From the Departments of Biology and
** Chemistry, Catholic University of America, Washington,
D. C. 20064, the ¶ Laboratory of Cell Biology, Center for Cancer
Research, NCI, National Institutes of Health, Bethesda, Maryland
20892-4254, and the
Department of Chemistry, University of
Wisconsin-Waukesha, Waukesha, Wisconsin 53188
Received for publication, October 24, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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The yeast (Saccharomyces
cerevisiae) multidrug transporter Pdr5p effluxes a broad range of
substrates that are variable in structure and mode of action. Previous
work suggested that molecular size and ionization could be important
parameters. In this study, we compared the relative sensitivity of
isogenic PDR5 and pdr5 strains toward putative
substrates that are similar in chemical structure. Three series were
used: imidazole-containing compounds, trialkyltin chlorides, and
tetraalkyltin compounds. We demonstrate that the Pdr5p transporter is
capable of mediating transport of substrates that neither ionize nor
have electron pair donors and that are much simpler in structure than
those transported by the human MDR1-encoded P-glycoprotein.
Furthermore, the size of the substrate is critical and independent of
any requirement for hydrophobicity. Substrates have surface volumes
greater than 90 Å3 with an optimum response at ~200-225
Å3 as determined by molecular modeling. Assays measuring
the efflux from cells of [3H]chloramphenicol and
[3H]tritylimidazole were used. A
concentration-dependent inhibition of chloramphenicol
transport was observed with imidazole derivatives but not with
either the organotin compounds or the antitumor agent doxorubicin. In
contrast, several of the organotin compounds were potent inhibitors of
tritylimidazole efflux, but the Pdr5p substrate tetrapropyltin was
ineffective in both assays. This argues for the existence of at least
three substrate-binding sites on Pdr5p that differ in behavior from
those of the mammalian P-glycoprotein. Evidence also indicates that
some substrates are capable of interacting at more than one site. The
surprising observation that Pdr5p mediates resistance to tetraalkyltins
suggests that one of the sites might use only hydrophobic interactions
to bind substrates.
In Saccharomyces cerevisiae, broad spectrum resistance
to structurally and mechanistically diverse inhibitors is mediated by
several members of the ATP-binding cassette
(ABC)1 superfamily of
transporters, including Pdr5p, a 160-kDa ABC transporter found in the
plasma membrane. Pdr5p is essential for basal levels of resistance to a
broad array of substrates, including antifungal and antitumor agents,
hormones, and ionophores (1-4). Compared with isogenic wild-type
strains, pdr5 loss-of-function mutants exhibit
hypersensitivity because of their inability to efflux inhibitors (3,
5).
Although an important study (3) provided strong indirect evidence for
the existence of more than one substrate-binding site, the chemical
basis of Pdr5p substrate specificity is unclear. One reason is that
many of the compounds analyzed to date are complex in chemical
structure. As an alternative approach, we searched for a series of
relatively simple compounds that were systematically related to each
other in structure but varied in their ability to serve as Pdr5p
substrates. By comparing several series, we hoped to identify shared
properties that are used in the Pdr5p-substrate interaction. In the
initial study, we compared the relative resistance of isogenic
PDR5 (wild-type) and pdr5 (loss-of-function
mutant) strains toward a series of tri-n-alkyltin chlorides
of increasing alkyl chain length. These compounds are potent inhibitors
of mitochondrial ATPases and are not substrates for at least two other
major ABC yeast transporters, YOR1 and SNQ2 (6).
Initial results suggested that size and ionization might be important
factors in Pdr5p-substrate interaction (6).
We tested these parameters further by comparing the ability of Pdr5p to
mediate resistance to two new series of substrates: nonionizable
tetraalkytins and aromatic derivatives of imidazole, a chemical
constituent of a number of clinically significant antifungal agents. The backbone structures of these compounds are shown in Fig.
1. The simple aromatic imidazole
derivatives used in this study differ dramatically in structure from
the organotin compounds, but they overlap in size and calculated log P
(ClogP) values. Our results show that although ionization is not
required for Pdr5p action, molecular size is an important
component of Pdr5p-substrate interaction for the three distinct series.
Furthermore, some of the features that appear necessary for mammalian
P-glycoprotein (P-gp)-substrate interaction, such as multiple electron
pair donors (7, 8), do not seem to be required for all Pdr5p
substrates. Assays using radiolabeled chloramphenicol and
tritylimidazole indicate that Pdr5p uses several sites for transporting
substrates.
Chemicals and Media--
Tripropyltin chloride was obtained from
Alfa Division (Danvers, MA). Tripentyltin chloride was purchased from
Organometallics, Inc. (E. Hampstead, NH). Tetrapropyl, tetrabutyl, and
tetrapentyltin chloride were purchased from Gelest (Talleytown, PA).
Doxorubicin was purchased from Calbiochem.
[3H]Tritylimidazole (25 mCi/mmol) was custom-synthesized
by American Radiolabeled Chemicals, Inc. (St. Louis, MO) by reduction
of clotrimazole (the purity of this compound was 99%).
[3H]Chloramphenicol (50 mCi/mmol) was obtained from
PerkinElmer Life Sciences. All other chemicals were purchased from
Sigma-Aldrich. The preparation of yeast extract, peptone, dextrose
medium (YPD) and yeast extract, peptone, glycerol medium (YPG) are
described elsewhere (1). Synthetic dextrose medium containing all of the necessary growth supplements was purchased from Bio 101 (Carlsbad, CA).
Synthesis of Tritylimidazole and
Phenylethylimidazole--
Tritylimidazole and phenylethylimidazole
were synthesized with the procedure of Aldabbagh and Bowman (9). The
structure of both compounds was verified by using mass spectroscopy and nuclear magnetic resonance. After most of our results were collected, tritylimidazole became commercially available through Sigma-Aldrich. The commercial preparation was compared with our synthesized one in the
transport assay described below and gave indistinguishable results.
Yeast Strains--
The yeast strains used in this study are
listed in Table I. The construction of
JG436 from RW2802 was previously described (1), as was the construction
of DYK2.1 from SEY6210 (10). The isogenic strains AD124567 and AD1-7
(3) were generously provided by Dr. Michel Ghislian with the kind
permission of Dr. A. DeCottiginies, who constructed them.
In Vivo Drug Sensitivity Assays--
Minimum inhibitory
concentrations (MICs) were derived on plates and in liquid culture.
Tests of cycloheximide, doxorubicin, and various imidazole derivatives
were carried out on YPD plates to which a specific concentration of
inhibitor was added after the medium was sterilized. Chloramphenicol
and organotin compounds were tested in YPG medium. To determine the MIC
of a particular compound for the strains used in this analysis, cells
were grown in YPD medium. They were washed in sterile water, and the
concentration was adjusted by dilution so that 5 × 104 cells were deposited on plates with a micropipettor in
a volume of 10 µl. As the concentration increases and the MIC of a
particular compound is approached, cell growth on plates is sometimes
diminished, although it remains confluent. The MIC is reported as a
range of values. The lower value is the highest concentration tested that allows confluent growth after 72-h incubation at 30 °C on YPD
or 96-h incubation on YPG. The higher value is the lowest concentration
tested on which no growth is observed under the same conditions.
Calculation of ClogP and Molecular Size Parameters--
The
calculation of ClogP was carried out with a BioByte program (6).
Molecular surface volume was calculated with CAChe 4.4 (Fujitsum SBA
Tokyo, Beaverton, OR), a computer-aided molecular modeling tool that
uses a force field calculation to determine the energy and geometry of
a molecule and arrive at its lowest energy confirmation, which is then
used to calculate volume.
Measurement of [3H]Chloramphenicol and
[3H]Tritylimidazole Efflux--
Because of the
relatively poor loading of chloramphenicol and its rapid efflux (5),
transport was measured under steady state conditions. Cells were grown
overnight in synthetic dextrose medium. They were washed twice with
sterile double-deionized water and once with 0.05 M Hepes
buffer (pH 7.0) before concentrating to ~2 × 107
cells/100 µl in 0.05 M Hepes (pH 7.0). Aliquots of 125 µl were prepared containing 1.25 × 106 cpm
[3H]chloramphenicol made up to 5 µM with
cold chloramphenicol. Inhibitors or 100 mM 2-deoxyglucose
were introduced, and the samples were incubated for 3 h at
30 °C. We collected 90-µl samples by vacuum filtration on GF/C
filters (Whatman International, Maidstone, UK). The filters were washed
twice with 10 ml of 0.05 M Hepes buffer before drying and
counting in a Beckman LS3801 liquid scintillation counter
(Beckman-Coulter, Columbia, MD). [3H]tritylimidazole
efflux was analyzed directly by measuring the efflux rather than by
using steady state conditions. The cells were loaded for 3 h with
the same general protocol described for [3H]chloramphenicol. Following this, samples were chilled
on ice for 5 min, centrifuged at 4 °C, and washed once in 1 ml of
cold 0.05 M Hepes buffer before resuspension in 250 µl of
this buffer containing 1 mM glucose in the presence or
absence of inhibitors. Samples were incubated for various times at
30 °C. Following this, 600 µl of cold buffer (pH 7.0) was added,
and the samples were placed on ice for 5 min. Because tritylimidazole
adheres to filters, the samples were centrifuged at 4 °C and washed
once in cold 0.05 M Hepes buffer before resuspension in 125 µl for counting. Under these conditions, background counts averaging
~2400 cpm were observed. This value was subtracted from the total to
calculate the amount of retained tritylimidazole.
Pdr5p Does Not Require Ionization of Substrates or Electron Pair
Donors--
The trialkyltin chlorides and many substrates mediated by
Pdr5p are capable of ionization to varying degrees. Nevertheless, Kolaczkowski et al. (3) showed that progesterone, with no
ionizable groups, is also a Pdr5p substrate. We confirmed the ability
of Pdr5p to confer resistance to nonionizing compounds by analyzing three tetraalkyltins: propyl, butyl, and pentyl (the methyl and ethyl
derivatives are not toxic in yeast at the highest concentrations that
we were able to use). These results are shown in Table
II. All three are strong Pdr5p
substrates. This was observed by comparing the MICs derived on solid
media for isogenic PDR5 and pdr5 strains. The
MICs for tetraalkyltins using the PDR5 strain were
10-50-fold higher than the pdr5-null mutant strain JG436.
The tetraalkyltins are simple structures without electron pair donors
and in this respect are unlike other Pdr5p substrates described to
date. Thus, the possibility that the hypersensitivity was caused by a
coincidental mutation in another gene rather than in PDR5
had to be considered and was investigated by genetic analysis. The
ura3, PDR5 (wild-type) strain RW2802 was mated to
DYK2.1 (10), which carries a URA3-marked deletion of
PDR5. The resulting diploid was sporulated and subjected to
tetrad analysis to determine whether hypersensitivity to tetrabutyltin cosegregated with the URA3 allele. There was complete
cosegregation in 17 tetrads scored for these phenotypes. Five petite
segregants (mitochondria deficient and unable to grow on
glycerol) were not scored. Of the remaining 55 segregants, 29 Ura+ (pdr5) spores were all hypersensitive and
failed to grow on plates with 10 mM tetrabutyltin, and the
26 Ura Relative Resistance of Aromatic Imidazoles and Organotin Compounds
in Isogenic Wild-type and pdr5 Mutant Strains--
Table II also
includes an analysis of imidazole derivatives and lists the MICs for
JG436 and RW2802. To verify that the phenotypic differences observed
with JG436 and RW2802 are caused by differences at PDR5,
another pair of isogenic strains, SC 4741 (PDR5) and SC2909
(pdr5::gentr), were
included in this study (data not shown). With two exceptions noted
below, the results with the latter pair of strains were similar or
identical to those obtained with JG436 and RW2802. In both pairs of
strains, significant differences were observed with some of the
aromatic imidazole compounds. Phenyl- and benzylimidazole are weak
substrates at best (MIC ratios in both sets of strains are 1-2), but
large differences are found with bifonazole, tritylimidazole, and
clotrimazole (Table II). MICs were also derived for organotin compounds
(Table II), some of which were used in our previous analysis (6). No
differences between these strains were observed for trimethyltin
chloride, an observation in accord with our initial study (6).
The large difference between PDR5 and pdr5
strains observed with tripentyltin chloride of ~200-300-fold was in
marked contrast to our initial report of no difference (6). There was
no obvious explanation for this other than the fact that the compound
used in this study was purchased from a new source and was probably purer than the previous sample. Thin layer chromatography indicated that the new material was ~97-98% pure, as
advertised.2 There was a
significant difference between the two sets of strains with regard to
their relative bifonazole and tricyclohexyltin chloride resistance.
With regard to the former, the JG436/RW2802 set gave a difference of
~80-fold, whereas the difference between SC4741 and SC2909 was only
~8-fold. Similarly, with tricyclohexyltin chloride, the RW2802/JG436
pair gave an MIC ratio of 50-fold. The SC4741/SC2909 set yielded a
ratio of 2. It remains to be determined whether this disparity reflects
differences at PDR5 or at some other gene.
The effect of single-deletion mutations in SNQ2 and
YOR1 on imidazole resistance was investigated in strains
that are isogenic to SC4741. We showed previously that the proteins
encoded by these genes do not mediate resistance to trialkyltin
chlorides (6). We observed no differences in the MICs for any of the
imidazoles. Thus, the values obtained for SC4741 are the MICs for
snq2 and yor1 mutants as well. For comparison,
data are shown (Table II) for compounds that are either used
extensively in the analysis of yeast multidrug resistance
(cycloheximide, chloramphenicol) or that are related in structure to
members of the organotin series (dibutyltin dichloride and triphenyltin chloride).
Relationship between Substrate Efficacy and Molecular
Size--
The data relating substrate efficacy and surface volume are
listed in Table III and are illustrated
as relative toxicity versus volume plots in Fig.
2 for the isogenic RW2802 and JG436
strains. Relative toxicity is represented as a ratio of the MICs for
wild type (numerator) and mutant (denominator). There is a strong
relationship between substrate efficacy and size as calculated with
CAChe 4.4. Compounds smaller than ~100 Å3 are always
poor substrates. As size increases, so does the efficacy, reaching a
maximum at ~200 Å3, although the ratio for tributyltin
chloride is unexpectedly low and causes the plot to have a noticeable
leveling and perhaps even a dip between ~145 and 200 Å3.
Studies with a reporter plasmid demonstrate that the observed differences are not due to differential induction of PDR5
transcription by substrates (data not shown).
Is the Size Requirement for Pdr5p-Substrate Interaction Independent
of Hydrophobicity?--
Evidence strongly indicates that
hydrophobicity, as measured by ClogP, is an important property in the
interaction between substrates and the mammalian multidrug resistance
transporters such as P-gp (7, 8). Because hydrophobicity often
increases with substrate size, the relationship of these two properties in a series of structurally related compounds is difficult to ascertain. The organotin compounds, however, offered such an
opportunity, because it was possible to find examples of similar size
and structure but different degrees of hydrophobicity. Thus,
tripropyltin chloride and dibutyltin dichloride have surface volumes
within 3 Å3 of each other, but their ClogP values differ
by an order of magnitude (Table III). The latter is considerably more
hydrophilic, because it has an additional polar chlorine group.
Nevertheless, the two compounds gave very similar MIC ratios
(~10-fold) between PDR5 and pdr5 strains,
suggesting that substrate size is of critical importance and is
independent of any hydrophobicity requirement in the interaction of
Pdr5p and its substrates. This supposition is reinforced by the
observation that the optimal substrates from each of the series
(tetraalkyltins, imidazoles, and trialkyltin chlorides) are all in
the range of 200-225 Å3, although their ClogP values vary
from 4.34 to 10 (Table III).
Relative Resistance of Imidazole and Organotin Chlorides in a
Strain That Overproduces Pdr5p--
Classic multidrug resistance is
often associated with overexpression of ABC transporters. AD124567, a
strain that has deletions of five important drug transporters but
overproduces Pdr5p because of a PDR1-3 mutation (2)
and is not isogenic to RW2802 was used in our transport assays.
Therefore, it was important to determine whether its substrate
specificity was different. MICs were derived from AD124567 and the
isogenic AD1-7 control stock, which also contains a pdr5
deletion. These data are found in Table
IV. Efficacy is expressed as in Table II.
Not surprisingly, moderate to strong inhibitors (bifonazole,
tritylimidazole, clotrimazole, tetrapropyltin, tripropyltin,
tributyltin, and tripentyltin chlorides) yielded MICs that were
considerably higher in the overproducing strain than in wild-type
strains. In contrast, the compounds that gave little or no difference
(2-fold or less) in MIC between wild-type and mutant strains also
yielded similar MICs when AD1-7 and AD124567 were compared. These
observations mean that the conclusions reached with the overexpressing
strain should be generally applicable to strains with wild-type levels
of Pdr5p. Examination of the MIC ratios for the
tri-n-alkyltin chlorides was also instructive. Unlike the
case for the wild-type strains, RW2802 and SC4741, the ratio increased
steadily as the number of carbons increased. The data also indicated
that the MICs derived for the organotin chlorides in the multiple ABC
transporter deletion strain AD1-7 were similar to single
pdr5-null mutants. Therefore, other ABC loci are not major
mediators of resistance to these compounds.
Effect of Substrates on Chloramphenicol Efflux--
In a previous
study (5), we used a pulse-chase experiment to demonstrate that a
pdr5::URA3 strain has reduced
[3H]chloramphenicol efflux compared with an isogenic
wild-type control. Most efflux occurs within 5 min. The null mutant
strain also accumulates more [3H]chloramphenicol under
steady state conditions. Because the hyperresistant strain AD124567
effluxes drug at an even faster rate than the wild-type strain used in
the original study,3 we
decided to employ steady state conditions. The assumption that the
steady state difference between the strains in chloramphenicol accumulation reflects Pdr5p efflux was confirmed in the experiment illustrated in Fig 3A, where
accumulation is plotted as a function of substrate concentration in
both the PDR5 overproducer and its isogenic pdr5
counterpart, AD1-7. Although the former accumulated 2-3-fold less
substrate, both strains exhibit uptake that is linear and unsaturable.
This indicated that influx is not carrier-dependent. Furthermore, the addition of 100 mM 2-deoxyglucose to
assays with the overproducing strain resulted in kinetics that were
indistinguishable from those obtained with the pdr5 mutant.
The addition of this inhibitor to pdr5 mutant cultures,
however, did not change the retention kinetics. Thus, the observed
difference between the two strains was the result of energy-driven
Pdr5p-mediated efflux.
To determine whether the imidazole derivatives and the organotin
compounds are competitive inhibitors of Pdr5p-mediated chloramphenicol efflux, the transport assay described under "Experimental
Procedures" was performed in the presence of clotrimazole,
tritylimidazole, tripentyltin chloride, tributyltin chloride,
tricyclohexyltin chloride, and triphenyltin chloride. In addition, the
effect on transport of tetrapropyltin and the antitumor agent
doxorubicin was also evaluated. The first three compounds are of
particular interest, because they are strong substrates of very similar
size. The results are shown in Fig. 3B. Several important
points bear mention. First, none of the organotin compounds caused
significant levels of inhibition (Fig. 3B), although the
concentrations used (50 µM) were considerably above the
MICs for the pdr5 mutant. Second, as indicated by the plot
in Fig. 3C, although tritylimidazole and clotrimazole gave
MIC ratios of 200-300 in the wild-type-mutant comparison, there was a
marked difference in their ability to inhibit chloramphenicol
transport. Clotrimazole was ~10-fold better than tritylimidazole as a
competitive inhibitor. The I50 for clotrimazole was a
~5-7.5 µM, and that of tritylimidazole was ~50-75
µM. Significantly, 50 µM doxorubicin did
not block chloramphenicol efflux, although this concentration was
lethal in the absence of Pdr5p (data not shown).
Transport Studies with [3H]Tritylimidazole--
The
difference between clotrimazole and tritylimidazole in the
chloramphenicol efflux assay suggested that they might have high
affinity for different Pdr5p substrate-binding sites because they are
equally effective Pdr5p substrates. To test this possibility, we
undertook a series of transport studies with
[3H]tritylimidazole. Because it is easier to load cells
with [3H]-tritylimidazole than with chloramphenicol,
efflux was directly measured with the assay described under
"Experimental Procedures." One representative experiment measuring
tritylimidazole efflux for up to 2 h is found in Fig.
4A. Although there was some
variation in the initial concentration of incorporated substrate from
experiment to experiment (five were carried out), a reproducible
pattern was observed. Following the loading of cells and prior to the addition of efflux buffer, the concentration of
[3H]tritylimidazole was ~30 pmol/107
cells, with slightly higher amounts in the mutant than in the overproducing Pdr5p strain (data not shown). Within the first 15 min
following suspension of cells in Hepes buffer without tritylimidazole, there was a significant drop in the concentration of substrate in both
strains. At least some of this was probably due to diffusion that
started as soon as cold efflux buffer was added to the cells on ice,
because the T0 point was always lower (6-15
pmol/107 cells) than the pre-efflux concentration. At
~15-30 min, the pdr5-deficient strain started to
reaccumulate substrate, while the Pdr5p strain continued to efflux drug
in an energy-dependent manner. Thus, samples with 100 mM 2-deoxyglucose had tritylimidazole levels similar to the
T0 values of the pdr5 strain when assayed at 60 min. At this time, there was 2-3-fold greater efflux in the Pdr5p
strain. During the last 1 h, there was a modest decrease in
retained tritylimidazole in the Pdr5p strain and little or no increase
in the pdr5 mutant. Although the increased efflux in the
Pdr5p strain was obvious by 30-60 min, it was 2-3-fold, whereas the
MIC differential between this strain and its isogenic pdr5
deletion is nearly 1000. This discrepancy was investigated further. An
experiment was carried out that compared the viability of strains under
assay conditions in 0.05 M Hepes buffer with and without
5.0 µM tritylimidazole. There were no significant differences between the strains, and viability remained high (
Data in Fig. 4B indicate the effects of 2-deoxyglucose and
various Pdr5p substrates on [3H]tritylimidazole efflux.
Unlike the results with chloramphenicol, all of the trialkyltin
chlorides showed some ability to inhibit efflux, as did clotrimazole.
In contrast, tetrapropyltin, which in the Pdr5p overproducer was a
5-fold better substrate in the MIC analysis than tripropyltin chloride,
showed no inhibition in the transport assay. The effect on efflux of
varying the concentration of tripropyltin chloride, tripentyltin
chloride, and clotrimazole was studied, and the resulting data are
found in Fig. 4, C and D. The I50 for
tripropyltin chloride is ~5.0 µM. Significantly, clotrimazole, which is a stronger Pdr5p substrate than is tripropyltin chloride, nevertheless showed relatively weaker inhibition of tritylimidazole efflux. These data are summarized in Table
V.
In yeast, a basal level of broad spectrum drug resistance is
mediated in large part by Pdr5p, an ABC transporter that has been
studied extensively (see Ref. 11 for a recent review). In this report,
we demonstrate that Pdr5p effluxes substrates that are much simpler in
structure than many of the previously studied antifungal or antitumor
agents (11). Our data suggest that molecular size is of critical
importance. Because hydrophobicity also increases with size, it could
be concluded that it is the former that drives the interaction between
Pdr5p and its substrates. Several pieces of evidence suggest, however,
that the size requirement is independent of any requirement for
hydrophobicity. The first is a comparison of two compounds:
tripropyltin chloride and dibutyltin dichloride. These are of similar
size but have ClogPs that vary by 10-fold. Nevertheless, the MIC ratios
for these substrates are not significantly different. A similar
conclusion is reached by comparing tributyltin chloride,
tetrapropyltin, and triphenyltin chloride. Although their ClogPs vary
from 3.56 to 7.9, their surface volumes are very similar (176 versus 183 Å3), and all three compounds yield
nearly the same MIC ratios. The second significant observation is that
the most effective compound in each series has a molecular size in the
200-225 Å3 range regardless of ClogP values (which range
from 4.34 to 10). A size dependence for P-gp drug binding capability
was also observed (12). In wild-type yeast strains, plots of
trialkyltin chloride substrate efficacy (MIC ratios) versus
surface volume (Fig. 2) show a modest initial
size-dependent increase followed by a leveling or even a
slight decline with tributyltin chloride followed by a second peak. The
Pdr5p-overproducing strain failed to show this behavior. Instead, there
is a steady size-dependent increase. It is possible that
another gene product present in pdr5 mutants mediates some
resistance to tributyltin chloride. In the Pdr5p-overproducing strain,
this as yet unidentified protein's effect would be masked.
It is known that Pdr5p mediates significant resistance to a variety of
antitumor agents, dyes, small peptides, and hormones (3, 11). Most of
these agents are significantly larger than even the biggest molecules
in the various series tested in this study. For instance, rhodamine 6-G
has a volume of 480 Å3. For this reason, models of Pdr5p
action should include the possibility that the distance between two
chemical moieties on a substrate rather than the length of the molecule
is the critical parameter. Large, structurally complex substrates could
have several sets of chemical groups capable of interacting with a
transporter and thus several binding options. How size influences the
efficiency of transport by Pdr5p is not clear. One possibility is that
some of the substrate-binding sites have an optimal binding space that maximizes hydrophobic and other interactions with Pdr5p. Another, less
likely, explanation is that the diffusion constant of the potential
substrate within the lipid bilayer, which is proportional to the size
and hydrophobicity of the substrate, is a critical determinant of the
initial interaction of the substrate and the transporter. Thus, small
hydrophobic substrates, which diffuse rapidly across the bilayer, would
have limited time to interact with the transporter. Larger, more slowly
diffusing substrates would have a greater chance of interaction. A
distinction between these models requires additional study, although
the nonlinear nature of the relative toxicity plot and the independence
of size from ClogP strongly support the first explanation.
The organotin compounds are markedly different from other Pdr5p (3) and
P-gp (7, 8) substrates. There was no difference in the killing curves
obtained for isogenic multidrug-resistant and -sensitive human cell
lines (KB-3-1 and KB-V1, respectively) when challenged with
tetrabutyltin and tripentyltin
chloride.5 Seelig (7) and
Seelig and Landwojtowicz (8) presented evidence indicating that an
important component of a P-gp substrate is the presence of at least two
electron pair donor groups at fixed distances from each other. The
importance of electron pair donors in P-gp-substrate interactions was
recently demonstrated for a series of eight anthracyclines that are
closely related to doxorubicin (13). Other studies with analogues of
verapamil and colchicine (reviewed in Ref. 14) support this idea. The
tetraalkyltins are extreme exceptions, since they have no electron pair
donors. At first glance, it is not surprising that these are not
competitive inhibitors of chloramphenicol, which resembles P-gp
substrates (although its volume is smaller than most). In fact, the
organotin compounds appear to define two additional binding sites on
Pdr5p: those like tripentyltin chloride that compete with
[3H]tritylimidazole and those like tetrapropyltin that do
not. The mode of substrate recognition at this putative third site
remains to be determined, although the extreme simplicity of the
tetraalkyltins suggests that a hydrophobic interaction may be critical.
How these sites are organized, of course, remains unknown. For
instance, they might be distinct or overlapping.
Several other observations should be emphasized. Although
tritylimidazole and clotrimazole are equally strong Pdr5p substrates, the latter is 10-fold more effective as a competitive inhibitor of
chloramphenicol efflux. In contrast, clotrimazole shows weaker inhibition in the tritylimidazole efflux assay when compared with tripropyltin chloride, a rather modest Pdr5p substrate. This is consistent with our observation that tritylimidazole and clotrimazole interact with Pdr5p at more than one site with varying affinity. It is
possible that although tripropyltin chloride is a strong inhibitor of
tritylimidazole efflux, it interacts at fewer binding sites than the
larger, more complicated substrates. The difference between the two
imidazoles is a single chlorine group. It would be tempting to argue
that the clotrimazole/chloramphenicol-binding site resembles that of
P-gp because both compounds have strong electron pair donor groups such
as chlorine and oxygen. Furthermore, P-gp is known to mediate
resistance to azole antifungals such as ketoconazole and fluconazole
(15), although these are more complex in structure than the imidazoles
used in this study. The inability of the strong P-gp substrate
doxorubicin to compete with chloramphenicol argues that the details of
Pdr5p-substrate interaction are different from those observed in
mammalian cells.
Leonard et al. (5) first noted the discrepancy between the
relative difference in the sensitivity between PDR5 and
pdr5 strains toward chloramphenicol (20-fold) compared with
that of efflux (4-fold). This discrepancy was observed in the present study to an even larger degree with tritylimidazole. A number of
obvious explanations were ruled out, including inadequate buffer conditions and saturating levels of substrate. It is also possible that
due to its hydrophobic nature, the efflux of tritylimidazole is underestimated.
In summary, our data demonstrate that Pdr5p-substrate interaction has a
size dependence that is independent of the requirement for
hydrophobicity. Assays measuring chloramphenicol and tritylimidazole efflux indicate the presence of at least three substrate-binding sites,
possibly overlapping, that differ from those posited for P-gp. One site
is shared by chloramphenicol, clotrimazole, and, to a lesser extent,
tritylimidazole, although not by doxorubicin or the organotin
compounds. A second site defined by the tritylimidazole efflux assay is
used by the trialkyltin chlorides. Because neither chloramphenicol nor
tritylimidazole efflux is inhibited by tetrapropyltin, a third site is
posited and may use hydrophobic interactions between substrate and Pdr5p.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Backbone structures of inhibitor series.
A, trialkyltin chloride, where R is an alkyl chain
containing 1-6 carbons. Tetraalkyltins have an additional R group
and no chlorine. B, imidazole series; C is a methylene or
ethylene group, and the number of phenyl groups is either one
(benzylimidazole) or three (clotrimazole, tritylimidazole, and
bifonazole).
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Yeast strains
RESULTS
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ABSTRACT
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(PDR5) spores all grew by 96 h of
incubation. Therefore, the drug hypersensitivity observed in DYK2.1 is
caused by the absence of Pdr5p. This result is further supported by our
subsequent observation that the four independent pdr5
knockout mutations in our collection are hypersensitive to
tetraalkyltin compounds compared with their isogenic controls (data not
shown).
Relative resistance of PDR5 and pdr5 strains to substrates:
tetraalkyltins, imidazoles, organotin chlorides, and other compounds
Molecular size, hydrophobicity (ClogP), and substrate efficacy
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Fig. 2.
Relative toxicity plot for trialkyltin
chlorides and imidazoles as a function of size. The MICs for
inhibitors are shown in Table III as a range of concentrations. The
midpoint of the MIC range derived for RW2802 (PDR5) and
JG436 (pdr5-deficient) was used to construct an MIC ratio
for each compound. , imidazoles;
, trialkyltin chlorides;
,
tetraalkyltins.
Specificity of PDR5-overexpressing strain AD124567
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Fig. 3.
Competitive inhibition of
[3H]chloramphenicol efflux by imidazole compounds.
A, the energy requirement for reduced retention in a
Pdr5p-overproducing strain is illustrated. The plots show
chloramphenicol retention as a function of concentration in the
presence and absence of 2-deoxyglucose. ,
PDR5-overproducing strain;
, pdr5 strain; ×,
PDR5 plus 2-deoxyglucose; *, pdr5 plus
2-deoxyglucose. B, relative inhibition by various
substrates. The pdr5 deletion strain AD1-7 serves as a
control for comparison. clo-20, 20 µM
clotrimazole; trit-50, 50 µM tritylimidazole;
c3-Cl, 50 µM tripropyltin chloride;
c5-Cl, 50 µM tripentyltin chloride;
c6-Cl, 50 µM tricyclohexyltin chloride;
phe-Cl, 50 µM triphenyltin chloride;
c3-tet, 60 µM tetrapropyltin; dox,
50 µM doxorubicin. C, comparison of inhibition
by clotrimazole, tritylimidazole, and doxorubicin. Both imidazole
compounds show concentration-dependent inhibition, although
clotrimazole has an I50 that is ~10-fold less.
Doxorubicin fails to show significant inhibition at concentrations as
high as 100 µM.
, clotrimazole;
, tritylimidazole;
, doxorubicin.
98%) for up to 5 h. Because the buffer conditions used in this study are commonly employed (3), it is unlikely that they contributed significantly to the difference between observed efflux and MIC values.
This was reinforced by the observation that under the same conditions
employed in the present study, rhodamine 6G efflux was at least 100 times greater in the Pdr5p
overproducer.4 Furthermore,
the concentration of tritylimidazole employed (5 µM) was
not saturating, because transport experiments carried out with 0.25 µM gave similar kinetics (data not shown), and the MIC of
the Pdr5p overproducer was greater than 80 µM.
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Fig. 4.
Transport studies with
[3H]tritylimidazole. A, efflux as a
function of time. All [3H]tritylimidazole efflux assays
were carried out as described under "Experimental Procedures." In
A-C, S.E. values are indicated by error
bars. Each point is the average of two samples. The
graph shows that retention of
[3H]tritylimidazole is greater in the pdr5
mutant ( ) than in the PDR5 (
) strain. B,
comparative inhibition of [3H]tritylimidazole by selected
compounds. Each compound is compared with the pdr5 mutant.
These data were collected at the 1-h time point of the experiment shown
in A. 2DG, 100 mM 2-deoxyglucose;
clo-20, 20 µM clotrimazole; C3-tet,
60 µM tetrapropyltin; C3-Cl, 20 µM tripropyltin chloride; C5-Cl, 20 µM tripentyltin chloride; C6-Cl, 20 µM tricyclohexyltin chloride. C,
inhibition of efflux as a function of tripropyltin chloride and
clotrimazole concentration.
, tripropyltin chloride has an
I50 of ~5 µM. Clotrimazole (
) appears to
have lower affinity for the tritylimidazole site. The addition of 20 µM of either substrate to the pdr5 strain does
not alter the amount of retained tritylimidazole, which in this
experiment was 14.2 pmol/107 cells at 1 h. The addition of 20 µM of either of these substrates to the pdr5
(control) assays did not alter the concentration of retained
[3H]tritylimidazole.
Summary of inhibition capacity for some Pdr5p substrates
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ACKNOWLEDGEMENTS |
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We thank Susanna Tchilibon for carrying out the mass spectroscopy of tritylimidazole and Carol O. Cardarelli for carrying out the tests of cytotoxicity of organotin compounds on human P-gp-expressing drug-resistant cells. We are very grateful to Trish Weisman for carefully editing several drafts of this paper. We greatly appreciate the help of Zuben Sauna with its submission.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ On sabbatical leave at the NCI, NIH. To whom correspondence may be addressed. Tel.: 202-319-5722; Fax: 202-319-5721; E-mail: golin@cua.edu.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M210908200
2 J. Sczepanski and L. May, unpublished observations.
3 J. Golin, unpublished observations.
4 J. Golin and S. Ambudkar, unpublished data.
5 C. Cardarelli, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: ABC, ATP-binding cassette; P-gp, P-glycoprotein; ClogP, calculated log P; MIC, minimum inhibitory concentration; YPD, yeast extract, peptone, dextrose medium; YPG, yeast extract, peptone, glycerol medium; tritylimidazole, 1-(triphenylmethylimidazole); bifonazole, diphenylbenzylimidazole; clotrimazole, 1[(2-chlorphenyl)diphenylmethyl]imidazole.
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