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• W2042974328 abstract "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. 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) 1The 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 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 (1Leppert G. McDevitt R. Falco S.C. Van Dyk T.K. Ficke M.B. Golin J. Genetics. 1990; 125: 13-20Google Scholar, 2Meyers S. Schauer W. Balzi E. Wagner M. Goffeau A. Golin J. Curr. Genet. 1992; 21: 431-436Google Scholar, 3Kolaczkowski M. Vanderest M. Cybularz-Kolaczkowski A. Soumilluion J. Konings W. Goffeau A. J. Biol. Chem. 1996; 271: 31543-31548Google Scholar, 4Egner R.F. Rosenthal A. Kralli D. Sanglard D. Kuchler K. Mol. Biol. Cell. 1998; 9: 523-543Google Scholar). Compared with isogenic wild-type strains, pdr5 loss-of-function mutants exhibit hypersensitivity because of their inability to efflux inhibitors (3Kolaczkowski M. Vanderest M. Cybularz-Kolaczkowski A. Soumilluion J. Konings W. Goffeau A. J. Biol. Chem. 1996; 271: 31543-31548Google Scholar,5Leonard P. Rathod P. Golin J. Antimicrob. Agents Chemother. 1994; 38: 2492-2494Google Scholar). Although an important study (3Kolaczkowski M. Vanderest M. Cybularz-Kolaczkowski A. Soumilluion J. Konings W. Goffeau A. J. Biol. Chem. 1996; 271: 31543-31548Google Scholar) 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 isogenicPDR5 (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 (6Golin J. Barkatt A. Cronin S. Eng G. May L. Antimicrob. Agents Chemother. 2000; 44: 134-138Google Scholar). Initial results suggested that size and ionization might be important factors in Pdr5p-substrate interaction (6Golin J. Barkatt A. Cronin S. Eng G. May L. Antimicrob. Agents Chemother. 2000; 44: 134-138Google Scholar). 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 (7Seelig A. Int. J. Clin. Pharmacol. Ther. 1998; 36: 50-54Google Scholar, 8Seelig A. Landwojtowicz E. Eur. J. Pharm. Sci. 2000; 1: 31-40Google Scholar), 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. 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 (1Leppert G. McDevitt R. Falco S.C. Van Dyk T.K. Ficke M.B. Golin J. Genetics. 1990; 125: 13-20Google Scholar). Synthetic dextrose medium containing all of the necessary growth supplements was purchased from Bio 101 (Carlsbad, CA). Tritylimidazole and phenylethylimidazole were synthesized with the procedure of Aldabbagh and Bowman (9Aldabbagh F. Bowman W.R. Tetrahedron. 1999; 55: 4109-4122Google Scholar). 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. The yeast strains used in this study are listed in Table I. The construction of JG436 from RW2802 was previously described (1Leppert G. McDevitt R. Falco S.C. Van Dyk T.K. Ficke M.B. Golin J. Genetics. 1990; 125: 13-20Google Scholar), as was the construction of DYK2.1 from SEY6210 (10Katzmann D. Burnett P.E. Golin J. Mahe Y. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 4653-4661Google Scholar). The isogenic strains AD124567 and AD1-7 (3Kolaczkowski M. Vanderest M. Cybularz-Kolaczkowski A. Soumilluion J. Konings W. Goffeau A. J. Biol. Chem. 1996; 271: 31543-31548Google Scholar) were generously provided by Dr. Michel Ghislian with the kind permission of Dr. A. DeCottiginies, who constructed them.Table IYeast strainsStrainGenotypeOriginRW2802MATa PDR5 leu2 met5 ura3Reed Wickner (1Leppert G. McDevitt R. Falco S.C. Van Dyk T.K. Ficke M.B. Golin J. Genetics. 1990; 125: 13-20Google Scholar)JG436Isogenic to RW2802 but pdr5∷Tn5Ref. 1Leppert G. McDevitt R. Falco S.C. Van Dyk T.K. Ficke M.B. Golin J. Genetics. 1990; 125: 13-20Google ScholarSC4741MATA PDR5 leu2 ura3 trpl his3 lys2Research GeneticsSC2909Isogenic to SC4741 butpdr5∷gent rResearch GeneticsAD 1–7MATα PDR1–3 ura3 hisl yor1Δpdr5snq2Δpdr10Δpdr11Δycf1Δpdr3ΔA. DeCottignies (3Kolaczkowski M. Vanderest M. Cybularz-Kolaczkowski A. Soumilluion J. Konings W. Goffeau A. J. Biol. Chem. 1996; 271: 31543-31548Google Scholar)AD124567Isogenic to AD1–7 but PDR5A. DeCottignies (3Kolaczkowski M. Vanderest M. Cybularz-Kolaczkowski A. Soumilluion J. Konings W. Goffeau A. J. Biol. Chem. 1996; 271: 31543-31548Google Scholar)DYK2.1MATαpdr5∷URA3 leu2 ura3 his3 trp1 lys2Ref. 10Katzmann D. Burnett P.E. Golin J. Mahe Y. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 4653-4661Google Scholar Open table in a new tab 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. The calculation of ClogP was carried out with a BioByte program (6Golin J. Barkatt A. Cronin S. Eng G. May L. Antimicrob. Agents Chemother. 2000; 44: 134-138Google Scholar). 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. Because of the relatively poor loading of chloramphenicol and its rapid efflux (5Leonard P. Rathod P. Golin J. Antimicrob. Agents Chemother. 1994; 38: 2492-2494Google Scholar), 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 × 107cells/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. The trialkyltin chlorides and many substrates mediated by Pdr5p are capable of ionization to varying degrees. Nevertheless, Kolaczkowski et al. (3Kolaczkowski M. Vanderest M. Cybularz-Kolaczkowski A. Soumilluion J. Konings W. Goffeau A. J. Biol. Chem. 1996; 271: 31543-31548Google Scholar) 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 TableII. 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 PDR5had to be considered and was investigated by genetic analysis. Theura3, PDR5 (wild-type) strain RW2802 was mated to DYK2.1 (10Katzmann D. Burnett P.E. Golin J. Mahe Y. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 4653-4661Google Scholar), which carries a URA3-marked deletion ofPDR5. 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 pdr5knockout mutations in our collection are hypersensitive to tetraalkyltin compounds compared with their isogenic controls (data not shown).Table IIRelative resistance of PDR5 and pdr5 strains to substrates: tetraalkyltins, imidazoles, organotin chlorides, and other compoundsCompoundMICaValues are μmexcept for the tetralkyltins, dibutyltin dichloride, phenyl, and benzylimidazole, where concentrations are mm. Values are the average of at least two experiments.MICbThe MIC ratio is the MIC of the wild-type strain (RW2802, PDR5) divided by the MIC of thepdr5 mutant (JG436, pdr5).ratioRW2802JG436TetraalkyltinsTetrapropyltin0.32–0.680.04–0.0710Tetrabutyltin90–1801.8–3.650Tetrapentyltin54–1103.2–5.420Aromatic imidazoles1-Phenylimidazole0.60–0.800.60–0.811-Benzylimidazole2.5–5.01.3–2.521-Phenylethylimidazole4.4–8.72.9–4.41–3Bifonazole190–3802.4–4.880Tritylimidazole6.5–12.90.02–0.08160–325Clotrimazole3.2–6.40.01–0.03213–320Alkyltin chloridesTrimethyltin chloride0.16–0.320.16–0.321Triethyltin chloride0.90–1.80.45–0.902Tripropyltin chloride1.2–2.40.12–0.2410Tributyltin chloride0.16–0.320.04–0.084Tripentyltin chloride40–800.13–0.25320Tricyclohexyltin chloride0.50–1001.0–2.050Other compoundsDibutyltin dichloride0.50–1.00.08–0.106.2–10Triphenyltin chloride0.75–1.500.08–0.1610Cycloheximide2.5–5.00.25–0.5010Chloramphenicol3.0–6.00.15–0.3020a Values are μmexcept for the tetralkyltins, dibutyltin dichloride, phenyl, and benzylimidazole, where concentrations are mm. Values are the average of at least two experiments.b The MIC ratio is the MIC of the wild-type strain (RW2802, PDR5) divided by the MIC of thepdr5 mutant (JG436, pdr5). Open table in a new tab 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 (6Golin J. Barkatt A. Cronin S. Eng G. May L. Antimicrob. Agents Chemother. 2000; 44: 134-138Google Scholar). No differences between these strains were observed for trimethyltin chloride, an observation in accord with our initial study (6Golin J. Barkatt A. Cronin S. Eng G. May L. Antimicrob. Agents Chemother. 2000; 44: 134-138Google Scholar). The large difference between PDR5 and pdr5strains observed with tripentyltin chloride of ∼200–300-fold was in marked contrast to our initial report of no difference (6Golin J. Barkatt A. Cronin S. Eng G. May L. Antimicrob. Agents Chemother. 2000; 44: 134-138Google Scholar). 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. 2J. Sczepanski and L. May, unpublished observations. 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 andYOR1 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 (6Golin J. Barkatt A. Cronin S. Eng G. May L. Antimicrob. Agents Chemother. 2000; 44: 134-138Google Scholar). We observed no differences in the MICs for any of the imidazoles. Thus, the values obtained for SC4741 are the MICs forsnq2 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). 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 PDR5transcription by substrates (data not shown).Table IIIMolecular size, hydrophobicity (ClogP), and substrate efficacyCompoundSurface volumeClogPMIC ratioaThe ratio of wild-type to mutant MIC is listed as the MIC ratio. Values are obtained from RW2802 (PDR5) and JG436 (pdr5) as described in Table II.Å3Trimethyltin chloride75.74−0.511Phenylimidazole89.421.881Benzylimidazole101.551.551.5Phenylethylimidazole112.791.931–3Triethyltin chloride112.121.082Dibutyltin dichloride143.641.566.2–10Tripropyltin chloride147.362.6610Chloramphenicol158.000.6920Cycloheximide158.00−0.4910Tetrapropyltin176.417.910Tributyltin chloride179.424.254Triphenyltin chloride183.33.5610Tritylimidazole200.334.34160–320Clotrimazole208.365.05213–320Tripentyltin chloride215.075.84320Tetrabutyltin224.011050Bifonazole247.814.7980Tricyclohexyltin chloride251.35.5850Tetrapentyltin275.5112.1325Inhibitors are listed in ascending order of molecular size. The ClogP and surface volume are calculated as described under “Experimental Procedures.”a The ratio of wild-type to mutant MIC is listed as the MIC ratio. Values are obtained from RW2802 (PDR5) and JG436 (pdr5) as described in Table II. Open table in a new tab Inhibitors are listed in ascending order of molecular size. The ClogP and surface volume are calculated as described under “Experimental Procedures.” 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 (7Seelig A. Int. J. Clin. Pharmacol. Ther. 1998; 36: 50-54Google Scholar, 8Seelig A. Landwojtowicz E. Eur. J. Pharm. Sci. 2000; 1: 31-40Google Scholar). 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). 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 (2Meyers S. Schauer W. Balzi E. Wagner M. Goffeau A. Golin J. Curr. Genet. 1992; 21: 431-436Google Scholar) 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 pdr5deletion. These data are found in TableIV. 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 singlepdr5-null mutants. Therefore, other ABC loci are not major mediators of resistance to these compounds.Table IVSpecificity of PDR5-overexpressing strain AD124567CompoundMICaAll of the compounds have MICs expressed as μm concentrations except for phenyl- and benzylimidazole, whose MICs are given in mmconcentrations.A/BAD124567 (A)AD1–7 (B)Aromatic imidazoles1-Phenylimidazole0.60–0.800.20–0.4021-Benzylimidazole2.5–5.01.25–2.521-Phenylethylimidazole>121.9–2.9>4Bifonazole1900–3800<12>316Tritylimidazole>80<0.08>1000Clotrimazole>240.02–0.03>800Organotin compoundsTetrapropyltin3.4–6.80.04–0.07100Trimethyltin chloride0.16–0.320.16–0.321Tripropyltin chloride4.8–9.60.24–0.4820Tributyltin chloride6.4–12.80.04–0.08160Tripentyltin chloride>20001.0>2000Dibutyltin dichloride>20.10–0.15>13a All of the compounds have MICs expressed as μm concentrations except for phenyl- and benzylimidazole, whose MICs are given in mmconcentrations. Open table in a new tab In a previous study (5Leonard P. Rathod P. Golin J. Antimicrob. Agents Chemother. 1994; 38: 2492-2494Google Scholar), we used a pulse-chase experiment to demonstrate that apdr5::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, 3J. Golin, unpublished observations. 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 3 A, where accumulation is plotted as a function of substrate concentration in both the PDR5 overproducer and its isogenic pdr5counterpart, 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, tricyclo" @default.
• W2042974328 created "2016-06-24" @default.
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• W2042974328 creator A5030334751 @default.
• W2042974328 creator A5046415907 @default.
• W2042974328 creator A5048991463 @default.
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• W2042974328 date "2003-02-01" @default.
• W2042974328 modified "2023-09-26" @default.
• W2042974328 title "Studies with Novel Pdr5p Substrates Demonstrate a Strong Size Dependence for Xenobiotic Efflux" @default.
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