Pyridines and Pyrimidines Mediating Activity against an Efflux-Negative Strain of Candida albicans through Putative Inhibition of Lanosterol Demethylase

Article abstract of DOI:10.1128/AAC.48.1.313-318.2004

The first step in ergosterol biosynthesis in Saccharomyces cerevisiae consists of the condensation of two acetyl coenzyme A (acetyl-CoA) moieties by acetoacetyl-CoA thiolase, encoded by ERG10. The inhibition of the sterol pathway results in feedback activ

Full text of DOI:10.1128/AAC.48.1.313-318.2004

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2004, p. 313–318
0066-4804/04/$08.00ϩ0 DOI: 10.1128/AAC.48.1.313–318.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 1
Pyridines and Pyrimidines Mediating Activity against an
Efflux-Negative Strain of Candida albicans through
Putative Inhibition of Lanosterol Demethylase
Ed T. Buurman,1* April E. Blodgett,1 Kenneth G. Hull,2 and Daniel Carcanague2
Department of Microbiology1 and Department of Chemistry,2 AstraZeneca R&D Boston,
Waltham, Massachusetts 02451
Received 21 July 2003/Returned for modification 2 September 2003/Accepted 8 October 2003
The first step in ergosterol biosynthesis in Saccharomyces cerevisiae consists of the condensation of two acetyl
coenzyme A (acetyl-CoA) moieties by acetoacetyl-CoA thiolase, encoded by ERG10. The inhibition of the sterol
pathway results in feedback activation of ERG10 transcription. A cell-based reporter assay, in which increased
ERG10 transcription results in elevated specific ␤-galactosidase activity, was used to find novel inhibitors of
ergosterol biosynthesis that could serve as chemical starting points for the development of novel antifungal
agents. A class of pyridines and pyrimidines identified in this way had no detectable activity against the major
fungal pathogen Candida albicans (MICs > 64 ␮g ⅐ ml؊1). However, a strain of C. albicans lacking the Cdr1p
and Cdr2p efflux pumps was sensitive to the compounds (with MICs ranging from 2 to 64 ␮g ⅐ ml؊1),
suggesting that they are efficiently removed from wild-type cells. Quantitative analysis of sterol intermediates
that accumulated during growth inhibition revealed the accumulation of lanosterol at the expense of ergosterol.
Furthermore, a clear correlation was found between the 50% inhibitory concentration at which the sterol
profile was altered and the antifungal activity, measured as the MIC. This finding strongly suggests that the
inhibition of growth was caused by a reduction in ergosterol synthesis. The compounds described here are a
novel class of antifungal pyridines and pyrimidines and the first pyri(mi)dines to be shown to putatively
mediate their antifungal activity against C. albicans via lanosterol demethylase.
The sterol biosynthesis pathway, which is taken here to in-
clude the mevalonate pathway, converts acetyl coenzyme A
(acetyl-CoA) into farnesyl-diphosphate, which subsequently
leads to the synthesis of ergosterol. This metabolic pathway has
many putative targets that vary in their degrees of genetic
conservation relative to fungal and human orthologs. Further-
more, the exploitation of many of these targets has led to
therapeutics for the treatment of human disease, and these
targets are therefore considered proper objects of drugs. The
therapeutics include drugs used for the treatment of fungal
infection (azoles, allylamines, thiocarbamates, and morpho-
lines, which all act against fungal targets that have human
homologs [21]) and also for the treatment of osteoporosis (2)
and hypercholesterolemia (e.g., reference 19).
Dimster-Denk and Rine (5) and Dixon et al. (7) developed
virtually identical gene reporter assays for Saccharomyces cer-
evisiae for the identification of fungal sterol biosynthesis inhib-
itors that could serve as chemical starting points for new drug
discovery programs. The attractiveness of this assay resides in
the fact that it can in principle identify inhibitors of any of the
essential steps in the pathway. Furthermore, since this is a
cell-based assay, all of these inhibitors are expected to have at
least some degree of antifungal activity. The use of this assay
has led to the identification of a new class of antifungal pyri-
dines and pyrimidines that is also distinct from the most closely
related class of antifungal pyrimidines, exemplified by triari-
mol. Furthermore, whereas triarimol-like pyrimidines have
been described as inhibitors of lanosterol demethylase in fun-
gal plant pathogens (20), the compounds described here are
the first examples of pyridines and pyrimidines inhibiting
lanosterol demethylase (Erg11p) of Candida albicans.
MATERIALS AND METHODS
Strains and media. Strains used in this study were S. cerevisiae FSB1 (MAT␣
leu2-3,112 ura3-52-pERG10-Escherichia coli lacZ-URA3 rme1 trp1 his4),
referred to as MEY133::pACoAT by Dixon et al. (7); C. albicans CAF 2-1
(SC5314 URA3/ura3::␭
ura3::␭imm434/ura3::␭
)
(9); and C. albicans DSY654 (SC5314
imm434
cdr1::hisG/cdr1::hisG cdr2::hisG-URA3-hisG/cdr2::
imm434
hisG) (17).
S. cerevisiae FSB1 was grown in uracil-deficient yeast minimal broth. One-liter
volumes of broth were prepared by adding yeast nitrogen base without amino
acids (6.7 g; Difco), adenine sulfate (40 mg), L-arginine-HCl (20 mg), L-methi-
onine (20 mg), L-tyrosine (30 mg), L-isoleucine (30 mg), L-lysine-HCl (30 mg),
L-phenylalanine (50 mg), L-glutamic acid (100 mg), L-aspartic acid (100 mg),
L-valine (150 mg), L-threonine (200 mg), and L-serine (400 mg). This broth was
brought to a pH value of 5.4 and a final volume of 900 ml and autoclaved. Before
the broth was used, 100 ml of filter-sterilized 20% glucose was added along with
200 ␮l of filter-sterilized stock solutions (10 g ⅐ literϪ1) of each histidine, tryp-
tophan, and leucine. YPD consisted of yeast extract (10 g ⅐ literϪ1), Bacto
Peptone (20 g ⅐ literϪ1), and glucose (20 g ⅐ literϪ1).
Susceptibility testing. The susceptibility of the isolates was determined ac-
cording to the NCCLS M-27A broth microdilution method (13).
Control inhibitors. The following control inhibitors were purchased from
commercial sources (in parentheses): alendronate (Calbiochem), amphotericin
B (Sigma), chlorhexidine (Sigma), cycloheximide (Calbiochem), fluconazole
(ICN Biomedicals), flucytosine (Aldrich), 5-fluoro-orotic acid (Acros Organics),
lovastatin (Sigma), terbinafine (TCI), and zaragozic acid (Sigma). Lovastatin was
activated by heating a 6-mg ⅐ mlϪ1 stock solution in 50% (vol/vol) ethanol–0.2 N
NaOH for 40 min at 65°C, after which 1 volume of 1 M Tris HCl (pH 8.0) was
added; this stock solution was stored at Ϫ20°C (6). A mock solution that did not
contain lovastatin but was treated identically was generated. This “lovastatin
control” did not contain antifungal activity and did not induce ␤-galactosidase
activity.
* Corresponding author. Mailing address: AstraZeneca R&D Bos-
ton, 35 Gatehouse Dr., Waltham, MA 02451. Phone: (781) 839-4592.
Fax: (781) 839-4800. E-mail: ed.buurman@astrazeneca.com.
313

314
BUURMAN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
Reporter assay. S. cerevisiae FSB1 was grown overnight in 25 ml of yeast
minimal broth in 125-ml flasks at 30°C at 120 rpm in a shaking incubator
(ISF-4-W; Ku¨hner, Birsfelden, Switzerland). The culture, typically with an opti-
cal density at 600 nm (OD600) of 1 to 3, was diluted to a final OD600 of 0.03 in
50-␮l volumes of yeast minimal broth containing a final concentration of 2%
(vol/vol) dimethyl sulfoxide (DMSO) and 10 subsequent twofold dilutions of the
compound of interest, starting at a maximum concentration of 64 ␮g ⅐ mlϪ1
(except for control compounds amphotericin B and 5-fluoroorotic acid, for which
the starting concentrations were 18 and 512 ␮g ⅐ mlϪ1, respectively). Samples
were prepared in quadruplicate and were distributed into 384-well plates
(Costar) and incubated for 24 h at 30°C without shaking (model 5025 incubator;
VWR Scientific). Subsequently, well contents were mixed and cell densities were
determined with a plate reader (Spectra Fluor Plus; Tecan). To this end, the
densities of one set of duplicate wells were measured without dilution; for the
other duplicate set, 25 ␮l of each well was diluted twofold into medium contain-
ing the compound, if any, at an identical concentration. This process resulted in
the establishment of a reference OD600 for the undiluted wells; wells with
OD600s higher than 1.5 or lower than 0.1 were rejected because the cultures in
these wells were too dense or too dilute, respectively, for reliable quantitative
measurement. To each well, 50 ␮l of reaction buffer (0.4 M Na2HPO4, 0.2 M
NaH2PO4, 0.05 M KCl, 0.01 M MgSO4, 0.135% [vol/vol] ␤-mercaptoethanol,
0.1% sodium dodecyl sulfate, 2.5 mM chlorophenol red-␤-D-galactopyranoside
[pH 7.0]) was added as described by Dixon et al. (7), and plates were incubated
at 30°C (model 5025 incubator; VWR Scientific). Color development was mon-
itored hourly by mixing well contents, followed by measurement of the A570. The
highest level of specific activity was typically found at the highest compound
concentration that allowed fungal growth.
Acetate incorporation and analysis of sterol intermediates. The method for
acetate incorporation and the analysis of sterol intermediates was based on that
of Ryder et al. (16). Strains of C. albicans were grown overnight in 25 ml of YPD
in 125-ml flasks at 30°C with shaking at 130 rpm (ISF-4-W; Ku¨hner), which
typically resulted in an OD600 of 5 to 10. Cultures were centrifuged at 4°C at
3,500 rpm for 10 min (Allegra 6R centrifuge; Beckman) and subsequently re-
suspended in 25 mM NaH2PO4–1% glucose (pH 6.5). Centrifugation was re-
peated, and the cells were resuspended at a final OD600 of 5.
One-milliliter volumes of resuspended cells were incubated in six-well plates at
30°C with shaking at 150 rpm (Environ shaker; Labline). To each well, 20 ␮l of
DMSO containing various amounts of test compound was added. After 30 min,
10 ␮l of 100 mM acetic acid was added along with 30 ␮l of [2-14C]acetate (6 ␮Ci
[60 mCi ⅐ mmolϪ1]; Amersham). Labeled acetate was allowed to incorporate for
90 min, after which the contents of each well were added to 2 ml of freshly
prepared 15% (wt/vol) ethanolic KOH in 15-ml Falcon tubes. Mixtures were
transferred to an 80°C water bath and kept there for 90 min, after which they
were cooled to room temperature.
One 3-ml volume of distilled water was added to each tube, followed by the
addition of 3 ml of petroleum ether (40 to 60°C, vol/vol). Samples were mixed,
and the petroleum ether layer was removed and retained. Another volume of
petroleum ether (40:60, vol/vol) was then added, mixed, and recovered. The two
aliquots were combined and dried by rotary evaporation (Universal Vacuum
System Plus, model UVS800DDA; Savant). The dry preparations were resus-
pended in 50 ␮l of hexanes. For thin-layer chromatographic analysis, samples
with equal amounts of radioactivity (10,000 to 50,000 dpm) were loaded on Silica
Gel 60 F254 plates (Merck, Darmstadt, Germany) and were developed in chlo-
roform. Plates were exposed to a low-energy phosphor screen (Molecular Dy-
namics) for 1 to 2 days and analyzed with a PhosphorImager (Molecular Dy-
namics).
In order to confirm the chromatographic properties of various metabolites as
they were reported by Ryder et al. (16), 10- to 50-nmol samples of purchased,
unlabeled intermediates were loaded and run on Silica Gel 60 plates and visu-
alized by spraying the plates with 40% (wt/vol) sulfuric acid in ethanol, followed
by baking for 10 min at 110°C. Rf values determined for intermediates were 0.08
to 0.13 for ergosterol (Sigma), 0.13 to 0.18 for farnesol (ICN), 0.17 to 0.22 for
lanosterol (ICN), 0.44 to 0.53 for oxidosqualene (Echelon Biosciences), and 0.72
to 0.75 for squalene (Sigma).
pounds under study. Pyridine derivatives (Fig. 1A) were prepared from 4-chlo-
ropyridine. Treatment with a strong base (lithium diisopropylamide [1 eq]-tet-
rahydrofuran [THF] at Ϫ70°C for 1.5 h) produced the 3-lithio species that was
reacted in situ with the required substituted benzaldehydes (R1R2PhCHO [1.1
eq] for 1.5 h at Ϫ70°C), affording the alcohol intermediates 1a through 1c (30 to
40% yield). Alcohols 1a through 1c were reacted with benzyl bromide (sodium
hydride [2.5 eq]-nBu4NI [cat]-PhCH2Br [2 eq]-dimethylformamide [DMF] at
0°C for 2 h; 80 to 90% yield) or diethyl carbamoylchloride (sodium hydride [3
eq]-diethyl carbamoylchloride [2 eq]-THF at 0 to 25°C for 1.5 h; 80 to 90% yield)
to give ethers 2a and 2b or carbamates 5a through 5c, respectively. The chloride
group of compound 2a was displaced with azide ion (sodium azide [5 eq]–18-
crown-6 [cat]–DMF-water [5:1, vol/vol] at 95°C for 18 h; 70% yield) or 2-mer-
captoethanol (2-mercaptoethanol [2 eq]–potassium carbonate [6 eq]–DMF at
80°C for 45 min; 80% yield) to yield the derivatives 3c and 4c, respectively.
Carbamate 5a was resolved into its component enantiomers via chiral prepara-
tive high-pressure liquid chromatography (Chiracel AD, 50 by 500 mm; isocratic
elution, 5% isopropanol–95% hexane at 100 ml ⅐ minϪ1; UV detection at 254
nm). The absolute configuration of the enantiomerically pure materials was not
determined.
Pyrimidine derivatives (Fig. 1B) were prepared from 4-(thiomethyl)-5-bro-
mopyrimidine 6 (4) (nBuLi [1.25 eq]–THF-diethyl ether [2:1, vol/vol] at Ϫ70°C
for 5 min; then R1R2PhCHO [1.3 eq] for 45 min at Ϫ70°C; 35 to 50% yield) or
5-bromopyrimidine (nBuLi [1.4 eq]–THF-diethyl ether [1:1, vol/vol] at Ϫ100°C
for 30 min; then 2-fluoro-6-trifluoromethylbenzaldehyde [1.4 eq] at Ϫ100 to 25°C
for 16 h; 65% yield). Alcohols 7a through 7c and 9 were subsequently prepared
and converted to the corresponding benzyl ethers, 8a through 8c (sodium hydride
[2.5 eq]-nBu4NI [cat]-PhCH2Br [2 eq]-DMF at 0°C for 2 h; 80 to 90% yield) and
10 (sodium hydride [3.5 eq]-THF, with reflux for 5 min; then PhCH2Br [1.4
eq]-NaI [cat] was added, with reflux for 30 min; then 25°C for 16 h; 35% yield),
respectively.
RESULTS
A number of years ago, Dimster-Denk and Rine (5) and
Dixon et al. (7) developed in parallel a cell-based reporter
assay for S. cerevisiae. This assay allows the detection of ele-
vated ␤-galactosidase activities resulting from the increased
transcription of ERG10. In principle, this assay identifies in-
hibitors of enzymes that make up the biosynthetic pathway that
leads to the synthesis of ergosterol from acetyl-CoA, which was
the purpose of our study. In this assay, compounds were typi-
cally tested at a maximum concentration of 64 ␮g ⅐ mlϪ1 and at
nine subsequent twofold dilutions to a lowest concentration of
0.125 ␮g ⅐ mlϪ1. As a first step, the assay was validated by using
positive and negative control inhibitors. Treatment of cells
with lovastatin, zaragozic acid, terbinafine, and fluconazole,
which each inhibit a different enzyme in the ergosterol biosyn-
thetic pathway (HMG-CoA reductase [8], squalene synthase
[14], squalene epoxidase [15], and lanosterol demethylase [11],
respectively), resulted in increased specific ␤-galactosidase ac-
tivities compared to that of the DMSO-only control (Table 1).
The exposure of cells to the negative-control inhibitors am-
photericin B (3), cycloheximide (18), and chlorhexidine (12),
which do not inhibit the synthesis of ergosterol but instead
disrupt membrane integrity or inhibit translation, did not lead
to increased specific ␤-galactosidase activities. Unexpectedly,
however, the negative-control inhibitor flucytosine, which in-
hibits DNA and RNA biosynthesis (21), also increased ␤-ga-
lactosidase activity (see Discussion).
Untreated cells were found to accumulate lanosterol (11%), 4-␣-methylated
sterols (13%), and ergosterol (76%), whereas the maximum inhibition of lanos-
terol demethylase resulted in 76% lanosterol, 15% 4-␣-methylated sterols, and
The reporter assay of Dixon et al. (7) was used to screen a
corporate compound collection to identify those compounds
that possibly mediated their antifungal activities via one of the
enzymes required for ergosterol biosynthesis. This assay re-
sulted in a hit rate of 0.3%, and one of the compounds iden-
tified in this way was compound 5b (Fig. 1). Compounds re-
lated to 5b were synthesized, and the structures and
9% 4-ergosterol. The IC50,
was therefore defined as the compound
lanosterol
concentration causing half the effect of maximum inhibition, i.e., resulting in a
lanosterol content of 45%, and the inhibition of lanosterol demethylase was
quantitated by determining the IC50, lanosterols of various compounds.
Chemical synthesis of pyridine and pyrimidine derivatives. Figure 1 outlines
the synthetic schemes and experimental conditions used to prepare the com-

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PYRIDINES AND PYRIMIDINES INHIBITING CaErg11p
315
FIG. 1. Preparation of pyridine (A) and pyrimidine (B) compounds (numbers in boldface refer to the various compounds). (A) Reaction a,
lithium diisopropylamide, THF, and (R1R2)PhCHO; reaction b, NaH, PhCH2Br and DMF; reaction c, NaH, diethyl carbamoylchloride, and THF;
reaction d, NaN3, DMF, and water; reaction e, 2-mercaptoethanol, K2CO3, and DMF; reaction f, chiral-phase preparative high-pressure liquid
chromatography. (B) Reaction a, nBuLi, THF, diethylether, and R1R2PhCHO; reaction b, NaH, PhCH2Br, and DMF; reaction c, nBuLi, THF,
diethylether; and 2-fluoro-6-trifluoromethylbenzaldehyde; reaction d, NaH, PhCH2Br, and THF.
antimicrobial activities of a representative subset of these com-
pounds are shown in Fig. 1 and Table 2, respectively. The
reporter assay identified about half of the compounds as pu-
tative inhibitors of sterol biosynthesis in S. cerevisiae, as could
be detected with maximum compound concentrations of 64 ␮g
⅐ mlϪ1 (Table 1).
None of the compounds were active against the actual
pathogen C. albicans, here exemplified by wild-type C. albicans
CAF2-1 (9), at concentrations lower than or equal to 64 ␮g ⅐
mlϪ1. However, when we tested C. albicans DSY654, a strain
isogenic with C. albicans CAF2-1 except for the removal of
CDR1 (both copies) and CDR2, both of which encode efflux
pumps (17), the compounds were found to display antifungal
activities in the concentration range tested (Table 2). This
finding suggests that this class of compounds is efficiently re-
moved from the wild-type fungal cell by these transporters.
This detectable activity against strain DSY654 allowed the
antifungal mode of action of these compounds in this strain to
be determined.
In order to determine which step in fungal ergosterol bio-
synthesis was inhibited, the fate of incorporated 14C-labeled
acetate was monitored as described by Ryder et al. (16). To
validate this assay, cells were treated with characterized sterol
biosynthesis inhibitors (Fig. 2). In the absence of inhibition,
three bands, corresponding to lanosterol, 4-␣-methylated ste-
rols, and ergosterol, were observed (16). Treatment with the
lanosterol demethylase inhibitor fluconazole led to the accu-
mulation of lanosterol, whereas the squalene epoxidase inhib-

316
BUURMAN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Effects of control inhibitors and pyri(mi)dines on the
resulted in high ␤-galactosidase activities, the effect of this
compound at a concentration of 64 ␮g ⅐ mlϪ1 on sterol biosyn-
thesis was evaluated, but no effect was found (82% ergosterol,
8% 4-␣-methylated sterols, and 8% lanosterol).
induction of specific ␤-galactosidase activity in S. cerevisiae FSB1a
␤-Galactosidase activity
Compound
(mA570 ⅐ minϪ1
⅐
Ϫ1
OD600
)
Since the uncharacterized band accumulated when cells
were treated with zaragozic acid (Fig. 2) but not when they
were treated with either the farnesyl-diphosphate synthase in-
hibitor alendronate (75% ergosterol, 10% 4-␣-methylated ste-
rols, and 14% lanosterol) or lovastatin, it is likely that the
metabolite was related to farnesyl-diphosphate. Commercially
available 3H-farnesyl-diphosphate was subjected to the same
treatment as cell extracts were, i.e., incubation in hot ethanolic
KOH and subsequent extraction with petroleum ether.
Whereas the nontreated control did not migrate upon thin-
layer chromatography in chloroform, the treated control prep-
aration contained a metabolite migrating to a position similar
to those of both the uncharacterized band and the commer-
cially available farnesol (data not shown). This finding suggests
that the presence of farnesol among nonsaponifiable lipids
obtained from zaragozic acid-treated cells of C. albicans is the
result of farnesol formation by the cells (10) and the conver-
sion of farnesyl-diphosphate into farnesol during saponifica-
tion.
Treatment of cells with the first-discovered compound, 5b,
and a number of similar compounds of that class all resulted in
the accumulation of lanosterol and thus a pattern identical to
that caused by fluconazole. In order to quantitate this inhibi-
tion, cells were treated with various concentrations of com-
pound, and as a result, IC50, lanosterols could be determined.
Untreated cells were found to accumulate lanosterol (11%),
4-␣-methylated sterols (13%), and ergosterol (76%), whereas
the maximum inhibition of lanosterol demethylase inhibition
resulted in 76% lanosterol, 15% 4-␣-methylated sterols, and
9% 4-ergosterol. The IC50, lanosterol was therefore defined as
the compound concentration causing half the effect of maxi-
mum inhibition, i.e., resulting in a lanosterol content of 45%.
Compounds were selected from a range of antifungal activities,
and the effects of their presence on ergosterol biosynthesis
were determined qualitatively and quantitatively. All com-
pounds caused the accumulation of lanosterol, and their
IC50, lanosterols were typically below the MICs of the com-
pounds (Table 2). In addition, a correlation between the
IC50, lanosterol and the MIC was obtained (Fig. 3). This finding
suggests that the inhibition of lanosterol demethylase is not
merely a secondary effect but that the inhibition of lanosterol
metabolism is the antifungal mode of action of these pyri(mi-
)dines.
Terbinafine .................................................................... 48 (64)
5-Fluorocytosine ........................................................... 44 (32)
Zaragozic acid............................................................... 16 (32)
Fluconazole ................................................................... 12 (64)
Lovastatin ...................................................................... 12 (32)
Amphotericin B ............................................................ 2 (9)
Cycloheximide............................................................... 0 (0.063)
Chlorhexidine................................................................ Ϫ2 (4)
DMSO............................................................................ 1 (NA)
2b.................................................................................... 3 (32)
3c .................................................................................... 12 (64)
4c .................................................................................... 2 (64)
5a(ϩ).............................................................................. 14 (64)
5a(Ϫ).............................................................................. 4 (64)
5b.................................................................................... 9 (64)
5c .................................................................................... 8 (64)
8a .................................................................................... 10 (32)
8b.................................................................................... 5 (64)
8c .................................................................................... 3 (32)
10 .................................................................................... 3 (64)
a The actual compound concentration (in micrograms per milliliter) at which
the maximum activity was obtained is indicated in parentheses. NA, not appli-
cable.
itor terbinafine caused the accumulation of squalene. As ex-
pected, the inhibition of squalene synthase by zaragozic acid
did not result in the accumulation of squalene but did lead to
the appearance of a previously described (16), uncharacterized
intermediate migrating with 4-␣-methylated sterol, which we
believe to be farnesol (see below). Since lovastatin acts in the
mevalonate pathway, the accumulation of sterol-like interme-
diates was not expected. At a lovastatin concentration of 64 ␮g
⅐ mlϪ1, the cellular levels of all intermediates were reduced
fivefold, but that left the relative amounts of each sterol (95%
ergosterol and 5% 4-␣-methylated sterols) virtually unchanged
relative to those in untreated cells. Finally, since flucytosine
TABLE 2. Activities of pyridines and pyrimidines against C.
albicans CAF2-1 and its efflux-negative derivative C. albicans
DSY654 and their potency in inducing lanosterol accumulation in C.
albicans DSY654
MIC (␮g ⅐ mlϪ1) for:
IC50, lanosterol (␮g ⅐ mlϪ1) for
Compounda
C. albicans
C. albicans
DSY654
C. albicans DSY654
CAF2-1
2b
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ64
16
2
32
16
64
4
1
DISCUSSION
3c
0.125
8
4c
With the reporter assay developed by Dimster-Denk and
Rine (5) and Dixon et al. (7), a positive signal can be obtained
with four antifungal compounds, each inhibiting a different
step in the biosynthesis of ergosterol (Table 1). However, it is
intriguing that the sizes of the signals varied considerably. The
rationale of the assay is that the inhibition of ergosterol bio-
synthesis leads to lowered levels of this sterol, thereby activat-
ing compensatory feedback mechanisms, one of which is the
increased transcription of ERG10. This mechanism is common
for all four inhibitors, and therefore they would be expected to
5a(ϩ)
5a(Ϫ)
5b
16
32
0.75
64
0.25
0.125
0.125
0.125
5c
64
2
8a
8b
4
8c
4
10
8
a For compound 5a, because the absolute configuration of the enantiomeri-
cally pure materials was not determined, they are referred to here as the (ϩ) and
(Ϫ) isomers.

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317
FIG. 2. Incorporation of 14C-labeled acetate into nonsaponifiable lipids of C. albicans DSY654 upon treatment with various concentrations of
ergosterol biosynthesis inhibitors. Concentrations of ergosterol (diamonds), lanosterol (triangles), squalene (asterisks), and 4␣-methylsterols/
farnesol (squares) are shown.
result in similar signals, albeit possibly at different compound
concentrations. The fact that they did not suggests that the
feedback mechanism that leads to the transcriptional activa-
tion of ERG10 is more complex and depends quantitatively
upon where in the pathway inhibition occurs.
is the possible accumulation of intermediates and their incor-
poration into the cell membrane. Treatment with zaragozic
acid or lovastatin led to very similar activities (12 to 16 mA570
⅐ minϪ1 ⅐ OD600Ϫ1) (Table 1). Since under these conditions no
accumulated intermediates are incorporated into the cell
membrane, this finding suggests that this signal was solely the
result of diminished ergosterol levels. Treatment with flucon-
azole also led to ␤-galactosidase activities of 12 mA570 ⅐ minϪ1
The most obvious factor differentiating the control inhibitors
Ϫ1
⅐ OD600 (Table 1), showing that the accumulation of lanos-
terol is of no consequence to the signal. Treatment with ter-
binafine, however, resulted in a three- to fourfold-higher signal
(48 mA570 ⅐ minϪ1 ⅐ OD600Ϫ1) (Table 1), which indicates that
the accumulation of squalene has a profound effect on the
feedback mechanism that leads to the transcription of ERG10.
The implication is that although this assay may in principle
detect inhibitors of each step in the pathway, screening results
will be biased towards inhibition of more sensitive steps, such
as the squalene epoxidase step.
A further complication of the assay is that flucytosine was a
strong inducer of the reporter, whereas its antifungal activity is
mediated by the inhibition of RNA and DNA biosynthesis
(21). In accordance with its mechanism, no alteration in sterol
biosynthesis was observed (data not shown). 5-Fluoroorotic
acid, which is converted in 5-fluorouracil and has a mode of
FIG. 3. Antifungal activities of pyridines and pyrimidines correlate
with their potency in reducing ergosterol biosynthesis in C. albicans
DSY654, as indicated by their IC50, lanosterols.

318
BUURMAN ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
3. Bolard, J. 1991. Mechanism of action of an anti-Candida drug: amphotericin
B and its derivatives, p. 214–238. In R. Prasad (ed.), Candida albicans.
Cellular and molecular biology. Springer-Verlag, Berlin, Germany.
4. Dietsche, T. J., D. B. Gorman, J. A. Orvik, G. A. Roth, and W. R. Shiang.
2000. Process chemistry related to the experimental rice herbicide 2,2-di-
methyl-1-(4-methylthio-5-pyrimidinyl)indane. Org. Process Res. Dev. 4:275–
285.
5. Dimster-Denk, D., and J. Rine. 1996. Transcriptional regulation of a sterol-
biosynthetic enzyme by sterol levels in Saccharomyces cerevisiae. Mol. Cell.
Biol. 16:3981–3989.
6. Dimster-Denk, D., M. K. Thorsness, and J. Rine. 1994. Feedback regulation
of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cer-
evisiae. Mol. Biol. Cell 5:655–656.
7. Dixon, G., D. Scanlon, S. Cooper, and P. Broad. 1997. A reporter gene assay
for fungal sterol biosynthesis inhibitors. J. Steroid Biochem. Mol. Biol. 62:
165–171.
8. Endo, A. 1988. Chemistry, biochemistry, and pharmacology of HMG-CoA
reductase inhibitors. Klin. Wochenschr. 66:421–427.
9. Fonzi, W. A., and M. Y. Irwin. 1993. Isogenic strain construction and gene
mapping in Candida albicans. Genetics 134:717–728.
10. Hornby, J. M., B. W. Kebaara, and K. W. Nickerson. 2003. Farnesol biosyn-
thesis in Candida albicans: cellular response to sterol inhibition by zaragozic
acid B. Antimicrob. Agents Chemother. 47:2366–2369.
11. Kelly, S. L., D. C. Lamb, A. J. Corran, B. C. Baldwin, and D. E. Kelly. 1995.
Mode of action and resistance to azole antifungals associated with the for-
mation of 14␣-methylergosta-8,24(28)-dien-3␤,6␣-diol. Biochem. Biophys.
Res. Commun. 207:910–915.
12. Maris, P. 1995. Modes of action of disinfectants. Rev. Sci. Tech. Off. Int.
Epizoot. 14:47–55.
13. National Committee for Clinical Laboratory Standards. 1997. Reference
method for broth microdilution antifungal susceptibility testing of yeasts:
approved standard. Document M27-A. National Committee for Clinical
Laboratory Standards, Wayne, Pa.
14. Ponpipom, M. M., N. N. Girotra, R. L. Bugianesi, C. D. Roberts, G. D.
Berger, R. M. Burk, R. W. Marquis, W. H. Parsons, K. F. Bartizal, J. D.
Bergstrom, M. M. Kurtz, J. C. Onishi, and D. J. Rew. 1994. Structure-activity
relationships of C1 and C6 side chains of zaragozic acid A derivatives.
J. Med. Chem. 37:4031–4051.
15. Ryder, N. S. 1985. Specific inhibition of fungal sterol biosynthesis by SF
86–327, a new allylamine antimycotic agent. Antimicrob. Agents Chemother.
27:252–256.
16. Ryder, N. S., G. Seidl, and P. F. Troke. 1984. Effect of the antimycotic drug
naftifine on growth of and sterol biosynthesis in Candida albicans. Antimi-
crob. Agents Chemother. 25:483–487.
17. Sanglard, D., F. Ischer, M. Monod, and J. Bille. 1997. Cloning of Candida
albicans genes conferring resistance to azole antifungals: characterization of
CDR2, a new multidrug ABC transporter gene. Microbiology 143:405–416.
18. Sto¨cklein, W., and W. Piepersberg. 1980. Binding of cycloheximide to ribo-
somes from wild-type and mutant strains of Saccharomyces cerevisiae. Anti-
microb. Agents Chemother. 18:863–867.
action similar to that of flucytosine, induced the reporter en-
zyme as well (9.4 mA570 ⅐ minϪ1 ⅐ OD600Ϫ1). Since both com-
pounds interfere with the availability of uridine metabolites for
RNA biosynthesis, it can be expected that genes involved in
uridine biosynthesis will be activated upon exposure to these
compounds. One of these genes, URA3, was used to integrate
the reporter construct (7), but it is not clear whether termina-
tor sequences that would prevent read-through from URA3
into ERG10-lacZ are present. One explanation for our results
is that such transcriptional read-through, leading to ␤-galacto-
sidase activity, might have occurred. It should be stated,
though, that recently published microarray analyses do not
provide evidence that an upregulation of URA3 upon the ex-
posure of S. cerevisiae to flucytosine does indeed occur (1, 22).
The induction of the reporter enzyme in the presence of
pyridines or pyrimidines suggested that these compounds in-
hibited sterol biosynthesis in S. cerevisiae. Hence, the effects of
this class of compounds on sterol biosynthesis in C. albicans
were evaluated in order to rule out interference with uridine
metabolism and assess the mode of action in this pathogen.
The mutant DSY654 was used because antifungal activity
could be observed only in the absence of efflux. Analysis of
sterol metabolites showed that lanosterol accumulated but that
ergosterol levels decreased. Quantification of this effect led to
the determination of IC50, lanosterols, which were typically well
below the MICs. This finding implies that when this strain of C.
albicans is incubated with compound concentrations at approx-
imately the MIC, sterol metabolism is severely compromised.
Furthermore, the antifungal potency of compounds in this
series ranks with the potency of inhibition of lanosterol de-
methylase, as assessed by lanosterol accumulation (Fig. 3).
This finding strongly suggests that the inhibition of this step in
sterol biosynthesis is the antifungal mode of action of these
compounds.
ACKNOWLEDGMENTS
We thank Shanta Bist and Julie Demeritt for the synthesis of various
compounds and Linda Otterson for antifungal susceptibility testing.
19. Teo, K. K., and J. R. Burton. 2002. Who should receive HMG CoA reductase
inhibitors? Drugs 62:1707–1715.
REFERENCES
20. Vanden Bossche, H. 1988. Mode of action of pyridine, pyrimidine and azole
antifungals, p. 79–119. In D. Berg and M. Plempel (ed.), Sterol biosynthesis
inhibitors: pharmaceutical and agrochemical aspects. Ellis Horwood, New
York, N.Y.
21. Vanden Bossche, H., P. Marichal, and F. C. Odds. 1994. Molecular mech-
anisms of drug resistance in fungi. Trends Microbiol. 2:393–400.
22. Zhang, L., Y. Zhang, Y. Zhou, Y. Zhao, Y. Zhou, and J. Cheng. 2002.
Expression profiling of the response of Saccharomyces cerevisiae to 5-fluo-
rocytosine using a DNA microarray. Int. J. Antimicrob. Agents 20:444–450.
1. Agarwal, A. K., P. D. Rogers, S. R. Baerson, M. R. Jacob, K. S. Barker, J. D.
Cleary, L. A. Walker, D. G. Nagle, and A. M. Clark. 2003. Genome-wide
expression profiling of the response to polyene, pyrimidine, azole, and echi-
nocandin antifungal agents in Saccharomyces cerevisiae. J. Biol. Chem. 278:
34998–35015.
2. Bergstrom, J. D., R. G. Bostedor, P. J. Masarachia, A. A. Reszka, and G.
Rodan. 2000. Alendronate is a specific, nanomolar inhibitor of farnesyl
diphosphate synthase. Arch. Biochem. Biophys. 373:231–241.