A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

Caixing Xiong; Dwight Baker; Patricia Pietrantonio

doi:10.3791/64505

Un test de fluorescence calcique « à double addition » pour le criblage à haut débit de récepteur...

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Biochemistry

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JoVE Journal Biochemistry

A “Dual-Addition” Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

Un test de fluorescence calcique « à double addition » pour le criblage à haut débit de récepteurs couplés à la protéine G recombinante

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08:46 min

December 2, 2022

DOI: 10.3791/64505-v

Caixing Xiong¹, Dwight Baker², Patricia Pietrantonio¹

¹Department of Entomology,Texas A&M University, ²Department of Biochemistry and Biophysics,Texas A&M University

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Overview

This study presents a high-throughput, intracellular calcium fluorescence assay designed for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs). The assay enables the identification of both agonists and antagonists using a dual-addition method with CHO-K1 cells expressing the kinin receptor from Rhipicephalus microplus.

Key Study Components

Area of Science

Neuroscience
Pharmacology
Cell Biology

Background

G protein-coupled receptors (GPCRs) play a crucial role in cellular signaling.
Calcium signaling is a key pathway for many GPCRs.
High-throughput screening (HTS) methods are essential for drug discovery.
The dual addition assay allows simultaneous detection of agonists and antagonists.

Purpose of Study

To develop a robust assay for screening small molecule ligands of GPCRs.
To optimize conditions for reproducibility in HTS.
To demonstrate the application of the assay using a specific GPCR target.

Methods Used

High-throughput calcium fluorescence assay in 384-well plates.
Use of CHO-K1 cells expressing the kinin receptor.
Dual-addition method for simultaneous detection of ligands.
Optimization of cell density, injection speed, and agonist concentration.

Main Results

Successful identification of novel small molecule ligands.
Demonstrated assay reproducibility under optimized conditions.
Fluorescence signal duration supports the dual addition approach.
Applicable to various GPCRs that signal through the calcium cascade.

Conclusions

The dual addition assay is a valuable tool for GPCR ligand screening.
Optimized conditions enhance assay reliability and efficiency.
This method can facilitate drug discovery in neuroscience and pharmacology.

Frequently Asked Questions

What is the main advantage of the dual addition assay?

The main advantage is the ability to detect both agonists and antagonists in a single assay using the same cells.

What type of cells are used in this assay?

CHO-K1 cells expressing the kinin receptor from Rhipicephalus microplus are used.

How does the assay contribute to drug discovery?

It allows for the identification of novel small molecule ligands that can be further developed as therapeutics.

What factors are important for assay reproducibility?

Optimizing cell density, injection speed, and agonist concentration are crucial for reproducibility.

Can this method be applied to other GPCRs?

Yes, it can be applied to any GPCR that signals through the calcium cascade.

Who demonstrated the procedure in the study?

Bianca Henriques-Santos, a post-doctoral research associate, demonstrated the procedure.

Dans ce travail, un test de fluorescence calcique intracellulaire à haut débit pour des plaques à 384 puits afin de cribler de petites banques de molécules sur des récepteurs couplés aux protéines G recombinantes (RCPG) est décrit. La cible, le récepteur kinine de la tique de la fièvre bovine, Rhipicephalus microplus, est exprimée dans les cellules CHO-K1. Ce test identifie les agonistes et les antagonistes utilisant les mêmes cellules dans un test de « double addition ».

Le test de double addition de fluorescence calcique à haut débit permet d’identifier de nouveaux ligands à petites molécules d’un récepteur couplé à la protéine G qui signale à travers la cascade de calcium intracellulaire. Le principal avantage du test à double addition est la détection de l’agoniste et de l’antagoniste HTS dans un seul test avec les mêmes cellules à condition que le signal de fluorescence dure deux minutes. Cette méthode peut être appliquée à n’importe quel RCPG qui signale à travers la cascade de calcium, qui comprend la plupart de la famille des peptides neuronaux arthropodes, un RCPG.

Pour la reproductibilité du test, il est important d’optimiser la densité cellulaire, la vitesse d’injection et la concentration de l’agoniste connu pour la deuxième édition. La démonstration de la procédure sera assurée par Bianca Henriques-Santos, associée de recherche postdoctorale dans mon laboratoire. Commencer l’essai de fluorescence calcique en retirant le milieu usé du ballon T-75 contenant de 70 à 90 % de BMLK confluents à trois cellules.

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