Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/04/11/dmd.116.069468.DC1

1521-009X/44/6/857–866$25.00 http://dx.doi.org/10.1124/dmd.116.069468 METABOLISM AND DISPOSITION Drug Metab Dispos 44:857–866, June 2016 Copyright ª 2016 by The American Society for and Experimental Therapeutics Cysteinyl Leukotriene 1/2 Antagonists Nonselectively Modulate Organic Anion Transport by Multidrug Resistance (MRP1-4) s

Mark A. Csandl, Gwenaëlle Conseil, and Susan P. C. Cole

Departments of Biomedical and Molecular Sciences (M.A.C., S.P.C.C.), and Pathology and Molecular (G.C., S.P.C.C.), Division of Cancer Biology and , Queen’s University Cancer Research Institute, Kingston, ON, Canada

Received January 13, 2016; accepted April 7, 2016

ABSTRACT

Active efflux of both and organic anion metabolites is class of antagonists showed any MRP selectivity. For E217bG Downloaded from mediated by the multidrug resistance proteins (MRPs). MRP1 uptake, LTM IC50s ranged from 1.2 to 26.9 mMandweremost (ABCC1), MRP2 (ABCC2), MRP3 (ABCC3), and MRP4 (ABCC4) have comparable for MRP1 and MRP4. The LTM rank order inhibitory partially overlapping substrate specificities and all transport 17b- potencies for E217bGversusLTC4 uptake by MRP1, and E217bG estradiol 17-(b-D-glucuronide) (E217bG). The cysteinyl leukotriene versus PGE2 uptake by MRP4, were also similar. Three of four receptor 1 (CysLT1R) antagonist MK-571 inhibits all four MRP CysLT1R-selective LTMs also stimulated MRP2 (but not MRP3) homologs, but little is known about the modulatory effects of newer transport and thus exerted a concentration-dependent biphasic leukotriene modifiers (LTMs). Here we examined the effects of effect on MRP2. The fourth CysLT1R antagonist, LY171883, only dmd.aspetjournals.org seven CysLT1R- and CysLT2R-selective LTMs on E217bG uptake stimulated MRP2 (and MRP3) transport but none of the MRPs were into MRP1–4-enriched inside-out membrane vesicles. Their effects stimulated by either CysLT2R-selectiveLTM.Weconcludethat,in on uptake of an additional physiologic solute were also measured contrast to their CysLTR selectivity, CysLTR antagonists show no for MRP1 [leukotriene C4 (LTC4)] and MRP4 [prostaglandin E2 MRP homolog selectivity, and data should be interpreted cau-

(PGE2)]. The two CysLT2R-selective LTMs studied were generally tiously if obtained from LTMs in systems in which more than one more potent inhibitors than CysLT1R-selective LTMs, but neither MRP is present. at ASPET Journals on October 1, 2021

Introduction Elevated MRP1/ABCC1 levels in tumor cells cause multidrug ’ Nine proteins composing a subset of the C subfamily of the ATP- resistance, consistent with MRP1 s ability to efflux chemotherapeutic binding cassette (ABC) superfamily are known as ABCC or multidrug agents (Cole et al., 1994; Cole, 2014a). MRP1 also protects normal resistance proteins (MRPs) (Slot et al., 2011). The four “short” MRPs tissues from xenobiotics when expressed at blood-tissue interfaces have a four-domain structure with two nucleotide binding domains, each (Wijnholds et al., 1998). In addition, MRP1 mediates the efflux of many preceded by a membrane-spanning domain (MSD1/2). The five “long” organic anions, including glutathione (GSH) and glucuronide-conjugated MRPs have an additional NH2-terminally located MSD0. MSD1 and metabolites (Cole, 2014a, b). The physiologic relevance of MRP1 is MSD2 form the pore through which solutes are translocated, powered by associated with its ability to efflux GSH, as well as cysteinyl leukotriene ATP binding and hydrolysis. (CysLT) C4 (LTC4)and17b-estradiol 17-(b-D-glucuronide) (E217bG) Each MRP has its own distinct, albeit somewhat overlapping, (Jedlitschky et al., 1996; Loe et al., 1996). substrate specificity and tissue distribution, and their roles in human The substrate specificities of MRP2/ABCC2 and MRP1 are similar, (patho) and pharmacology vary accordingly (Slot et al., but unlike MRP1, MRP2 is found predominantly in the liver, where it 2011; Keppler, 2011). The long MRP1–3 and the short MRP4 are the helps maintain biliary homeostasis through elimination of conjugated most pharmacologically relevant MRPs and can influence pharmacoki- metabolites (Nies and Keppler, 2007). In addition, MRP2 deficiency is netics, play important roles in tissue defense, and participate in signaling the underlying cause of Dubin-Johnson syndrome, a disorder charac- pathways (Tang et al., 2013; van der Schoor et al., 2015). terized by conjugated hyperbilirubinemia (Kartenbeck et al., 1996). MRP3/ABCC3 is most closely related to MRP1, but like MRP2 it has a narrower tissue distribution and substrate specificity. Unlike MRP1 and MRP2, MRP3 does not transport GSH and only poorly transports This work was supported by the Canadian Institutes of Health Research [MOP- LTC and other GSH conjugates although it shares an ability to transport 106513, MOP-133584]. M.A.C. was the recipient of the Eldon Boyd Fellowship 4 b from Queen’s University. S.P.C.C. is Canada Research Chair in Cancer Biology E217 G (Kool et al., 1999). Hepatic MRP3 has a role in the disposition and Bracken Chair in Genetics and Molecular Medicine. of certain drugs and/or their metabolites (van de Wetering et al., 2007). dx.doi.org/ 10.1124/dmd.116.069468. The substrate specificity of the short MRP4/ABCC4 overlaps to a s This article has supplemental material available at dmd.aspetjournals.org. lesser extent with the long MRP1–3. Thus MRP4 is involved in the

ABBREVIATIONS: ABC, ATP-binding cassette; CysLT, cysteinyl leukotriene; CysLTR, CysLT receptor; E217bG, 17b-estradiol 17-(b-D- glucuronide); GSH, glutathione; HEK, human embryonic kidney; HRPase, horseradish peroxidase; LTC4, leukotriene C4; LTM, leukotriene modifier; mAb, monoclonal antibody; MRP, multidrug resistance ; PGE2, prostaglandin E2. 857 858 Csandl et al. tissue distribution and elimination of used to treat The development of antagonists or leukotriene modifiers (LTMs) that viral and neoplastic diseases (Schuetz et al., 1999; Slot et al., 2011; Park can distinguish between CysLT1R and CysLT2R raises the possibility et al., 2014). It is also distinct in its ability to efflux prostanoids that, unlike MK-571, one or more of them might also be selective for [e.g., prostaglandin E2 (PGE2)] and cyclic nucleotides (Reid et al., 2003; one of the MRP homologs. The goal of the present study was to test this Lin et al., 2008; Jin et al., 2014). However, like MRP1–3, MRP4 can idea by comparing the effects of seven CysLT1R-selective, CysLT2R- transport E217bG. selective, and non-CysLTR-selective LTMs on organic anion transport There has been a long-standing interest in identifying small-molecule by MRP1–4. inhibitors of MRP1-mediated drug efflux in human tumors (Boumendjel et al., 2005; Cole, 2014a). This interest extended to other MRPs as their roles in drug distribution and elimination have been elucidated. MK-571 Materials and Methods was originally developed as a CysLT receptor (CysLTR) antagonist to Materials. AMP, ATP, and E217bG were from Sigma-Aldrich (Oakville, ON, treat asthma (Jones et al., 1989). However, it also sensitizes tumor cells Canada). LTC4 was from Calbiochem (San Diego, CA). Dulbecco’s modified expressing MRP1, and inhibits MRP1-mediated transport of organic Eagle’s medium (DMEM), OptiMEM, and Lipofectamine 2000 were from Gibco/ Life Technologies (Burlington, ON) and protease inhibitors were from Roche anions including LTC4 (Gekeler et al., 1995; Cole, 2014a). MK-571 also modulates transport by other MRPs (Keppler, 2011), other ABC (Mississauga, ON). MK-571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phe- transporters (Matsson et al., 2009), the solute carrier OATP1B3/ nyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid) (Fig. 1A), (1-[[[(1R)-1-[3-(1E)-2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3- SLCO1B3 (Letschert et al., 2005) and flavonol conjugation (Barrington [2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]-methyl]-cyclopropaneacetic acid)) et al., 2015). Despite its well documented lack of selectivity, MK-571 is (Fig. 1A), pranlukast (N-[4-oxo-2-(1H-tetrazol-5-yl)-4H-1-benzopyran-8-yl]-4-(4- Downloaded from still the most widely used small-molecule MRP inhibitor today. phenylbutoxy)-benzamide) (Fig. 1A), LY171883 (1-[2-hydroxy-3-propyl-4-[4- The CysLTs LTC4 and LTD4 act on two classes of -coupled (1H-tetrazol-5-yl)butoxy]phenyl]-ethanone) (Fig. 1A), BAY-u9773 (4-[[(1R, CysLTRs (Haeggström and Funk, 2011). CysLT1R has a prominent role 2E,4E,6Z,9Z)-1-[(1S)-4-carboxy-1-hydroxybutyl]-2,4,6,9-pentadecatetraenyl] in the pathogenesis of asthma, and is selectively targeted by the thio]-benzoic acid) (Fig. 1C), and PGE2 were from Cayman Chemical (Ann experimental agents MK-571 and LY171883, and the clinically used Arbor, MI). HAMI 3379 (3-[[(3-carboxycyclohexyl)amino]carbonyl]-4-[3-[4- [4-(cyclohexyloxy)butoxy]phenyl]propoxy]-benzoic acid) (Fig. 1B) and BayCysLT2

montelukast and pranlukast (Fig. 1A) (Lynch et al., 1999; Sarau et al., dmd.aspetjournals.org (3-[[(3-carboxycyclohexyl)amino]carbonyl]-4-[3-[4-(4-phenoxybutoxy)phenyl] 1999) . The role of CysLT2R in human health and disease is less well propoxy]-benzoic acid) (Fig. 1B) were kind gifts from Dr. Colin Funk understood. It is found in immune cells and the vasculature of several 3 21 (Queen’s University, Kingston, ON). [6,7- H(N)]E217bG (42 Ci mmol ), tissues, including brain and heart (Heise et al., 2000; Nothacker 3 21 3 [14,15,19,20- H(N)]LTC4 (166.3 Ci mmol ), and [5,6,8,11,12,14,15- H(N)] et al., 2000). Two experimental CysLT2R-selective inhibitors are 21 PGE2 (153.7 Ci mmol ) were from PerkinElmer Life Sciences (Boston, MA). available, HAMI 3379 and BayCysLT2 (Fig. 1B) (Wunder et al., Murine monoclonal antibody (mAb) M2Ι-4 (anti-MRP2) (ALX-801-015) and rat 2010; Ni et al., 2011). mAb M4Ι-10 (anti-MRP4) (ALX-801-038) were from Enzo (Farmingdale, NY). + + Rabbit anti-Na /K -ATPase a (H-300) (SC-28800) and murine mAb M3ΙΙ-9 (anti-MRP3) (SC-59613) were from Santa Cruz Biotechnology (Dallas, TX). at ASPET Journals on October 1, 2021 The human MRP1-specific mAb QCRL-1 was generated in this laboratory (Hipfner et al., 1994). Horseradish peroxidase (HRPase)-conjugated goat anti- mouse antibody (BML-SA204) was from Pierce Biotechnology (Rockford, IL), HRPase-conjugated goat anti-rabbit antibody (CED-CLAS10-667) was from Cedarlane (Burlington, ON), and HRPase-conjugated goat anti-rat antibody (AP136P) was from EMD Millipore (Billerica, MA). Western Lightning Plus- enhanced chemiluminescence blotting reagents were from PerkinElmer. Cell Culture and Transfection. The stably transfected MRP1-expressing human embryonic kidney (HEK) 293 cell line has been previously described (Myette et al., 2013). The stably transfected MRP2- and MRP3-expressing HEK293 cell lines were established using pCEBV7-based expression vectors generated by moving the MRP coding sequences from pcDNA3.1(–)-MRP2 (Ito et al., 2001) and pcDNA3.1(+)-MRP3 (Oleschuk et al., 2003) into a modified pCEBV7 vector as described (Cole et al., 1994). HEK293T cells transiently expressing human MRP4 were generated using pcDNA3.1(–)-MRP4 and Lipofectamine 2000 as described (Myette et al., 2013). All cell lines were grown

in DMEM/7.5% fetal bovine serum at 37Cin5%CO2/95% air. After collection, cells were overlaid with homogenization buffer (250 mM sucrose/50 mM Tris pH

7.4/0.25 mM CaCl2) with protease inhibitors, and cell pellets stored at –80Cuntil needed. Preparation of MRP-Enriched Membrane Vesicles and Immunoblotting. Membrane vesicles were prepared from MRP-transfected HEK cells as previously described (Loe et al., 1996). Membrane vesicle protein was quantified using a Bradford assay and the presence of MRP1, MRP2, MRP3, and MRP4 in the membrane vesicles was confirmed by immunoblotting as before (Létourneau et al., 2007; Myette et al., 2013). Blots were incubated overnight at 4Cwiththe appropriate MRP-specific antibodies as well as with anti-Na+/K+-ATPase as a membrane-protein-loading control. Bound antibodies were detected using HRPase-conjugated secondary antibodies followed by chemiluminescence blotting reagents and exposure of blots to X-ray film. 3 Fig. 1. Chemical structures of the LTMs investigated for their MRP modulatory ATP-Dependent MRP-Mediated Uptake of H-Labeled Organic Anions 3 properties in this study. The LTMs shown are selective antagonists of (A) CysLT1R, into Inside-Out Membrane Vesicles. The ATP-dependent uptake of H-labeled (B) CysLT2R, or (C) both (CysLT1/2R). E217bG, LTC4, and PGE2 into inside-out vesicles was measured using a modified Transport by MRP1–4 Modulated by CysLT Receptor Antagonists 859 rapid filtration method adapted to a 96-well plate format (Loe et al., 1996; Létourneau et al., 2007). Stock solutions of LTMs prepared in either dimethyl sulfoxide or were diluted so that the final vehicle concentration in the reaction mixture was ,1%. Transport reactions were carried out in a final volume of 30 ml consisting of 50 mM Tris/250 mM sucrose buffer (TSB), pH 7.4, 4 mM

ATP (or AMP), 10 mM MgCl2, the desired concentration of LTM, membrane vesicle protein, and 3H-labeled substrate as follows: MRP1, 2 mgprotein, 3 3 [ H]E217bG(400nM,20nCi)or[H]LTC4 (50 nM, 10 nCi); MRP2, 4 mg 3 3 protein, [ H]E217bG (400 nM, 40 nCi); MRP3, 4 mg protein, [ H]E217bG 3 3 (1 mM, 80 nCi); MRP4, 5 mg protein, [ H]E217bG (30 mM, 60 nCi), or [ H]PGE2 (5 mM, 100 nCi) (Létourneau et al., 2007; Myette et al., 2013). Assays were 3 3 carried out in duplicate at 37Cfor[H]E217bG and [ H]PGE2 uptake and at 3 23Cfor[ H]LTC4 uptake (Loe et al., 1996). The optimal (linear) uptake times for each MRP and organic anion substrate were determined in preliminary experiments to be as follows: MRP1, E217bG, 3 minutes; MRP2, E217bG, 7 minutes; MRP3, E217bG, 7 minutes; MRP4, E217bG, 10 minutes; MRP1, LTC4,1minutes;MRP4,PGE2,10minutes.Uptakewasstoppedbydilutionin ice-cold TSB and the reaction contents filtered onto a Unifilter-96 GF/B filter plate (Perkin-Elmer) using a FilterMate Harvester apparatus (Packard BioScience

Company, Meriden, CT). Tritium associated with the vesicles was quantified using Downloaded from a TopCount NXT Microplate Scintillation and Luminescence Counter (Perkin- Elmer). ATP-dependent uptake of 3H-labeled organic anions was calculated by subtracting uptake in the presence of AMP from uptake in the presence of ATP; results are expressed as a percent of uptake in the absence of the LTM.

Half-maximal inhibitory (IC50) and half-maximal stimulatory (SC50) values were determined by curve-fitting with nonlinear regression using GraphPad Prism 6.0 software (GraphPad, San Diego, CA). Experiments were repeated two or more dmd.aspetjournals.org times using at least two independent membrane vesicle preparations, and the mean

SC50 and/or IC50 values (6 S.D.) determined. One-way analysis of variance with a Tukey’s multiple comparisons posthoc test was performed using GraphPad Prism 6.0; P values , 0.05 were considered significant.

Results

3 at ASPET Journals on October 1, 2021 All LTMs Inhibit Vesicular [ H]E217bG Transport by MRP1. To compare the relative abilities of the seven LTMs depicted in Fig. 1 to modulate MRP1-mediated transport, vesicular uptake assays using the 3 common MRP substrate [ H]E217bG were performed using MRP1- enriched membrane vesicles prepared from stably transfected HEK cells. The presence of MRP1 in the membrane vesicles was first confirmed Fig. 2. Immunoblots of MRP-enriched membrane vesicles. Levels of MRP1, by immunoblot analysis, and as expected, a single band at 190 kDa MRP2, MRP3, and MRP4 proteins were detected in membrane vesicles prepared corresponding to MRP1 was observed (Fig. 2A). from transfected HEK293 cells by immunoblotting. Membrane vesicles derived from untransfected HEK293 cells were used as a control. (A) MRP1, (B) MRP2, (C) The IC50 values of the seven LTMs were then determined by MRP3, and (D) MRP4 were detected with mAbs QCRL-1, M2I-4, M3II-9, and + + measuring their effects on E217bG uptake at 6–7 LTM concentrations M4I-10, respectively; anti-Na /K -ATPase was used as a protein loading control. (range 0.01–40 mM) and sigmoid curves fitted to the datasets. Representative concentration-response curves are shown in Fig. 3 and the mean IC50 values obtained from multiple independent experiments the membrane vesicles was first confirmed by immunoblotting, and as are summarized in Table 1. For MRP1-mediated E217bG uptake, the expected a single band at 190 kDa corresponding to MRP2 was observed IC50s of the LTMs ranged from 1.6 to 16.1 mM and no stimulation was (Fig. 2B). 3 observed at any of the concentrations tested. The IC50s (1.8–16.1 mM) of MRP2-mediated [ H]E217bG transport was then measured in the the four CysLT1R-selective LTMs (MK-571, montelukast, pranlukast, presence of the LTMs at 6–14 concentrations (range 0.1–175 mM). As LY171883) (Fig. 1A) were generally greater and broader than those of shown in Fig. 4 (and summarized in Table 2), three of four CysLT1R- the CysLT2R-selective HAMI 3379 and BayCysLT2 (Fig. 1B) (IC50s 1.6 selective LTMs (MK-571, montelukast, and pranlukast) had biphasic and 1.9 mM, respectively). However, the IC50s of the most potent effects on E217bG transport with stimulation of uptake by MRP2 at CysLT1R-selective LTMs (MK-571 and pranlukast, 1.8 and 2.8 mM, lower LTM concentrations (1 to ;10 mM), and inhibition of uptake respectively) were comparable to the IC50s of the CysLT2R-selective at higher concentrations (Fig. 4, A–C). The maximal stimulation of LTMs and the nonselective BAY-u9773 (Fig. 1C) (1.6–1.9 mM) (P . E217bG uptake observed ranged from 1.3- to 1.6-fold (at 2–6 mM 0.05) (Table 1). Overall, the data indicate that the CysLTR selectivity of LTM). the LTMs does not correlate with their potencies as inhibitors of MRP1- The fourth CysLT1R-selective LTM, LY171883, only stimulated mediated E217bG uptake. MRP2-mediated E217bG transport (maximal 4-fold stimulation at LTMs Can Stimulate, Inhibit, or Have a Biphasic Effect on 100 mM, 3.2-fold at 30 mM) (Fig. 4D; Table 2). This contrasts with its 3 MRP2-Mediated Vesicular [ H]E217bG Transport. To determine effect on MRP1 transport activity, where only inhibition was observed the ability of the LTMs to modulate MRP2-mediated E217bG transport, over the same concentration range (Table 1). The SC50 value of LY171883 uptake assays were performed using MRP2-enriched membrane vesicles was 11.3 mM, which was significantly higher than the SC50sforMK-571, prepared from stably transfected HEK cells. The presence of MRP2 in montelukast, and pranlukast (range 1.0–2.6 mM) (P , 0.05) (Table 2). 860 Csandl et al. Downloaded from dmd.aspetjournals.org

Fig. 3. The inhibitory effects of LTMs on E217bG transport by MRP1. Representative concentration-response curves illustrating the inhibitory effects of increasing LTM concentrations on ATP-dependent MRP1-mediated E217bG uptake into membrane vesicles: (A) MK-571, (B) montelukast, (C) pranlukast, (D) HAMI 3379, (E) BayCysLT2, and (F) BAY-u9773. Each data point is the mean of duplicate determinations in a single experiment. IC50 values derived from multiple independent experiments are summarized in Table 1 (LTM concentration range tested 0.01–40 mM). at ASPET Journals on October 1, 2021 As inhibitors of MRP2 activity, MK-571, montelukast, and pranlukast E217bG uptake was observed, the CysLT1R-selective LTMs (with the were 2- to 6-fold less potent (IC50s 21.2, 26.9, and 23.7 mM, exception of LY171883) were significantly less potent inhibitors (up to respectively) than the CysLT2R-selective and nonselective LTMs 6-fold) than the CysLT2R-selective and nonselective LTMs. 3 (P , 0.05) (Fig. 4, A–C, versus Fig. 4, E–G; Table 2). On the other LTMs Can Both Inhibit and Stimulate [ H]E217bG Uptake by hand, the IC50s for the CysLT2R-selective HAMI 3379 and BayCysLT2 MRP3 but Do Not Elicit a Biphasic Response. To further explore and the nonselective BAY-u9773 were comparable to each other at their possible MRP selectivity, the effects of the LTMs on E217bG 4.3 mM, 12.2 mM, and 8.0 mM, respectively (P . 0.05) (Fig. 4, E–G; uptake by MRP3 were determined using MRP3-enriched membrane Table 2). vesicles prepared from stably transfected HEK cells. MRP3 levels were Together these data indicate that CysLTR selectivity of the LTMs first confirmed by immunoblotting as before (Fig. ). Two closely may weakly correlate with their ability to modulate MRP2 transport migrating immunoreactive bands at ;190 kDa corresponding to the activity since 1) stimulation of E217bG uptake was only observed in molecular weight of MRP3 were observed and are presumed to represent the presence of CysLT1R-selective LTMs; and 2) when inhibition of variably glycosylated forms of the transporter. The LTMs were tested at six concentrations (range 0.1–175 mM), and TABLE 1 in all instances with one exception, they inhibited MRP3-mediated b – Inhibition of MRP1-mediated vesicular uptake of E217bG and LTC4 by LTMs E217 G uptake and no stimulation was observed (Fig. 5A, B; D F; Table 2). The exception was LY171883, which only stimulated E217bG a IC50 uptake by MRP3 at concentrations up to 30 mM (Fig. 5C; Table 2), as it CysLTR Selectivity LTM b E217bG LTC4 did for MRP2-mediated E217 G uptake (Fig. 4C; Table 2). Thus, in mM mM contrast to MRP2 and when compared over the same concentration CysLT1R MK-571 1.8 6 0.7 (4)† 2.7 [2.1, 3.2] ranges, none of the LTMs had a biphasic effect on MRP3 transport. 6 Montelukast 7.2 1.9 (3)* 10.2 [9.0, 11.4] The CysLT1R-selective montelukast and pranlukast inhibited MRP3- 6 † Pranlukast 2.8 1.0 (3) 3.3 [3.8, 2.8] mediated E 17bG uptake with comparable IC sof20.3mMand LY171883 16.1 6 1.3 (3)b,‡ 52.2 [61.4, 43.0] 2 50 19.2 mM(P . 0.05), respectively, whereas MK-571 inhibited uptake by CysLT2R HAMI 3379 1.6 6 0.4 (3)† 3.3 [3.0, 3.5] BayCysLT2 1.9 6 0.6 (5)† 3.4 [2.3, 4.3] 57.0 6 4.0% at 30 mM and by 96.3 6 0.2% at 100 mM (Fig. 5, A and B; CysLT R BAY-u9773 1.9 6 0.4 (4)† 4.3 [4.7, 4.0] 1/2 Table 2). The CysLT2R-selective HAMI 3379 and BayCysLT2, and the nonselective BAY-u9773 were more potent inhibitors (;2.6- to 4.9- *IC50 is significantly different from † and ‡ (P , 0.05). †IC50s are not significantly different . ‡ , from one another (P 0.05) but are significantly different from * and (P 0.05). IC50 is fold) with IC50sof4.3mM, 7.7 mM, and 4.0 mM, respectively (P , significantly different from * and † (P , 0.05) – aValues represent the means 6 S.D. of results obtained from the number of independent 0.05) (Fig. 5, D F; Table 2). In contrast, the CysLT1R-selective experiments indicated in parentheses; when only two independent experiments were performed, LY171883 stimulated MRP3-mediated E217bG transport by a maxi- individual results are given in square brackets. Statistical significance was determined using a m m one-way analysis of variance with a Tukey’s multiple comparisons posthoc test. mum of approximately 2-fold at 30 M(SC50 8.1 M) (Fig. 5C; b m IC50 from Conseil and Cole (2013). Table 2), which was similar to observations for MRP2 (SC50 11.3 M). Transport by MRP1–4 Modulated by CysLT Receptor Antagonists 861 Downloaded from Fig. 4. The inhibitory, stimulatory, and biphasic modulatory effects of LTMs on E217bG transport by MRP2. Representative concentration-response curves illustrating the modulatory effects of increasing LTM concentrations on MRP2-dependent E217bG uptake into membrane vesicles: (A) MK-571, (B) montelukast, (C) pranlukast, (D) LY171883, (E) HAMI 3379, (F) BayCysLT2, and (G) BAY-u9773. Biphasic modulatory effects (A and B) are dmd.aspetjournals.org defined as stimulation of E217bG uptake by MRP2 at lower LTM concentrations followed by an inhibition of E217bG uptake at higher concentrations. Each data point is the mean of duplicate determinations in a single experiment. IC50 and SC50 values derived from multiple independent experiments are summarized in Table 2 (LTM concentration range tested 0.03–175 mM). at ASPET Journals on October 1, 2021

Thus, as shown for MRP2 (but again with the exception of LY171883), The seven LTMs were tested at six concentrations (range 0.1–100 3 the CysLT1R-selective LTMs were less potent inhibitors of MRP3 mM) for their ability to modulate MRP4-mediated [ H]E217bG uptake, transport activity (up to 5-fold) than the CysLT2R-selective and and inhibition was observed in all cases (Fig. 6, A–G). Thus, in contrast nonselective LTMs. to MRP2 and MRP3, but like MRP1, none of the seven LTMs stimulated 3 All LTMs Can Inhibit MRP4-Mediated [ H]E217bG Uptake. MRP4-mediated E217bG uptake at any of the concentrations tested. Because the LTMs had differential effects on E217bG uptake by the Mean IC50 values from multiple independent experiments are summa- long MRP1–3, it was of interest to determine their effects on E217bG rized in Table 3. uptake by the more distantly related short MRP4. Thus, vesicular uptake As with MRP1, the IC50 values for the CysLT1R-selective LTMs assays were performed using membrane vesicles prepared from trans- (montelukast, pranlukast, LY171883) (3.3–18.5 mM) were generally fected HEK cells enriched for MRP4 as demonstrated by immunoblot- greater and broader than the IC50s for the CysLT2R-selective HAMI ting (Fig. 2D). As reported earlier, two immunoreactive bands close to 3379 and BayCysLT2 (1.2 and 1.9 mM, respectively). However, the the expected molecular mass of MRP4 (170 kDa) were detected and are IC50s of the most potent CysLT1R-selective LTMs (pranlukast, 3.3 mM; presumed to be variably glycosylated forms of the transporter (Myette MK-571, 2.2 mM) were comparable to the IC50s of the CysLT2R- et al., 2013). selective LTMs (1.2 and 1.9 mM) as well as the nonselective BAY-u9773 862 Csandl et al.

TABLE 2

Modulation of MRP2- and MRP3-mediated vesicular uptake of E217bG by LTMs

MRP2 MRP3 CysLTR selectivity LTM a a a a SC50 IC50 SC50 IC50 mM mM mM mM b c CysLT1R MK-571 1.0 [1.0, 0.9] 21.2 [20.0, 22.4] ND ND Montelukast 2.4 6 0.4 (3)c,x 26.9 6 4.1 (3)c,* — 20.3 6 1.2 (3)† Pranlukast 2.6 6 0.8 (3)c,x 23.7 6 3.2 (3)c,* — 19.2 6 4.8 (3)† LY171883 11.3 6 5.3 (3){ — 8.1 6 2.4 (3) — CysLT2R HAMI 3379 — 4.3 6 1.8 (5)** — 4.3 6 0.7 (3)† BayCysLT2 — 12.2 6 0.8 (3)** — 7.7 6 1.8 (3)‡ CysLT1/2R BAY-u9773 — 8.0 6 2.2 (3)** — 4.0 6 0.6 (3)‡

*IC50s are not significantly different from one another (P . 0.05) but are significantly different from **(P , 0.05). †IC50s are not significantly different from one another (P . 0.05) but are significantly different from ‡(P , 0.05). {IC50 is significantly different from x(P , 0.05). aValues represent the means 6 S.D. of results obtained from the number of independent experiments indicated in parentheses; when only two independent experiments were performed, the individual results are given in square brackets. Statistical significance was determined using a one-way analysis of variance with a Tukey’s multiple comparisons posthoc test b ND, IC50 not determined; MK-571 inhibited MRP3-mediated E217bG uptake by 57.0 6 4.0% at 30 mM and by 96.3 6 0.2% at 100 mM(n =3). c Downloaded from Where both an IC50 and SC50 are reported, the LTM modulates MRP2 transport activity in a biphasic manner

(2.6 mM) (Table 3) (P . 0.05). Thus, these data indicate that, as LTMs Do Not Modulate MRP1- and MRP4-Mediated Organic observed for MRP1 (Table 1), the CysLTR selectivity of the LTMs Anion Transport in a Substrate-Specific Manner. To compare the does not correlate with their as inhibitors of MRP4-mediated ability of the LTMs to inhibit E217bG transport with their ability to

E217bG transport. inhibit the transport of a more MRP homolog–specific organic anion dmd.aspetjournals.org Analysis of the MRP Selectivity of the LTMs. To better illustrate substrate, MRP1-mediated uptake of its physiologic and highest the effects of an individual LTM on MRP1–4 transport activities, the affinity substrate LTC4 was measured in the presence of LTMs at 10 3 IC50s for the [ H]E217bG uptake data as summarized in Tables 1–3 were concentrations (range 0.01–175 mM). IC50s were determined as replotted against the four different MRP homologs (Supplemental Fig. before and the results are summarized in Table 1. All seven LTMs 1). When the data are presented in this fashion, it becomes more apparent inhibited LTC4 uptake as they did MRP1-mediated E217bG uptake, that the CysLT1R-selective LTMs are generally less potent than the with mean IC50 values ranging from 2.7 to 52.2 mM(forE217bG CysLT2R-selective and dual-selective BAY-u9773 LTMs, and that IC50 transport, the range was 1.6–16.1 mM). The general rank order at ASPET Journals on October 1, 2021 values for inhibition of E217bG transport by MRP1 and MRP4 are potencies of inhibition by the LTMs for LTC4 and E217bG uptake by closer than those for transport by MRP2 and MRP3. MRP1 were also similar (Table 1). These data indicate that the LTMs

Fig. 5. The inhibitory and stimulatory effects of LTMs on E217bG transport by MRP3. Representative concentration-response curves illustrating the modulatory effects of increasing LTM concentrations on MRP3-dependent E217bG uptake into membrane vesicles: (A) montelukast, (B) pranlukast, (C) LY171883, (D) HAMI 3379, (E) BayCysLT2, and (F) BAY-u9773. Each data point is the mean of duplicate determinations in a single experiment. IC50 and SC50 values derived from multiple independent experiments are summarized in Table 2 (LTM concentration range tested was 0.1–175 mM). Transport by MRP1–4 Modulated by CysLT Receptor Antagonists 863 Downloaded from

Fig. 6. The inhibitory effects of LTMs on E217bG transport by MRP4. Shown are representative concentration-response curves illustrating the inhibitory effects of increasing LTM concentra- tions on MRP4-dependent E217bG uptake into membrane

vesicles. (A) MK-571, (B) montelukast, (C) pranlukast, (D) dmd.aspetjournals.org LY171883, (E) HAMI 3379, (F) BayCysLT2, and (G) BAY- u9773. Each data point is the mean of duplicate determinations in a single experiment. IC50 values derived from multiple independent experiments are summarized in Table 3 (LTM concentration range tested 0.1–100 mM). at ASPET Journals on October 1, 2021

do not differentially affect the transport of these two organic anion inhibitory potencies for PGE2 uptake and E217bG uptake by MRP4 substrates of MRP1. were also essentially the same, with the three CysLT2R-selective and The seven LTMs were also tested for their ability to modulate uptake nonselective LTMs tending to be more potent inhibitors than the four 3 of [ H]PGE2 by MRP4, a physiologic substrate of this short MRP that CysLT1R-selective LTMs (Table 3). These data indicate that the LTMs is not transported by MRP1, 2, or 3 (Reid et al., 2003; Lin et al., 2008). do not distinguish between at least these two organic anion substrates of

Thus PGE2 uptake into MRP4-enriched membrane vesicles was MRP4. measured in the presence of the LTMs at six concentrations (range

0.1–100 mM) and IC50s determined as before. As summarized in Discussion Table 3, all seven LTMs inhibited PGE2 uptake as they did MRP4- b mediated E217 G uptake (Fig. 6, Table 3). The mean IC50s for PGE2 CysLT1R is the primary molecular target of MK-571 which blocks the m b transport ranged from 1.1 to 9.9 M (for MRP4-mediated E217 G LTD4-mediated of this receptor at low nanomolar concentra- transport, the range was 1.2–18.5 mM). The rank order of LTM tions (Lynch et al., 1999; Heise et al., 2000), whereas low-to-mid 864 Csandl et al.

TABLE 3 The comparable sensitivities of MRP1 and MRP4 to the CysLT1R-

Inhibition of MRP4-mediated vesicular uptake of E217bG and PGE2 by LTMs selective LTMs is of interest because of the substantial structural and substrate specificity differences between the long MRP1 and short a IC50 CysLTR Selectivity LTM MRP4. The sequence similarity between MRP1 and MRP4 is just 31%, b E217 G PGE2 whereas the sequence similarities among the long MRP1–3are mM mM significantly greater (46–56%). The chemical properties of the solutes CysLT1R MK-571 2.1 [2.1, 2.2] 7.0 [8.7, 5.3] transported by MRP4 are also more distinct than those transported by Montelukast 12.6 6 0.9 (3)* 9.9 [10.2, 9.6] – 6 † MRP1 3, and the few substrates that MRP1 and MRP4 do have in Pranlukast 3.3 0.6 (3) 6.1 [5.3, 6.9] b LY171883 18.5 6 4.9 (3) ‡ 8.5 [8.2, 8.7] common (e.g., E217 G) differ considerably in their transport kinetics m CysLT2R HAMI 3379 1.2 6 0.1 (3) † 1.1 [0.6, 1.5] (Km(app) 1.7 versus 17 M, respectively) (Jedlitschky et al., 1996; 6 † BayCysLT2 1.9 0.1 (3) 1.4 [1.3, 1.5] Wittgen et al., 2012). It seems improbable that MRP1 and MRP4 have a 6 † CysLT1/2R BAY-u9773 2.6 1.1 (4) 1.7 [1.6, 1.8] common set of contact amino acids for these LTMs that enables them to † ‡ , † b *IC50 is significantly different from and (P 0.05). IC50s are not significantly different inhibit transport of E217 G (as well as their more homolog-specific . ‡ , ‡ from one another (P 0.05) but are significantly different from * and (P 0.05). IC50 is substrates LTC and PGE , respectively) with comparable potency. A significantly different from * and † (P , 0.05). 4 2 aValues represent the means 6 S.D. of results obtained from the number of independent better explanation may be more forthcoming when more detailed experiments indicated in parentheses; when only two independent experiments were performed, structural information on the MRPs becomes available. the individual results are shown in square brackets. Statistical significance was determined using a one-way analysis of variance with a Tukey’s multiple comparisons posthoc test. The biphasic modulatory effects observed with the CysLT1R-

selective MK-571, montelukast, and pranlukast on E217bG uptake by Downloaded from MRP2 and the stimulatory effects observed with LY171883 on E217bG micromolar MK-571 concentrations are needed to inhibit transport by uptake by both MRP2 and MRP3 are reminiscent of earlier reports on MRP1/ABCC1. Regardless of these differences in potency, the ability of these transporters from our group and others. Thus a structurally diverse MK-571 to inhibit MRP1 (and other transporters) is somewhat un- array of compounds, including type 1 (CB1) receptor expected given the lack of sequence homology and structural similarity antagonists (e.g., rimonabant), ethinyl estradiol conjugates, sulfinpyra- between CysLT1RandtheMRPs.Thedatapresentedhereand

zone, indomethacin, and chalcogenopyrylium dyes have been reported dmd.aspetjournals.org elsewhere show that the sensitivity of MRP1 and its homologs to to modulate organic anion transport by MRP2 (and MRP3) in a complex b modulation by MK-571 varies substantially when E217 G is used as a fashion (Bakos et al., 2000; Bodo et al., 2003; Zelcer et al., 2003; Chu – b probe for transport activity (Tables 1 3). Thus, E217 Guptakeby et al., 2004; Gerk et al., 2004; Wittgen et al., 2011; Myette et al., 2013). MRP3/ABCC3 appears to be least sensitive to inhibition by MK-571, To explain the biphasic response to some modulators, it has been and MRP1 and MRP4/ABCC4 most sensitive, with MRP2/ABCC2 proposed that the transporter contains both a transport site and an in between. MK-571 is most often used with the intent of fully allosteric modulatory site whereby the modulator stimulates the trans- inhibiting MRP activity, and for this purpose our data indicate that porter allosterically at low probe (E217bG) concentrations and competes . at ASPET Journals on October 1, 2021 the MK-571 concentrations needed to achieve 80% inhibition are for the E217bG at higher concentrations (Zelcer et al., 2003; 5 mMforMRP1,30mMforMRP2,100mMforMRP3,and5mMfor Bodo et al., 2003). The observation that LY171883 only stimulates MRP4. In intact cell or whole organism systems in which modulators E217bG transport by MRP2 and MRP3 suggests that this LTM can must be taken up into the cell to exert their effects, the concentrations occupy only the allosteric site on these transporters. MRP4 is also required to achieve MRP1–4 inhibition can be expected to be signifi- thought to contain multiple allosteric substrate binding sites (Van Aubel cantly higher. However, if 5 mM MK-571 is used to inhibit MRP1 in a et al., 2005), but we saw no evidence of an allosteric interaction of the vesicular transport system where the other homologs are present, MRP4 LTMs with the E217bG or PGE2 probes used here. Whether the would also be inhibited, but MRP2 transport activity would be CysLT1R-selective LTMs (and the CysLT2R-selective LTMs discussed stimulated (see below). below) are themselves actively transported by the MRPs or simply Newer CysLT1R-selective LTMs also inhibit the transport of various occupy a substrate or allosteric modulatory binding site remains to be MRP1 substrates but far less is known about their abilities to inhibit determined. other MRP homologs (Nagayama et al., 1998; van Brussel et al., 2004; Drug-drug interactions have been implicated with the concomitant Conseil and Cole, 2013). To address this deficiency we investigated the use of montelukast and an array of other drugs, including efavirenz and effects of three additional CysLT1R-selective LTMs on the MRP1–4- ivacaftor, and have been attributed to alterations in oxidative metabolism mediated transport of E217bG. The patterns of inhibition of MRP1 and (Ibarra-Barrueta et al., 2014; Schneider et al., 2015). However, the MRP4 by the antiasthmatic agents pranlukast and montelukast were metabolites of montelukast and pranlukast are excreted primarily via the comparable to the pattern observed for MK-571, whereas LY171883 hepatobiliary elimination route, where modulation of MRP2 activity was less potent (Tables 1 and 3). The responses of MRP2 and MRP3 to may have pharmacokinetic consequences (Keam et al., 2003; Diamant the CysLT1R-selective LTMs were more complex. Thus, MRP2 was et al., 2009). Thus, our observations with the CysLT1R-selective LTMs inhibited by MK-571, montelukast, and pranlukast only at higher suggest a potential role for the MRPs in drug-drug interactions in concentrations, but at lower concentrations (1–;10 mM), MRP2- patients taking this class of antiasthma medication. Further studies using mediated E217bG uptake was stimulated (up to 1.6-fold). In contrast, intact cell assays and appropriate Abcc knockout mice may be helpful in these same three CysLT1R-selective LTMs exerted only inhibitory evaluating this possibility. effects on MRP3. However, this was not the case for LY171883, a The CysLT2R-selective LTMs have only recently become available, CysLT1R antagonist that can also act as a phosphodiesterase inhibitor but their usefulness in elucidating the role of CysLT2R in multiple (Fleisch et al., 1985) and a peroxisome-proliferating agent (Foxworthy cellular processes including vascular permeability and ischemic injury is et al., 1990). Thus, LY171883 only stimulated E217bG uptake by MRP2 already well established (Ni et al., 2011; Zhang et al., 2013). HAMI 3379 and MRP3 (up to 4-fold and 2-fold, respectively) and did not exert the is reported to be 10,000-fold more selective and BayCysLT2 500-fold biphasic actions of the other CysLT1R-selective LTMs, at least at the more selective for CysLT2R than CysLT1R (Wunder et al., 2010; Ni concentrations tested, which include those likely to be used in vesicular et al., 2011). However, until now, the (possible) effects of the CysLT2R- transport studies in vitro. selective LTMs on the transport activities of the MRPs have not been Transport by MRP1–4 Modulated by CysLT Receptor Antagonists 865 investigated. Our data show that, despite their ability to distinguish Boumendjel A, Baubichon-Cortay H, Trompier D, Perrotton T, and Di Pietro A (2005) Anticancer multidrug resistance mediated by MRP1: recent advances in the discovery of reversal agents. between the two CysLTR isoforms, HAMI 3379 and BayCysLT2 show Med Res Rev 25:453–472. little or no such selectivity with respect to inhibiting E217bG transport Cheung L, Flemming CL, Watt F, Masada N, Yu DMT, Huynh T, Conseil G, Tivnan A, Polinsky A, and Gudkov AV, et al. (2014) High-throughput screening identifies Ceefourin 1 and Cee- by the four MRP homologs. However, like the CysLT1R-selective fourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4). Biochem – LTMs, the two CysLT2R-selective LTMs more potently inhibited Pharmacol 91:97 108. b , m Chu X-Y, Huskey S-EW, Braun MP, Sarkadi B, Evans DC, and Evers R (2004) Transport of E217 G transport by MRP1 and MRP4 (IC50s 2 M) than by ethinylestradiol glucuronide and ethinylestradiol sulfate by the multidrug resistance proteins – MRP2 and MRP3 (IC50s , 12 mM). In contrast, unlike the CysLT1R- MRP1, MRP2, and MRP3. J Pharmacol Exp Ther 309:156 164. Cole SPC, Sparks KE, Fraser K, Loe DW, Grant CE, Wilson GM, and Deeley RG (1994) Phar- selective LTMs, neither HAMI 3379 nor BayCysLT2 stimulated or macological characterization of multidrug resistant MRP-transfected human tumor cells. Cancer caused a biphasic effect on MRP2 and MRP3 transport activity. Finally, Res 54:5902–5910. Cole SPC (2014a) Targeting multidrug resistance protein 1 (MRP1, ABCC1): past, present, and as an MRP modulator, the nonselective or dual CysLTR antagonist future. Annu Rev Pharmacol Toxicol 54:95–117. BAY-u9773 behaved much more like HAMI 3379 and BayCysLT2 than Cole SPC (2014b) Multidrug resistance protein 1 (MRP1, ABCC1), a “multitasking” ATP-binding it did the CysLT R-selective LTMs, despite the fact that BAY-u9773 is cassette (ABC) transporter. J Biol Chem 289:30880–30888. 1 Conseil G and Cole SPC (2013) Two polymorphic variants of ABCC1 selectively alter drug 20- to 500-fold less potent at blocking LTC4-mediated CysLT2R resistance and inhibitor sensitivity of the multidrug and organic anion transporter multidrug activation (Ni et al., 2011). resistance protein 1. Drug Metab Dispos 41:2187–2196. Dantzig AH, Shepard RL, Pratt SE, Tabas LB, Lander PA, Ma L, Paul DC, Williams DC, Peng The data presented here and elsewhere highlight the ongoing need S-B, and Slapak CA, et al. (2004) Evaluation of the binding of the tricyclic isoxazole photoaffinity for more selective small-molecule inhibitors of the individual MRP label LY475776 to multidrug resistance associated protein 1 (MRP1) orthologs and several ATP- binding cassette (ABC) drug transporters. Biochem Pharmacol 67:1111–1121. homologs. The tricyclic isoxazole LY475776 and ceefourin-2 have Diamant Z, Mantzouranis E, and Bjermer L (2009) Montelukast in the treatment of asthma and – been identified as relatively selective inhibitors of MRP1 and MRP4, beyond. Expert Rev Clin Immunol 5:639 658. Downloaded from Fleisch JH, Rinkema LE, Haisch KD, Swanson-Bean D, Goodson T, Ho PPK, and Marshall WS respectively (Dantzig et al., 2004; Cheung et al., 2014), but to our (1985) LY171883, 1-,2-hydroxy-3-propyl-4-,4-(1H-tetrazol-5-yl) butoxy.phenyl.ethanone, knowledge no MRP2 or MRP3 selective inhibitors have been reported. an orally active leukotriene D4 antagonist. J Pharmacol Exp Ther 233:148–157. Foxworthy PS, Perry DN, Hoover DM, and Eacho PI (1990) Changes in hepatic lipid metabolism However, it seems evident that both existing and newly developed MRP associated with lipid accumulation and its reversal in rats given the peroxisome proliferator inhibitors require extensive testing for activity against a broad range of LY171883. Toxicol Appl Pharmacol 106:375–383. Gekeler V, Ise W, Sanders KH, Ulrich WR, and Beck J (1995) The leukotriene LTD4 receptor both efflux and import transporters in a variety of intact cell and cell-free antagonist MK571 specifically modulates MRP associated multidrug resistance. Biochem Bio- phys Res Commun 208:345–352. assay systems to substantiate all claims of MRP homolog selectivity. dmd.aspetjournals.org Gerk PM, Li W, and Vore M (2004) Estradiol 3-glucuronide is transported by the multidrug In conclusion, the comparative studies reported here clearly illustrate resistance-associated protein 2 but does not activate the allosteric site bound by estradiol the nonselectivity of both classes of CysLTR antagonists with respect to 17-glucuronide. Drug Metab Dispos 32:1139–1145. – Haeggström JZ and Funk CD (2011) Lipoxygenase and leukotriene pathways: , bi- their ability to modulate organic anion transport by MRP1 4, and the ology, and roles in disease. Chem Rev 111:5866–5898. consequent limitations of these LTMs as small-molecule inhibitors in Heise CE, O’Dowd BF, Figueroa DJ, Sawyer N, Nguyen T, Im DS, Stocco R, Bellefeuille JN, Abramovitz M, and Cheng R, et al. (2000) Characterization of the human cysteinyl leukotriene 2 studies of these transporters. 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