J Antimicrob Chemother 2011; 66: 820 826

doi:10.1093/jac/dkq530 Advance Access publication 28 January 2011

Novel high-throughput screen against Candida albicans identifies

antifungal potentiators and agents effective against biofilms
Michael D. LaFleur 1*, Edinson Lucumi 2, Andrew D. Napper 2, Scott L. Diamond 2 and Kim Lewis 1
1

Antimicrobial Discovery Center and Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, MA, USA;
2
Penn Center for Molecular Discovery and Department of Chemical and Biomolecular Engineering, University of Pennsylvania,
Philadelphia, PA, USA
*Corresponding author. Present address: Arietis Corporation, 650 Albany St., Room 130, Boston, MA 02118, USA. Tel: +1-617-638-0370;
Fax: +1-617-358-5001; E-mail: [email protected]
Present address: Nemours Center for Childhood Cancer Research, Wilmington, DE, USA.

Received 11 June 2010; returned 23 August 2010; revised 10 December 2010; accepted 15 December 2010
Objectives: Microbial adhesion and biofilms have important implications for human health and disease.
Candida albicans is an opportunistic pathogen which forms drug-resistant biofilms that contribute to the recalcitrance of disease. We have developed a high-throughput screen for potentiators of clotrimazole, a common
therapy for Candida infections, including vaginitis and thrush. The screen was performed against C. albicans biofilms grown in microtitre plates in order to target the most resilient forms of the pathogen.
Methods: Biofilm growth, in individual wells of 384-well plates, was measured using the metabolic indicator alamarBluew and found to be very consistent and reproducible. This assay was used to test the effect of more than
120000 small molecule compounds from the NIH Molecular Libraries Small Molecule Repository, and compounds
that enhanced the activity of clotrimazole or acted on the biofilms alone were identified as hits.
Results: Nineteen compounds (0.016% hit rate) were identified and found to cause more than 30% metabolic
inhibition of biofilms compared with clotrimazole alone, which had a modest effect on biofilm viability at the concentration tested. Hits were confirmed for activity against biofilms with doseresponse measurements. Several
compounds had increased activity in combination with clotrimazole, including a 1,3-benzothiazole scaffold
that exhibited a .100-fold improvement against biofilms of three separate C. albicans isolates. Cytotoxicity experiments using human fibroblasts confirmed the presence of lead molecules with favourable antifungal activity
relative to cytotoxicity.
Conclusions: We have validated a novel approach to identify antifungal potentiators and completed a highthroughput screen to identify small molecules with activity against C. albicans biofilms. These small molecules
may specifically target the biofilm and make currently available antifungals more effective.
Keywords: clotrimazole, alamarBluew, drug resistance

Introduction
Candida albicans is a common member of the human gastrointestinal microbiota and an important opportunistic pathogen.
Biofilms of C. albicans often form on indwelling devices, such
as blood and urinary catheters and heart valves.1 12 Antifungals
are unable to effectively treat biofilm infections and infected
prosthetics require device removal to prevent the dissemination
of disease. Systemic invasive fungal infections are a cause of
high mortality, approaching 40%, despite a variety of antifungal
therapeutic options.13 15 Biofilm resistance to antimicrobials
may be due to up-regulation of drug efflux pumps,16 18 drug

binding to the extracellular matrix,19,20 high cell density within

the biofilm21 or the presence of persister cells.22,23
Other important diseases caused by C. albicans are vaginitis
and oral thrush. These infections are difficult to treat and recurrent. Recurrence of infection may be due to the presence of biofilms. Most females will develop vaginitis due to C. albicans at
some point in their lives.24 Usually cases are treatable with
azoles, but 5%8% resist therapy, producing a chronic
disease.25 Weekly prophylactic fluconazole decreased relapse,
but did not cure patients.26,27
Oral thrush is a serious disease for immunocompromised
patients. Topical clotrimazole and miconazole are standard

# The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected]

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High-throughput Candida biofilm screen

treatments, but are usually only first-line treatments and temporarily effective.28 Recalcitrance of these infections to antimicrobials is not obvious, since planktonic populations of the
same strain can be highly susceptible to a range of antifungals,
including azoles, echinocandins and amphotericin B.
Traditionally, antifungals have been identified and validated
for efficacy by determining the MIC or the minimal fungicidal
concentration against a rapidly growing planktonic population
of cells. MIC determination is useful in many regards, for
instance, to track the emergence of drug resistance. However,
in vivo infection is considerably more complex, and therapeutic
failure does not always correlate with increases in MIC for the
pathogen. In vivo, pathogens may grow as biofilms, where they
are unlikely to experience rapid, exponential growth. This may
limit the utility of an in vitro assay to predict the efficacy of a
drug in vivo. Indeed, non-growing, slow-growing and biofilm
populations are considerably more tolerant to antimicrobials
compared with growing cells.29,30 Biofilms may also limit the
components of the immune system from the pathogen, further
complicating treatment.31,32 Currently, high doses of topically
applied clotrimazole cream or troches are used as an alternative
to systemic fluconazole treatment for vaginitis and oral thrush.
Fluconazole has no microbicidal activity against C. albicans,
and resistance is common. Even the most effective cidal antifungals, such as amphotericin B and caspofungin, are not completely active against C. albicans biofilms.22 We reasoned that a
molecule that synergizes with a conventional antifungal may
overwhelm the biofilm and lead to a more effective therapeutic.
Thus, we developed a screen for potentiators of the antifungal
clotrimazole, which is based on testing biofilms growing in a
microtitre plate with the viability dye alamarBluew. Hits from
the screen were subsequently validated for their ability to
inhibit biofilms alone, and in the presence of clotrimazole.

(145 mM). At the time of screening, four separate compound pin transfers
from a 1536-well compound plate were made into a single 384-well
intermediate plate to yield a mixture of four compounds per well. A
volume of 114 nL of compound (2.5 mM in DMSO) was transferred into
25 mL of RPMI 1640 medium with clotrimazole to yield a final concentration of 11.4 mM per compound. Next, 20 mL of this mixture was transferred into the biofilm-containing plates using a PerkinElmer Evolution P3
pipetting platform, yielding a final concentration of 7.6 mM per compound. Each HTS plate contained mixtures of compounds in columns
3 22 (experimental), biofilms containing clotrimazole and no compound
in columns 2 and 24 (negative control), and biofilms containing a lethal
mixture of clotrimazole (145 mM) and chlorhexidine (400 mM) in columns
1 and 23 (positive control). Biofilms were incubated for 48 h at 378C.
After incubation, medium was removed manually without biofilm disruption and replaced with 1% alamarBluew in PBS. Biofilms were incubated
at 378C for 2 h and alamarBluew reduction was measured using a PerkinElmer Envision microplate fluorimeter with excitation and emission at
535 and 590 nm, respectively.

Data analysis and retesting

Data were analysed in ActivityBase (IDBS, Guildford, UK). Biofilm metabolic inhibition, measured by alamarBluew reduction, was calculated for
each compound using the following equation:
Percentage inhibition =
 

(experimentalpositive control average)
100 1
(negative control averagepositive control average)
Wells were scored as hits if percentage inhibition was .30%. Individual
compounds from the mixtures of four were retested and a compound
was scored as a hit if the percentage inhibition was greater than 30%
in the retest.

IC50 testing

Materials and methods

Biofilm growth conditions
Wild-type C. albicans strain CAF2-1 was streaked onto YPD agar medium
(10 g/L yeast extract and 20 g/L Bacto peptone) and incubated at 308C
for 48 h. A single colony from the YPD plate was used to inoculate
100 mL liquid YPD in a 1 L baffled Erlenmeyer flask and the culture was
incubated at 308C for 24 h. Cells were concentrated by centrifugation
and resuspended in 15% glycerol in phosphate-buffered saline. Stocks
of the suspension in 15% glycerol were divided into single-use aliquots
and frozen in liquid nitrogen and stored at 2808C. Twenty-four hour biofilms were formed by standard methods according to Ramage et al.33
Briefly, cells from the frozen stock were resuspended in RPMI 1640 with
L-glutamine and 0.165 M MOPS (BioWhittaker). The OD600 of the suspension was adjusted to 0.1, approximately 1106 cfu/mL. Suspensions of
30 mL were aliquoted into wells of flat-bottom 384-well (Greiner
7 81086) microtitre plates. The plates were incubated for 24 h at 378C,
allowing biofilm formation.

High-throughput screen (HTS)

C. albicans biofilms were grown in 384-well plates. Biofilm growth
medium was removed by inverting and discarding. The medium was
replaced with 10 mL of fresh RPMI 1640 growth medium containing clotrimazole (145 mM). Compounds from the Molecular Libraries Small Molecule Repository (MLSMR) were initially transferred into a separate
intermediate 384-well plate containing RPMI 1640 and clotrimazole

Biofilms were grown in 384-well plates as described previously.

C. albicans strain SC5314 and a fluconazole-resistant strain (29E) isolated
from a cancer patient23 were tested in addition to CAF2-1. Hits from the
MLSMR were resupplied directly from their commercial vendor and dissolved in DMSO. Compounds were serially diluted (2-fold from 100 mM
to 3 mM) using a PerkinElmer Evolution P3 pipetting platform. From this
dilution plate, 108 nL of each compound was transferred into a separate
intermediate 384-well plate containing 25 mL RPMI 1640 alone (columns
1 and 23) and RPMI 1640 with clotrimazole (145 mM) (columns 2 22 and
24). Biofilm growth medium was removed by inversion and discarded.
The medium was replaced with 10 mL of fresh RPMI 1640 growth
medium and medium containing 145 mM clotrimazole. Next, 20 mL of
medium containing clotrimazole and compound was transferred from
the intermediate plate to each well of the biofilm-containing plates.
Final concentrations for each compound ranged from 288 mM to 8 nM
and each compound was tested for activity against biofilms alone and
along with clotrimazole. Biofilms were incubated and assayed for metabolic activity as described previously. The concentration of each compound that inhibited biofilm metabolic activity by 50% was calculated
by non-linear regression (four-parameter logistic fit) using XLfit (IDBS,
Guildford, UK) and reported as the IC50.

Cytotoxicity
Cryopreserved adult human fibroblasts were obtained from Lifeline Cell
Technology (Walkersville, MD, USA). Fibroblasts were suspended in
Eagles Minimum Essential Medium (ATCC catalogue no. 30-2003) with

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LaFleur et al.

Results
HTS optimization
In order to determine whether biofilms could be grown reproducibly in 384-well plates, C. albicans cells were seeded and incubated for 24 h, in a manner similar to biofilm growth in 96-well
plates.33 Visual and microscopic inspection of these plates
revealed that attached biofilms were present in each well.
Biofilm metabolic activity was measured with alamarBluew
(1%) in order to determine biofilm growth and variability. Fluorescent intensity readings, corresponding to biofilm metabolic
activity, were measured every 20 min for 4 h. On average, alamarBluew was found to be almost completely reduced after
2 h of incubation, with only a minimal increase in fluorescence
detected thereafter. Next, we calculated the variability of
biofilm metabolic activity between individual wells of the microplate. Biofilms were found to grow very consistently and reproducibly in 384-well plates. In fact, the coefficient of variation,
defined as standard deviation/mean100, was found to be
only 3.3%. The consistency of growing biofilms in the 384-well
microplate format was quantified by calculation of the Z -factor,
which is a standard measure of the robustness and feasibility of
an HTS. The Z -factor determines the magnitude of the difference
between the positive and negative controls relative to the sum of
the respective standard deviations:


(3SD+ +3SD )
Z = 1
(Ave+ Ave )
where SD+ positive control standard deviation, SD2 negative
control standard deviation, Ave+ positive control average and
Ave2 negative control average. In our assay, the positive
control consisted of microtitre wells seeded with C. albicans biofilms in the presence of a combination of clotrimazole and chlorhexidine, while negative control wells contained biofilms and
clotrimazole alone (Figure 1). Clotrimazole was added to the
negative control wells at 145 mM, since this concentration
caused a mid-range reduction of alamarBluew signal compared
with positive and negative controls. Chlorhexidine is known to
kill biofilms, so it was added to clotrimazole as the positive
control. The Z was found to be 0.90, well above the Z .0.5

822

30
25
RFU

10% fetal bovine serum and seeded into a 75 cm2 tissue culture flask
according to the manufacturers recommendations. Cells were incubated
at 378C and 10% CO2 until nearly confluent. Fibroblasts were trypsinized
and passaged into clear-bottomed, black 96-well plates (Corning Costar
3603) and grown until 70% confluence. Experimental compounds were
diluted into a separate plate, using a 1:2 ratio in fresh medium, from concentrations ranging from 16 to 500 mM. After dilution, the fibroblast
medium was aspirated from the fibroblast plate and replaced with
fresh medium containing the experimental compounds. The plates
were incubated at 378C and 10% CO2. After 24 h, fibroblasts were
washed three times with fresh medium and incubated for an additional
18 h. Next, 10% alamarBluew was added to each well and the plates
were incubated for 2 h. The percentage metabolic activity of fibroblasts
in each well was calculated based on measurements from a fluorescent
plate reader and negative control wells without experimental compounds. The highest concentration of each compound that caused
greater than 50% reduction in metabolic activity was reported as the
cytotoxic concentration.

20
15
10
5
0

100

200
Well number

300

400

Figure 1. Fluorescent intensity of untreated biofilms (squares), those

treated with clotrimazole alone (circles) and those treated with a
combination of clotrimazole and chlorhexidine (crosses). Biofilms were
grown for 24 h, challenged with antimicrobials for 48 h and assayed
with alamarBluew for 2 h.

generally regarded as being suitable for an HTS. In addition to

Z , a variety of other standard HTS assay performance parameters, including coefficient of variation (,10) and
signal-to-background ratio (.10), were measured to determine
suitability for HTS. The parameters determined for the 384-well
alamarBluew assay showed it to be suitable for HTS, comparing
favourably with criteria for HTS accepted into the NIH Molecular
Libraries Screening Center Network.

HTS
More than 120 000 compounds were screened as mixtures of
four compounds per well in 384-well plates. Individual wells
that showed activity were parsed and retested to attribute
activity to a specific compound. In total, only 19 compounds
were identified that inhibited biofilm metabolic activity more
than 30%. The overall hit rate from the screen was surprisingly
low, approximately 0.016%. The results from the entire screen
are included in an Excel file (available as Supplementary data
at JAC Online), along with the PubChem identification number
of each compound.

Validation of HTS hits

Of the 19 hits, 5 were unavailable for immediate resupply and
were excluded from further analysis. The structures of the
other hits are detailed, along with the average percentage
metabolic inhibition scores from the primary screen and retest
validations (Figure 2). It should be noted that the HTS did not
discriminate between compounds with direct activity on the
biofilm and potentiator compounds that had no effect alone,
since each small molecule was tested in combination with clotrimazole. To distinguish between these two possibilities, hits
from the HTS were tested in a dose response experiment for
efficacy against biofilms alone and in the presence of 145 mM
clotrimazole. Dose response curves were generated against
biofilms of three strains of C. albicans, including a
fluconazole-resistant strain isolated from a cancer patient23
(strain 29E). The concentration of each compound required to
inhibit biofilm metabolic activity by 50%, the IC50, was calculated from each doseresponse using non-linear regression

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High-throughput Candida biofilm screen

NH2
N

NH2

F
F

N+

HO

Br

OH

S
S

NH

O
O

N
O
(1)
49.4%

(2)
43.3%

(3)
37.6%

(4)
42.3%
Br
N+

Br
N

N+

Br

N+
OH

Br
(5)
37.4%

(7)
41.1%

(6)
50%
Cl
N N
S

N+

H
N
Cl

O S O O

(10)
41.5%

40.9%

Cl

(8)
36.7%

(12)
51.8%

N
N
N+

N+

(11)
49.9%

OH
(9)

OH

N+

S
N

N+

Cl
(13)
69.4%

(14)
34.3%

Figure 2. Compounds that inhibited the metabolic activity of C. albicans biofilms, tested in the presence of clotrimazole. For each compound, the
average percentage metabolic inhibition from the primary screen and retest confirmation experiments is given.

(Table S1, available as Supplementary data at JAC Online).

Several compounds had greatly improved activity when tested
in combination with clotrimazole compared with the compound

alone based on the IC50 (Figure 3). In particular, compounds 2,

7 and 12 14 demonstrated a greater than 8-fold increase in
activity together with clotrimazole, across the three strains. As

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LaFleur et al.

Untreated

Clotrimazole

IC50 (M)

(a) 300
250

Compound

200

1
2
3
4
5
6
7
8
9
10
11
12
13
14

150
100
50
0

7 8 9 10 11 12 13 14
Compound

(b) 300
250
IC50 (M)

Table 1. Cytotoxic concentration of each hit tested against human

fibroblasts

200

Cytotoxic concentration (mM)

500
125
62
250
62
31
125
62
16
500
125
16
16
31

150

produced by strain 29E revealed sparse biomass and reduced

hyphae compared with the other two strains.

100
50

Cytotoxicity
0

7 8 9 10 11 12 13 14
Compound

7 8 9 10 11 12 13 14
Compound

(c) 300

IC50 (M)

250
200
150
100
50
0

Figure 3. Effects of each hit on biofilm metabolism. Dose response

curves were generated from biofilms exposed to a range of
concentrations from 288 mM to 8 nM of each compound. Biofilms of
C. albicans strains CAF2-1 (a) and SC5314 (b) and fluconazole-resistant
clinical isolate 29E (c) were tested with or without clotrimazole for 48 h
and the IC50 for each compound was determined.

expected, some compounds caused decreases in biofilm metabolism alone. Most compounds had similar relative effects on
biofilms produced by three separate strains of Candida
(Figure 3a c). However, differences in susceptibilities between
strains were noted for individual compounds. Surprisingly, a
fluconazole-resistant strain appeared to have greater overall
susceptibilities (Figure 3c) compared with two wild-type
strains. Subsequent microscopic examination of biofilms

824

HTS libraries often contain toxic, promiscuous or generally reactive molecules which do not make good drug candidates. We
reasoned that molecules that act in synergy with conventional
antifungals and do not have activity alone are not likely to be
toxic. In order to confirm this hypothesis, we tested the HTS
hits for cytotoxicity against human cell cultures. Fibroblasts
were chosen due to their ubiquitous nature and widespread
use in cytotoxicity testing. Fibroblasts were grown in 96-well
plates and exposed to increasing doses (2-fold increments) of
each hit compound for 24 h. After exposure, fibroblast metabolic
activity was measured and used as an indicator of cell viability.
The concentration of each compound that caused a 50%
reduction of fibroblast metabolic activity was reported as the
cytotoxic concentration (Table 1). Compounds 1214, which
exhibited dramatically improved potency along with clotrimazole, were also cytotoxic at concentrations at or below 31 mM.
The most promising molecules to emerge from the HTS were
compounds 2, 7, 10 and 11. These molecules had cytotoxic concentrations at or above 125 mM and had biofilm inhibitory activities less than 10 mM for at least one strain of C. albicans.

Discussion
Fungal infections are often recurrent and difficult to treat; thus
there is an urgent need for novel antifungal therapeutics and
treatment strategies. Currently available antifungals may be
growth inhibitory or cidal to exponentially growing cells, but
are largely ineffective against biofilms. We have utilized the
intrinsic drug-resistant nature of the biofilm growth state to
establish acceptable conditions to screen for antifungal potentiators. The primary screen did not discriminate between compounds that acted directly on the biofilm or those that
potentiated clotrimazole, and the overall low hit rate of the

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High-throughput Candida biofilm screen

screen confirms the notion that biofilms are extremely difficult to

eradicate.
Clotrimazole troches (10 mg) and creams (1%) are used to
treat oral and vaginal candidiasis. The clotrimazole potentiators
identified in this screen have the potential to improve dosing
regimens, decrease the acting concentration of drug for these
indications, and combat resistance. Also, since the hits identified
in our screen have activity against biofilms, they may be
especially useful in treating biofilm-related fungal infections.
Recent evidence suggests C. albicans forms biofilms on the oral
and vaginal mucosa during infection.34,35 Compounds that are
particularly effective against biofilm pathologies may prove
useful in clearing infections or preventing relapse.
Clotrimazole, like other azole antifungals, is known to target
a-14 lanosterol demethylase, causing ergosterol depletion and
growth arrest. However, a fluconazole-resistant strain, 29E, was
found to produce poor biofilms and this strain had increased susceptibilities to certain molecules (compounds 2, 4, 7 and 10 13)
compared with wild-type strains that formed more robust biofilms. Thus, a high level of antifungal drug resistance was conferred by the extent to which a strain was able to form a
biofilm. Furthermore, if a susceptible strain with a low MIC is
able to form a robust biofilm, the biofilm may provide even
greater protection compared with a strain with a higher MIC
that cannot form a robust biofilm. Strain 29E did exhibit
increased resistance to the combination of compound 3 or 5
and clotrimazole. These compounds may be particularly good
substrates of fungal multidrug efflux transporters, which are
known to be up-regulated in the presence of azoles36 and in
drug-resistant strains.37
Several of the most potent clotrimazole potentiators that
were identified in the screen contain a 1,3-benzothiazole scaffold
(compounds 6, 12 and 14). This motif is responsible for the biological activity of luciferin,38 found in fireflies, and the pharmaceutical riluzole,39 used to treat amyotrophic lateral sclerosis.
Antifungal activities of benzothiazole compounds have been
reported40 and 6-amino-2-n-pentylthiobenzothiazole was
found to inhibit Candida filamentation,41 which is required for
biofilm formation. We are planning to test whether the
1,3-benzothiazole compounds identified in this screen also
inhibit filamentation. The apparent cytotoxic liabilities of the
1,3-benzothiazole hits may be irrelevant for topical applications
or it may be possible to optimize this scaffold for reduced cytotoxicity by synthesizing and testing chemical analogues of this
moiety. Other azole potentiators identified in the literature are
known to function by diverse mechanisms, including inhibiting
calcineurin,42 HSP9043 or drug efflux pumps.44 The targets and
mechanisms of action of the potentiator compounds identified
in the screen are currently under investigation in order to determine whether they function by known or novel mechanisms.
We have demonstrated the feasibility of performing an HTS
against C. albicans biofilms. As proof of principle, we identified
clotrimazole potentiators, which may prove useful for topical
applications such as vaginitis or oral thrush. It is likely that
screens for potentiators of systemic antifungals could be conducted in a similar manner. For example, the identification of
potentiators of caspofungin, which targets the fungal cell wall,
may shed light on Candida cell wall biology and lead to improved
treatment of systemic fungal infections. These probes may also
reveal the extent to which biofilms are responsible for

untreatable or recurrent fungal infection in vivo. The small molecule approach to specifically target biofilms that we have
described may have important therapeutic and industrial
applications.

Funding
This work was supported by NIH grants GM061162 to K. L. and HG003915
to S. L. D. The funder had no role in study design, data collection and
analysis, decision to publish or preparation of the manuscript.

Transparency declarations
None to declare.

Disclaimer
The content is solely the responsibility of the authors and does not
necessarily represent the official views of the NIH.

Supplementary data
The Excel file and Table S1 are available as Supplementary data at JAC
Online (http://jac.oxfordjournals.org/).

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