Direct Automated MALDI Mass Spectrometry Analysis of Cellular Transporter Function: Inhibition of OATP2B1 Uptake by 294 Drugs.

OATP2B1, a member of the solute carrier (SLC) transporter family, is an important mechanism of substrate drug uptake in the intestine and liver and therefore a determinant of clinical pharmacokinetics and site of drug-drug interactions. Other SLC transporters have emerged as pharmacology targets. Studies of SLC transporter uptake to-date relied on radioisotope- or fluorescence-labeled reagents or low-throughput quantification of unlabeled compounds in cell lysate. In this study, we developed a cell-based MALDI MS workflow for investigation of OATP2B1 cellular uptake by optimizing the substrate, matrix, matrix-analyte ratio, and matrix application and normalization method. This workflow was automated and applied to characterize substrate transport kinetics and to test 294 top-marketed drugs for OATP2B1 inhibition and quantify inhibitory potencies necessary for extrapolation of clinical drug-drug interaction potential. Intra-assay reproducibility of this MALDI MS method was high (CV < 10%), and results agreed well (83% overlap) with previously published radioisotope assay data. Our results indicate that fast and robust MALDI MS cellular assays could emerge as a high-throughput label-free alternative for direct assessment of drug transporter function in DDIs and toxicities as well as enable drug discovery for transporters as pharmacology targets.

Impact of Mistletoe Triterpene Acids on the Uptake of Mistletoe Lectin by Cultured Tumor Cells.

Complementary treatment possibilities for the therapy of cancer are increasing in demand due to the severe side effects of the standard cytostatics used in the first-line therapy. A common approach as a complementary treatment is the use of aqueous extracts of Viscum album L. (Santalaceace). The therapeutic activity of these extracts is attributed to Mistletoe lectins which are Ribosome-inactivating proteins type II. Besides these main constituents the extract of Viscum album L. comprises also a mixture of lipophilic ingredients like triterpene acids of the oleanane, lupane and ursane type. However, these constituents are not contained in commercially available aqueous extracts due to their high lipophilicity and insolubility in aqueous extraction media. To understand the impact of the extract ingredients in cancer therapy, the intracellular uptake of the mistletoe lectin I (ML) by cultured tumor cells was investigated in relation to the mistletoe triterpene acids, mainly oleanolic acid. Firstly, these hydrophobic triterpene acids were solubilized using cyclodextrins ("TT" extract). Afterwards, the uptake of either single compounds (isolated ML and the aqueous "viscum" extract) or in combination with the TT extract (ML+TT, viscumTT), was analyzed. The uptake of ML was studied inTHP-1-, HL-60-, 143B- and Ewing TC-71-cells and determined after 30, 60 and 120 minutes by an enzyme linked immunosorbent assay which quantifies the A-chain of the hololectin. It could be shown that the intracellular uptake after 120 minutes amounted to 20% in all cell lines after incubation with viscumTT. The studies further revealed that the uptake in THP-1-, HL-60- and Ewing TC-71-cells was independent of the addition of TT extract. Interestingly, the uptake of ML by 143B-cells could only be measured after addition of triterpenes pointing to resistance to mistletoe lectin.