Methotrexate Transport Research Articles

Dexamethasone reduces methotrexate biliary elimination and potentiates its hepatotoxicity in rats

Increased hepatotoxicity of methotrexate has been reported during dexamethasone therapy in humans. Despite the observed inducing effect of dexamethasone on some methotrexate transporting proteins in the liver, the kinetic aspects of this interaction have not been studied yet. Thus, the aim of the present study was to evaluate the influence of dexamethasone on the hepatic and overall pharmacokinetics of methotrexate. Pharmacokinetics of methotrexate was evaluated in rats during an in vivo steady-state clearance study after either single intravenous dose of dexamethasone or its four-day oral administration in a dose optimized for transport proteins induction. Dexamethasone oral pretreatment reduced biliary clearance of methotrexate by 53%. Although liver tissue concentration of methotrexate increased only slightly in these animals, a significant increase in liver weights produced by dexamethasone pretreatment revealed a marked increase in liver content of the drug. An evaluation of plasma liver enzyme activities measured before and after methotrexate administration demonstrated a potentiation of corticosteroid hepatotoxicity by the cytostatic. Analysis of methotrexate transporter expression in the liver showed up-regulation of Mrp2, Oatp1a4, and Oat2, and down-regulation of Mrp3. These observations comply with increased biliary excretion and reduced plasma concentrations of their endogenous substrate, conjugated bilirubin. In contrast, single intravenous bolus of dexamethasone did not influence any pharmacokinetic parameter of methotrexate. In conclusion, these results indicate that hepatocellular impairment associated with reduced biliary elimination of methotrexate, and its raised liver content may contribute to increased hepatotoxicity of the drug when co-administered with dexamethasone. Moreover, an influence of dexamethasone on protein expression of anionic drugs transporters in the liver and kidney was demonstrated.

Effects of Putative Catalytic Base Mutation E211Q on ABCG2-Mediated Methotrexate Transport

ABCG2 is a half-ATP binding cassette (ABC) drug transporter that consists of a nucleotide binding domain (NBD) followed by a transmembrane domain. This half-ABC transporter is thought to form a homodimer in the plasma membrane where it transports anticancer drugs across the biological membranes in an ATP-dependent manner. Substitution of the putative catalytic residue E211 with a nonacidic amino acid glutamine (E211Q) completely abolished its ATPase activity and ATP-dependent methotrexate transport, suggesting that ATP hydrolysis is required for the ATP-dependent solute transport. However, whether one ATP hydrolysis or two ATP hydrolyses in the homodimer of ABCG2 with the NBD.ATP.ATP.NBD sandwich structure is/are required for the ATP-dependent solute transport is not known yet. To address this question, we have made an YFP/ABCG2 fusion protein and expressed this 99 kDa fusion protein alone or along with the 70 kDa E211Q-mutated ABCG2 in BHK cells. Although membrane vesicles prepared from BHK cells expressing YFP/ABCG2 exert higher ATPase activity than that of wt ABCG2, the dATP-dependent methotrexate transport activities of these two proteins are the same. Interestingly, membrane vesicles prepared from BHK cells expressing both YFP/ABCG2 and E211Q-mutated ABCG2 (with a ratio of 1:1) form homodimers and heterodimer and exert 55% of wt ABCG2 ATPase activity that can be further enhanced by anticancer drugs, suggesting that the wt NBD in the heterodimer of YFP/ABCG2 and E211Q may be able to hydrolyze ATP. Furthermore, the membrane vesicles containing both YFP/ABCG2 and E211Q exert approximately 79% of wt ABCG2-mediated methotrexate transport activity, implying that the heterodimer harboring YFP/ABCG2 and E211Q may be able to transport the anticancer drug methotrexate across the biological membranes.

The critical role of alpha‐folate receptor in the resistance of melanoma to methotrexate

Although methotrexate (MTX) is an effective drug for several types of cancer, it is not active against melanoma. Experiments following methotrexate treatment indicated a reduced accumulation of the drug in the cytosolic compartment in melanoma cells, suggesting that the mechanisms that control the transport and retention of this drug could be altered in melanoma. For this reason, we analyzed the presence and function of folate receptor-alpha (FRalpha) in melanoma cells. In this study, we have identified the presence of FRalpha in normal and pathological melanocytes and demonstrated that MTX is preferentially transported through this receptor in melanoma cells. FRalpha-induced endocytic transport of MTX, together with drug melanosomal sequestration and cellular exportation, ensures reduced accumulation of this cytotoxic compound in intracellular compartments. The critical role of FRalpha in this mechanism of resistance and the therapeutic consequences of these findings are also discussed.

Physiological and molecular characterization of methotrexate transport by Malpighian tubules of adult Drosophila melanogaster

A radioisotope tracer technique and quantitative PCR were used to study the mechanisms and regulation of transepithelial transport of the type II organic anion methotrexate (MTX) by the Malpighian tubules of Drosophila melanogaster. Transport of MTX was saturable and Na +-independent; the kinetic parameters J max and K t were 437 fmol min −1 and 23.5 μM, respectively. The transport of MTX was competitively inhibited by phenol red and probenecid; non-competitively inhibited by salicylate, verapamil and MK-571; and uncompetitively inhibited by Texas Red. Dietary exposure to 0.1 mM MTX led to dramatic increases in gene expression for several members of the ABC family of transporters in both the Malpighian tubules and the gut. Our results suggest that multiple transporters are upregulated in response to dietary exposure to MTX. Increased levels of the protein products which may result from expression of these genes may enhance elimination of toxic compounds such as MTX or its metabolites.

Polymorphisms in Methotrexate Pathways: What Is Clinically Relevant, What Is Not, and What Is Promising

Methotrexate (MTX) is a folate inhibitor that is used successfully in treating a great variety of malignancies and autoimmune diseases. However, there are certain drawbacks to its use, as a significant percentage of patients do not respond to the therapy or develop serious adverse effects. In the last ten years there has been a growing body of evidence pointing to the clinical significance of polymorphisms in genes that are involved in the metabolism, transport, and function of folates and MTX. Indeed, many studies have shown that both the toxicity and the efficacy of this drug can be modified by the presence of these genetic alterations. However, there is also a great deal of contradictory evidence that has as yet prevented the acceptance of any definitive conclusions. This review aims to summarize current knowledge on the pharmacogenetics of MTX, dealing with the reasons for numerous reported discrepancies. The particular focus will be on studies analyzing interactions between single nucleotide polymorphisms (SNPs) along a certain pathway rather than following a single-SNP design. New approaches and future directions in the study of MTX pharmacogenetics will also be discussed.

Molecular Basis for Reduced Estrone Sulfate Transport and Altered Modulator Sensitivity of Transmembrane Helix (TM) 6 and TM17 Mutants of Multidrug Resistance Protein 1 (ABCC1)

Multidrug resistance protein 1 (MRP1) confers drug resistance and also mediates cellular efflux of many organic anions. MRP1 also transports glutathione (GSH); furthermore, this tripeptide stimulates transport of several substrates, including estrone 3-sulfate. We have previously shown that mutations of Lys(332) in transmembrane helix (TM) 6 and Trp(1246) in TM17 cause different substrate-selective losses in MRP1 transport activity. Here we have extended our characterization of mutants K332L and W1246C to further define the different roles these two residues play in determining the substrate and inhibitor specificity of MRP1. Thus, we have shown that TM17-Trp(1246) is crucial for conferring drug resistance and for binding and transport of methotrexate, estradiol glucuronide, and estrone 3-sulfate, as well as for binding of the tricyclic isoxazole inhibitor N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo-[4,3-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide (LY465803). In contrast, TM6-Lys(332) is important for enabling GSH and GSH-containing compounds to serve as substrates (e.g., leukotriene C(4)) or modulators (e.g., S-decyl-GSH, GSH disulfide) of MRP1 and, further, for enabling GSH (or S-methyl-GSH) to enhance the transport of estrone 3-sulfate and increase the inhibitory potency of LY465803. On the other hand, both mutants are as sensitive as wild-type MRP1 to the non-GSH-containing inhibitors (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]-ethanone (LY171883), and highly potent 6-[4'-carboxyphenylthio]-5[S]-hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid (BAY u9773). Finally, the differing abilities of the cysteinyl leukotriene derivatives leukotriene C(4), D(4), and F(4) to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys(332) is involved in the recognition of the gamma-Glu portion of substrates and modulators containing GSH or GSH-like moieties.

Functional Reconstitution of Human ABCC3 into Proteoliposomes Reveals a Transport Mechanism with Positive Cooperativity

ABCC3 (MRP3) is a member of the family of multidrug resistance-associated proteins (MRP), which belong to the largest family of membrane transport proteins, namely, the ATP binding cassette (ABC) transporters. Members of this family contribute to the excretion of several organic anions from cells and play a critical role in conferring resistance against drugs used in the treatment of cancer. The overexpression of ABCC3 in the yeast Pichia pastoris and its subsequent purification made possible the study of substrate-dependent ATPase activity [Chloupkova, M., et al. (2007) Biochemistry 46, 7992-8003]. Here we describe the successful reconstitution of purified ABCC3 in proteoliposomes and ABCC3-dependent uptake of the anticancer drug methotrexate (MTX), as well as the physiological substrate leukotriene C(4) (LTC(4)). Our results show specific transport in a cell-free environment and in the absence of other proteins, revealing positive allosteric cooperativity for ABCC3-mediated substrate translocation. The ABCC3-mediated transport of MTX indicates a Hill coefficient of 2.3 +/- 1.7, a maximum transport rate (V(max)) of >2 micromol min(-1) mg(-1), and a K(M) in the millimolar range, whereas the translocation of LTC(4) into proteoliposomes displayed a Hill coefficient of 2.3 +/- 0.5 with a maximum transport rate of 4.7 +/- 0.8 nmol min(-1) mg(-1), and a K(M) in the micromolar range (1.7 +/- 0.3 microM). The transport of both substrates, MTX and LTC(4), was inhibited by etoposide, confirming a higher affinity of ABCC3 for LTC(4) than for MTX. The technical advances described in this report represent the basis for the extended and detailed kinetic characterization of ABCC3 with a wide range of implications for the investigation of other human ABC transporters.

Nilotinib (AMN107, Tasigna ®) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters

Nilotinib, a BCR-Abl tyrosine kinase inhibitor (TKI), was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia positive chronic myelogenous leukemia. Recently, it was shown that several human multidrug resistance (MDR) ATP-binding cassette (ABC) proteins could be modulated by specific TKIs. MDR can produce cancer chemotherapy failure, typically due to overexpression of ABC transporters, which are involved in the extrusion of therapeutic drugs. Here, we report for the first time that nilotinib potentiates the cytotoxicity of widely used therapeutic substrates of ABCG2, such as mitoxantrone, doxorubicin, and ABCB1 substrates including colchicine, vincristine, and paclitaxel. Nilotinib also significantly enhances the accumulation of paclitaxel in cell lines overexpressing ABCB1. Similarly, nilotinib significantly increases the intracellular accumulation of mitoxantrone in cells transfected with ABCG2. Furthermore, nilotinib produces a concentration-dependent inhibition of the ABCG2-mediated transport of methotrexate (MTX), as well as E 217βG a physiological substrate of ABCG2. Uptake studies in membrane vesicles overexpressing ABCG2 have indicated that nilotinib inhibits ABCG2 similar to other established TKIs as well as fumitremorgin C. Nilotinib is a potent competitive inhibitor of MTX transport by ABCG2 with a K i value of 0.69 ± 0.083 μM as demonstrated by kinetic analysis of nilotinib. Overall, our results indicate that nilotinib could reverse ABCB1- and ABCG2-mediated MDR by blocking the efflux function of these transporters. These findings may be used to guide the design of present and future clinical trials with nilotinib, elucidating potential pharmacokinetic interactions. Also, these findings may be useful in clinical practice for cancer combination therapy with nilotinib.

Alteration of Methotrexate Biliary and Renal Elimination during Extrahepatic and Intrahepatic Cholestasis in Rats

Methotrexate (MTX), an important anticancer and immunosuppressive agent, has been suggested for the treatment of primary biliary cirrhosis. However, the drug's pharmacodynamics and toxicity is dependent on its concentrations in plasma which in turn are directly related to MTX's elimination in the liver and kidney. Therefore, the aim of this study was to evaluate changes in MTX biliary and renal excretion during either intrahepatic or obstructive cholestasis in rats. The steady state pharmacokinetic parameters of MTX were evaluated in rats one (BDO1) or seven (BDO7) days after bile duct obstruction (BDO) or 18 h after administration of lipopolysaccharide (LPS). In comparison to the respective control groups, biliary and total clearances of MTX were decreased to 12% and 49% in the BDO1 group, to 5% and 56% in the BDO7 animals, and to 42% and 43% in the LPS group, respectively. Renal clearance of MTX was unchanged in BDO groups, but decreased to 23% of controls in the LPS animals. The serum biochemistry and expression of main hepatic MTX transporters (Mrp2, Mrp3, Mrp4, Bcrp, Oatp1a1, Oatp1a4 and Oatp1b2) confirmed the pathological cholestatic changes in the liver and partly elucidated the cause of changes in MTX pharmacokinetic parameters. In conclusion, this study is the first describing marked alteration of MTX hepatic and renal elimination induced by cholestasis in rats. Moreover, the reported changes in MTX pharmacokinetics and respective transporter expression suggest important mechanistic differences between the two widely used cholestatic models.

Co‐administration of proton pump inhibitors delays elimination of plasma methotrexate in high‐dose methotrexate therapy

To assess whether or not co-administration of proton pump inhibitors (PPIs) is a risk factor for delayed elimination of plasma methotrexate (MTX) in high-dose MTX (HDMTX) therapy for malignant diseases. To assess the effects of PPI co-administration on elimination of plasma MTX, we examined plasma MTX concentration data on 171 cycles of HDMTX therapy performed in 74 patients. We performed multiple logistic regression analysis to evaluate PPI co-administration as a risk factor. Inhibitory potencies of omeprazole, lansoprazole, rabeprazole and pantoprazole on MTX transport via breast cancer resistance protein (BCRP, ABCG2) were also investigated in an in vitro study using membrane vesicles expressing human BCRP. We identified co-administration of PPIs as a risk factor for delayed elimination (odds ratio 2.65, 95% confidence interval 1.03, 6.82) as well as renal and liver dysfunction. All four PPIs inhibited BCRP-mediated transport of MTX, with half-maximal inhibitory concentrations of 5.5-17.6 microM--considerably higher than the unbound plasma concentrations of the PPIs. Our results support previous findings suggesting that PPI co-administration is associated with delayed elimination of plasma MTX in patients with HDMTX therapy. This drug interaction, however, cannot be explained solely by the inhibitory effects of PPIs on BCRP-mediated MTX transport.

Lack of association between HLA-G 14 bp insertion/deletion polymorphism and response to long-term therapy with methotrexate response in rheumatoid arthritis

Pharmacogenetic studies examining methotrexate (MTX) response/toxicity have focused on enzymes involved in MTX transport and folate metabolism.1 However, recently it has been reported that patients with rheumatoid arthritis (RA) with...

Interaction of genes from influx-metabolism-efflux pathway and their influence on methotrexate efficacy in rheumatoid arthritis patients among Indians

Methotrexate (MTX) is the drug of choice for rheumatoid arthritis (RA) but is effective only in around 60% of treated patients. Bioavailability of MTX may be a major determinant of response status and this may be governed by variations in MTX receptor and transporter genes and genes responsible for polyglutamation and deconjugation. We investigated the contribution of single nucleotide polymorphisms (SNPs) in RFC, FOLR1, FPGS, GGH and MDR1 genes to MTX response in RA patients from North India. RA patients recruited using American College of Rheumatology criteria, were categorized into good and poor responders to MTX, based on disease activity score. A total of 17 SNPs from the above mentioned genes were genotyped and tested for association with MTX response using [chi]2 test; logistic regression along with clinical variables; and gene-gene interaction using multifactor dimensionality reduction (MDR). One novel synonymous SNP Ala324Ala (972 G > A) was identified in RFC gene. The CT genotype of C3435T in MDR1 gene conferred almost twice the risk of poor response [[chi]2 = 5.85, P = 0.01, odds ratio (95% confidence interval) = 1.97 (1.13-3.42)] and was retained in binary logistic regression [B = 0.66, P = 0.025, adjusted odds ratio (95% confidence interval) = 1.93(1.09-3.42)]. Significant interaction between SNPs in GGH and MDR1 genes seems promising. Interactions between genes coding for deconjugation and transporter seem to be important determinants of MTX response in RA but replication and functional studies would be confirmatory.

A Whole Genome Analysis Identifies SLCO1B1 as a Determinant of Methotrexate Pharmacokinetics and Adverse Effects

Methotrexate is a major component in every treatment protocol for childhood acute lymphoblastic leukemia (ALL). Both beneficial and detrimental effects of methotrexate in ALL have been clearly related to methotrexate plasma pharmacokinetics, which vary substantially among patients. However the genetic basis of such variability remains largely unknown. Herein, we surveyed 600,000 germline single nucleotide polymorphisms (SNPs) to determine how inherited genetic variation affects the disposition of methotrexate among 434 children with ALL who received 3014 courses of methotrexate at 2 to 5 g/m2. Adjusting for age, race, gender and methotrexate regimen, the most significant SNPs associated with methotrexate clearance were annotated to a plausible gene, the organic anion transporter polypeptide, SLCO1B1. The three top SNPs included rs11045879 (P = 1.7 × 10−10), rs4149081 (P = 1.7 × 10−9), and rs2900478 (P = 2.8 × 10−8). Linkage disequilibrium (LD) was observed among these three SLCO1B1 SNPs (r2=1) and with a known functional polymorphism in SLCO1B1, T521C (rs4149056, r2 = 0.86). The top two SLCO1B1 SNPs rs11045879 and rs4149081 were further validated (P = 0.018 and P = 0.017) in an independent cohort of 206 patients with ALL. Additional SNPs annotated to SLCO1B1 were identified and further validated. In a stepwise multiple linear regression analysis, SLCO1B1 genetic variation remained significant and explained clearance variability comparable to that of other non-genetic factors including treatment regimen. SNPs in SLCO1B1 were also associated with methotrexate-related gastrointestinal toxicity (P= 0.03 to 0.0005, odds ratio 8.3 to 16.4). In summary, we have identified a candidate gene, SLCO1B1, which is strongly associated with the pharmacokinetics of methotrexate, an anticancer drug with a low therapeutic index in multiple treatment regimens. Although SLCO1B1 is widely recognized as having a strong impact on the disposition of many drugs in clinical use, based on in vitro data, it was not thought to have a major role in methotrexate transport or disposition. Our study demonstrates proof of principle that genome-wide tools in clinical pharmacologic problems can lead to the discovery of important and novel pharmacogenetic links between inherited genomic variation and drug response in humans.

Transport of Diclofenac by Breast Cancer Resistance Protein (ABCG2) and Stimulation of Multidrug Resistance Protein 2 (ABCC2)-Mediated Drug Transport by Diclofenac and Benzbromarone

Diclofenac is an important analgesic and anti-inflammatory drug, widely used for treatment of postoperative pain, rheumatoid arthritis, and chronic pain associated with cancer. Consequently, diclofenac is often used in combination regimens and undesirable drug-drug interactions may occur. Because many drug-drug interactions may occur at the level of drug transporting proteins, we studied interactions of diclofenac with apical ATP-binding cassette (ABC) multidrug efflux transporters. Using Madin-Darby canine kidney (MDCK)-II cells transfected with human P-glycoprotein (P-gp; MDR1/ABCB1), multidrug resistance protein 2 (MRP2/ABCC2), and breast cancer resistance protein (BCRP/ABCG2) and murine Bcrp1, we found that diclofenac was efficiently transported by murine Bcrp1 and moderately by human BCRP but not by P-gp or MRP2. Furthermore, in Sf9-BCRP membrane vesicles diclofenac inhibited transport of methotrexate in a concentration-dependent manner. We next used MDCK-II-MRP2 cells to study interactions of diclofenac with MRP2-mediated drug transport. Diclofenac stimulated paclitaxel, docetaxel, and saquinavir transport at only 50 microM. We further found that the uricosuric drug benzbromarone stimulated MRP2 at an even lower concentration, having maximal stimulatory activity at only 2 microM. Diclofenac and benzbromarone stimulated MRP2-mediated transport of amphipathic lipophilic drugs at 10- and 250-fold lower concentrations, respectively, than reported for other MRP2 stimulators. Because these concentrations are readily achieved in patients, adverse drug-drug interactions may occur, for example, during cancer therapy, in which drug concentrations are often critical and stimulation of elimination via MRP2 may result in suboptimal chemotherapeutic drug concentrations. Moreover, stimulation of MRP2 activity in tumors may lead to increased efflux of chemotherapeutic drugs and thereby drug resistance.

Lapatinib (Tykerb, GW572016) Reverses Multidrug Resistance in Cancer Cells by Inhibiting the Activity of ATP-Binding Cassette Subfamily B Member 1 and G Member 2

Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (Her-1 or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABC subfamily B member 1 (ABCB1) and ABC subfamily G member 2 (ABCG2) transporters. Our results showed that lapatinib significantly enhanced the sensitivity to ABCB1 or ABCG2 substrates in cells expressing these transporters, although a small synergetic effect was observed in combining lapatinib and conventional chemotherapeutic agents in parental sensitive MCF-7 or S1 cells. Lapatinib alone, however, did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in sensitive and resistant cells. Additionally, lapatinib significantly increased the accumulation of doxorubicin or mitoxantrone in ABCB1- or ABCG2-overexpressing cells and inhibited the transport of methotrexate and E(2)17betaG by ABCG2. Furthermore, lapatinib stimulated the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner. However, lapatinib did not affect the expression of these transporters at mRNA or protein levels. Importantly, lapatinib also strongly enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBv200 cell xenografts in nude mice. Overall, we conclude that lapatinib reverses ABCB1- and ABCG2-mediated MDR by directly inhibiting their transport function. These findings may be useful for cancer combinational therapy with lapatinib in the clinic.

Genetic determinants of methotrexate toxicity in rheumatoid arthritis patients: a study of polymorphisms affecting methotrexate transport and folate metabolism

Methotrexate (MTX) is a disease-modifying antirheumatic drug used in the treatment of rheumatoid arthritis (RA). Genetic polymorphisms of reduced folate carrier (RFC1 A80G), P-glycoprotein (MDR1 G2677T>A/C and C3435T), 5,10-methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), thymidylate synthase (TS 2R-->3R), methionine synthase (MS A2756G) and methionine synthase reductase (MTRR A66G) modify MTX transport and metabolic effects and may influence the treatment response. A genotyping approach was used to determine the studied polymorphisms in 213 RA patients. We observed that 56 (26.3%) patients discontinued MTX treatment due to poor response and/or toxicity. RFC1 A80G and MDR1 C3435T polymorphisms increased the risk for overall MTX toxicity (P = 0.039, OR = 3.574, 95% CI = 1.065-11.993 and P = 0.032, OR = 7.801, 95% CI = 1.194-50.960 respectively), while MTHFR A1298C polymorphism had a protective effect on overall MTX toxicity (P = 0.027, OR = 0.170, 95% CI = 0.035-0.820). Our results suggest that genetic polymorphisms in the folate metabolic pathway and MTX transporters modify the toxicity but not the efficacy of MTX treatment.

Emerging Subspecialties: Neurorehabilitation

The ATP-binding cassette multidrug resistance protein 1 MRP1 (ABCC1) mediates the cellular efflux of organic anions including conjugated metabolites, chemotherapeutic agents, and toxicants. We previously described a mutation in cytoplasmic loop 7 (CL7) of MRP1, Pro1150Ala, which reduced leukotriene C₄ (LTC₄) transport but increased 17β-estradiol 17β-d-glucuronide (E₂17βG) and methotrexate (MTX) transport. Vanadate-induced trapping of [α-³²P]8N₃ADP by the Pro1150Ala mutant in the absence of substrate was also greatly reduced compared with wild-type MRP1 suggesting an uncoupling of ATP hydrolysis and transport activity. To determine whether the functional importance of MRP1-Pro¹¹⁵⁰ is conserved, the analogous Pro¹¹⁵⁸ and Pro¹¹⁴⁷ residues in the MRP2 and MRP3 transporters, respectively, were mutated to Ala. Expression levels of the three mutants were unaffected; however, the vesicular transport activity of at least one organic anion substrate was significantly altered. As observed for MRP1-Pro1150Ala, LTC₄ transport by MRP2-Pro1158Ala was decreased. However, E₂17βG and MTX transport was comparable with that of wild-type MRP2 rather than increased as was observed for MRP1-Pro1150Ala. In the case of MRP3-Pro1147Ala, LTC₄ transport was increased, whereas E₂17βG transport was unaffected. MTX transport by MRP3-Pro1147Ala was also increased but to a lesser extent than for MRP1-Pro1150Ala. In contrast, all three mutants showed a marked reduction in levels of vanadate-induced trapped [α-³²P]8N₃ADP. We conclude that MRP1-Pro¹¹⁵⁰, MRP2-Pro¹¹⁵⁸, and MRP3-Pro¹¹⁴⁷ in CL7 differ in their influence on substrate specificity but share a common role in the nucleotide interactions of these transporters.

Leflunomide and its metabolite A771726 are high affinity substrates of BCRP: implications for drug resistance

Background:Earlier publications have suggested a possible role for the efflux transporter breast cancer resistance protein (BCRP) in acquired resistance to disease-modifying antirheumatic drugs (DMARDs) such as leflunomide and its metabolite...

Role of proline 1150 in functional interactions between the membrane spanning domains and nucleotide binding domains of the MRP1 (ABCC1) transporter

The ATP-binding cassette multidrug resistance protein 1 (MRP1) mediates ATP-dependent cellular efflux of drugs and organic anions. We previously described a mutant, MRP1-Pro1150Ala, which exhibits selectively increased estradiol glucuronide (E 217βG) and methotrexate transport as well as altered interactions with ATP. We have now further explored the functional importance of MRP1-Pro 1150 at the interface of transmembrane helix 15 and cytoplasmic loop 7 (CL7) by replacing it with Gly, Ile, Leu and Val. All four mutants exhibited a phenotype similar to MRP1-Pro1150Ala with respect to organic anion transport and [γ 32P]8N 3ATP photolabeling. They also displayed very low levels of substrate-independent vanadate-induced trapping of [α 32P]8N 3ADP. To better understand the relationship between the altered nucleotide interactions and transport activity of these mutants, [α 32P]8N 3ADP trapping experiments were performed under different conditions. Unlike leukotriene C 4, E 217βG decreased [α 32P]8N 3ADP trapping by both wild-type and mutant MRP1. [α 32P]8N 3ADP trapping by MRP1-Pro1150Ala could be increased by using Ni 2+ instead of Mg 2+, and by decreasing temperature; however, the transport properties of the mutant remained unchanged. We conclude that the reduced [α 32P]8N 3ADP trapping associated with loss of Pro 1150, or the presence of E 217βG, is due to enhanced ADP release following ATP hydrolysis rather than a reduction in ATP hydrolysis itself. We hypothesize that loss of Pro 1150 alters the role of CL7 as a coupling helix that mediates signaling between the nucleotide binding domains and some substrate binding sites in the membrane spanning domains of MRP1.

Role of Lysine 411 in Substrate Carboxyl Group Binding to the Human Reduced Folate Carrier, as Determined by Site-Directed Mutagenesis and Affinity Inhibition

Reduced folate carrier (RFC) is the major membrane transporter for folates and antifolates in mammalian tissues. Recent studies used radioaffinity labeling with N-hydroxysuccinimide (NHS)-[(3)H]methotrexate (MTX) to localize substrate binding to residues in transmembrane domain (TMD) 11 of human RFC. To identify the modified residue(s), seven nucleophilic residues in TMD11 were mutated to Val or Ala and mutant constructs expressed in RFC-null HeLa cells. Only K411A RFC was not inhibited by NHS-MTX. By radioaffinity labeling with NHS-[(3)H]MTX, wild-type (wt) RFC was labeled; for K411A RFC, radiolabeling was abolished. When Lys411 was replaced with Ala, Arg, Gln, Glu, Leu, and Met, only K411E RFC showed substantially decreased transport. Nine classic diamino furo[2,3-d]pyrimidine antifolates with unsubstituted alpha- and gamma-carboxylates (1), hydrogen- or methyl-substituted alpha-(2,3) or gamma-(4,5) carboxylates, or substitutions of both alpha- and gamma-carboxylates (6-9) were used to inhibit [(3)H]MTX transport with RFC-null K562 cells expressing wt and K411A RFCs. For wt and K411A RFCs, inhibitory potencies were in the order 4 > 5 > 1 > 3 > 2; 6 to 9 were poor inhibitors. Inhibitions decreased in the presence of physiologic anions. When NHS esters of 1, 2, and 4 were used to covalently modify wt RFC, inhibitory potencies were in the order 2 > 1 > 4; inhibition was abolished for K411A RFC. These results establish that Lys411 participates in substrate binding via an ionic association with the substrate gamma-carboxylate; however, this is not essential for transport. An unmodified alpha-carboxylate is required for high-affinity substrate binding to RFC, whereas the gamma-carboxyl is not essential.