Elsevier

Biochemical Pharmacology

Volume 67, Issue 6, 15 March 2004, Pages 1111-1121
Biochemical Pharmacology

Evaluation of the binding of the tricyclic isoxazole photoaffinity label LY475776 to multidrug resistance associated protein 1 (MRP1) orthologs and several ATP-binding cassette (ABC) drug transporters

https://doi.org/10.1016/j.bcp.2003.11.006Get rights and content

Abstract

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB100 cells to both vincristine and doxorubicin. LY475776 photolabeled 170Ā kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190Ā kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C4, vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.

Introduction

The ABC transporter superfamily is comprised of seven families of transport proteins containing āˆ¼50 human transporters, many of which actively extrude substrates from cells. Alterations in several have been linked to human diseases [1]; for example, cystic fibrosis transmembrane conductance regulator CFTR is defective in cystic fibrosis patients, cholesterol efflux regulatory protein ABCA1 has an important role in arteriosclerosis, MRP6 (ABCC6) is mutated in a connective tissue disorder called Pseudoxanthoma elasticum, and MRP2 (ABCC2) is mutated in Dubinā€“Johnson syndrome resulting in hyperbilirubinemia [2], [3], [4], [5], [6]. This superfamily also contains several efflux transporters that are capable of conferring drug resistance to anticancer and/or antiviral agents when overexpressed in drug-sensitive cell lines. These transporters include P-glycoprotein (ABCB1) in the ā€œBā€ family, BCRP (ABCG2) a member of the ā€œWhiteā€ family and seven MRP1ā€“5 (ABCC1-5) members of the ā€œCā€ family as well as a recently identified MRP7 (ABCC10) and MRP8 (ABCC11) [7], [8], [9]. P-glycoprotein confers resistance to vinca alkaloids, anthracyclines, podophyllotoxins, and taxanes; the MRPs 1, 2, 3, and 6 confer resistance to anthracyclines, Vinca alkaloids, methotrexate, and both MRP4 and MRP5 give resistance to nucleoside antiviral and anticancer agents [10], [11], [12]. Overexpression of BCRP (ABCG2) confers resistance to topotecan and camptothecin [13], [14], [15]. Despite the overlapping substrate specificity of certain superfamily members, the homology of transporters with similar function is 15% or less [16]. Interestingly, differences in substrate specificity have been observed in orthologs of MRP1. Even though the identity of human, murine, canine, monkey is 88% or greater, only human MRP1 confers resistance to anthracyclines, rodent (both murine and rat) and canine MRP1 do not; and monkey has a reduced ability to do so [17], [18], [19], [20], [21], [22]. There appear to be important differences in the binding site(s) within these MRP1 orthologs for this clinically important class of anticancer agents.

Selective transport inhibitors can be used to study the physiological role of each transporter in the laboratory as well as to determine their clinical importance. Because Pgp is overexpressed in numerous tumor types and effluxes drugs from four major classes of oncolytics, inhibitors of Pgp have long been sought [23], [24], [25]. Several inhibitors (LY335979, ZosuquidarĀ·3HCl); XR9576 (tariquidar) and OC144-093 are more selective for Pgp than MRP1 and other family members [25]. MRP1 confers resistance to many of the same drugs and is overexpressed in lung tumors and neuroblastomas [26], [27]. We have reported previously that the tricyclic isoxazole compounds inhibit MRP1-mediated transport of its physiological substrate, LTC4 and sensitize MRP1-expressing transfectants to anticancer drugs that are effluxed by this transporter [28]. In addition, a [125I]-radiolabeled tricyclic isoxazole photoactiveable analog LY475776 derived from LY465803 shown in Fig. 1 labels MRP1 in a GSH-dependent fashion and binds to the COOH-terminal half of MRP1 in the third membrane spanning domain (MSD3) [29]. Coincubation of MRP1 with LY475776 and vanadate inhibits photolabeling suggesting that an ATP-dependent conformation of MRP1 is labeled. Additional studies done with the murine mrp1 have indicated that this ortholog binds LY475776 less well than the human form [30]. This is particularly interesting since murine mrp1 does not confer resistance to anthracyclines unlike human MRP1 [17], [18], [31]. Mutational analysis and hybrids of human MRP1 and murine mrp1 identified a likely binding region for anthracycline and LY475776 to be within the same 572 amino acid region (residues 959ā€“1531) [19], [29], [31].

The present study was undertaken to explore the binding of this photolabel to two other orthologs of MRP1. Canine MRP1 does not confer anthracycline resistance while monkey confers reduced anthracycline resistance relative to human MRP1 [20], [21]. The specificity of photolabeling by LY475776 for several other ABC drug efflux transporters is also examined.

Section snippets

Materials

The ECL detection kit was purchased from Amersham Pharmacia Biotech. E.M. Science was the supplier for MgCl2. The pRev-TRE retroviral vector and tetracycline-system approved FBS was purchased from Clontech while the other serums were purchased from Hyclone laboratories. Improved minimum essential medium was purchased from BioSource International. Gibco was the supplier of all other media and media components. Lipofectamine 2000 was purchased from Invitrogen. The Pierce BCA Protein Assay Reagent

Effect on cytotoxicity of MRP1 and Pgp expressing cell lines

The ability of LY475776 (Fig. 1) to modulate the cytotoxicity of two anticancer agents that are pumped by MRP1 or Pgp was examined [43]. As shown in Fig. 2, the presence of 1Ā Ī¼M LY475776 enhanced the cytotoxicity of vincristine (Fig. 2A) and doxorubicin (Fig. 2B), to MRP1-transfected HeLa cells (HeLa-T5) by 13- and 7.8-fold, respectively, and close to the drug sensitivity of the vector-transfected HeLa cells, HeLa-C1. In addition, when the effect of this MRP1 modulator was examined on

Conclusions

Photoaffinity probes have been useful tools in the identification of transport proteins and in the characterization of substrates of a number of ABC transporter proteins [50]. Although Pgp and MRP1 confer resistance to many of the same anticancer agents, clear differences in the substrate binding properties of these two transporters became apparent when photoactiveable derivatives of doxorubicin, vinca alkaloids, or of the modulators verapamil or azidopine, that label Pgp quite effectively did

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