  
Author(s):  
Cristina Lo Giudice ◽  
Haonan Zhang ◽  
Beili Wu ◽  
David Alsteens
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Mechanochemical Activation ◽  
Peptide Ligand ◽  
G Protein Coupled Receptor ◽  
Class B ◽  
G Protein Coupled

Improved ligand-binding- and signaling-competent human NK2R yields in yeast using a chimera with the rat NK2R C-terminus enable NK2R-G protein signaling platform

2019 ◽  
Vol 32 (10) ◽  
pp. 459-469 ◽  
Author(s):  
Abhinav R Jain ◽  
Zachary T Britton ◽  
Chester E Markwalter ◽  
Anne S Robinson
Keyword(s):  
Protein Engineering ◽  
Ligand Binding ◽  
G Protein ◽  
Mammalian Cells ◽  
Protein Signaling ◽  
Ligand Interaction ◽  
Downstream Signaling ◽  
Drug Candidates ◽  
C Terminus ◽  
Engineering Strategy

Abstract The tachykinin 2 receptor (NK2R) plays critical roles in gastrointestinal, respiratory and mental disorders and is a well-recognized target for therapeutic intervention. To date, therapeutics targeting NK2R have failed to meet regulatory agency approval due in large part to the limited characterization of the receptor-ligand interaction and downstream signaling. Herein, we report a protein engineering strategy to improve ligand-binding- and signaling-competent human NK2R that enables a yeast-based NK2R signaling platform by creating chimeras utilizing sequences from rat NK2R. We demonstrate that NK2R chimeras incorporating the rat NK2R C-terminus exhibited improved ligand-binding yields and downstream signaling in engineered yeast strains and mammalian cells, where observed yields were better than 4-fold over wild type. This work builds on our previous studies that suggest exchanging the C-termini of related and well-expressed family members may be a general protein engineering strategy to overcome limitations to ligand-binding and signaling-competent G protein-coupled receptor yields in yeast. We expect these efforts to result in NK2R drug candidates with better characterized signaling properties.

scholarly journals Model Organisms in G Protein–Coupled Receptor Research

2015 ◽  
Vol 88 (3) ◽  
pp. 596-603 ◽  
Author(s):  
Tobias Langenhan ◽  
Maureen M. Barr ◽  
Michael R. Bruchas ◽  
John Ewer ◽  
Leslie C. Griffith ◽  
...  
Keyword(s):  
G Protein ◽  
Model Organisms ◽  
G Protein Coupled Receptor ◽  
Receptor Research ◽  
G Protein Coupled
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