Flóra Szeri

The role of multidrug transporters in drug availability, metabolism and toxicity

Multidrug resistance is frequently observed when treating cancer patients with chemotherapeutic agents. A variety of ATP binding cassette (ABC) transporters, localized in the cell membrane, cause this phenomenon by extruding a variety of chemotherapeutic agents from the tumor cells. However, the major physiological role of the multidrug transporters is the protection of our cells and tissues against xenobiotics, and these transporters play a key role in drug availability, metabolism and toxicity. Three major groups of ABC transporters are involved in multidrug resistance: the classical P-glycoprotein MDR1, the multidrug resistance associated proteins (MRP1, MRP2, and probably MRP3, MRP4 and MRP5), and the ABCG2 protein, an ABC half-transporter. All these proteins were shown to catalyze an ATP-dependent active transport of chemically unrelated compounds. MDR1 (P-glycoprotein) and ABCG2 preferentially extrude large hydrophobic, positively charged molecules, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water-soluble anionic compounds. By examining the interactions of the multidrug transporters with pharmacological and toxic agents, a prediction for the cellular and tissue distribution of these compounds can be achieved. Oral bioavailability, entering the blood-brain and blood-CSF barrier, reaching the fetus through the placenta, liver and kidney secretion, cellular entry for affecting intracellular targets, are all questions, which can be addressed by basic in vitro studies on the multidrug resistance proteins. Investigation of the substrate interactions and modulation of multidrug transporters may pave the way for predictive toxicology and pharmacogenomics. Here we show that by using in vitro assay systems it is possible to measure the interactions of multidrug transporters with various drugs and toxic agents. We focus on the characterisation of the MRP1 and MRP3 proteins, their relevance in chemoresistance of cancer and in drug metabolism and toxicity.

Differential modulation of the human liver conjugate transporters MRP2 and MRP3 by bile acids and organic anions

The multidrug resistance proteins MRP2 (ABCC2) and MRP3 (ABCC3) are key primary active transporters involved in anionic conjugate and drug extrusion from the human liver. The major physiological role of MRP2 is to transport conjugated metabolites into the bile canaliculus, whereas MRP3 is localized in the basolateral membrane of the hepatocytes and transports similar metabolites back to the bloodstream. Both proteins were shown to interact with a large variety of transported substrates, and earlier studies suggested that MRPs may work as co-transporters for different molecules. In the present study we expressed the human MRP2 and MRP3 proteins in insect cells and examined their transport and ATPase characteristics in isolated, inside-out membrane vesicles. We found that the primary active transport of estradiol-17-β-d-glucuronide (E217βG), a major product of human steroid metabolism, was differently modulated by bile acids and organic anions in the case of human MRP2 and MRP3. Active E217βG transport by MRP2 was significantly stimulated by the organic anions indomethacin, furosemide, and probenecid and by several conjugated bile acids. In contrast, all of these agents inhibited E217βG transport by MRP3. We found that in the case of MRP2, ATP-dependent vesicular bile acid transport was increased by E217βG, and the results indicated an allosteric cross-stimulation, probably a co-transport of bile acids and glucuronate conjugates through this protein. There was no such stimulation of bile acid transport by MRP3. In conclusion, the different transport modulation of MRPs by bile acids and anionic drugs could play a major role in regulating physiological and pathological metabolite fluxes in the human liver.

ABCC6–Mediated ATP Secretion by the Liver Is the Main Source of the Mineralization Inhibitor Inorganic Pyrophosphate in the Systemic Circulation—Brief Report

Objective— Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PPi). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PPi, and whether human PXE ptients have low plasma PPi concentrations. Approach and Results— Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PPi. The absence of functional ABCC6 in patients with PXE leads to strongly reduced plasma PPi concentrations. Conclusions— Hepatic ABCC6-mediated ATP release is the main source of circulating PPi, revealing an unanticipated role of the liver in systemic PPi homeostasis. Patients with PXE have a strongly reduced plasma PPi level, explaining their mineralization disorder. Our results indicate that systemic PPi is relatively stable and that PXE, generalized arterial calcification of infancy, and other ectopic mineralization disorders could be treated with PPi supplementation therapy.

ABCC6 is a basolateral plasma membrane protein

Rationale: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum and general arterial calcification of infancy. To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein. Objective: In a recent article in Circulation Research, ABCC6 was reported to localize to the mitochondria-associated membrane and not the plasma membrane. As the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment. Methods and Results: We performed immunofluorescent labeling of frozen mouse and human liver sections, as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and is not associated with the mitochondria, mitochondria-associated membrane, or the endoplasmic reticulum. Conclusions: Our findings support the model that ABCC6 is in the basolateral membrane, mediating the sinusoidal efflux of a metabolite from the hepatocytes to systemic circulation.

The dMRP/CG6214 gene of Drosophila is evolutionarily and functionally related to the human multidrug resistance-associated protein family

ATP‐binding cassette (ABC) transporters are involved in the transport of substrates across biological membranes and are essential for many cellular processes. Of the fifty‐six Drosophila ABC transporter genes only white, brown, scarlet, E23 and Atet have been studied in detail. Phylogenetic analyses identify the Drosophila gene dMRP/CG6214 as an orthologue to the human multidrug‐resistance associated proteins MRP1, MRP2, MRP3 and MRP6. To study evolutionarily conserved roles of MRPs we have initiated a characterization of dMRP. In situ hybridization and Northern analysis indicate that dMRP is expressed throughout development and appears to be head enriched in adults. Functional studies indicate that DMRP is capable of transporting a known MRP1 substrate and establishes DMRP as a high capacity ATP‐dependent, vanadate‐sensitive organic anion transporter.

Oral administration of pyrophosphate inhibits connective tissue calcification

Various disorders including pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI), which are caused by inactivating mutations in ABCC6 and ENPP1, respectively, present with extensive tissue calcification due to reduced plasma pyrophosphate (PPi). However, it has always been assumed that the bioavailability of orally administered PPi is negligible. Here, we demonstrate increased PPi concentration in the circulation of humans after oral PPi administration. Furthermore, in mouse models of PXE and GACI, oral PPi provided via drinking water attenuated their ectopic calcification phenotype. Noticeably, provision of drinking water with 0.3 mM PPi to mice heterozygous for inactivating mutations in Enpp1 during pregnancy robustly inhibited ectopic calcification in their Enpp1−/− offspring. Our work shows that orally administered PPi is readily absorbed in humans and mice and inhibits connective tissue calcification in mouse models of PXE and GACI. PPi, which is recognized as safe by the FDA, therefore not only has great potential as an effective and extremely low‐cost treatment for these currently intractable genetic disorders, but also in other conditions involving connective tissue calcification.

Inhibition of DNA methyltransferase leads to increased genomic 5-hydroxymethylcytosine levels in hematopoietic cells

5‐Hydroxymethylcytosine (5hmC) is produced from 5‐methylcytosine (5mC) by Ten‐eleven translocation (TET) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC‐MS/MS‐based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5‐aza‐2′‐deoxycytidine without significantly influencing 5mC levels.

In vitro transport of methotrexate by Drosophila Multidrug Resistance-associated Protein

Methotrexate (MTX) is a widely used chemotherapeutic agent, immune suppressant and antimalarial drug. It is a substrate of several human ABC proteins that confer multidrug resistance to cancer cells and determine compartmentalization of a wide range of physiological metabolites and endo or xenobiotics, by their primary active transport across biological membranes. The substrate specificity and tissue distribution of these promiscuous human ABC transporters show a high degree of redundancy, providing robustness to these key physiological and pharmacological processes, such as the elimination of toxins, e.g. methotrexate from the body. A similar network of proteins capable of transporting methotrexate has been recently suggested to exist in Drosophila melanogaster. One of the key players of this putative network is Drosophila Multidrug-resistance Associated Protein (DMRP). DMRP has been shown to be a highly active and promiscuous ABC transporter, capable of transporting various organic anions. Here we provide the first direct evidence that DMRP, expressed alone in a heterologous system lacking other, potentially functionally overlapping D. melanogaster organic anion transporters, is indeed able to transport methotrexate. Our in vitro results support the hypothesized but debated role of DMRP in in vivo methotrexate excretion.

Boosted coupling of ATP hydrolysis to substrate transport upon cooperative estradiol-17-β-D-glucuronide binding in a Drosophila ATP binding cassette type-C transporter

ATP binding cassette type‐C (ABCC) transporters move molecules across cell membranes upon hydrolysis of ATP; however, their coupling of ATP hydrolysis to substrate transport remains elusive. Drosophila multidrug resistance‐associated protein (DMRP) is the functional ortholog of human long ABCC transporters, with similar substrate and inhibitor specificity, but higher activity. Exploiting its high activity, we kinetically dissected the catalytic mechanism of DMRP by using E2‐D‐glucuronide (E2G), the physiologic substrate of human ABCC. We examined the DMRP‐mediated interdependence of ATP and E2G in biochemical assays. We observed E2G‐dependent ATPase activity to be biphasic at subsaturating ATP concentrations, which implies at least 2 E2G binding sites on DMRP. Furthermore, transport measurements indicated strong nonreciprocal cooperativity between ATP and E2G. In addition to confirming these findings, our kinetic modeling with the Complex Pathway Simulator indicated a 10‐fold decrease in the E2G‐mediated activation of ATP hydrolysis upon saturation of the second E2G binding site. Surprisingly, the binding of the second E2G allowed for substrate transport with a constant rate, which tightly coupled ATP hydrolysis to transport. In summary, we show that the second E2G binding—similar to human ABCC2—allosterically stimulates transport activity of DMRP. Our data suggest that this is achieved by a significant increase in the coupling of ATP hydrolysis to transport.—Karasik, A., Ledwitch, K. V., Arányi, T., Varadi, A., Roberts, A., Szeri, F. Boosted coupling of ATP hydrolysis to substrate transport upon cooperative estradiol‐17‐β‐D‐glucuronide binding in a Drosophila ATP binding cassette type‐C transporter. FASEB J. 32, 669‐680 (2018). www.fasebj.org