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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

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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)
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   A  NTIMICROBIAL   A  GENTS AND  C HEMOTHERAPY ,0066-4804/01/$04.00  0 DOI: 10.1128/AAC.45.2.439–446.2001Feb. 2001, p. 439–446 Vol. 45, No. 2Copyright © 2001, American Society for Microbiology. All Rights Reserved. High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a  Leishmania tropica  P-Glycoprotein-LikeTransporter and Chemosensitization of a Multidrug-ResistantParasite to Daunomycin JOSE´ M. PE´REZ-VICTORIA, 1 F. JAVIER PE´REZ-VICTORIA, 1 GWENAE¨LLE CONSEIL, 2 MATHIAS MAITREJEAN, 3 GILLES COMTE, 3 DENIS BARRON, 3  ATTILIO DI PIETRO, 2 SANTIAGO CASTANYS, 1  AND  FRANCISCO GAMARRO 1 *  Instituto de Parasitologia y Biomedicina “Lo´pez-Neyra,” Consejo Superior de Investigaciones Cientı´ficas, Granada,Spain, 1  and Institut de Biologie et Chimie des Prote´ines, UPR 412 du CNRS, Lyon, 2  and Laboratoire des Produits Naturels, UMR-CNRS 5013, Universite´ Claude Bernard de Lyon, Villeurbanne, 3  France Received 25 May 2000/Returned for modification 5 September 2000/Accepted 8 November 2000 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 compoundslacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-bindingdomain of the  L. tropica  P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promas-tigote forms of a multidrug-resistant  L. tropica  line overexpressing the transporter. Oxidation of the flavanonolsilybin 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 thebinding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domaincorrelated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydro-silybin effectively sensitized multidrug-resistant  Leishmania  spp. to daunomycin. The cytosolic domains aretherefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters. Drug resistance has become a major impediment to thetreatment of diseases caused by protozoan parasites, whichthreaten the life of nearly one-quarter of the human popula-tion. Among parasitic infections, World Health Organizationstatistics show that the incidence of leishmaniasis has increased42-fold between 1985 and 1998 and has become the secondleading cause of death (18) worldwide. Chemotherapy remainsthe only effective way to control infections. The conventionalclinical drugs, pentavalent antimonials in the form of Glucan-time and Pentostam (reviewed in reference 27), are not veryefficient due to their toxicity and the increased appearance of drug resistance (14). ATP-binding cassette (ABC) transportershave been found to be involved in  Leishmania  species in vitroselected for metal resistance (reviewed in reference 30). Themultidrug resistance (MDR) phenotype due to P-glycoprotein(Pgp)-like transporters has been extensively characterized incancer cells (1, 13) and protozoan parasites (41), including  Plasmodium  (45) and  Leishmania  (4, 5, 17) spp. Pgp is an ATP-dependent pump that exports a wide range of drugs fromthe cell, decreasing their intracellular concentration and pre- venting their cytotoxic activity. Pgp belongs to the ABC super-family of transporters. It consists of two homologous halves,each comprising a transmembrane domain involved in drugefflux, and a cytosolic nucleotide-binding domain (NBD) re-sponsible for ATP binding and hydrolysis. Pgp can be inhibitedin vitro by agents such as verapamil and cyclosporine, whichcompete with drug binding to the transmembrane domains(16). However, most of these inhibitors are also pumped sub-strates and therefore require high concentrations for effectiveinhibition. These concentrations produce undesirable side ef-fects. In addition, these classical modulators of drug efflux incancer cells only poorly sensitize the MDR phenotype in  Leish- mania  parasites (4, 17, 33). Thus, new classes of more specific,nontransported inhibitors of Pgp-like transporters with lowerhost toxicity need to be developed. Recently, it has been de-scribed that NBDs can be the target for inhibitors of Pgp-liketransporters (7, 11, 33). Flavonoids, which constitute a well-known class of natural inhibitors of different ATP-binding pro-teins (28), with contradictory modulation effects on differentMDR cells (8, 15, 33, 37– 39), bind to transporter NBDs. Theyinteract with both the ATP-binding site and a vicinal hydro-phobic region (7, 9, 33), inhibiting drug efflux and reversing theresistance phenotype of an  L. tropica  MDR line (33). Theirefficient modulation of drug efflux has been correlated withtheir affinity binding to the transporter cytosolic domain (33).Silymarin is a mixture of flavanolignans isolated from themedicinal plant  Silybum marianum , with silybin (or silybinin)(Fig. 1A) as the main component (31). These natural com-pounds are well-established hepatoprotectants and are used inEurope and Asia for the clinical treatment of liver diseases with different aetiologies (reviewed in references 25 and 32).Silymarin is well tolerated as a therapeutic agent and is largelydevoid of adverse effects (25, 32). It has been recently mar-keted in the United States and in Europe as a nutritionalsupplement. Silymarin may directly affect cholesterol metabo- * Corresponding author. Mailing address: Instituto de Parasitologia y Biomedicina “Lo´pez-Neyra,” Consejo Superior de InvestigacionesCientificas, c/Ventanilla 11, 18001 Granada, Spain. Phone: 34-958-805185. Fax: 34-958-203323. E-mail: gamarro@ipb.csic.es.439   on O c  t   o b  er 4  ,2  0 1  5  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om   lism and is therefore considered as a potential hypocholester-olemic (41). In vitro studies indicate that silymarin and silybinmay help to prevent and treat breast, prostate, skin and ovariancancers (32, 36, 46). Silybin also appeared to be synergistic withdoxorubicin in a doxorubicin-resistant cell line, probably byinhibiting Pgp function (36). Thus, silybin, either alone or incombination with other cytotoxic drugs, is currently beingtested in patients with advanced ovarian cancer (36).The binding of flavonoids to Pgp (7, 33) and their modula-tion of drug efflux in an  L. tropica  MDR line are class depen-dent (33). Flavones and flavonols display better binding affin-ities than the corresponding isoflavones and flavanones. Thepresent study has tested the ability of the flavanonol silybin,plus its oxidized and hydrophobically substituted derivatives, tobind to purified recombinant C-terminal NBD (NBD2) of a  Leishmania  Pgp-like transporter and to sensitize the MDRphenotype. The oxidation of silybin to its corresponding flavo-nol and the prenylation of the latter, especially at position 8,dramatically increased the binding affinity for NBD2 and thesensitization of an  L. tropica  MDR line overexpressing thetransporter, thereby inhibiting growth in the presence of thedaunomycin. MATERIALS AND METHODSChemical compounds.  HECAMEG [6- O -[(  N  -heptylcarbamoyl)methyl]-  - D -glucopyranoside] was purchased from Calbiochem, daunomycin was obtainedfrom Pharmacia & Upjohn (Barcelona, Spain), and imidazole (catalog referenceI 0250) was from Sigma. Commercial flavonoids were obtained from either Aldrich (galangin) or Sigma (chrysin and silybin). 1,1-DMA-chrysin (3), 1,1-DMA-galangin (2) and the derivatives of silybin (26) were synthesized as de-scribed. 3,3-DMA-chrysin was from the Natural Products Laboratory collectionsof compounds. Synthesis of 8-(3,3-DMA)-galangin.  To a stirred solution of 1.2 g of galangin(4.4 mmol) and 1.6 g of tetraethylammonium iodide (6.2 mmol) in 70 ml of 10%aqueous tetramethylammonium hydroxide was added 1 ml of prenyl bromide(8.7 mmol) dropwise at room temperature. After a 90-min reaction, the medium was acidified to pH 1 (with HCl, 1 N) and extracted with ethyl acetate. Isolationof 8-(3,3-DMA)-galangin (0.19 g, 0.6 mmol, 14%) from the ethyl acetate extract was carried out by medium pressure liquid chromatography on a C 18  reversed-phase column using a gradient of methanol in water as solvent. The  1 H nuclearmagnetic resonance (acetone-d 6 , 300 MHz) variables were as follows:    12.02(1H, s, 5-OH),  8.30 (2H, m, H-2  6  ),  7.55 (3H, m, H-3  4  5  ),  6.40 (1H,s, H-6),    5.30 (1H, brt,  J     6.5 Hz, H-2),    3.59 (2H, d,  J     6.5 Hz, H-1 ),    1.83(3H, s, H-5 ), and    1.67 (3H, s, H-4 ). For electron impact mass spectrometry(EIMS) (70 eV), the  m/z  (rel. int.) values were 338 [M]  (94), 323 (100), 283(72), and 270 (42). For high-resolution EIMS, the  m/z  value was 338.1155 (ascalculated for C 20 H 18 O 5    338.1154). Parasite culture and in vitro experiments.  The wild-type  L. tropica  LRC strain was a clone obtained by agar plating (19). An  L. tropica  line highly resistant to FIG. 1. Chemical structures of silybin and derivatives. (A) The flavanol silybin with a reduced 2,3-bond, a hydroxyl group at position 3 and themonolignol unit (rings D and E) adjacent to ring B. (B) The flavonol DHS with oxidized 2,3-bond and derivatives substituted by 3,3-DMA orgeranyl groups at either position 6 (R1) or 8 (R2) of ring A. (C) The flavonol galangin, lacking the monolignol unit, and derivatives substitutedat position 8 (R3). (D) The flavone chrysin, lacking both the monolignol unit and the hydroxyl group at position 3, and 8-substituted derivatives(R3).440 PE´REZ-VICTORIA ET AL. A  NTIMICROB . A  GENTS  C HEMOTHER .   on O c  t   o b  er 4  ,2  0 1  5  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om   daunomycin (DNM-R150) was maintained in the presence of 150   M dauno-mycin and used as previously described (4). This resistant line had an MDRphenotype similar to that of tumor cells, with cross-resistance to several drugsand an overexpressed drug efflux Pgp-like transporter (4). Promastigote forms were grown at 28°C in RPMI 1640-modified medium (Gibco) (20) and supple-mented with 20% heat-inactivated fetal bovine serum (Gibco). The growthsensitivity of wild-type and drug-resistant parasites to modulators of drug efflux  was ascertained as described earlier (33, 34). Overexpression, protein purification, and interaction of   Leishmania  NBD2 with silybin and derivatives.  The recombinant NBD2 from  Leishmania  Pgp-liketransporter was overexpressed in  E. coli  M15 (pREP4) cells and purified byaffinity chromatography (33). Fluorescence experiments were performed at25.0    0.1°C using an SLM-Aminco 8000C spectrofluorimeter with spectralbandwidths of 2 and 4 nm for excitation and emission, respectively. The mea-surements were corrected for wavelength dependence on the excitation lightintensity by using rhodamine B in the reference channel. All spectra were cor-rected for buffer Raman effect and for dilution. The intrinsic fluorescence of NBD2 (0.2 to 0.5  M final concentration) was measured in 1 ml of diluting buffer(50 mM potassium phosphate, pH 8.0; 1 M NaCl; 20% [wt/vol] glycerol, 0.05%[wt/vol] HECAMEG; 1 mM   -mercaptoethanol; 10 mM imidazole), with in-creasing concentrations of silybin or derivatives dissolved in dimethyl sulfoxide.The emission spectrum was measured in the range of 300 to 350 nm uponexcitation at 288 nm (a wavelength which minimized imidazole interference [33,34]). Ligand binding was monitored by the quenching of emission fluorescenceproduced upon addition of increasing ligand concentrations. Corrections for theinner-filter effect and dimethyl sulfoxide addition (up to a 2% final concentra-tion) were determined under the same conditions, by using a mixture of   N  -acetyltryptophanamide and  N  -acetyltyrosinamide in the same ratio (3:7) as thetryptophan and tyrosine residues present in NBD2. Curve fitting of ligand bind-ing-related fluorescence quenching was analyzed with the Grafit program (Er-ithacus Software) (12). This allowed the determination of the apparent dissoci-ation constant (  K   d ) and maximal quenching of fluorescence. RESULTSInteraction of NBD2 with silybin and derivatives.  Incuba-tion of recombinant NBD2 with the naturally occurring fla- vanolignan silybin (Fig. 1A) produced the concentration-de-pendent quenching of protein intrinsic fluorescence illustratedin Fig. 2A. Detailed analysis of binding (Fig. 2B) gave anapparent dissociation constant (  K   d ) of 9.2  1.0  M. Oxidationof the 2,3-bond of ring C in silybin to the corresponding fla- vonol dehydrosilybin (DHS) (Fig. 1B), gave a fourfold-higherbinding affinity for the domain (Fig. 2B, with a  K   d  of 2.3    0.2  M). A further 3.5- to 21-fold increase in binding affinity wasproduced by addition of hydrophobic substituents (dimethyl-allyl [DMA] or geranyl) at either position 6 or position 8 of ring A, with  K   d  values in the nanomolar range (Table 1). Theeffect was dependent on both the nature and the position of thehydrophobic substituent. Thus, a 3,3-DMA group at position 6or 8 of ring A increased the binding affinity a further 2.5- to3-fold more, respectively, than a geranyl group at the samepositions (Table 1). In addition, the hydrophobic substitutionat position 8 of ring A by either prenyl or geranyl substituentgave twofold-greater affinity than substitution at position 6(Table 1). Thus, the 8-(3,3-DMA)-DHS derivative gave thehighest binding affinity, with a  K   d  of 0.11  0.02  M. This value was 85-fold less than that obtained with unmodified silybin.The effect of prenylation at position 8 was further studied within the compounds galangin and chrysin (Fig. 1C and D),lacking the monolignol unit. When compared to DHS, thebinding affinity for NBD2 was reduced fourfold for galanginand eightfold for chrysin that also lacks an hydroxyl group atposition 3 of ring C (Table 2). In both cases, prenylation by3,3-DMA at position 8 also markedly increased (6- to 15-fold)the binding affinity. A double increase (12- to 28-fold) was evenobtained with the 1,1-DMA substituent. Sensitization of promastigote forms by silybin and its de-rivatives.  The resistance to daunomycin in the MDR  L. tropica line is mainly due to the overexpression of a Pgp-like trans-porter involved in drug efflux (4) which limits drug accumula-tion. We tested whether the binding of silybin derivatives to thecytosolic domain of the Pgp-like transporter could inhibit drugpumping and overcome drug resistance. Potential modulators were assessed for their ability to inhibit the growth of resistantparasites in the presence of daunomycin, in comparison with wild-type parasites in absence of drug. Figure 3 shows that a72-h incubation of resistant parasites with 150  M daunomycin FIG. 2. Interaction of recombinant  Leishmania  NBD2 with silybinand derivatives monitored by the quenching of protein intrinsic fluo-rescence. (A) Spectral modification upon interaction with silybin. Thefluorescence spectrum of 0.5   M NBD2 was recorded after excitationat 288 nm, in the absence (continuous line) or presence of either 4   M(broken line) or 16   M (dotted line) silybin; the traces were obtainedby buffer subtraction before correction for inner-filter effect. (B) Theconcentration-dependent binding of silybin and derivatives was ana-lyzed by quenching of NBD2 intrinsic fluorescence, determined byspectral integration from 300 to 350 nm, and corrected for the inner-filter effect in the presence of increasing concentrations of either sily-bin ( Œ ), DHS (  ), or 8-(3,3-DMA)-DHS ( F ) added as a dimethylsulfoxide solution.V OL  . 45, 2001 INHIBITION OF  L. TROPICA  MDR BY SILYBIN DERIVATIVES 441   on O c  t   o b  er 4  ,2  0 1  5  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om   in the presence of different silybin derivatives gave differentialdose-dependent growth inhibition (GI). These studies demon-strate the importance of silybin oxidation to DHS and theprenylation of the latter. Consistent with the binding analysis,8-(3,3-DMA)-DHS was the most efficient sensitizer. It gavemore than 95% GI at 10   M and showed only a minor toxiceffect in the wild-type line. The other hydrophobically substi-tuted derivatives also showed considerable sensitization of thecells (89 to 98% GI in the resistant line) at a threefold-higherconcentration (30  M). However, this concentration of geranylderivatives gave significant cytotoxicity in the wild-type para-sites (31 to 62% GI). In contrast, unmodified silybin only gavemodest sensitization, even at much higher concentrations (100to 300   M). Silymarin also did not reverse the resistant phe-notype at high concentrations such as 250   g/ml (data notshown). Finally, DHS showed considerable cytotoxicity on wild-type parasite (ca. 62% GI at 20   M). This hamperedstudies of its sensitization of the MDR phenotype.The reversal of parasite resistance to daunomycin was alsotested with prenylated derivatives of galangin and chrysin. Fig-ure 4A shows that higher concentrations of galangin deriva-tives were required to produce ca. 80% GI in the resistant line[20   M 8-(1,1-DMA)-galangin or 50   M 8-(3,3-DMA)-galan-gin] compared with 8-(3,3-DMA)-DHS (5 to 10   M), whilenonprenylated galangin produced a slight sensitization at 75  M. The chrysin derivatives were even less efficient (Fig. 4B):30   M 8-(1,1-DMA)-chrysin produced a 88% GI in the resis-tant line, and 40   M 8-(3,3-DMA)-chrysin gave 71% GI, butthis concentration was relatively cytotoxic for the wild-typeline. The nonsubstituted chrysin produced a small effect at 75  M. Interestingly, in both galangin and chrysin derivatives, the1,1-DMA substitution gave better sensitivity to daunomycinthat the 3,3-DMA substitution. DISCUSSION We show here that oxidized and prenylated derivatives of the therapeutic agent silybin exhibit high binding affinity to therecombinant cytosolic domain of   Leishmania  Pgp-like trans-porter and revert the MDR of an  L. tropica  line that overex-presses the transporter, drawing attention to the importance of the monolignol unit within the above compounds.Silybin is a natural flavanolignan with many positive thera-peutic properties and few adverse effects in animals and hu-mans. Although its cellular target is unknown, it increased thecytotoxicity of doxorubicin in a doxorubicin-resistant cell line(36). In order to circumvent MDR phenotype in  Leishmania , we have studied silybin binding to recombinant NBD2 of the  L.tropica  multidrug transporter, the effects of its oxidation toDHS, and the effects of different hydrophobic substitutions onring A, previously described as critical for the increase thebinding of flavones to the cytosolic domain of   Leishmania Pgp-like transporter (33). These results, together with compar-ative data for derivatives lacking the monolignol unit, provideimportant structure-activity information. (i) First of all, theseresults demonstrate the significance of the oxidation of 2,3-bond within the silybin ring C to its corresponding flavonolDHS, which may reinforce the mimicry of the adenine moietyof ATP, as previously suggested from the higher binding affin-ity of the flavone apigenin compared to its reduced analoguenaringenin (7, 33). Conversely, the reduction of the 2,3-doublebond of flavones to give flavanones resulted in a decrease of the competitive inhibition of H  ,K   -ATPase with respect to ATP (29). (ii) They demonstrate the importance of the addi-tion of a prenyl or geranyl hydrophobic substituent on ring A that could increase the interaction with the hydrophobic region vicinal to the ATP site (33). (iii) The monolignol unit (rings Dand E in Fig. 1A and B) within flavanolignans producessignificant effects with a five- to sixfold-higher affinity for8-(3,3-DMA)-DHS with respect to 8-(3,3-DMA)-galangin anda fourfold higher affinity for DHS with respect to galangin. Additional studies are needed to determine its specific role inthe interaction with the domain. (iv) These results furtherdemonstrate the preference for hydrophobic substitution atposition 8 over position 6 of ring A, suggesting some differ-ences in binding orientation of the differently substituted com-pounds. (v) These results show the more efficient effect of prenylation compared to geranylation despite a lower hydro-phobicity. (vi) Finally, these studies show the systematicallymore efficient effect of the 1,1-DMA compared to 3,3-DMA.Similar in vitro results with some of these silybin derivativeshave been obtained in parallel studies with the cytosolic do-main of mammalian Pgp (26), except that the geranyl substi-tution was more efficient than the prenyl one. These differen-tial results may indicate some differences between the cytosolicdomains of   Leishmania  and mammalian transporters, possiblyat the level of the hydrophobic interacting region.The same sequence in efficiency has also been obtained fromthe in vitro sensitization studies in an MDR  Leishmania  line,so that compounds that display higher binding affinity for therecombinant NBD2 most efficiently sensitize the MDR pheno-type. Thus, although the reversing effects of the compounds TABLE 1. Effects of DHS prenylation on the affinity of binding to  Leishmania  NBD2  a DHS derivative  K   d (  M    SD)% Maximalquenching    SD 6-Geranyl-DHS 0.67    0.06 56.7    1.48-Geranyl-DHS 0.31    0.05 72.0    2.26-(3,3-Dimethylallyl)-DHS 0.27    0.05 76.1    2.48-(3,3-Dimethylallyl)-DHS 0.11    0.02 76.1    1.7  a The NBD2 domain was incubated, under the conditions of Fig. 2B, with theindicated hydrophobic derivatives of DHS. The dissociation constant and max-imal quenching values were determined by fitting with the Graft program usingthe Erithacus software (see Materials and Methods). TABLE 2. Binding affinities of different prenylated compoundslacking the monolignol unit  a - Compound  K   d (  M    SD)% Maximalquenching   SDSource orreference Galangin 9.2    1.0 75.9    3.9 338-(3,3-Dimethylallyl)-galangin 0.62    0.24 80.0    6.4 This study8-(1,1-Dimethylallyl)-galangin 0.34    0.09 85.0    4.0 This studyChrysin 17.6    5.9 81.3    8.6 338-(3,3-Dimethylallyl)-chrysin 2.9    0.3 84.3    2.0 This study8-(1,1-Dimethylallyl)-chrysin 1.4    0.2 75.4    2.7 33  a The NBD2 domain was incubated, under the conditions of Fig. 2B, with theindicated compounds, and the binding analyses were performed as described inTable 1. 442 PE´REZ-VICTORIA ET AL. A  NTIMICROB . A  GENTS  C HEMOTHER .   on O c  t   o b  er 4  ,2  0 1  5  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om   could in some cases be partially covered by their intrinsiccytotoxicity, as was observed for geranyl-DHS, the importanceof the monolignol unit is evident from the higher reversion of resistance obtained with 8-(3,3-DMA)-DHS with respect to8-(3,3-DMA)-galangin, as well as the role of prenylation, es-pecially 1,1-DMA, at position 8 of ring A. Thus, 8-(3,3-DMA)-DHS is the most active MDR-sensitizing agent ever describedfor  Leishmania . Work is in progress to synthesize the 8-(1,1-DMA)-DHS derivative that would probably bind with evenhigher affinity to the domain and sensitize MDR at even lowerconcentrations. The single exception to the correlation be-tween binding to NBD2 and MDR sensitization was the ab-sence of any reversion by DHS. This appears to be due to thehigh cytotoxicity caused by this compound (ca. 62% GI in wild-type parasites at 20   M) compared to silybin (29% at 300  M). Similarly, a fourfold-higher GI for DHS compared tothat for silybin has been described in a human ovarian carci-noma cell line (36). This effect could be due to higher mimicry with ATP after oxidation of the silybin 2,3-bond, thereby fa- voring additional binding to other ATP-binding proteins. In-deed, flavonols such as DHS, with a hydroxyl at position 3 anda 2,3-double bond in addition to the hydroxyl at position 5 andthe ketone at position 4, contain all the requirements to bind to ATP-binding site, as previously shown not only for  L. tropica (33) and mammalian (7, 11) multidrug transporters but also forcrystallized CDK2 (10) and HcK (40).We propose that prenylation of the ring A within thesecompounds might generate more specific inhibitors of Pgp-liketransporters by strengthening the interaction with its cytosolicdomains, possibly with the hydrophobic region vicinal to the ATP site. This would, on the one hand, increase the reversalefficiency on the MDR parasites and, on the other hand, lowerthe affinity for other cellular ATP-binding proteins, as deducedfrom the significant decrease of the cytotoxicity on wild-typeparasites after prenylation (ca. 10% GI for 40   M 6-prenyl-DHS compared to nearly 100% GI for DHS at the sameconcentration; data not shown) A number of observations have indicated flavonoid antago-nism toward ATP: (i) the binding of kaempferide or derivativesto recombinant NBD2 from either mammalian (7) or  Leish- mania  (33) multidrug transporter was partly prevented or dis-placed by ATP; (ii) flavonoid inhibition of several ATP-utiliz-ing enzymes involves competitive interaction at the ATP-binding site (28), as studied in detail for H  ,K   -ATPase (29)and as clearly demonstrated by crystallization experiments withthe protein kinases CDK2 (10) and Hck (40); and (iii) theflavonol quercetin, on the one hand, competitively inhibitedthe ATPase activity of a recombinant NBD2 from the cysticfibrosis transmembrane conductance regulator (another ABCprotein) (35) and, on the other hand, prevented Hoechst 33342transport by mammalian Pgp, partly by inhibiting its ATPaseactivity (38). We show here that the sensitization caused by FIG. 3. Sensitization by silybin derivatives in a daunomycin-resistant  L. tropica  line. Cell growth of either wild-type or resistant parasites wasdetermined after incubation at 28°C for 72 h. Wild-type parasites (  ) were incubated in the presence of different concentrations of silybinderivatives. Resistant parasites were incubated with the same concentrations of silybin derivatives in the presence of 150   M daunomycin. Theresults are expressed as the percentage of growth inhibition observed in each cell line compared to the absence of modulator (control cells). Thedata are means with standard deviations for three experiments performed in duplicate.V OL  . 45, 2001 INHIBITION OF  L. TROPICA  MDR BY SILYBIN DERIVATIVES 443   on O c  t   o b  er 4  ,2  0 1  5  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om 
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