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

J Pharmacol Toxicol Methods. Jul-Aug 2020;104:106882. doi: 10.1016/j.vascn.2020.106882. Epub 2020 May 28.

Abstract

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.

Keywords: ABC transporter activity; ABCC1; Blood-brain barrier; Cell-based assay; Drug screening; Methods; Multidrug resistance-associated protein 1 (MRP1); Plant extract testing.

Publication types

  • Validation Study

MeSH terms

  • Alzheimer Disease / physiopathology
  • Benzbromarone / pharmacology
  • Cell Line
  • Fluorescent Dyes / metabolism*
  • Humans
  • Multidrug Resistance-Associated Proteins / drug effects*
  • Multidrug Resistance-Associated Proteins / metabolism
  • Real-Time Polymerase Chain Reaction / methods*
  • Vanadates / pharmacology

Substances

  • Fluorescent Dyes
  • Multidrug Resistance-Associated Proteins
  • Vanadates
  • Benzbromarone
  • multidrug resistance-associated protein 1