Numerous natural products are widely used as first-line/alternative therapeutics and dietary supplements in both western and eastern society. However, the safety and efficacy profiles for herbal products are still limited. Organic anion transporters (OATs; SLC22 family) are expressed in many barrier organs and mediate in vivo body disposition of a broad array of endogenous substances and clinically important drugs. As some dietary flavonoids and phenolic acids were previously demonstrated to interact with OATs, it is necessary to explore the potential interaction of such components found in natural products in order to avoid potential OAT-mediated drug-drug interactions (DDIs). The inhibitory effects of 23 natural products were assessed on the function of human (h) OATs, hOAT1 (SLC22A6), hOAT3 (SLC22A7), and hOAT4 (SLC22A11) and/or the murine (m) orthologs mOat1 and mOat3. For compounds exhibiting marked inhibition at initial screening, dose-response curves (IC50 values) and DDI indices were determined. At the initial screening concentrations, 14, 19, and 2 test compounds exhibited significant inhibition on hOAT1, hOAT3, and hOAT4, respectively. Additionally, all test Danshen (a Chinese herbal medicine) hydrophilic components significantly reduced mOat1- and mOat3-mediated substrate uptake at 1 mM. For selected compounds, the IC50 and Ki values were estimated to be in the micromolar or even nanomolar range. Considering the clinical plasma concentration and unbound fraction in plasma, DDI indices for gallic acid, gentisic acid, lithospermic acid, protocatechuic acid, rosmarinic acid, salvianolic acid B, and tanshinol indicated DDIs may occur in vivo in situations of co-administration of these compounds and clinical therapeutics known to be OAT substrates. Finally, a new, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify gallic acid and gentisic acid in cell lysates in order to measure cellular uptake of these compounds in mOat1- or mOat3-expressing cells. Significant cellular uptake of gallic acid was observed in mOat1-expressing cells, compared with background control cells. The absorptive uptake was completely blocked by probenecid (known OAT inhibitor) at 1 mM. These results indicate that gallic acid is a substrate for mOat1 and suggest that human OAT1 might be involved in the active renal secretion of gallic acid.

The organic cation transporters (OCTs) play a critical role in the absorption, distribution and elimination of many drugs, hormones, herbal medicines, and environmental toxins. Given the broad substrate specificity of OCTs, they fall victim to the high susceptibility for contributing to harmful drug-drug interactions. Further defining how human (h)OCTs mechanistically bind to its broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCTs. The goal of the current study was to elucidate the critical amino acid residues for transporter-substrate binding interactions on human (h)OCT1 and 2 utilizing in silico molecular modeling techniques (homology modeling and automated docking), as well as in vitro mutagenesis and kinetic transport experiments. Three-dimensional homology models were generated for hOCT1 and 2 using Piriformospora indica phosphate transporter (PiPT) serving as template. A putative binding pocket was identified and used to dock the prototypical substrate MPP+. Docking studies revealed five residues for each transporter (hOCT1 and hOCT2) that may be critical for substrate-transporter interactions. The in silico data was used to guide subsequent in vitro site-directed mutagenesis and kinetic analysis. Four hOCT1 mutants (Gln241Lys, Thr245Lys, Tyr361Ala, and Glu447Lys) and three hOCT2 mutants (Gln242Lys, Tyr362Phe, and Tyr362Ala) showed complete loss of MPP+ transporter activity. Decreased affinity for MPP+ was observed for Phe244Ser and Thr245Ser in hOCT1, and Tyr245Ala in hOCT2. All amino acid residues highlighted in the in vitro experiments may be potentially critical for substrate-transporter interactions particularly Tyr361, Phe244 and Thr245 in hOCT1; and Tyr362 and Tyr245 in hOCT2. Docking of known structurally divergent hOCT1 and hOCT2 substrates revealed similar binding interactions as that identified for MPP+, albeit with some unique residues, suggesting the presence of a large central cavity within both transporters. Through the combination of in silico and in vitro experiments, a putative binding pocket was defined and several residues important for substrate-transporter interaction were identified and verified for hOCT1 and hOCT2. Further defining how OCTs biochemically interact with their broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCT1 and OCT2.