Increased Degree of Unsaturation in the Lipid of Antifungal Cationic Amphiphiles Facilitates Selective Fungal Cell Disruption

Article abstract of DOI:10.1021/acsinfecdis.7b00272

Antimicrobial cationic amphiphiles derived from aminoglycosides act through cell membrane permeabilization but have limited selectivity for microbial cell membranes. Herein, we report that an increased degree of unsaturation in the fatty acid segment of antifungal cationic amphiphiles derived from the aminoglycoside tobramycin significantly reduced toxicity to mammalian cells. A collection of tobramycin-derived cationic amphiphiles substituted with C₁₈ lipid chains varying in degree of unsaturation and double bond configuration were synthesized. All had potent activity against a panel of important fungal pathogens including strains with resistance to a variety of antifungal drugs. The tobramycin-derived cationic amphiphile substituted with linolenic acid with three cis double bonds (compound 6) was up to an order of magnitude less toxic to mammalian cells than cationic amphiphiles composed of lipids with a lower degree of unsaturation and than the fungal membrane disrupting drug amphotericin B. Compound 6 was 12-fold more selective (red blood cell hemolysis relative to antifungal activity) than compound 1, the derivative with a fully saturated lipid chain. Notably, compound 6 disrupted the membranes of fungal cells without affecting the viability of cocultured mammalian cells. This study demonstrates that the degree of unsaturation and the configuration of the double bond in lipids of cationic amphiphiles are important parameters that, if optimized, result in compounds with broad spectrum and potent antifungal activity as well as reduced toxicity toward mammalian cells.

Full text of DOI:10.1021/acsinfecdis.7b00272

Article
pubs.acs.org/journal/aidcbc
Cite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX
Increased Degree of Unsaturation in the Lipid of Antifungal Cationic
Amphiphiles Facilitates Selective Fungal Cell Disruption
Kfir B. Steinbuch,† Raphael I. Benhamou,† Lotan Levin,‡ Reuven Stein,‡ and Micha Fridman*,†
†School of Chemistry, Raymond and Beverley Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, Israel 6997801
‡Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel 6997801
S
* Supporting Information
ABSTRACT: Antimicrobial cationic amphiphiles derived from aminoglycosides act through cell membrane permeabilization but
have limited selectivity for microbial cell membranes. Herein, we report that an increased degree of unsaturation in the fatty acid
segment of antifungal cationic amphiphiles derived from the aminoglycoside tobramycin significantly reduced toxicity to
mammalian cells. A collection of tobramycin-derived cationic amphiphiles substituted with C18 lipid chains varying in degree of
unsaturation and double bond configuration were synthesized. All had potent activity against a panel of important fungal
pathogens including strains with resistance to a variety of antifungal drugs. The tobramycin-derived cationic amphiphile
substituted with linolenic acid with three cis double bonds (compound 6) was up to an order of magnitude less toxic to
mammalian cells than cationic amphiphiles composed of lipids with a lower degree of unsaturation and than the fungal
membrane disrupting drug amphotericin B. Compound 6 was 12-fold more selective (red blood cell hemolysis relative to
antifungal activity) than compound 1, the derivative with a fully saturated lipid chain. Notably, compound 6 disrupted the
membranes of fungal cells without affecting the viability of cocultured mammalian cells. This study demonstrates that the degree
of unsaturation and the configuration of the double bond in lipids of cationic amphiphiles are important parameters that, if
optimized, result in compounds with broad spectrum and potent antifungal activity as well as reduced toxicity toward mammalian
cells.
KEYWORDS: amphiphilic aminoglycosides, membrane disruption, antifungal drugs, lipid unsaturation
pressure.8 Azoles such as fluconazole (FLC, Figure 1) inhibit
ungal infections such as candidiasis are a major cause of
Fnosocomial infections and are especially prevalent among the fungal cytochrome P450 enzyme 14α-demethylase involved
in the biosynthesis of the fungal membrane sterol ergosterol.6
Toxic side effects caused by antifungal drugs, especially
polyenes, and evolutionary pressure that has enhanced the
emergence of resistance to the current repertoire of clinically
approved antifungal drugs highlight the urgent need for new
additional antifungal agents.9−11
In recent years, we as well as other research groups have
developed antimicrobial cationic amphiphiles derived from
amino-sugars or aminoglycoside (AG) antibiotics or poly-β-
peptides. Several of the reported cationic amphiphiles display
broad spectrum and potent antibacterial12−21 and/or anti-
patients with compromised immune systems.1,2 The high
evolutionary similarity between fungal and mammalian cells
greatly limits the number of specific targets available for
antifungal drug development, and there are currently very few
classes of antifungal drugs in clinical use (Figure 1).3 Among
these antifungal drugs are polyenes such as amphotericin B
(AmB, Figure 1). These drugs are potent and last resort
antifungal drugs that directly bind to ergosterol, an essential
fungal membrane lipid that has a similar function to that of
cholesterol in mammalian cells.4−7 Echinocandins such as
caspofungin (CASP, Figure 1) are a lipopeptide class of
antifungal agents that act through inhibition of the (1−3)-β-D-
glucan synthase complex involved in the biosynthesis of the
(1−3)-β-D-glucan layer that protects fungal cells from osmotic
Received: December 20, 2017
Published: February 8, 2018
© XXXX American Chemical Society
A
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Figure 1. Structures of major clinically used antifungal drugs.
Scheme 1. Synthesis of TOB-Derived Cationic Amphiphiles
fungal22−29 activities against strains with high levels of
resistance to clinically used antimicrobial drugs; however,
limited membrane selectivity prevents most of these com-
pounds from being considered for further clinical development
for systemic use. We recently reported that conversion of the
primary amines of cationic amphiphiles derived from the AG
nebramine to the corresponding imidazoles resulted in
compounds with potent antifungal activity against Candida
glabrata, an important fungal pathogen.30 Notably, compared to
other types of AG-derived antimicrobial cationic amphiphiles,
the reported imidazole-decorated cationic amphiphiles caused
only minor damage to mammalian red blood cells (RBCs). In
search of strategies to further improve the safety profile of AG-
derived cationic amphiphiles through chemical modifications,
our attention was drawn to the degree of unsaturation and the
configuration of the double bond in the lipid segment of these
antimicrobial agents. It is well established that the degree of cis
unsaturation of membrane lipids regulates fundamental
biophysical and biological properties such as fluidity,
permeability, endo- and exocytosis, cell division, signal
transduction, and membrane protein activities.31−35 Saturated
fatty acids increase membrane rigidity, and cis unsaturated fatty
acids increase membrane fluidity.33,34 We therefore hypothe-
sized that displacement of the saturated lipid chains in AG-
derived cationic amphiphiles with cis unsaturated lipids would
lead to a higher selectivity in the membrane permeabilizing
activity of these antimicrobial agents, potentially reducing their
toxicity to the host tissues. To test our hypothesis, we
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a
Table 1. Antifungal Activity: Minimal Inhibitory Concentration (MIC) Values [μg/mL]
Compounds
Strains
1
2
3
4
5
6
FLC
AmB
CASP
TOB
A. C. albicans ATCC 90028
B. C. albicans ATCC 24433
C. C. albicans SC5314
8
4
4
8
2
4
4
4
4
4
4
4
4
4
8
8
8
2
2
8
8
4
8
4
4
4
4
4
4
8
4
4
4
8
8
8
4
4
8
4
4
8
4
4
4
4
4
4
4
4
4
4
8
8
8
4
4
8
8
8
8
4
4
4
4
4
4
8
4
4
4
8
4
8
2
2
8
8
4
8
4
4
4
4
4
4
4
8
4
4
8
8
8
4
4
8
8
8
8
4
8
4
4
8
8
4
8
8
8
8
4
8
4
4
0.5
1
0.25
0.125
0.125
0.25
0.25
0.5
0.25
0.25
0.5
2
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
1
D. C. parapsilosis ATCC 90018
E. C. tropicalis Clinical isolate 660
1
b
0.5
1
1
b
F. C. dubliniensis Wu284
0.125
0.25
0.5
0.25
0.25
0.25
0.5
1
b
G. C. guilliermondii B3163
0.25
0.25
0.25
0.5
b
H. C. albicans Clinical isolate 280
I. C. glabrata ATCC 66032
32
>64
32
b
J. C. glabrata Clinical isolate 50
0.25
0.25
0.25
0.125
0.25
0.125
1
K. C. glabrata ATCC 2001
>64
>64
>64
>64
>64
1
b
L. C. glabrata 195
M. C. albicans ATCC 96901
N. C. albicans ATCC 64124
O. C. albicans ATCC 11651
0.5
0.5
64
b
P. C. albicans Clinical isolate 2066
b
Q. C. albicans Clinical isolate 2068
R. C. neoformans ATCC 32045
S. C. neoformans ATCC 66031
0.5
1
64
0.5
0.25
0.25
8
0.25
8
a
All MICs were determined using the broth double-dilution method. The highest concentration tested was 64 μg/mL. Cells were grown in RPMI
1640 medium at 37 °C under 5% CO2. The experimental duration was 24 to 48 h for Candida strains and 72 h for the C. neoformans strains. Each
b
concentration was tested in triplicate, and results were reproduced in at least two independent experiments. Strains received from Prof. J. Berman,
George S. Wise Faculty of Life Sciences, Tel Aviv University.
synthesized a collection of cationic amphiphiles derived from
the AG tobramycin (TOB) differing in the degree of
unsaturation and the double-bond configuration in their lipid
segment and evaluated their antifungal activity and effects on
mammalian cells.
neoformans strains (Table 1). C. neoformans causes a severe
form of meningitis and meningoencephalitis in those with
compromised immune systems.39 These fungal pathogens are
among the most common causes of opportunistic fungal
infections worldwide.40
Of the 19 tested strains, eight (strains H−O) are resistant to
the commonly used azole drug FLC (for information about the
strains, also see Table S3, Supporting Information). Two strains
(P and Q) are highly resistant to the echinocandin drug CASP;
this resistance is conferred by a mutation in the FKS1 gene.41
FLC, CASP, and the fungal membrane disrupting polyene AmB
were used as controls. Notably, the degree of cis unsaturation
had little to no effect on the antifungal activity of cationic
amphiphiles 1−6 against the tested panel of fungal pathogens.
MIC values ranged from 2 to 8 μg/mL (Table 1).
Next we evaluated the antibacterial activities of compound 1,
which has a fully saturated lipid segment, and 6, which has the
highest degree of unsaturation in its lipid segment of the
derivatives tested against three Gram positive and three Gram
negative pathogens. Both compounds displayed poor anti-
bacterial activities against the tested strains (MIC range of 32
μg/mL to >64 μg/mL, see Table S4 in the Supporting
Information). These results indicate that the reported cationic
amphiphiles are selective antifungal agents.
Addition of double bonds to a lipid chain results in π-bonded
electrons that are less tightly held by the nucleus and are
therefore potentially available as hydrogen bond donors. The
calculated log D values of the free base forms of compounds 1−
6 (Table 2) revealed that each addition of a double bond to the
lipid incrementally decreased the log D value. For the TOB-
derived cationic amphiphiles in this study, there was no
correlation between log D value and antifungal activity.
Effect of the degree of unsaturation and double bond
configuration on membranes of mammalian red blood
cells. One of the major drawbacks in considering cationic
RESULTS AND DISCUSSION
■
Synthesis of tobramycin-derived cationic amphi-
philes containing unsaturated fatty acids. We synthesized
a collection of six cationic amphiphiles derived from the AG
antibiotic TOB, which served as the positively charged
headgroup, and a fatty acid containing a C18 chain. Six fatty
acids differing in the degree of unsaturation were chosen
(Scheme 1): the fully saturated octadecanoic acid known as
stearic acid (compound 1), the monounsaturated ω-9 elaidic
acid with a single trans double bond (compound 2), the
monounsaturated ω-9 oleic acid with one cis double bond
(compound 3), the diunsaturated ω-6 linolelaidic acid with two
trans double bonds positioned at C-9 and C-12 (compound 4),
the diunsaturated ω-6 linoleic acid with two cis double bonds
positioned at C-9 and C-12 (compound 5), and the ω-3
linolenic acid with three cis double bonds positioned at the C-9,
C-12, and C-15 of the fatty acid carbon chain (compound 6).
Synthesis was accomplished by forming an amide bond
between the free fatty acids and the 6′-amine of the TOB-
derived precursor 1a36,37 using HATU to afford the Boc-
protected compounds in 57−79% isolated yields. These were
deprotected by treatment with TFA to yield the desired
compounds 1−6 in quantitative yields as penta-TFA salts
(Scheme 1). The purity of compounds 1−6 was >95% as
determined by ultraliquid chromatography−mass spectroscopy
(sections 1−3, Supporting Information).
Antifungal activity. The antifungal activity of cationic
amphiphiles 1−6 was evaluated using the broth double-dilution
assay38 against a panel of Candida spp. and Cryptococcus
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a
2). Notably, the HC50 of cationic amphiphile 6, the least
hemolytic of the cationic amphiphiles in this study, was at least
2 orders of magnitude higher than that of AmB. Furthermore,
the configuration of the double bond in the lipid also had a
substantial effect on the hemolysis. The HC50 of compound 2,
containing a single trans double bond in its lipid, was
approximately 2.4-fold lower than that of compound 3, which
contains the cis double bond isomer. Comparison between the
HC50 values of compound 4, composed of the diunsaturated ω-
6 linolelaidic acid with two trans double bonds positioned at C-
9 and C-12, and compound 5, with the corresponding
diunsaturated ω-6 linoleic acid with two cis double bonds,
revealed that the latter was less hemolytic. With this pair, the
double bond configuration effect was less pronounced than
when compounds 2 and 3 were compared (Figure 2). Our
results are in agreement with the previously reported
demonstrations that introduction of an unsaturated lipid
chain in lipo-γ-AA peptides and in lysine-based cationic
amphiphiles significantly decreases their hemolytic activity.43,44
We calculated the selectivity index between RBC hemolysis
and antifungal MIC (HC50 divided by the highest MIC value
measured for the tested panel of fungi) for compound 1 with a
fully saturated fatty acid and compound 6 with the highest
degree of cis unsaturation. Comparison between the selectivity
index values revealed that the increase in unsaturation resulted
in a 12-fold increase in the selectivity index of compound 6
compared to that of compound 1: For compound 1 the
selectivity index was 2, and for compound 6 the index was 24.
The results of the hemolysis tests indicated that an increase
in the degree of cis unsaturation of the lipid segment reduced
undesired mammalian cell membrane damage without a
significant effect on the ability of the cationic amphiphiles to
disrupt yeast cell membranes. Thus, in designing antifungal
cationic amphiphiles, introduction of lipids carrying a high
degree of cis unsaturation can lead to a significant increase in
the specificity to the fungal cell membrane and reduce severe
RBC membrane damage that can lead to side effects such as
hemolytic anemia.
Table 2. Partition Coefficients of Compounds 1−6
Compound
Log D (pH = 7.4)
1
2
3
4
5
6
−6.55
−6.91
−6.91
−7.27
−7.27
−7.63
a
Log D values were calculated in MarvinSketch software (version
6.3.1) with default parameters. Electrolyte concentration: [Cl−] =
[Na+] = [K+] = 0.1 M.
amphiphiles for systemic antimicrobial treatment is that these
compounds nonselectively damage mammalian cell mem-
branes. Evaluation of RBC hemolysis enables quantitative
evaluation of mammalian cell membrane permeabilization
activity and has therefore become the most commonly applied
method for determination of the membrane selectivity of
antimicrobial cationic amphiphiles.42 The effect of the degree of
lipid unsaturation and double bond configuration of cationic
amphiphiles 1−6 on RBC hemolysis was evaluated using RBCs
isolated from laboratory rats. The concentrations at which 50%
of RBCs were lysed (HC50) by TOB-derived cationic
amphiphiles are summarized in Figure 2.
Effects on the viability of immortalized and primary
mammalian cells. Mammalian RBC hemolysis serves as an
indication of mammalian membrane damage. However, cationic
amphiphiles may have additional toxic effects on mammalian
cells that reduce their viability. To further assess the potential
toxicity of cationic amphihiles 1−6 toward mammalian cells,
effects on cell viability were tested on three nucleated
mammalian cells lines. The chosen lines were HepG2 cells,
an immortalized human liver hepatocellular carcinoma cell line,
3T9MEFs, an immortalized mouse embryonic fibroblast cell
line, and HEK 293 cells, an immortalized human embryonic
kidney cell line. These cell lines were treated with each of the
cationic amphiphiles at concentrations up to 256 μg/mL for 24
h, after which cell viability was evaluated using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay (Figure 3).
Since transformed and immortalized cell lines often differ
genetically and phenotypically from their tissues of origin, we
also evaluated the impact of cationic amphiphiles 1−6 on the
viability of primary dermal fibroblasts obtained from newborn
mice. Similar to the results of the hemolysis assay, there was a
correlation between the degree of cis unsaturation and the
ability of the compounds to reduce cell viability (as indicated by
the MTT cell viability assay). The highest reduction in viability
was displayed by the fully saturated stearic acid-derived cationic
Figure 2. Hemolysis of RBCs. Rat erythrocytes were incubated with
AmB (black), 1 (orange), 2 (gray), 3 (red), 4 (blue), 5 (green), 6
(purple), and FLC (brown) for 1 h at 37 °C. Released hemoglobin
absorbance was measured at 550 nm. Results are expressed as the
percentage of hemoglobin released relative to Triton X100 (100%
hemolysis). HC50 values are given in the legend. Each concentration
was tested in triplicate, and the results are expressed as means
standard error from two independent experiments.
The potent and last resort antifungal drug AmB, which is
known to cause extensive RBCs hemolysis,5 was used as a
positive control, and the azole antifungal FLC, which does not
directly permeabilize the yeast cell membrane, was used as a
negative control.6 Whereas the degree of unsaturation and
double bond configuration in the lipids of cationic amphiphiles
1−6 had no significant influence on antifungal activity against
the tested panel of yeast strains, a significant differential effect
on rat RBC hemolysis was observed (p < 0.0004−0.03 for
compound 6 in comparison to each of compounds 1−4,
Student’s t test). Of the cationic amphiphiles 1−6, compound
1, derived from the fully saturated stearic acid, was the most
hemolytic, and the ω-3 linolenic acid-derived cationic
amphiphile 6 with the highest degree of cis unsaturation was
more than 1 order of magnitude less hemolytic than 1 (Figure
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Hence, at concentrations significantly higher than the MIC
range against the tested yeast strains, compound 6 did not
reduce the viability of immortalized and primary mammalian
cell lines. Notably, the extent of the effects of the lipid double
bond configuration on the nucleated mammalian cells viability
was cell-type specific. For example, in 3T9MEFs, the IC50 of
compound 2, which is substituted with elaidic acid with a single
trans double bond, was about 2.2-fold lower than that of
compound 3, which is composed of oleic acid with a cis double-
bonded isomer. In the other tested cell lines, however, the
differences in IC50 values were more modest.
We then calculated the selectivity index between antifungal
activity and the effect on mammalian cell viability (lowest IC50
value measured for the tested cell lines divided by the highest
MIC value measured for a tested panel of fungi) for
compounds 1 and 6. The selectivity index values were 4.5 for
compound 1 and 27 for compound 6. Compound 6 with the
unsaturated lipid had a 6-fold higher selectivity index than
compound 1 with the fully saturated fatty acid segment. The
results of the mammalian cell viability assay and the selectivity
index calculations indicated that, as observed for hemolytic
activity, an increase in the degree of unsaturation reduces the
toxicity of antifungal cationic amphiphiles.
Figure 3. Effects of compounds 1−6 on the viability of mammalian
cells. Cells were plated in 96-well microtiter plates. After 24 h, the
tested compounds (at concentrations up to 256 μg/mL) were added.
After an additional 24 h, cell viability was determined by MTT assay.
The percentage of viability was determined relative to control
untreated cells. Each concentration was tested in triplicate, and the
results are expressed as means SD of the IC50 values from at least
three independent experiments. In columns marked with asterisks, the
decrease in viability did not reach 50% even at the highest
concentration tested, 256 μg/mL.
Inhibition of the growth of C. albicans in a coculture
with mammalian cells. To date, the antimicrobial activity of
synthetic antifungal cationic amphiphiles has not been
evaluated in cultures containing both the pathogen and
mammalian cells. The efficacy of antimicrobial cationic
amphiphiles is reduced due to nonspecific interactions with
numerous lipophilic and anionic moieties found in the cell
culture.45−48 We therefore investigated the antifungal activity
and cell-selectivity of cationic amphiphiles 1 and 6 in a
amphiphile 1, and the lowest reduction was displayed by the
linolenic acid-derived cationic amphiphile 6 with three cis
double bonds in its lipid chain. The concentration of
compound 1 that led to a 50% reduction in cell viability
(IC50) ranged between approximately 36 μg/mL for 3T9MEFs
to about 90 μg/mL for HEK 293 cells, whereas the IC50 values
of compound 6 were approximately 216 μg/mL for HepG2
cells and above the maximal concentration tested (256 μg/mL)
for the other cell types (Table S6, Supporting Information).
Figure 4. Effect of compounds 1 and 6 on a coculture of HEK 293 cells with C. albicans SC5314. HEK 293 cells alone (A−F) and HEK 293 cells
cocultured with C. albicans (G−L) were left untreated (A, B, G, H) or treated with compounds 1 (C, D, I, J) or 6 (E, F, K, L). Bright field images
and images of samples stained with phalloidin (red) and Hoechst dye (blue) were visualized with an Olympus motorized inverted fluorescent
microscope.
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coculture of both pathogen and mammalian cells (Figure 4).
To form the coculture, C. albicans SC5314 cells were added to a
culture of HEK 293 cells at a multiplicity of infection of 2. After
30 min of incubation, cells were treated with 128 μg/mL of
compound 1 or 6 or untreated. Notably, this concentration is
32 and 16 times higher than the MIC values of 1 and 6 against
C. albicans SC5314, respectively (Table 1). The progress of the
experiment was monitored by light microscopy. After 4 h of
incubation, a thick network of Candida cells (hyphae form) and
highly damaged mammalian cells appeared in untreated control
wells, and the experiments were therefore terminated. Cells
were then stained with Hoechst 33258, a DNA binding
fluorescent dye that labels the nuclei of dead cells with damaged
plasma membrane but not of healthy cells. Next, the cells were
fixed and stained with phalloidin, which fluorescently labels F-
actin fibers, a major constituent of the cell cytoskeleton. This
staining paradigm enabled visualization of HEK 293 cell
viability as reflected by cell morphology, exclusion of Hoechst
33258 from healthy cells, and cytoskeleton integrity.
values of cationic amphiphiles 1 and 6 and of the control
antifungal agents at the inoculum used for the flow cytometry
analysis, which was 100-fold higher than that used for the MIC
experiment (Table S7, Supporting Information). We incubated
C. glabrata ATCC 66032 (Strain F, Table 1), which exists only
in the yeast form, with compounds 1 or 6 at the MIC under
these conditions and monitored yeast cell membrane integrity
every 60 min for 6 h. At each time point, a sample was
withdrawn from the culture, stained with PI, and analyzed by
flow cytometry. From the obtained graphs of percentage of
membrane damaged cells vs time, we extracted T1/2 values,
which were defined as the time at which the membranes of 50%
of the cells were damaged (Figure 5). As positive controls we
As shown in Figure 4A and 4B, in control cultures consisting
of HEK 293 cells alone, the untreated cells were alive and
healthy, as indicated by their morphology, their phalloidin
staining pattern, and the exclusion of Hoechst 33258 dye.
Treatment with compound 1 led to substantial HEK 293 cell
death, as indicated by damaged cytoskeletons, abnormal
morphologies, and Hoechst 33258-stained nuclei (Figure 4C
and 4D). These results are in line with the results obtained
when cell viability was assessed by the MTT assay (Figure 3).
In contrast to compound 1, compound 6 did not cause any
apparent cytotoxic effect on HEK 293 cells (Figures 4E and
4F). Co-culturing HEK 293 cells with C. albicans resulted in the
appearance of a network of fungal cells and damaged HEK 293
cells (Figure 4G and 4H). Treatment of the cocultures with
compound 1 or 6 substantially reduced the numbers of fungal
cells (Figures 4I and 4K, respectively). This reduction was
accompanied by extensive damage of compound 1-treated
(Figures 4I and 4J), but not compound 6-treated, HEK 293
cells (Figures 4K and 4L).
Figure 5. C. glabrata ATCC 66032 cell cultures in stationary phase
were incubated with compound 1 or 6 for 6 h. Samples were removed
every 60 min, stained with PI, and analyzed using flow cytometry. Each
data point is based on analysis of 10,000−20,000 cells. Results shown
are averages of two independent experiments.
Taken together these results indicate that compound 6
targets the fungal cells selectively without affecting the viability
of mammalian cells. These observations are in agreement with
our findings that of the six antimicrobial cationic amphiphiles
synthesized, compound 1, composed of the saturated stearic
acid, displayed potent antifungal activity but was also the most
hemolytic and cytotoxic to the tested mammalian cells. In
contrast, compound 6, composed of linolenic acid with the
highest degree of cis unsaturation evaluated in this study,
displayed potent antifungal activity against the tested yeast
strains as well as the lowest levels of hemolysis and mammalian
cell cytotoxicity.
Cell membrane permeabilization activity. The intrigu-
ing observation that raising the degree of cis unsaturation in the
lipid segment of the antifungal cationic amphiphiles did not
affect antifungal potency but significantly reduced the toxicity
toward mammalian cells raised the question of whether or not,
like other cationic amphiphiles, compound 6 acts through
disruption of the fungal membrane. The cell permeabilizing
effects of compounds 1 and 6 were therefore evaluated using a
propidium iodide (PI) dye exclusion assay with the number of
PI-stained membrane-damaged cells quantified by flow
cytometry as previously described.49,50
used ethanol (100% permeability), and the fungal cell
membrane disrupting compound AmB. As negative controls
we sampled untreated culture and cultures treated with CASP,
which does not directly affect the fungal cell membrane.
As expected, membrane integrity was not altered in samples
treated with CASP, whereas AmB rapidly permeabilized yeast
cell membranes (Figure 5, T1/2 value of 179.7 min). The T1/2
values of compounds 1 and 6 were almost identical (72.1 and
72.3 min, respectively); thus, the results of the PI assay support
our hypothesis that, at MIC, both cationic amphiphiles 1 and 6
exert their antifungal activity by permeabilizing the fungal cell
membrane and that the degree of lipid unsaturation does not
have an effect on the mode of action or the kinetics of the in
vitro antifungal activity. Notably, compound 6 disrupted the
yeast cell membranes about 2.5-fold more rapidly than clinically
used AmB.
Finally, the results of the PI assay suggest that the reason for
the improved selectivity of antifungal cationic amphiphile 6,
with the highest degree of cis-unsaturation in its fatty acid
segment, relative to compound 1 does not result from a
difference in mode of action on fungi. The observed selectivity
presumably stems from the fundamental differences in
compositions of the plasma membranes of fungal and
mammalian cells.24 The mammalian cell plasma membrane
contains cholesterol, whereas that of the fungal cell contains
Since the antimicrobial activity of cationic amphiphiles is
affected by the inoculum,51 we initially determined the MIC
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ergosterol.31 The percentage of negatively charged lipids such
as phosphatidylinositol, which are known to interact with
antimicrobial cationic peptides, is higher in the plasma
membrane of fungal cells than in the membrane of mammalian
cells.31,52,53 These fundamental differences in membrane
composition may be the cause for the observed improvement
in the selective antifungal activity of compound 6.
were obtained from commercial sources. Compounds were
purified using flash chromatography on silica gel columns
(Merck, Kieselgel 60). The purity of compounds 1−6 was
≥95% as determined by ULC-ESI-MS. The chromatographic
separation was achieved by using a Waters e2695 HPLC
Separation Module and an XBridge Amide, 3.5 μm, 3.0 × 100
mm column. Sample aliquots of 10 μL were injected onto the
column at a flow rate of 300 μL/min. HPLC separation
conditions, and the MS data are described in the Supporting
Information.
CONCLUSIONS
■
The structures of antifungal tobramycin-derived cationic
amphiphiles were systematically altered by the attachment of
fatty acid chains differing in the degree of unsaturation and/or
double-bond configuration. We demonstrated that the degree
of unsaturation and double bond configuration had no
significant effect on antifungal activity. As the degree of
unsaturation increased, however, there was a significant
reduction in hemolysis of mammalian red blood cells and in
the harmful effect on nucleated immortalized and primary
mammalian cells’ viability. We showed that the cationic
amphiphile composed of a fatty acid with cis double bonds
caused less hemolysis than the corresponding cationic
amphiphiles composed of a fatty acid with the corresponding
trans double bonds.
Compound 1. Stearic acid (47 mg, 0.16 mmol), HATU
(78.6 mg, 0.21 mmol), and DIPEA (85 μL, 0.47 mmol) were
dissolved in DMF (2 mL) under argon atmosphere, stirred for
15 min at ambient temperature, and cooled in an ice bath.
Compound 1a (100 mg, 0.10 mmol) was then added to the
cold mixture, and upon dissolution the temperature was
allowed to reach ambient temperature. Propagation of the
reaction was monitored by TLC (EtOAc/petroleum ether,
1:1). Upon completion the reaction mixture was diluted with
EtOAc (20 mL). The organic layer was washed with 1.0 M HCl
(2 × 15 mL), saturated NaHCO3 (2 × 15 mL), and brine (2 ×
15 mL) then dried over MgSO4 and concentrated under
reduced pressure. Purification by column chromatography
(SiO2 using EtOAc/petroleum ether) gave the corresponding
NHBoc-protected product. TFA (0.5 mL) was added at
ambient temperature and, after 3 min, was removed under
reduced pressure. The residue was dissolved in a minimal
volume of H2O and freeze-dried to afford compound 1 as a
white solid (80.0 mg, 62.7%). HRESI-MS, m/z calculated for
We demonstrated the efficacy of antifungal cationic
amphiphiles in a coculture of C. albicans and mammalian
cells (HEK 293). Notably, at a concentration 16-fold higher
than its MIC against the C. albicans strain used for the coculture
experiment, cationic amphiphile 6 effectively protected the
mammalian cells from damage by Candida cells and caused lysis
of the fungal pathogen cells. Quantitative evaluation of the
membrane integrity of Candida cells treated with the reported
cationic amphiphiles revealed that these compounds rapidly
permeabilized the yeast cells and that increased degree of lipid
unsaturation did not alter the rapid membrane permeabilizing
activity of this series of compounds. The results reported herein
demonstrate that by optimizing the degree of unsaturation and
the configuration of the double bond in the lipid segment of
cationic amphiphiles it is possible to retain potent and broad
spectrum antifungal activity while reducing significantly the
membrane damage and cytotoxic effects toward mammalian
cells. We expect that these finding will lead to identification of
clinically useful broad spectrum antifungal cationic amphiphiles.
C36H73N6O9 , 733.5439; found for [M + H]+, 733.5432. H
NMR (500 MHz, D2O) δ 5.84 (d, J = 2.9 Hz, H-1′, 1H), 5.06
(d, J = 2.3 Hz, H-1’’, 1H), 4.03 (dd, J = 9.6 Hz, H-4, 1H),
3.97−3.92 (m, H-5′, H-5′’, H-2’’, 3H), 3.85 (dd, J = 9.0 Hz, H-
6, 1H), 3.79 (dd, J = 9.5 Hz, H-5, 1H), 3.72−3.39 (m, H-1, H-
3, H-2′, H-4′, H-6′, H-3′’, H-4’’, H-6’’ (2H), 9H), 3.21 (dd, J =
13.4, 7.56 Hz, H-6′, 1H), 2.56 (m, H-2eq, 1H), 2.33−2.23 (m,
stearyl chain (2H), H-3′eq, 3H), 2.06 (ddd, J = 11.3 Hz, H-
3′ax, 1H) 1.98 (ddd, J = 12.5 Hz, H-2ax, 1H), 1.56 (m, stearyl
chain, 2H), 1.28 (m, stearyl chain, 28H), 0.88 (t, J = 6.4 Hz,
stearyl chain, 3H). 13C NMR (100 MHz, D2O) δ 178.9, 164.2
(q, JCF = 35.4 Hz, CF3CO2H), 117.7 (q, JCF = 290.1 Hz,
CF3CO2H), 102.3, 95.2, 84.9, 78.6, 75.6, 72.5, 71.8, 69.5, 67.8,
66.0, 55.9, 51.2, 49.8, 49.2, 41.4, 40.3, 37.2, 33.0, 30.8, 30.5,
30.4, 30.3, 30.2, 29.1, 26.9, 23.8, 15.0.
+
1
EXPERIMENTAL SECTION
Compound 2. Elaidic acid (47 mg, 0.16 mmol), HATU
(71.0 mg, 0.19 mmol), and DIPEA (72 μL, 0.41 mmol) were
dissolved in DMF (2 mL) under argon atmosphere, stirred for
15 min at ambient temperature, and cooled in an ice bath.
Compound 1a (100 mg, 0.10 mmol) was then added to the
cold mixture, and upon dissolution the temperature was
allowed to reach ambient temperature. Propagation of the
reaction was monitored by TLC (EtOAc/petroleum ether,
1:1). Upon completion the reaction mixture was diluted with
EtOAc (20 mL). The organic layer was washed with 1.0 M HCl
(2 × 15 mL), saturated NaHCO3 (2 × 15 mL), and brine (2 ×
15 mL) then dried over MgSO4 and concentrated under
reduced pressure. Purification by column chromatography on
SiO2 using EtOAc/petroleum ether (4:10) as eluent gave the
corresponding NHBoc-protected product. TFA (0.5 mL) was
added at ambient temperature and, after 3 min, was removed
under reduced pressure. The residue was dissolved in a minimal
volume of H2O and freeze-dried to afford compound 2 as a
white solid (79.6 mg, 62.5%). HRESI-MS, m/z calculated for
■
General chemistry methods. Instrumentation and
reagents. 1H NMR and 1D-TOCSY experiments were
recorded on Bruker Avance 400 or 500 MHz spectrometers,
and chemical shifts (reported in ppm) were calibrated to D2O
or CD3OD (d = 4.79 and 3.31 ppm, respectively). 13C NMR
experiments were recorded on Bruker Avance 400 or 500 MHz
spectrometers at 100 or 125 MHz. Multiplicities are reported
using the following abbreviations: br. = broad, s = singlet, d =
doublet, dd = doublet of doublets, ddd = doublet of doublet of
doublets, t = triplet, q = quartet, m = multiplet. Coupling
constants (J) are given in Hertz. High-resolution electrospray
ionization (HRESI) mass spectra were measured on a Waters
Synapt instrument. Chemical reactions were monitored by
TLC (Merck, Silica gel 60 F254). Visualization was achieved
using a cerium molybdate stain [(NH4)2Ce(NO3)6 (5 g),
(NH4)6Mo7O24·4H2O (120 g), H2SO4 (80 mL), H2O (720
mL)]. All reactions were carried out in an argon atmosphere
with anhydrous solvents unless otherwise noted. All chemicals
G
DOI: 10.1021/acsinfecdis.7b00272
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
C36H71N6O9 , 731.5283; found for [M + H]+, 731.5286. H
NMR (500 MHz, D2O) δ 5.84 (d, J = 2.0 Hz, H-1′, 1H), 5.42
(m, elaidyl chain, 2H), 5.07 (br. s, H-1’’, 1H), 4.04 (dd, J = 9.5
Hz, H-4, 1H), 3.97−3.92 (m, H-5′, H-5′’, H-2’’, 3H), 3.85 (dd,
J = 8.8 Hz, H-6, 1H), 3.79 (dd, J = 9.7 Hz, H-5, 1H), 3.72−3.38
(m, H-1, H-3, H-2′, H-4′, H-6′, H-3′’, H-4’’, H-6’’ (2H), 9H),
3.21 (dd, J = 13.3, 7.55 Hz, H-6′, 1H), 2.56 (m, H-2eq, 1H),
2.32−2.23 (m, elaidyl chain (2H), H-3′eq, 3H), 2.06 (ddd, J =
11.7 Hz, H-3′ax, 1H) 1.97 (m, elaidyl chain (4H), H-2ax, 5H),
1.56 (m, elaidyl chain, 2H), 1.28 (m, elaidyl chain, 20H), 0.87
(t, J = 6.7 Hz, elaidyl chain, 3H). 13C NMR (125 MHz, D2O) δ
178.8, 164.2 (q, JCF = 35.0 Hz, CF3CO2H), 131.8, 131.7, 117.8
(q, JCF = 290.1 Hz, CF3CO2H), 102.3, 95.3, 84.9, 78.7, 75.6,
72.6, 71.9, 69.5, 67.8, 66.1, 56.0, 51.2, 49.8, 49.2, 41.4, 40.2,
37.2, 33.7, 33.1, 32.7, 31.1, 30.7, 30.6, 30.5, 30.3, 30.2, 30.1,
29.1, 26.9, 23.8, 15.1.
pressure. Purification by column chromatography on SiO2 using
DCM/MeOH (97:3) as eluent gave the corresponding
NHBoc-protected product. TFA (0.5 mL) was added at
ambient temperature and, after 3 min, was removed under
reduced pressure. The residue was dissolved in a minimal
volume of H2O and freeze-dried to afford compound 4 as a
white solid (99.0 mg, 78.7%). HRESI-MS, m/z calculated for
+
1
C36H69N6O9 , 729.5126; found for [M + H]+, 729.5136. H
NMR (400 MHz, D2O) δ 5.68 (d, J = 2.9 Hz, H-1′, 1H), 5.40
(m, linolelaidyl chain, 4H), 4.95 (d, J = 2.7 Hz, H-1’’, 1H),
3.87−3.75 (m, H-4, H-5′, H-2’’, H-5′’, H-6’’, 5H), 3.71 (dd, J =
8.9 Hz, H-5, 1H), 3.67−3.48 (m, H-6, H-4′, H-2’ 3H), 3.47−
3.28 (m, H-1, H-3, H-6′, H-3′’, H-4’’, H-6’’, 6H), 3.14 (dd, J =
6.3 Hz, H-6′, 1H), 2.57 (m, linolelaidyl chain, 2H), 2.36 (ddd, J
= 12.1, 3.7 Hz, H-2eq, 1H), 2.23−2.11 (m, linolelaidyl chain
(2H), H-3′eq, 3H), 1.96−1.85 (m, linolelaidyl chain (4H), H-
3′ax, 5H),1.74 (ddd, J = 12.4 Hz, H-2ax, 1H), 1.47 (m,
linolelaidyl chain, 2H), 1.18 (m, linolelaidyl chain, 14H), 0.75
(t, J = 6.6 Hz, linolelaidyl chain, 3H). 13C NMR (100 MHz,
D2O) δ 179.4, 164.3 (q, JCF = 34.4 Hz, CF3CO2H), 133.2,
133.1, 130.4, 130.3, 117.8 (q, JCF = 292.6 Hz, CF3CO2H),
102.2, 95.5, 85.4, 79.9, 75.7, 72.4, 71.6, 69.6, 68.1, 66.0, 57.9,
56.1, 51.4, 50.3, 49.9, 49.3, 41.4, 40.4, 37.2, 36.3, 33.2, 32.1,
31.0, 30.1, 29.9, 29.8, 29.7, 29.6, 26.8, 23.4, 14.9.
+
1
Compound 3. Oleic acid (47 mg, 0.16 mmol), HATU (78.6
mg, 0.21 mmol), and DIPEA (85 μL, 0.47 mmol) were
dissolved in DMF (2 mL) under argon atmosphere, stirred for
15 min at ambient temperature, and cooled in an ice bath.
Compound 1a (100 mg, 0.10 mmol) was then added to the
cold mixture, and upon dissolution the temperature was
allowed to reach ambient temperature. Propagation of the
reaction was monitored by TLC (EtOAc/petroleum ether,
1:1). Upon completion the reaction mixture was diluted with
EtOAc (20 mL). The organic layer was washed with 1.0 M HCl
(2 × 15 mL), saturated NaHCO3 (2 × 15 mL), and brine (2 ×
15 mL) then dried over MgSO4 and concentrated under
reduced pressure. Purification by column chromatography on
SiO2 using EtOAc/petroleum ether (7:10) as eluent gave the
corresponding NHBoc-protected product. TFA (0.5 mL) was
added at ambient temperature and, after 3 min, was removed
under reduced pressure. The residue was dissolved in a minimal
volume of H2O and freeze-dried to afford compound 3 as a
white solid (73.0 mg, 57.4%). HRESI-MS, m/z calculated for
Compound 5. Linoleic acid (70 mg, 0.25 mmol), HATU
(94.4 mg, 0.25 mmol), and DIPEA (144 μL, 0.83 mmol) were
dissolved in DMF (3 mL) under argon atmosphere, stirred for
15 min at ambient temperature, and cooled in an ice bath.
Compound 1a (200 mg, 0.21 mmol) was then added to the
cold mixture, and upon dissolution the temperature was
allowed to reach ambient temperature. Propagation of the
reaction was monitored by TLC (EtOAc/petroleum ether,
1:1). Upon completion the reaction mixture was diluted with
EtOAc (20 mL). The organic layer was washed with 1.0 M HCl
(2 × 15 mL), saturated NaHCO3 (2 × 15 mL), and brine (2 ×
15 mL) then dried over MgSO4 and concentrated under
reduced pressure. Purification by column chromatography on
SiO2 using EtOAc/petroleum ether (7:10) as eluent gave the
corresponding NHBoc-protected product. TFA (0.5 mL) was
added at ambient temperature and, after 3 min, was removed
under reduced pressure. The residue was dissolved in a minimal
volume of H2O and freeze-dried to afford compound 5 as a
white solid (144.8 mg, 56.9%). HRESI-MS, m/z calculated for
+
1
C36H71N6O9 , 731.5283; found for [M + H]+, 731.5285. H
NMR (400 MHz, D2O) δ 5.81 (d, J = 3.3 Hz, H-1′, 1H), 5.47
(m, oleyl chain, 2H), 5.06 (d, J = 3.8 Hz, H-1’’, 1H), 4.00−3.89
(m, H-4, H-5′, H-2’’, H-5′’, 4H), 3.84 (dd, J = 9.0 Hz, H-6,
1H), 3.78−3.64 (m, H-5, H-2′, H-4′, H-6’’, 4H), 3.62−3.41 (m,
H-1, H-3, H-6′, H-4’’, H-6’’, 6H), 3.28 (dd, J = 13.7, 6.9 Hz, H-
6′, 1H), 2.54 (ddd, J = 12.7, 3.9 Hz, H-2eq, 1H), 2.34−2.25 (m,
oleyl chain (2H), H-3′eq, 3H), 2.08−1.99 (m, oleyl chain
(4H), H-3′ax, 5H), 1.92 (ddd, J = 12.7 Hz, H-2ax, 1H) 1.58
(m, oleyl chain, 2H), 1.29 (m, oleyl chain, 20H), 0.86 (t, J = 6.8
Hz, oleyl chain, 3H). 13C NMR (100 MHz, D2O) δ 178.8,
164.2 (q, JCF = 35.2 Hz, CF3CO2H), 131.4, 131.3, 117.8 (q, JCF
= 290.2 Hz, CF3CO2H), 102.3, 95.3, 84.9, 78.7, 75.6, 72.6, 71.9,
69.5, 67.8, 66.1, 56.0, 51.2, 49.8, 49.2, 41.4, 40.2, 37.2, 33.1,
32.7, 30.9, 30.8, 30.7, 30.6, 30.5, 30.4, 30.3, 30.2, 29.2, 28.4,
28.3, 26.9, 23.8, 15.1.
+
1
C36H69N6O9 , 729.5126; found for [M + H]+, 729.5115. H
NMR (400 MHz, CD3OD) δ 5.86 (d, J = 3.2 Hz, H-1′, 1H),
5.34 (m, linoleyl chain, 4H), 5.03 (d, J = 3.6 Hz, H-1’’, 1H),
4.00−3.88 (m, H-4, H-5′, H-5′’, 3H), 3.80−3.74 (m, H-2’’, H-6,
2H), 3.70 (dd, J = 9.5 Hz, H-5, 1H), 3.64 (dd, J = 14.4, 3.4 Hz,
H-6’’, 1H), 3.59−3.50 (m, H-2′, H-4′, 2H), 3.49−3.35 (m, H-1,
H-3, H-6′, H-3′’, H-4’’, H-6’’, 6H), 3.07 (dd, J = 13.3, 8.4 Hz,
H-6′, 1H), 2.77 (dd, J = 6.3 Hz, linoleyl chain, 2H), 2.46 (ddd,
J = 12.4, 4.1 Hz, H-2eq, 1H), 2.29−2.19 (m, linoleyl chain
(2H), H-3′eq, 3H), 2.11−1.96 (m, linoleyl chain (4H), H-2ax,
H-3′ax, 6H), 1.61 (m, linoleyl chain, 2H), 1.34 (m, linoleyl
chain, 14H), 0.91 (t, J = 7.0 Hz, linoleyl chain, 3H). 13C NMR
(100 MHz, CD3OD) δ 177.8, 163.3 (q, JCF = 34.4 Hz,
CF3CO2H), 131.0, 130.8, 129.1, 129.0, 118.1(q, JCF = 290.7 Hz,
CF3CO2H), 102.4, 95.4, 85.6, 78.9, 76.1, 73.1, 71.7, 70.2, 68.8,
66.8, 56.3, 51.4, 50.0, 41.8, 40.8, 37.0, 32.6, 31.4, 30.8, 30.5,
30.4, 30.37, 30.3, 29.5, 28.2, 27.1, 26.5, 23.6, 14.4.
Compound 4. Linolelaidic acid (48 μL, 0.12 mmol), HATU
(47.2 mg, 0.12 mmol), and DIPEA (72 μL, 0.41 mmol) were
dissolved in DMF (1 mL) under argon atmosphere, stirred for
15 min at ambient temperature, and cooled in an ice bath.
Compound 1a (100 mg, 0.10 mmol) was then added to the
cold mixture, and upon dissolution the temperature was
allowed to reach ambient temperature. Propagation of the
reaction was monitored by TLC (DCM/MeOH, 95:5). Upon
completion the reaction mixture was diluted with EtOAc (20
mL). The organic layer was washed with 1.0 M HCl (2 × 15
mL), saturated NaHCO3 (2 × 15 mL), and brine (2 × 15 mL)
then dried over MgSO4 and concentrated under reduced
Compound 6. Linolenic acid (70 mg, 0.25 mmol), HATU
(94.4 mg, 0.25 mmol) and DIPEA (144 μL, 0.83 mmol) were
dissolved in DMF (3 mL) under argon atmosphere, stirred for
H
DOI: 10.1021/acsinfecdis.7b00272
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
15 min at ambient temperature, and cooled in an ice bath.
Compound 1a (200 mg, 0.21 mmol) was then added to the
cold mixture, and upon dissolution the temperature was
allowed to reach ambient temperature. Propagation of the
reaction was monitored by TLC (EtOAc/petroleum ether,
1:1). Upon completion the reaction mixture was diluted with
EtOAc (20 mL). The organic layer was washed with 1.0 M HCl
(2 × 15 mL), saturated NaHCO3 (2 × 15 mL), and brine (2 ×
15 mL) then dried over MgSO4 and concentrated under
reduced pressure. Purification by column chromatography on
SiO2 using EtOAc/petroleum ether (7:10) as eluent gave the
corresponding NHBoc-protected product. TFA (0.5 mL) was
added at ambient temperature and, after 3 min, was removed
under reduced pressure. The residue was dissolved in a minimal
volume of H2O and freeze-dried to afford compound 6 as a
white solid (173.0 mg, 68.1%). HRESI-MS, m/z calculated for
Tested compounds were added to RPMI 1640 media to form
the mother liquors (32 μL of stock solution in 1218 μL of
RPMI 1640) at a concentration of 128 μg/mL. Next, 100 μL
serial double dilutions of the tested compounds (128, 64, 32,
16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/mL) were prepared in
RPMI 1640 in flat-bottomed 96-well microplates.
An equal volume (100 μL) of yeast cell suspension in RPMI
1640 was added to each well for a final volume of 200 μL. Final
inoculum was typically between 5 × 104 and 5 × 105 CFU/mL.
Control wells containing only RPMI 1640 media (blanks) or
only fungal cells in RPMI 1640 media were also prepared. After
incubation at 37 °C in 5% CO2 (24 h for Candida species and
72 h for C. neoformans), MTT (50 μL of a 1 mg/mL solution in
H2O) was added to each well followed by additional incubation
at 37 °C for 2 h. The MIC value was defined as the lowest
concentration of compound in which no fungal growth was
observed. Results were obtained from two independent
experiments, and each drug concentration was tested in
triplicate in each experiment.
Minimal inhibitory concentration test for antibacterial
activity. MICs were determined for compounds 1 and 6 against
Staphylococcus epidermidis ATCC 12228 (A), S. aureus ATCC
9144 (B), Enterococcus faecalis ATCC 29212 (C), Pseudomonas
aeruginosa Migula ATCC 47085 (D), Acinetobacter baumannii
ATCC 19606 (E), and E. coli ATCC 25922 (F) (Table S4,
Supporting Information). Compounds were tested using the
broth double-dilution method in 96-well plates (Corning). For
inoculum preparation, starter cultures were incubated for 24 h
(37 C, 5% CO2, aerobic conditions) and then diluted in fresh
Lysogeny Broth (LB) to obtain an optical density of 0.008
(OD600).
Tested compounds were added to LB to form mother liquors
(32 μL of stock solution in 1218 μL of LB) at a concentration
of 128 μg/mL. Next, 100 μL serial double dilutions of the
tested compounds (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and
0.125 μg/mL) were prepared in LB in flat-bottomed 96-well
microplates. An equal volume (100 μL) of bacterial suspension
in LB was added to each well for a final volume of 200 μL and a
final OD600 of 0.004. Control wells containing only LB (blanks)
or only bacteria in LB were also prepared. After 24 h, MTT (50
μL of a 1 mg/mL solution in H2O) was added to each well
followed by additional incubation at 37 °C for 2 h. The MIC
value was defined as the lowest concentration of compound in
which no bacterial growth was observed. Results were obtained
from two independent experiments, and each drug concen-
tration was tested in triplicate in each experiment.
C36H67N6O9 , 727.4970; found for [M + H]+, 727.4964. H
NMR (400 MHz, CD3OD) δ 5.90 (d, J = 3.5 Hz, H-1′, 1H),
5.34 (m, linolenyl chain, 6H), 5.03 (d, J = 3.6 Hz, H-1’’, 1H),
4.09 (dd, J = 9.1, 10.0 Hz, H-4, 1H), 3.97−3.88 (m, H-5′, H-5′’,
2H), 3.81−3.77 (m, H-2’’, H-6, 2H), 3.72 (dd, J = 9.8 Hz, H-5,
1H), 3.64 (dd, J = 14.6, 3.3 Hz, H-6’’, 1H), 3.59−3.46 (m, H-1,
H-3, H-2′, H-4′, 4H), 3.43−3.35 (m, H-6′, H-3′’, H-4’’, H-6’’,
4H), 3.08 (dd, J = 13.3, 8.4 Hz, H-6′, 1H), 2.82−2.79 (m,
linolenyl chain, 4H), 2.52 (ddd, J = 12.5, 4.2 Hz, H-2eq, 1H),
2.25−2.19 (m, linolenyl chain (2H), H-3′eq, 3H), 2.16−2.05
(m, linolenyl chain (4H), H-2ax, H-3′ax, 6H), 1.60 (m, linoleyl
chain, 2H), 1.33 (m, linolenyl chain, 8H), 0.97 (t, J = 7.5 Hz,
linolenyl chain, 3H). 13C NMR (100 MHz, CD3OD) δ 177.8,
163.3 (q, JCF = 34.5 Hz, CF3CO2H), 132.7, 131.0, 129.2, 129.1,
128.9, 128.2, 118.1(q, JCF = 290.8 Hz, CF3CO2H), 102.4, 95.4,
85.5, 78.8, 76.1, 73.1, 71.8, 70.2, 68.7, 66.7, 56.3, 51.4, 50.0,
41.8, 40.8, 37.0, 31.3, 30.7, 30.4, 30.3, 30.2, 29.4, 28.2, 27.1,
26.5, 26.4, 21.5, 14.6.
General biological evaluation methods. Preparation of
test compound stock solutions. The antifungal agents FLC,
CASP, and AmB were purchased from Sigma-Aldrich and were
dissolved in anhydrous absolute ethanol, H2O (18 MΩ), and
DMSO, respectively, to give final concentrations of 5 mg/mL.
Compounds 1-6 were dissolved in H2O (18 MΩ) to give final
concentrations of 5 mg/mL.
Fungal strains. The yeast strains (Table 1) C. albicans
ATCC 90028 (A), C. albicans ATCC 24433 (B), C. parapsilosis
ATCC 90018 (D), C. glabrata ATCC 66032 (I), C. albicans
ATCC 96901 (M), C. albicans ATCC 64124 (N), C. albicans
ATCC 11651 (O), C. neoformans ATCC 32045 (R), and C.
neoformans ATCC 66031 (S), were purchased from the ATCC.
The yeast strains C. albicans ATCC SC5314 (C), C. tropicalis
clinical isolate 660 (E), C. dubliniensis Wu284 (F), C.
guilliermondii B3163 (G), C. albicans clinical isolate 280 (H),
C. glabrata clinical isolate 50 (J), C. glabrata ATCC 2001 (K),
C. glabrata 195 (L), C. albicans clinical isolate 2066 (P), and C.
albicans clinical isolate 2068 (Q) were provided by Prof. Judith
Berman from the George S. Wise Faculty of Life Sciences, Tel
Aviv University.
+
1
Erythrocyte hemolysis assay. Laboratory rat RBCs (2% w/
w) were incubated with each dose of the tested compounds for
1 h at 37 °C in 5% CO2. Compounds were tested in 96-well
plates (Corning) using the double-dilution method starting at a
concentration of 192 or 128 μg/mL. The negative control was
PBS (without compounds), and the positive control was a 1%
v/v solution of Triton X100 (100% hemolysis). Following
centrifugation (2000 rpm, 10 min, 10 °C), 100 μL of the
supernatant was transferred into a 96-well plate, and
absorbance at 550 nm was measured using a microplate reader
(Genios, TECAN). The results are expressed as percentage of
hemoglobin released relative to the positive control (Triton
X100). Each concentration was tested in triplicate, and the
Minimal inhibitory concentration test for antifungal
activity. Compounds were tested using the broth double-
dilution method in 96-well plates (Corning). For inoculum
preparation, starter cultures of yeast strains, Candida or C.
neoformans spp., were incubated in RPMI 1640 for 24 or 72 h
respectively (37 °C, 5% CO2, aerobic conditions) and then
diluted 1:100 into fresh medium.
results are expressed as means
standard error from two
independent experiments. Each independent experiment was
performed on a blood sample taken from a different laboratory
rat.
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Mammalian cell viability assay. Cell lines (HEK 293,
HepG2, and 3T9MEF) and primary cells (mouse primary
dermal fibroblasts) were grown in DMEM supplemented with
10% FCS and 1% penicillin−streptomycin in a humidified
chamber of 95% air, 5% CO2 at 37 °C. For the MTT assay, cells
were seeded into 96-well microtiter plates after calibrating the
amount of cells which yield subconfluent culture and an
appropriate MTT signal. Accordingly 48 × 103 or 24 × 103 cells
per well for cell lines or primary cells, respectively, were seeded
in 200 μL/well of medium and allowed to adhere for 24 h.
Thereafter, solutions containing the tested compounds (10 μL
per well) were added to the cells at final concentrations ranging
from 2 to 256 μg/mL, and cultures were incubated for an
additional 24 h. Cell viability was determined by the MTT assay
as follows: MTT was added to the medium at a final
concentration of 0.25 mg/mL, followed by a 2-h incubation.
The medium was then removed by aspiration, and DMSO (100
μL per well) was added. Absorbance at 570 nm in each well was
determined using a Tecan Spectra Fluor microplate reader with
a reference wavelength of 630 nm. Each compound was tested
in triplicate, and the results are expressed as means standard
error from at least three independent experiments.
300 μL/well). After incubation for 30 min at room
temperature, wells were washed twice with Buffer A. The
coverslips were then taken out of the wells and mounted
onto microscope slides using ProLong Gold Antifade
Mountant. The cells were photographed with an
Olympus motorized inverted research microscope
Model IX81 (60× magnification).
Propidium iodide-based membrane permeabilization
assay. C. glabrata ATCC 66032 cells were grown in RPMI at
37 °C, 5% CO2 overnight. Tested drugs and synthesized
compounds were added to 500 μL of cell culture at final
concentrations of 2, 4, 16, and 64 μg/mL for CASP, Amp B,
compound 1, and compound 6, respectively. Cultures were
incubated with the compounds for an additional 6 h at 37 °C.
An aliquot of 10 μL was collected from each of the culture
tubes every 60 min and added to 80 μL of TE 50:50 buffer (50
mM Tris HCl pH 8.0, 50 mM EDTA) and 10 μL of propidium
iodide stain at a final PI concentration of 1 mg/mL. Cells
treated with ethanol (which results in 100% cell permeability)
were prepared as follows: 500 μL of the cell suspension were
centrifuged for 4 min at 10,000 rpm (Eppendorf, Centrifuge
5424). The supernatant was removed and resuspended in 100
μL of 96% ethanol. After 5 min, the ethanol suspension was
centrifuged for 4 min at 10,000 rpm. The supernatant (ethanol)
was removed, and the pellet was resuspended in 500 μL of
RPMI. Flow cytometry data were collected from 10,000 to
20,000 cells per time point using B2 laser excitation (excitation
at 488 nm and emission at 614/50 nm) on a MACSQuant flow
cytometer. Analysis was performed using FlowJo 8.7 software.
Coculture of HEK 293 and C. albicans cells.
A Coating microscope cover glass with poly-D-lysine (PDL).
Microscope coverslips (18 mm diameter and 0.13−0.17
mm thickness) were placed in a 12-well plate (one slide
in each well), covered with PDL solution (1 mL/well, 50
mg/mL), and incubated for 45 min. The PDL was then
removed by aspiration, and the coverslips were sterilized
and dried under UV light for an additional 20 min.
Finally coverslips were rinsed with PBS (1 mL/well).
B Fungal cell growth conditions. C. albicans SC5314 cells
were grown in the yeast form on YPAD agar plates (yeast
peptone dextrose plus 40 mg/L adenine and 80 mg/L
uridine) at 30 °C overnight. An aliquot of 1 mL DMEM
was then inoculated with a loopful of cells from the agar
plate, and number of cells was determined by measuring
OD600 (OD600 value of 1 equals 3 × 107 cells/mL).
C Mammalian cell growth conditions. HEK 293 cells were
grown on PDL-coated microscope coverslips (6 × 105
cells per well) in DMEM supplemented with 10% FCS
and 1% penicillin-streptomycin in a humidified chamber
of 95% air, 5% CO2 at 37 °C. The HEK 293 cells were
allowed to adhere for 24 h.
D Incubation of mammalian cells with fungi cells. After the
adherence of the HEK 293 cells to the coverslips, C.
albicans SC5314 cells at a multiplicity of infection of 2
(i.e., 12 × 105 cells per well) were added on top of the
HEK 293 cells. After 30 min of incubation the coculture
was treated with compound 1 or 6 or left untreated and
was further incubated for 4 h.
E Cell staining. Hoechst 33258 was added to each well at a
final concentration of 1 μg/mL, and the plate was
incubated for 10 min at 37 °C. Medium was removed by
aspiration, and wells were gently rinsed with PBS.
Paraformaldehyde (4%, 0.5 mL) was then added to each
well, and after 30 min of incubation at room temperature
it was removed, and wells were washed twice with Buffer
A (TBS X1, 2 mM CaCl2). Thereafter, TritonX 0.1% in
buffer A (0.5 mL) was added, and after 10 min of
incubation at room temperature wells were washed twice
with Buffer A. Finally phalloidin was added (1 μg/mL,
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsinfec-
dis.7b00272.
■
S
1H and 13C NMR spectra, ULC-MS traces, compound
purity information, HC50 and IC50 values with SD,
antibacterial activity data, inoculum dependent MIC data
and mammalian cell viability curves are provided as
Supporting Information. (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone +972 3 6408687; E-mail: mfridman@post.tau.ac.il.
ORCID
■
Micha Fridman: 0000-0002-2009-7490
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Israel Science Foundation Grant
6/14 and by the Israel Ministry of Science Technology &
Space, Grant 48966.
ABBREVIATIONS USED
■
AG, aminoglycoside; AmB, amphotericin B; CASP, caspofun-
gin; FLC, fluconazole; TIBSCl, 2,4,6-triisopropylbenzenesul-
fonyl chloride; HC50, concentrations at which 50% of RBCs
were lysed; IC50, concentration toxic to 50% of cells; log P,
partition coefficient; MIC, minimal inhibitory concentration;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
J
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interactions with lipopolysaccharides. Antimicrob. Agents Chemother. 58
(8), 4420−4430.
(19) Berkov-Zrihen, Y., Herzog, I. M., Benhamou, R. I., Feldman, M.,
Steinbuch, K. B., Shaul, P., Lerer, S., Eldar, A., and Fridman, M. (2015)
Tobramycin and Nebramine as Pseudo-oligosaccharide Scaffolds for
the Development of Antimicrobial Cationic Amphiphiles. Chem. - Eur.
J. 21 (11), 4340−4349.
(20) Benhamou, R. I., Shaul, P., Herzog, I. M., and Fridman, M.
(2015) Di-N-Methylation of Anti-Gram-Positive Aminoglycoside-
Derived Membrane Disruptors Improves Antimicrobial Potency and
Broadens Spectrum to Gram-Negative Bacteria. Angew. Chem., Int. Ed.
54 (46), 13617−13621.
(21) Zimmermann, L., Das, I., Desire, J., Sautrey, G., Vinicius Barros,
R. S., El Khoury, M., Mingeot-Leclercq, M. P., and Decout, J. L.
(2016) New Broad-Spectrum Antibacterial Amphiphilic Aminoglyco-
sides Active against Resistant Bacteria: From Neamine Derivatives to
Smaller Neosamine Analogues. J. Med. Chem. 59 (20), 9350−9369.
(22) Chang, C.-W. T., Fosso, M., Kawasaki, Y., Shrestha, S., Bensaci,
M. F., Wang, J., Evans, C. K., and Takemoto, J. Y. (2010) Antibacterial
to antifungal conversion of neamine aminoglycosides through alkyl
modification. Strategy for reviving old drugs into agrofungicides. J.
Antibiot. 63 (11), 667−672.
bromide; PI, propidium iodide; RBC, red blood cells; TOB,
tobramycin; T1/2, time at which the membranes of 50% of the
cells were damaged.
REFERENCES
■
(1) Delaloye, J., and Calandra, T. (2014) Invasive candidiasis as a
cause of sepsis in the critically ill patient. Virulence 5 (1), 161−169.
(2) Perlroth, J., Choi, B., and Spellberg, B. (2007) Nosocomial fungal
infections: epidemiology, diagnosis, and treatment. Med. Mycol. 45,
321−346.
(3) Benhamou, R. I., Bibi, M., Steinbuch, K. B., Engel, H., Levin, M.,
Roichman, Y., Berman, J., and Fridman, M. (2017) Real-Time Imaging
of the Azole-Class of Antifungal Drugs in Live Candida Cells. ACS
Chem. Biol. 12 (7), 1769−1777.
(4) Holz, R., and Finkelstein, A. (1970) The Water and
Nonelectrolyte Permeability Induced in Thin Lipid Membranes by
the Polyene Antibiotics Nystatin and Amphotericin B. J. Gen. Physiol.
56, 125−145.
(5) Bolard, J. (1986) How do the polyene macrolide antibiotics affect
the cellular membrane properties? Biochim. Biophys. Acta, Rev.
Biomembr. 864, 257−304.
(23) Liu, R., Chen, X., Hayouka, Z., Chakraborty, S., Falk, S. P.,
Weisblum, B., Masters, K. S., and Gellman, S. H. (2013) Nylon-3
polymers with selective antifungal activity. J. Am. Chem. Soc. 135 (14),
5270−5273.
(6) Ghannoum, M. A., and Rice, L. B. (1999) Antifungal agents:
mode of action, mechanisms of resistance, and correlation of these
mechanisms with bacterial resistance. Clin. Microbiol. Rev. 12 (4), 501−
517.
(24) Shrestha, S., Grilley, M., Fosso, M. Y., Chang, C. W. T., and
Takemoto, J. Y. (2013) Membrane Lipid-Modulated Mechanism of
Action and Non-Cytotoxicity of Novel Fungicide Aminoglycoside
FG08. PLoS One 8 (9), 3−10.
(7) Szpilman, A. M., and Carreira, E. M. (2010) Probing the biology
of natural products: Molecular editing by diverted total synthesis.
Angew. Chem., Int. Ed. 49 (50), 9592−9628.
(8) Letscher-Bru, V., and Herbrecht, R. (2003) Caspofungin: The
first representative of a new antifungal class. J. Antimicrob. Chemother.
51 (3), 513−521.
(9) Ngo, H. X., Garneau-Tsodikova, S., and Green, K. D. (2016) A
complex game of hide and seek: the search for new antifungals.
MedChemComm 7 (7), 1285−1306.
(10) Gauwerky, K., Borelli, C., and Korting, H. C. (2009) Targeting
virulence: A new paradigm for antifungals. Drug Discovery Today 14
(3−4), 214−222.
(11) Anderson, J. B. (2005) Evolution of antifungal-drug resistance:
mechanisms and pathogen fitness. Nat. Rev. Microbiol. 3 (7), 547−556.
(12) Bera, S., Zhanel, G. G., and Schweizer, F. (2008) Design,
synthesis, and antibacterial activities of neomycin-lipid conjugates:
Polycationic lipids with potent gram-positive activity. J. Med. Chem. 51
(19), 6160−6164.
(13) Zhang, J., Chiang, F.-I., Wu, L., Przemyslaw Greg Czyryca, D. L.,
and Chang, C.-W. T. (2008) Surprising Alteration of Antibacterial
Activity of 5′’-Modified Neomycin against Resistant Bacteria. J. Med.
Chem. 51 (23), 7563−7573.
(14) Bera, S., Zhanel, G. G., and Schweizer, F. (2010) Antibacterial
activities of aminoglycoside antibiotics-derived cationic amphiphiles.
Polyol-modified neomycin B-, Kanamycin A-, Amikacin-, and Ne-
amine-based amphiphiles with potent broad spectrum antibacterial
activity. J. Med. Chem. 53 (9), 3626−3631.
(15) Bera, S., Zhanel, G. G., and Schweizer, F. (2010) Antibacterial
activity of guanidinylated neomycin B- and kanamycin A-derived
amphiphilic lipid conjugates. J. Antimicrob. Chemother. 65 (6), 1224−
1227.
(25) Shrestha, S. K., Fosso, M. Y., and Garneau-Tsodikova, S. (2015)
A combination approach to treating fungal infections. Sci. Rep. 5
(17070), 17070.
(26) Fosso, M. Y., Shrestha, S. K., Green, K. D., and Garneau-
Tsodikova, S. (2015) Synthesis and Bioactivities of Kanamycin B-
Derived Cationic Amphiphiles. J. Med. Chem. 58 (23), 9124−9132.
́
(27) Mingeot-Leclercq, M.-P., and Decout, J.-L. (2016) Bacterial
lipid membranes as promising targets to fight antimicrobial resistance,
molecular foundations and illustration through the renewal of
aminoglycoside antibiotics and emergence of amphiphilic aminoglyco-
sides. MedChemComm 7 (4), 586−611.
(28) Shrestha, S. K., Grilley, M., Anderson, T., Dhiman, C., Oblad, J.,
Chang, C. W. T., Sorensen, K. N., and Takemoto, J. Y. (2015) In vitro
antifungal synergy between amphiphilic aminoglycoside K20 and
azoles against Candida species and Cryptococcus neoformans. Med.
Mycol. 53 (8), 837−844.
(29) Zhang, Q., Alfindee, M. N., Shrestha, J. P., Nziko, V. D. P. N.,
Kawasaki, Y., Peng, X., Takemoto, J. Y., and Chang, C. W. T. (2016)
Divergent Synthesis of Three Classes of Antifungal Amphiphilic
Kanamycin Derivatives. J. Org. Chem. 81 (22), 10651−10663.
(30) Benhamou, R. I., Steinbuch, K. B., and Fridman, M. (2016)
Antifungal Imidazole-Decorated Cationic Amphiphiles with Markedly
Low Hemolytic Activity. Chem. - Eur. J. 22 (32), 11148−11151.
(31) van Meer, G., Voelker, D. R., and Feigenson, G. W. (2008)
Membrane lipids: where they are and how they behave. Nat. Rev. Mol.
Cell Biol. 9 (2), 112−124.
(32) Escriba,
J., Vigh, L., San
Horvath, I., and Barcelo-
point for lipids, proteins and therapies: Translational Medicine. J. Cell.
Mol. Med. 12 (3), 829−875.
(33) Los, D. A., and Murata, N. (2004) Membrane fluidity and its
roles in the perception of environmental signals. Biochim. Biophys. Acta,
Biomembr. 1666 (1−2), 142−157.
́
P. V., Gonzal
chez-Magraner, L., Fernan
Coblijn, G. (2008) Membranes: A meeting
́
ez-Ros, J. M., Goni, F. M., Kinnunen, P. K.
̃
́
́
dez, A. M., Busquets, X.,
(16) Dhondikubeer, R., Bera, S., Zhanel, G. G., and Schweizer, F.
(2012) Antibacterial activity of amphiphilic tobramycin. J. Antibiot. 65
(10), 495−498.
́
́
(17) Zimmermann, L., Bussier
Jolivalt, C., Mingeot-Leclercq, M. P., and Dec
the antibacterial activity of amphiphilic neamine derivatives and
comparison to paromamine homologues. J. Med. Chem. 56 (19),
7691−7705.
̀
e, A., Ouberai, M., Baussanne, I.,
out, J. L. (2013) Tuning
́
(34) Quinn, P. J., Joo, F., and Vigh, L. (1989) the Role of
Unsaturated Lipids in Membrane-Structure and Stability. Prog. Biophys.
Mol. Biol. 53 (2), 71−103.
(35) Spector, A. A., and Yorek, M. A. (1985) Membrane lipid
composition and cellular function. J. Lipid Res. 26 (9), 1015−1035.
(18) Sautrey, G., Zimmermann, L., Deleu, M., Delbar, A., Souza
Machado, L., Jeannot, K., Van Bambeke, F., Buyck, J. M., Decout, J.-L.,
and Mingeot-Leclercq, M.-P. (2014) New amphiphilic neamine
derivatives active against resistant Pseudomonas aeruginosa and their
K
DOI: 10.1021/acsinfecdis.7b00272
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
(36) Herzog, I. M., Green, K. D., Berkov-Zrihen, Y., Feldman, M.,
Vidavski, R. R., Eldar-Boock, A., Satchi-Fainaro, R., Eldar, A., Garneau-
Tsodikova, S., and Fridman, M. (2012) 6″-thioether tobramycin
analogues: Towards selective targeting of bacterial membranes. Angew.
Chem., Int. Ed. 51 (23), 5652−5656.
(37) Herzog, I. M., Feldman, M., Eldar-Boock, A., Satchi-Fainaro, R.,
and Fridman, M. (2013) Design of membrane targeting tobramycin-
based cationic amphiphiles with reduced hemolytic activity.
MedChemComm 4 (1), 120−124.
(38) Rodríguez-Tudela, J. L., Barchiesi, F., Bille, J., Chryssanthou, E.,
Cuenca-Estrella, M., Denning, D., Donnelly, J. P., Dupont, B., Fegeler,
W., Moore, C., et al. (2003) Method for the determination of
minimum inhibitory concentration (MIC) by broth dilution of
fermentative yeasts. Clin. Microbiol. Infect. 9 (8), 1−8.
(39) Brown, G. D., Denning, D. W., Gow, N. A. R., Levitz, S. M.,
Netea, M. G., and White, T. C. (2012) Hidden Killers: Human Fungal
Infections. Sci. Transl. Med. 4 (165), 165rv13−165rv13.
(40) Pfaller, M. A., and Diekema, D. J. (2007) Epidemiology of
invasive candidiasis: A persistent public health problem. Clin.
Microbiol. Rev. 20 (1), 133−163.
(41) Balashov, S. V., Park, S., and Perlin, D. S. (2006) Assessing
resistance to the echinocandin antifungal drug caspofungin in Candida
albicans by profiling mutations in FKS1. Antimicrob. Agents Chemother.
50 (6), 2058−2063.
(42) Schmitt, M. A., Weisblum, B., and Gellman, S. H. (2007)
Interplay among folding, sequence, and lipophilicity in the antibacterial
and hemolytic activities of α/β-peptides. J. Am. Chem. Soc. 129 (2),
417−428.
(43) Niu, Y., Padhee, S., Wu, H., Bai, G., Qiao, Q., Hu, Y.,
Harrington, L., Burda, W. N., Shaw, L. N., Cao, C., et al. (2012) Lipo-
γ-AApeptides as a new class of potent and broad-spectrum
antimicrobial agents. J. Med. Chem. 55 (8), 4003−4009.
(44) Konai, M. M., and Haldar, J. (2015) Lysine-Based Small
Molecules That Disrupt Biofilms and Kill both Actively Growing
Planktonic and Nondividing Stationary Phase Bacteria. ACS Infect. Dis.
1 (10), 469−478.
(45) Svenson, J., Brandsdal, B. O., Stensen, W., and Svendsen, J. S.
(2007) Albumin binding of short cationic antimicrobial micropeptides
and its influence on the in vitro bactericidal effect. J. Med. Chem. 50
(14), 3334−3339.
(46) Lopes, J. P., Stylianou, M., Nilsson, G., and Urban, C. F. (2015)
Opportunistic pathogen Candida albicans elicits a temporal response
in primary human mast cells. Sci. Rep. 5, 12287.
́ ́
(47) Moreno-Ruiz, E., Galan-Díez, M., Zhu, W., Fernandez-Ruiz, E.,
D’Enfert, C., Filler, S. G., Cossart, P., and Veiga, E. (2009) Candida
albicans internalization by host cells is mediated by a clathrin-
dependent mechanism. Cell. Microbiol. 11 (8), 1179−1189.
(48) Lischewski, A., Amann, R. I., Harmsen, D., Merkert, H., Hacker,
J., and Morschhauser, J. (1996) Specific detection of Candida albicans
̈
and Candida tropicalis by fluorescent in situ hybridization with an 18S
rRNA-targeted oligonucleotide probe. Microbiology 142 (10), 2731−
2740.
(49) Pina-Vaz, C., Sansonetty, F., Rodrigues, A. G., Costa-Oliveira, S.,
Tavares, C., and Martinez-De-Oliveira, J. (2001) Cytometric approach
for a rapid evaluation of susceptibility of Candida strains to antifungals.
Clin. Microbiol. Infect. 7 (11), 609−618.
(50) Shapiro, H. M. Practical Flow Cytometry; Wiley-Liss, 2003.
(51) Chin, J. N., Rybak, M. J., Cheung, C. M., and Savage, P. B.
(2007) Antimicrobial activities of ceragenins against clinical isolates of
resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 51 (4),
1268−1273.
(52) Makovitzki, A., Avrahami, D., and Shai, Y. (2006) Ultrashort
antibacterial and antifungal lipopeptides. Proc. Natl. Acad. Sci. U. S. A.
103 (43), 15997−16002.
́
(53) Ingolfsson, H. I., Melo, M. N., Van Eerden, F. J., Arnarez, C.,
Lopez, C. A., Wassenaar, T. A., Periole, X., De Vries, A. H., Tieleman,
D. P., and Marrink, S. J. (2014) Lipid organization of the plasma
membrane. J. Am. Chem. Soc. 136 (41), 14554−14559.
L
DOI: 10.1021/acsinfecdis.7b00272
ACS Infect. Dis. XXXX, XXX, XXX−XXX