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Antimicrobial Agents and Chemotherapy, June 2005, p. 2558-2560, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2558-2560.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Utilization of Target-Specific, Hypersensitive Strains of Saccharomyces cerevisiae To Determine the Mode of Action of Antifungal Compounds

Ed T. Buurman,1* Beth Andrews,2 April E. Blodgett,1 Jini S. Chavda,2,{dagger} and Norbert F. Schnell2,{ddagger}

Departments of Microbiology,1 Molecular Sciences, AstraZeneca R&D Boston, Waltham, Massachusetts 024512

Received 14 December 2004/ Returned for modification 18 January 2005/ Accepted 31 January 2005


   ABSTRACT
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Target-specific hypersusceptible strains of Saccharomyces cerevisiae were used to screen antifungal compounds. Two novel Erg7p inhibitors were identified, providing proof of principle of the approach taken. However, observed hypersensitivities to antifungals acting via other targets imply that use of this tool to identify the mode of action requires significant deconvolution.


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Overexpression of a target gene can decrease the cell's susceptibility to inhibitors acting via that target (28, 30). Conversely, underexpression of a target can lead to an increased susceptibility to its inhibitors (2, 5). Here we describe how the well-studied GAL system of Saccharomyces cerevisiae (11) can be used for the identification of target-inhibitor pairs using target-specific, hypersusceptible strains of S. cerevisiae.

Genes encoding putative targets that have been shown to be essential for growth of S. cerevisiae were selected (18, 26). Target-specific hypersensitive, or "switch-down," strains were constructed as previously described (17) using S. cerevisiae MEY121 (MATa his3 leu2-3,112 ura3-52 trp1 rme1) (derived from JK9-3da [14]) and a HIS3-GAL1 promoter integration cassette; correct integration was verified by a number of diagnostic PCRs. As a result, when a switch-down strain was grown at 30°C in YP (1)-2% galactose medium and transferred to YP-2% glucose medium, growth continued unabated for a number of generations until the cellular pool of target protein was presumed depleted. Since this number was constant for each target, the final density of the culture was proportional to its initial density.

When wild-type strains of S. cerevisiae were incubated with growth inhibitors at sub-MICs, the growth rates of the strains were lowered but the final optical density at 600 nm was unchanged. Switch-down strains growing in the presence of glucose were expected to behave identically except when they were incubated with a compound that mediated its antifungal effect via the down-regulated target. At its 50% inhibitory concentration (IC50), a target-specific compound would decrease the cellular target activity by 50% and the number of doublings the culture could undergo before the growth arrest would be one fewer. This would result in a 50% lower final optical density at 600 nm compared to growth arrest in the absence of the compound. To test this hypothesis, eight switch-down strains (ERG9 [10], ERG11 [12], ERG1 [9], LCB1 [20], AUR1 [19], PKC1 [15], ERG7 [4], and ERG8 [29]) were grown in the presence of the following six control compounds: zaragozic acid (22), fluconazole (13), terbinafine (23, 24), myriocin (3), aureobasidin A (8), and staurosporine (31). Invariably, down-regulation of a target led to hypersusceptibility to its genuine inhibitor (Table 1), thus providing proof of principle of the approach taken. However, in some cases hypersensitivity to other compounds resulted as well (Table 1); the most striking example is the ERG1 switch-down strain that was hypersensitive to both terbinafine and fluconazole but not to any other control compound.


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  		TABLE 1. Target-specific hypersusceptible strains of S. cerevisiae show various degrees of increased susceptibility to control compounds

 
In order to assess the utility of switch-down strains in cell-based screening of antifungal compounds, a set of 34 switch-down strains was tested against a library of 2,500 antifungal compounds with IC50s below 400 µM against wild-type S. cerevisiae and Candida albicans. Dose responses of each compound against the panel of switch-down strains were determined by testing the compounds at a maximum test concentration and seven twofold serial dilutions. The maximum test concentration was set around 25 to 50% of the previously determined growth IC50 against wild-type strains. As a result, only strains hypersusceptible to the compound would be expected to show growth inhibition. Hits were defined as compounds that showed a growth IC50 against one or more switch-down strains.

As a first step in the analysis, the number of hits against each target was determined (data not shown). ERG11, ERG7, and ERG1 stood out among the 34 targets with 520, 448, and 103 hits, respectively. Many of these hits (77%, 37%, and 72%, respectively) consisted of azoles and 4-pyrrolidinopyridines that are well established Erg11p and Erg7p inhibitors, respectively (7, 13). This indicated that the screening method identified compounds with known modes of action by using the appropriate target-specific hypersusceptible strain. Two compounds that only inhibited the ERG7 switch-down strain (Fig. 1A and B) were further investigated; many other compounds inhibited multiple strains. Using NCCLS methods (21), no antifungal activity was detected against C. albicans CAF-2 (6) or S. cerevisiae (MIC > 64 µg ml–1). However, each compound showed an MIC of 16 µg ml–1 against C. albicans DSY654, a strain from which genes encoding efflux pump CDR1 and CDR2 have been deleted (27). Therefore the mode of action of both compounds was assessed by analyzing the alterations that occurred in the sterol composition of C. albicans DSY654 upon treatment with these inhibitors (1, 25). Compound A resulted in a decrease of ergosterol level and accumulation of 2,3-oxidosqualene, whereas compound B showed an accumulation of both lanosterol and 2,3-oxidosqualene (Fig. 1C and D). The compound concentrations at which these changes occur, with ergosterol IC50s of 10 to 20 µg ml–1, and their MICs are in line with the previously established quantitative relationship between antifungal activity and inhibition of sterol synthesis in C. albicans DSY654 in this assay (1). This strongly suggests that compound A mediates its antifungal activity via inhibition of Erg7p whereas compound B seems to inhibit both Erg7p and Erg11p. These compounds show that the use of target-specific hypersusceptible strains of S. cerevisiae can lead to the identification of novel antifungal compounds with a mode of action via that target.



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  		FIG. 1. (A and B) Growth inhibition caused by two identified hits, compounds A and B, respectively, that showed greater potency against the ERG7 switch-down strain of S. cerevisiae (triangles) but not against PKC1 (circles) or ERG1 (diamonds) switch-down strains. (C and D) Incorporation of 14C-labeled acetate into nonsaponifiable lipids of C. albicans DSY654 upon treatment with various concentrations of compounds A and B, respectively. Concentrations of ergosterol (diamonds), lanosterol (triangles), squalene (asterisks), 4{alpha}-methylsterols (squares), and 2,3-oxidosqualene (circles) are shown.

 
Although proof of principle was obtained and novel inhibitors of Erg7p were discovered, the results reflect the fact that most, if not all, targets are part of an intricate cellular network of biochemical and biological interactions and that care should be taken when interpreting data obtained using hypersensitive strains. Although this approach may establish the mode of action in the down-regulated strain, the mode of action in a wild-type strain with relatively high target levels may be different, as shown for the antibacterial thiopyridine FabI inhibitors (16). Furthermore, downregulation of proteins like LpxC that affect permeability of the bacterial cell envelope will lead to hypersusceptibility to a range of compounds (D. Young, N. Rafanan, P. Margolis, and J. Trias, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2024, 2000). As a rule, hypersensitivity of a strain is usually not limited to inhibitors of the target whose level has been altered but can include inhibitors of other targets, which sometimes can be anticipated. Therefore, additional mode-of-action studies are needed to confirm the results obtained with switch-down strains.


   ACKNOWLEDGMENTS
 
We thank Helen Butcher and Carolyn Britton for high-throughput screening of the switch-down strains, Linda Otterson for fungal susceptibility testing, and John Rosamond for intellectual support.


   FOOTNOTES
 
* Corresponding author. Mailing address: AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451. Phone: (781) 839-4592. Fax: (781) 839-4800. E-mail: Ed.Buurman{at}astrazeneca.com. Back

{dagger} Present address: Cancer Bioscience, AstraZeneca, Alderley Park, Macclesfield SK10 4TG, United Kingdom. Back

{ddagger} Present address: Fachhochschule Aalen, Beethovenstrasse 1, Aalen 73430, Germany. Back


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Antimicrobial Agents and Chemotherapy, June 2005, p. 2558-2560, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2558-2560.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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