Determination of metabolomic signatures of drug transporter inhibitors in cellular models
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Embargo End Date
2025-05-25
ICR Authors
Authors
Naseem, A
Document Type
Thesis or Dissertation
Date
2024-11-25
Date Accepted
Abstract
Drug transporter interactions are thoroughly investigated in drug discovery to evaluate the mechanism of clearance, drug distribution and to identify the risk of potential drug-drug interactions associated with pre-clinical entities. We hypothesized that a metabolomics signature of transporter inhibition could be identified in cellular models expressing uptake and efflux transporters that are available in our laboratory. These include the Caco-2 monolayer assay for P-glycoprotein (Pgp), Breast Cancer Resistance Protein (BCRP), Multidrug Drug Resistance Protein 2 (MRP2) evaluation and Organic Anion Transporting Polypeptide 1B1 (OATP1B1), OATP1B3 and OATP2B1 transfected Human Embryonic Kidney (HEK) 293 cells. In Caco-2 monolayer assay using selective inhibitors and transporter knock-out (KO) cells and a targeted Liquid Chromatography tandem Mass Spectrometry (LC-MS) method, we identified eleven metabolites increased in cells with depleted Pgp activity, four metabolites were altered with BCRP inhibition, and nine metabolites were identified in the MRP2 signature. A scoring system was created that could discriminate among the three transporters and tested with additional inhibitors and substrates of these transporters. Network analysis of the metabolites associated with efflux transporter inhibition led us to investigate changes of enzymes in one-carbon metabolism (folate and methionine cycle) where changes in key enzymes were identified with Pgp and MRP2 inhibition. In addition, the methionine cycle is also affected by Pgp and MRP2 inhibition. In HEK293 overexpressing cells, non-significant marginal changes were observed with the OATPs inhibition, or the metabolite changes were identified to be potentially related to the pharmacological activity of the inhibitors. Therefore, metabolomics signatures are not suitable to identify inhibition of these OATP transporters in the current format of the uptake assay. In summary, we demonstrated that targeted LC-MS metabolomics applied in routine DMPK assays has the potential to identify inhibitors of efflux transporters and to reveal potential new interactors.
Citation
2024
DOI
Source Title
Publisher
Institute of Cancer Research (University Of London)
ISSN
eISSN
Collections
Research Team
Clinical Pharma & Trials