Studies with Novel Pdr5p Substrates Demonstrate a Strong Size Dependence for Xenobiotic Efflux*

John GolinDagger §, Suresh V. Ambudkar, Michael M. Gottesman, Asif Dominic Habib||, John Sczepanski**, William ZiccardiDagger , and Leopold May**

From the Departments of Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

                              
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Table I
Yeast strains

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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- (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).

                              
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Table II
Relative resistance of PDR5 and pdr5 strains to substrates: tetraalkyltins, imidazoles, organotin chlorides, and other compounds

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).

                              
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Table III
Molecular size, hydrophobicity (ClogP), and substrate efficacy
Inhibitors are listed in ascending order of molecular size. The ClogP and surface volume are calculated as described under "Experimental Procedures."


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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. black-square, imidazoles; black-triangle, trialkyltin chlorides; open circle , tetraalkyltins.

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.

                              
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Table IV
Specificity of PDR5-overexpressing strain AD124567

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.


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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. diamond , PDR5-overproducing strain; black-triangle, 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; black-triangle, tritylimidazole; open circle , doxorubicin.

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 (>= 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 (diamond ) than in the PDR5 (black-square) 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. black-triangle, tripropyltin chloride has an I50 of ~5 µM. Clotrimazole (open circle ) 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.

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.

                              
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Table V
Summary of inhibition capacity for some Pdr5p substrates
A plus sign indicates complete or nearly complete inhibition at 20 µM and an I50 of ~5 µM. A minus sign indicates no inhibition at 50 µM (60 µM in the case of tetrapropyltin). A plus/minus sign indicates that significant but incomplete inhibition was observed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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