Using a qPCR device to screen for modulators of ABC transporter activity: A step-by-step protocol

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.