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

Abstract

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.

Hydroxylated Tetramethoxyflavone Affects Intestinal Cell Permeability and Inhibits Cytochrome P450 Enzymes

“…These cells can express morphological and functional characteristics of the small intestine, such as brush borders, tight junctions, intestinal efflux, and uptake transporters, which affect the passage of drugs and food extracts from the intestinal lumen to the bloodstream [ 22 ]. The transport studies using Caco-2 cell monolayers have revealed essential information about intestinal permeability, the transport method (paracellular, transcellular, or active carrier), the function of intestinal metabolism, and the impact of the P-glycoprotein, breast cancer resistance, and multidrug drug-resistance protein efflux system [ 23 ]. The minimum solubility required for an orally active drug is also determined by its intestinal permeability.…”

Metabolite transport across central nervous system barriers

“…Lastly, in vitro transporter knock-outs may be compensated by upregulation of another transporter. 19 Moreover, for many drug transporters, 107 possible metabolite substrates are not yet known. Despite these limitations, this manuscript is the first attempt to evaluate influx and efflux routes of metabolites in the CNS considering factors influencing metabolite concentrations in the CSF.…”