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Updated: Dec 20, 2025
High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes
Published on: March 3, 2015
Using a qPCR device to screen for modulators of ABC transporter activity: A step-by-step protocol
Luisa Möhle1, Barbora Schwarzová2, Markus Krohn2
1Department of Neuro-/Pathology, University of Oslo & Oslo University Hospital, Oslo, Norway; Department of Bioorganic Chemistry, Leibniz-Institute of Plant Biochemistry, Halle, Germany.
Introduction:
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are transmembrane proteins which actively transport a large variety of substrates across biological membranes. ABC transporter overexpression can be the underlying cause of multidrug resistance in oncology. Moreover, it has been revealed that increased ABCC1 transporter activity can ameliorate behavioural changes and Aβ pathology in a rodent model of Alzheimer's disease and it is currently tested in AD patients.
Methods:
Finding substances that modulate ABC transporter activity (inhibitors and activators) is of high relevance and thus, different methods have been developed to screen for potential modulators. For this purpose, we have developed a cell-based assay to measure the kinetics of ABCC1-mediated efflux of a fluorescent dye using a common qPCR device (Agilent AriaMx).
Results:
We validated the specificity of our method with vanadate and benzbromarone controls. Furthermore, we provide a step-by-step protocol including statistical analysis of the resulting data and suggestions how to modify the protocol specifically to screen for activators of ABCC1.
Discussion:
Our approach is biologically more relevant than cell-free assays. The continuous detection of kinetics allows for a more precise quantification compared with assays with single end-point measurements.
Insights
This study introduces a novel cell-based assay for measuring ABCC1 transporter kinetics using a qPCR device. This method allows for precise quantification of potential drug modulators for conditions like Alzheimer's disease.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are crucial transmembrane proteins involved in substrate transport.
- Overexpression of ABC transporters, such as ABCC1, is linked to multidrug resistance in cancer and implicated in Alzheimer's disease (AD) pathology.
- Modulating ABCC1 activity shows therapeutic potential for AD, necessitating effective screening methods.
Purpose of the Study:
- To develop and validate a novel cell-based assay for measuring the kinetics of ABCC1 transporter activity.
- To enable the screening of substances that modulate ABCC1 function, including inhibitors and activators.
- To provide a robust and biologically relevant method for drug discovery targeting ABCC1.
Main Methods:
- A cell-based assay was developed to measure the kinetics of ABCC1-mediated efflux of a fluorescent dye.
- The assay utilizes a standard quantitative polymerase chain reaction (qPCR) device (Agilent AriaMx) for continuous detection.
- Assay specificity was confirmed using vanadate and benzbromarone as controls.
Main Results:
- The developed assay successfully measured the kinetics of ABCC1 transporter activity.
- Specificity was validated, and a step-by-step protocol with statistical analysis was provided.
- The protocol includes modifications for screening ABCC1 activators.
Conclusions:
- The cell-based kinetic assay offers higher biological relevance compared to cell-free assays.
- Continuous detection provides more precise quantification of ABCC1 activity than single end-point measurements.
- This assay is a valuable tool for identifying modulators of ABCC1 for therapeutic applications, particularly in Alzheimer's disease research.
