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
Introduction
The ABC transporter superfamily is comprised of seven families of transport proteins containing ā¼50 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 DubināJohnson 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 āBā family, BCRP (ABCG2) a member of the āWhiteā family and seven MRP1ā5 (ABCC1-5) members of the āCā 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 []-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 959ā1531) [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
References (60)
- et al.
The human ATP-binding cassette (ABC) transporter superfamily
J. Lipid Res.
(2001) - et al.
Cellular cholesterol efflux
Biochim. Biophys. Acta
(2001) - et al.
Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6)
J. Biol. Chem.
(2002) - et al.
MRP8 ATP-binding cassette C11 (ABCC11) is a cyclic nucleotide efflux pump and a resistance factor for fluoropyrimidines 2ā²,3ā²-dideoxycytidine and 9ā²-(2ā²-phosphonylmethoxyethyl)adenine
J. Biol. Chem.
(2003) - et al.
Therapeutic and biological importance of getting nucleotides out of cells: a case for the ABC transporters, MRP4 and 5
Adv. Drug Deliv. Rev.
(2002) - et al.
Breast cancer resistance protein directly confers SN-38 resistance of lung cancer cells
Biochem. Biophys. Res. Commun.
(2001) - et al.
Transport of 7-ethyl-10-hydroxycamptothecin (SN-38) by breast cancer resistance protein ABCG2 in human lung cancer cells
Biochem. Biophys. Res. Commun.
(2001) - et al.
Toxicological relevance of the multidrug resistance protein 1 MRP1 (ABCC1) and related transporters
Toxicology
(2001) - et al.
Increased efflux of vincristine, but not of daunorubicin, associated with the murine multidrug resistance protein (MRP)
Biochem. Pharmacol.
(1996) - et al.
Considerations in the design and development of transport inhibitors as adjuncts to drug therapy
Adv. Drug Deliv. Rev.
(2003)
Tricyclic isoxazoles are novel inhibitors of the multidrug resistance protein (MRP1)
Bioorg. Med. Chem. Lett.
GSH-dependent photolabeling of multidrug resistance protein MRP1 (ABCC1) by []LY475776. Evidence of a major binding site in the COOH-proximal membrane spanning domain
J. Biol. Chem.
Photolabeling of human and murine multidrug resistance protein 1 with the high affinity inhibitor []LY475776 and azidophenacyl-[]glutathione
J. Biol. Chem.
Localization of a substrate specificity domain in the multidrug resistance protein
J. Biol. Chem.
Methanodibenzosuberyl piperazines as potent multidrug resistance reversal agents
Bioogr. Med. Chem. Lett.
A high throughput assay for measurement of multidrug resistance protein (MRP1)-mediated transport of leukotriene C4 into membrane vesicles
Anal. Biochem.
The leukotriene LTD4 receptor antagonist MK571 specifically modulates MRP associated multidrug resistance
Biochem. Biophys. Res. Commun.
Photoaffinity analogs for multidrug resistance-related transporters and their use in identifying chemosensitizers
Drug Resistance Updates
The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structurally related conjugates
J. Biol. Chem.
Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. Demonstration of glutathione-dependent vincristine transport
J. Biol. Chem.
Glutathione-dependent binding of a photoaffinity analog of agosterol A to the C-terminal half of human multidrug resistance protein
J. Biol. Chem.
Cystic fibrosis and CFTR
Pflugers Arch.
Cholesterol efflux regulatory protein, Tangier disease and familial high-density lipoprotein deficiency
Curr. Opin. Lipidol.
A common DubināJohnson syndrome mutation impairs protein maturation and transport activity of MRP2 (ABCC2)
Am. J. Physiol. Gastrointest Liver Physiol.
Impaired 2ā²,3ā²-dideoxy-3ā²-thiacytidine accumulation in T-lymphoblastoid cells as a mechanism of acquired resistance independent of multidrug resistant protein 4 with a possible role for ATP-binding cassette C11
Biochem. J.
Characterization of the transport properties of human multidrug resistance protein 7 (MRP7, ABCC10)
Mol. Pharmacol.
Role of MRP4 and MRP5 in biology and chemotherapy
AAPS PharmSci.
Characterization of the drug resistance and transport properties of multidrug resistance protein 6 (MRP6, ABCC6)
Cancer Res.
Reversal of breast cancer resistance protein-mediated drug resistance by estrogen antagonists and agonists
Mol. Cancer Ther.
Overexpression of the multidrug resistance-associated protein (MRP) gene in vincristine and not doxorubicin-selected multidrug-resistant murine erythroleukemia cells
Cancer Res.
Cited by (31)
Targeting multidrug resistance-associated protein 1 (MRP1)-expressing cancers: Beyond pharmacological inhibition
2021, Drug Resistance UpdatesGlioblastoma Therapy in the Age of Molecular Medicine
2019, Trends in CancerBiochemistry and Pharmacology of Human ABCC1/MRP1 and Its Role in Detoxification and in Multidrug Resistance of Cancer Chemotherapy
2012, Recent Advances in Cancer Research and TherapyRole of proline 1150 in functional interactions between the membrane spanning domains and nucleotide binding domains of the MRP1 (ABCC1) transporter
2008, Biochemical PharmacologyCitation Excerpt :Ī±32P]8N3ATP (12 Ci mmolā1) and [Ī³32P]8N3ATP (10.6 Ci mmolā1) were purchased from Affinity Labeling Technologies Inc. (Lexington, KY). LY465803 was a gift from Eli Lilly (Indianapolis, IN) [14], and MK571 was purchased from Cayman Chemicals (Ann Arbor, MI). MAbs MRPm6 and MRPr1 were kind gifts from Drs. R.J. Scheper and G.L. Scheffer (Amsterdam, Netherlands).
Transport of glutathione and glutathione conjugates by MRP1
2006, Trends in Pharmacological SciencesA macrocyclic peptide inhibitor traps MRP1 in a catalytically incompetent conformation
2023, Proceedings of the National Academy of Sciences of the United States of America