Marios Stylianou_Paper III_Antifungal application of nonantifungal drugs.

Antifungal Application of Nonantifungal Drugs
Marios Stylianou,a,b
Evgeny Kulesskiy,c
José Pedro Lopes,a,b
Margareta Granlund,a
Krister Wennerberg,c
Constantin F. Urbana,b
Department of Clinical Microbiology, Umeå University, Umeå, Swedena
; Laboratory for Molecular Infection Medicine, Sweden (MIMS), and Umeå Centre for Microbial
Research, Umeå University, Umeå, Swedenb
; Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finlandc
Candida species are the cause of 60% of all mycoses in immunosuppressed individuals, leading to ϳ150,000 deaths annually due
to systemic infections, whereas the current antifungal therapies either have toxic side effects or are insufficiently efficient. We
performed a screening of two compound libraries, the Enzo and the Institute for Molecular Medicine Finland (FIMM) oncology
collection library, for anti-Candida activity based on the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) guidelines. From a total of 844 drugs, 26 agents showed activity against Candida albicans. Of those, 12 were standard
antifungal drugs (SADs) and 7 were off-target drugs previously reported to be active against Candida spp. The remaining 7 off-
target drugs, amonafide, tosedostat, megestrol acetate, melengestrol acetate, stanozolol, trifluperidol, and haloperidol, were
identified with this screen. The anti-Candida activities of the new agents were investigated by three individual assays using opti-
cal density, ATP levels, and microscopy. The antifungal activities of these drugs were comparable to those of the SADs found in
the screen. The aminopeptidase inhibitor tosedostat, which is currently in a clinical trial phase for anticancer therapy, displayed
a broad antifungal activity against different Candida spp., including Candida glabrata. Thus, this screen reveals agents that were
previously unknown to be anti-Candida agents, which allows for the design of novel therapies against invasive candidiasis.
The number of immunocompromised patients is increasing
worldwide, and these individuals are at high risk for acquiring
severe microbial infections, which are frequently caused by Can-
dida spp. (1). Most commonly, cases of bloodstream infections
with Candida spp. (candidemia) are related to surgery, intensive
care, solid tumors, or hematological malignancies (2). The mor-
tality rate that is directly attributable to candidemia ranges from 5
to 71%, depending on the clinical cohort (3, 4). Particularly high
mortalities occur in patients with solid tumors (65%) and hema-
tologic malignancies (46%) (5). Severe mycoses are frequently
caused by species of the Candida clade, such as C. albicans, C.
dubliniensis, and C. glabrata (6, 7). Although these species cause
severe invasive infections in immunocompromised persons, they
are also common as part of the commensal flora on mucous mem-
branes. C. albicans is the most common human fungal pathogen
and is able to switch back and forth from yeast to hyphal growth.
This revertible morphogenetic switch plays a key role in the viru-
lence of C. albicans (8–10). C. albicans and C. dubliniensis are
phylogenetically closely related, sharing a polymorphic and oblig-
atory diploid nature (11). However, C. dubliniensis differs in vir-
ulence-associated gene families, such as the agglutinin-like se-
quences (ALS), which render C. dubliniensis less virulent than C.
albicans (12). Nevertheless, C. dubliniensis causes candidemia as-
sociated with equally high mortality rates (13). C. glabrata is a
haploid yeast and is often referred to as the second most frequent
yeast causing candidemia (7). Importantly, C. glabrata has de-
creased in vitro susceptibility against fluconazole. This suggests an
increased risk for C. glabrata infections due to prophylactic flu-
conazole treatment of patients at high risk for invasive candidiasis
(14).
Despite the urgent requirement for efficient antifungal thera-
pies, the available standard antifungal drugs (SADs) are few and
have a restricted set of fungal targets. Polyenes, azoles, allylamines,
morpholines, antimetabolites, and echinocandins are the 6 major
antifungal drug categories (15). The first three directly or indi-
rectly target ergosterol, a major fungal membrane component.
The long-term use of drugs that target ergosterol, such as flucona-
zole or amphotericin B, can result in renal and liver toxicity (16).
The following two categories interfere with DNA/RNA synthesis.
Antimetabolites are known human carcinogens, causing liver tox-
icity and bone marrow depression and thus are less frequently
used (16, 17). The relatively newly introduced echinocandins in-
hibit 1,3-␤-glucan synthesis in the cell wall and have few notable
side effects. The emergence of echinocandin-resistant isolates,
however, is a cause for clinical concern (18). Taken together, there
is a demand for new antifungal substances, preferably those with
novel fungal targets.
The scope of this study was to find previously unknown anti-
fungal activities in agents from the Enzo drug library and the In-
stitute for Molecular Medicine Finland (FIMM) oncology collec-
tion (19). We rationalized that knowledge about antifungal
activity against common human fungal pathogens in off-patent
drugs from the Enzo library should be beneficial to increase the
treatment possibilities for severe mycoses. Moreover, we screened
the FIMM oncology collection, which contains antineoplastic
drugs, with the purpose of identifying agents that affect both pri-
mary immune-suppressive cancer disease and a possible second-
ary Candida infection, which occurs frequently in cancer patients.
This additional information is beneficial for patients if a choice of
therapy is possible. We performed the screen with C. albicans and
confirmed antifungal activities for 19 drugs that have been previ-
ously described for their antimycotic capacities, approving the
validity of our methods. We identified 7 novel agents previously
unknown to inhibit the growth of C. albicans (Table 1). Notably,
Received 21 May 2013 Returned for modification 12 July 2013
Accepted 22 November 2013
Published ahead of print 25 November 2013
Address correspondence to Constantin F. Urban, constantin.urban@mims.umu.se.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01087-13
February 2014 Volume 58 Number 2 Antimicrobial Agents and Chemotherapy p. 1055–1062 aac.asm.org 1055
onAugust21,2015byUMEAUNIVERSITYLIBRARYhttp://aac.asm.org/Downloadedfrom

the susceptibility of C. albicans to SADs was comparable to the
antifungal effect of the seven agents identified in this screen.
MATERIALS AND METHODS
Drugs and fungal strains. The in vitro susceptibility of C. albicans strain
SC5314 was tested against 844 drugs from the Enzo FDA-approved drug
library (640 drugs) and the FIMM oncology collection (19) (FDA-ap-
proved anticancer drugs [n ϭ 119] and preclinical compounds [n ϭ 85]).
Thirteen FDA-approved antifungal drugs, 12 of which were active against
C. albicans SC5314, and five nonantifungal drugs with antifungal activity
served as controls. The screen was performed with C. albicans SC5314,
and hits were further confirmed with the type strains C. dubliniensis
CD36/CBS7987 and C. glabrata ATCC 90030, as well as with unrelated
clinical strains of C. albicans UBC3-7922, C. glabrata UCB3-7268, and C.
dubliniensis UCB-3892 from the strain collection of Norrland’s University
Hospital, Umeå, Sweden.
Media and antifungal microdilution testing. Cell concentration and
drug microdilution analyses were performed according to the European
Committee on Antimicrobial Susceptibility Testing (EUCAST) guide-
lines, with modifications (20). Candida yeast cells were grown overnight
at 30°C with shaking in yeast peptone medium plus 2% glucose (YPD).
Subcultures of 107
cells/ml in YPD grew for 4 h at 30°C. Drugs in the
amounts of 15 to 150 nl from the Enzo and FIMM oncology collections
were distributed by a liquid handling platform (Labcyte Echo 550 acoustic
dispenser) in black 96-well plates with clear bottoms in six different con-
centrations from 0.17 nM to 10 ␮M. Subsequently, 50 ␮l RPMI 1640 was
added to each well and the start plates were shaken (30 rpm) prior to the
assay for 1 h to ensure equal distribution of the agents within the well. The
yeast suspension, 100 ␮l of 5 ϫ 105
cells/ml in RPMI 1640 without phenol
red, and 10 mM HEPES (Lonza) were transferred to the 96-well plates
containing medium and agents using a robotic device (Matrix WellMate;
Thermo Scientific), resulting in a final volume of 150 ␮l in each well. The
plates were incubated at 37°C, 5% CO2, for 6 or 24 h.
Determination of fungal growth using absorbance. The growth of C.
albicans SC5314 was analyzed using a microdilution plate assay according
to EUCAST recommendations (20). One-hundred-microliter suspen-
sions of yeasts (5 ϫ 105
cells/ml) in RPMI 1640 were incubated in the
presence or absence of drugs in a total volume of 150 ␮l at 37°C, 5% CO2,
for 6 h and 24 h. The optical densities at 450 nm (OD450) in the plates were
determined using a plate reader (Tecan Infinite F200). ODs of Ͻ0.1 for 6
h and 0.2 for 24 h for the 100% growth control were considered to repre-
sent poor growth and were not taken into account for the evaluation. As
described above, 100% and 0% growth controls were included with every
plate. All assays were performed at least as two biological replicates in
triplicate (n ϭ 2 [3]).
Determination of fungal viability using ATP levels. In order to de-
termine the viability of the C. albicans, C. glabrata, and C. dubliniensis
strains, the CellTiter-Glo luminescent cell viability kit (Promega) was
used. One hundred-microliter suspensions of yeasts (5 ϫ 105
cells/ml) in
RPMI 1640 were incubated in the presence or absence of drugs in a total
volume of 150 ␮l at 37°C, 5% CO2, for 6 h and 24 h. An equal volume of
the CellTiter-Glo reagent was added to the medium and incubated for 15
min at room temperature with shaking at 900 rpm. The luminescent sig-
nals after 6 h and 24 h were detected using a luminometer (Tecan Infinite
F200). The resulting signal intensity corresponds to ATP amounts and
thus to the number of viable microbial cells upon drug exposure. In all
96-well plates, 100% and 0% growth controls were included as microbes
plus dimethyl sulfoxide (DMSO) (0.1%) and microbes plus benzetho-
nium chloride (BzCl) (100 ␮M), respectively. All assays were performed at
least as two biological replicates in triplicate (n ϭ 2 [3]).
Microscopic analysis of morphological changes occurring upon
drug treatment. For a morphological analysis of C. albicans SC5314
treated with antifungal agents (1 ␮M), an IncuCyte automated micro-
scope was used (Essen Bioscience). The plates were incubated at 37°C
under 5% CO2. After the indicated time points, prior to analysis, the fungi
were fixed with 2% paraformaldehyde (PFA) and phase-contrast images
were captured. In this study, 4 pictures per well were taken from two
biological and three technical replicates.
Statistical and data analysis. Percent growth inhibition (%Inh) was
calculated from the ATP and OD measurements resulting from the mean
values from all biological replicates, using the equation %Inh ϭ 100 Ϫ
(valuesample/valuecontrol) ϫ 100. The %Inh values (y axis) were plotted
against the drug concentration (x axis), and the according trend line of the
dose-response curve was defined and the resulting linear equation was
applied to calculate the MICs using Microsoft Office Excel 2007. The MIC
was the lowest drug concentration resulting in Ն50% growth inhibition
compared to that of the drug-free control according to the EUCAST
guidelines for flucytosine, azole antifungal agents, and echinocandins
(20). Additionally, we defined MIC0.3 as the lowest drug concentration
resulting in Ն30% growth inhibition compared to that of the drug-free
control.
The data were analyzed and evaluated from 3 biological replicates in
triplicate (n ϭ 3 [3]) (Tables 2 and 3), as well as from 4 biological repli-
cates in triplicate (n ϭ 4 [3]) (Table 4). The strains C. dubliniensis CD36/
CBS7987 and C. glabrata ATCC 90030 shown in Table 4 were analyzed in
2 biological replicates in triplicate (n ϭ 2 [3]). The R2
values for all dose-
response curves ranged between 0.87 and 0.92. Additionally, the coeffi-
cients of variation (the ratio of the standard deviation to the mean) ex-
pressed as a percentage (also referred to as relative standard deviation) for
all biological replicates ranged from 13 to 28%.
RESULTS
Outline of the study. Our main goal was to identify antifungal
activities in drugs that were designed for other purposes. Two
collection libraries, Enzo and FIMM oncology, comprising a total
of 844 agents, were screened for activity against C. albicans. A
TABLE 1 All drugs with antifungal activity identified in this study
(n ϭ 26)
Identified drug
Previously
described as
antifungal
Previously
described as
anti-Candida
Therapeutic
use
Reference
no. or
source
Haloperidol HCl Yes No Antipsychotic 21, this
study
Trifluperidol 2HCl No No Antipsychotic This study
Stanozolol No No Anemia,
angioedema
This study
Melengestrol acetate No No Anticancer This study
Megestrol acetate No No Anticancer This study
Tosedostat No No Anticancer This study
Amonafide No No Anticancer This study
Methiothepin
maleate
Yes Yes Antipsychotic 25
Rapamycin Yes Yes Anticancer 26
Auranofin Yes Yes Antirheumatic 27
Bleomycin sulfate Yes Yes Anticancer 40
Disulfiram Yes Yes Anticancer 41
Artemisinin Yes Yes Antimalarial 42
Tamoxifen citrate Yes Yes Anticancer 43
Tioconazole Yes Yes Antifungal NAa
Oxiconazole nitrate Yes Yes Antifungal NA
Ketoconazole Yes Yes Antifungal NA
Climbazole Yes Yes Antifungal NA
Miconazole Yes Yes Antifungal NA
Myclobutanil Yes Yes Antifungal NA
Fluconazole Yes Yes Antifungal NA
Amorolfine Yes Yes Antifungal NA
Bifonazole Yes Yes Antifungal NA
Sertaconazole Yes Yes Antifungal NA
Itraconazole Yes Yes Antifungal NA
Terbinafine HCl Yes Yes Antifungal NA
a
NA, not applicable.
Stylianou et al.
1056 aac.asm.org Antimicrobial Agents and Chemotherapy

major challenge for screenings with C. albicans is the characteristic
of the fungus to grow as hyphal filaments (8). Filamentation com-
plicates assessments of growth using OD, for instance, since the
number of individual cells does not increase and hyphae tend to
clump excessively. Therefore, we used a luciferase-based quanti-
fication of ATP to assess fungal viability. We additionally con-
firmed the screening results by quantifying fungal growth using
OD measurements. Both methods resulted in highly comparable
results for all tested drugs.
Seven off-target drugs revealed to have anti-Candida activi-
ties. The screen identified a total of 26 agents that are active against
C. albicans (Table 1). Of those, 12 were SADs and 7 were off-target
drugs with known antifungal activities. Additionally, the screen
revealed 7 drugs from 4 different categories of therapy with pre-
viously unidentified potent anti-Candida activities (Table 2). Two
are antipsychotic (haloperidol and trifluperidol), one is used for
the treatment of anemia (stanozolol), and 4 are used for cancer
therapy (melengestrol acetate, megestrol acetate, tosedostat, and
amonafide). Haloperidol, but not trifluperidol, has previously
been identified in a chemical-genetic screen to have antimicrobial
activity against Saccharomyces cerevisiae (21). Four agents are
FDA-approved drugs and 2 are anticancer agents (amonafide and
tosedostat) that are currently being tested in clinical trials (22, 23).
Although it has been applied in animal husbandry, of the identi-
fied drugs, only melengestrol acetate is not currently used in hu-
mans (24). Moreover, we identified the antipsychotic drug me-
thiothepin maleate, which only very recently has been identified in
a repurposing screen for anticryptococcal agents (25). We used
the immunosuppressant drug rapamycin and the antirheumatic
drug auranofin as references for the antifungal activities of the
newly identified agents (Table 2). Interestingly, rapamycin was
originally identified as an antifungal agent (26), and gold (I) com-
plexes, such as auranofin, have been recognized for their antimi-
crobial activities (27).
We determined the MIC and MIC0.3 values for C. albicans by
OD and ATP measurements. As mentioned above, the methods
resulted in highly similar values, and thus one value for each agent
is presented (Table 2). In general, the MICs were slightly lower
after 6 h than after 24 h of incubation. However, the activities of
the 7 compounds against C. albicans were stable over a period of
24 h (Table 2). Importantly, in this screen, we did not use concen-
TABLE 2 MIC and MIC0.3 values against Candida albicans type straina
Antifungal agent
This study
Other studiesc
Concn range
(␮g/ml)
ATP level and OD450
b
MIC at: MIC0.3 at:
6 h 24 h 6 h 24 h Cmax (␮g/ml) Ref. for Cmax
Haloperidol HCl 6.4 ϫ 10Ϫ5
to 3.76 0.38 3.76 0.04 0.35 2.00–3.00 44
Trifluperidol 2HCl 7 ϫ 10Ϫ5
to 4.00 4.00 4.00 0.40 0.40 UAd
UA
Stanozolol 3.3 ϫ 10Ϫ5
to 3.29 3.29 Ͼ3.29 0.30 0.30 0.007 45
Melengestrol acetate 6.8 ϫ 10Ϫ5
to 3.97 2.20 3.97 0.40 0.22 0.01 46
Megestrol acetate 6 ϫ 10Ϫ5
to 3.85 2.10 3.85 0.39 0.40 0.50–0.70 47
Tosedostat 4 ϫ 10Ϫ3
to 4.00 Ͼ4.00 Ͼ4.00 4.00 4.00 0.15 23
Amonafide 2.8 ϫ 10Ϫ3
to 2.83 Ͼ2.83 Ͼ2.83 1.50 Ͼ2.83 4.00 22
Methiothepin maleatee
7 ϫ 10Ϫ5
to 3.57 0.35 3.57 0.044 0.25 UA UA
Auranofine
1 ϫ 10Ϫ4
to 6.78 0.70 0.38 0.007 0.07 6.60 48
Rapamycine
1.55 ϫ 10Ϫ5
to 9.14 0.001 0.005 1 ϫ 10Ϫ5
1 ϫ 10Ϫ5
0.01–0.21 49
a
The data were determined from three biological replicates in triplicate (n ϭ 3 [3]). MIC, minimal concentration of drug resulting in Ն50% growth inhibition; MIC0.3, minimal
concentration of drug resulting in Ն30% growth inhibition.
b
OD450, optical density at 450 nm.
c
Cmax, plasma peak concentrations reachable in humans upon first dose of the drugs; Ref., literature reference.
d
UA, unavailable.
e
The anti-Candida albicans activities of these drugs were demonstrated previously.
TABLE 3 Comparison of SADs with off-target antifungal agents
identified in this study at a concentration of 1 ␮Ma
Drugs MIC MIC0.3
Standard antifungalb
Tioconazole 0.39 ␮g/ml
Oxiconazole nitrate 0.40 ␮g/ml
Ketoconazole 0.50 ␮g/ml
Climbazole 0.29 ␮g/ml
Miconazole 0.40 ␮g/ml
Fluconazole 0.30 ␮g/ml
Amorolfine 0.32 ␮g/ml
Myclobutanil 0.29 ␮g/ml
Bifonazole 0.30 ␮g/ml
Sertaconazole 0.40 ␮g/ml
Itraconazole 0.70 ␮g/ml
Terbinafine HCl Ͼ1 ␮M
Nystatin Ͼ1 ␮M
Off-target antifungal
Haloperidol HCl 0.38 ␮g/ml
Methiothepin maleate 0.36 ␮g/ml
Auranofin 0.68 ␮g/ml
Trifluperidol 2HCl 0.40 ␮g/ml
Stanozolol 0.30 ␮g/ml
Melengestrol acetate 0.40 ␮g/ml
Megestrol acetate 0.39 ␮g/ml
Tosedostat Ͼ1 ␮M
Amonafide Ͼ1 ␮M
a
SADs, standard antifungal drugs. C. albicans SC5314 was challenged with SADs and
antifungal agents identified in this study.
b
MIC, minimal concentration of drug resulting in Ն50% growth inhibition; MIC0.3,
minimal concentration of drug resulting in Ն30% growth inhibition. The MIC and
MIC0.3 were determined by ATP measurement after 6 h of incubation. Nystatin did not
show any activity against C. albicans SC5314 in this assay. The data are determined
from three biological replicates in performed triplicate (n ϭ 3 [3]).
Dual Use of Known Drugs as Antimycotics
February 2014 Volume 58 Number 2 aac.asm.org 1057

trations of Ͼ10 ␮M (corresponding to 3 to 10 ␮g/ml, depending
on the molecular weight of the agent), since in the therapy of
systemic mycoses, maximal peak blood serum concentrations
above this level are unlikely to be reached. Haloperidol, trifluperi-
dol, stanozolol, melengestrol acetate, and megestrol acetate
showed MIC values of Ͻ4 ␮g/ml. For tosedostat and amonafide,
the MIC0.3 values were determined to be 4 and 2.8 ␮g/ml, respec-
tively. All 7 substances displayed a dose-dependent effect on C.
albicans SC5314. The antifungal activities of amonafide and tose-
dostat (Table 2) increased slowly over a wide concentration range,
from approximately 3 ϫ 10Ϫ3
␮g to 4 ␮g/ml.
Novel antifungal off-target drugs and SADs have similar
anti-Candida activities. We next compared the antifungal activ-
ities of the 7 identified agents to 13 established SADs present in the
Enzo library. Notably, the novel candidates were inhibitory
against C. albicans at a level similar to those of 12 of the SADs at a
concentration of 1 ␮M, ranging from 0.3 ␮g to 0.7 ␮g/ml, depend-
ing on individual molecular weights (Table 3). Terbinafine HCl,
tosedostat, and amonafide had an MIC0.3 at a concentration of Ͼ1
␮M. At this concentration, nystatin was the only SAD that lacked
anti-Candida activity after 6 h. Additionally, five off-target drugs
with previously known antifungal activities were also identified in
this screen, confirming that the applied methods were suitable to
identify antifungal activity against C. albicans (Table 5).
Microscopic analysis of morphological changes in C. albicans
occurring upon treatment with newly identified agents. The an-
tifungal effects of tosedostat and amonafide were milder than
those of other drugs (Tables 2 and 3). To verify the possible effects
of the selected agents identified in this study on C. albicans, we
additionally performed a direct microscopic investigation of
treated C. albicans (Fig. 1). DMSO- and BzCl-treated C. albicans
served as 100% and 0% growth controls, respectively (Fig. 1A and
B). Haloperidol and trifluperidol (Fig. 1E and G) show a very
similar effect as fluconazole (Fig. 1C). The hyphae are consider-
ably shorter, with the tendency to form branches more frequently
than with untreated control hyphae. Notably, tosedostat and
amonafide (Fig. 1F and H) caused similar morphological changes
as those observed in the samples treated with rapamycin (Fig. 1D).
The hyphae are significantly shorter, with the germ tubes having a
curved shape. The control hyphae, in contrast, are longer and
straight. Thus, our screen identified substances with comparable
effects on C. albicans morphology as the well-known antifungal
agent fluconazole or the immunosuppressant drug with antifun-
gal activity, rapamycin. This indicates that the identified agents
indeed inhibit the growth of C. albicans.
Confirmation of antifungal activities of identified drugs on
clinical isolates from different Candida spp. To assess whether
the 7 new antifungal candidate agents were also effective against
other clinical isolates of C. albicans, as well as other Candida spe-
cies, we compared C. albicans SC5314 to other clinical isolates
from C. albicans, C. dubliniensis, and C. glabrata (Table 4). C.
albicans SC5314 and the off-target drugs with known antifungal
TABLE 4 MIC and MIC0.3 values of antifungal agents for type strains and clinical isolates of Candida spp.a
Antifungal agent
Concn range
(␮g/ml)
C. albicans C. dubliniensis C. glabrata
SC5314 (type
strain)b
UBC3-7922 (clinical
strain)
CD36/CBS7987
(type strain)c
UBC3-3892 (clinical
strain)
ATCC 90030
(type strain)c
UBC3-7268
(clinical strain)
MIC MIC0.3 MIC MIC0.3 MIC MIC0.3 MIC MIC0.3 MIC MIC0.3 MIC MIC0.3
Haloperidol HCl 6.4 ϫ 10Ϫ3
to 3.76 3.76 0.46 3.76 0.38 3.76 0.38 Ͼ 3.76 0.38 Ͼ3.76 3.76 Ͼ3.76 Ͼ3.76
Trifluperidol 2HCl 7 ϫ 10Ϫ3
to 4.00 4.00 0.40 Ͼ4.00 0.40 Ͼ4.00 Ͼ4.00 Ͼ 4.00 0.40 Ͼ4.00 Ͼ4.00 Ͼ4.00 Ͼ4.00
Stanozolol 3.3 ϫ 10Ϫ3
to 3.29 Ͼ3.29 0.33 Ͼ3.29 0.33 Ͼ3.29 3.29 Ͼ3.29 3.29 Ͼ3.29 Ͼ3.29 Ͼ3.29 3.29
Melengestrol acetate 6.8 ϫ 10Ϫ3
to 3.97 3.97 0.37 3.97 0.40 Ͼ3.97 3.97 Ͼ3.97 1.80 Ͼ3.97 3.97 Ͼ3.97 3.97
Megestrol acetate 6 ϫ 10Ϫ3
to 3.85 3.85 0.39 3.85 0.39 Ͼ3.85 3.85 Ͼ3.85 3.85 Ͼ3.85 3.85 Ͼ3.85 3.85
Tosedostat 4 ϫ 10Ϫ3
to 4.00 Ͼ4.00 4.00 Ͼ4.00 4.00 Ͼ4.00 4.00 Ͼ4.00 4.00 Ͼ4.00 4.00 Ͼ4.00 2.00
Amonafide 2.8 ϫ 10Ϫ3
to 2.83 Ͼ2.83 1.40 Ͼ2.83 2.83 Ͼ2.83 2.83 Ͼ2.83 1.40 Ͼ2.83 Ͼ2.83 Ͼ2.83 Ͼ2.83
Methiothepin maleate 7 ϫ 10Ϫ3
to 3.57 3.30 0.31 3.30 0.36 Ͼ3.57 0.36 Ͼ3.57 0.36 3.57 0.36 3.57 0.36
Auranofin 4 ϫ 10Ϫ3
to 6.78 0.68 0.08 0.61 0.07 0.68 0.04 0.62 0.04 1.10 0.62 Ͼ3.73 3.73
Rapamycin 9 ϫ 10Ϫ3
to 9.14 0.002 Ͻ9 ϫ 10Ϫ3
0.002 Ͻ9 ϫ 10Ϫ3
0.009 Ͻ9 ϫ 10Ϫ3
0.01 Ͻ9 ϫ 10Ϫ3
0.50 0.04 0.09 0.009
a
Candida clinical strains were tested with the 7 identified drugs.
b
MIC, minimal concentration of drug resulting in Ն50% growth inhibition; MIC0.3, minimal concentration of drug resulting in Ն30% growth inhibition. MIC and MIC0.3 were
determined by ATP measurement after 24 h of incubation. The data were analyzed and evaluated from 4 biological replicates in triplicate (n ϭ 4 [3]).
c
The type strains C. dubliniensis CD36/CBS7987 and C. glabrata ATCC 90030 were analyzed in 2 biological replicates in triplicate (n ϭ 2 [3]).
TABLE 5 Nonantifungal drugs with known antifungal activitya
Antifungal agent
This study Previous studies
Reference(s)
Tested concn
(␮g/ml)
Incubation
times (h)
Tested concn
(␮g/ml)
Incubation
times (h)
Rapamycin 1.55 ϫ 10Ϫ5
to 9.14 6 and 24 0.09–100 48 and 72 26, 38
Auranofin 1 ϫ 10Ϫ4
to 6.78 6 and 24 12.5–200 48 27
Methiothepin maleate 7 ϫ 10Ϫ5
to 3.57 6 and 24 64 48 25
Bleomycin sulfate 2.6 ϫ 10Ϫ4
to 15 6 and 24 1.56 6 and 12 40
Disulfiram 5.1 ϫ 10Ϫ5
to 2.97 6 and 24 1–8 24 41
Artemisinin 4.8 ϫ 10Ϫ5
to 2.82 6 and 24 8–50 24 42
Tamoxifen citrate 9.7 ϫ 10Ϫ5
to 5.63 6 and 24 8–32 24 43
a
The tested concentrations of off-target drugs with previously demonstrated antifungal activity used in this study were compared to concentrations used in previous studies with
similar incubation times.
Stylianou et al.

activity, methiothepin maleate, rapamycin, and auranofin, were
included. Since the MICs calculated from the OD and ATP mea-
surements were very similar, we exclusively applied ATP measure-
ment. Rapamycin was effective against all tested strains (MIC,
Ͻ0.1 ␮g/ml). The C. albicans strains SC5314 and clinical isolate
UBC3-7922 were affected by the 10 agents to a similar extent (Ta-
ble 4). A slightly lower level of inhibition was seen for UBC3-7922
than for SC5314. Auranofin was efficient against both C. dublini-
ensis strains tested. Haloperidol, in contrast, inhibited the C. dub-
liniensis type strain (MIC, 3.76 ␮g/ml) but inhibited the UBC3-
3892 isolate less efficiently. Together, the C. dubliniensis strains
were more resistant against the tested agents than the C. albicans
strains. The two C. glabrata strains were affected to an even lower
extent; however, they were inhibited by 6 of the 10 agents tested.
Remarkably, methiothepin maleate reached an MIC of 3.57 ␮g/ml
in both C. glabrata strains (Table 4).
DISCUSSION
Therapy against invasive fungal infections remains a challenge in
health care. Many patients in surgery, intensive care, oncology, or
hematology wards suffer from bloodstream infections caused by
Candida spp. However, the dreary outcomes for severe mycoses
do not stem solely from a lack of efficient antifungal drugs (28).
Disease progression is also determined by the immune status of
the afflicted host. Our screen aimed to identify previously un-
known anti-Candida activities in drugs already in use with known
pharmacokinetics. We believe that this knowledge for a large
amount of available pharmaceutical agents may enable (i) the de-
velopment of new applications as antimycotic therapy for these
types of agents, (ii) a future evaluation of the potential synergistic
effects between these drugs and SADs, and (iii) therapy for pa-
tients with a primary disease, such as cancer, with drugs that have
additional known antifungal activities to reduce the risk of severe
secondary mycosis. Of course, the latter is only possible in cases in
which a choice between several drugs is amenable.
The screening of 844 approved drugs or agents in clinical trials
revealed 26 substances that are active against Candida spp., 7 of
which were newly identified. We showed these antifungal activi-
ties in three independent assays, metabolic activity measurement,
optical densitometry according to EUCAST guidelines, and mi-
croscopy, to validate our screening results. The indicated drugs
have anti-Candida activities at 6 h and 24 h (Table 2). The MICs of
the tested agents against C. albicans SC5314 were determined by
OD and ATP measurements, and the assays correlated well. Lu-
ciferase-based quantification of ATP concentrations recorded the
growth of C. albicans in a similar fashion as the tetrazolium dye
XTT (data not shown). XTT has been widely used by many
groups, including ours, to quantify fungal viability (29, 30),
whereas ATP quantification is more sensitive than the determina-
tion of XTT metabolism, allowing for the use of fewer fungal cells
per assay. This indicates that the determination of metabolic ac-
tivity is a reliable measure of antifungal activity. ATP measure-
ment has, to our knowledge, not been used in antifungal drug
screening; however, it was successfully applied in an S. cerevisiae-
based small-molecule screen published in the PubChem BioAssay
database (31). We confirmed the validity of our screen by identi-
fying the FDA-approved antifungal drugs in the Enzo library (Ta-
ble 3), with the exception of nystatin. In agreement with this find-
ing, nystatin-resistant C. albicans isolates were reported (32–34).
At a concentration of 0.3 to 0.7 ␮g/ml, the SADs and the newly
identified agents showed comparable degrees of inhibition against
C. albicans, such as with fluconazole (MIC, 0.3 ␮g/ml) and halo-
peridol (MIC, 0.38 ␮g/ml). Our focus was to apply drug concen-
trations that are likely to be reached in human therapy, and there-
fore, we used maximal concentrations of 10 ␮M. The blood
plasma peak concentrations (Table 2, Cmax and references) and
the concentrations for C. albicans inhibition of the novel antifun-
gal candidate agents were within a similar range. This suggests that
therapeutic antifungal concentrations for the treatment of sys-
temic mycoses are accomplishable. For the trifluperidol and me-
thiothepin maleate, such data were not obtained.
FIG 1 C. albicans SC5314 challenged for 6 h with novel and control drugs. The
pictures are taken from an IncuCyte microscope with a 20ϫ objective lens; the
scale bar corresponds to 200 ␮m. The drug solvent DMSO (A) and the anti-
septic BzCl (B) correspond to the 100% and 0% growth controls, respectively.
Fluconazole (C) and rapamycin (D) are representative control drugs. Repre-
sentative images from C. albicans SC5314 were treated with haloperidol (E),
trifluperidol (G), tosedostat (F), and amonafide (H). Morphological changes
in C. albicans upon treatment with haloperidol (E) and trifluperidol (G) re-
sembled those caused by fluconazole (C), whereas morphological changes in
C. albicans upon treatment with tosedostat (F) and amonafide (H) resembled
those of rapamycin (D).

To broaden the impact of our study, we included additional
Candida strains. We tested the 7 novel agents with reference
strains and clinical isolates of C. albicans from bloodstream infec-
tions. The C. albicans strains showed similar susceptibilities to-
ward all 7 novel candidate agents, confirming that our findings are
valid beyond common laboratory strains. The C. dubliniensis and
C. glabrata strains were more resistant against treatment with
these agents (Table 4). C. glabrata is known to be less susceptible to
antifungal therapy (14). In agreement with this, C. glabrata was
less susceptible to auranofin than the other two Candida spp. No-
tably, methiothepin maleate and tosedostat were active against
both C. glabrata strains. This possibly suggests a broad anti-Can-
dida activity for methiothepin maleate and tosedostat.
The cellular targets of the identified drugs are known in hu-
mans. The dopaminergic drugs haloperidol and trifluperidol act
on G-protein-coupled receptors (GPCRs) (Table 6) (35, 36). Hal-
operidol has previously been described as a potential antifungal
agent in a S. cerevisiae-based chemical-genetic screen to identify
molecular targets for off-target drugs (21). In this screen, we iden-
tified the derivative drug trifluperidol and demonstrated the anti-
Candida activities of both peridols. GPCRs also exist in Candida
spp.; however, the major cellular pathways targeted by dopami-
nergic drugs are amino acid biosynthesis and metabolism (21).
The antineoplastic agent tosedostat is an aminopeptidase inhibi-
tor (Table 6) (23). The targeted cellular pathways of this drug in
eukaryotic cell lines are amino acid metabolism and reduced ac-
tivity of target of rapamycin (TOR) kinases (37). Remarkably,
rapamycin targets TOR kinases in C. albicans (38), and we re-
corded a very similar morphological effect for tosedostat and
rapamycin (Fig. 1), indicating that the drugs might have the same
target against fungi. Amonafide is a topoisomerase inhibitor (22).
It is therefore likely, though not proven, that amonafide targets
topoisomerases in fungi. Gene knockout of topoisomerase II in S.
cerevisiae resulted in severely attenuated DNA replication (39).
Stanozolol, melengestrol, and megestrol acetate are all pseudoste-
roids, and thus, their molecular targets are less predictable (Ta-
ble 6).
In summary, three independent assays identified 7 novel drugs
with dosage-dependent activities against C. albicans with MICs of
Յ4 ␮g/ml. This study conclusively adds a new pharmacological
approach to these drugs, and more importantly, it may help phy-
sicians select antineoplastic therapeutics with the antifungal activ-
ities identified here for groups of patients that are at high risk for
acquiring invasive candidiasis.
ACKNOWLEDGMENTS
This work was supported by grants to C.F.U. from the Swedish Research
Council VR-M (grant no. K2012-99X-21961-01-3), the Laboratory for
Molecular Medicine Sweden (MIMS), the Medical Faculty Umeå (grant
no. 316-886-10), and the Cancer Research Foundation in Northern Swe-
den (grant no. AMP 11-684).
Personnel at the High-Throughput Biomedicine Unit (FIMM Tech-
nology Centre, Helsinki, Finland) are acknowledged for their expert tech-
nical support. We thank Laura Turunen for her excellent assistance with
drugging compounds. Research infrastructure support was provided by
Biocenter Finland.
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