Amphiphilic tobramycin analogues as antibacterial and antifungal agents

Article abstract of DOI:10.1128/AAC.00229-15

In this study, we investigated the in vitro antifungal activities, cytotoxicities, and membrane-disruptive actions of amphiphilic tobramycin (TOB) analogues. The antifungal activities were established by determination of MIC values and in time-kill studie

Full text of DOI:10.1128/AAC.00229-15

Amphiphilic Tobramycin Analogues as Antibacterial and Antifungal
Agents
Sanjib K. Shrestha, Marina Y. Fosso, Keith D. Green, Sylvie Garneau-Tsodikova
Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky, USA
In this study, we investigated the in vitro antifungal activities, cytotoxicities, and membrane-disruptive actions of amphiphilic
tobramycin (TOB) analogues. The antifungal activities were established by determination of MIC values and in time-kill studies.
Cytotoxicity was evaluated in mammalian cell lines. The fungal membrane-disruptive action of these analogues was studied by
using the membrane-impermeable dye propidium iodide. TOB analogues bearing a linear alkyl chain at their 6؆-position in a
thioether linkage exhibited chain length-dependent antifungal activities. Analogues with C12 and C14 chains showed promising
antifungal activities against tested fungal strains, with MIC values ranging from 1.95 to 62.5 mg/liter and 1.95 to 7.8 mg/liter,
respectively. However, C4, C6, and C8 TOB analogues and TOB itself exhibited little to no antifungal activity. Fifty percent inhib-
itory concentrations (IC50s) for the most potent TOB analogues (C12 and C14) against A549 and Beas 2B cells were 4- to 64-fold
and 32- to 64-fold higher, respectively, than their antifungal MIC values against various fungi. Unlike conventional aminoglyco-
side antibiotics, TOB analogues with alkyl chain lengths of C12 and C14 appear to inhibit fungi by inducing apoptosis and dis-
rupting the fungal membrane as a novel mechanism of action. Amphiphilic TOB analogues showed broad-spectrum antifungal
activities with minimal mammalian cell cytotoxicity. This study provides novel lead compounds for the development of antifun-
gal drugs.
he frequency of invasive fungal infections, such as candidiasis, drug TOB itself (7). However, the antifungal activities of these
T
has dramatically increased due to the rising populations of analogues are yet to be determined. It was recently reported that
the incorporation of a C8 linear alkyl chain at the O-4Љ-position
and of an octanesulfonyl chain at the O-6=-position of kanamycins
A and B, respectively, led to the discovery of new applications of
these AGs as antifungal agents that lack bacterial activity (8–11).
This prompted us to investigate the antifungal properties of our
6Љ-thioether TOB analogues with linear alkyl chains with chain
lengths between C4 and C14 (here, we are referring to these as C4,
C6, C8, C10, C12, and C14) (Fig. 1) and to further investigate the
extent of the antibacterial spectra of these analogues against resis-
tant clinical bacterial isolates that we had not previously tested.
We report the antibacterial and antifungal activities as well as the
cytotoxicities of our C4 to C14 TOB analogues.
immunocompromised patients as a result of AIDS and cancer
therapy, as well as bone marrow and organ transplantations (1).
Candida spp. are known to cause the majority of fungal infections
and are the fourth most common cause of nosocomial blood in-
fection in the United States (2). However, other fungal pathogens,
such as Candida glabrata, Candida parapsilosis, Candida tropicalis,
Candida lusitaniae, Crytococcus neoformans, and Aspergillus spp.,
are also on the rise and are causing threats to human and animal
health (3). Currently, a number of antifungal drugs, such as azoles,
echinocandins, and amphotericin B (AmB), are used to treat in-
vasive fungal infections, including candidiasis, in humans. Grow-
ing fungal resistance and host side effects, however, limit their
therapeutic efficacies. There is therefore a growing need to de-
velop novel antifungal drugs.
MATERIALS AND METHODS
Aminoglycosides (AGs) are compounds that consist of two or
more amino sugars that are connected to a 2-deoxystreptamine
scaffold via glycosidic bonds. AGs, such as tobramycin (TOB), are
well-known antibiotics that are used to treat bacterial infections in
humans, but they do not inhibit the growth of fungi. They bind to
the prokaryotic 16S rRNA in the decoding A-site region, which
induces codon misreading and inhibits translocation (4, 5). Al-
though AGs are predominantly known for their antibacterial ac-
tivities, some conventional AGs have been reported to have anti-
fungal activities against fungal oomycetes and other true fungi (6).
Despite being potent antibiotics, emerging bacterial resistance
against AGs has compromised their therapeutic use. However,
there has been a growing interest in structural modifications of
AGs that could revive the efficacy of these drugs against resistant
bacterial strains. We recently demonstrated that modifying TOB
at the 6Љ-position by incorporating linear alkyl chains (C6 to C22)
in a thioether linkage resulted in chain length-dependent antibac-
terial activities against vancomycin-resistant enterococci (VRE)
and TOB-resistant Escherichia coli, with resistance to the parent
Materials. TOB was purchased from AK Scientific (Union City, CA). All
other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and
used without further purification. Compound 1 (Fig. 1) was prepared as
previously described (7). Chemical reactions were monitored via thin-
layer chromatography (TLC; with silica gel 60, F250; Merck). Visualization
was achieved by using one of the following methods: H2SO4 stain (5% in
methanol) or KMnO4 stain (1.5 g KMnO4, 10 g K2CO3, 1.25 ml 10%
NaOH, 200 ml H2O). Compounds were purified by SiO2 flash chroma-
Received 28 January 2015 Returned for modification 7 March 2015
Accepted 28 May 2015
Accepted manuscript posted online 1 June 2015
Citation Shrestha SK, Fosso MY, Green KD, Garneau-Tsodikova S. 2015.
Amphiphilic tobramycin analogues as antibacterial and antifungal agents.
Antimicrob Agents Chemother 59:4861–4869. doi:10.1128/AAC.00229-15.
Address correspondence to Sylvie Garneau-Tsodikova, sylviegtsodikova@uky.edu.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.00229-15
August 2015 Volume 59 Number 8
Antimicrobial Agents and Chemotherapy
aac.asm.org 4861

Shrestha et al.
FIG 1 Scheme for the synthesis of the 6Љ-thioether TOB analogues C4 to C14 used in this study.
tography (Flash silica gel, 32 to 63 ␮m; Dynamic Adsorbents Inc.). 1H and UT, USA). The yeast strains C. albicans ATCC MYA-90819 (D), C. albi-
13C nuclear magnetic resonance (NMR) spectra were recorded on a Var- cans ATCC MYA-2310 (E), C. albicans ATCC MYA-1237 (F), C. albicans
ian 400-MHz spectrometer. TOB analogues (C4 to C14) were dissolved in
double-distilled water (ddH2O) at a final concentration of 10 g/liter.
The Eis (12), AAC(6=)-Ib= (13), AAC(6=)-Ie/APH(2Љ)-Ia [used only
for its AAC(6=) activity] (14), AAC(3)-IV (14), AAC(2=)-Ic (12), and
ANT(4=) (15) resistance enzymes were purified as previously described.
5,5=-Dithiobis(2-nitrobenzoic acid) (DTNB), ATP, acetyl coenzyme A,
and inorganic pyrophosphatase were bought from Sigma-Aldrich and
used without further purification. The determinations of the activities of
these resistance enzymes against C4 were performed as previously re-
ported for a group of C6 to C14 antimicrobial agents (Fig. 2) (7). 3-(4,5-
Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide was pur-
chased from TCI America (Portland, OR, USA). Spectrophotometric and
colorimetric assays were performed on a multimode SpectraMax M5 plate
reader (Molecular Devices, Sunnyvale, CA) using 96-well plates (Fisher
Scientific).
The antifungal agents posaconazole (POS), itraconazole (ITC), and
fluconazole (FLC) were obtained from AK Scientific, Inc. (Union City,
CA). POS, ITC, and FLC were dissolved in dimethyl sulfoxide (DMSO) at
a final concentration of 5 g/liter.
The yeast strains Candida albicans ATCC 10231 (designated strain A
here), C. albicans ATCC 64124 (B), and C. albicans ATCC MYA-2876 (C)
were kindly provided by Jon Y. Takemoto (Utah State University, Logan,
ATCC MYA-1003 (G), and Cryptococcus neoformans MYA-895 (H) were
obtained from the American Type Culture Collection (ATCC, Manassas,
VA, USA). The filamentous fungal strains Aspergillus nidulans ATCC
38163 (I) and Fusarium graminearum 053 (J) were kindly provided by Jon
S. Thorson and Lisa J. Vaillancourt (University of Kentucky, Lexington,
KY, USA), respectively. Filamentous fungi and yeasts were cultivated at
35°C (except for F. graminearum 053 [J], which was grown at room tem-
perature [RT]) in RPMI 1640 (catalog number R6504; Sigma-Aldrich
Chemical Co., St. Louis, MO) buffered to pH 7.0 with 0.165 M morpho-
linepropanesulfonic acid (MOPS) buffer (Sigma-Aldrich Chemical Co.).
The bacterial strains used in this study were obtained from various
sources. Mycobacterium parascrofulaceum ATCC BAA-614 (Dϩ) and
Haemophilus influenzae ATCC 51907 (FϪ) were purchased from ATCC.
Methicillin-resistant Staphylococcus aureus (MRSA; Cϩ) and S. aureus
NorA (Fϩ) were a gift from David H. Sherman (University of Michigan,
Ann Arbor, MI, USA). Mycobacterium smegmatis MC2 155 (Eϩ) was a gift
from Sabine Ehrt (Weill Cornell Medical College, New York, NY, USA).
Enterococcus faecium BM4105-RF (Aϩ), Listeria monocytogenes ATCC
19115 (Bϩ), S. aureus NRS22 (Gϩ), E. coli MC1061 (EϪ), Klebsiella
pneumoniae ATCC 27736 (GϪ), and Pseudomonas aeruginosa PAO1 (IϪ)
were gifts from Paul J. Hergenrother (University of Illinois at Urbana-
Champaign, Champaign, IL, USA). Staphylococcus epidermidis ATCC
12228 (Hϩ), Streptococcus pyogenes ATCC 12384 (Iϩ), Escherichia coli
ATCC 25922 (AϪ), and P. aeruginosa ATCC 27853 (HϪ) were generously
provided by Dev P. Arya (Clemson University, Clemson, SC, USA). E. coli
⌬7 wild type (wt; BϪ), E. coli ⌬7 A1408G (CϪ), and E. coli ⌬7 G1491U
(DϪ) were gifts from Kurt Frederick (Ohio State University, Columbus,
OH, USA). Shigella flexneri 2475 pgm-24 (JϪ) was obtained from An-
thony T. Maurelli (Uniformed Services University of the Health Sciences,
Bethesda, MD, USA).
The human lung carcinoma epithelial cell line A549 (ATCC CCL-185)
and the normal human bronchial epithelial cell line Beas 2B (ATCC CRL-
9609) were kindly provided by David K. Orren (University of Kentucky,
Lexington, KY, USA).
Protocol for formation of the Boc-protected thioether TOB ana-
logues. The amphiphilic tert-butyloxycarbonyl (Boc)-protected TOB an-
alogues C6 to C14 (compounds 2b to 2f) were synthesized from TOB as
previously described (Fig. 1) (7). For the preparation of the novel ana-
logue 2a (Boc-protected C4), 1-butanethiol (0.13 ml, 1.22 mmol) was
added to a solution of analogue 1 (0.30 g, 0.24 mmol) and Cs2CO3 (0.12 g,
0.36 mmol) in freshly distilled dimethylformamide (3 ml). The mixture
was stirred at RT overnight. Progress of the reaction was monitored by
TLC (hexanes:ethyl acetate [EtOAc] ratio, 2:3; Rf, 0.60). Upon comple-
tion, the reaction mixture was diluted with EtOAc (10 ml) and washed
with H2O (5 ml). The aqueous layer was extracted with EtOAc (twice, 10
ml). The combined organic layers were washed again with H2O (twice, 10
FIG 2 Bar graph showing the relative activities of the listed AMEs against C4,
normalized to that of TOB (100%).
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Antifungal Tobramycin Analogues
ml) and brine (10 ml), dried over anhydrous MgSO4, and filtered. The
solvents were removed under reduced pressure, and the crude product
obtained was purified by flash column chromatography (SiO2; pure
hexanes to hexanes:EtOAc in a 2:3 ratio) to afford compound 2a (0.19 g;
Antifungal susceptibility testing. In vitro MIC values for the C4 to C14
TOB analogues against yeast cells were evaluated in 96-well plates as de-
scribed in CLSI document M27-A3 (18) with minor modifications. Yeast
cells were grown in RPMI 1640 for 48 h, counted using a hemocytometer,
and diluted to a concentration of 5 ϫ 104 cells/ml in fresh RPMI 1640. Cell
suspensions (200 ␮l) containing 0.97 to 125 mg/liter of TOB analogue,
0.97 to 62.5 mg/liter of POS, 0.97 to 62.5 mg/liter of ITC, or 0.97 to 125
mg/liter of FLC were added to the wells of a 96-well microtiter plate and
incubated for 48 h at 35°C. The final concentration of DMSO was ensured
to be Ͻ2% in all experiments. Growth of C. albicans 10231 (strain A) was
not affected by this concentration of DMSO. The MIC values of TOB
analogues were defined as the minimum drug concentration that yielded
complete inhibition, or the MIC-0. MIC values of azoles for yeasts were
determined as the lowest drug concentration that produced at least 50%
growth inhibition (i.e., MIC-2) compared with the growth control well. It
is important to note that at the lowest concentration tested, the growth of
C. neoformans MYA-895 (H) was completely inhibited. For testing azoles
against molds, on the other hand, the MIC-0 endpoint was used.
1
76%) as a white solid: H NMR (400 MHz, CD3OD) ␦ 5.07 (br s, 1H,
H-1=), 5.02 (br s, 1H, H-1Љ), 4.05 (m, 1H, H-5Љ), 3.70 to 3.28 (m, 13H,
H-1, H-3, H-4, H-5, H-6, H-2=, H-4=, H-5=, H-6= (2H), H-2Љ, H-3Љ, H-4Љ),
2.97 (br dd, J1 ϭ 14.4 Hz, J2 ϭ 2.2 Hz, 1H, H-6Љ), 2.62 (m, 1H, H-6Љ), 2.58
[t, J ϭ 7.6 Hz, 2H, SCH2(CH2)2CH3], 2.12 (m, 1H, H-2eq), 1.99 (m, 1H,
H-3=eq), 1.66 to 1.24 [m, 51H, H-2ax, H-3=ax, 5 ϫ CO2(CH3)3,
SCH2(CH2)2CH3], 0.90 [t, J ϭ 7.6 Hz, 3H, SCH2(CH2)2CH3]; 13C NMR
(100 MHz, CD3OD) ␦ 158.0, 157.9, 156.5, 156.4 (2 carbons), 98.6 (ano-
meric carbon), 98.1 (anomeric carbon), 82.9, 81.1, 79.3, 79.0 (2 carbons),
78.9, 78.7, 75.8, 72.5 (2 carbons), 72.1, 70.7, 65.0, 55.7, 50.0, 49.8, 49.6,
40.5, 34.4, 33.3, 32.9, 32.4, 31.6, 27.4 to 27.3 (15 carbons), 21.6, 12.7.
ProtocolforBocdeprotection[e.g., synthesisofcompound3a(C4)].
The amphiphilic TOB analogues C6 to C14 (compounds 3b to f) were
synthesized from 2b to f as previously described (Fig. 1) (7). For the
preparation of the novel analogue 3a (C4), compound 2a (46 mg, 0.044
mmol) was treated at RT with neat trifluoroacetic acid (TFA; 1 ml) for
3 min. The TFA was removed under reduced pressure, and the residue
was dissolved in a minimal volume of H2O and freeze-dried to afford
the novel 6Љ-thioether TOB derivative 3a (C4; 49 mg, 98%) as a white
foam: 1H NMR (400 MHz, D2O) ␦ 5.74 (d, J ϭ 3.6 Hz, 1H, H-1=), 5.10
(d, J ϭ 4.0 Hz, 1H, H-1Љ), 4.05 to 3.93 (m, 4H, H-4, H-5=, H-2Љ, H-5Љ),
3.89 (app. t, J1 ϭ J2 ϭ 9.2 Hz, 1H, H-5), 3.82 to 3.54 (m, 6H, H-1, H-3,
H-6, H-2=, H-4=, H-4Љ), 3.46 (app. t, J1 ϭ J2 ϭ 10.4 Hz, 1H, H-3Љ), 3.43
(dd, J1 ϭ 13.6 Hz, J2 ϭ 3.6 Hz, 1H, H-6=), 3.28 (dd, J1 ϭ 13.6 Hz, J2 ϭ
6.4 Hz, 1H, H-6=), 3.05 (dd, J1 ϭ 14.4 Hz, J2 ϭ 2.4 Hz, 1H, H-6Љ), 2.78
(dd, J1 ϭ 14.4 Hz, J2 ϭ 8.0 Hz, 1H, H-6Љ), 2.63 [t, J ϭ 7.6 Hz, 2H,
SCH2(CH2)2CH3], 2.57 (app. dt, J1 ϭ 12.4 Hz, J2 ϭ J3 ϭ 4.4 Hz, 1H,
H-2eq), 2.57 (app. dt, J1 ϭ 12.0 Hz, J2 ϭ J3 ϭ 4.4 Hz, 1H, H-3=eq), 2.03
(app. q, J1 ϭ J2 ϭ J3 ϭ 12.4 Hz, 1H, H-2ax), 1.95 (app. q, J1 ϭ J2 ϭ
J3 ϭ 12.8 Hz, 1H, H-3=ax), 1.58 (p, J ϭ 7.6 Hz, 2H, SCH2CH2CH2CH3), 1.39
(sextet, J ϭ 7.6 Hz, 2H, SCH2CH2CH2CH3), 0.89 [t, J ϭ 7.6 Hz, 3H,
SCH2(CH2)2CH3]; 13C NMR (100 MHz, D2O) ␦ 100.5 (anomeric carbon),
94.5 (anomeric carbon), 83.8, 77.6, 74.1, 72.5, 70.2, 68.0, 67.8, 64.2, 54.5, 49.3,
48.1, 47.8, 39.7, 32.2, 32.0, 30.9, 29.2, 27.7, 21.1, 12.7. Notethatcompounds3a
to f were stored at Ϫ20°C as a 10-g/liter stock solution in ddH2O.
Antibacterial susceptibility testing. MIC values of TOB analogues
(C4 to C14) were determined by using the microdilution broth method
for aerobic and anaerobic organisms according to CLSI standardized
methodology (16, 17) with minor modifications. A variety of Gram-
positive and Gram-negative bacteria were diluted 1:1,000 from over-
night cultures into fresh medium and grown to an optical density of
ϳ0.4 at 600 nm, or on average, for 4 h. To prepare bacteria for MIC
determinations, an additional 1:1,000 dilution of turbid culture was
performed. Briefly, solutions of the studied compounds were added to
the optimal medium for each bacterial strain (i.e., LB for MRSA [Bϩ],
S. aureus NorA [Fϩ], S. epidermidis ATCC 12228 [Hϩ], S. pyogenes
ATCC 12384 [Iϩ], E. coli ⌬7 wt [BϪ], E. coli ⌬7 A1408G [CϪ], E. coli ⌬7
G1491U [DϪ], E. coli MC1061 [EϪ], K. pneumoniae ATCC 27736 [GϪ],
and S. flexniri 2475T pgm-24 [JϪ]; brain heart infusion (BHI) for E.
faecium BM4105-RF [Aϩ], L. monocytogenes ATCC 19115 [Bϩ], and S.
aureus NRE22 USA600 [Gϩ]; supplemented BHI for H. influenzae ATCC
51907 [FϪ]; tryptic soy medium for E. coli ATCC 25922 [AϪ], P. aerugi-
nosa ATCC 27583 [HϪ], and P. aeruginosa PAO1 [IϪ]; 7H9 for M. smeg-
matis MC2 155 [Eϩ]; 7H9 with ADC for M. parascrofulaceum ATCC
BAA-614 [Dϩ]), and a double dilution was performed (100 ␮l) in a mi-
crotiter plate. The diluted bacterial cultures were then added to these
medium-drug mixtures (100 ␮l). The microtiter plate cultures were
grown for 16 to 20 h and visually inspected for growth. MIC values were
defined as the concentration of the drug in the last well showing no bac-
terial growth.
The minimal fungicidal concentration (MFC) values for the C12 and
C
14 TOB analogues were determined as previously described (19, 20) with
minor modifications. To determine MFCs, yeast cells of 1 ϫ 104 CFU/ml
were used to perform broth microdilution assays as described above. After
48 h of incubation, all of the MIC wells with no visible growth were
homogenized with a micropipette, and aliquots of 20 ␮l cell content were
spread on Sabouraud’s dextrose agar (SDA; Difco, BD, Franklin Lakes, NJ,
USA). The plates were incubated for 24 to 48 h at 35°C for colony counts.
The MFC was defined as the lowest drug concentration that killed 99% of
the final inoculum (with Յ3 colonies on SDA plates, in agreement with a
previous report [21]). Each test was performed in triplicate.
In vitro MIC values for these analogues against A. nidulans ATCC
38163 (I) and F. graminearum 053 (J) were conducted as previously de-
scribed in CLSI document M38-A2 (22). Spores were collected from spo-
rulating cultures growing in potato dextrose agar (PDA) by filtration
through sterile glass wool and enumerated using a hemocytometer to
obtain the desired inoculum size. Serial dilutions of TOB analogues as well
as POS, ITC, and FLC were made in sterile 96-well plates in the ranges of
0.97 to 125 mg/liter (for TOB analogues), 0.097 to 12.5 mg/liter (for POS
and ITC), and 0.97 to 62.5 mg/liter (for FLC) using RPMI, and spore
suspensions were added to make a final concentration of 5 ϫ 105 CFU/ml.
The plates were incubated at 35°C for 48 h (except for F. graminearum 053
[J], which was incubated at room temperature). The MIC values of azoles
and TOB analogues against molds were based on the complete inhibition
of growth compared to the growth control, or the MIC-0 (18, 22). Each
test was performed in triplicate.
Antifungal carryover and time-kill studies. Prior to performing
time-kill studies, antifungal carryover effects were evaluated as previously
described (19). C. albicans ATCC 64124 (B) cell suspensions were pre-
pared to achieve an inoculum of approximately 1 ϫ 105 to 4 ϫ 105 CFU/
ml. An aliquot of 100 ␮l of each suspension was added to 900 ␮l of sterile
ddH2O (control) or to sterile ddH2O plus TOB analogue (C12 or C14) at
concentrations of 8, 16, or 32 mg/liter and 2, 4, or 8 mg/liter, respectively.
Immediately after addition of the fungal suspension, tubes were vortexed
and 100 ␮l of suspension was removed and spread on PDA for colony
count determinations. Antifungal carryover was defined as a reduction in
colony counts of a sample by Ͼ25% compared to controls. Time-kill
curve studies were performed against the azole-resistant C. albicans strain
ATCC 64124 (B) as previously described (23) with modifications. The
initial inoculum used for this assay was 1 ϫ 105 CFU/ml in liquid RPMI
1640 medium at 35°C in 50 ml Falcon tubes (10-ml volume) with contin-
uous agitation (200 rpm). TOB analogues (C12 and C14) at concentrations
of 8, 16, or 32 mg/liter and 2, 4, or 8 mg/liter, respectively, were tested
against C. albicans ATCC 64124 (B). Test solutions were incubated at 200
rpm at 35°C. At 0, 3, 9, 12, and 24 h, 100-␮l aliquots were removed from
each solution and serially diluted in sterile ddH2O. Fifty microliters of
each dilution was spread onto a potato dextrose agar plate and incubated
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Shrestha et al.
at 35°C. FLC (16 mg/liter) and AmB (1 mg/liter) were used as controls in The strains tested ranged from completely resistant to TOB (Ͼ150
this assay. Colony counts were determined after 48 h of incubation. The
experiment was performed in triplicate. The lower limit for accurate and
reproducible quantification was 50 CFU/ml (23).
In vitro cytotoxicity assay. The in vitro cytotoxicity assay was per-
formed as previously described (24) with minor modifications. Human
lung carcinoma epithelial A549 cells and normal human bronchial epi-
thelial cells Beas 2B were grown in F-12K and Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% an-
tibiotics, respectively. The confluent cells were then trypsinized with
0.05% trypsin–0.53 mM EDTA and resuspended in fresh medium (F-12K
mg/liter, strains E. faecium BM4105-RF [Aϩ], MRSA [Cϩ], S.
aureus NRS22 USA600 [Gϩ], E. coli ⌬7 A1408G [CϪ], E. coli ⌬7
G1491U [DϪ], and P. aeruginosa ATCC 27853 [HϪ]) to very
susceptible to TOB (Ͻ0.3 mg/liter; strain M. smegmatis MC2 155
[Eϩ]). The C4 analogue showed excellent antibacterial activity
(2.3 to 4.7 mg/liter) against four Gram-positive strains (L. mono-
cytogenes ATCC 19115 [Bϩ], M. smegmatis MC2 155 [Eϩ], S.
aureus NorA [Fϩ], and S. epidermidis ATCC 12228 [Hϩ]) and
one Gram-negative strain (H. influenzae ATCC 51907 [FϪ]), and
or DMEM). The cells were transferred into 96-well microtiter plates at a there were only 10 strains (E. faecium BM4105-RF [Aϩ], MRSA
density of 3,000 cells/well and were grown overnight. The following day,
the media were replaced with 100 ␮l of fresh culture medium containing
serially diluted TOB analogues at final concentrations of 0.97 to 125 mg/
liter or sterile ddH2O (negative control). The cells were incubated for an
additional 24 h at 37°C with 5% CO2 in a humidified incubator. To eval-
uate cell survival, each well was treated with 10 ␮l (25 mg/liter) of resaz-
urin sodium salt (Sigma-Aldrich) for 3 h. Metabolically active cells can
convert the blue nonfluorescent dye resazurin to the pink and highly
fluorescent dye resorufin, which can be detected at wavelengths of 560 nm
(excitation) and 590 nm (emission) by using a SpectraMax M5 plate
[Cϩ], M. parascrofulaceum ATCC BAA-614 [Dϩ], S. aureus
NRS22 USA600 [Gϩ], E. coli ATCC 25922 [AϪ], E. coli ⌬7
A1408G [CϪ], E. coli ⌬7 G1491U [DϪ], K. pneumoniae ATCC
27736 [GϪ], P. aeruginosa ATCC 27853 [HϪ], and P. aeruginosa
PAO1 [IϪ]) against which it was ineffective (MIC, 75 to Ͼ150
mg/liter). For the C6 analogue, MIC values ranged from Ͼ150
mg/liter to 18.8 mg/liter, and for the C8 compound the values
ranged from Ͼ150 mg/liter to 37.5 mg/liter against all Gram-
positive and Gram-negative strains tested. Compounds with the
reader. Triton X-100 (1%, vol/vol) resulted in complete loss of cell viabil- C10, C12, or C14 modification had none to very good activity, with
ity and was used as the positive control. The percent cell survival was
calculated as follows: [(control value – test value) ϫ 100]/(control value),
where the control value represents results for cells plus resazurin but with-
out drug and the test value represents cells plus resazurin plus the drug.
Propidium iodide staining. A single colony of C. albicans ATCC
64124 (B) was used to inoculate 5 ml of potato dextrose broth (PDB) in a
Falcon tube and was grown overnight at 35°C at 200 rpm. The overnight
culture was further diluted by transferring 200 ␮l of the cell suspension to
800 ␮l of RPMI 1640 medium. Fifty microliters of cell suspension was
then added to the RPMI medium containing no drug (negative control) or
MIC values ranging from Ͼ150 mg/liter to 2.3 mg/liter (C10), Ͼ150
mg/liter to 0.6 mg/liter (C12), and Ͼ150 mg/liter to Ͻ0.3 mg/liter
(C14). On a broader scale, all compounds displayed better MIC
values against Gram-positive strains than against Gram-negative
strains.
The main mechanism of resistance to aminoglycoside antibi-
otics is their modification by resistance enzymes termed amin-
oglycoside-modifying enzymes (AMEs) that can be acquired by
bacteria (27). There are three types of AMEs: the aminoglycoside
C12 or C14 TOB analogues at concentrations of 15.6 (0.5ϫ MIC), 31.2 (1ϫ N-acetyltransferases (AACs), the aminoglycoside O-phospho-
MIC), and 62.5 mg/liter (2ϫ MIC), or 3.9 (0.5ϫ MIC), 7.8 (1ϫ MIC), and
15.6 (2ϫ MIC), respectively. TOB (125 mg/liter) and POS (62.5 mg/liter)
were used as controls. The cell suspensions were then treated for 1 h at
35°C with continuous agitation (200 rpm). After incubation, treated cells
were centrifuged and resuspended in 500 ␮l of phosphate-buffered saline
(PBS; pH 7.2). Subsequently, cells were treated with propidium iodide
(PI; 9 ␮M, final concentration) and incubated for 5 min at room temper-
ature in the dark (25). Glass slides prepared with 15 ␮l of each mixture
were observed in bright-field and fluorescence modes (using a Texas Red
filter set, with excitation and emission wavelengths of 535 and 617 nm,
transferases (APHs), and the aminoglycoside O-nucleotidyltrans-
ferases (ANTs). Recently, a unique AAC, the enhanced intracellu-
lar survival (Eis) protein from a variety of bacterial species, was
found capable of multiacetylating aminoglycosides (12, 28–36).
Knowing that our compounds had good antibacterial activity, we
tested the novel C4 compound to see if it became modified by
AMEs (Fig. 2). All other compounds in this study had previously
been tested against these AMEs (7). The rates at which AAC(6=)-
Ib=, AAC(6=)-Ie, and Eis modified C4 were only 50% or less of the
respectively) using a Zeiss Axiovert 200M fluorescence microscope. Data rate at which TOB was modified by these enzymes. The converse
were obtained from at least two independent experiments.
Annexin V and PI staining. We chose one of the potent TOB ana-
logues, C14, to perform the assay with Annexin V and PI staining. C.
was true for the AAC(3)-IV, AAC(2=)-Ic, and ANT(4=) enzymes,
as C4 was modified at a rate 1.5 to 2 times that of TOB with the
same enzymes.
albicans ATCC 64124 (B) was treated with C14 at a subinhibitory concen-
In vitro antifungal assay. The MIC values for TOB and its C4
tration (4 mg/liter) for 1 h. Cells were washed in PBS and digested at 30°C
to C14 analogues as well as those of three known antifungal azoles
for 30 min in 0.1 M potassium phosphate buffer that contained 150 mg/
(POS, ITC, and FLC) were determined against various fungal
liter Zymolyase 20T, 40 mM 2-mercaptoethanol, and 2.4 M sorbitol at pH
strains (Table 2). TOB analogues C12 and C14 showed broad-spec-
7.2 (26). Protoplasts were washed in modified Annexin binding buffer (10
trum antifungal activities against yeast and filamentous fungi. The
MIC values for C12 and C14 against tested fungal strains ranged
mM HEPES-NaOH [pH 7.4], 40 mM NaCl, 50 mM CaCl2, and 1.2 M
sorbitol), resuspended in the same buffer along with 5 ␮l of fluorescein
isothiocyante (FITC)-Annexin V, 2 ␮l of PI (20 mg/liter), and RNase (250 from 1.95 to 31.2 mg/liter and 1.95 to 7.8 mg/liter, respectively.
mg/liter), and incubated at 37°C for 30 min. Cells were analyzed in bright-
field and fluorescence modes (using Texas Red and FITC filter sets) using
a Zeiss Axiovert 200M fluorescence microscope.
The C10 TOB analogue exhibited good to no activity against vari-
ous fungal strains, with MICs ranging from 3.9 to 125 mg/liter.
The parent TOB and its analogues C4, C6, and C8 exhibited no
antifungal activities except for C10 against F. graminearum 053
(strain J; 3.9 mg/liter) and only low activity against C. neoformans
RESULTS
In vitro antibacterial assay. The new C4 compound along with MYA-895 (H; 15.6 mg/liter). It should be noted that the fungal
the C6 to C14 compounds previously evaluated (7) were tested for strains used in this study showed better sensitivity to C12 and C14
antibacterial activity (MIC value determinations) against several than all three azoles, with the exception of two yeast strains, C.
strains of Gram-positive and Gram-negative bacteria (Table 1). neoformans MYA-895 (H) and C. albicans 10231 (A), and two
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TABLE 1 MICs for TOB and its analogues
MIC (mg/liter) for compoundb
Bacterial straina
C4
C6
C8
C10
C12
C14
TOB
Gram-positive strains
Aϩ
Bϩ
Cϩ
Dϩ
Eϩ
Fϩ
Gϩ
Hϩ
Iϩ
Ͼ150
2.3
Ͼ150
75
2.3
4.7
Ͼ150
2.3
37.5
Ͼ150
37.5 (75)
Ͼ150
150
18.8
150 (Ͼ150)
Ͼ150
75 (75)
150
Ͼ150
75 (Ͼ150)
Ͼ150
150
Ͼ150
Ͼ150 (150)
150
150 (75)
37.5
75
9.4
4.7 (18.8)
18.8
4.7
2.3 (9.4)
2.3
Ͼ150
18.8 (37.5)
Ͼ150
37.5
Ͼ150
18.8 (37.5)
9.4
1.2 (4.7)a
Ͼ150
75
9.4
1.2
Ͻ0.3
1.2 (9.4)
Ͼ150
Ͻ0.3 (0.3)
9.4
Ͼ150
4.7 (9.4)
2.3
1.2 (2.3)
0.6
Ͼ150
0.6 (9.4)
1.2
0.6 (1.2)
Ͻ0.3
9.4 (9.4)
2.3
Gram-negative strains
AϪ
BϪ
CϪ
DϪ
EϪ
FϪ
GϪ
HϪ
IϪ
75
Ͼ150
Ͼ150
Ͼ150
Ͼ150
150 (150)
18.8
Ͼ150
Ͼ150
Ͼ150
150
Ͼ150
150
150
75
75
75
18.8 (9.4)
75
Ͼ150
150
37.5
37.5
18.8
37.5
4.7
4.7
9.4
2.3
Ͼ150
Ͼ150
2.3 (9.4)
1.2
2.3
Ͼ150
1.2
18.8
Ͼ150
Ͼ150
9.4
2.3
150
Ͼ150
Ͼ150
18.8
Ͼ150
Ͼ150
9.4
9.4 (18.8)
Ͼ150
75
18.8
18.8
2.3
Ͼ150
Ͼ150
75 (75)
Ͼ150
Ͼ150
Ͼ150
150
4.7
9.4 (4.7)
Ͼ150
18.8
18.8
37.5
2.3
JϪ
150
4.7
a Gram-positive strains: Aϩ, E. faecium BM4105-RF; Bϩ, L. monocytogenes ATCC 19115; Cϩ, MRSA; Dϩ, M. parascrofulaceum ATCC BAA-614; Eϩ, M. smegmatis MC2 155; Fϩ,
S. aureus NorA; Gϩ, S. aureus NRS22 USA600; Hϩ, S. epidermidis ATCC 12228; Iϩ, S. pyogenes ATCC 12384. Gram-negative strains: AϪ, E. coli ATCC 25922; BϪ, E. coli ⌬7 wt;
CϪ, E. coli ⌬7 A1408G; DϪ, E. coli ⌬7 G1491U, EϪ, E. coli MC1061; FϪ, H. influenzae ATCC 51907; GϪ, K. pneumoniae ATCC 27736; HϪ, P. aeruginosa ATCC 27853; IϪ, P.
aeruginosa PA01; JϪ, S. flexneri 2475T pgm-24.
b MICs were determined for TOB and its analogues with various alkyl chain lengths (C4 to C10) against a variety of Gram-positive and Gram-negative bacterial strains. The values in
parentheses were previously reported in reference 7 for a different batch of these molecules.
filamentous strains, A. nidulans 38163 (I) and F. graminearum 053 and C14 compounds and were found to be 62.5 mg/liter and 15.6
(J) (Table 2). However, only C. neoformans MYA-895 (H) showed mg/liter, respectively, in almost all strains tested, with the excep-
higher sensitivity to C8, C12, and C14, with low MIC values of 1.95 tion of C14 against C. albicans ATCC 64124, for which the MFC
mg/liter.
was 7.8 mg/liter (Table 3). A trailing growth effect was observed
The MFC values were also determined for the most active C12 for all three azoles against all yeast strains except against C. albi-
TABLE 2 MICs determined for TOB and its analogues with various alkyl chain lengths (C4 to C14) and for three control antifungal agents (POS,
ITC, and FLC) against various yeast strains and filamentous fungi
Yeast or
fungal
straina
MIC (mg/liter) for compoundb
C4
C6
C8
C10
C12
C14
TOB
POS
ITC
FLC
Yeast strains
A
B
C
D
E
F
G
H
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
31.25
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
7.8
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
1.95
31.2
62.5
62.5
125
125
125
125
15.6
31.2
31.2
15.6
31.2
31.2
15.6
31.2
1.95
7.8
7.8
7.8
7.8
7.8
7.8
7.8
1.95
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
Ͼ125
62.5
0.5
Ͼ62.5
7.8
31.2
31.2
15.6
15.6
Ͻ0.975
0.5
Ͼ62.5
7.8
31.2
31.2
31.2
31.2
Ͻ0.975
62.5
Ͼ125
15.6
Ͼ125
Ͼ125
62.5
62.5
Ͻ0.975
Filamentous
fungi
I
J
Ͼ125
62.5
125
31.2
125
31.2
62.5
3.9
7.8
3.9
7.8
3.9
Ͼ125
62.5
0.195
Յ1.56
0.195
Ͻ1.95
62.5
Ͼ125
a Yeast strains: A, C. albicans ATCC 10231; B, C. albicans ATCC 64124; C, C. albicans ATCC MYA-2876(S); D, C. albicans ATCC 90819(R); E, C. albicans ATCC MYA-2310(S); F, C.
albicans ATCC MYA-1237(R); G, C. albicans ATCC MYA-1003(R); H, C. neoformans MYA-895. Note that here the S or R following a strain name indicates that ATCC reports the
strain susceptible (S) or resistant (R) to ITC and FLC. Filamentous fungi: I, Aspergillus nidulans ATCC 38163; J, Fusarium graminearum 053.
b For yeast strains, MIC-0 values are reported for TOB and its analogues, whereas MIC-2 values are reported for azoles. For filamentous fungi, MIC-0 values are reported for all
compounds.
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Shrestha et al.
TABLE 3 MFCs determined for TOB analogues with an alkyl chain
length of C12 or C14 against various yeast strains
Phosphatidylserine (PS) is distributed asymmetrically in the inner
leaflet of the lipid bilayer of plasma membranes of yeast cells (38).
Externalization of PS on the outer surface of the plasma mem-
brane is an early symptom of apoptosis or programmed cell death
that can be detected by FITC-conjugated Annexin V staining
(green) in yeast cells. Similarly, PI dye only stains necrotic cells, as
intact fungal membranes are impermeable. As shown in Fig. 6,
C14, at subinhibitory doses, induced both early apoptosis (ϳ13%)
and late apoptosis/necrosis (ϳ40%) of C. albicans ATCC 64124
(strain B) after 1 h of exposure. However, untreated yeast cells did
not show any Annexin V or PI staining.
MFC (mg/liter) for analogue
Yeast straina
C12
C14
A
B
C
D
E
F
G
H
62.5
62.5
62.5
62.5
62.5
62.5
62.5
3.9
15.6
15.6
7.8
15.6
15.6
15.6
15.6
3.9
a Yeast strains: A, C. albicans ATCC 10231; B, C. albicans ATCC 64124; C, C. albicans
ATCC MYA-2876 (S); D, C. albicans ATCC 90819 (R); E, C. albicans ATCC MYA-2310
(S); F, C. albicans ATCC MYA-1237 (R); G, C. albicans ATCC MYA-1003 (R); H, C.
neoformans MYA-895. The S or R following a strain name indicates that ATCC reports
the strain susceptible (S) or resistant (R) to ITC and FLC.
DISCUSSION
Previously, we showed chain length-dependent antibacterial ac-
tivities of TOB analogues derivatized at the 6Љ-position with linear
alkyl chains (C6 to C22) against VRE and E. coli with resistance to
the parent compound TOB itself (7). In the current study, we
further expanded the antibacterial spectra of these analogues
along with that of a newly synthesized C4 TOB analogue against
various clinically important bacterial isolates. Unlike TOB ana-
logues with longer alkyl chains, C4 demonstrated strong antibac-
terial activities against L. monocytogenes ATCC 19115 (strain Bϩ;
2.3 mg/liter) and H. influenzae ATCC 51907 (FϪ; 2.3 mg/liter)
that were also comparable to the activity of TOB itself (1.2 mg/
liter) against these strains (Table 1). Among all of these analogues,
cans 10231 (A) and C. neoformans (H). Since the majority of azoles
did not completely inhibit the growth of yeast at the tested con-
centrations, MFC values were not determined for them.
Antifungal carryover and time-kill studies. An antifungal
carryover effect was not observed for TOB analogues with linear
alkyl chain lengths of C12 and C14 at 1, 2, or 4ϫ MICs against C.
albicans ATCC 64124 (B). The time-kill course of the most potent
TOB analogues, C12 and C14, and of FLC against the representative
strain C. albicans ATCC 64124 (B) over a 24-h period is shown in
Fig. 3. C12 at 32 mg/liter (MIC) and C14 at 8 mg/liter (MIC) rapidly
reduced the CFU of C. albicans ATCC 64124 (B) by Ն2 log10 after
6 and 9 h of treatment, but complete killing was observed only
after 24 h for C12 and after 12 h for C14 (Fig. 3). However, FLC at
16 mg/ml showed a fungistatic effect against C. albicans ATCC
64124 (B) for the first 6 h of growth, and after that the trend of cell
growth was similar to that of control cells. Likewise, AmB at a
subinhibitory concentration (1 mg/liter) maintained its fungi-
static activity against C. albicans ATCC 64124 (B) for 24 h.
In vitro cytotoxicity assay. The cytotoxicity assay results with
TOB analogues with linear alkyl chain lengths of C4 to C14 against
A549 and Beas 2B cells are shown in Fig. 4. The IC50s of C14 against
the A549 and Beas 2B cell lines were Ͼ62.5 mg/liter and 125 mg/
liter, respectively (Fig. 4). This was a 32- to 64-fold-higher con-
centration than the antifungal MICs against fungi (Table 2). Sim-
ilarly, the IC50s of C12 against both cell lines were Ն125 mg/liter, 4-
to 64-fold higher than its antifungal MICs. However, C4, C6, C8,
C10, and TOB itself show little to no toxicity against these cell lines.
Effects of C12 and C14 on fungal membrane integrity. PI was
used to investigate the effects of TOB analogues (C12 and C14) on
the fungal membrane integrity of C. albicans ATCC 64124 (strain
B). PI, a membrane-impermeable dye, cannot enter an intact cell
unless the cell membrane is compromised, and it fluoresces upon
binding to nucleic acids (37). When yeast cells were exposed to
TOB analogues C12 and C14 at 15.6, 31.2, or 62.5 mg/liter and 3.9,
7.8, or 15.6 mg/liter, respectively, 6, 46, or 93% and 10, 36, or 92%
staining of the yeast cells was observed, respectively. However,
cells treated with TOB (2% staining) or POS (5%) or untreated
(1%) showed negligible staining (Fig. 5).
C
12 and C14 exhibited moderate to strong antibacterial activities
FIG 3 Representative time-kill studies of 6Љ-thioether TOB analogues with
12 and C14 linear alkyl chains against azole-resistant C. albicans ATCC 64124
C
Annexin V and PI staining analysis. Annexin V and PI stain-
ing assays were performed to determine whether the killing effect
(strain B). (a) Cultures were exposed to C12 at 8, 16, or 32 mg/liter. (b) Cultures
were exposed to C14 at 2, 4, or 8 mg/liter. In both panels, cultures were exposed
of TOB analogues involves apoptosis or necrosis in yeast cells. to FLC at 16 mg/liter and AmB at 1 mg/liter or to a no-drug control.
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Antifungal Tobramycin Analogues
FIG 4 Cytotoxicity of 6Љ-thioether C4 to C14 TOB analogues against mamma-
lian cells. A549 cells (a) and Beas 2B cells (b) were incubated at 37°C for 24 h in
a CO2 incubator with various concentrations of TOB or its 6Љ-thioether ana-
logues with C4, C6, C8, C10, C12, and C14 linear alkyl chains.
against most of the bacterial strains, except against M. smegmatis
MC2 155 (Eϩ; Ͼ150 mg/liter) and H. influenzae ATCC 51907
(FϪ; Ͼ150 mg/liter) (Table 1). The MIC values of C6 to C14 de-
termined in this study were mostly consistent with previously re-
ported MIC values of these analogues against the specific strains of
bacteria (7).
Recently, the amphiphilic kanamycin A and B derivatives K20
and FG08, respectively, with C8 linear alkyl chains were shown to
have promising antifungal properties with no or mild antibacte-
rial properties, respectively (8–10). This report has motivated us
to explore the potential antifungal activities of our C4 to C14 TOB
analogues that were yet to be determined. The C4 to C14 analogues
demonstrated a parabolic pattern of chain length-dependent an-
tifungal activity, and C12 and C14 were found to be the most potent
among them all. A similar pattern was also observed for the anti-
bacterial properties of these analogues (7). Both C12 and C14
showed moderate growth-inhibitory effects against all tested fun-
gal strains, with a relatively high degree of inhibition against C.
neoformans MYA-895 (strain H) and against two filamentous
fungi, A. nidulans 38163 (I) and F. graminearum 053 (J). Intrigu-
ingly, the C8 TOB analogue showed no activity against fungi tested
in this study, except against C. neoformans MYA-895 (H). In con-
trast to kanamycin derivatives FG08 and K20, our TOB analogues
did not require an optimal chain length of C8 to show antifungal
FIG 5 (a) Representative dose-dependent membrane permeabilization effects
of 6Љ-thioether TOB analogues with C12 or C14 linear alkyl chains on azole-
resistant C. albicans ATCC 64124 (strain B). From top to bottom: PI dye
uptake by yeast cells without drug, with POS (62.5 mg/liter), with TOB (125
mg/liter), with C12 (0.5ϫ, 1ϫ, or 2ϫ MIC) or with C14 (0.5ϫ, 1ϫ, or 2ϫ
MIC). (b) Quantitative representation of the images shown in panel a.
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Shrestha et al.
that the possible cause of killing may be associated with a direct
effect on fungal membranes. The membrane primary or second-
ary effects of TOB analogues against fungi are not known. PS
externalization on the outer surface of the lipid bilayer of plasma
membranes is a sign of early apoptosis in yeast cells. The yeast cells
that were treated with C14 at a subinhibitory concentration did
show both early apoptosis (Fig. 6, indicated by the letter a), late
apoptosis/necrosis (indicated by the letter c), and no apoptosis (as
indicated by the letter b), as demonstrated in the Annexin V and PI
assay. We speculate that the induction of early apoptosis by C14
causes externalization of phosphatidylserine on the plasma mem-
brane by making the surface highly negatively charged, which
could then react with the polycationic amphiphilic aminoglyco-
sides generated. However, thorough experimental analysis, out-
side the scope of this study, is desirable for the future to elucidate
the exact target of these analogues on fungal membranes.
FIG 6 Annexin V and propidium iodide stains of C. albicans ATCC 64124
(strain B) cells treated with 6Љ-thioether C14 TOB analogue (4 mg/liter) for 1 h
(right panel) and the bright-field image (left panel). Note: in the right panel,
the letter a indicates early apoptotic cells that stained only green, the letter b
indicates healthy cells that did not stain, and the letter c indicates late apop-
totic/necrotic cells that stained green and orange.
In conclusion, C12 and C14 showed broad-spectrum antifungal
properties (9, 19). Not only were the MIC values of C12 and C14 at activities against yeasts and filamentous fungi as they targeted fun-
least 1- to 2-fold lower than amphiphilic kanamycin derivatives gal plasma membranes. They were less hemolytic and showed low
FG08 and K20, but also C12 and C14 were active against A. nidulans mammalian cell toxicities. This study provides novel lead com-
38163 (I) while FG08 and K20 were completely inactive against pounds for the development of antifungal drugs, which we are
Aspergillus strains (Table 2) (9, 19). It is also worth noting that the currently following by synthesizing and investigating the proper-
MIC values of C12 and C14 against resistant fungal strains, except ties of additional AGs derivatized in 6Љ-thioether form with long
for C. albicans 10231 (strain A), C. neoformans MYA-895 (H), and linear alkyl chains.
A. nidulans 38163 (I), were lower than those of POS, ITC, and FLC
ACKNOWLEDGMENTS
(Table 2). These results not only indicate that C12 and C14 may
have different modes of action than the conventional azoles
against fungi, but also it appears that they may circumvent the
azole resistance mechanisms of resistant fungi such as C. albicans
ATCC 64124 (B), which has mutations in its ERG11 sequences
(39, 40).
This work was supported by National Institutes of Health (NIH) grant
AI090048 (S.G.-T.) and by startup funds from the College of Pharmacy at
the University of Kentucky (S.G.-T.).
We thank Gregory A. Graf and Markos Leggas (University of Ken-
tucky) for letting us use their fluorescence microscope and cell culture
facilities, respectively. We thank David H. Sherman (University of Mich-
igan), Sabine Ehrt (Weill Cornell Medical College), Paul J. Hergenrother
(University of Illinois at Urbana-Champaign), Dev P. Arya (Clemson
University), Kurt Frederick (Ohio State University), and Anthony T.
Maurelli (Uniformed Services University of Health Sciences) for provid-
ing some of the bacterial strains used in our work. We also thank Lisa J.
Vaillancourt and Jon S. Thorson (University of Kentucky), as well as Jon
Y. Takemoto (Utah State University), for providing some of the fungal
strains used in our study.
Another noteworthy feature of the C12 and C14 analogues is
that they showed a fungicidal effect on the azole-resistant strain C.
albicans ATCC 64124 (strain B), inhibiting it completely after 24 h
(Fig. 3). However, as expected, FLC was fungistatic against C.
albicans ATCC 64124 (B) and completely lost its activity after 6 h,
which was consistent with previous reports (41). Also, C12 and C14
lysed 50% of mouse erythrocytes at concentrations that were 4- to
32-fold and 8- to 32-fold higher than their antifungal MICs, re-
spectively (data not shown), and these results were consistent with
the results published previously for these analogues (7). However,
in this study, we expanded the reports of effects of C12 and C14 on
nucleated mammalian cells. C12 and C14 did not affect A549 (hu-
man lung carcinoma epithelial cells) or Beas 2B (normal human
bronchial epithelial cells) proliferation even at concentrations
higher than their antifungal MIC values, thus suggesting a selec-
tive antifungal activity (Fig. 4).
It has been shown that C14 targets bacterial membranes over
the ribosomes, as does the parent drug (7), but the effect of C14 on
fungal membranes was yet to be determined. We chose the most
potent TOB analogues, C12 and C14, to explore their abilities to
affect fungal membrane integrity by measuring PI dye influx into
C12- or C14-treated C. albicans ATCC 64124 (strain B). Both C12
and C14, at their MIC and at 2ϫ MIC, triggered rapid influx of PI
by yeast cells after 1 h of exposure (Fig. 5). It is noteworthy that C12
and C14, even at 0.5ϫ MIC, caused more PI staining of yeast cells
than POS (62.5 mg/liter) after 1 h of exposure, which further
supports our proposed statement of different modes of action of
TOB analogues than that of azoles against fungi. The rapidity of PI
dye influx into C12- or C14-treated yeast cells, however, suggests
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Antimicrobial Agents and Chemotherapy
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