The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation.

Several members of different families of the ATP-binding cassette (ABC) superfamily of transport proteins are capable of transporting an extraordinarily structurally diverse array of endo- and xenobiotics and their metabolites across cell membranes. Together, these transporters play an important role in the absorption, disposition and elimination of these chemicals in the body. In tumor cells, increased expression of these drug transporters is associated with resistance to multiple chemotherapeutic agents. In this review, current knowledge of the biochemical, physiological and pharmacological properties of nine members of the multidrug resistance protein (MRP)-related ABCC family (MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, MRP7, ABCC11 and ABCC12) as well as the G family member, ABCG2/BCRP, are summarized. A focus is placed on the structural similarities and differences of these drug transporters as well as the molecular determinants of their substrate specificities and transport activities. Factors that regulate expression of the MRP-related proteins and ABCG2/BCRP are also reviewed.

RU49953: a non-hormonal steroid derivative that potently inhibits P-glycoprotein and reverts cellular multidrug resistance.

Progesterone and the antiprogestin RU38486 have been reported as non-transported modulators of P-glycoprotein-mediated drug efflux. However, their hormonal properties limit their potential for clinical trials. The present work shows that some derivatives from either progesterone/RU38486 or estradiol, displaying differential interaction with hormone receptors, bind to P-glycoprotein and chemosensitize the growth of MDR1-transfected cells to vinblastine more strongly than does RU38486. Structure comparison of the compounds indicates that the highly hydrophobic estradiol derivative RU49953, which does not interact with any hormone receptor, inhibits P-glycoprotein-mediated drug efflux very efficiently, as monitored by flow cytometry, and prevents drug site photoaffinity labeling by azidopine. It induces a much higher chemosensitization than the well-known P-glycoprotein modulator verapamil, which is itself more efficient than RU38486. RU49953 therefore constitutes a promising new lead for steroid-type modulators of multidrug resistance.

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters.

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding.

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.

Protein kinase C effectors bind to multidrug ABC transporters and inhibit their activity.

P-Glycoprotein and homologous multidrug transporters contain a phosphorylatable linker sequence that was proposed to control drug efflux on the basis that it was indeed phosphorylated in vitro and in vivo, and that inhibitors of protein kinase C (PKC) inhibited both P-glycoprotein phosphorylation and activity. However, site-directed mutagenesis of all phosphorylatable residues did not alter the drug resistance. The present work shows that PKC effectors are able to bind directly to multidrug transporters, from either cancer cells (mouse P-glycoprotein), yeast (Saccharomyces cerevisiae Pdr5p), or protozoan parasite (Leishmania tropica ltmdr1), and to inhibit their energy-dependent drug-efflux activity. The binding of staurosporine and derivatives such as CGP 41251 is prevented by preincubation with ATP, suggesting at least partial interaction at the ATP-binding site. In contrast, more hydrophobic compounds such as calphostin C and CGP 42700 bind outside the ATP-binding site and strongly interfere with drug interaction. A direct correlation is obtained between the efficiencies of PKC effectors to inhibit energy-dependent interaction of rhodamine 6G with yeast Pdr5p, to promote intracellular drug accumulation in various multidrug resistant cells, and to chemosensitize growth of resistant cells. The noncompetitive inhibition by PKC effectors of rhodamine 6G interaction with Pdr5p suggests that the binding might interfere with signal transduction between nucleotide hydrolysis and drug interaction. The overall results indicate that the multidrug transporters from different species display common features for interaction with PKC inhibitors. The hydrophobic derivative of staurosporine, CGP 42700, constitutes a potentially powerful modulator of P-glycoprotein-mediated multidrug resistance.

C-Isoprenylation of flavonoids enhances binding affinity toward P-glycoprotein and modulation of cancer cell chemoresistance.

Previous studies have shown that flavones bind to P-glycoprotein (Pgp) with higher affinity than isoflavones, flavanones, and glycosylated derivatives. In the present work, a series of C- or O-substituted hydrophobic derivatives of chrysin were synthesized to further investigate structural requirements of the A ring toward Pgp modulation. Increasing hydrophobicity at either position 6, 8, or 7 increased the affinity of in vitro binding to a purified cytosolic domain of Pgp, but only benzyl and 3,3-dimethylallyl C-substitution produced a high maximal quenching of the protein intrinsic fluorescence. Inhibition of membrane Pgp within leukemic cells, characterized by intracellular drug accumulation, was specifically produced by isoprenylated derivatives, with 8-(3,3-dimethylallyl)chrysin being even more efficient than the commonly used cyclosporin A.

High-affinity binding of silybin derivatives to the nucleotide-binding domain of a Leishmania tropica P-glycoprotein-like transporter and chemosensitization of a multidrug-resistant parasite to daunomycin.

In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters.

B-ring substituted 5,7-dihydroxyflavonols with high-affinity binding to P-glycoprotein responsible for cell multidrug resistance.

Starting from the interaction of galangin (3,5,7-trihydroxyflavone) with a cytosolic nucleotide-binding domain of P-glycoprotein, a series of flavonol derivatives was synthesized and tested for their binding affinity towards the same target. The 5,7-dihydroxy-4'-iodoflavonol and 5,7-dihydroxy-4'-n-octylflavonol derivatives displayed much higher binding affinities, with respective increases of 6- and 93-fold as compared to galangin.

Prenylated xanthones as potential P-glycoprotein modulators.

Dimethylallyl (DMA) derivatives of a naturally occurring xanthone (decussatin 1) were prepared. Their activity as potential P-glycoprotein inhibitors was monitored by affinity of direct binding and compared to that of corresponding DMA-flavones. Both classes of compounds exhibited the same structure-activity relationships. Decreasing polarity enhanced the binding affinity for the P-glycoprotein C-terminal cytosolic domain since DMA derivatives were more active, but unsubstituted hydroxyl group close to the carbonyl was required for efficient activity.

Prenyl-flavonoids as potent inhibitors of the Pdr5p multidrug ABC transporter from Saccharomyces cerevisiae.

The Pdr5p multidrug ABC ("ATP-binding cassette) transporter was highly overexpressed in plasma membranes from a yeast strain exhibiting both pdr1-3 gain-of-function mutation in the transcription factor-encoding gene PDR1 and disruption of genes encoding other plasma membrane ABC transporters. Solubilized and purified Pdr5p displayed a tryptophan-characteristic intrinsic fluorescence, whose quenching was used to monitor interactions with substrates and effectors. The transporter exhibited a magnesium-dependent binding affinity for ATP and its fluorescent analogue 2'(3')-N-methylanthraniloyl-ATP, producing a marked fluorescence resonance-energy transfer. It also bound a series of known drug substrates and modulators. Interestingly, yeast Pdr5p interacted with flavonoids recently found to bind to cancer cell P-glycoprotein and to the protozoan parasite multidrug transporter. The extent of high-affinity binding of prenyl-flavonoids to purified Pdr5p was correlated to their efficiency to inhibit energy-dependent quenching of rhodamine 6G fluorescence catalyzed by Pdr5p-enriched plasma membranes. The hydrophobic flavonoid derivative 6-(3, 3-dimethylallyl)galangin was the most efficient, with a K(i) of 0.18 microM for competitive inhibition of the MgATP-dependent quenching of rhodamine 6G fluorescence. In contrast, inhibition of either ATP or UTP hydrolysis occurred at much higher concentrations and appeared to be noncompetitive. Prenyl-flavonoids therefore behave as potent inhibitors of drug binding to the yeast Pdr5p ABC transporter.

The flavanolignan silybin and its hemisynthetic derivatives, a novel series of potential modulators of P-glycoprotein.

A new series of potential flavonoidic modulators of P-glycoprotein activity has been prepared. The flavanolignan silybin was first oxidised to dehydrosilybin and then C-alkylated with either prenyl or geranyl bromide. The resulting isoprenoid dehydrosilybins were shown to display high in vitro affinities for direct binding to P-glycoprotein, which ranged them among the best flavonoids ever tested.

P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships.

Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.

Correlation between the affinity of flavonoids binding to the cytosolic site of Leishmania tropica multidrug transporter and their efficiency to revert parasite resistance to daunomycin.

The C-terminal nucleotide-binding domain (NBD2) of a P-glycoprotein-like transporter, encoded by the ltrmdr1 gene in Leishmania tropica and involved in parasite multidrug resistance (MDR), was overexpressed in Escherichia coli as a hexahistidine tagged protein and purified. The L. tropica recombinant domain efficiently bound fluorescent derivatives of ATP, the hydrophobic steroid analogue RU 486, and different classes of flavonoids with the following efficiency: flavone > flavanone > isoflavone > glucorhamnosyl-flavone > chromone. The affinity for flavones was dependent on the presence of hydroxyl groups at positions 5 and 3 and was further increased by a hydrophobic 1,1-dimethylallyl substituent at position 8. When flow cytometry was used to measure daunomycin accumulation in a MDR L. tropica line, a reversing effect was observed with flavones such as dimethylallyl-kaempferide at low concentration or apigenin at higher concentration, but neither with the glucorhamnosyl derivative rutin nor with the isoflavone genistein. The in vivo reversing effect of dimethylallyl-kaempferide was correlated to a high inhibition of MDR cell growth in the presence of daunomycin. The results suggest that flavone inhibition of both daunomycin efflux and parasite growth in the presence of the drug correlates to direct binding of the compound to cytosolic domain of the P-glycoprotein-like transporter.

Synthesis and biological activity of 4-alkoxy chalcones: potential hydrophobic modulators of P-glycoprotein-mediated multidrug resistance.

A series of 4-alkoxy-2',4',6'-trihydroxychalcones have been synthesized and evaluated for their ability to inhibit P-glycoprotein-mediated multidrug resistance (MDR) by direct binding to a purified protein domain containing an ATP-binding site and a modulator-interacting region. The introduction of hydrophobic alkoxy groups at position 4 led to much more active compounds as compared to the parent chalcone. The binding affinity increased as a function of the chain length, up to the octyloxy derivative for which a K(D) of 20 nM was obtained.

Halogenated chalcones with high-affinity binding to P-glycoprotein: potential modulators of multidrug resistance.

Previous studies have shown that flavonoids are modulators of the transmembrane P-glycoprotein (P-gp) which mediates cell multidrug resistance. Some structural elements have been identified which seem to contribute to these compounds' activity. In the present study, a series of halogenated chalcones was prepared to further explore the structural requirements for the P-gp modulation. Four halogenated chalcones have been synthesized and evaluated as potential modulators of P-gp-mediated multidrug resistance of cancer cells by in vitro assays using a purified recombinant domain of the transporter containing the modulator binding site. Halogenated chalcones exhibited high-affinity binding, the 2',4', 6'-trihydroxy-4-iodochalcone behaving as the most potent compound with a KD value in the nanomolar range.

Flavonoids: a class of modulators with bifunctional interactions at vicinal ATP- and steroid-binding sites on mouse P-glycoprotein.

A hexahistidine-tagged C-terminal nucleotide-binding domain (H6-NBD2) from mouse P-glycoprotein was designed, overexpressed, and purified as a highly soluble recombinant protein. Intrinsic fluorescence of its single tryptophan residue allowed monitoring of high-affinity binding of 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP), a fluorescent ATP derivative that induces a marked quenching correlated to fluorescence resonance-energy transfer. H6-NBD2 also bound all flavonoids known to modulate the multidrug resistance phenotype of P-glycoprotein-positive cancer cells, with similar affinities and relative efficiencies. Flavones (like quercetin or apigenin) bound more strongly than flavanones (naringenin), isoflavones (genistein), or glycosylated derivatives (rutin). Kaempferide, a 4'-methoxy 3,5,7-trihydroxy flavone, was even more reactive and induced a complete quenching of H6-NBD2 intrinsic fluorescence. Kaempferide binding was partly prevented by preincubation with ATP, or partly displaced upon ATP addition. Interestingly, kaempferide was also able to partly prevent the binding of the antiprogestin RU 486 to a hydrophobic region similar to that recently found, close to the ATP site, in the N-terminal cytosolic domain. Conversely, RU 486 partly prevented kaempferide binding, the effect being additive to the partial prevention by ATP. Furthermore, MANT-ATP binding, which occurred at the ATP site and extended to the vicinal steroid-interacting hydrophobic region, was completely prevented or displaced by kaempferide. All results indicate that flavonoids constitute a new class of modulators with bifunctional interactions at vicinal ATP-binding site and steroid-interacting region within a cytosolic domain of P-glycoprotein.