In vitro screening model for compound interactions with human and dairy animal BCRP orthologs.

Orthologs of breast cancer resistance protein (BCRP/ABCG2), an ATP-binding cassette (ABC) efflux transmembrane transporter, are present in several species. The list of compounds known to interact with BCRP is growing, and many questions remain concerning species-specific variations in substrate specificity and affinity and the potency of inhibitors. As the most abundant efflux transporter known to be present in the blood-milk barrier, BCRP can increase the elimination of certain xenobiotics to milk, posing a risk for suckling offspring and dairy product consumers. Here we developed a model that can be employed to investigate species-specific differences between BCRP substrates and inhibitors. Membrane vesicles were isolated from transiently transduced human embryonic kidney (HEK) 293 cells, overexpressing BCRP, with human, bovine, caprine, and ovine cDNA sequences. To confirm BCRP transport activity in the transduced cells, D-luciferin efflux was measured and to confirm transport activity in the membrane vesicles, [3H] estrone-3-sulfate ([3H]E1S) influx was measured. We also determined the Michaelis-Menten constant (Km) and Vmax of [3H]E1S for each species. We have developed an in vitro transport model to study differences in compound interactions with BCRP orthologs from milk-producing animal species and humans. BCRP transport activity was demonstrated in the species-specific transduced cells by a reduced accumulation of D-luciferin compared with the control cells, indicating BCRP-mediated efflux of D-luciferin. Functionality of the membrane vesicle model was demonstrated by confirming ATP-dependent transport and by quantifying the kinetic parameters, Km and Vmax for the model substrate [3H]E1S. The values were not significantly different between species for the model substrates tested. This model can be insightful for appropriate inter-species extrapolations and risk assessments of xenobiotics in lactating woman and dairy animals.

Effects of allopurinol and febuxostat on uric acid transport and transporter expression in human umbilical vein endothelial cells.

Uric acid induces radical oxygen species formation, endothelial inflammation, and endothelial dysfunction which contributes to the progression of atherosclerosis. Febuxostat inhibits BCRP- and allopurinol stimulates MRP4-mediated uric acid efflux in human embryonic kidney cells. We hypothesized that endothelial cells express uric acid transporters that regulate intracellular uric acid concentration and that modulation of these transporters by febuxostat and allopurinol contributes to their different impact on cardiovascular mortality. The aim of this study was to explore a potential difference between the effect of febuxostat and allopurinol on uric acid uptake by human umbilical vein endothelial cells. Febuxostat increased intracellular uric acid concentrations compared with control. In contrast, allopurinol did not affect intracellular uric acid concentration. In line with this observation, febuxostat increased mRNA expression of GLUT9 and reduced MRP4 expression, while allopurinol did not affect mRNA expression of these uric acid transporters. These findings provide a possible pathophysiological pathway which could explain the higher cardiovascular mortality for febuxostat compared to allopurinol but should be explored further.

Exome sequencing of ATP1A3-negative cases of alternating hemiplegia of childhood reveals SCN2A as a novel causative gene.

Alternating hemiplegia of childhood (AHC) is a rare neurodevelopment disorder that is typically characterized by debilitating episodic attacks of hemiplegia, seizures, and intellectual disability. Over 85% of individuals with AHC have a de novo missense variant in ATP1A3 encoding the catalytic α3 subunit of neuronal Na+/K+ ATPases. The remainder of the patients are genetically unexplained. Here, we used next-generation sequencing to search for the genetic cause of 26 ATP1A3-negative index patients with a clinical presentation of AHC or an AHC-like phenotype. Three patients had affected siblings. Using targeted sequencing of exonic, intronic, and flanking regions of ATP1A3 in 22 of the 26 index patients, we found no ultra-rare variants. Using exome sequencing, we identified the likely genetic diagnosis in 9 probands (35%) in five genes, including RHOBTB2 (n = 3), ATP1A2 (n = 3), ANK3 (n = 1), SCN2A (n = 1), and CHD2 (n = 1). In follow-up investigations, two additional ATP1A3-negative individuals were found to have rare missense SCN2A variants, including one de novo likely pathogenic variant and one likely pathogenic variant for which inheritance could not be determined. Functional evaluation of the variants identified in SCN2A and ATP1A2 supports the pathogenicity of the identified variants. Our data show that genetic variants in various neurodevelopmental genes, including SCN2A, lead to AHC or AHC-like presentation. Still, the majority of ATP1A3-negative AHC or AHC-like patients remain unexplained, suggesting that other mutational mechanisms may account for the phenotype or that cases may be explained by oligo- or polygenic risk factors.

Transporter-Mediated Cellular Distribution of Tyrosine Kinase Inhibitors as a Potential Resistance Mechanism in Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a hematologic neoplasm characterized by the expression of the BCR::ABL1 oncoprotein, a constitutively active tyrosine kinase, resulting in uncontrolled growth and proliferation of cells in the myeloid lineage. Targeted therapy using tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, dasatinib, bosutinib, ponatinib and asciminib has drastically improved the life expectancy of CML patients. However, treatment resistance occurs in 10-20% of CML patients, which is a multifactorial problem that is only partially clarified by the presence of TKI inactivating BCR::ABL1 mutations. It may also be a consequence of a reduction in cytosolic TKI concentrations in the target cells due to transporter-mediated cellular distribution. This review focuses on drug-transporting proteins in stem cells and progenitor cells involved in the distribution of TKIs approved for the treatment of CML. Special attention will be given to ATP-binding cassette transporters expressed in lysosomes, which may facilitate the extracytosolic sequestration of these compounds.

Perfluoroalkyl substances (PFASs) are substrates of the renal human organic anion transporter 4 (OAT4).

Poly- and perfluoroalkyl substances (PFASs) are omnipresent in the environment and have been shown to accumulate in humans. Most PFASs are not biotransformed in animals and humans, so that elimination is largely dependent on non-metabolic clearance via bile and urine. Accumulation of certain PFASs in humans may relate to their reabsorption from the pre-urine by transporter proteins in the proximal tubules of the kidney, such as URAT1 and OAT4. The present study assessed the in vitro transport of 7 PFASs (PFHpA, PFOA, PFNA, PFDA, PFBS, PFHxS and PFOS) applying URAT1- or OAT4-transfected human embryonic kidney (HEK) cells. Virtually no transport of PFASs could be measured in URAT1-transfected HEK cells. All PFASs, except PFBS, showed clear uptake in OAT4-transfected HEK cells. In addition, these in vitro results were further supported by in silico docking and molecular dynamic simulation studies assessing transporter-ligand interactions. Information on OAT4-mediated transport may provide insight into the accumulation potential of PFASs in humans, but other kinetic aspects may play a role and should also be taken into account. Quantitative information on all relevant kinetic processes should be integrated in physiologically based kinetic (PBK) models, to predict congener-specific accumulation of PFASs in humans in a more accurate manner.

SLC7A8 coding for LAT2 is associated with early disease progression in osteosarcoma and transports doxorubicin.

Background: Despite (neo) adjuvant chemotherapy with cisplatin, doxorubicin and methotrexate, some patients with primary osteosarcoma progress during first-line systemic treatment and have a poor prognosis. In this study, we investigated whether patients with early disease progression (EDP), are characterized by a distinctive pharmacogenetic profile. Methods and Findings: Germline DNA from 287 Dutch high-grade osteosarcoma patients was genotyped using the DMET Plus array (containing 1,936 genetic markers in 231 drug metabolism and transporter genes). Associations between genetic variants and EDP were assessed using logistic regression models and associated variants (p <0.05) were validated in independent cohorts of 146 (Spain and United Kingdom) and 28 patients (Australia). In the association analyses, EDP was significantly associated with an SLC7A8 locus and was independently validated (meta-analysis validation cohorts: OR 0.19 [0.06-0.55], p = 0.002). The functional relevance of the top hits was explored by immunohistochemistry staining and an in vitro transport models. SLC7A8 encodes for the L-type amino acid transporter 2 (LAT2). Transport assays in HEK293 cells overexpressing LAT2 showed that doxorubicin, but not cisplatin and methotrexate, is a substrate for LAT2 (p < 0.0001). Finally, SLC7A8 mRNA expression analysis and LAT2 immunohistochemistry of osteosarcoma tissue showed that the lack of LAT2 expression is a prognostic factor of poor prognosis and reduced overall survival in patients without metastases (p = 0.0099 and p = 0.14, resp.). Conclusion: This study identified a novel locus in SLC7A8 to be associated with EDP in osteosarcoma. Functional studies indicate LAT2-mediates uptake of doxorubicin, which could give new opportunities to personalize treatment of osteosarcoma patients.

The Effect of Single Nucleotide Variations in the Transmembrane Domain of OATP1B1 on in vitro Functionality.

PURPOSE: Organic Anion Transporting Polypeptide 1B1 (OATP1B1) mediates hepatic influx and clearance of many drugs, including statins. The SLCO1B1 gene is highly polymorphic and its function-impairing variants can predispose patients to adverse effects. The effects of rare genetic variants of SLCO1B1 are mainly unexplored. We examined the impact of eight naturally occurring rare variants and the well-known SLCO1B1 c.521C > T (V174A) variant on in vitro transport activity, cellular localization and abundance. METHODS: Transport of rosuvastatin and 2,7-dichlorofluorescein (DCF) in OATP1B1 expressing HEK293 cells was measured to assess changes in activity of the variants. Immunofluorescence and confocal microscopy determined the cellular localization of OATP1B1 and LC-MS/MS based quantitative targeted absolute proteomics analysis quantified the amount of OATP1B1 in crude membrane fractions. RESULTS: All studied variants, with the exception of P336R, reduced protein abundance to varying degree. V174A reduced protein abundance the most, over 90% compared to wild type. Transport function was lost in G76E, V174A, L193R and R580Q variants. R181C decreased activity significantly, while T345M and L543W retained most of wild type OATP1B1 activity. P336R showed increased activity and H575L decreased the transport of DCF significantly, but not of rosuvastatin. Decreased activity was interrelated with lower absolute protein abundance in the studied variants. CONCLUSIONS: Transmembrane helices 2, 4 and 11 appear to be crucial for proper membrane localization and function of OATP1B1. Four of the studied variants were identified as loss-of-function variants and as such could make the individual harboring these variants susceptible to altered pharmacokinetics and adverse effects of substrate drugs.

Food Additives as Inhibitors of Intestinal Drug Transporter OATP2B1.

Food additives are compounds that are added to food and beverage to improve the taste, color, preservation, or composition. Generally, food additives are considered safe for human use due to safety evaluations conducted by food safety authorities and high safety margins applied to permitted usage levels. However, the interaction potential of food additives with simultaneously administered medication has not received much attention. Even though many food additives are poorly absorbed into systemic circulation, high concentrations could exist in the intestinal lumen, making intestinal drug transporters, such as the uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1), a possible site of food additive-drug interactions. In the present work, we aimed to characterize the interaction of a selection of 25 food additives including colorants, preservatives, and sweeteners with OATP2B1 in vitro. In human embryonic kidney 293 (HEK293) cells transiently overexpressing OATP2B1 or control, uptake of dibromofluorescein was studied with and without 50 µM food additive at pH 7.4. As OATP2B1 displays substrate- and pH-dependent transport functions and the intraluminal pH varies along the gastrointestinal tract, we performed the studies also at pH 5.5 using estrone sulfate as an OATP2B1 substrate. Food additives that inhibited OATP2B1-mediated substrate transport by ≥50% were subjected to dose-response studies. Six colorants were identified and validated as OATP2B1 inhibitors at pH 5.5, but only three of these were categorized as inhibitors at pH 7.4. One sweetener was validated as an inhibitor under both assay conditions, whereas none of the preservatives exhibited ≥50% inhibition of OATP2B1-mediated transport. Extrapolation of computed inhibitory constants (Ki values) to estimations of intestinal food additive concentrations implies that selected colorants could inhibit intestinal OATP2B1 also in vivo. These results suggest that food additives, especially colorants, could alter the pharmacokinetics of orally administered OATP2B1 substrate drugs, although further in vivo studies are warranted to understand the overall clinical consequences of the findings.

Rifampicin Transport by OATP1B1 Variants.

Single nucleotide polymorphisms in the OATP1B1 transporter have been suggested to partially explain the large interindividual variation in rifampicin exposure. HEK293 cells overexpressing wild-type (WT) or OATP1B1 variants *1b, *4, *5, and *15 were used to determine the in vitro rifampicin intrinsic clearance. For OATP1B1*5 and *15, a 36% and 42% reduction in intrinsic clearance, respectively, compared to WT was found. We consider that these differences in intrinsic clearance most likely have minor clinical implications.

Human multidrug resistance protein 4 (MRP4) is a cellular efflux transporter for paracetamol glutathione and cysteine conjugates.

Paracetamol (acetaminophen, APAP) overdose is a leading cause of acute drug-induced liver failure. APAP hepatotoxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI is inactivated by conjugation with glutathione (GSH) to APAP-GSH, which is further converted into its cysteine derivative APAP-CYS. Before necrosis of hepatocytes occurs, APAP-CYS is measurable in plasma of the affected patient and it has been proposed as an early biomarker of acetaminophen toxicity. APAP-GSH and APAP-CYS can be extruded by hepatocytes, but the transporters involved are unknown. In this study we examined whether ATP-binding cassette (ABC) transporters play a role in the cellular efflux of APAP, APAP-GSH, and APAP-CYS. The ABC transport proteins P-gp/ABCB1, BSEP/ABCB11, BCRP/ABCG2, and MRP/ABCC1-5 were overexpressed in HEK293 cells and membrane vesicles were produced. Whereas P-gp, BSEP, MRP3, MRP5, and BCRP did not transport any of the compounds, uptake of APAP-GSH was found for MRP1, MRP2 and MRP4. APAP-CYS appeared to be a substrate of MRP4 and none of the ABC proteins transported APAP. The results suggest that the NAPQI metabolite APAP-CYS can be excreted into plasma by MRP4, where it could be a useful biomarker for APAP exposure and toxicity. Characterization of the cellular efflux of APAP-CYS is important for its development as a biomarker, because plasma concentrations might be influenced by drug-transporter interactions and upregulation of MRP4.

Efflux transport of nicotine, cotinine and trans-3'-hydroxycotinine glucuronides by human hepatic transporters.

Nicotine is the addiction causing alkaloid in tobacco, and it is used in smoking cessation therapies. Although the metabolic pathways of nicotine are well known and mainly occur in the liver, the transport of nicotine and its metabolites is poorly characterized. The highly hydrophilic nature and urinary excretion of nicotine glucuronide metabolites indicate that hepatic basolateral efflux transporters mediate their excretion. We aimed here to find the transporters responsible for the hepatic excretion of nicotine, cotinine and trans-3'-hydroxycotinine (OH-cotinine) glucuronides. To this end, we tested their transport by multidrug resistance-associated proteins 1 (MRP1, ABCC1) and MRP3-6 (ABCC3-6), which are located on the basolateral membranes of hepatocytes, as well as MRP2 (ABCC2), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MDR1, P-gp, ABCB1) that are expressed in the apical membranes of these cells. ATP-dependent transport of these glucuronides was evaluated in inside-out membrane vesicles expressing the transporter of interest. In addition, potential interactions of both the glucuronides and parent compounds with selected transporters were tested by inhibition assays. Considerable ATP-dependent transport was observed only for OH-cotinine glucuronide by MRP3. The kinetics of this transport activity was characterized, resulting in an estimated Km value of 895 µmol/L. No significant transport was found for nicotine or cotinine glucuronides by any of the tested transporters at either 5 or 50 µmol/L substrate concentration. Furthermore, neither nicotine, cotinine nor OH-cotinine inhibited MRP2-4, BCRP or MDR1. In this study, we directly examined, for the first time, efflux transport of the three hydrophilic nicotine glucuronide metabolites by the major human hepatic efflux transporters. Despite multiple transporters studied here, our results indicate that an unknown transporter may be responsible for the hepatic excretion of nicotine and cotinine glucuronides.

Uremic solutes modulate hepatic bile acid handling and induce mitochondrial toxicity.

Chronic kidney disease (CKD) is accompanied by accumulating levels of uremic solutes in the circulation. Changes in the size and composition of the bile acid pool have also been observed. We investigated via which mechanisms uremic solutes may interfere with hepatocyte function and thus contribute to altered bile acid handling. We studied interference on the level of bile acid synthesis by cytochrome P450 7A1 (CYP7A1), explored effects on hepatic bile acid transporters, and investigated effects on mitochondrial function. In HEK293 cells overexpressing bile salt transporters, we observed that p-cresyl sulfate inhibited Na+-taurocholate cotransporting polypeptide (NTCP)-mediated uptake of taurocholic acid (TCA), whereas organic anion-transporting polypeptide 1B1 (OATP1B1)-mediated TCA uptake was increased. Assays in transporter-overexpressing membrane vesicles revealed that kynurenic acid inhibited TCA transport via the bile salt efflux pump (BSEP), whereas p-cresyl glucuronide and hippuric acid increased TCA efflux via multidrug resistance-associated protein 3 (MRP3). Moreover, indoxyl sulfate decreased mRNA expression of NTCP, OATP1B3 and CYP7A1 in primary human hepatocytes. Transport studies confirmed a decreased TCA uptake in indoxyl sulfate-exposed hepatocytes. Decreased hepatocyte viability was found for all seven uremic solutes tested, whereas five out of seven also decreased intracellular ATP levels and mitochondrial membrane potential. In conclusion, uremic solutes affect hepatic bile acid transport and mitochondrial function. This can contribute to the altered bile acid homeostasis observed in CKD patients.

Transmembrane Domain Single-Nucleotide Polymorphisms Impair Expression and Transport Activity of ABC Transporter ABCG2.

PURPOSE: To study the function and expression of nine naturally occurring single-nucleotide polymorphisms (G406R, F431L, S441N, P480L, F489L, M515R, L525R, A528T and T542A) that are predicted to reside in the transmembrane regions of the ABC transporter ABCG2. METHODS: The transport activity of the variants was tested in inside-out membrane vesicles from Sf9 insect and human derived HEK293 cells overexpressing ABCG2. Lucifer Yellow and estrone sulfate were used as probe substrates of activity. The expression levels and cellular localization of the variants was compared to the wild-type ABCG2 by western blotting and immunofluorescence microscopy. RESULTS: All studied variants of ABCG2 displayed markedly decreased transport in both Sf9-ABCG2 and HEK293-ABCG2 vesicles. Impaired transport could be explained for some variants by altered expression levels and cellular localization. Moreover, the destructive effect on transport activity of variants G406R, P480L, M515R and T542A is, to our knowledge, reported for the first time. CONCLUSIONS: These results indicate that the transmembrane region of ABCG2 is sensitive to amino acid substitution and that patients harboring these ABCG2 variant forms could suffer from unexpected pharmacokinetic events of ABCG2 substrate drugs or have an increased risk for diseases such as gout where ABCG2 is implicated.

Digitalis-like Compounds Facilitate Non-Medullary Thyroid Cancer Redifferentiation through Intracellular Ca2+, FOS, and Autophagy-Dependent Pathways.

Up to 20%-30% of patients with metastatic non-medullary thyroid cancer have persistent or recurrent disease resulting from tumor dedifferentiation. Tumor redifferentiation to restore sensitivity to radioactive iodide (RAI) therapy is considered a promising strategy to overcome RAI resistance. Autophagy has emerged as an important mechanism in cancer dedifferentiation. Here, we demonstrate the therapeutic potential of autophagy activators for redifferentiation of thyroid cancer cell lines. Five autophagy-activating compounds, all known as digitalis-like compounds, restored hNIS expression and iodide uptake in thyroid cancer cell lines. Upregulation of hNIS was mediated by intracellular Ca2+ and FOS activation. Cell proliferation was inhibited by downregulating AKT1 and by induction of autophagy and p21-dependent cell-cycle arrest. Digitalis-like compounds, also designated as cardiac glycosides for their well-characterized beneficial effects in the treatment of heart disease, could therefore represent a promising repositioned treatment modality for patients with RAI-refractory thyroid carcinoma. Mol Cancer Ther; 16(1); 169-81. ©2016 AACR.

Inhibitory Potential of Antifungal Drugs on ATP-Binding Cassette Transporters P-Glycoprotein, MRP1 to MRP5, BCRP, and BSEP.

Inhibition of ABC transporters is a common mechanism underlying drug-drug interactions (DDIs). We determined the inhibitory potential of antifungal drugs currently used for invasive fungal infections on ABC transporters P-glycoprotein (P-gp), MRP1 to MRP5, BCRP, and BSEP in vitro Membrane vesicles isolated from transporter-overexpressing HEK 293 cells were used to investigate the inhibitory potential of antifungal drugs (250 µM) on transport of model substrates. Concentration-inhibition curves were determined if transport inhibition was >60%. Fifty percent inhibitory concentrations (IC50s) for P-gp and BCRP were both 2 µM for itraconazole, 5 and 12 µM for hydroxyitraconazole, 3 and 6 µM for posaconazole, and 3 and 11 µM for isavuconazole, respectively. BSEP was strongly inhibited by itraconazole and hydroxyitraconazole (3 and 17 µM, respectively). Fluconazole and voriconazole did not inhibit any transport for >60%. Micafungin uniquely inhibited all transporters, with strong inhibition of MRP4 (4 µM). Anidulafungin and caspofungin showed strong inhibition of BCRP (7 and 6 µM, respectively). Amphotericin B only weakly inhibited BCRP-mediated transport (127 µM). Despite their wide range of DDIs, azole antifungals exhibit selective inhibition on efflux transporters. Although echinocandins display low potential for clinically relevant DDIs, they demonstrate potent in vitro inhibitory activity. This suggests that inhibition of ABC transporters plays a crucial role in the inexplicable (non-cytochrome P450-mediated) DDIs with antifungal drugs.

Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System.

P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed proteins can be isolated. Insect cells are a perfect system to study P-type ATPases as they have little or no endogenous Na,K-ATPase activity and other ATPase activities can be inhibited easily. Here we describe in detail the expression and isolation of Na,K-ATPase and H,K-ATPase isoforms with the baculovirus expression system.

ATPase Activity Measurements Using Radiolabeled ATP.

ATP provides the energy that is essential for all P-type ATPases to actively transport their substrates against an existing gradient. This ATP hydrolysis can be measured using different methods. Here, we describe a method that uses radiolabeled [γ-(32)P]ATP, which is hydrolyzed by P-type ATPases to ADP and (32)Pi. Activated charcoal is used to bind the excess of [γ-(32)P]ATP, which can be separated from the unbound (32)Pi by centrifugation. With this method, a wide range (0.1 µM-10 mM) of ATP can be used. In addition, we also describe in detail how ATP hydrolysis is translated into ATPase activity.

Inhibitory potential of tuberculosis drugs on ATP-binding cassette drug transporters.

BACKGROUND: Multiple-drug therapy for tuberculosis (TB) and TB-associated co-morbidity increase the likelihood of drug-drug interactions (DDIs). Inhibition of membrane transporters is an important mechanism underlying DDIs. In this study, we assessed the in vitro inhibitory potential of currently used first and second-line TB drugs and of proposed mycobacterial efflux pump inhibitors (EPIs) on the major ABC transporters relevant to drug transport, namely P-gp, BCRP, BSEP and MRP1-5. METHODS: Membrane vesicles isolated from transporter-overexpressing HEK293 cells were used to study the inhibitory action of TB drugs and EPIs on the transport of model substrates [(3)H]-NMQ (P-gp); [(3)H]-E1S (BCRP); [(3)H]-TCA (BSEP); [(3)H]-E217ßG (MRP1, 3 and 4) and [(3)H]-MTX (MRP2 and 5). RESULTS: A strong inhibition (IC50 value <15 µM) was observed for clofazimine (P-gp, BCRP and MRP1), thioridazine (BCRP), timcodar (P-gp, BSEP and MRP1) and SQ109 (P-gp and BCRP). Rifampicin inhibited all transporters, but less potently. CONCLUSIONS: Co-administration of clofazimine, thioridazine, timcodar, SQ109 and possibly rifampicin with drugs that are substrates for the inhibited transporters may lead to DDIs. The mycobacterial EPIs potently inhibited a wider range of human ABC transporters than previously reported. These vesicular transport data are especially valuable considering the current emphasis on development of TB drug regimens.

PfMDR2 and PfMDR5 are dispensable for Plasmodium falciparum asexual parasite multiplication but change in vitro susceptibility to anti-malarial drugs.

BACKGROUND: Membrane-associated ATP binding cassette (ABC) transport proteins hydrolyze ATP in order to translocate a broad spectrum of substrates, from single ions to macromolecules across membranes. In humans, members from this transport family have been linked to drug resistance phenotypes, e.g., tumour resistance by enhanced export of chemotherapeutic agents from cancer cells due to gene amplifications or polymorphisms in multidrug resistance (MDR) protein 1. Similar mechanisms have linked the Plasmodium falciparum PfMDR1 transporter to anti-malarial drug resistance acquisition. In this study, the possible involvement of two related MDR proteins, PfMDR2 and PfMDR5, to emerging drug resistance is investigated by a reverse genetics approach. METHODS: A homologous double crossover strategy was used to generate P. falciparum parasites lacking the Pfmdr2 (PfΔmdr2) or Pfmdr5 (PfΔmdr5) gene. Plasmodium lactate dehydrogenase activity was used as read-out for sensitivity to artemisinin (ART), atovaquone (ATO), dihydroartemisinin (DHA), chloroquine (CQ), lumefantrine (LUM), mefloquine (MQ), and quinine (QN). Differences in half maximal inhibitory concentration (IC50) values between wild type and each mutant line were determined using a paired t-test. RESULTS: Both PfΔmdr2 and PfΔmdr5 clones were capable of asexual multiplication. Upon drug exposure, PfΔmdr2 showed a marginally decreased sensitivity to ATO (IC50 of 1.2 nM to 1.8 nM), MQ (124 nM to 185 nM) and QN (40 nM to 70 nM), as compared to wild type (NF54) parasites. On the other hand, PfΔmdr5 showed slightly increased sensitivity to ART (IC50 of 26 nM to 19 nM). CONCLUSION: Both Pfmdr2 and Pfmdr5 are dispensable for blood stage development while the deletion lines show altered sensitivity profiles to commonly used anti-malarial drugs. The findings show for the first time that next to PfMDR2, the PfMDR5 transport protein could play a role in emerging drug resistance.