Comparison of in vitro P-glycoprotein screening assays: recommendations for their use in drug discovery

J Med Chem. 2003 Apr 24;46(9):1716-25. doi: 10.1021/jm021012t.


The ATP-dependent drug efflux pump P-glycoprotein (P-gp) affects the absorption and disposition of many compounds. P-gp may also play role in clinically significant drug-drug interactions. Therefore, it is important to find potential substrates or inhibitors of P-gp early in the drug discovery process. To identify compounds that interact with this transporter, several P-gp assays were validated and compared by testing a set of 28 reference compounds, including inhibitors of cytochrome P450 3A4 (CYP3A4). The assays included in silico predictions, inhibition assays (based on cellular uptake of rhodamine-123 or calcein AM), and functional assays (ATPase activity assay and transcellular transport assay, the latter for a subset of compounds). In addition, species differences were studied in an indirect fluorescence indicator screening assay and test systems expressing porcine, mouse, or human P-gp. Our results suggest that several P-gp assays should be used in combination to classify compounds as substrates or inhibitors of P-gp. Recommendations are given on screening strategies which can be applied to different phases of the drug discovery and development process.

Publication types

  • Comparative Study
  • Validation Study

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / analysis*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / antagonists & inhibitors
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • Adenosine Triphosphatases / analysis
  • Animals
  • Biological Transport
  • Cells, Cultured
  • Drug Evaluation, Preclinical / methods*
  • Fluoresceins
  • Fluorescent Dyes
  • Humans
  • Indicators and Reagents
  • Mice
  • Models, Molecular
  • Rhodamines
  • Species Specificity
  • Swine


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Fluoresceins
  • Fluorescent Dyes
  • Indicators and Reagents
  • Rhodamines
  • calcein AM
  • Adenosine Triphosphatases