BACKGROUND
the last decades have seen an increase in the immunocompromised population for several reasons including as a result of treatment of malignant diseases, hiv infection, as well as advances in organ transplantation procedures.
in this scenario opportunistic infections, especially those caused by fungi, have become a serious public health problem  <cit> – <cit> .
candidiasis is the most common fungal infection among patients with a condition that leads to immunosuppression  <cit> , <cit> .
azoles, especially fluconazole, have been commonly used to treat fungal infections  <cit> .
however, overexpression of membrane efflux pumps by fungal cells is an important mechanism that causes azole resistance  <cit> .
some of these efflux pumps belong to the pleiotropic drug resistance  sub-family of atp-binding cassette  transporters, and they lead to active transport of drugs using energy derived from atp hydrolysis  <cit> .
saccharomyces cerevisiae can express several abc transporters, and of these, pdr5p has been the best studied  <cit> .
this efflux pump causes the extrusion of several drugs that are used to treat fungal infections.
also, it exhibits a profile of substrates and inhibitors that is similar to those of other abc transporters that are expressed by pathogenic fungi  <cit> .
these features make pdrp <dig> a good experimental model for the study of antifungal resistance mediated by abc transporters.
one strategy for overcoming drug resistance mediated by efflux pumps is the use of compounds that can function as chemosensitizers.
these compounds potentiate the efficacy of existing azoles, such as fluconazole, by inhibiting these abc transporters  <cit> .
thus, the development of novel azole chemosensitizers that increase the potency of these drugs against both sensitive and resistant fungi may allow the use of previously ineffective antifungal to treat fungal infections  <cit> .
some studies have already reported compounds that are capable of reversing the resistance phenotype, such as d-octapeptides  <cit> , enniatin  <cit> , isonitrile  <cit>  and gallic acid derivatives  <cit> .
recently, interest in organic compounds containing tellurium  or selenium  has increased and several studies have been published demonstrating biological properties for both elements.
despite the relative toxicity conferred by organic compounds containing tellurium  <cit> , some studies have shown that these molecules may have immunomodulatory and anti-inflammatory properties  <cit> , antioxidant abilities  <cit> , and anti-proliferative actions against certain tissues  <cit> .
selenium is a nutritionally essential trace element for mammals.
studies have shown that some organic compounds derived from this chalcogenide exhibit antinociceptive, hepatoprotective, neuroprotective, anti-inflammatory and anti-carcinogenic properties  <cit> .
furthermore, some organochalcogenides containing te or se are capable of inhibiting the atpase activity of the na+/k+ atpase that is present in rat brains  <cit>  and can inhibit the atpase activity of p-glycoprotein and vinblastine efflux mediated by this neoplasic cell multidrug transporter  <cit> .
finally, te and se containing compounds can inhibit the plasma membrane h+-atpase from s. cerevisiae <cit> .
although several biological properties have already been described in the literature for chalcogenides and their derivatives, molecules containing selenium or tellurium with the capacity to reverse efflux pump-mediated azole resistance have not yet been reported.
we were interested in studying the effects of organic compounds containing tellurium or selenium on pdr5p, which is a well-known experimental model for the study of fungal resistance mediated by efflux pumps.
in this study, we evaluated  <dig> synthetic compounds; some of which contained tellurium  or selenium , and others that were devoid of both chalcogenides.
methods
chemicals
reagents were purchased from sigma-usa  or tecoland-usa  unless otherwise stated.
all reagents purchased were of highest available standard.
synthetic compounds used in this study
the compounds listed in figure  <dig> were synthesized according to procedures that had been previously developed by our group; synthetic and spectroscopic information about these compounds can be found in the original publications  <cit> – <cit> .
all of the compounds were kept in a desiccator at 4°c, and the stock solutions were prepared using dimethyl sulfoxide  as a solvent.
strains and culture conditions
in this study, two mutant strains of saccharomyces cerevisiae were used.
the first strain ad <dig>  overexpresses pdr5p, while the genes encoding the pdr3p regulator and the other five abc transporters  have been deleted.
the second one ad <dig>  contains deletions of the same six genes, as well as the gene that encodes the pdr5p transporter  <cit> .
the yeast strains were grown in ypd medium  at 30°c with agitation and were harvested in the exponential phase of growth.
one fluconazole resistant strain of candida albicans, isolated from urine sample, was also used .
in this case, the yeast were cultivated in sabouraud medium , at 37°c under agitation .
preparation of plasma membranes
yeast plasma membrane isolates from the s. cerevisiae mutant strain pdrp5+ and from the null mutant pdr5p- were obtained as previously described by rangel et al.
<cit> .
the plasma membrane preparations were stored in liquid nitrogen and thawed immediately prior to use in the pdr5p atpase activity assays.
atpase activity assay
the effect of the compounds on the atpase activity of pdr5p was quantified by incubating pdr5p-containing membranes  in a 96-well plate at 37°c for 60 min in a reaction medium containing 100 mm tris–hcl , 4 mm mgcl <dig>  75 mm kno <dig>   <dig>  mm nan <dig>   <dig>  mm ammonium molybdate and 3 mm atp in the presence of the synthetic compounds.
after incubation, the reaction was stopped by the addition of 1% sds, as described previously by dulley  <cit> .
the amount of released inorganic phosphate  was measured as previously described by fiske & subbarrow  <cit> .
preparations containing plasma membranes obtained from the null mutant strain ad <dig>  were used as controls.
the difference between the atpase activity of the pdr5p + and pdr5p- membranes represents the atpase activity that is mediated by pdr5p.
effect of compounds on the growth of s. cerevisiae strains
this assay was conducted according to niimi et al.
<cit> .
the effect of the compounds on the growth of both mutant strains of s.cerevisiae used in this work was determined by microdilution assays using 96-well microplates.
the cells were inoculated into ypd medium at a concentration of  <dig> ×  <dig> cells per well and incubated at 30°c for 48 h with agitation  in the presence of different concentrations of the compounds.
controls were performed using dmso at a final concentration of 1% to verify the toxicity of the solvent used to solubilize the compounds.
cell growth was determined using a microplate reader at 600 nm .
lytic effect of compounds on human erythrocytes
this assay was conduct as described by niimi et al.
<cit> .
human erythrocytes were previously washed three times and resuspended in phosphate-buffered saline .
red blood cells  were then incubate in the presence of different concentrations of the synthetic compounds for 60 min at 37°c.
after incubation, the cells were pelleted by centrifugation at  <dig>  g for 5 min and aliquots of 100 μl of the supernatant were transferred to the wells of a microplate.
the absorbance of the hemoglobin released from the erythrocytes was measured at 540 nm.
a control of 100% hemolysis was performed incubating the cells in the presence of pbs containing 1% triton x- <dig>
evaluation of fluconazole resistance reversion by the synthetic compounds
the “spot test” was used as a measure of growth as previously described by rangel et al.
<cit> .
for s. cerevisiae strain pdr5p+, 5 μl samples of fivefold serially diluted yeast cultures  were spotted on ypd agar in  <dig> well sterile polystyrene plates.
they were incubated in the presence of synthetic compounds  only or associated with fluconazole at 120 μg/ml.
controls were performed using ypd alone and ypd supplemented with: 120 μg/ml fluconazole, 120 μg/ml fluconazole +  <dig> % dmso, 120 μg/ml fluconazole + 10 μm fk <dig>  plates were incubated at 30°c for 48 h.
in the case of c. albicans, the same methodology was used, but with some adaptations: 5 μl of a five-fold serial dilution from a yeast suspension containing  <dig> ×  <dig> cells/ml was spotted on sabouraud agar supplemented with the compounds at 100 μm alone or combined with fluconazole at 64 μg/ml.
the incubation of the six well plates was carried at 37°c for 48 h.
checkerboard assay with compounds and fluconazole using candida strain from clinical isolate
candida albicans cells, in exponential growth phase  were incubated in presence of different combinations of fluconazole and compound at 37°c for 48 hours in rpmi  <dig>  using 96-well plates under stirring.
cell growth was determined using a plate reader  at a wavelength of 600 nm.
the mic value was referred to concentration capable of causing 80% growth inhibition .
possible synergism between fluconazole and tested compounds was determined based on the fractional inhibition concentration index .
synergic, indifferent and antagonistic interactions were defined by a fici of < <dig> ,  <dig> - <dig>  or  <dig>  respectively  <cit> .
statistical analysis
all experiments were performed in triplicate.
data were presented as mean ± standard error.
a probability level of 5%  in student’s t -test was considered significant.
RESULTS
atpase activity
pdr5p is an abc transporter and as such the inhibition of its atpase activity could significantly affect the efflux of fluconazole and contribute to the reversal of resistance against this antifungal.
thus, a screening assay was performed to identify synthetic compounds that could promote inhibition of atp hydrolysis catalyzed by pdr5p .
of the  <dig> compounds tested only four  were capable of inhibiting pdr5p atpase activity by more than 90% .
all four compounds contained a butyl-tellurium residue, a lateral hydrocarbon chain and an amide group, that were absent in the other tested compounds.
this suggests that these chemical structure could have an important role in the inhibitory process.
the four active compounds  were selected for further investigation.
dose–response curves and a double reciprocal plot were performed .
the results confirm that these compounds are strong inhibitors of pdr5p atpase activity and exhibit ic <dig> values lower than 2 μm .
furthermore, the double reciprocal plot for compound  <dig> demonstrated an uncompetitive pattern of inhibition .
compounds  <dig>   <dig> and  <dig> demonstrated the same mechanism of inhibition .
50
values of the compounds against the atpase activity of pdr5p
50
the data represent the means ± standard error of three independent experiments.
until now, there have been no reports in the literature of organic synthetic compounds containing tellurium that inhibit pdr5p atpase activity.
however, many other molecules, of synthetic or natural origin, also exhibit this ability.
silva et al.
<cit>  demonstrated that oroidin, a derivative of a compound from a sponge, is able to inhibit the catalytic activity of this multidrug transporter with an ic <dig> of 20 μm.
rangel et al.
<cit> , while studying gallic acid derivatives, observed that decyl gallate has an ic <dig> value of  <dig>  μm.
both compounds competitively inhibit the enzyme activity of pdr5p.
competitive inhibition is a more common characteristic than the uncompetitive inhibition shown by the four organotellurides.
as mentioned by cannon et al.
<cit> , inhibition of plasma membrane h+-atpase activity could contribute to the reversal of abc transporter-mediated azole resistance, by depleting the intracellular atp concentration.
to investigate this, the effects of the four organotellurides  on the plasma membrane h+-atpase of s. cerevisiae were evaluated.
the organotellurides leaded a powerful inhibition of the h+-atpase activity  and exhibited ic <dig> values of approximately  <dig>  μm .
chan and colleagues  <cit>  previously demonstrated that ebselen, a well-known organoselenium compound, was also able to inhibit the activity of s. cerevisiae plasma membrane h+-atpase in a dose dependent manner.
ebselen was also shown to be toxic for s. cerevisiae at a concentration of 10 μm, unlike the organotellurides investigated in this study.
effect of the compounds on the growth of pdr5p+ and pdr5p- mutant s. cerevisiae strains
the organotellurides  <dig>   <dig>   <dig> and  <dig> that inhibited pdr5p activity did not affect the growth of the pdr5p+ strain at concentrations up to 200 μm .
however, the growth of the pdr5p- null mutant was significantly impaired after incubation with 200 μm of compound  <dig> .
this indicates that this compound could work as substrate and atpase activity inhibitor of pdr5p such as fk <dig>  a classical and potent pdr5p inhibitor  <cit> .
evaluation of cytotoxicity against human erythrocytes
the active compounds were tested for their hemolytic activity on human erythrocytes .
as shown in figure  <dig>  even at the highest concentration used in this assay  the four compounds promoted the release of around 4% of erythrocyte hemoglobin.
there was no significant difference between the hemolysis caused by the compounds and that observed in pbs  and dmso  controls.
therefore, all four active compounds showed another a desirable feature of a compound to reverse fluconazole resistance that is a low toxicity for a mammal cell line.
fluconazole resistance reversion by the synthetic organotellurides
the spot assay shown in figure 6a demonstrates that pdr5p+ strain, which is resistant to fluconazole , was able to grow on a medium containing fluconazole at 120 μg/ml as well as in presence of compounds at 100 μm.
however, an evident reduction in growth was observed when this strain was incubated in presence of fluconazole  associated with any of the four organotellurides .
thus, it was possible to demonstrate that these synthetic compounds were able to reverse the fluconazole resistance mediated by pdr5p in a manner similar to the reversion promoted by fk <dig>  a control using the pdr5p- null mutant ) was performed to confirm that the presence of pdr5p is responsible for the fluconazole resistance of the ad <dig> strain.
the same experiment was performed using a fluconazole resistant candida albicans clinical isolate because overexpression of efflux pumps is a possible mechanism of resistance to azoles in this yeast also.
however, the level of expression of the c. albicans abc transporter  is lower in comparison to the s. cerevisiae strains used in the present work that were genetically modified to overexpress the efflux pump .
thus, the hypothesis is that would be possible to reverse the resistance in pathogenic yeast, as resistant c. albicans from a clinical isolate, with lower concentration of azole in comparison with ad <dig> strain.
the c. albicans clinical isolate was able to grow in presence of fluconazole at 64 μg/ml  that is considered as resistant strain.
the active compounds alone  did not affect growth of c. albicans, but when associated with fluconazole  were able to promote a complete growth inhibition in comparison with inhibition obtained in the presence of fk <dig> .
this data reinforces the results obtained with s. cerevisiae and provides further evidence that blocking efflux pumps represents a valid therapy measure for treatment of resistant fungal strains.
this strategy becomes more evident using the checkerboard assay where compounds and fluconazole were tested in different concentrations .
all compounds tested were able to act synergistically with fluconazole since they showed fici values lower than  <dig>   <cit> .
this proves the efficiency of the use of those organotellurides in combination with azoles in reversion of resistance due to overexpression of efflux pumps in pathogenic fungi such as c. albicans.
candida albicans
strain
a
b
*this assay was done with organotellurides and fluconazole isolated or combined.
mics were determined by a microdilution technique based on 80% reduction of growth.
afic = fractional inhibitory concentration; bfici = fractional inhibitory concentration index.
CONCLUSIONS
compounds  <dig>   <dig>   <dig> and  <dig> are synthetic compounds that have some similarities.
firstly, all they contain a butyl tellurium residue, secondly, they have a lateral hydrocarbon chain and finally, they all possess an amide group.
all they were able to reverse the fluconazole resistance mediated by pdr5p from s. cerevisiae.
a likely mechanism for this reversal is the direct inhibition of the atpase activity of pdr5p and the indirect inhibition of the plasma membrane h+-atpase.
furthermore, the compounds could also overcome fluconazole resistance expressed by a clinical isolate of candida albicans.
the reversal of fluconazole resistance was obtained using 100 μm of the compounds.
this concentration did not demonstrate toxicity against human erythrocytes or fungal cells.
in conclusion, these compounds could be promising candidates for the reversal of resistance mediated by drug efflux pumps, act synergistically with fluconazole and could serve as prototypes for the synthesis of other molecules that could be capable of inhibiting efflux pumps with greater efficiency.
availability of supporting data
the data sets supporting the results of this article are included within the article.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
lfrs: carried out the conception and design the experiments; the acquisition, analysis and interpretation of data.
he also drafts the manuscript.
ft: carried out the synthesis of the compounds used in this work and was involved in revising the manuscript critically.
bas: carried out the synthesis of the compounds used in this work, and was involved in revising the manuscript critically.
acg: carried out the synthesis of the compounds used in this work, and was involved in revising the manuscript critically.
act: had made contributions to analysis and interpretation of data and was involved in revising the manuscript critically.
epw: carried out the synthesis of the compounds used in this work, and was involved in revising the manuscript critically.
jvc: carried out the supervision of the students involved in the synthesis of the compounds used in this work, and was involved in revising the manuscript critically.
aas: designed the synthesized compounds and carried out the supervision of the students involved in the synthesis of the compounds used in this work, and was involved in revising the manuscript critically.
he was involved in revising the manuscript critically and gave final approval of the final version.
afp: helped with the conception and design the experiments; with analysis and interpretation of data and draft the manuscript.
he was involved in revising the manuscript critically and gave final approval of the version to be published.
all authors read and approved the final manuscript.
acknowledgments
the authors thank faperj , fapesp , cnpq  and capes for financial support and scholarships.
the authors are grateful for the financial and structural support offered by the university of são paulo through the nap-catsinq .
the authors thank also to our lab assistant, mrs. geralda rodrigues almeida for her great support and dr. louise kemp for your critical reading of this manuscript.

 the last decades have seen an increase in the immunocompromised population for several reasons including as a result of treatment of malignant diseases, hiv infection, as well as advances in organ transplantation procedures. in this scenario opportunistic infections, especially those caused by fungi, have become a serious public health problem –. candidiasis is the most common fungal infection among patients with a condition that leads to immunosuppression,. azoles, especially fluconazole, have been commonly used to treat fungal infections. however, overexpression of membrane efflux pumps by fungal cells is an important mechanism that causes azole resistance. some of these efflux pumps belong to the pleiotropic drug resistance sub-family of atp-binding cassette transporters, and they lead to active transport of drugs using energy derived from atp hydrolysis. saccharomyces cerevisiae can express several abc transporters, and of these, pdr5p has been the best studied. this efflux pump causes the extrusion of several drugs that are used to treat fungal infections. also, it exhibits a profile of substrates and inhibitors that is similar to those of other abc transporters that are expressed by pathogenic fungi. these features make pdrp a good experimental model for the study of antifungal resistance mediated by abc transporters. one strategy for overcoming drug resistance mediated by efflux pumps is the use of compounds that can function as chemosensitizers. these compounds potentiate the efficacy of existing azoles, such as fluconazole, by inhibiting these abc transporters. thus, the development of novel azole chemosensitizers that increase the potency of these drugs against both sensitive and resistant fungi may allow the use of previously ineffective antifungal to treat fungal infections. some studies have already reported compounds that are capable of reversing the resistance phenotype, such as d-octapeptides, enniatin, isonitrile and gallic acid derivatives. recently, interest in organic compounds containing tellurium or selenium has increased and several studies have been published demonstrating biological properties for both elements. despite the relative toxicity conferred by organic compounds containing tellurium, some studies have shown that these molecules may have immunomodulatory and anti-inflammatory properties, antioxidant abilities, and anti-proliferative actions against certain tissues. selenium is a nutritionally essential trace element for mammals. studies have shown that some organic compounds derived from this chalcogenide exhibit antinociceptive, hepatoprotective, neuroprotective, anti-inflammatory and anti-carcinogenic properties. furthermore, some organochalcogenides containing te or se are capable of inhibiting the atpase activity of the na+/k+ atpase that is present in rat brains and can inhibit the atpase activity of p-glycoprotein and vinblastine efflux mediated by this neoplasic cell multidrug transporter. finally, te and se containing compounds can inhibit the plasma membrane h+-atpase from s. cerevisiae. although several biological properties have already been described in the literature for chalcogenides and their derivatives, molecules containing selenium or tellurium with the capacity to reverse efflux pump-mediated azole resistance have not yet been reported. we were interested in studying the effects of organic compounds containing tellurium or selenium on pdr5p, which is a well-known experimental model for the study of fungal resistance mediated by efflux pumps. in this study, we evaluated synthetic compounds; some of which contained tellurium or selenium, and others that were devoid of both chalcogenides. methods chemicals reagents were purchased from sigma-usa or tecoland-usa unless otherwise stated. all reagents purchased were of highest available standard. synthetic compounds used in this study the compounds listed in figure  were synthesized according to procedures that had been previously developed by our group; synthetic and spectroscopic information about these compounds can be found in the original publications –. all of the compounds were kept in a desiccator at 4°c, and the stock solutions were prepared using dimethyl sulfoxide as a solvent. strains and culture conditions in this study, two mutant strains of saccharomyces cerevisiae were used. the first strain ad overexpresses pdr5p, while the genes encoding the pdr3p regulator and the other five abc transporters have been deleted. the second one ad contains deletions of the same six genes, as well as the gene that encodes the pdr5p transporter. the yeast strains were grown in ypd medium at 30°c with agitation and were harvested in the exponential phase of growth. one fluconazole resistant strain of candida albicans, isolated from urine sample, was also used. in this case, the yeast were cultivated in sabouraud medium, at 37°c under agitation. preparation of plasma membranes yeast plasma membrane isolates from the s. cerevisiae mutant strain pdrp5+ and from the null mutant pdr5p- were obtained as previously described by rangel et al.. the plasma membrane preparations were stored in liquid nitrogen and thawed immediately prior to use in the pdr5p atpase activity assays. atpase activity assay the effect of the compounds on the atpase activity of pdr5p was quantified by incubating pdr5p-containing membranes in a 96-well plate at 37°c for 60 min in a reaction medium containing 100 mm tris–hcl, 4 mm mgcl 75 mm kno  mm nan  mm ammonium molybdate and 3 mm atp in the presence of the synthetic compounds. after incubation, the reaction was stopped by the addition of 1% sds, as described previously by dulley. the amount of released inorganic phosphate was measured as previously described by fiske & subbarrow. preparations containing plasma membranes obtained from the null mutant strain ad were used as controls. the difference between the atpase activity of the pdr5p + and pdr5p- membranes represents the atpase activity that is mediated by pdr5p. effect of compounds on the growth of s. cerevisiae strains this assay was conducted according to niimi et al.. the effect of the compounds on the growth of both mutant strains of s.cerevisiae used in this work was determined by microdilution assays using 96-well microplates. the cells were inoculated into ypd medium at a concentration of × cells per well and incubated at 30°c for 48 h with agitation in the presence of different concentrations of the compounds. controls were performed using dmso at a final concentration of 1% to verify the toxicity of the solvent used to solubilize the compounds. cell growth was determined using a microplate reader at 600 nm. lytic effect of compounds on human erythrocytes this assay was conduct as described by niimi et al.. human erythrocytes were previously washed three times and resuspended in phosphate-buffered saline. red blood cells were then incubate in the presence of different concentrations of the synthetic compounds for 60 min at 37°c. after incubation, the cells were pelleted by centrifugation at  g for 5 min and aliquots of 100 μl of the supernatant were transferred to the wells of a microplate. the absorbance of the hemoglobin released from the erythrocytes was measured at 540 nm. a control of 100% hemolysis was performed incubating the cells in the presence of pbs containing 1% triton x- evaluation of fluconazole resistance reversion by the synthetic compounds the “spot test” was used as a measure of growth as previously described by rangel et al.. for s. cerevisiae strain pdr5p+, 5 μl samples of fivefold serially diluted yeast cultures were spotted on ypd agar in well sterile polystyrene plates. they were incubated in the presence of synthetic compounds only or associated with fluconazole at 120 μg/ml. controls were performed using ypd alone and ypd supplemented with: 120 μg/ml fluconazole, 120 μg/ml fluconazole + % dmso, 120 μg/ml fluconazole + 10 μm fk plates were incubated at 30°c for 48 h. in the case of c. albicans, the same methodology was used, but with some adaptations: 5 μl of a five-fold serial dilution from a yeast suspension containing × cells/ml was spotted on sabouraud agar supplemented with the compounds at 100 μm alone or combined with fluconazole at 64 μg/ml. the incubation of the six well plates was carried at 37°c for 48 h. checkerboard assay with compounds and fluconazole using candida strain from clinical isolate candida albicans cells, in exponential growth phase were incubated in presence of different combinations of fluconazole and compound at 37°c for 48 hours in rpmi using 96-well plates under stirring. cell growth was determined using a plate reader at a wavelength of 600 nm. the mic value was referred to concentration capable of causing 80% growth inhibition. possible synergism between fluconazole and tested compounds was determined based on the fractional inhibition concentration index. synergic, indifferent and antagonistic interactions were defined by a fici of <, - or respectively. statistical analysis all experiments were performed in triplicate. data were presented as mean ± standard error. a probability level of 5% in student’s t -test was considered significant.  atpase activity pdr5p is an abc transporter and as such the inhibition of its atpase activity could significantly affect the efflux of fluconazole and contribute to the reversal of resistance against this antifungal. thus, a screening assay was performed to identify synthetic compounds that could promote inhibition of atp hydrolysis catalyzed by pdr5p. of the compounds tested only four were capable of inhibiting pdr5p atpase activity by more than 90%. all four compounds contained a butyl-tellurium residue, a lateral hydrocarbon chain and an amide group, that were absent in the other tested compounds. this suggests that these chemical structure could have an important role in the inhibitory process. the four active compounds were selected for further investigation. dose–response curves and a double reciprocal plot were performed. the results confirm that these compounds are strong inhibitors of pdr5p atpase activity and exhibit ic values lower than 2 μm. furthermore, the double reciprocal plot for compound demonstrated an uncompetitive pattern of inhibition. compounds and demonstrated the same mechanism of inhibition. 50 values of the compounds against the atpase activity of pdr5p 50 the data represent the means ± standard error of three independent experiments. until now, there have been no reports in the literature of organic synthetic compounds containing tellurium that inhibit pdr5p atpase activity. however, many other molecules, of synthetic or natural origin, also exhibit this ability. silva et al. demonstrated that oroidin, a derivative of a compound from a sponge, is able to inhibit the catalytic activity of this multidrug transporter with an ic of 20 μm. rangel et al., while studying gallic acid derivatives, observed that decyl gallate has an ic value of  μm. both compounds competitively inhibit the enzyme activity of pdr5p. competitive inhibition is a more common characteristic than the uncompetitive inhibition shown by the four organotellurides. as mentioned by cannon et al., inhibition of plasma membrane h+-atpase activity could contribute to the reversal of abc transporter-mediated azole resistance, by depleting the intracellular atp concentration. to investigate this, the effects of the four organotellurides on the plasma membrane h+-atpase of s. cerevisiae were evaluated. the organotellurides leaded a powerful inhibition of the h+-atpase activity and exhibited ic values of approximately  μm. chan and colleagues previously demonstrated that ebselen, a well-known organoselenium compound, was also able to inhibit the activity of s. cerevisiae plasma membrane h+-atpase in a dose dependent manner. ebselen was also shown to be toxic for s. cerevisiae at a concentration of 10 μm, unlike the organotellurides investigated in this study. effect of the compounds on the growth of pdr5p+ and pdr5p- mutant s. cerevisiae strains the organotellurides and that inhibited pdr5p activity did not affect the growth of the pdr5p+ strain at concentrations up to 200 μm. however, the growth of the pdr5p- null mutant was significantly impaired after incubation with 200 μm of compound. this indicates that this compound could work as substrate and atpase activity inhibitor of pdr5p such as fk a classical and potent pdr5p inhibitor. evaluation of cytotoxicity against human erythrocytes the active compounds were tested for their hemolytic activity on human erythrocytes. as shown in figure  even at the highest concentration used in this assay the four compounds promoted the release of around 4% of erythrocyte hemoglobin. there was no significant difference between the hemolysis caused by the compounds and that observed in pbs and dmso controls. therefore, all four active compounds showed another a desirable feature of a compound to reverse fluconazole resistance that is a low toxicity for a mammal cell line. fluconazole resistance reversion by the synthetic organotellurides the spot assay shown in figure 6a demonstrates that pdr5p+ strain, which is resistant to fluconazole, was able to grow on a medium containing fluconazole at 120 μg/ml as well as in presence of compounds at 100 μm. however, an evident reduction in growth was observed when this strain was incubated in presence of fluconazole associated with any of the four organotellurides. thus, it was possible to demonstrate that these synthetic compounds were able to reverse the fluconazole resistance mediated by pdr5p in a manner similar to the reversion promoted by fk a control using the pdr5p- null mutant ) was performed to confirm that the presence of pdr5p is responsible for the fluconazole resistance of the ad strain. the same experiment was performed using a fluconazole resistant candida albicans clinical isolate because overexpression of efflux pumps is a possible mechanism of resistance to azoles in this yeast also. however, the level of expression of the c. albicans abc transporter is lower in comparison to the s. cerevisiae strains used in the present work that were genetically modified to overexpress the efflux pump. thus, the hypothesis is that would be possible to reverse the resistance in pathogenic yeast, as resistant c. albicans from a clinical isolate, with lower concentration of azole in comparison with ad strain. the c. albicans clinical isolate was able to grow in presence of fluconazole at 64 μg/ml that is considered as resistant strain. the active compounds alone did not affect growth of c. albicans, but when associated with fluconazole were able to promote a complete growth inhibition in comparison with inhibition obtained in the presence of fk. this data reinforces the results obtained with s. cerevisiae and provides further evidence that blocking efflux pumps represents a valid therapy measure for treatment of resistant fungal strains. this strategy becomes more evident using the checkerboard assay where compounds and fluconazole were tested in different concentrations. all compounds tested were able to act synergistically with fluconazole since they showed fici values lower than. this proves the efficiency of the use of those organotellurides in combination with azoles in reversion of resistance due to overexpression of efflux pumps in pathogenic fungi such as c. albicans. candida albicans strain a b *this assay was done with organotellurides and fluconazole isolated or combined. mics were determined by a microdilution technique based on 80% reduction of growth. afic = fractional inhibitory concentration; bfici = fractional inhibitory concentration index.  compounds and are synthetic compounds that have some similarities. firstly, all they contain a butyl tellurium residue, secondly, they have a lateral hydrocarbon chain and finally, they all possess an amide group. all they were able to reverse the fluconazole resistance mediated by pdr5p from s. cerevisiae. a likely mechanism for this reversal is the direct inhibition of the atpase activity of pdr5p and the indirect inhibition of the plasma membrane h+-atpase. furthermore, the compounds could also overcome fluconazole resistance expressed by a clinical isolate of candida albicans. the reversal of fluconazole resistance was obtained using 100 μm of the compounds. this concentration did not demonstrate toxicity against human erythrocytes or fungal cells. in conclusion, these compounds could be promising candidates for the reversal of resistance mediated by drug efflux pumps, act synergistically with fluconazole and could serve as prototypes for the synthesis of other molecules that could be capable of inhibiting efflux pumps with greater efficiency. availability of supporting data the data sets supporting the results of this article are included within the article. competing interests the authors declare that they have no competing interests. authors’ contributions lfrs: carried out the conception and design the experiments; the acquisition, analysis and interpretation of data. he also drafts the manuscript. ft: carried out the synthesis of the compounds used in this work and was involved in revising the manuscript critically. bas: carried out the synthesis of the compounds used in this work, and was involved in revising the manuscript critically. acg: carried out the synthesis of the compounds used in this work, and was involved in revising the manuscript critically. act: had made contributions to analysis and interpretation of data and was involved in revising the manuscript critically. epw: carried out the synthesis of the compounds used in this work, and was involved in revising the manuscript critically. jvc: carried out the supervision of the students involved in the synthesis of the compounds used in this work, and was involved in revising the manuscript critically. aas: designed the synthesized compounds and carried out the supervision of the students involved in the synthesis of the compounds used in this work, and was involved in revising the manuscript critically. he was involved in revising the manuscript critically and gave final approval of the final version. afp: helped with the conception and design the experiments; with analysis and interpretation of data and draft the manuscript. he was involved in revising the manuscript critically and gave final approval of the version to be published. all authors read and approved the final manuscript. acknowledgments the authors thank faperj, fapesp, cnpq and capes for financial support and scholarships. the authors are grateful for the financial and structural support offered by the university of são paulo through the nap-catsinq. the authors thank also to our lab assistant, mrs. geralda rodrigues almeida for her great support and dr. louise kemp for your critical reading of this manuscript.
these four compounds were capable of inhibiting pdr5p atpase activity by more than 90%, they demonstrated ic values less than 2 μm and had an uncompetitive pattern of pdr5p atpase activity inhibition. these organotellurides did not demonstrate cytotoxicity against human erythrocytes or s. cerevisiae mutant strains even in concentrations above 100 μm. resistance to fluconazole, a commonly used azole antifungal, is a challenge for the treatment of fungal infections. when tested at 100 μm, they could reverse the fluconazole resistance expressed by both the s. cerevisiae mutant strain that overexpress pdr5p and a clinical isolate of candida albicans. resistance can be mediated by overexpression of abc transporters, which promote drug efflux that requires atp hydrolysis. the pdr5p abc transporter of saccharomyces cerevisiae is a well-known model used to study this mechanism of antifungal resistance. we have identified four organotellurides that are promising candidates for the reversal of drug resistance mediated by drug efflux pumps. among the tested compounds, four contained a tellurium-butane group and shared structural similarities that were absent in the other tested compounds: a lateral hydrocarbon chain and an amide group.
 resistance to fluconazole, a commonly used azole antifungal, is a challenge for the treatment of fungal infections. resistance can be mediated by overexpression of abc transporters, which promote drug efflux that requires atp hydrolysis. the pdr5p abc transporter of saccharomyces cerevisiae is a well-known model used to study this mechanism of antifungal resistance. the present study investigated the effects of synthetic compounds on pdr5p.  among the tested compounds, four contained a tellurium-butane group and shared structural similarities that were absent in the other tested compounds: a lateral hydrocarbon chain and an amide group. these four compounds were capable of inhibiting pdr5p atpase activity by more than 90%, they demonstrated ic values less than 2 μm and had an uncompetitive pattern of pdr5p atpase activity inhibition. these organotellurides did not demonstrate cytotoxicity against human erythrocytes or s. cerevisiae mutant strains even in concentrations above 100 μm. when tested at 100 μm, they could reverse the fluconazole resistance expressed by both the s. cerevisiae mutant strain that overexpress pdr5p and a clinical isolate of candida albicans.  we have identified four organotellurides that are promising candidates for the reversal of drug resistance mediated by drug efflux pumps. these molecules will act as scaffolds for the development of more efficient and effective efflux pump inhibitors that can be used in combination therapy with available antifungals. organotelluridespdr5pfluconazole resistancesaccharomyces cerevisiae 