Antifungal Activity of Amiodarone Is Mediated by Disruption of Calcium Homeostasis

Abstract

The antiarrhythmic drug amiodarone was recently demonstrated to have novel broad range fungicidal activity. We provide evidence that amiodarone toxicity is mediated by disruption of Ca2+ homeostasis in Saccharomyces cerevisiae. In mutants lacking calcineurin and various Ca2+ transporters, including pumps (Pmr1 and Pmc1), channels (Cch1/Mid1 and Yvc1), and exchangers (Vcx1), amiodarone sensitivity correlates with cytoplasmic calcium overload. Measurements of cytosolic Ca2+ by aequorin luminescence demonstrate a biphasic response to amiodarone. An immediate and extensive calcium influx was observed that was dose-dependent and correlated with drug sensitivity. The second phase consisted of a sustained release of calcium from the vacuole via the calcium channel Yvc1 and was independent of extracellular Ca2+ entry. To uncover additional cellular pathways involved in amiodarone sensitivity, we conducted a genome-wide screen of nearly 5000 single-gene yeast deletion mutants. 36 yeast strains with amiodarone hypersensitivity were identified, including mutants in transporters (pmr1, pdr5, and vacuolar H+-ATPase), ergosterol biosynthesis (erg3, erg6, and erg24), intracellular trafficking (vps45 and rcy1), and signaling (ypk1 and ptc1). Of three mutants examined (vps45, vma3, and rcy1), all were found to have defective calcium homeostasis, supporting a correlation with amiodarone hypersensitivity. We show that low doses of amiodarone and an azole (miconazole, fluconazole) are strongly synergistic and exhibit potent fungicidal effects in combination. Our findings point to the potentially effective application of amiodarone as a novel antimycotic, particularly in combination with conventional antifungals. The antiarrhythmic drug amiodarone was recently demonstrated to have novel broad range fungicidal activity. We provide evidence that amiodarone toxicity is mediated by disruption of Ca2+ homeostasis in Saccharomyces cerevisiae. In mutants lacking calcineurin and various Ca2+ transporters, including pumps (Pmr1 and Pmc1), channels (Cch1/Mid1 and Yvc1), and exchangers (Vcx1), amiodarone sensitivity correlates with cytoplasmic calcium overload. Measurements of cytosolic Ca2+ by aequorin luminescence demonstrate a biphasic response to amiodarone. An immediate and extensive calcium influx was observed that was dose-dependent and correlated with drug sensitivity. The second phase consisted of a sustained release of calcium from the vacuole via the calcium channel Yvc1 and was independent of extracellular Ca2+ entry. To uncover additional cellular pathways involved in amiodarone sensitivity, we conducted a genome-wide screen of nearly 5000 single-gene yeast deletion mutants. 36 yeast strains with amiodarone hypersensitivity were identified, including mutants in transporters (pmr1, pdr5, and vacuolar H+-ATPase), ergosterol biosynthesis (erg3, erg6, and erg24), intracellular trafficking (vps45 and rcy1), and signaling (ypk1 and ptc1). Of three mutants examined (vps45, vma3, and rcy1), all were found to have defective calcium homeostasis, supporting a correlation with amiodarone hypersensitivity. We show that low doses of amiodarone and an azole (miconazole, fluconazole) are strongly synergistic and exhibit potent fungicidal effects in combination. Our findings point to the potentially effective application of amiodarone as a novel antimycotic, particularly in combination with conventional antifungals. The emergence of drug-resistant fungi poses an increasing threat to the treatment of opportunistic fungal infections in patients with compromised immune systems, as occurs commonly in cancer and AIDS. This can only be countered by the discovery of new antifungal agents, particularly those that target different molecular pathways, and a better understanding of their mode of action. Recently, the antiarrhythmic drug amiodarone (AMD) 1The abbreviations used are: AMD, amiodarone; BAPTA, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetracetic acid; FLUC, fluconazole; MIC, miconazole; RLU, relative luminescence unit(s); WT, wild type; SC, synthetic complete.1The abbreviations used are: AMD, amiodarone; BAPTA, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetracetic acid; FLUC, fluconazole; MIC, miconazole; RLU, relative luminescence unit(s); WT, wild type; SC, synthetic complete. was shown to have potent fungicidal activity against not only Saccharomyces but also pathogenic yeasts such as Candida, Cryptococcus, Fusarium, and Aspergillus (1Courchesne W.E. J. Pharmacol. Exp. Ther. 2002; 300: 195-199Crossref PubMed Scopus (81) Google Scholar). Viability of Cryptococcus neoformans after exposure to 10 μm AMD fell dramatically, with a 10-fold loss of viable cells after1hof treatment and only 0.001% viable cells remaining after 1 day. The cytotoxic effect of AMD appeared to be mediated by calcium, as evidenced by the ability of high (millimolar) concentrations of extracellular Ca2+ to ameliorate drug toxicity (1Courchesne W.E. J. Pharmacol. Exp. Ther. 2002; 300: 195-199Crossref PubMed Scopus (81) Google Scholar) and by an increase in cytosolic Ca2+ upon drug application (2Courchesne W.E. Ozturk S. Mol. Microbiol. 2003; 47: 223-234Crossref PubMed Scopus (75) Google Scholar). There is emerging evidence that cytosolic Ca2+ entry in yeast is critical for survival under a variety of cell stresses, including hyper- and hypo-osmotic shock, protein unfolding agents, and antifungal drugs (3Cruz M.C. Goldstein A.L. Blankenship J.R. Del Poeta M. Davis D. Cardenas M.E. Perfect J.R. McCusker J.H. Heitman J. EMBO J. 2002; 21: 546-559Crossref PubMed Scopus (272) Google Scholar, 4Matsumoto T.K. Ellsmore A.J. Cessna S.G. Low P.S. Pardo J.M. Bressan R.A. Hasegawa P.M. J. Biol. Chem. 2002; 277: 33075-33080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 5Bonilla M Nastase K.K. Cunningham K.W. EMBO J. 2002; 21: 2343-2353Crossref PubMed Scopus (207) Google Scholar, 6Edlind T. Smith L. Henry K. Katiyar S. Nickels J. Mol. Microbiol. 2002; 46: 257-268Crossref PubMed Scopus (88) Google Scholar). The release of calcium from intracellular stores must be compensated by stimulation of extracellular calcium influx, a phenomenon commonly known as capacitative calcium entry (7Locke E.G. Bonilla M. Liang L. Takita Y. Cunningham K.W. Mol. Cell. Biol. 2000; 20: 6686-6694Crossref PubMed Scopus (178) Google Scholar). Thus, one possibility is that the cytosolic Ca2+ increase in response to AMD promotes cell survival and is due to capacitative calcium entry, as has been suggested by Courchesne and Ozturk (2Courchesne W.E. Ozturk S. Mol. Microbiol. 2003; 47: 223-234Crossref PubMed Scopus (75) Google Scholar). Paradoxically, excessive or unregulated levels of calcium in the cytoplasm also lead to cell death and are implicated in the cytotoxicity of several drugs (8Andjelic S. Khanna A. Suthanthiran M. Nikolic-Zugic J. J. Immunol. 1997; 158: 2527-2534PubMed Google Scholar) as well as fungal toxins (9Kurzweilova H. Sigler K. Folia Microbiol. 1993; 38: 524-526Crossref PubMed Scopus (8) Google Scholar, 10Lecourieux D. Mazars C. Pauly N. Ranjeva R. Pugin A. Plant Cell. 2002; 14: 2627-2641Crossref PubMed Scopus (305) Google Scholar). To better understand the role of calcium in AMD toxicity, it is important to distinguish between a capacitative calcium entry mechanism triggered by store depletion or a loss in calcium homeostasis, possibly due to an effect of the drug channels and The in and to the cellular of AMD We a of AMD Ca2+ homeostasis in Saccharomyces cerevisiae. The of different Ca2+ transporters, including pumps (Pmr1 and Pmc1), channels and Yvc1), and exchangers (Vcx1), were in cells lacking and to levels of is an that Ca2+ and the and cellular homeostasis under A. R. J. Biol. Cell. 1997; Google Scholar, Y. R. PubMed Scopus Google Scholar). of are in including and and the of D. C. R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). is the yeast Ca2+ to that is under calcium K.W. Mol. Cell. Biol. PubMed Scopus Google Scholar) or in the of Y. R. PubMed Scopus Google Scholar) and to Ca2+ by the calcium is also by the K.W. J. Biol. PubMed Scopus Google Scholar, Mol. Cell. Biol. PubMed Scopus Google Scholar, A. R. L. PubMed Scopus Google the by the vacuolar release from the vacuole is mediated by a Ca2+ channel J. C. Y. S. A. PubMed Scopus Google Scholar). is the yeast of calcium channels with the Ca2+ channel a Ca2+ entry channel the M. S. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). There is evidence for one calcium influx channel of molecular M Nastase K.K. Cunningham K.W. EMBO J. 2002; 21: 2343-2353Crossref PubMed Scopus (207) Google Scholar, E.G. Cunningham K.W. PubMed Google Scholar). cellular calcium levels and are critical for of the calcium signaling of and the of the and protein are to lead to a of and that a variety of different cellular to extracellular including cell and the protein cell in PubMed Scopus Google and Heitman J. 2002; PubMed Scopus Google Scholar). The of several of the calcium with yeast an to the role of calcium signaling in AMD The of of deletion mutants point to a for Ca2+ homeostasis in the response to to drug (3Cruz M.C. Goldstein A.L. Blankenship J.R. Del Poeta M. Davis D. Cardenas M.E. Perfect J.R. McCusker J.H. Heitman J. EMBO J. 2002; 21: 546-559Crossref PubMed Scopus (272) Google Scholar, 6Edlind T. Smith L. Henry K. Katiyar S. Nickels J. Mol. Microbiol. 2002; 46: 257-268Crossref PubMed Scopus (88) Google we also demonstrate a critical role for calcineurin in AMD of cytoplasmic Ca2+ levels by aequorin luminescence a correlation between cytosolic Ca2+ increase and AMD to Ca2+ influx the cell and release from including the To additional that drug sensitivity, we nearly 5000 single-gene deletion mutants for hypersensitivity to We 36 implicated in known and novel pathways that be important for drug and The of that AMD yeast by a mechanism different from that used by conventional such as the and the we show that low concentrations of AMD and an azole and fluconazole) are synergistic in that of conventional antifungal treatment with AMD be a of the effect of the effective In findings provide important the cellular effects of AMD and in the treatment of fungal and of yeast were all from and have been K.W. J. Biol. PubMed Scopus Google Scholar). The S. deletion was from The and of have been D. T. R. J. Biol. Cell. 2000; Scholar, Y. J. A. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). 1993; PubMed Google Scholar) was from the of The of and C. neoformans M.C. Goldstein A.L. Blankenship J. Poeta Perfect J.R. McCusker J.M. Cardenas M.E. Heitman J. PubMed Scopus Google Scholar) was a from the of Heitman yeast strains were in synthetic or yeast or as in the to and between amiodarone and in S. cerevisiae. wild S. was in in the or of AMD or or in combination. the cells were with and as under Viability was as the of cells by the of cells as a The of three is and wild cells were in and with drugs and with μm for 1 concentrations of AMD and were μm and 1 was and was and between amiodarone and in pathogenic C. was in 1 of with μm and μm AMD in the or of of cells were of and for was as a of are of with C. neoformans was in of for The μm AMD with or of cells to AMD or of cells to drugs were 10 of and for was as and as a of are of with to or for was from in as a and 10 of were in of different concentrations of AMD were in a for and was by To the effect of AMD sensitivity was in the of or 10 the 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetracetic was to concentrations of and To the role of calcineurin was from a of to a of 1 and to cell in and were from a of or In the screen with C. the was by and to 1 of different drug concentrations in a a of cells were of drugs and was by C. of was to of with various drug were for of cells to AMD or of cells to drugs were in 10 and to for was by AMD of with μm AMD was to a and with of phase yeast from single-gene deletion strains from the deletion an of The were for a of were with a and was by the in a The relative of was as a of of the with sensitivity to AMD were of or relative to strains were for sensitivity in and μm strains were with T.K. Ellsmore A.J. Cessna S.G. Low P.S. Pardo J.M. Bressan R.A. Hasegawa P.M. J. Biol. Chem. 2002; 277: 33075-33080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) the aequorin and to phase in lacking were with in the for and in and for to AMD was to of cells in a an and luminescence was second for 10 in a The luminescence was as M.C. Goldstein A.L. Blankenship J. Poeta Perfect J.R. McCusker J.M. Cardenas M.E. Heitman J. PubMed Scopus Google Scholar). cells were with in the of 1 and a of was was with the a The calcium of in and was and that of was Viability cells in with and the concentrations of AMD and were with from a of cells were under a cells yeast was from of cells in the or of drugs in were in for 1 with μm was in from a of μm in after the cells were examined under a with an The was by the of was by of and or cells was with of the to the role of various calcium pathways in drug sensitivity, we the of deletion mutants in with concentrations of shown in the was to in the a but to the relative to wild of deletion mutants of the vacuolar and all known Ca2+ channels in yeast and a of AMD sensitivity in and mutants but not in the that of Ca2+ in by the calcium is important in the toxicity of calcium has been to a dose-dependent of to fungal in the yeast J. M. D. Smith Mol. Microbiol. PubMed Scopus Google Scholar). 10 or appeared to cell against AMD toxicity, BAPTA, a AMD toxicity, with the of Ca2+ the effect of increasing Ca2+ concentrations AMD sensitivity was in strains lacking in the known of extracellular Ca2+ for J. R. S. Mol. Biol. Cell. PubMed Scopus Google Scholar). can be a for Ca2+ in a of but was in not and were to have or effect AMD toxicity to yeast (1Courchesne W.E. J. Pharmacol. Exp. Ther. 2002; 300: 195-199Crossref PubMed Scopus (81) Google Scholar). of the of in the high of Ca2+ and the A. R. J. Biol. Cell. 1997; Google Scholar, Y. J. A. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google it was of to the sensitivity of to AMD was a of a loss of Ca2+ or We have mutants of and show nearly of Ca2+ or D. T. R. J. Biol. Cell. 2000; Scholar, Y. J. A. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). mutants were in a yeast of Ca2+ with the additional deletion of calcineurin to A. R. J. Biol. Cell. 1997; Google Scholar, K.W. J. Biol. PubMed Scopus Google Scholar). loss of Ca2+ in in loss of to AMD, to the lacking and In loss of of not AMD sensitivity. are with the of extracellular to against AMD toxicity, and the role of Ca2+ in the cellular effects of the of of has been shown to be for but critical for survival in C. The AMD sensitivity of the relative to the suggested that loss of calcineurin be synergistic with loss of the Ca2+ To we the effect of a potent of the AMD sensitivity of wild and strains the of the AMD sensitivity of the the lacking sensitivity to AMD concentrations of the drug the of calcineurin in the Ca2+ signaling involved in drug sensitivity. in we AMD calcium entry yeast and calcium entry correlated with drug sensitivity. luminescence of the was in the drug In all strains application of AMD in a biphasic of cytoplasmic calcium that was AMD The of calcium and was by a sustained that for the of the The of as well as the of the was AMD with increase observed between 10 and μm of cytosolic calcium was found to with AMD the of 10 μm AMD the luminescence in the the relative to wild cytosolic calcium concentrations of the the under μm in wild and and to 1 μm in the second sustained μm for and but μm for in to AMD from and was of to the relative of extracellular and intracellular Ca2+ stores to the biphasic of cytosolic Ca2+ observed in we used the to extracellular the of aequorin luminescence in wild cells was that calcium to the in cytoplasmic calcium a a sustained was observed in cells that to the second phase of luminescence in the of that Ca2+ release from intracellular stores must to cytosolic of Ca2+ that Ca2+ entry from extracellular is not to release from The channel Yvc1 has been shown to Ca2+ release from the vacuole in response to J. Biol. 2002; PubMed Scopus Google Scholar). We the of vacuolar Ca2+ release by aequorin luminescence in the The in intracellular Ca2+ in the were to wild in the of The of wild cells and that of was The second sustained in strains from to μm of treatment Ca2+ in the that the of cytosolic Ca2+ from the from extracellular and vacuolar Courchesne and Ozturk that be the channel calcium entry that the Ca2+ in was the wild not (2Courchesne W.E. Ozturk S. Mol. Microbiol. 2003; 47: 223-234Crossref PubMed Scopus (75) Google Scholar). In we show that the of the calcium channel or the of Ca2+ but not the the from that the in luminescence from extracellular the increase is from extracellular and intracellular concentrations of extracellular Ca2+ or also the possibly due to of the channel and the of cytosolic Ca2+ the after AMD In the of calcium is and the is that Ca2+ the by the in to the of aequorin the vacuole is known to be the store for cellular calcium, we a of vacuolar Ca2+ in response to calcium we that in the of known vacuolar Ca2+ transporters, and the to extracellular Ca2+ was with an of μm under This is with a role for vacuolar in the Ca2+ The disruption of all three known Ca2+ pathways, and is known to be K.W. Mol. Cell. Biol. PubMed Scopus Google due to a loss of cytosolic Ca2+ of a for S. deletion a for the of new to AMD is a novel with in it was of to additional to AMD sensitivity that lead to the cellular mechanism of We a of nearly 5000 for hypersensitivity to AMD mutants were in the and were found in the second were all several to their The of from genome-wide screen independent of the shown in We also found pdr5, a lacking the drug and known to be to a variety of cytotoxic H. K. PubMed Scopus Google Scholar). is to be involved in of AMD by drug from the of of the vacuolar vma3, and the vacuolar in is to vacuolar calcium The fell that mutants in of the ergosterol biosynthesis intracellular trafficking pathways, signaling and and with of strains have been shown to have sensitivity to drugs and be involved with AMD toxicity in a and mutants have in of and are known to be to R. R. A. 2000; Google Scholar) and and D. D. 2000; Google The ergosterol biosynthesis is the target of S. Microbiol. PubMed Scopus Google and disruption of the of the cell was not to mutants (erg3, erg6, and with sensitivity to the discovery of mutants defective in various of intracellular trafficking pathways, such as and was We that in trafficking and signaling pathways Ca2+ homeostasis, in hypersensitivity to screen of for pdr5, erg6, in a new we have suggested a correlation between AMD sensitivity and Ca2+ levels in the We to by luminescence of aequorin in three mutants from the genome-wide screen for AMD hypersensitivity. shown in all three mutants and of cytosolic Ca2+ upon the of AMD the in their cellular the of the vacuolar J.R. Mol. 1993; PubMed Scopus (81) Google is a vacuolar protein defective in or H. T. EMBO J. PubMed Scopus Google and is important for the A. S. C. R. H. J. Biol. 2000; PubMed Scopus Google Scholar). In the and upon the of AMD the of aequorin luminescence is to μm and that in the and of the vacuole ability to Ca2+ The a response to AMD, in luminescence such that the an Ca2+ of was not the second phase To the second of calcium in from we examined aequorin luminescence in the of BAPTA, to extracellular in strains we that the of cytosolic Ca2+ was upon but the sustained of Ca2+ Our release of Ca2+ from possibly the be from the cytoplasm in the from the the of the protein for between AMD and azole such as and cell and are in the treatment with is the of AMD has been to have broad range fungicidal particularly against Cryptococcus (1Courchesne W.E. J. Pharmacol. Exp. Ther. 2002; 300: 195-199Crossref PubMed Scopus (81) Google the concentrations be high to be for treatment We the effect of low concentrations of AMD cell of Saccharomyces in the of doses of of drug effect cell as by The synergistic effect of the drugs was all concentrations and is shown μm cell survival fell from to in AMD or to in the of To the of cells were with the the cells were and to the in the of AMD and for nearly all cells and a in the cytoplasm findings were observed the pathogenic yeasts C. and C. neoformans (1Courchesne W.E. J. Pharmacol. Exp. Ther. 2002; 300: 195-199Crossref PubMed Scopus (81) Google AMD of an effective of or cell a exposure to cells to low levels of AMD or or to or in to levels In the combination of drugs of C. and C. neoformans by of an effective loss of be that levels of μm have been for AMD and (1Courchesne W.E. J. Pharmacol. Exp. Ther. 2002; 300: 195-199Crossref PubMed Scopus (81) Google that doses be effective in antifungal AMD is an important used in the treatment of and 2002; PubMed Scopus Google Scholar). AMD and are in their and their broad range of In AMD channels such as Ca2+ channels and and the of K. J. J. Full Text Full Text PDF PubMed Scopus Google Scholar). the of AMD are well effects in the and the There is evidence that AMD in T. M. J. 2000; PubMed Scopus Google Scholar) and R. A. R. T. J. 2000; Google Scholar). with AMD show in the after the and to the A. S. M. A. C. D. D. A. J. Google Scholar). Of to are the of AMD exposure to a sustained cytoplasmic Ca2+ in D. Pharmacol. PubMed Scopus Google Scholar) and in or cells D. M. Pharmacol. 2000; PubMed Scopus Google Scholar, R. D. Pharmacol. PubMed Scopus Google Scholar). Our a correlation between AMD toxicity and cytoplasmic Ca2+ levels in S. the of Ca2+ and signaling pathways between yeast and a better understanding of the cellular of AMD toxicity in is to understanding and the effects of the drug in We show that doses of AMD, in the range is Ca2+ a channel that is by Ca2+ channel such as and and by with Courchesne and Ozturk (2Courchesne W.E. Ozturk S. Mol. Microbiol. 2003; 47: 223-234Crossref PubMed Scopus (75) Google Scholar) have suggested that one for Ca2+ entry is the channel found that the was to AMD that loss of to we that the sensitivity to AMD to that of the The for is not and We provide new evidence for Ca2+ release from the vacuole via the with a of the role of channel in the response to J. Biol. 2002; PubMed Scopus Google it that Yvc1 is the release mechanism for intracellular stores of Ca2+ in depletion of extracellular Ca2+ or the of Ca2+ channel the immediate of cytosolic not the release from vacuolar to increase the of the of calcium the in cells were to to and μm in response to 10 μm In strains to AMD, such as to μm and We that it is the second sustained of cytoplasmic Ca2+ that to that the of calcium entry from extracellular that from intracellular stores in we can a role for capacitative calcium entry in the immediate response to AMD, such are to to the sustained phase of calcium is well known that excessive cytosolic Ca2+ is S. 1997; PubMed Scopus Google and we show a correlation between hypersensitivity to AMD and excessive of cytosolic in the of the Ca2+ Ca2+ levels in response to AMD in a with the important role of in the of cellular Ca2+ the deletion strains that we only the was to AMD and was to have of cytosolic relative to wild Paradoxically, high levels of extracellular Ca2+ or the effects of AMD (1Courchesne W.E. J. Pharmacol. Exp. Ther. 2002; 300: 195-199Crossref PubMed Scopus (81) Google Scholar) in to is that the Ca2+ influx a Ca2+ is in the of of extracellular response has been observed in cells to toxins D. Mazars C. Pauly N. Ranjeva R. Pugin A. Plant Cell. 2002; 14: 2627-2641Crossref PubMed Scopus (305) Google in calcium influx and with increasing extracellular Ca2+ In to the molecular of AMD in we have cellular pathways and that drug sensitivity upon disruption The genome-wide were not and it is that mutants were are we have several mutants with the molecular of AMD toxicity as well as calcium homeostasis in The sensitivity of trafficking mutants be due to in drug Ca2+ via the or vacuolar the is defective in from an has been to from the and pathways A. S. C. R. H. J. Biol. 2000; PubMed Scopus Google Scholar). AMD toxicity in the be due to defective of or with Ca2+ or in the of AMD to the the a or H. T. EMBO J. PubMed Scopus Google and vma3, a lacking the of the vacuolar J.R. Mol. 1993; PubMed Scopus (81) Google are defective in vacuolar and that Ca2+ be The drug sensitivity observed in signaling mutants and is also and a in the of the cell an vacuole by M. A. Mol. Cell. Biol. Scopus Google that vacuolar is compromised in is a of the high and has been shown to the activity of J. J. J. D. Mol. Cell. Biol. 21: PubMed Scopus Google Scholar). findings to the between AMD toxicity and as well as in the to be AMD and a Ca2+ channel or an intracellular signaling that to cytosolic Ca2+ entry. We that in to cellular of AMD and and of A. R. L. R. Mol. Pharmacol. 47: Google of and H. Pharmacol. 1993; PubMed Scopus Google and and of Davis Davis J. PubMed Scopus Google Scholar, D. Pharmacol. PubMed Scopus Google Scholar) as well as protein D. Google Scholar). In the possibility of AMD as a broad range that is to be effective against drug-resistant pathogenic not of the cellular pathways and in screen for mutants to drug are to be in the pathogenic of and fungal infections are the in and fungi have drugs such as the are only that are such as the have effects that treatment M.E. A. Microbiol. 2002; PubMed Scopus Google Scholar). The target of and is ergosterol and biosynthesis mechanism of drug is the of a in the of a A. R. M. Del M. S. Mol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of the is to and the was in screen for AMD sensitivity that cytotoxicity of AMD occurs by a mechanism from that of the conventional antifungals. to an antifungal AMD potentially the by cells is only by the In against pathogenic fungi be by the and toxicity be by To be the low concentrations of AMD that are synergistic with have toxicity to the of different of is used or and be A. with doses of to N. R.A. 2000; PubMed Scopus Google Scholar). combination of and or in of the the ergosterol biosynthesis the cell M.E. A. Microbiol. 2002; PubMed Scopus Google Scholar). the between or and AMD demonstrated in a potentially effective against and fungal S. S.G. C. M. C. and R. of for the We of for the of and C. and J. Heitman for C.