Amino acid-derived 1,2-benzisothiazolinone derivatives as novel small-molecule antifungal inhibitors: identification of potential genetic targets.

Abstract

We have identified four synthetic compounds (DFD-VI-15, BD-I-186, DFD-V-49, and DFD-V-66) from an amino acid-derived 1,2-benzisothiazolinone (BZT) scaffold that have reasonable MIC(50) values against a panel of fungal pathogens. These compounds have no structural similarity to existing antifungal drugs. Three of the four compounds have fungicidal activity against Candida spp., Cryptococcus neoformans, and several dermatophytes, while one is fungicidal to Aspergillus fumigatus. The kill rates of our compounds are equal to those in clinical usage. The BZT compounds remain active against azole-, polyene-, and micafungin-resistant strains of Candida spp. A genetics-based approach, along with phenotype analysis, was used to begin mode of action (MOA) studies of one of these compounds, DFD-VI-15. The genetics-based screen utilized a homozygous deletion collection of approximately 4,700 Saccharomyces cerevisiae mutants. We identified mutants that are both hypersensitive and resistant. Using FunSpec, the hypersensitive mutants and a resistant ace2 mutant clustered within a category of genes related directly or indirectly to mitochondrial functions. In Candida albicans, the functions of the Ace2p transcription factor include the regulation of glycolysis. Our model is that DFD-VI-15 targets a respiratory pathway that limits energy production. Supporting this hypothesis are phenotypic data indicating that DFD-VI-15 causes increased cell-reactive oxidants (ROS) and a decrease in mitochondrial membrane potential. Also, the same compound has activity when cells are grown in a medium containing glycerol (mitochondrial substrate) but is much less active when cells are grown anaerobically.

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Published Ahead of Print 11 June 2012.
10.1128/AAC.00477-12.
2012, 56(9):4630. DOI:
Antimicrob. Agents Chemother.
and Richard Calderone
Thampi, Dengfeng Dou, Aileen Mooney, William Groutas
Deepu Alex, Francoise Gay-Andrieu, Jared May, Linta
Identification of Potential Genetic Targets
Novel Small-Molecule Antifungal Inhibitors:
1,2-Benzisothiazolinone Derivatives as
Amino Acid-Derived
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Amino Acid-Derived 1,2-Benzisothiazolinone Derivatives as Novel
Small-Molecule Antifungal Inhibitors: Identification of Potential
Genetic Targets
Deepu Alex,aFrancoise Gay-Andrieu,a,cJared May,aLinta Thampi,aDengfeng Dou,bAileen Mooney,aWilliam Groutas,band
Richard Calderonea
Georgetown University Medical Center, Washington, DC, USAa; Wichita State University, Department of Chemistry, Wichita, Kansas, USAb; and Nantes Atlantique
Universities, EA1155-IICiMed, Nantes, Francec
We have identified four synthetic compounds (DFD-VI-15, BD-I-186, DFD-V-49, and DFD-V-66) from an amino acid-de-
rived 1,2-benzisothiazolinone (BZT) scaffold that have reasonable MIC50values against a panel of fungal pathogens. These
compounds have no structural similarity to existing antifungal drugs. Three of the four compounds have fungicidal activ-
ity against Candida spp., Cryptococcus neoformans, and several dermatophytes, while one is fungicidal to Aspergillus fu-
migatus. The kill rates of our compounds are equal to those in clinical usage. The BZT compounds remain active against
azole-, polyene-, and micafungin-resistant strains of Candida spp. A genetics-based approach, along with phenotype analy-
sis, was used to begin mode of action (MOA) studies of one of these compounds, DFD-VI-15. The genetics-based screen
utilized a homozygous deletion collection of approximately 4,700 Saccharomyces cerevisiae mutants. We identified mu-
tants that are both hypersensitive and resistant. Using FunSpec, the hypersensitive mutants and a resistant ace2 mutant
clustered within a category of genes related directly or indirectly to mitochondrial functions. In Candida albicans, the
functions of the Ace2p transcription factor include the regulation of glycolysis. Our model is that DFD-VI-15 targets a re-
spiratory pathway that limits energy production. Supporting this hypothesis are phenotypic data indicating that DFD-
VI-15 causes increased cell-reactive oxidants (ROS) and a decrease in mitochondrial membrane potential. Also, the same
compound has activity when cells are grown in a medium containing glycerol (mitochondrial substrate) but is much less
active when cells are grown anaerobically.
C
oropharyngeal (OPC) diseases occur at high frequencies in hu-
mans (20, 48, 53). As for other fungal infections, Aspergillus fu-
migatus is one of the most common causes of mold infections of
humans (48). For these and other fungal infections, the cost-bur-
den analysis is estimated to be ?$2.6 billion per year (21, 36, 37,
44, 60). Fungal infections are also of global importance. Oropha-
ryngeal candidiasis (OPC) in the HIV/AIDS patient is a heavy
health burden in countries of Africa as well as in India and China
(7,11,12,29,43,62).CryptococcalmeningitisinHIV/AIDSpop-
ulations exceeds that of other kinds of bacterial meningitis, in-
cludingtuberculosis,andisamongthemostreportedinfectionsin
HIV/AIDSpatientsindevelopingcountries(42,43,58).Theinci-
dence of these diseases is a compelling reason to continue efforts
into developing new preventative therapies.
Antifungaldrugsthatarecurrentlyusedinpatienttreatmentof
candidiasis include the polyene amphotericin B (AmpB), azoles,
andtheechinocandins(45).Thesecompoundsbindtomembrane
ergosterol (AmpB) and perturb membrane functions, inhibit er-
gosterol synthesis (azoles), or inhibit cell wall ?-1,3-glucan syn-
thesis(echinocandins).Theuseofazolesandespeciallytriazolesis
not without problems, as resistance to fluconazole has resulted in
an increase in several non-albicans species of Candida, such as C.
parapsilosis, C. glabrata, and C. krusei, as well as A. fumigatus, as
pathogens (23, 32, 34, 47–49, 54, 59).
It is quite clear that progress toward the identification of new
classesofantifungalshasbeenslow.Newertrizolesareremodeled
existingdrugs.Thisissimilartothesituationinantibacterialdrug
andidaspeciesrankfourthamongthecausesofbacterial/fun-
gal nosocomial infectious diseases, and Candida vaginitis and
development, such that phrases like “bad bugs, no drugs” now
reflectthecurrentscenariowherepathogensresistanttonearlyall
availableantibacterialdrugsarebeingencounteredinbothhospi-
tal and community settings (8).
We have identified a series of 4-amino-acid-derived-1,2-
benzisothiazolinone (BZT) derivatives that have broad activity
against diverse fungal pathogens (18) (see Fig. S1 in the supple-
mentalmaterial).Thecurrentdatainthispaperfurtherdefinethe
activityofthesecompoundsandsuggestamodeofaction(MOA)
for one of the active compounds.
MATERIALS AND METHODS
Compound library. A series of four broad-spectrum antifungal agents
based on the 1,2-benzisothiazol-3(2H)-one (BZT) scaffold were used in
this study. We have previously reported their structures, shown in Fig. S1
in the supplemental material (18).
Strains. For MIC50screening experiments with each of the four com-
pounds, we used Candida albicans (CAF2 and SC5314) as well as C.
glabrata (RC-201), Candida lusitaniae (RC-301), Candida guilliermondii
Received 1 March 2012 Returned for modification 20 April 2012
Accepted 6 June 2012
Published ahead of print 11 June 2012
Address correspondence to Richard Calderone, calderor@georgetown.edu.
D.A. and F.G.-A. contributed equally to the manuscript.
Supplemental material for this article may be found at http://aac.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.00477-12
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(RC-401), Candida tropicalis (RC-501), C. parapsilosis (RC-601), and
Candida apicola (RC-701), A. fumigatus (AF-294), Cryptococcus neofor-
mans strains (H-99 and JEC-21), Trichophyton rubrum (22403), Tricho-
phyton mentagrophytes (22402), Epidermophyton floccosum (1252), and
Microsporum canis (22349). S. cerevisiae BY4743 was used in phenotype
profilingagainstoneofthefourcompounds(DFD-VI-15).TheRCseries
of strains was obtained from the Microbiology and Immunology Labora-
tory of the Georgetown University-MedStar Hospital, Washington, DC.
MIC susceptibility assays also included strains that were resistant to flu-
conazole, micafungin, or amphotericin B (see below).
MIC determinations (broth microdilution method) and growth
curves. The MIC50of all strains was determined by following the
guidelines in CLSI standards M27-A3 (14) and M38-A2 (15). A 50%
growth inhibition was used as an endpoint. A total of 1,000 cells in
RPMI were added to each well of 96-well microtiter plates to a final
volume of 200 ?l of RPMI. All assays were read after 24 h for Candida
spp., 48 h for A. fumigatus, 72 h for C. neoformans, and 5 to 7 days for
dermatophyte cultures. The dermatophytes were initially grown on
Sabouraud agar and then screened according to the method described
above. Growth curves were plotted by measuring the optical density at
595 nm (OD595) of the test plate wells every 30 min for 48 h in treated
and untreated cultures.
MFC determinations. The in vitro minimum fungicidal concentra-
tions (MFC) were determined by subculturing 10 ?l of a 103cell sus-
pension with or without compounds from each microtiter plate well
on YPD agar plates (1% yeast extract, 2% peptone, 2% glucose). How-
ever, to rule out low-cell-density effects that could influence MFC
determinations, we also used the entire inoculum of 1,000 cells with
compounds. Concentrations in wells that showed complete inhibition
in the MIC assays as well as the last well that indicated growth were
determined. Cultures were incubated at 30°C for 48 h. The MFC was
defined as the lowest drug concentration that resulted either in no
growth or in fewer than five colonies. If the ratio of the MFC50to the
MIC50concentration was ?4, the compound was designated fungi-
cidal, and if higher, fungistatic (24).
Time-kill assays. C. albicans CAF2 cells from log growth phase were
usedataconcentrationof104cells/mlin10mlofRPMIwithandwithout
compounds.Atotalof100?lofcellsampleswastakenaftermixingevery
hourfor14handplatedonYPDagarfor24to48hat30°C.Theviablecell
concentrationwascalculatedfromthenumberofCFUafter48h(10).The
maximumkillratewascalculatedatthe6-to8-htimeperiodpointwhich
represents the average time-kill rate.
Toxicity assays. Cell viability was measured by neutral red and MTT
(dimethyl diphenyl tetrazolium) assays in tissue culture with each of the
four BZT compounds at defined concentrations in the human hepatoma
cell line HepG2, as described by Mosmann et al. (40) and Repetto et al.
(50), respectively. The 50% cell cytotoxicity (CC50) concentrations were
calculated for each compound after 24, 48, and 72 h of incubation. Data
are indicated as CC50(?g/ml per compound). CC50/MIC50ratios were
calculated to determine fold changes in toxicity.
Synergy experiments. In vitro synergy interactions between the BZT
compoundsandcurrentantifungalssuchasmicafungin,fluconazole,and
amphotericin B were determined. C. albicans CAF2 was screened using a
checkerboardassaymethoddescribedbyothers(4,26,55).Thefractional
inhibitory concentration index (FICI) value of each pair of compounds
wascalculatedandclassifiedasfollows:antagonism,?2;additive,0.5to2;
andsynergy,?0.5.Interactionsbetweenthefourcompoundsandknown
antifungals were determined.
S. cerevisiae screens for hypersensitive mutants with DFD-VI-15.
We used the YSC Homozygous Diploid library (YSC1056; Thermo
Scientific) in susceptibility assays against DFD-VI-15. The library con-
tains ?4,700 null mutants in nonessential genes in 96-well microtiter
plates. Individual deletion strains (5 ?l) were transferred from frozen
stocks to 200 ?l of YPD (10 g yeast extract, 20 g peptone, and 20 g
glucose per liter) for overnight growth. Then cultures were transferred
to fresh YPD in 96-well plates for dilutions to achieve a 1:40 dilution of
cells. Following this, each mutant was printed (pin replicator) onto
YPD agar plates (150-mm diameter) with or without compound (6
?g/ml) (see Fig. S2 in the supplemental material). We chose a concen-
tration that yielded approximately 1 to 5% mutants per 96-well plate
with a hypersensitive phenotype, according to previously published
protocolandpreliminarydata(5).Thegrowthofeachdeletionmutant
was determined at 48 h and 72 h and compared to that of the same
TABLE 1 MIC50s of the BZT compounds against human pathogenic fungia
MIC50(?g/ml)
Strain DFD-VI-15 BD-I-186DFD-V-49DFD-V-66 FluconazoleMicafungin AmpB
C. albicans CAF2
C. glabrata RC-201
C. tropicalis RC-501
C. parapsilosis RC-601
C. lusitaniae RC-301
C. guilliermondii RC-401
C. apicola RC-701
A. fumigatus AF-294
C. neoformans H99
C. neoformans JEC-21
1.6
1.6
1.6
1.6
1.6
1.6
1.6
12.5
1.6
1.6
1.6
0.8
1.6
0.8
0.8
1.6
3.2
6.4
1.6
0.8
1.6
3.2
3.2
3.2
1.6
3.2
3.2
6.4
6.4
6.4
1.6
1.6
3.2
3.2
3.2
3.2
3.2
3.2
3.2
1.6
0.2
3.2
16
0.4
3.2
6.4
0.2
16
?64
?64
0.016
0.016
0.016
0.5
0.03
0.5
0.016
1.0
>2
>2
0.25
0.25
0.5
0.25
0.25
0.25
0.25
0.5
>10
>10
aMIC50s (?g/ml) of the BZT compounds are shown against a panel of human pathogenic fungi. The antifungal activities of the compounds were also compared to those of
fluconazole, micafungin, and amphotericin B (AmpB) against the same strains. Results for fungicidal compounds are indicated in bold numbers. (Fungicidal, MFC/MIC50?4;
fungistatic, MFC/MIC50?4.) Data are shown as averages of results from five experiments.
TABLE 2 MIC50s of the BZT compounds against species of
dermatophytic fungia
Strain
MIC50(?g/ml)
DFD-
VI-15
BD-
I-186
DFD-
V-66
DFD-
V-49Ketoconazole
Trichophyton rubrum
(22403)
Trichophyton mentagrophytes
(22402)
Epidermophyton floccosum
(1252)
Microsporum canis (22349)
3.2
3.2
3.23.2
0.5
3.23.2 3.2 3.2
0.25
3.20.8
3.2
12
0.13
0.8
0.83.2
3.20.5
aThe activities of all compounds were compared to that of ketoconazole. Results for
fungicidal compounds are shown in bold numbers as in Table 1. All data are averages of
results from three experiments.
Benzisothiazolinone as Antifungals
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mutant grown in the absence of compound. Mutants that displayed
hypersensitivity after 48 h and confirmed after 72 h were considered
hypersensitive. These experiments were done three times with the en-
tire yeast library. Hypersensitive mutants were identified, and a sec-
ondary screen was done with the hypersensitive mutants using the
MIC microtiter dilution method but in YPD broth, since S. cerevisiae
BY4743 grows less in RPMI broth. This screen was used to rule out
falsepositivesthatmayhaveoccurredduringtheprimaryscreensofall
mutants. As all mutants are coded, we identified strains and their
corresponding deleted genes that resulted in either hypersensitivity or
resistance to DFD-VI-15 (see below). Screens included the parental
strain S. cerevisiae BY4743.
FIG 1 The growth curves of C. albicans CAF2 in untreated (CAF2 only) and DFD-VI-15-treated cultures are shown. A concentration-dependent inhibition is
observed. The OD595at 24 h for 1.6 ?g/ml (MIC50) of DFD-VI-15 is approximately half that of untreated cells. Complete inhibition of growth was seen at 6.4
?g/ml (?4? MIC50). Similar profiles were observed for the other BZT compounds.
FIG2 Time-killexperimentswithDFD-VI-15.Dataareshownaslog10viablecells/ml.CellcountsareshownfordifferentconcentrationsofDFD-VI-15against
C. albicans CAF2. The maximum kill concentration was 3.2 to 6.4 ?g/ml after 24 h of incubation with DFD-VI-15.
Alex et al.
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S. cerevisiae screens for resistant mutants with DFD-VI-15. The
samemethodologyforidentifyinghypersensitivemutantswasusedfor
resistant mutants (described above), except that solid agar plates were
replaced by 96-well plates with YPD broth containing 15 ?g/ml of
DFD-VI-15. This concentration was fungicidal for S. cerevisiae
BY4743. Growth was determined at 24 and 48 h, and the resistant
mutants were identified visually as those that grew in the presence of
DFD-VI-15. The screen was performed in triplicate with fresh liquid
cultures. The MIC50of the resistant mutants was determined in YPD
medium. Mutants resistant to 12 to 24 ?g/ml were further analyzed
phenotypically as described below.
Data analysis. FunSpec (51) was used to identify the functional
categoriesthatwererepresentedbythemutantswhoseabsencerenders
cells hypersensitive or resistant to DFD-VI-15. The corresponding list
ofthegeneswhoseabsenceresultedinthesephenotypestoDFD-VI-15
were imported into FunSpec, and, based on prior knowledge, data
were integrated in the Munich Information Center for Protein Se-
quence (MIPS) functional categories. Cell functions were searched
using the Saccharomyces Genome Database (SGD) Gene Ontology
(GO) Term Finder. The P values in FunSpec represent the probability
that the intersection of a given list with any functional category occurs
by chance. The Bonferroni correction divides the P value threshold
that would be deemed significant for an individual test by the number
of tests conducted and thus accounts for spurious significance due to
multiple testing over the categories of a database. After the Bonferroni
correction, the only categories displayed are those for which the
chance probability of enrichment is lower than P/CD, where CD is the
number of categories in the selected database.
Drop plates of CAF2 and BY4743 with DFD-VI-15 under aerobic
andanaerobicconditions.ThesensitivitiesofC.albicansstrainCAF2and
S. cerevisiae strain BY4743 to DFD-VI-15 were tested by plating serial
dilutionsof5?101to5?106cells(eachinatotalof5?l)ontoYPDagar
plates containing 12 (S. cerevisiae) or 24 (C. albicans) ?g/ml of the com-
pound. Yeast cells were obtained from overnight cultures grown in YPD
brothat30°Candstandardizedbyhemocytometercounts.Thegrowthof
each strain was evaluated for sensitivity after 48 h of incubation at 35°C.
Strains were also evaluated for their growth on YPG (yeast extract-pep-
tone-glycerol) agar containing 1% yeast, 2% peptone, and 2% glycerol
and in anaerobic conditions (using Anaerobic Packs) with and without
DFD-VI-15.
Flow cytometry assays. (i) Intracellular ROS. Reactive oxidant spe-
cies (ROS) production was determined by staining cells with the fluores-
cent dye DCFDA (2=-7=-dichlorofluorescein diacetate; Sigma) using a
FACScanflowcytometer(488nm;Becton,Dickinson)(3,33).C.albicans
SC5314 cells were grown at 30°C overnight in YPD medium. After 16 h,
the pellets were collected and suspended to 1 ? 106cells in 15 ml of YPD
andseparatedintothreetubes.Twoofthetubesweretreatedwith12or24
?g/mlofDFD-VI-15for4h,sincemorecellswereneededfortheseassays.
The third tube was used as the untreated control. After 4 h, cells were
collectedfromeachconditionandwashedtwicewithphosphate-buffered
saline (PBS). The pellets were suspended to 1 ? 106cells in 1 ml of PBS
andtreatedwithorwithout25?MDCFDAfor30minat30°Cinthedark.
Cell fluorescence in the absence of DCFDA was used to verify that back-
ground fluorescence was similar for treated and untreated strains. Cells
thatweretreatedwithDCFDAwerethencollectedandwashedtwicewith
PBS. Then propidium iodide (PI) was added to each sample to exclude
deadcellspriortotheDCDFAassays.ThemeanfluorescenceforROSwas
quantified only in live and similar-sized cells.
(ii) Mitochondrial membrane potential. Mitochondrial membrane
potential was detected by staining cells with JC-1 (Cayman Chemical
Company), a cationic dye used to assess variation in mitochondrial po-
tential, using a FACScan flow cytometer (488 nm; Becton, Dickinson).
JC1monomersdisplayagreencytoplasmicfluorescenceat525nm,when
cells are excited at 490 nm. In cells with physiological mitochondrial
membranepotential,JC-1formscomplexesknownasJC1aggregateswith
intenseredfluorescence.Incellswithdysfunctionalmitochondrialmem-
brane potential, JC-1 remains in a monomeric form and displays a green
fluorescence. C. albicans SC5314 cells that had been grown overnight at
30°C were treated with 12 and 24 ?g/ml of DFD-VI-15 for 4 h. After
washing, cells were treated with JC-1 according to the manufacturer’s
recommendations (33). JC-1 fluorescence was quantified only in live and
standard-sized cells by adding 7-amino-actinomycin D (7-AAD) to ex-
clude dead cells in assays. JC1 fluorescence at 488 nm was collected in the
TABLE 3 Maximum kill rates (KRmax) against C. albicans CAF2a
Compound KRmax(per h)b
0.85 ? 0.05
0.75 ? 0.07
0.80 ? 0.03
0.67 ? 0.08
0.25 ? 0.10
0.90 ? 0.04
0.80 ? 0.08
DFD-VI-15
BD-I-186
DFD-V-66
DFD-V-49
Fluconazole
Micafungin
Amphotericin B
aThe maximum kill rates for DFD-VI-15, BD-I-186, DFD-V-66, DFD-V-49,
fluconazole, micafungin, and amphotericin B against C. albicans CAF2 were compared.
The BZT derivatives have comparable maximum kill rates to amphotericin B and
micafungin but those rates are significantly higher than that to fluconazole. Greater kill
rates are indicated by higher KRmaxvalues.
bKRmax(per h) data are averages of results from three experiments.
TABLE 4 Activity (MIC50) of BZT compounds against azole-resistant strains of C. albicansa
StrainMechanism of resistanceb
MIC50of BZT
compounds
MIC50of
fluconazole
Source or
referencec
CAF2/SC5314
95-68
FH5
96-25
C17 (12–99)
DSY1764
DSY296
B4
CAAL-61
CAAL-74
CAAL-75
Sensitive
CDR overexpression
CDR overexpression
MDR ? ERG11 overexpression
CDR ? MDR ? ERG11 overexpression
Erg11/erg11erg3/erg3
TAC1-5/TAC1-5 ERG11-5/ERG11-5
Mrr1p mutation (G878E/G878E)
AA G307S, Y447H
AA Y132F, G448V
AA Y132H, K143R
1.6
1.6–3.2
1.6
1.6
1.6–3.2
1.6–3.2
1.6–3.2
1.6–3.2
1.6–3.2
1.6–3.2
1.6–3.2
0.2
?64
64
32
?64
?128
?64
12.5
?64
?64
?64
This paper
35, 59
35, 59
35, 59
35, 59
16, 17
16, 17
39
38
38
38
aStrain numbers, mechanisms of resistance, and MIC50values are shown. The MIC50values reflect the range of concentrations for all BZT compounds against all strains.
bAA, amino acid point mutants of erg11.
cReferences describe the source and/or resistance mechanisms.
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FL1channel(greenfluorescence)andintheFL2channel(orangefluores-
cence).
RESULTS
MIC determinations using the broth microdilution method. In
the course of screening for antifungal compounds in accordance
with the guidelines of the CLSI standards M27-A3 and M38-A2
(broth microdilution method), four synthetic compounds be-
longing to the benzisothiazolinone (BZT) scaffold were discov-
ered to have potent antifungal activity (see Fig. S1 in the supple-
mental material). These compounds have no structural similarity
to known antifungal compounds. The compounds (DFD-VI-15,
BD-I-186, DFD-V-49, and DFD-V-66) show antifungal activity
against Candida spp., A. fumigatus, C. neoformans, and four der-
matophyte species at MIC50concentrations in the range of 0.8 to
12.5 ?g/ml (Tables 1 and 2). Of the four BZT compounds, DFD-
VI-15 and BD-I-186 have relatively better activity against all test
organisms (0.8 to 3.2 ?g/ml) except A. fumigatus. Candida spp.,
such as C. glabrata and C. parapsilosis, that often are resistant to
triazoles such as fluconazole are susceptible to the BZT com-
pounds.ForallspeciestestedexceptA.fumigatus,thecompounds
are fungicidal, while only DFD-V-66 displayed fungicidal activity
against A. fumigatus (Table 1). Fungicidal activity is defined as an
MFC50?4-fold the MIC50(24). Likewise, the BZT compounds
are more often fungicidal against four dermatophytic fungi (Ta-
ble 2).
Growthcurvesandtime-killexperiments.GrowthofC.albi-
cans CAF2 was measured for 48 h in the presence or absence of
DFD-VI-15 at concentrations ranging from 0 to 6.4 ?g/ml
(Fig. 1). Growth was inhibited in a concentration-dependent
manner for DFD-VI-15 and the other BZT compounds to a sim-
ilar extent (data not shown for other BZT compounds). To mea-
sureviabilityinthepresenceofcompounds,time-killexperiments
weredone.Thetime-killrateisdefinedasthereductionincolony
count(log10)dividedbyexposuretimecomparedtothegrowthof
untreated cultures (Fig. 2). Viable cell counts were determined at
2-hintervals,andthe6-to8-htimepointwasusedtocalculatekill
rates of C. albicans CAF2 at different concentrations of DFD-VI-
15.FungicidalactivityofDFD-VI-15wasnotedinitiallyat2to4h
with 3.2 to 6.4 ?g/ml and then increased to a maximum kill at 12
h at both concentrations. We estimate the fungicidal concentra-
tiontobe3.2to6.4?g/ml.WhileonlyDFD-VI-15dataareshown,
the other BZT compounds had similar killing profiles (data not
shown).
ThemaximumkillratesofDFD-VI-15,BD-I-186,DFD-V-66,
and DFD-V-49 were compared to those of other antifungal drugs
and found to be similar to those of amphotericin B and micafun-
gin while greater than that of fluconazole (Table 3).
Activity against drug-resistant strains. Although the BZT
compounds were active against a panel of fungal pathogens de-
scribed above, we next screened the BZT compounds against a
varietyofclinicalorlaboratory-constructedantifungaldrug-resis-
tantstrainsofC.albicansandC.glabrataand,whenpossible,their
parental strains. Strains were selected to represent some of the
mechanisms that are commonly found in clinically resistant iso-
lates(10,16,17,19,22,34,38,39,46,56,59)(Tables4and5).The
MIC50valuesofallBZTcompoundsremainedunchangedagainst
C. albicans strains resistant to fluconazole. Similar results were
foundwhenthecompoundsweretestedagainstmicafungin-resis-
tantisolatesofC.albicansandC.glabrataandasingleisolateofC.
glabrata resistant to amphotericin B (Table 5). We suggest that
since these strains were resistant to fluconazole, micafungin, or
amphotericin B, their sensitivity may indicate unique targets for
the BZT compounds.
Synergy experiments can partially resolve common or unique
targetsofunknowncompounds.Toaddressthisissueandthedata
from Tables 4 and 5, synergy experiments using the standard
checkerboard method were done. The BZT compounds were
tested in combination with fluconazole, micafungin, or ampho-
tericin B against C. albicans CAF2. Fractional inhibitory concen-
tration index values (FICI) are indicated in Table 6. Two of the
four compounds, BD-I-186 and DFD-V-49, synergized with mi-
cafungin (?-1,3-glucan synthesis inhibitor) and amphotericin B
(membrane ergosterol), respectively. Synergy may indicate that
each compound of a pair of compounds inhibits a different target
to increase the activity of one compound.
Toxicity studies. We compared each of the four BZT com-
TABLE 5 Activity (MIC50) of BZT compounds against micafungin- and
amphotericin-resistant strains of C. albicans and C. glabrataa
Strainb
Mechanism of
resistancec
MIC50of
BZT
compounds
(?g/ml)
MIC50of
micafungin/
AmB
(?g/ml)
Source or
referenced
C. albicans
ATCC
90028
DSP11
DSP12
DSP14
DSP15
1.6 0.016/0.25This paper
AA (FKS1) S645F
AA (FKS1) S645Y
AA (FKS1) F641S
AA (FKS1) S645P
3.2
3.2
3.2
3.2
2
2
?2
2
22, 46
22, 46
22, 46
22, 46
C. glabratae
RC-201
DSP16
DSP17
DSP18
DSP19
DSP20
0.8–3.2
1.6–3.2
1.6–3.2
0.8–3.2
1.6–3.2
1.6–3.2
0.016/0.25
1
?2
?2
?2
1
This paper
22, 46
22, 46
22, 46
22, 46
22, 46
AA (FKS2) S663P
AA (FKS1) S645P
AA (FKS1) F659V
AA (FKS2) S663P
AA (FKS1) D666E
aStrain numbers, mechanisms of resistance, and MIC50values are shown. The MIC50
values reflect the range of MIC50values for all BZT compounds against all strains.
bDSP 11–15, C. albicans strains. DSP 16–20 and strain 21230, C. glabrata strains.
cAA, point mutants of FKS1 or FKS2.
dReferences that describe the source and/or resistance mechanisms are included.
eC. glabrata 21230 was also resistant to amphotericin B (56 and data not shown).
TABLE 6 Results of checkerboard experimentsa
Compound
Interaction type (FICI)
Fluconazole Micafungin Amphotericin B
DFD-VI-15
BD-I-186
DFD-V-66
DFD-V-49
Additive (1.0)
Additive (1.5)
Additive (1.2)
Additive (1.0)
Additive (0.5)
Synergy (0.3)
Additive (0.8)
Additive (1.0)
Additive (0.6)
Additive (1.3)
Additive (1.0)
Synergy (0.25)
aCheckerboard experiments identify synergy. The in vitro interactions of the active
compounds with fluconazole, micafungin, and amphotericin B were evaluated against
those for C. albicans CAF2. The FICI (fractional inhibitory concentration) values are
calculated, and the interactions are grouped. BD-I-186 has a synergistic interaction with
micafungin, and DFD-V-49 has a synergistic interaction with amphotericin B. All other
interactions were additive. No antagonistic interactions were observed. See text for
definitions.
Alex et al.
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pounds for toxicity in vitro against the HepG2 human hepatocar-
cinomacellline(Fig.3).TheneutralredandMTTassaysmeasure
the viability of cells by incorporation of dye in lysosomes (neutral
red) or mitochondria (MTT), respectively. Dose-response curves
were done for each compound at 24, 48, and 72 h, and the con-
centration that resulted in 50% cell growth inhibition was deter-
mined. Of the four BZT compounds, DFD-VI-15 displayed the
most significant toxicity at ?4- to 10-fold higher than the MIC50
forthatcompound,whileBD-I-186andDFD-V-49hadCC50val-
ues of 100 ?g/ml, or about 60-fold of the MIC50of each com-
pound (CC50/MIC50ratios). Less toxicity was seen with DFD-
V-66 than with DFD-VI-15.
Identification of S. cerevisiae mutants sensitive to DFD-VI-
15. DFD-VI-15-sensitive strains were identified by screening the
BY4743 S. cerevisiae parental strain (sensitive to DFD-VI-15;
MIC50, 6 ?g/ml) and a set of 4,700 nonessential yeast gene null
mutants with 6 ?g/ml of compound. In these assay conditions,
hypersensitivemutantsonagarplateassaysappearat1to5%.Two
representative agar plate assays of ?96 mutants are shown in Fig.
S2 in the supplemental material. Also, these concentrations were
chosen for the screen since preliminary studies indicated that
about 1 to 5% of mutants were hypersensitive, an amount re-
ported by others (5). The entire library screen was done three
times on YPD agar with and without compound. Strains were
labeled sensitive to DFD-VI-15 if growth inhibition occurred
from three mutant library screens. This analysis identified 96
strains whose gene deletion caused sensitivity to DFD-VI-15. Mi-
crotiterplatedilutionassayswereusedtoverifythehypersensitiv-
FIG 3 Cell cytotoxicity of DFD-VI-15, BD-I-186, DFD-V-66, and DFD-V-49 against the human HepG2 cell line. Viability assays were used to determine the
CC50concentrationwithneutralred(upper)andMTT(lower)stains.Assayswereperformedat24,48,and72h.TheCC50sforBD-I-186andDFD-V-49are100
?g/ml, or about 50- to 60-fold higher than the MIC50, for each compound. For DFD-VI-15 and DFD-V-66, the CC50s were 10- to 20-fold and 30- to 50-fold
greater, respectively.
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ity of mutants selected on agar plate assays. Of the 96 hypersensi-
tive mutants representing 85 annotated genes, 31 mutants were
verifiedtohaveMIC50valueslessthanthatofS.cerevisiaeBY4743,
?6?g/ml.Elevenofthesemutants,asdefinedbyGOannotation,
fall into a cluster represented by direct or indirect loss of mito-
chondrial functions (P ? 0.05) (Table 7). The second largest cat-
egory of mutants was deleted in genes encoding amino acid syn-
thesis functions.
Identification of S. cerevisiae mutants resistant to DFD-VI-
15. DFD-VI-15-resistant strains were identified by screening
the same mutant collection with 15 ?g/ml of compound, fol-
lowed by a second screen in YPD broth containing 6 to 48
?g/ml. Two replicates of the entire library screen were done in
96-well microtiter plates as stated above. Three mutants
showed resistance at 48 h (data not shown). The three mutants
had deletions of gene CNE1, HLJ1 (both 12 ?g/ml), or ACE2
(24 ?g/ml). Hlj1p is a cochaperone for Hsp40p and promotes
endoplasmic reticulum (ER)-associated degradation of inte-
gral membrane proteins. Cne1p is an integral ER membrane
protein that is part of the quality control of glycoprotein fold-
ing (FunSpec). The Ace2p transcription factor is required for
many activities in S. cerevisiae, C. glabrata, and C. albicans,
including polarized growth, virulence, and morphogenesis
and, interestingly, is a positive regulator of glycolysis (6, 30, 31,
41). We suggest that the hypersensitive and resistant mutants
may reflect respiratory dysfunction in treated cells; to demon-
strate this, the following experiments were done.
Dropplateexperiments.Thehypersensitiveandresistantmu-
tantsofS.cerevisiaesuggestthatDFD-VI-15mayhaveactivitythat
results in detrimental energy loss. To determine if this is correct,
three types of phenotypic profiling analyses were pursued in
strains treated with this compound. On YPD (glucose; glycolytic
respiration) as well as YPG (glycerol; mitochondrial respiration)
agar, cell growth was measured using drop plate assays in the
presenceorabsenceofcompound.Wefoundthatparentalstrains
CAF2(C.albicans)(Fig.4,upperpanel)andBY4743(S.cerevisiae)
(Fig. 4, lower panel) were inhibited by DFD-VI-15 in aerobic cul-
tures containing glucose (YPD) but also on glycerol (YPG), a mi-
tochondrial substrate. Under anaerobic conditions on YPD agar,
compoundinhibitionwasnotobservedwithS.cerevisiaeBY4743,
whileC.albicansCAF2waslessaffectedthanS.cerevisiaeBY4743,
which was inhibited.
ROSproductionisincreasedinCAF2cellstreatedwithDFD-
VI-15. Mitochondrial dysfunction is associated with an in-
crease in cell ROS (33). To assess the levels of ROS in untreated
and treated cells, we used an inoculum of 106cells/ml, com-
pared to the amount in MIC assays (103cells/well). Moreover,
preliminary experiments were performed to check the cell via-
bility during the assay with various concentrations of DCFDA.
The flow cytometry analysis is performed only on viable cells,
with propidium iodide detecting dead cells. Quantitative flow
cytometry experiments were done to measure the total amount
of intracellular ROS following treatment with DFD-VI-15. The
DCFDA stain diffuses into the cell, is hydrolyzed into 2=,7=-
dichlorofluorescein (DCFH) by esterases, and accumulates in
viable cells. Treated cells had significantly higher levels of ROS
than untreated cells (Table 8). GOA31, a C. albicans mutant
strain with defects in mitochondrial function and higher ROS
levels, was used as a positive-control strain to measure ROS
levels and mitochondrial potential (below) as we have demon-
strated previously (3, 33).
DFD-VI-15inhibitsmitochondrialmembranepotential.We
demonstratethatmitochondrialmembranepotentialdecreasesin
C. albicans in the presence of DFD-VI-15 in a dose-dependent
manner (Fig. 5). We were unable to measure a membrane poten-
tial in untreated or treated cultures of S. cerevisiae (data not
shown). These data indicate that DFD-VI-15 is at least partially
directed against mitochondrial functions.
DISCUSSION
The novelty of our studies starts first with the structures of active
compounds,heretoforenotreported(18).Further,fromourcur-
rent data, we have established that compounds with a BZT core
structure have antifungal activity against a variety of human
pathogen isolates, including dermatophytic fungi (Tables 1 and
2).Thecompoundsareforthemostpartfungicidal,havekill-time
ratios that compare favorably to those of amphotericin B and mi-
cafungin, and are inhibitory against a variety of clinical isolates of
Candida spp. that are resistant to triazoles, micafungin, and am-
photericin B.
We realize that the BZT compounds have only modest activity
(?g/ml) against our panel of fungal pathogens, but their fungi-
cidal activity compelled us to understand the MOA of one (DFD-
VI-15). Initial experiments on MOA employed macromolecular
synthesis determinations in treated and untreated cultures. How-
ever, inhibition of protein, RNA, or ergosterol synthesis by DFD-
VI-15 was not observed after 1 to 3 h of treatment (data not in-
cluded). Also, while from the same scaffold, unpublished data
indicate that BD-I-186 may target cell separation, suggesting a
different MOA.
In this paper, we placed considerable importance on chemo-
genomicstounderstandtheMOAofDFD-VI-15.Chemogenom-
ics is the application of genomics with chemistry (5, 9, 25, 27, 52)
in the pursuit of target discovery. As DFD-VI-15 was significantly
active against S. cerevisiae (MIC50? 6 ?g/ml, YPD broth), the
TABLE 7 Functions of selected genes deleted in the S. cerevisae BY4743
DFD-VI-15 hypersensitive mutantsa
Biological process Gene(s)
Mitochondrial morphologyERG6, ERG4, ERG24, ERG3,
SAC1
ABF2
PET123, MRP51
YME1
ISA2
GLR1
TRP1, ARO2, ILV1, TRP5,
THR1
AFT1
RPN4
URE2
ZAP1
UME6
LTE1
VPS34, RPP1A, SEC66
SNF2
ELP6
Mitochondrial DNA replication
Mitochondrial ribosomal protein
Mitochondrial inner membrane
Mitochondrial protein maturation
Mitochondrial glutathione reductase
Amino acid biosynthesis
Transcription factor, iron utilization
Transcription factor, stress response
Nitrogen catabolite repression
Zinc-regulated transcription factor
Meiotic gene regulation
Mitotic cell cycle spindle orientation
Protein sorting, ribosome stalk
Chromatin remodeling
tRNA synthesis
aFunctional annotation of C. albicans CAF2 orthologs. For C. albicans, ZAP1, UME6,
and VPS34 are required for hyphal development; ZAP1 is also a regulator of biofilm
formation.
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availability of mutant libraries of yeast provided an attractive ap-
proachtoMOAstudies.Wechoseinitiallytousethehomozygous
null, diploid library of ?4,700 nonessential gene mutants. The
prevailing paradigm is that nontarget gene products are believed
to buffer or protect (or even compensate for) an inhibited target
(52). Mutants lacking these genes have a hypersensitivity pheno-
type. Oppositely, resistant mutants can represent the target since
the lack of a target confers resistance. Susceptibility assays identi-
fied hypersensitive and resistant mutants. Cluster analysis using
FunSpec and phenotype experiments have helped us construct a
hypothetical MOA model.
Our screens yielded 31 mutants that were hypersensitive to
DFD-VI-15, and of these mutants, 11 lacked mitochondrial or
mitochondrium-relatedencodinggenes.Thenextlargestcategory
of hypersensitive mutants (five) lacked genes involved in amino
acid biosynthesis. Other categories include regulators of hyphal
growth, protein sorting, iron utilization, chromatin remodeling,
and stress adaptation. Annotation data for 27 mutants are shown
in Table 7. The alignment of 11 genes associated directly or indi-
rectly with mitochondrial functions follows that of other investi-
gators. For example, Altmann and Westermann (2) utilized a li-
braryof768S.cerevisiaemutantslackingessentialgenestoidentify
those genes that were required for normal mitochondrial mor-
phogenesis.Ofthese,119essentialgenesthatclusteredinessential
cell pathways that included ergosterol biosynthesis, mitochon-
drial import and assembly, vesicular trafficking and secretion, ac-
tin cytoskeleton-dependent transport, and ubiqutin/26S prote-
some-dependent protein degradation were described. Our list of
genes of hypersensitive mutants includes those functional anno-
tationsthatarerequiredfornormalmitochondrialmorphologyas
described previously (2).
We also identified three mutants that were resistant to DFD-
VI-15,includingonelackingACE2.InC.albicanstheace2mutant
has been also implicated in a number of functions, and it is im-
portant to our current observations, as it is a positive regulator of
glycolysis (31, 41). Genes encoding glycolytic metabolism were
downregulated while respiratory genes associated with the tricar-
boxylic acid (TCA) cycle, oxidative phosphorylation, and ATP
synthesis were upregulated in the C. albicans ace2 null mutant
(41). The resistance of the ACE2 mutant to DFD-VI-15 may be
explained by a compensatory change that elevates mitochondrial
functions and, consequently, survival of treated cells. However,
our phenotype data suggest that DFD-VI-15 inhibits mitochon-
drial respiration, as parental cells have mitochondrial defects, in-
cluding increased ROS and membrane depolarization. Further,
anaerobically grown cells (especially S. cerevisiae) are more resis-
tanttothecompound,pointingtoaroleforaerobicrespirationas
inhibited by DFD-VI-15.
Weadvocatemitochondriaasrichsourcesofantifungaltargets
for several reasons. First, there are significant fungus- and even
Candida-specific proteins and pathways of mitochondrial respi-
ration. For example, most fungi, but not S. cerevisiae, have an
alternative oxidase (AOX) pathway and a parallel pathway (PAR)
not found in mammalian cells (1). Most, but not all, of the com-
plex I proteins of the electron transport chain (ETC) are highly
conserved.Second,mitochondriaareessentialforenergyproduc-
tionand,consequently,manycellfunctions.Therefore,inhibition
of fungus-specific proteins should affect numerous cell activities.
Third,publisheddataindicatethatinhibitionofrespiratorycom-
plexes and the AOX pathway by antimycin and benzohydroxam-
ate (BHAM), respectively, convert caspofungin-resistant strains
ofC.parapsilosistoahypersensitivityphenotype(13,61).Fourth,
FIG 4 Drop plate assays with DFD-VI-15. Both C. albicans CAF2 (upper
panel)andS.cerevisiaeBY4743(lowerpanel)wereinhibitedbythecompound
at 2? the MIC50. The concentrations of DFD-VI-15 used were 12 and 24
?g/mlforBY4743andCAF2,respectively.Bothstrainsshowedhypersensitiv-
ity with the compound on yeast extract-peptone-glucose (YPD) or yeast ex-
tract-peptone-glycerol (YPG) medium (aerobic respiration). The antifungal
activityofthecompoundwasmuchlessapparentwhenthestrainsweregrown
anaerobically (An).
TABLE 8 ROS measurementsa
Strain and treatmentMean fluorescence SDP valueb
SC5314, untreated
SC5314, 12 ?g/ml DFD-VI-15
SC5314, 24 ?g/ml DFD-VI-15
GOA31
13.6
18.1
19.6
24.9
1.1
0.8
1.2
1.2
0.005*
0.003*
aThe mean fluorescence of DCFDA was compared between nontreated and treated (12
and 24 ?g/ml of DFD-VI-15) cells of C. albicans SC5314 and GOA31. There was
significantly higher fluorescence in treated strains than in untreated strains. These data
show that there is a higher accumulation of intracellular ROS upon treatment with
DFD-VI-15. Strain C. albicans GOA31, which is known to overproduce ROS following
deletion of its GOA1, was used as a positive control (33).
bP value compared to untreated SC5314 calculated by unmatched t test. *, statistically
significant (P ? 0.01).
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inhibitors of mitochondrial targets are even suggested for the
treatment of human diseases such as type 2 diabetes, certain can-
cers, and neurodegenerative diseases (28, 57, 63). Exploitation of
mitochondrial targets is compelling, especially given the lack of
new antifungal targets.
ACKNOWLEDGMENTS
The experiments were supported by a grant from the NIH-NIAID
(AI09029). We also thank the Biomedical Graduate Research Organiza-
tionoftheGeorgetownUniversityMedicalCenterfortheirgrantsupport.
The yeast homozygous null mutant library was provided by Anne
Rosenwald, Biology Department, Georgetown University. We also
thank Steven Peters, Ted White, Joachim Morschhäuser, Dominique
Sanglard, David Perlin, Patrice Le Pape, Jean-Philippe Bouchara, Ger-
aldine Butler, and Mahmoud Ghannoum for providing strains for our
studies as well as the Lombardi Cancer Center Core Facility of George-
town University for assisting with the flow cytometry experiments.
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