The model of the fruit fly Drosophila melanogaster as a novel tool for characterization of human membrane transporters

Abstract

Numerous studies have demonstrated that membrane transporters are critical determinants for drug disposition, drug absorption, therapeutic efficacy, and adverse drug reactions. In the human genome, more than 400 membrane transporters have been annotated and been divided into two superfamilies: ATPbinding cassette (ABC) family and solute carrier (SLC) family. Many ABC and SLC transporters not only contribute to the influx of metabolites and drugs but are also involved in their efflux. In vitro and in vivo, various animal models including mice, rats, rabbits, monkeys, and different cell lines were used to study the relationship between clinical pharmacokinetic drug-drug interaction (DDI) and human transporters. However, these studies are usually expensive and timeconsuming. Therefore, we planned to use the fruit fly Drosophila melanogaster as a rapid testing and low-cost animal model to study the function of human membrane transporters. At first, I sought to study the function of the organic anion transporters hOATP1B1 (encoded by SLCO1B1), hOATP2B1 (SLCO2B1) and hOATP1B3 (SLCO1B3), which are clinically relevant uptake transporters in the sinusoidal membrane of human hepatocytes. For this purpose, I generated flies expressing these human uptake transporters in the salivary glands. They did not localize to the target tissue, but were instead found in excretory cells suggesting that they were eliminated because of potential cell toxic properties. Arguing that undetectable levels of hOATP1B1, hOATP2B1 and hOATP1B3 may nevertheless be localized correctly, I performed tracer transport assays by confocal microscopy. My results suggested that fluorescent substrates of hOATP1B1, hOATP2B1 and hOATP1B3 (Mercury dibromofluorescein disodium salt, MDBF; 4,5- Dibromofluorescein, DBF; Cholyl-Lysyl-Fluorescein, CLF) were transported to the lumen of the salivary glands of embryos independently of these human transporters. Further results failed to demonstrate that fly organic anion-transporting polypeptides (Oatps) may assist fluorescent substrate transfer across the cell membrane into the lumen of the salivary glands. Rather, my data suggested that dyes were transported by transcytosis to the lumen of this organ. Thus, contrary to our5 expectation, the fruit fly is not a suitable model for pharmacological studies of OATPs. In a second project, I established a test system for DDI involving hOCT1, a further clinically relevant uptake transporter in human hepatocytes. hOCT1 was expressed in various organs and tissues in embryos produced by females that were fed with the cytotoxin and hOCT1 substrate cisplatin. My results suggested that cisplatin was transported into embryos by hOCT1 and was embryonic lethal. Interestingly, cimetidine, another substrate of hOCT1, reduced the toxicity of cisplatin in embryos that expressed hOCT1 in the nervous system. Thus, this setup allows studying successfully DDI in D. melanogaster and may ultimately serve to identify and evaluate new hOCT1 interacting molecules in pharmaceutical experiments.