Intracellular Metabolomics Identifies Efflux Transporter Inhibitors in a Routine Caco-2 Cell Permeability Assay-Biological Implications.
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Date
2022-10-19Author
Naseem, A
Pal, A
Gowan, S
Asad, Y
Donovan, A
Temesszentandrási-Ambrus, C
Kis, E
Gaborik, Z
Bhalay, G
Raynaud, F
Type
Journal Article
Metadata
Show full item recordAbstract
Caco-2 screens are routinely used in laboratories to measure the permeability of compounds and can identify substrates of efflux transporters. In this study, we hypothesized that efflux transporter inhibition of a compound can be predicted by an intracellular metabolic signature in Caco-2 cells in the assay used to test intestinal permeability. Using selective inhibitors and transporter knock-out (KO) cells and a targeted Liquid Chromatography tandem Mass Spectrometry (LC-MS) method, we identified 11 metabolites increased in cells with depleted P-glycoprotein (Pgp) activity. Four metabolites were altered with Breast Cancer Resistance (BCRP) inhibition and nine metabolites were identified in the Multidrug Drug Resistance Protein 2 (MRP2) signature. A scoring system was created that could discriminate among the three transporters and validated with additional inhibitors. Pgp and MRP2 substrates did not score as inhibitors. In contrast, BCRP substrates and inhibitors showed a similar intracellular metabolomic signature. Network analysis of signature metabolites led us to investigate changes of enzymes in one-carbon metabolism (folate and methionine cycles). Our data shows that methylenetetrahydrofolate reductase (MTHFR) protein levels increased with Pgp inhibition and Thymidylate synthase (TS) protein levels were reduced with Pgp and MRP2 inhibition. In addition, the methionine cycle is also affected by both Pgp and MRP2 inhibition. In summary, we demonstrated that the routine Caco-2 assay has the potential to identify efflux transporter inhibitors in parallel with substrates in the assays currently used in many DMPK laboratories and that inhibition of efflux transporters has biological consequences.
Collections
Subject
MTHFR
Multidrug Drug Resistance Protein 2
P-glycoprotein
breast cancer resistance
folate metabolism
protein
Humans
Caco-2 Cells
ATP Binding Cassette Transporter, Subfamily G, Member 2
Multidrug Resistance-Associated Proteins
Thymidylate Synthase
Methylenetetrahydrofolate Reductase (NADPH2)
ATP Binding Cassette Transporter, Subfamily B, Member 1
Neoplasm Proteins
Multidrug Resistance-Associated Protein 2
Membrane Transport Proteins
ATP Binding Cassette Transporter, Subfamily B
Permeability
Folic Acid
Methionine
Carbon
Research team
Clinical Pharma & Trials
Pharma & Stress Response
Language
eng
Date accepted
2022-10-17
License start date
2022-10-19
Citation
Cells, 2022, 11 (20), pp. 3286 -
Publisher
MDPI
Except where otherwise noted, this item's license is described
as
http://creativecommons.org/licenses/by/4.0/
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