A Kinetic Fluorescence-based Ca<sup>2+</sup> Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

Sandra Claes; Thomas D'huys; Anneleen Van Hout; Dominique Schols; Tom Van Loy

doi:10.3791/56780

운동 형광 기반 Ca2 + 동원 분석 결과 G 단백질 결합 된 수용 체 주 작동 근, 길 항 근, 그...

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A Kinetic Fluorescence-based Ca²⁺ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

운동 형광 기반 Ca2 + 동원 분석 결과 G 단백질 결합 된 수용 체 주 작동 근, 길 항 근, 그리고 Allosteric 변조기를 식별 하

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07:41 min

February 20, 2018

DOI: 10.3791/56780-v

Sandra Claes¹, Thomas D'huys¹, Anneleen Van Hout¹, Dominique Schols¹, Tom Van Loy¹

¹Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research,KU Leuven

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Overview

This cell-based assay is designed to identify and characterize molecules that can stimulate or inhibit G protein-coupled receptor-mediated intracellular calcium mobilization. It allows for the screening of agents that interact with CXC chemokine receptor 4 (CXCR4) and can modify receptor-mediated intracellular calcium release.

Key Study Components

Area of Science

Neuroscience
Pharmacology
Cell Biology

Background

G protein-coupled receptors (GPCRs) are critical therapeutic targets.
Intracellular calcium mobilization is a key signaling pathway.
Identifying modulators of GPCRs can lead to novel drug candidates.
Assays can differentiate between agonistic and antagonistic properties.

Purpose of Study

To develop a method for screening CXCR4-interacting agents.
To facilitate drug discovery for GPCR-related diseases.
To enhance understanding of receptor-mediated signaling.

Methods Used

Cell-based assay using a 96-well plate format.
Coating wells with gelatin to support cell attachment.
Using trypsin EDTA for cell detachment and resuspension.
Screening for molecules that affect intracellular calcium levels.

Main Results

Identification of compounds that can either inhibit or stimulate calcium release.
Demonstration of the assay's ability to screen for both agonists and antagonists.
Potential for discovering new therapeutic agents targeting GPCRs.
Validation of the assay's effectiveness in drug discovery processes.

Conclusions

The assay provides a robust platform for GPCR drug discovery.
It enables the identification of diverse pharmacological agents.
This method may contribute to advancements in treating diseases linked to GPCRs.

Frequently Asked Questions

What is the significance of CXCR4 in drug discovery?

CXCR4 is a GPCR involved in various diseases, making it a key target for therapeutic intervention.

How does the assay differentiate between agonists and antagonists?

The assay measures intracellular calcium levels, allowing for the identification of compounds that either stimulate or inhibit receptor activity.

What type of cells are used in this assay?

The assay utilizes cells that express CXCR4, enabling the study of receptor interactions.

Can this method be applied to other GPCRs?

Yes, the assay can be adapted to study other GPCRs beyond CXCR4.

What are the advantages of using a 96-well plate format?

The 96-well format allows for high-throughput screening of multiple compounds simultaneously.

Is this assay suitable for early-stage drug discovery?

Yes, it is designed to facilitate early-stage screening of potential drug candidates.

설명된 세포 분석 결과 CXC chemokine 수용 체 4 (CXCR4)의 식별을 위해 설계 되었습니다-상호 작용 하거나 에이전트는 억제 자극, 경쟁적으로 또는 allosterically, 세포내 캘리포니아^{2 +}릴리스 CXCR4 활성화에 의해 시작.

이 세포 기반 분석의 전반적인 목표는 G 단백질 결합 수용체 매개 세포 내 칼슘 동원을 자극하거나 억제할 수 있는 분자를 식별하고 특성화하는 것입니다. 이 방법은 수용체 매개 세포 내 칼슘체를 변형하는 분자를 스크리닝하는 데 사용할 수 있기 때문에 G 단백질 결합 수용체 약물 발견에 유용한 분석법이 될 수 있습니다. 이 기술의 주요 장점은 작용 작용 또는 길항 특성을 가진 분자를 동일한 분석 내에서 식별할 수 있다는 것입니다.

이 분석은 많은 인간 질병에서 역할을 하는 유망한 치료 표적을 약속하는 G 단백질 결합 수용체에 작용하는 새로운 약물 후보를 발견하는 데 도움이 될 수 있습니다. 세포 배양 당일, 바닥이 투명한 검은색 폴리스티렌 96웰 플레이트의 각 웰에 0.1% 젤라틴 용액 100마이크로리터로 코팅하고 실온에서 2시간 동안 플레이트를 배양합니다. 젤라틴이 응고되는 동안 트립신 EDTA 용액을 사용하여 배양 플라스크 바닥에서 관심 세포를 들어 올리고 세포가 분리되면 50ml 원추형 튜브에서 신선한 성장 배지에 세포를 재현탁시킵니다.

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