Ins and outs of the ABCG2 multidrug transporter: An update on in vitro functional assays

Advanced Drug Delivery Reviews 61 (2009) 47–56

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Advanced Drug Delivery Reviews j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a d d r

Ins and outs of the ABCG2 multidrug transporter: An update on in vitro functional assays☆ Csilla Hegedűs a, Gergely Szakács b, László Homolya a, Tamás I. Orbán a, Ágnes Telbisz a, Márton Jani c, Balázs Sarkadi a,⁎ a b c

Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, and National Blood Center, Budapest, Hungary Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary SOLVO Biotechnology, Budapest/Szeged, Hungary

a r t i c l e

i n f o

Article history: Received 18 August 2008 Accepted 3 September 2008 Available online 24 December 2008 Keywords: ABCG2 Multidrug resistance ADME-Tox Membrane-based assays Cellular assays Conformation sensitive antibody Fluorescent proteins

a b s t r a c t The major aim of this chapter is to provide a critical overview of the in vitro methods available for studying the function of the ABCG2 multidrug transporter protein. When describing the most applicable assay systems, in each case we present a short overview relevant to ABC multidrug transporters in general, and then we concentrate on the tools applicable to analysis of substrate-drug interactions, the effects of potential activators and inhibitors, and the role of polymorphisms of the ABCG2 transporter. Throughout this chapter we focus on recently developed assay systems, which may provide new possibilities for analyzing the pharmacological aspects of this medically important protein. © 2008 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basic in vitro assay systems for the investigation of ABCG2 function . . . . . . . . . . . 2.1. Membrane-based assay systems . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. ATPase assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Vesicular transport assay . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Photoaffinity labeling . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cell-based assay systems. . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Cytotoxicity assay . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Monolayer assay . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Cell surface reacting monoclonal antibodies to investigate ABCG2 function . . . . . . 4. Fluorescence-based assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Transport of fluorescent dyes . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Application of fusion proteins for studying MDR-ABC multidrug transporters . . 5. Effect of membrane lipids/cholesterol on ABCG2 multidrug transporter function . . . . 5.1. The impact of cholesterol on membrane-based ABCG2 functional assay systems . 5.2. The impact of cholesterol on cell-based ABCG2 functional assay systems . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: ABC,ATP Binding Cassette; ABCP placenta-specific ABC transporter; ADME-Tox, absorption, distribution, metabolism, excretion, and toxicology; BCRP, breast cancer resistance protein; MXR, mitoxantrone resistance protein; MDR, Multidrug Resistance; NBD, Nucleotide Binding Domain; Pgp, P-glycoprotein; TMD, Transmembrane Domain. ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “The Role of Human ABC Transporter ABCG2 (BCRP) in Pharmacotherapy”. ⁎ Corresponding author. Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, and National Blood Center, 1113 Budapest, Diószegi u. 64, Hungary. Tel./fax: +36 1 372 4353. E-mail address: [email protected] (B. Sarkadi). 0169-409X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2008.09.007

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1. Introduction As described in greater detail in other chapters of this thematic issue, ABC transporters are large membrane-bound proteins with evolutionarily conserved structure-function features. They are built from a combination of characteristic domains, namely from cytoplasmic globular ATP-binding cassette (ABC) domains (also called nucleotide binding domains, NBDs) and helical transmembrane domains (TMDs). Comprising the catalytic unit, NBDs are capable of binding and hydrolyzing ATP, thus providing the energy for the active transport of ABC substrate compounds across biological membranes. The sites interacting with the transported substrates are most likely located within the TMDs. The molecular link promoting communication and signal transmission between the NBDs and the TMDs is still unclear. In eukaryotes, ABC proteins almost exclusively transport compounds from the cytosol, either to the extracellular space or into intracellular organelles, such as the endoplasmic reticulum or vacuoles. ABC transporters need at least two NBDs and two TMDs to function. These four required domains can be present in a single polypeptide chain, as in the case of the so-called ‘full transporters’. In contrast, ‘half transporters’ possess only a single NBD and a single TMD, and the fully functional transporter is assembled via homo- or heterodimerization (or oligomerization) (for extensive reviews see [1–5]). ABC multidrug transporters form a functional class within these proteins, although with somewhat variable structures, and they belong to various subclasses of ABC proteins (B, C and G). They have the unique ability to transport a wide variety of large hydrophobic or amphipatic molecules, various xenobiotics and endobiotics. In humans, the major ABC multidrug transporters are ABCB1 (MDR1/P-glycoprotein), ABCC1 (MRP1) and its close homologs (ABCC2, C3, C4, and possibly some other members of the ABCC subfamily), and ABCG2 [2]. ABCG2 (also known as BCRP/MXR/ABCP), the second member of the ABCG subfamily, is a 655 residue half-transporter protein. To function properly, ABCG2 must form homodimers/homooligomers. In tumor cells, ABCG2 overexpression may be responsible for the emergence of so-called multidrug resistance (MDR). This phenomenon is mediated by the protein's ability to extrude a wide range of anticancer agents. Furthermore, ABCG2 is believed to play a major role at the pharmacological barriers of the human body, where its transport activity has a major influence on the ADME-Tox (absorption, distribution, metabolism excretion, and toxicology) properties of numerous pharmacological agents and natural compounds [5]. ABCG2 substrates include a vast array of chemically and targetwise unrelated compounds, such as antifolates (e.g., mitoxantrone), tyrosine kinase inhibitors (e.g., Imatinib and Gefitinib), and topoisomerase I inhibitors (e.g., topotecan and irinotecan). Soon after the discovery of ABCG2 in cancer cells, reports describing its presence in normal tissues followed. Abundant physiological expression of ABCG2 has been demonstrated in the small intestine, colon, liver, lung, kidney, capillary endothelium, central nervous system, testis, and placenta [2,6]. Based on its Hoechst33342 dye extrusion capacity, ABCG2 has been identified as a marker protein of the side population (SP) of stem cells [7]. The systemic localization of ABCG2 suggests its physiological role in the body's general protection against xenobiotics [8,9]. Bearing in mind its above-mentioned ‘omnivorous’ property and its overlapping substrate specificity, ABCG2, together with ABCB1 (MDR1/Pgp) and ABCC1 (MRP1), has been suggested to be a key element of the body's chemoimmunity defense system [2]. As a result of the promiscuity of the MDR-ABC transporters, it has been relatively easy to find high affinity substrates or inhibitors that block transport. An important aspect in studying ABCG2 function is the availability of specific, selective, high affinity inhibitors (acting in nanomolar concentrations) of this protein. As will be detailed in the subsequent sections, Fumitremorgin C (FTC) and its derivatives, Ko143 or Ko133, are especially useful for selectively blocking ABCG2 function both in vitro and in vivo [10,11].

Regarding both the expression and assay systems used for studying ABCG2 function, it is worth mentioning that ABCG2 is glycosylated on its third extracellular loop [12]. However, since glycosylation is not required for either the proper localization or function of the protein, expression systems lacking a full glycosylation apparatus are also suitable for the functional study of ABCG2 [13–15]. As mentioned above, the ABCG2 transporter works as a homodimer. The two polypeptide chains of the functional transporter are covalently linked by a disulfide bond. Interestingly, the presence of this disulfide bond is not essential for the expression and functionality of the transporter, as proven by mutation studies [16–18]. In an initial study carried out by Honjo et al., it was observed that various drug-selected human tumor cell lines expressed different ABCG2 variants, having arginine, glycine, or threonine at position 482 [19]. While R482 is the wild-type form of ABCG2, R482G and R482T were suggested to be gain-of-function mutations acquired during the course of drug selection. Following the initial observation, detailed studies confirmed that single amino-acid changes at position 482 indeed cause an altered drug resistance profile and substrate specificity compared to the wild type ABCG2 transporter. Only the wild-type ABCG2 is able to extrude the antifolate methotrexate (MTX) [20–23]. In contrast, doxorubicin and rhodamine123 are transported by the R482G and R482T protein variants, whereas they are not substrates for wtABCG2 [11,19,23,24]. Mitoxantrone, Hoechst33342, and BODIPY-Prazosin are pumped by each of the ABCG2 forms mentioned above [11,19,23,24]. Further detailed analyses also underscored the key role of position 482 in the substrate handling of the ABCG2 transporter [23,25,26]. Interestingly, to date, none of the R482 mutant ABCG2 variants have been found in the human population [2,3]. Besides some missense and frameshift mutations, more than 50 single nucleotide polymorphisms (SNPs) have been identified in the ABCG2 gene. C421A, resulting in the Q141K amino acid change, has been examined most extensively, due to its high frequency in Asian and Caucasian populations [27,28]. It is important to note that ABCG2 knock-out mice are viable and fertile [29,30], and to date, no human disease has been linked to mutations or lack of ABCG2. As ABCG2 orchestrates the systemic and cellular fate of various compounds (xenobiotics, metabolites, anticancer agents) in the body, and it is also a marker protein of stem cells, sensitive and reliable methods for analysis of its function remain of great interest. In the following sections, we provide a critical overview of the in vitro methods widely used to investigate ABCG2 transporter function and the transporter's interaction with various compounds. We focus on two recently developed assay systems, namely the cholesterol loaded membrane based assay system and the use of GFP-tagged ABCG2. 2. Basic in vitro assay systems for the investigation of ABCG2 function ABCG2 and other ABC multidrug transporters play a significant role in cancer multidrug resistance and the body's protection against xenobiotics [2,3]. Located at important pharmacological barriers, they were shown to significantly influence the ADME-Tox properties of drugs [5,31,32]. These discoveries gradually increased the pharmaceutical industry's interest in these transporters, as investigation of transporter-drug interactions is generally believed to lead to predictions of the distribution of active compounds both at cellular and systemic levels. Since transport function-related modifying effects have great influence on late phase drug development, high-throughput assay systems were required to screen for potential transporterinteracting partners. In order to gain better insight into ABCG2 transporter function, ABCG2 knock-out mice have also been generated [29,30]. However, in vivo studies are relatively expensive and low-throughput; moreover, special differences between human and mouse ABC transporters may significantly limit their human applicability. In the following sections, we describe several key in vitro assay systems employing both isolated

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membranes and intact cells, applicable for the determination of ABC multidrug transporter interactions and modulations. These assay systems, in appropriate combinations, can be used as rapid, highthroughput, valuable prediction methods, and are capable of providing a low-cost alternative to excessive animal testing. 2.1. Membrane-based assay systems One possible option for low cost and high-throughput analysis of ABC multidrug transporters is the application of isolated membrane (or even isolated protein) preparations from cells overexpressing the protein of interest [33,34]. Transfected [35], virus-infected [36], and drug-selected cell lines [37] have successfully been employed as a source of ABC transporter-expressing membranes. Due to their relatively high protein expression yields, baculovirus-infected insect ovary cells (Spodoptera frugiperda, Sf9) are widely used to attain membranes overexpressing various ABC transporters. As a caveat, transporters in Sf9 membrane vesicles may be functionally impaired due to the low cholesterol content of the insect cell membranes (see Section 6) or because of improper protein glycosylation in these cells. It has been shown that differences in the glycosylation status of several ABC multidrug transporters, including ABCG2, do not affect functionality [13–15,38,39]. Also, the abundant expression of functional membrane proteins in these heterologous cells and the relative ease of applying this transient, viral expression system make it a unique choice for studying ABC transporter function. The use of low ionic strength buffers, containing no bivalent cations, throughout the membrane preparation steps promotes the formation of open membrane sheets and/or inside-out vesicles (IOVs) [40,41]. This membrane arrangement is crucial, since the substrate binding sites have to be accessible to both ATP and the investigated molecules, and would be hidden in a right side-out vesicle (ROV) configuration. Numerous techniques for the enrichment of IOVs have also been described (e.g., dextrane or sucrose gradient centrifugation, concanavalin-A affinity chromatography). Nitrogen cavitation is another well-established method used to obtain membrane vesicles in the proper orientation [42]. Transporter-expressing membranes produced by any of these techniques can be collected by ultracentrifugation; the pellet is then suspended in hypotonic or isotonic buffer and stored at -80 °C or in liquid nitrogen until use. Vesicle composition of the membrane preparation may be assessed by enzymatic reactions assaying proteins located exclusively in the inner or the outer membrane leaflet. It should be mentioned that in various membrane preparations, particles can be formed from the plasma membrane, as well as from the endoplasmic reticulum and other intracellular membranes. This distribution may yield transporters with different maturation states (e.g., with different glycosylation or other secondary modifications), and also a mixture of membranes with different lipid assemblies. In order to avoid such problems and to reduce the possible heterogeneity of membrane-based assay systems, the isolation and reconstitution of the relevant ABC transporters is sometimes a method of choice (see further chapters in this volume) [43]. 2.1.1. ATPase assay ABC multidrug transporters utilize the chemical energy of ATP to promote the transport of substrates across the cell membrane. According to a general consensus, the transport process is tightly linked to ATP cleavage, which is corroborated by the fact that ABC protein-mediated drug transport into membrane vesicles is strictly ATP-dependent [44]. Moreover, in intact cells, a forced ABC transporter function combined with ATP synthesis inhibition results in intracellular ATP depletion [45]. In vitro measurement of ABC transporter ATPase activity can be performed either in isolated membranes containing the given transporter or in reconstituted ABC protein preparations. The first

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documentation of the applicability of such an assay system was with the ABCB1 protein (MDR1-P-gp), using both isolated membranes [46,47] and protein preparations [48]. In the case of transported substrates, an increased turnover and related ATP hydrolysis can be monitored, for example, by a simple colorimetric detection of inorganic phosphate produced throughout the process [46]. Because the disposition of the substrate molecules is not directly monitored in this ATPase assay, ABC proteins both in the inside-out vesicles and in open membrane lamellae contribute to the detected signal. By now, it has been amply documented that the modulation of ABC transporter-specific ATP hydrolysis by a given test-compound is a clear sign of its interaction with the protein. In addition, in most cases, the obtained ATPase activity profile closely reflects the nature of the interaction of the transporter with the compound of interest. Activation of the turnover suggests the presence of an actively transported compound, while inhibition (studied, for example, in a fully activated transporter) indicates a low transport rate or direct interference with the transporter function. However, it is clear that the ATPase assay system is not always suitable for distinguishing among potential substrates, inhibitors, or other types of transporter modulators [34]. ABCG2-ATPase activity is most often studied using the insect-cell membrane expression system. ABCG2 expressed in these membranes displays relatively high baseline ATPase activity, which may reflect the presence of endogenous substrates in these membranes or a partially uncoupled form of the protein [36]. Still, numerous substrates of the protein, for which a transport assay may not be applicable, have been identified based on increased ABCG2-ATPase activity. These compounds include some of the most hydrophobic substrates of ABCG2, including prazosine and various tyrosine kinase inhibitors. A new approach for increasing the sensitivity of the assay in the insect membranes is cholesterol-loading, as discussed in Section 6. Still, some of the ABCG2 substrates, transported at a low rate, may not generate an appreciable increase in inorganic phosphate liberation, and thus a false negative result would be obtained. One possible way to reduce the number of false negative hits is to apply the inhibition-type assay setup, where high ATPase activity of the transporter is triggered by a high turnover substrate, and then the modulatory effect of the test compound is monitored. In this setup, several ABCG2-interacting compounds will inhibit the increased ATPase activity caused by the other established ABCG2 substrate. Notably, even when using this configuration, distinguishing inhibitors and slowly transported substrates remains a challenge [2,5,34]. Furthermore, both stimulation and inhibition of the ATPase activity can occur at increasing drug concentrations, resulting in a bell-shaped ATPase/substrate concentration curve. One possible explanation for this phenomenon may be the existence of secondary, low-affinity substrate binding site(s) on the transporter. Once such a secondary binding site is saturated with the substrate at high concentrations, the transport process might become inhibited. If the secondary binding site has a lower affinity by several orders of magnitude, MichaelisMenten type kinetics can be observed. When a new compound is studied, it is therefore important to investigate a wide concentration range with dense sampling. 2.1.2. Vesicular transport assay Measurement of the ATP-dependent direct transport of drugs into inside-out membrane vesicles is another possibility for characterization of the nature of ABC multidrug transporter-drug interactions in vitro [44,49,50]. By using the assay in a direct setup, an increased accumulation of a transported substrate in the MDR-ABC-expressing inside-out membrane vesicles can be detected. Similarly to the ATPase assay, the vesicular transport assay can also be used in an indirect (inhibition-type) setup, where the modulatory effect of the test drug on the transport of an established ABC transporter substrate (probe) is studied [51].

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It is important to emphasize that the hydrophobic nature of the test compounds, quite characteristic for the substrates of ABC multidrug transporters, can be a major obstacle in a vesicular transport assay. Since hydrophobic compounds bearing medium or high passive membrane permeability cannot be trapped inside the membrane vesicles, no net accumulation can be measured, and that may result in false negative hits. Another possible drawback of this system might be that it requires radioactively or fluorescently labeled test-compounds. Alternatively, intravesicularly accumulated unlabeled compounds can be detected by sensitive analytical methods, such as HPLC/MS. In the case of ABCG2, several relatively polar, hydrophilic transported substrates (e.g., methotrexate, estrone-sulfate, sulfasalazine, or lucifer yellow) are available. Low passive permeability of these “probe” compounds makes them ideal for studying transportersubstrate interactions, especially in indirect (inhibitory-type) measurements [51]. The indirect assay setup is not sensitive to the passive permeability of the test compound, and therefore is not likely to produce false negative hits. However, as seen in the case of the ATPase assay, inhibitors and substrates cannot be distinguished using the indirect assay setup. A combination of the ATPase and vesicular transport assays are often employed successfully by the pharmaceutical industry to screen for potential ABCG2 substrate drugs. One advantage of the ATPase assay system over the vesicular transport assay is that it is much less expensive and is not sensitive to the passive permeability of the investigated compounds. However, because the disposition of substrates cannot be monitored in the ATPase assays, the preferred method in industry often remains the indirect vesicular transport assay with an appropriate probe compound. 2.1.3. Photoaffinity labeling There are two major types of photoaffinity labeling assays widely applied for studying ABC multidrug transporter function. The first type detects direct substrate/modulator binding to the ABC transporter proteins. Briefly, ABC transporter-expressing membranes or isolated proteins are incubated with labeled photoaffinity compounds (e.g., potential transported compounds), and then irradiated to promote covalent linkage of the labeled compound to the protein. Radioactively labeled ABC transporters are then solubilized, and separated by gel electrophoresis, and protein labeling (drug-binding) is then visualized and quantitated by autoradiography. Selective binding conditions, binding sites, and structural details can be appropriately investigated by this technology. A general drawback of these studies is that ABC multidrug transporters form low-affinity interactions with a great variety of hydrophobic compounds, and the interaction sites and intensities may directly depend on the test drug, as well as on the actual conformation of the transporter. As first demonstrated by Shukla et al., the photoaffinity analogs of prazosin [(125)I]Iodoarylazidoprazosin (IAAP) and [(3)H]azidopine are transported substrates of ABCG2, and they can be effectively used in ABCG2 photolabeling experiments [52]. Importantly, [(3)H]azidopine can covalently link to both wtABCG2 and to the ABCG2 R482T mutant variant. In numerous studies, IAAP has been demonstrated to serve as a useful probe in assay setups, when potential substrates or modulators of ABCG2 are screened and interacting sites on the protein are defined [52–60]. Generally, drugs interacting with the substratebinding site of ABCG2 effectively inhibit IAAP labeling in photolabeling experiments. Another kind of photoaffinity test system, applied to the analysis of the catalytic cycle and drug interactions with ABC transporters, is based on the use of a radioactively labeled ATP analog, 8-azido-ATP. Documented first for the ABCB1 (MDR1) multidrug transporter, both ATP binding and transition state intermediate formation during ATP hydrolysis can be investigated by this technology [61]. Under nonhydrolytic conditions, labeled 8-azido-ATP binding can be directly

followed by UV-irradiation, size fractionation, and autoradiography. Under hydrolytic conditions permitting ATP hydrolysis, the binding and release of an ATP analog is generally too rapid to be followed. However, in the presence of a phosphate-mimicking transport inhibitor (vanadate, fluoro-aluminate, or beryllium-fluoride), a transition state complex of the ABC transporter is stabilized, carrying an already hydrolyzed “trapped” nucleotide analog, stably occluded in the nucleotide binding pocket [62,63]. Therefore, by using 8-azido-[α-32P]-ATP and a “trapping” phosphate analog, the rate of the formation of this transition state can be determined after stopping the catalytic reaction by excess ATP and UV cross-linking. In general, the formation of the trapped nucleotide is proportional to the rate of transport, and thus, increased nucleotide trapping can be observed when efficiently transported substrates are present [64]. In the case of ABCG2, only cobalt complexes of the ATP analogs were found to be suitable for such studies [11]. In Section 6, we again emphasize the use of this technique for studies of the substrate handling and cholesterol-modulation of ABCG2. Since both direct photoaffinity labeling and nucleotide trapping experiments are technologically complex and difficult procedures, they are relatively inferior to the ATPase and vesicular transport assays with regard to their throughput capacity. Moreover, direct photolabeling is generally not suitable for distinguishing between substrates and inhibitors. Accordingly, although important tools for studying details of the molecular mechanism, these methods are not routinely applied by the pharmaceutical industry. 2.2. Cell-based assay systems 2.2.1. Cytotoxicity assay The most widely applied cell-based approach for investigating ABC multidrug transporter function is the cytotoxicity assay, in which the extrusion of drugs bearing cytotoxic or antiproliferative effects can be tested. Following the incubation of ABC protein expressing cells with the test compound, the number of living cells is determined, and by assessing the drug concentration resulting in 50% cell death, the IC50 values are calculated. In a direct assay setup, ABC transporter-expressing cells show elevated IC50 values for the actively extruded compounds, compared to the parental cells. In an indirect setup, combined treatment with a well-defined cytotoxic transporter substrate and a potential transporter inhibitor or competing substrate (test compound) is performed. In most cases, ABC transporter interaction with the test compound results in a decreased IC50 value for the cytotoxic ABC transporter substrate. As an example, the cytotoxicity assay proved to be an excellent method when interaction of ABCG2 with small molecule protein kinase inhibitors (PKI) was investigated. Forced expression of ABCG2 was achieved in a cell line in which the target protein kinase was a key player in driving cellular proliferation and survival processes. Compared to the parental counterparts, PKI-treated ABCG2-expressing cells showed elevated IC50 values if the given drug was a transported substrate of ABCG2. This was indeed shown to be the case for Iressa/ Gefitinib/ZD1839 [65] and Gleevec/Imatinib/STI-571 [66,67]. 2.2.2. Monolayer assay The cell-based monolayer assay is an in vitro tool for investigating vectorial transport across tight cell layers. Pharmacological barriers contain at least one tight cell layer, which is deterministic of the barrier's substance trafficking properties. Therefore, a representative immortalized cell line in a monolayer transport setup is believed to be an excellent in vitro tool for modeling pharmacological barriers [68–70]. Development of porous plastic materials with surfaces suitable for cell culture were integral to the advent of higher throughput monolayer transport experiments. At this time, ready-to-use solutions fitted in a common microplate were available and were complemented

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with inserts with a thin filter on a plastic frame. In addition to the range of 6- to 96-well plate layouts, pore size, pore density, and filter material can also be varied to fit the needs of the selected cell line. The insert effectively splits the well into two compartments, thus the solute can only pass through the filter and the cells which grow on it. Therefore, transport processes occurring between the two compartments are determined by the characteristics of the cell layer. Passive restriction in movement of a test compound by the filter membrane also has to be taken into account when calculating the specific absolute transport values. The two compartments are commonly designated as apical and basolateral, denoting the membrane orientation of polarized cell layers. The difference between the basolateral-to-apical and the apical-to-basolateral transport is assessed and the calculated ratio is referred to as ‘efflux ratio’. Compounds producing an efflux ratio greater than 2 are considered to be subjects to active efflux processes. In the case of some efficiently transported compounds, the efflux ratio falls in the range of hundreds [71]. Passive permeability of the test compounds is an important factor influencing the design of monolayer experiments, since it may counteract active transport processes. Moreover, parameters, such as incubation time and concentration range, have to be accurately chosen and tightly controlled when studying extremely hydrophobic compounds. Concentration equilibrium between the two compartments is reached rapidly and sample retrieval in the saturation phase might significantly underestimate the speed of the transport. It is important to note that without proper kinetic measurements, referring only to ratios achieved at a steady-state, active transport is often masked by high passive permeability producing false negative hits. Decreasing the initial concentration might increase the overall impact of transporters on the net transport, but this might also strain analytical capabilities. In contrast, low passive permeability compounds that are not actively transported require long incubation periods and high initial concentrations to ensure detectable final amounts in the receptor compartment [72]. ABCG2-specific monolayer assays require ABCG2-transfected mammalian cells that are capable of forming a polarized tight cell layer. MDCKII (Madin-Darby canine kidney), a cell line derived from canine kidney proximal tubule epithelial cells, is the most prominent and widely-used for this purpose [35,73,74]. Importantly, polarized MDCKII cells may express intrinsic transporters, the substrate specificities of which may overlap with those of the transfected transporter [75]. It is therefore crucial to run parallel experiments using the parental and mock-transfected cell line. In MDCKII/ABCG2 cells, the transporter localizes to the apical membrane fraction. To actually reach the intracellular substrate binding site, the compound has to cross the basolateral cell membrane; therefore, it is advised to choose a medium passive permeability probe, such as prazosin, or to incorporate appropriate exchange transporters into the basolateral membrane [76]. Absorption through the gut wall is modeled by the Caco-2 (human colon adenocarcinoma) cell line-based monolayer assay, which is an extensively used tool in the pharmaceutical industry. ABCG2, along with several other transporters, was shown to be expressed in the apical membrane of Caco-2 cells [77,78]. Therefore, in this model system, presence of transporters bearing overlapping substrate specificities (e.g., MDR1) makes exploration of ABCG2-specific interactions difficult. However, application of selective ABCG2 inhibitors [78] may pinpoint ABCG2-mediated efflux through Caco-2 monolayers [79–81]. 3. Cell surface reacting monoclonal antibodies to investigate ABCG2 function Monoclonal antibodies directed against extracellular epitopes of MDR-ABC transporters allow for both the sensitive detection and various structure-function studies of the given protein. Among MDR-

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ABC transporters, MDR1 was the first to be targeted by cell surface reacting monoclonal antibodies [82–85]. Functional modifications of MDR1 by monoclonal antibodies were first shown by Hamada et al., who generated the MDR1-specific antibodies MRK16 and MRK17 by immunizing mice with adriamycin-resistant human K562 cells [82]. In their study, Hamada et al. demonstrated that MRK16 modulated vincristine and actinomycin D transport in resistant cells and in upcoming studies, MRK16 was shown to inhibit the growth of human drug-resistant tumor cells in xenograft model systems [86–88]. Another MDR1-specific cell surface reacting monoclonal antibody, UIC2, was developed by Mechetner et al. by immunizing mice with murine BALB/c 3T3 fibroblasts engineered to express human MDR1 [85]. UIC2 was found to effectively inhibit the efflux of various MDR1 substrates, and it also increased the cytotoxicity of all the tested MDRassociated drugs. By performing UIC2-MDR1 binding experiments, it was also implied that MDR1 exists in different conformations through the different steps of the catalytic cycle, and UIC2-mediated MDR1 inhibition is mediated by the antibody's ability to trap the transporter in a transient conformation [89]. Sensitive detection and potential functional analysis of the human ABCG2 protein expressed on the cell surface became possible with the generation of the 5D3 monoclonal antibody. 5D3 was prepared by immunizing mice with human ABCG2-expressing intact murine fibroblasts [7], and the antibody was reported to inhibit ABCG2 transporter function [90,91]. A detailed study reported that 5D3 binding strongly depends on the actual conformation of the ABCG2 protein [91]. Experiments performed on both intact cells and isolated membrane fragments expressing ABCG2 revealed that various steps of the catalytic cycle could be analyzed through changes in the 5D3 binding capacity of ABCG2. Stabilization of ABCG2 in the prehydrolytic state and the formation of the catalytic intermediate by vanadate both represented low-affinity 5D3 binding forms. In contrast, transport inhibition by Ko143 or inhibitory concentrations of the ABCG2substrate flavopiridol, as well as cellular ATP depletion, resulted in the appearance of the high-affinity 5D3 binding form. Furthermore, in accordance with literature data suggesting such an effect [16,17], in a recent report [92], it was documented that the presence of an intact intramolecular S-S bridge in the third extracellular loop of ABCG2 (mediated by Cys592 and Cys608) plays a crucial role in modulating the appropriate conformation for both the 5D3 antibody and substrate binding of the transporter. In contrast, the presence of an intermolecular S-S bridge in the ABCG2 dimer is not required for 5D3 binding. These results suggest that the ABCG2-specific 5D3 monoclonal antibody is an excellent candidate for multiple applications: besides allowing for fast-and-easy flow-cytometry detection and sorting of ABCG2-expressing intact cells, it is also a tool for further structure-function studies and investigations of specific drug interactions with ABCG2. 4. Fluorescence-based assays 4.1. Transport of fluorescent dyes Transported substrates show lower accumulations in cells expressing MDR-ABC transporters, and inhibitors increase drug accumulation up to the level observed in parental cells. Cellular concentrations of compounds with intrinsic fluorescence can be directly measured, and their steady state accumulation, net efflux or uptake, can be determined. However, cellular sequestration, “membrane leakage”, intracellular binding, and dependence of fluorescence on the intracellular milieu of the substrates complicate quantification. Therefore, indirect transport assays, where substrates and inhibitors are identified by following the transport of a reporter substrate, have been developed. Reporter substrates should generally not be toxic; their cellular fate should be well-characterized. Several indirect assays that use fluorescent compounds meeting the above criteria

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have been developed. In case of MDR1 and MRP1, calcein-AM proved to be an ideal reference substrate [2,93]. Detailed investigations proved that the calcein transport assay correlates with functional MDR1 expression and is applicable for the prediction of multidrug resistance in acute leukemia [94]. ABCG2 does not transport calcein-AM. Instead, the accumulation of various other fluorescent dyes may be followed for detection of ABCG2 function and in drug screening projects [23]. ABCG2 transports several fluorescent substrates, including topotecan, flavopiridol, BODIPYprazosin, and mitoxantrone. In a systematic study, Robey et al. screened several fluorescent compounds in a series of ABCG2 overexpressing and control cell lines, aiming to establish a functional assay for ABCG2. The authors found that the FTC-inhibitable efflux of mitoxantrone and prazosin was detectable even in cell lines expressing low levels of ABCG2, while rhodamine123 and LysoTracker could not be reliably detected [24]. However, since the transport of these compounds is not specific for ABCG2, the combined use of ABCG2specific inhibitors is advocated. The use of specific inhibitors enables the simultaneous analysis of the three major MDR-ABC transporters [95]. A cell-based multiplex assay combines high-throughput flow cytometry with color coding of cells to allow simultaneous determination of compound efficacy and selectivity in a HTS format. The “duplex MDR assay” consists of a pair of overlapping transporter assays, one using JC1 to quantify MDR1 and ABCG2 activity and the other using CaAM to quantify MDR1 and MRP1 activity. Pheophorbide a (PhA), a chlorophyll catabolite, was shown to be an ABCG2 substrate based on Abcg2(-/-) knockout mouse experiments [29]. Based on the phenotype of the knockout mice, an in vitro functional assay was developed to measure ABCG2 function. The pheophorbide-assay measures the FTC-sensitive accumulation of PhA in cell lines overexpressing ABCG2. PhA is a specific ABCG2 substrate, since its transport was only observed in cell lines overexpressing ABCG2, and not MDR1 or MRP1. FTC-inhibitable PhA transport was found to correlate with cell surface ABCG2 expression, as measured by the anti-ABCG2 antibody 5D3 [96]. The pheophorbide assay proved suitable for the characterization of ABCG2 function as well as the highthroughput search for novel inhibitors of ABCG2 [58,97]. ABCG2 was shown to transport several other photosensitizers with structures similar to that of pheophorbide A, such as pyropheophorbide A methyl ester, chlorin e6, and protoporphyrin IX [98]. The so called “side population (SP)” of progenitor cells represent pluripotent stem cells in a variety of tissues and can be identified based on their ability to actively extrude the fluorescent Hoechst 33342 dye [7]. Similarly, there is a distinct side population of cells with high drug efflux capacity in human tumor cells [99]. Elegant work from several laboratories has proven that the efflux of Hoechst 33342 is mediated by ABCG2, which defines the SP stem cell phenotype, suggesting that the physiological function of the transporter is to protect stem cells from cytotoxic substrates [7,100]. While ABCG2overexpressing cells can be readily identified and characterized by flow cytometry analysis of Hoechst 33342 efflux, the assay requires an ultraviolet (UV) laser source, usually necessitating an expensive and maintenance-intensive argon- or krypton-ion gas laser. As noted earlier, both substrates and inhibitors can hinder the accumulation of a fluorescent reporter substrate. Therefore, the indirect setup does not distinguish between substrates and inhibitors. 4.2. Application of fusion proteins for studying MDR-ABC multidrug transporters Creating a specific tool to study various functional aspects of multidrug transporter proteins is generally a challenge, as the available methods are usually suitable for only limited types of assays. Besides using specific antibodies, a widely applied method involves generation of a fusion species with a bioluminescent molecule. One advantage of this technique is that it provides an opportunity to

examine dynamic processes in living cells, such as subcellular protein trafficking, changes in protein localization, or protein degradation. In addition, for an ABC transporter, such a fusion molecule would provide an easy-to-use cell-based assay system for identifying substrates or inhibitors, as well as examining drug-transporter interactions. The problem with tagged membrane transporters, however, is that the generated fusion species often becomes inactive or mislocalized, thus greatly hindering its use in functional or localization studies. Due to these technical difficulties, to date, only a limited number of publications have described either functional MDR-ABC transporters tagged with a fluorescent protein [101] or partially active transporters suitable for certain types of studies [14]. It has been shown that the N-terminally GFP-tagged ABCG2 transporter is fully functional, whereas the C-terminally tagged species is not correctly expressed and/or localized (unpublished data). These results are consistent with the findings that removal of 5-10 amino acids from the N-terminal part of ABCG2 is not harmful, whereas similar truncations at the C-terminal end can drastically alter the function of the protein [14]. The N-terminally GFP-tagged ABCG2 (GFPABCG2) is fully functional in drug-stimulated ATPase and in vesicular transport assays; moreover, it shows the same subcellular localization and similar cell surface epitope conformational changes (“5D3-shift”) as the wild type ABCG2 [102]. After transient transfection of cells with plasmids expressing GFP-ABCG2 or the nonfunctional GFP-ABCG2 K86M mutant protein, the fluorescent fusion species were found to be well-suited for rapid flow cytometry or fluorescence microscopy applications [102]. Both protein detection and activity measurements can be performed rapidly: ABCG2-expressing cells can be sorted even after a short post-transfection period, without the usual timeconsuming laboratory practice of cloning and selection. GFP-ABCG2 is also suitable for studying drug transport by flow cytometry, as demonstrated by measuring the transport of mitoxantrone, a known ABCG2 substrate [102]. The assay can be performed as early as 48 hours post-transfection, since GFP-positive ABCG2expressing cells can be distinguished. Using a specific inhibitor, either Ko143 or FTC [10], and comparing the results with those of the GFPtagged K86M species, the transport characteristics of the applied fluorescent drug can be rapidly and reliably assessed. This double control system makes the assay especially reliable and informative, allowing the investigator to identify modulators or inhibitors of the transporter, or confirm potential substrates in a short period of time. The major advantage of a GFP-ABCG2-based assay is that the method can be further refined for a high-throughput setup using the modern microplate-based fluorescence devices, such as FLIPR [103]. Furthermore, image-analysis software make such a cell-based kinetic assay feasible for automated and rapid functional drug screening in a 96- or 384-well layout. 5. Effect of membrane lipids/cholesterol on ABCG2 multidrug transporter function Membrane lipids, especially cholesterol, have been implicated in the regulation of various membrane proteins, including several ABC transporters. Membrane micro-domains with increased cholesterol content have also been suggested to play an important role in such regulation [104–107]. ABCG2 was shown to be located in rafts of mammalian cell membranes, and it could be co-immunoprecipitated with caveolin-1. Furthermore, it has been shown that destroying membrane rafts by cholesterol depletion causes about a 40% decrease in ABCG2 activity [108]. ABCG2 transport activity depends on the cholesterol content of its membrane environment, and this phenomenon actually affects all types of assay systems used to study ABCG2 function [109]. It has been shown that sulfated conjugates of bile acids and steroids can be transported by ABCG2 expressed in mammalian cells [35,110,111]. Furthermore, unconjugated neutral steroids can be transported by ABCG2 expressed in Lactococcus lactis [112,113]. Sterol

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binding elements were also identified in ABCG2 [114]. In the following paragraphs, we discuss the modifying effect of cholesterol on ABCG2 function, as assessed in the in vitro assays described above. 5.1. The impact of cholesterol on membrane-based ABCG2 functional assay systems As mentioned in Section 3.A., ABC transporter-expressing insect Sf9 membranes are widely used to perform the general membranebased experiments for high-throughput screening of transporter-drug interactions. Nevertheless, membranes prepared from insect cells have a low level of intrinsic cholesterol [115–117], which might result in certain difficulties and discrepancies when studying ABCG2 function in Sf9 membranes. When expressed in Sf9 membranes, human ABCG2 shows a relatively high intrinsic ATPase activity that can only be moderately stimulated by transported compounds (the best substrates show only a mere 20% stimulatory effect). In contrast, Sf9 membranes engineered to overexpress the ABCG2 R482G or ABCG2 R482T mutants show much higher substrate stimulation. When expressed in mammalian membrane vesicles, the activity of the wild-type ABCG2 protein is comparable to that of the mutant variants; however, the low protein expression yield of this system greatly counterbalances this advantage. Therefore, to screen for potential ABCG2 substrates, the ABCG2 R482 mutant species expressed in Sf9 membranes are generally used; however, these variants show somewhat altered substrate specificity [2]. To overcome these problems, the cholesterol content of the transporter-expressing Sf9 membranes may be increased [39,109]. In biological systems, the most widespread tool for cholesterol loading is the cholesterol-cyclodextrin complex. Cyclodextrin (CD) is a barrel-like sugar molecule that can trap different hydrophobic compounds fitting in its cavity. The non-specific binding between CD and the test compound is low and is mostly determined by the size of the hydrophobic compound. The cholesterol-CD complex can be applied directly to Sf9 membranebased assays in a 1-5 mM concentration. Another option is loading membranes with cholesterol during the course of the membrane preparation procedure. The advantage of this setup is the ability to remove excess CD and cholesterol-CD complexes by centrifugation. By utilizing the cholesterol-CD complex, transporter-expressing Sf9 membranes could be loaded to obtain different cholesterol levels. Cholesterol loading of the Sf9 membrane vesicles resulted in a

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significantly increased substrate-effect on the ABCG2 ATPase. Cholesterol loading of the membranes resulted in higher Vmax values for several compounds, while the Km values for both ATP and the transported substrates were unchanged. Several ABCG2 substrates (such as SN-38, flavopiridols, and doxorubicin), which do not cause a measurable stimulation of ABCG2-ATPase activity in untreated Sf9 cell membranes, produce detectable ATPase stimulation in cholesterol-loaded membranes. Interestingly, the substrate-stimulated ATPase activity of the ABCG2 R482G and ABCG2 R482T mutant variants cannot be further increased by cholesterol loading. Cholesterol loading of Sf9 membranes also increased the sensitivity of the other two membrane-based assay systems for studying ABCG2, namely the vesicular transport and photoaffinity labeling systems. The methotrexate or estradiol-glucuronide transport capacity of ABCG2 in cholesterol-loaded membranes increased by up to 20fold (again mostly affecting the Vmax), and an increase in the substrate stimulation of ABCG2 photoaffinity labeling by azido-ATP was also observed. 5.2. The impact of cholesterol on cell-based ABCG2 functional assay systems Cholesterol makes up to 20% of the total lipids in mammalian cell membranes and is unequally distributed, accumulating in membrane microdomains (‘rafts’). Partial cholesterol depletion can be achieved by using cyclodextrins (CD), widely applied for the investigation of raftlocated membrane proteins [118]. Mild CD treatment typically decreases cellular cholesterol levels by 20-40%, extracting cholesterol mostly from the plasma membrane. It is also possible to increase cellular cholesterol levels by treating the cells with cholesterol-loaded CD. In ABCG2 expressing mammalian cell lines, mild cholesterol depletion, not affecting cell survival, significantly increased the intracellular accumulation of fluorescent ABCG2 substrates (Hoechst33342, mitoxantrone, and pheophorbide A) [109]. The loss of ABCG2 transport function was further supported by the finding that dye accumulation in cholesterol-depleted cells was not significantly increased in the presence of ABCG2 inhibitors FTC or Ko143. Elevation of normal cellular cholesterol levels by treatment with a cholesterol-CD complex was relatively ineffective in fluorescent dye accumulation assays. All these in vitro studies suggest that membrane cholesterol significantly modulates the transport capacity and the turnover rate of the ABCG2 multidrug transporter. Whether cholesterol is an additional

Fig. 1. Application of the in vitro assay systems discussed in this chapter for ABCG2 multidrug transporter and drug interaction screening. Summary of the membrane-based assays and cellular assays, and the relative potential throughput of the assay systems. The throughput of an assay system is roughly inversely correlated with the cost of its application in drug discovery / screening.

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substrate for ABCG2 or rather a co-factor or allosteric modulator promoting its proper transport function remains an open question. 6. Conclusions Human ABCG2 is a key multidrug transporter, and in vitro functional assays for studying this protein may significantly help with ongoing drug research and development. The major advantage of these assays is that selected combinations are able to characterize the affinity and the nature of the transporter-drug interactions; furthermore, these assays can be performed at a relatively low cost and highthroughput for a large number of new compounds (see Fig. 1). More detailed studies, especially those required for lead compounds or agents already near clinical applications, can also be performed using a broader combination of more sophisticated membrane-based or specific cellular assays. Since the wild type ABCG2 and its polymorphic variants have species-dependent and cholesterol-modulated interaction profiles, in vitro assays using the human proteins, reflecting the cholesterol composition of mammalian cell membranes, are often used. Some of the new assays that are described here or are currently under development may further increase the applicability of this important set of tools. Acknowledgments This work has been supported by OTKA (AT 048986, PF60435, K68936 and NK72057) NKFP, FP6-INTHER, FP6-MEMTRANS, NEDO, ETT, NIH (R01TW007250) and Marie Curie Grants (046560, 041547). Gergely Szakács is the recipient of a János Bolyai Scholarship and a Special Fellow Award from the Leukemia and Lymphoma Society. References [1] M. Dean, T. Fojo, S. Bates, Tumour stem cells and drug resistance, Nat. Rev., Cancer 5 (2005) 275–284. [2] B. Sarkadi, L. Homolya, G. Szakacs, A. Varadi, Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system, Physiol. Rev. 86 (2006) 1179–1236. [3] P. Krishnamurthy, J.D. Schuetz, Role of ABCG2/BCRP in biology and medicine, Annu. Rev. Pharmacol. Toxicol. 46 (2006) 381–410. [4] G. Szakacs, J.K. Paterson, J.A. Ludwig, C. Booth-Genthe, M.M. Gottesman, Targeting multidrug resistance in cancer, Nat. Rev. Drug Discov. 5 (2006) 219–234. [5] G. Szakacs, A. Varadi, C. Ozvegy-Laczka, B. Sarkadi, The role of ABC transporters in drug absorption, distribution, metabolism, excretion and toxicity (ADME-Tox), Drug Discov. Today 13 (2008) 379–393. [6] M. Maliepaard, G.L. Scheffer, I.F. Faneyte, M.A. van Gastelen, A.C. Pijnenborg, A.H. Schinkel, M.J. van De Vijver, R.J. Scheper, J.H. Schellens, Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues, Cancer Res. 61 (2001) 3458–3464. [7] S. Zhou, J.D. Schuetz, K.D. Bunting, A.M. Colapietro, J. Sampath, J.J. Morris, I. Lagutina, G.C. Grosveld, M. Osawa, H. Nakauchi, B.P. Sorrentino, The ABC transporter Bcrp1/ ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype, Nat. Med. 7 (2001) 1028–1034. [8] B. Sarkadi, C. Ozvegy-Laczka, K. Nemet, A. Varadi, ABCG2 – a transporter for all seasons, FEBS Lett. 567 (2004) 116–120. [9] J. Cervenak, H. Andrikovics, C. Ozvegy-Laczka, A. Tordai, K. Nemet, A. Varadi, B. Sarkadi, The role of the human ABCG2 multidrug transporter and its variants in cancer therapy and toxicology, Cancer Lett. 234 (2006) 62–72. [10] J.D. Allen, A. van Loevezijn, J.M. Lakhai, M. van der Valk, O. van Tellingen, G. Reid, J.H. Schellens, G.J. Koomen, A.H. Schinkel, Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C, Mol. Cancer Ther. 1 (2002) 417–425. [11] C. Ozvegy, A. Varadi, B. Sarkadi, Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation, J. Biol. Chem. 277 (2002) 47980–47990. [12] T. Litman, U. Jensen, A. Hansen, K.M. Covitz, Z. Zhan, P. Fetsch, A. Abati, P.R. Hansen, T. Horn, T. Skovsgaard, S.E. Bates, Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2, Biochim. Biophys. Acta 1565 (2002) 6–16. [13] N.K. Diop, C.A. Hrycyna, N-Linked glycosylation of the human ABC transporter ABCG2 on asparagine 596 is not essential for expression, transport activity, or trafficking to the plasma membrane, Biochemistry 44 (2005) 5420–5429. [14] T. Takada, H. Suzuki, Y. Sugiyama, Characterization of polarized expression of point- or deletion-mutated human BCRP/ABCG2 in LLC-PK1 cells, Pharmacol. Res. 22 (2005) 458–464.

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