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

Article abstract of DOI:10.1128/AAC.45.2.439-446.2001

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.

Full text of DOI:10.1128/AAC.45.2.439-446.2001

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2001, p. 439–446
Vol. 45, No. 2
0
066-4804/01/$04.00ϩ0 DOI: 10.1128/AAC.45.2.439–446.2001
Copyright © 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-Like
Transporter and Chemosensitization of a Multidrug-Resistant
Parasite to Daunomycin
1
1
2
´
´
´
¨
JOSE M. PEREZ-VICTORIA, F. JAVIER PEREZ-VICTORIA, GWENAELLE CONSEIL,
3
3
3
2
MATHIAS MAITREJEAN, GILLES COMTE, DENIS BARRON, ATTILIO DI PIETRO,
1
1
SANTIAGO CASTANYS, AND FRANCISCO GAMARRO *
Instituto de Parasitologia y Biomedicina “L o´ pez-Neyra,” Consejo Superior de Investigaciones Cient ´ı ficas, Granada,
1
2
Spain, and Institut de Biologie et Chimie des Prot ´e ines, UPR 412 du CNRS, Lyon, and Laboratoire des Produits
3
Naturels, UMR-CNRS 5013, Universit ´e Claude Bernard de Lyon, Villeurbanne, 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 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 promas-
tigote 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)-dehydro-
silybin 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.
Drug resistance has become a major impediment to the
treatment of diseases caused by protozoan parasites, which
threaten the life of nearly one-quarter of the human popula-
tion. Among parasitic infections, World Health Organization
statistics show that the incidence of leishmaniasis has increased
in vitro by agents such as verapamil and cyclosporine, which
compete with drug binding to the transmembrane domains
(16). However, most of these inhibitors are also pumped sub-
strates and therefore require high concentrations for effective
inhibition. These concentrations produce undesirable side ef-
fects. In addition, these classical modulators of drug efflux in
cancer 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 lower
host toxicity need to be developed. Recently, it has been de-
scribed that NBDs can be the target for inhibitors of Pgp-like
transporters (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 different
MDR cells (8, 15, 33, 37– 39), bind to transporter NBDs. They
interact with both the ATP-binding site and a vicinal hydro-
phobic region (7, 9, 33), inhibiting drug efflux and reversing the
resistance phenotype of an L. tropica MDR line (33). Their
efficient modulation of drug efflux has been correlated with
their affinity binding to the transporter cytosolic domain (33).
Silymarin is a mixture of flavanolignans isolated from the
medicinal plant Silybum marianum, with silybin (or silybinin)
4
2-fold between 1985 and 1998 and has become the second
leading cause of death (18) worldwide. Chemotherapy remains
the only effective way to control infections. The conventional
clinical drugs, pentavalent antimonials in the form of Glucan-
time and Pentostam (reviewed in reference 27), are not very
efficient due to their toxicity and the increased appearance of
drug resistance (14). ATP-binding cassette (ABC) transporters
have been found to be involved in Leishmania species in vitro
selected for metal resistance (reviewed in reference 30). The
multidrug resistance (MDR) phenotype due to P-glycoprotein
(Pgp)-like transporters has been extensively characterized in
cancer 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 from
the 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 drug
efflux, and a cytosolic nucleotide-binding domain (NBD) re-
sponsible for ATP binding and hydrolysis. Pgp can be inhibited
(Fig. 1A) as the main component (31). These natural com-
pounds are well-established hepatoprotectants and are used in
Europe 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 largely
devoid of adverse effects (25, 32). It has been recently mar-
keted in the United States and in Europe as a nutritional
supplement. Silymarin may directly affect cholesterol metabo-
*
Corresponding author. Mailing address: Instituto de Parasitologia
y Biomedicina “L o´ pez-Neyra,” Consejo Superior de Investigaciones
Cientificas, c/Ventanilla 11, 18001 Granada, Spain. Phone: 34-958-
8
05185. Fax: 34-958-203323. E-mail: gamarro@ipb.csic.es.
4
39

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PEREZ-VICTORIA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
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 the
monolignol 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 or
geranyl groups at either position 6 (R1) or 8 (R2) of ring A. (C) The flavonol galangin, lacking the monolignol unit, and derivatives substituted
at position 8 (R3). (D) The flavone chrysin, lacking both the monolignol unit and the hydroxyl group at position 3, and 8-substituted derivatives
(R3).
lism and is therefore considered as a potential hypocholester-
olemic (41). In vitro studies indicate that silymarin and silybin
may help to prevent and treat breast, prostate, skin and ovarian
cancers (32, 36, 46). Silybin also appeared to be synergistic with
doxorubicin in a doxorubicin-resistant cell line, probably by
inhibiting Pgp function (36). Thus, silybin, either alone or in
combination with other cytotoxic drugs, is currently being
tested in patients with advanced ovarian cancer (36).
MATERIALS AND METHODS
Chemical compounds. HECAMEG [6-O-[(N-heptylcarbamoyl)methyl]-␣-D-
glucopyranoside] was purchased from Calbiochem, daunomycin was obtained
from Pharmacia & Upjohn (Barcelona, Spain), and imidazole (catalog reference
I 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 collections
of compounds.
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. The
present study has tested the ability of the flavanonol silybin,
plus its oxidized and hydrophobically substituted derivatives, to
bind to purified recombinant C-terminal NBD (NBD2) of a
Leishmania Pgp-like transporter and to sensitize the MDR
phenotype. 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 the
sensitization of an L. tropica MDR line overexpressing the
transporter, thereby inhibiting growth in the presence of the
daunomycin.
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. Isolation
of 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 C18 reversed-
phase column using a gradient of methanol in water as solvent. The H nuclear
magnetic resonance (acetone-d
1
6
, 300 MHz) variables were as follows: ␦ 12.02
(1H, s, 5-OH), ␦ 8.30 (2H, m, H-2Јϩ6Ј), ␦ 7.55 (3H, m, H-3Јϩ4Јϩ5Ј), ␦ 6.40 (1H,
s, H-6), ␦ 5.30 (1H, brt, J ϭ 6.5 Hz, H-2), ␦ 3.59 (2H, d, J ϭ 6.5 Hz, H-1 ), ␦ 1.83
(3H, s, H-5 ), and ␦ 1.67 (3H, s, H-4 ). For electron impact mass spectrometry
ϩ
(
(
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 (as
calculated for C20H18O5 ϭ 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

VOL. 45, 2001
INHIBITION OF L. TROPICA MDR BY SILYBIN DERIVATIVES
441
daunomycin (DNM-R150) was maintained in the presence of 150 ␮M dauno-
mycin and used as previously described (4). This resistant line had an MDR
phenotype similar to that of tumor cells, with cross-resistance to several drugs
and 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 growth
sensitivity 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-like
transporter was overexpressed in E. coli M15 (pREP4) cells and purified by
affinity chromatography (33). Fluorescence experiments were performed at
2
5.0 Ϯ 0.1°C using an SLM-Aminco 8000C spectrofluorimeter with spectral
bandwidths of 2 and 4 nm for excitation and emission, respectively. The mea-
surements were corrected for wavelength dependence on the excitation light
intensity 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 upon
excitation at 288 nm (a wavelength which minimized imidazole interference [33,
3
4]). Ligand binding was monitored by the quenching of emission fluorescence
produced upon addition of increasing ligand concentrations. Corrections for the
inner-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 the
tryptophan 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.
RESULTS
Interaction 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 illustrated
in Fig. 2A. Detailed analysis of binding (Fig. 2B) gave an
FIG. 2. Interaction of recombinant Leishmania NBD2 with silybin
and derivatives monitored by the quenching of protein intrinsic fluo-
rescence. (A) Spectral modification upon interaction with silybin. The
fluorescence spectrum of 0.5 ␮M NBD2 was recorded after excitation
at 288 nm, in the absence (continuous line) or presence of either 4 ␮M
apparent dissociation constant (K ) of 9.2 Ϯ 1.0 ␮M. Oxidation
d
of the 2,3-bond of ring C in silybin to the corresponding fla-
vonol dehydrosilybin (DHS) (Fig. 1B), gave a fourfold-higher
(
broken line) or 16 ␮M (dotted line) silybin; the traces were obtained
binding affinity for the domain (Fig. 2B, with a K of 2.3 Ϯ 0.2
d
by buffer subtraction before correction for inner-filter effect. (B) The
concentration-dependent binding of silybin and derivatives was ana-
lyzed by quenching of NBD2 intrinsic fluorescence, determined by
spectral 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 dimethyl
sulfoxide solution.
␮M). A further 3.5- to 21-fold increase in binding affinity was
produced by addition of hydrophobic substituents (dimethyl-
allyl [DMA] or geranyl) at either position 6 or position 8 of
ring A, with K values in the nanomolar range (Table 1). The
d
effect was dependent on both the nature and the position of the
hydrophobic substituent. Thus, a 3,3-DMA group at position 6
or 8 of ring A increased the binding affinity a further 2.5- to
3
-fold more, respectively, than a geranyl group at the same
positions (Table 1). In addition, the hydrophobic substitution
at position 8 of ring A by either prenyl or geranyl substituent
gave twofold-greater affinity than substitution at position 6
the binding affinity. A double increase (12- to 28-fold) was even
obtained 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 the
cytosolic domain of the Pgp-like transporter could inhibit drug
pumping and overcome drug resistance. Potential modulators
were assessed for their ability to inhibit the growth of resistant
parasites in the presence of daunomycin, in comparison with
wild-type parasites in absence of drug. Figure 3 shows that a
72-h incubation of resistant parasites with 150 ␮M daunomycin
(Table 1). Thus, the 8-(3,3-DMA)-DHS derivative gave the
highest binding affinity, with a K of 0.11 Ϯ 0.02 ␮M. This value
d
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, the
binding affinity for NBD2 was reduced fourfold for galangin
and eightfold for chrysin that also lacks an hydroxyl group at
position 3 of ring C (Table 2). In both cases, prenylation by
3
,3-DMA at position 8 also markedly increased (6- to 15-fold)

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PEREZ-VICTORIA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Effects of DHS prenylation on the affinity of binding to
TABLE 2. Binding affinities of different prenylated compounds
a
a
Leishmania NBD2
lacking the monolignol unit -
K
d
% Maximal
% Maximal
DHS derivative
K
d
Source or
reference
(
␮M Ϯ SD)
quenching Ϯ SD
Compound
quenching
(
␮M Ϯ SD)
Ϯ SD
6
8
6
8
-Geranyl-DHS
-Geranyl-DHS
-(3,3-Dimethylallyl)-DHS
-(3,3-Dimethylallyl)-DHS
0.67 Ϯ 0.06
0.31 Ϯ 0.05
0.27 Ϯ 0.05
0.11 Ϯ 0.02
56.7 Ϯ 1.4
72.0 Ϯ 2.2
76.1 Ϯ 2.4
76.1 Ϯ 1.7
Galangin
9.2 Ϯ 1.0
75.9 Ϯ 3.9 33
8-(3,3-Dimethylallyl)-galangin 0.62 Ϯ 0.24 80.0 Ϯ 6.4 This study
8-(1,1-Dimethylallyl)-galangin 0.34 Ϯ 0.09 85.0 Ϯ 4.0 This study
Chrysin
8
8
17.6 Ϯ 5.9
2.9 Ϯ 0.3
1.4 Ϯ 0.2
81.3 Ϯ 8.6 33
84.3 Ϯ 2.0 This study
75.4 Ϯ 2.7 33
a
The NBD2 domain was incubated, under the conditions of Fig. 2B, with the
-(3,3-Dimethylallyl)-chrysin
-(1,1-Dimethylallyl)-chrysin
indicated hydrophobic derivatives of DHS. The dissociation constant and max-
imal quenching values were determined by fitting with the Graft program using
the Erithacus software (see Materials and Methods).
a
The NBD2 domain was incubated, under the conditions of Fig. 2B, with the
indicated compounds, and the binding analyses were performed as described in
Table 1.
in the presence of different silybin derivatives gave differential
dose-dependent growth inhibition (GI). These studies demon-
strate the importance of silybin oxidation to DHS and the
prenylation of the latter. Consistent with the binding analysis,
we have studied silybin binding to recombinant NBD2 of the L.
tropica multidrug transporter, the effects of its oxidation to
DHS, and the effects of different hydrophobic substitutions on
ring A, previously described as critical for the increase the
binding 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, provide
important structure-activity information. (i) First of all, these
results demonstrate the significance of the oxidation of 2,3-
bond within the silybin ring C to its corresponding flavonol
DHS, which may reinforce the mimicry of the adenine moiety
of ATP, as previously suggested from the higher binding affin-
ity of the flavone apigenin compared to its reduced analogue
naringenin (7, 33). Conversely, the reduction of the 2,3-double
bond of flavones to give flavanones resulted in a decrease of
8
-(3,3-DMA)-DHS was the most efficient sensitizer. It gave
more than 95% GI at 10 ␮M and showed only a minor toxic
effect in the wild-type line. The other hydrophobically substi-
tuted derivatives also showed considerable sensitization of the
cells (89 to 98% GI in the resistant line) at a threefold-higher
concentration (30 ␮M). However, this concentration of geranyl
derivatives gave significant cytotoxicity in the wild-type para-
sites (31 to 62% GI). In contrast, unmodified silybin only gave
modest sensitization, even at much higher concentrations (100
to 300 ␮M). Silymarin also did not reverse the resistant phe-
notype at high concentrations such as 250 ␮g/ml (data not
shown). Finally, DHS showed considerable cytotoxicity on
wild-type parasite (ca. 62% GI at 20 ␮M). This hampered
studies of its sensitization of the MDR phenotype.
ϩ
ϩ
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 D
and E in Fig. 1A and B) within flavanolignans produces
significant effects with a five- to sixfold-higher affinity for
8-(3,3-DMA)-DHS with respect to 8-(3,3-DMA)-galangin and
a fourfold higher affinity for DHS with respect to galangin.
Additional studies are needed to determine its specific role in
the interaction with the domain. (iv) These results further
demonstrate the preference for hydrophobic substitution at
position 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 systematically
more efficient effect of the 1,1-DMA compared to 3,3-DMA.
Similar in vitro results with some of these silybin derivatives
have 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 cytosolic
domains of Leishmania and mammalian transporters, possibly
at the level of the hydrophobic interacting region.
The reversal of parasite resistance to daunomycin was also
tested 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), while
nonprenylated galangin produced a slight sensitization at 75
␮M. The chrysin derivatives were even less efficient (Fig. 4B):
3
0 ␮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, but
this concentration was relatively cytotoxic for the wild-type
line. The nonsubstituted chrysin produced a small effect at 75
␮
1
M. Interestingly, in both galangin and chrysin derivatives, the
,1-DMA substitution gave better sensitivity to daunomycin
that the 3,3-DMA substitution.
DISCUSSION
We show here that oxidized and prenylated derivatives of
the therapeutic agent silybin exhibit high binding affinity to the
recombinant 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 the
cytotoxicity of doxorubicin in a doxorubicin-resistant cell line
The same sequence in efficiency has also been obtained from
the in vitro sensitization studies in an MDR Leishmania line,
so that compounds that display higher binding affinity for the
recombinant NBD2 most efficiently sensitize the MDR pheno-
type. Thus, although the reversing effects of the compounds
(36). In order to circumvent MDR phenotype in Leishmania,

VOL. 45, 2001
INHIBITION OF L. TROPICA MDR BY SILYBIN DERIVATIVES
443
FIG. 3. Sensitization by silybin derivatives in a daunomycin-resistant L. tropica line. Cell growth of either wild-type or resistant parasites was
determined after incubation at 28°C for 72 h. Wild-type parasites (ء) were incubated in the presence of different concentrations of silybin
derivatives. Resistant parasites were incubated with the same concentrations of silybin derivatives in the presence of 150 ␮M daunomycin. The
results are expressed as the percentage of growth inhibition observed in each cell line compared to the absence of modulator (control cells). The
data are means with standard deviations for three experiments performed in duplicate.
could in some cases be partially covered by their intrinsic
cytotoxicity, as was observed for geranyl-DHS, the importance
of the monolignol unit is evident from the higher reversion of
resistance obtained with 8-(3,3-DMA)-DHS with respect to
We propose that prenylation of the ring A within these
compounds might generate more specific inhibitors of Pgp-like
transporters by strengthening the interaction with its cytosolic
domains, possibly with the hydrophobic region vicinal to the
ATP site. This would, on the one hand, increase the reversal
efficiency on the MDR parasites and, on the other hand, lower
the affinity for other cellular ATP-binding proteins, as deduced
from the significant decrease of the cytotoxicity on wild-type
parasites after prenylation (ca. 10% GI for 40 ␮M 6-prenyl-
DHS compared to nearly 100% GI for DHS at the same
concentration; data not shown)
8
-(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 described
for Leishmania. Work is in progress to synthesize the 8-(1,1-
DMA)-DHS derivative that would probably bind with even
higher affinity to the domain and sensitize MDR at even lower
concentrations. 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 the
high cytotoxicity caused by this compound (ca. 62% GI in
wild-type parasites at 20 ␮M) compared to silybin (29% at 300
A number of observations have indicated flavonoid antago-
nism toward ATP: (i) the binding of kaempferide or derivatives
to 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-
␮M). Similarly, a fourfold-higher GI for DHS compared to
that 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 and
a 2,3-double bond in addition to the hydroxyl at position 5 and
the ketone at position 4, contain all the requirements to bind to
ATP-binding site, as previously shown not only for L. tropica
ϩ
ϩ
binding site (28), as studied in detail for H ,K -ATPase (29)
and as clearly demonstrated by crystallization experiments with
the protein kinases CDK2 (10) and Hck (40); and (iii) the
flavonol quercetin, on the one hand, competitively inhibited
the ATPase activity of a recombinant NBD2 from the cystic
fibrosis transmembrane conductance regulator (another ABC
protein) (35) and, on the other hand, prevented Hoechst 33342
transport by mammalian Pgp, partly by inhibiting its ATPase
activity (38). We show here that the sensitization caused by
(33) and mammalian (7, 11) multidrug transporters but also for
crystallized CDK2 (10) and HcK (40).

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PEREZ-VICTORIA ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 4. Sensitization of growth caused by prenylated derivatives lacking the monolignol unit. Wild-type (ء) and resistant parasites were
incubated under the conditions of Fig. 3 with different prenylated derivatives of galangin (A) and chrysin (B). The data are means with standard
deviations for three experiments performed in duplicate.
silybin derivatives in an MDR L. tropica line correlates with
their affinity of binding to the cytosolic NBD2, which is con-
sistent with a previous correlation between the binding affinity
of other flavonoids for NBD2 and the increase of intracellular
daunomycin accumulation monitored by flow cytometry (33).
All of these results suggest that the reversion of drug resistance
caused by silybin derivatives was produced by direct interaction
with the nucleotide-binding domain of the Pgp-like trans-
porter. However, other factors such as interaction with mem-
brane phospholipids, altering the lipid packing density and the
diffusion rate of the drug (6), or modulation of the multidrug
transporter expression (22) may contribute to the observed
sensitization of MDR. In addition, it is possible that prenylated
derivatives might mainly interact at the hydrophobic region
characterized within the NBDs or bind to the drug-binding
sites within the transmembrane domains of the Pgp-like mul-

VOL. 45, 2001
INHIBITION OF L. TROPICA MDR BY SILYBIN DERIVATIVES
445
tidrug transporter. Quercetin has indeed been proposed to
interact at the mammalian Pgp site where the transported
drugs Hoechst 33342 and colchicine bind, which stimulated the
transport of anthracyclines (38). Additional work is therefore
required to further characterize the flavonoid-binding site(s).
In India, recent clinical studies showed that approximately
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establish structure-function relationship. Braz. J. Med. Biol. Res. 32:925–
5
0% of patients with leishmaniasis fail to achieve parasite
clearance after a standard dose of pentavalent antimonials
42). The in vivo data are supported by in vitro monitoring of
(
9
39.
drug sensitivity of fresh clinical isolates obtained at the same
study site (24). The high rate of therapeutic failure to these
usual drugs in cases of leishmaniasis call for new rational
approaches to develop alternative drugs. Some pharmaceutical
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considered as potential leishmanicidal agents, such as doxoru-
bicin (23), taxol (21) and alkyl lysophospholipids (44), are
known substrates of Pgps or other ABC transporters and thus
could induce an MDR phenotype. The development of inhib-
itors which block the MDR mechanism is a promising potential
way to circumvent resistance to drugs. Our studies also provide
useful models for understanding how similar defense mecha-
nisms can be overcome in other protozoan parasites such as
Plasmodium, Entamoeba, and Trichomonas spp., where ABC
transporters have been associated with drug resistance.
1
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Michel, B. Faugere, and H. Dumon. 1997. In vitro and in vivo resistance of
Leishmania infantum to meglumine antimoniate: a study of 37 strains col-
lected from patients with visceral leishmaniasis. Antimicrob. Agents Che-
mother. 41:827–830.
5. Fert ´e , J., J. M. K u¨ hnel, G. Chapuis, Y. Rolland, G. Lewin, and M. A.
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ACKNOWLEDGMENTS
This work was supported by the Spanish Grants PM97-0139 (F.G.),
PM98-0115 (S.C.), and CICYT-FEDER IFD97-0747-C04-03 (S.C.)
and the Convenio CSIC-CNRS between F.G. and A.D.P. (1997–2000).
J.M.P.-V. received a fellowship from the Junta de Andaluc ´ı a (Spain),
F.J.P.-V. received a fellowship from the Ministerio de Educaci o´ n y
Cultura (Spain), G.C. received fellowship from the Ligue Nationale
contre le Cancer (Haute-Savoie, France), and M.M. received a fellow-
ship from the French Ministry of National Education and Technolog-
ical Research. Financial support from the Association pour la Recher-
che sur le Cancer (ARC 9147) and the Ligue Nationale contre le
Cancer (Rh oˆ ne 1999) is also acknowledged.
19. Iovannisci, D. M., and B. Ullman. 1983. High efficiency plating method for
Leishmania promastigotes in semi-defined or completely-defined medium. J.
Parasitol. 69:633–636.
2
0. Jackson, P. R., J. M. Lawrie, J. M. Stiteler, D. W. Hawkins, J. A. Wohlhieter,
and E. D. Rowtin. 1986. Detection and characterization of Leishmania spe-
cies and strains from mammals and vectors by hybridization and restriction
endonuclease digestion of kinetoplast DNA. Vet. Parasitol. 20:195–215.
1. Kapoor, P., M. Sachdeva, and R. Madhubala. 1999. Effect of the microtu-
bule stabilising agent taxol on leishmanial protozoan parasites in vitro.
FEMS Microbiol. Lett. 176:429–435.
2
22. Kioka, N., N. Hosokawa, T. Komano, K. Hirayoshi, K. Nagata, and K. Ueda.
1992. Quercetin, a bioflavonoid, inhibits the increase of human multidrug
resistance gene (MDR1) expression caused by arsenite. FEBS Lett. 301:307–
We thank Pilar Navarro for the help in parasite culture. We also
acknowledge Pharmacia & Upjohn (Barcelona, Spain) for providing
the daunomycin used in this study and Brian Monk for improving the
English of the manuscript.
3
09.
2
3. Kole, L., L. Das, and P. K. Das. 1999. Synergistic effect of interferon-gamma
and mannosylated liposome-incorporated doxorubicin in the therapy of ex-
perimented visceral leishmaniasis. J. Infect. Dis. 180:811–820.
4. Lira, R., S. Sundar, A. Makharia, R. Kenney, A. Gam, E. Saraiva, and D.
Sacks. 1999. Evidence that the high incidence of treatment failures in Indian
kala-azar is due to the emergence of antimony-resistant strains of Leishma-
nia donovani. J. Infect. Dis. 180:564–567.
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