Intracellular Metabolomics Identifies Efflux Transporter Inhibitors in a Routine Caco-2 Cell Permeability Assay-Biological Implications

• Published in: Cells (Basel, Switzerland) Switzerland), 2022-10, Vol.11 (20), p.3286
• Main Authors: Naseem, Afia, Pal, Akos, Gowan, Sharon, Asad, Yasmin, Donovan, Adam, Temesszentandrási-Ambrus, Csilla, Kis, Emese, Gaborik, Zsuzsanna, Bhalay, Gurdip, Raynaud, Florence
• Format: Article
• Language: English
• Subjects: ABC transporters; ATP Binding Cassette Transporter, Subfamily B - metabolism; ATP Binding Cassette Transporter, Subfamily B, Member 1 - metabolism; ATP Binding Cassette Transporter, Subfamily G, Member 2; Bioavailability; Biological assay; Biological transport; Breast cancer; breast cancer resistance; Caco-2 Cells; Carbon - metabolism; Cell culture; Cell lines; Cell metabolism; Cell permeability; Cell research; Cells; Chemical inhibitors; Drug interactions; Drug resistance; Experiments; folate metabolism; Folic Acid; Humans; Identification and classification; Intracellular; Laboratories; Liquid chromatography; Mass spectroscopy; Membrane Transport Proteins; Metabolism; Metabolites; Metabolomics; Methionine; Methods; Methylenetetrahydrofolate reductase; Methylenetetrahydrofolate Reductase (NADPH2); MTHFR; Multidrug Drug Resistance Protein 2; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins - metabolism; Neoplasm Proteins - metabolism; P-Glycoprotein; Permeability; Pharmacokinetics; Physiological aspects; protein; Thymidylate synthase; Thymidylate Synthase - metabolism
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells11203286

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.