Antibacterial to antifungal conversion of neamine aminoglycosides through alkyl modification. Strategy for reviving old drugs into agrofungicides

Article abstract of DOI:10.1038/ja.2010.110

Many Actinomycetes aminoglycosides are widely used antibiotics. Although mainly antibacterials, a few known aminoglycosides also inhibit yeasts, protozoans and important crop pathogenic fungal oomycetes. Here we show that attachment of a C8 alkyl chain to

Full text of DOI:10.1038/ja.2010.110

The Journal of Antibiotics (2010) 63, 667–672
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ORIGINAL ARTICLE
Antibacterial to antifungal conversion of neamine
aminoglycosides through alkyl modification. Strategy
for reviving old drugs into agrofungicides
Cheng-Wei T Chang1, Marina Fosso1, Yukie Kawasaki2, Sanjib Shrestha2, Mekki F Bensaci2, Jinhua Wang1,
Conrad K Evans2 and Jon Y Takemoto2
Many Actinomycetes aminoglycosides are widely used antibiotics. Although mainly antibacterials, a few known aminoglycosides
also inhibit yeasts, protozoans and important crop pathogenic fungal oomycetes. Here we show that attachment of a C8 alkyl
chain to ring III of a neamine-based aminoglycoside specifically at the 400-O position yields a broad-spectrum fungicide (FG08)
without the antibacterial properties typical for aminoglycosides. Leaf infection assays and greenhouse studies show that FG08 is
capable of suppressing wheat fungal infections by Fusarium graminearum—the causative agent of Fusarium head blight—at
concentrations that are minimally phytotoxic. Unlike typical aminoglycoside action of ribosomal protein translation miscoding,
FG08’s antifungal action involves perturbation of the plasma membrane. This antibacterial to antifungal transformation could
pave the way for the development of a new class of aminoglycoside-based fungicides suitable for use in crop disease
applications. In addition, this strategy is an example of reviving a clinically obsolete drug by simple chemical modification to
yield a new application.
The Journal of Antibiotics (2010) 63, 667–672; doi:10.1038/ja.2010.110; published online 6 October 2010
Keywords: FG08; fungicide; Fusarium head blight; kanamycinB
INTRODUCTION
Many crop, horticultural and turf diseases are due to fungal and
Among the oldest and most widely used antibiotics are certain oomycetes mold infections which globally cause enormous economic
aminoglycosides produced by Actinomycetes. They include the classical losses.10 Direct application of fungicides remains the prevalent strategy
aminoglycosides streptomycin, neomycin and kanamycin and newer to control these diseases.10 However, concerns with inconsistent and
versions amikacin, gentamicin and tobramycin. They act by binding to declining effectiveness due to resistance, environmental impacts,
the aminoacyl-transfer RNA decoding A site in ribosomal 16S ribo- animal and human toxicity and costs continue to challenge their
somal RNA to decrease the fidelity of protein translation. Although use. Heavy use of triazole agrofungicides are linked to recent rises in
generally known as antibacterials, certain classical aminoglycosides are human cases of invasive aspergillosis resistant to antifungal azoles.11
reported to inhibit important crop pathogenic fungal oomycetes,1 and An example of the limitations of current agrofungicide use is illu-
structurally unusual ones inhibit yeasts and protozoans.2,3 The appli- strated by Fusarium head blight (FHB)—a destructive and costly
cation of aminoglycosides to combat crop diseases, however, is disease of wheat, barley and other small grains caused by F. grami-
controversial as the impact it may have on broader antibiotic nearum.12 There is a serious and persistent need for new agrofungi-
resistance is debated and uncertain.4,5 The antibiotic aminoglycosides cides that are efficacious, less toxic, cost effective and that do not
are generally viewed to act against bacteria.6 However, unusual bicyclic promote resistance. Although potentially useful as agrofungicides,1 the
ring-containing aminoglycosides are additionally active against application of currently available aminoglycosides to combat crop
yeasts.7–9 A recent report revealed inhibition of plant pathogenic diseases is discouraged as such practices are predicted to promote
oomycetes Phytophora and Pythium sp. by neomycin, paramomycin, bacterial resistance to therapeutically used antibiotics.5
ribostamycin and streptomycin, although they showed comparatively
Kanamycins A, B and C produced by the soil microbe Streptomyces
little activity against several other fungal genera.1 Consequently, kanamyceticus are among the most successful antibacterial amino-
aminoglycosides are more accurately antibacterial or antibacterial glycosides.13,14 They are structurally based on sugar rings I and II
and antifungal depending on the nature of chemical tailoring of the composed of 4-O-(6-amino-6-deoxy-a-D-glucopyranosyl)-2-deoxy-
constituent amino sugars. Here, we demonstrate the third possibility streptamine, neamine and paromamine, respectively, with an attached
of antifungal but not antibacterial capabilities.
ring III of O-6-linked kanosamine. Ototoxic and nephrotoxic at high
1Department of Chemistry and Biochemistry, Utah State University, Logan, UT, USA and 2Department of Biology, Utah State University, Logan, UT, USA
Correspondence: Dr JY Takemoto, Department of Biology, 5305 University Hill, Utah State University, Logan, UT 84322, USA.
E-mail: jon.takemoto@usu.edu
Received 25 July 2010; revised 8 September 2010; accepted 9 September 2010; published online 6 October 2010

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doses, the original natural product kanamycins are now mostly Organisms and culture conditions
Fusarium graminearum strains B-4-5A and Butte86 (ADA-11) were obtained
obsolete and confined to treating mycobacterial infections. They
have been largely replaced by semi-synthetic analogs (for example,
amikacin, dibekacin and arbekacin) that are chemically modified
in the 2-4-O-(6-amino-6-deoxy-a-D-glucopyranosyl)-2-deoxystrepta-
mine and ring I aminosugar moieties and now broadly used clini-
cally.15–18 Earlier attempts to improve antibiotic efficacy with
modifications in the ring III moiety were limited to chemical varia-
tions at the C600 position.19,20
Recently, we developed protocols to stereoselectively attach a-
glycosyl donors to an ‘optimized’ disaccharide neamine core to
produce novel kanamycin class aminoglycosides.21–23 With this cap-
ability, glycosyl donors with structural variations can be used to create
a library of kanamycin analogs altered in ring III. Structure-bioactivity
screens of libraries of these analogs uncovered a few compounds that
inhibited the growth of fungi and yeasts. One compound, FG08,
described here in detail, displayed broad spectrum fungicidal activity
with concomitant loss of antibacterial activity and a novel mechanism
of action for antimicrobial aminoglycosides.
from the Small Grain Pathology Program, University of Minnesota, Minnea-
polis. Escherichia coli ATCC25922, Staphylococcus aureus ATCC6538, Pseudo-
monas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), Klebsiella
pneumoniae (ATCC 138883), Klebsiella pneumomiae (ATCC 700603), Rhodo-
torula pilimanae strains ATCC9449 and ATCC 26423 and Candida albicans
ATCC10231 were obtained from the American Type Culture Collection
(Manassas, VA, USA). Curvularia brachyspora was obtained from A De Lucca
(Southern Regional Research Center, USDA, ARS, New Orleans, LA, USA) and
E. coli strain B from the microbial culture collection of the Department of
Biology, Utah State University, Logan, UT, USA. Rhizopus stolonifer, Fusarium
oxysporum, Ulocladium spp., Pythium irregulare, Pythium ultimum, Phy-
tophthora parasitica, Phoma spp., Bortrytis cinerea and Cladosporium cladospor-
ioides were obtained from Dr B Kropp, Department of Biology, Utah State
University. R. pilimanae Hedrick and Burke (ATCC 26423) was grown and
maintained as previously described.24 S. cerevisiae W303 was grown on yeast-
extract peptone dextrose medium24 at 281C. All other fungi and oomyctes were
cultivated in potato dextrose broth (Difco, BD, Franklin Lakes, NJ,
USA)+4 g lꢀ1 casamino acids (PDB-CA medium)24 at 28–30 1C. Bacterial
strains were grown at 37 1C for 24h on Luria–Bertani medium25 except S.
aureus ATCC6538 which was grown in Mueller–Hinton medium (Difco).
MATERIALS AND METHODS
Chemical syntheses
In vitro antibacterial and antifungal tests
Microbroth dilution assays for determination of minimal inhibitory concen-
trations (MICs) were conducted using the protocols of the Clinical and
Laboratory Standards Institute.26,27 Bacteria were grown in Luria–Bertani broth
medium and fungi were grown in PDB-CA medium. For disk diffusion growth
inhibitory assays, cultures or conidia were spread on agar plate medium
surfaces. Paper disks (0.5cm diameter) were placed on the surfaces, and 8ml
aliquots of test solutions were applied to the disks. Clear zones of inhibition
were observed after incubation at 371C (bacteria) or 281C (fungi).
Compound FG08 and its analogs (Supplementary Figure 1) were synthesized
through successive regio- and stereoselective glycosylation of an azido neamine
core, deacetylation, Staudinger reduction of azido groups and hydrogenolysis of
benzyl groups using previously described methods21 (Figure 1). Analogs JL022,
JL029 and JL033 and the alkylated glycosyl donor (phenyl 2,3-di-O-benzyl-6-
deoxy-4-O-n-octyl-1-thio-b-D-glucopyranoside) for FG08 were synthesized as
described previously.23 Details of the general synthetic procedures are presented
in Supplementary Information.
Figure 1 Scheme for synthesis of FG08, FG01 and FG02. Neamine derivative compound 3 is described by Li et al.22
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Leaf infection assay
RESULTS AND DISCUSSION
Suspensions of F. graminearum macroconidia (2.0ꢁ104 mlꢀ1) were prepared in
sterile solution of 0.25% (wtvolꢀ1) agar and 0.2% (by volume) of Tween 20
and mixed with equal volumes of FG08 or kanamycin B made in the same
solution. Leaf infection assay methods were previously described.28
An analog of kanamycin B, FG08, was synthesized with phenyl 2,3-
di-O-benzyl-6-deoxy-4-O-n-octyl-1-thio-b-D-glucopyranoside as the
a-glycosyl donor (Figure 2). Related analogs were synthesized with
kanosamine modifications that included placement of an axial or
equatorial hydroxyl group at the 400 position and deoxygenation at the
600 position (Supplementary Figures 1B and D and 3). Also, analogs
were synthesized that were deaminated at the 300 position (Supple-
mentary Figures 1B and D and 3), deoxygenated at the 600 position or
with replacement of the kanosamine 600-CH2OH with a hydrogen
atom (Supplementary Figures 1B–F and 4), and with attachment of
hydroxyaminoalkyl chains in place of the C8 alkyl chain at the 400
position (Supplementary Figures 1C and E).
FHB disease suppression
Rapid-maturing cultivar Apogee29,30 was grown for 5–6 weeks in a greenhouse
to the flowering stage. Florets (one per spikelet) were treated with a solution of
FG08 (10 ml, 5 and 1080 mg mlꢀ1, and then inoculated with suspension of
F. graminearum macroconidia (10 ml, 105 conidia mlꢀ1). After 4 days, the
spikelets were visually inspected for disease symptoms (chlorosis, spikelet
curling and dehydration).
FG08 showed little or no growth inhibition of Gram-positive and
Gram-negative bacteria (Figure 3, Table 1) with MICs that were at
least 125-fold higher than shown by kanamycin B (Table 1). Instead,
FG08 was fungicidal to a variety of yeasts, oomycetes and true fungi
with MICs ranging between 3.9 and 31.3mg mlꢀ1 (Table 1, Figure 3).
Like kanamycin B, all synthesized analogs of FG08 lacked antifungal
activity (Supplementary Table 1). Therefore, the antibacterial to
Mechanism of action
Fungal membrane permeability was assessed using SYTOX green.31 Haemolytic
activity was determined using sheep erythrocytes according to previously
described methods.32
Detailed procedural descriptions accompany this paper (Supplementary
Information).
NH2
4’
NH2
4’
HO
HO
O
HO
HO
O
1’
1’
3’
3’
4
4
H2N
HO
H2N
HO
NH2
NH2
NH2
NH2
O
O
6
6
O
O
1
1
5’’
5’’
1’’
1’’
CH3
OH
O
O
HO
HO
OH
3’’
3’’
4’’
O
4’’
C8H17
H2N
OH
Figure 2 Structures of kanamycin B and FG08. FG08 differs from kanamycin B with C8 alkyl, hydroxyl and H vs H, amino and OH groups at the 400, 300 and
500 positions, respectively.
S. a.
E. c.
S. c.
C. c.
FG08
kanB
FG08 concentration x 1mg mL-1
2-3 2-4 2-5 2-6
Ca
Cb
20
2-1
2-2
2-7
2-8
2-9
Figure 3 Antimicrobial activities of FG08 and kanamycin B. (Upper panel) Disk agar diffusion assays show that FG08 is antifungal, but not antibacterial in
contrast to kanamycin B (kanB). Water solutions of FG08, kanamycin B, at concentrations of 10.9, 10.0 mgmlꢀ1, respectively (each of volume 8 ml), were
applied to paper disks. (Lower panel) FG08 has an antifungal MIC of 31.3 mg mlꢀ1 against F. graminearum as revealed by microbroth dilution inhibition
assays (also see Table 1). Controls are Ca (medium only) and Cb (no compound). S.a., Staphylococcus aureus; E.c., Escherichia coli;
S. c., Saccharomyces cereviseae; C.c., Cladosporium cladosporioides.
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Table 1 Minimal inhibitory concentrations (MICs) of FG08 and
kanamycin B against bacteria and fungi
a
MIC (mg mlꢀ1
)
Organism
FG08
Kanamycin B
Bacteria
Escherichia coli TG1
125–250
250
1.95
o0.98
1.95
Staphylococcus aureus ATC 25923
Pseudomonas aeruginosa (ATCC 27853)
Enterococcus faecalis (ATCC 29212)
Klebsiella pneumoniae (ATCC 138883)
Klebsiella pneumomiae (ATCC 700603)
250
125–250
250
o0.98
1.95
250
1.95
Fungi
Rhodotorula pilimanae (ATCC26423)
Candida albicans (ATCC10231)
Saccharomyces cerevisiae W303
Fusarium graminearum B-4-5A
Fusarium oxysporum
Ulocladium spp.
7.8
31.3
3.9
4250
4250
4250
4250
4250
NDb
ND
31.3
7.8
7.8
Pythium irregulare
15.6
15.6
15.6
31.3
31.3
31.3
31.3
31.3
Pythium ultimum
ND
Phytophthora parasitica
Rhizopus stolonifer
ND
ND
Cladosporium cladosporioides
Curvularia brachyspora
Bortrytis cinerea
ND
ND
ND
Phoma spp.
ND
aMicrobroth dilution assays were performed at least twice, and each in triplicate.
bNot determined.
antifungal activity ‘switch’ in the FG08 analog appears to be mainly
due to the occurrence of a C8 alkyl chain at the 400 position. Finally,
structure-bioactivity analyses were conducted as a function of the
length of the 400 alkyl chain. Analogs with shorter (C4) or longer (C12)
alkyl chains had lower antifungal activities than FG08 (Supplementary
Figure 2). Therefore, the C8 alkyl chain imparts optimal antifungal Figure 4 FG08 suppression of wheat leaf infection after exposure to
F. graminearum (F. g.). Two-leaf stage FHB-susceptible Alsen (blank bars) or
activity.
FHB-resistant Frontana (black bars) wheat leaves were inoculated with
The fungus F. graminearum—the causative agent of FHB—was
F. graminearum macroconidia and treated with FG08 (10ml drop at the
susceptible to FG08 as observed with in vitro susceptibility tests
(Figure 3, lower panel). FG08 was investigated for its ability to control
inoculation site) 24 h later. (Upper panel) Cut segments from leaves treated
with 30mg mlꢀ1 FG08 did not show subsequent mycelial growth when
FHB of wheat. First, leaf infection assays with seedlings (2 leaves, 14
days post germination) that correlate with FHB disease potential28
were used to assess FG08’s ability to suppress in planta F. graminearum
infection. Suspensions of F. graminearum (strain B-4-5A) macroconi-
dia were premixed with FG08 or kanamycin B each at 30 and
180 mg mlꢀ1 concentrations, 0.25% agar and Tween 20 (0.2%, by
volume) and 10 ml of the mixture applied on the surface at the middle
of primary leaves of FHB susceptible (Alsen) and resistant (Frontana)
incubated on potato dextrose agar medium, and (middle panel) this level of
FG08 caused a 5-fold decrease in leaf lesions caused by F. graminearum
infection. (Middle panel) FG08 at 30 mg mlꢀ1 was not phytotoxic as seen by
no increase in lesion area with both FHB susceptible (Alsen) and resistant
(Frontana) cultivars and (lower panel) no decrease in chlorophyll (Chl)
content. FG08 at 180mg mlꢀ1 showed phytotoxicity in this assay.
cultivar seedlings at the two-leaf growth stage. With the Alsen cultivar, not influenced by exposure to FG08 (Figure 4, upper and middle
leaves inoculated with mixtures containing FG08 at 30mg mlꢀ1 panels). Subsequent incubation of macroconidia-inoculated and FG08
showed 5- and 1.25-fold less leaf lesion area and degree of chlorosis, (30 mg mlꢀ1)-treated leaf segments on potato dextrose agar medium
respectively, when compared with non-FG08 treated controls (25 1C, 5 days) showed no mycelia growth indicating that FG08 at this
(Figure 4). At 180 mg mlꢀ1, macroconidia-induced lesion formation concentration was fungicidal (Figure 4, upper panel). Altogether, these
was not measurable because of possible phytotoxicity at this concen- results show that FG08 at concentrations equal to their in vitro MICs
tration as suggested by controls with no macroconidia inoculation afforded in planta protection against F. graminearum infection when
(Figure 4, lower panel). In contrast, kanamycin B did not suppress applied to leaves of susceptible wheat seedlings.
lesion formation at 180 mgmlꢀ1. As expected, leaves of resistant cultivar
Second, the ability of FG08 to suppress FHB symptoms of wheat
Frontana showed relatively low levels of lesion formation and chlorosis head spikelets exposed to F. graminearum macroconidia was exam-
with or without macroconidia inoculation, and these parameters were ined. Cultivar Apogee—a rapidly maturing and FHB-susceptible
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Figure 6 FG08 effects on membrane permeability. (a) Addition of FG08 at
30 and 10 mg mlꢀ1 (10min) increased the permeability of C. albicans (C. a.)
cells and F. graminearum (F. g.) hyphae, respectively, to SYTOX green
(0.2 mM). (b) FG08 is more effective than kanamycin B (kanB) (both with
10 min exposure) in causing SYTOX green dye permeation in C. albicans
cells. At 30mg mlꢀ1, FG08 was B12 times more effective than kanamycin
B. Triton X100 (1%) gave 100% dye permeation (data not shown). Numbers
above the range bars indicate number of cells analyzed. (c) Neither FG08
Figure 5 FG08 suppression of FHB disease in wheat spikelet florets. Florets
(one per spikelet) of early flowering heads (cultivar Apogee) were pre-treated
with FG08 (10 ml, 5–1080 mg mlꢀ1 followed by inoculation with
F. graminearum (F.g.) macroconidia (10 ml, 105 conidia mlꢀ1) 24h later.
(Upper panel) Without FG08 pretreatment, FHB symptoms (chlorosis and
(gray bars) or kanamycin
B (blank bars) caused hemolysis of sheep
curled spikes) were evident within
pretreatment. (Lower panel) Suppression was observed at FG08
concentrations between
and 1080 mg mlꢀ1 with reduced disease
4 days, but suppressed with
erythrocytes at concentrations equal to their antifungal or antibacterial MICs,
respectively.
5
incidences between 27 and 82%, respectively. Uninoculated spikelets had
visually healthy florets following FG08 treatments at concentrations ranging
between 5 and 1080 mg mlꢀ1. Numbers in the bars indicate the numbers of
florets examined (8–12 florets per spikelet), and the florets were scored as
diseased or not diseased on the basis of visual inspection.
of action differs from that of kanamycin B and appears to involve the
perturbation of fungal membranes. Whether FG08’s membrane effects
are direct (for example, pore formation) or indirect or both are not
known. The rapidity of FG08’s influence on dye permeability (10 min),
however, suggests a role for direct membrane effects. In contrast, FG08
did not lyse more than 20% of erythrocytes at 313 mg mlꢀ1 (10-fold
higher than the antifungal MIC) and only 2% at 28mg mlꢀ1
(Figure 6c). Therefore, at antifungal MICs, FG08 does not seem to
act as a surface-active agent that non-specifically disrupts membranes.
In summary, a novel neamine aminoglycoside analog, FG08—with
a linear C8 alkyl chain as a major structural feature—is antifungal in
contrast to its prototype analog kanamycin B that is predominantly
antibacterial. FG08 also lacks antibacterial activity and appears to have
effects on fungal membrane permeability. These findings suggest that
FG08 and its next generation analogs may have agrofungicidal and
possibly therapeutic applications. With a different mechanism of
action, it may be speculated that FG08 would not be subject to the
resistance mechanisms that have evolved for the classical aminoglyco-
sides. The overall finding of this study, whereby a chemically simple
modification of an obsolete drug (for example, kanamycin B) yields a
potentially useful one, suggests a novel drug development strategy.
variety of wheat—was used.29,30 FG08 pre-treatment of Apogee
spikelet florets at concentrations as low as 5 and 30mg mlꢀ1 reduced
the rate of spikelet infection by 29 and 49%, respectively, over controls
that were not pretreated with FG08 (Figure 5). FG08 pretreatments at
1080mg mlꢀ1 reduced disease incidence by 82%. FG08 alone did not
show toxicity in spikelets as observed in controls that were not
inoculated with macroconidia, even at the highest concentration
tested (1080 mg mlꢀ1). These results show that FG08 pretreatment of
wheat spikelets protects against F. graminearum infection with no
phytotoxicity. As, this procedure mimics the spread of FHB in the
field, these findings are particularly relevant to FG08’s potential for
controlling this disease.
The C8 alkyl chain should confer amphipathic properties to FG08.
This raises the prospect that its antifungal activity is because of
perturbation of membrane function. Studies with fluorescent dye
SYTOX green (Molecular Probes, Invitrogen, Carlsbad, CA USA)
were conducted to determine if FG08 increases cell surface membrane
permeability of fungi. At levels close to the fungal MICs, FG08 allowed
dye permeation of C. albicans cells and F. graminearum hyphae
(Figure 6a). With C. albicans, FG08 was about 12 times more effective
than kanamycin B (Figure 6b). The results suggest that FG08’s mode
ACKNOWLEDGEMENTS
We acknowledge LTakemoto and H HLiu for technical assistance, B Kropp and
A DeLucca for fungal strains, and B Bugbee for Apogee wheat seeds. The
research was funded by Baicor LC, the National Institutes of Health (Grant no.
AI053138 to CW T Chang), the Utah Science Technology and Research
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Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics