Applying molecular dynamics simulations to validate OCT1 substrates uncovers vitamin K1 as a high-affinity ligand.

OCT1 is a hepatic membrane uptake transporter of clinical importance in drug metabolism and pharmacokinetics. OCT1 exhibits broad substrate specificity, mediating uptake of diverse compounds, including the neurotransmitter serotonin and the antidiabetic drug metformin. Numerous compounds have been reported as OCT1 substrates using in vitro model cell lines overexpressing the transporter and computational prediction approaches, often benchmarked against the prototypical substrate tetraethylammonium. The reported substrate affinities span a wide kinetic range, with K m values from 0.48 µM for prostaglandin F2α and prostaglandin E2 to 33,900 µM for trimethylamine N-oxide, highlighting the potential for clinically relevant drug-drug competition during cellular uptake. In this study, we establish an integrated framework to evaluate OCT1 substrates using in silico docking and molecular dynamics simulations, complemented by in vitro transport assays. The computational analysis concurs with previous in vitro cell line studies that ethidium bromide is a high-affinity substrate for OCT1, and herein, we validated experimentally that OCT1-deficient HEK293 cells showed negligible uptake of the compound. Competitive uptake experiments further demonstrated that metformin significantly inhibits ethidium bromide transport, indicating that under these conditions, metformin could be a high-affinity OCT1 substrate. In addition, our approach revealed for the first time that vitamin K1 potently inhibits OCT1-mediated transport of ethidium bromide in HEK293 cells. The integration of computational modeling with experimental validation provides a robust strategy for identifying and characterizing high-affinity OCT1 ligands and highlights the importance of OCT1 in drug transport, nutrient uptake, and potential drug-drug interactions.