Meleagrin, a New FabI Inhibitor from Penicillium chryosogenum with at Least One Additional Mode of Action
a New FabI Inhibitor from Penicillium chryosogenum with at Least One Additional Mode of
Action. PLoS ONE 8(11): e78922. doi:10.1371/journal.pone.0078922
Meleagrin, a New FabI Inhibitor from Penicillium chryosogenum with at Least One Additional Mode of Action
Chang Ji Zheng 0
Mi-Jin Sohn 0
Sangku Lee 0
Won-Gon Kim 0
Martin Pavelka, University of Rochester, United States of America
0 Superbacteria Research Center, Korea Research Institute of Bioscience and Biotechnology , Yusong, Daejeon , Republic of Korea
Bacterial enoyl-acyl carrier protein reductase (FabI) is a promising novel antibacterial target. We isolated a new class of FabI inhibitor from Penicillium chrysogenum, which produces various antibiotics, the mechanisms of some of them are unknown. The isolated FabI inhibitor was determined to be meleagrin by mass spectroscopy and nuclear magnetic resonance spectral analyses, and its more active and inactive derivatives were chemically prepared. Consistent with their selective inhibition of Staphylococcus aureus FabI, meleagrin and its more active derivatives directly bound to S. aureus FabI in a fluorescence quenching assay, inhibited intracellular fatty acid biosynthesis and growth of S. aureus, and increased the minimum inhibitory concentration for fabI-overexpressing S. aureus. The compounds that were not effective against the FabK isoform, however, inhibited the growth of Streptococcus pneumoniae that contained only the FabK isoform. Additionally no resistant mutant to the compounds was obtained. Importantly, fabK-overexpressing Escherichia coli was not resistant to these compounds, but was resistant to triclosan. These results demonstrate that the compounds inhibited another target in addition to FabI. Thus, meleagrin is a new class of FabI inhibitor with at least one additional mode of action that could have potential for treating multidrug-resistant bacteria.
Funding: This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of
Education, Science and Technology (2012R1A2A2A01014821) and the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of
Education, Science and Technology (2011-0031944). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
Multidrug-resistant bacteria such as methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci, and
vancomycin-resistant S. aureus have become an important global
health concern [1,2]. One approach to combat antibiotic
resistance is to identify new drugs that can function through novel
mechanisms of action. One such target is bacterial type 2 fatty acid
synthesis (FASII), which is essential for bacterial cell growth .
FASII is conducted by a set of individual enzymes, whereas
mammalian fatty acid synthesis is mediated by a single
multifunctional enzyme-acyl carrier protein (ACP) complex
referred to as type I. Enoyl-ACP reductase catalyzes the final
and rate-limiting step of the chain-elongation process of the
FASII. Four isoforms have been reported for enoyl-ACP
reductase. FabI is highly conserved among most bacteria,
including S. aureus and Escherichia coli. Streptococcus pneumoniae
contains only FabK, whereas Enterococcus faecalis and Pseudomonas
aeruginosa contain both FabI and FabK, and Bacillus subtilis
contains both FabI and FabL. Recently, the FabV isoform was
isolated from Vibrio cholera, Pseudomonas aeruginosa, and Burkholderia
mallei [6,7]. No analogue protein is present in mammals for
similar transformation; thus, FabI inhibitors should not interfere
with mammalian fatty acid synthesis. Because of these properties,
FabI is an attractive target for antibacterial drug development
[8,9]. As drugs with single targets such as rifampicin and
fosfomycin are particularly vulnerable to mutational resistance
, FabI-specific inhibitors also have a tendency to develop
resistance in bacteria by mutations that alter the drug-binding
site. FabI is known to be the main target for triclosan and
isoniazid, which have been used in consumer products and for
treating tuberculosis, respectively [11,12]. Triclosan-resistant
bacteria and isoniazid-resistant M. tuberculosis are highly prevalent
because of point mutations in their FabI genes . In
addition, rapid mutation development has been often reported in
synthetic FabI inhibitors . Thus, it has been recently
emphasized that ideal antibiotics should bind to multiple targets
Many FabI inhibitors have been reported from
high-throughput screening of existing compound libraries. However, most are
not suitable for the development of new antibiotics because of
their lack of permeability into cell membranes and efflux in
addition to their high mutational frequency . The problem
with such screening results lies in the compound libraries, which
are systematically biased. Microorganisms produce diverse
antibiotics that function in an antagonistic capacity in nature
where they have competition. Most antibacterial agents in
clinical use today are either microbial products or analogs .
A few FabI inhibitors have been reported from microorganisms
, and most of these are phenolic compounds. Therefore,
more unique FabI inhibitors need to be obtained from
During our continued screening for FabI inhibitors from
microbial metabolites, we found meleagrin (1) with a druggable
structure during solid-state fermentation of a seashore
slimederived Penicillium chrysogenum, a penicillin-producing species
(Figure 1). Here, we report the isolation and analog preparation
of meleagrin, in addition to its inhibition of FabI isoforms and
whole cells of various pathogenic bacteria, target validation, and
its multitarget effect.
Materials and Methods
The bacterial strains used in the antibacterial activity assays
were obtained from the Culture Collection of Antimicrobial
Resistant Microbes of Korea and the Korean Collection for
Type Cultures. The pump-negative (tolC) E. coli EW1b was
obtained from the E. coli Genetic Stock Center of Yale
Screening and isolation of compound 1
Over 25,000 microbial extracts composed of actinomycetes
and fungi were screened against S. aureus FabI and confirmed
through a target-based whole cell assay by using
fabI-overexpressing S. aureus. This analysis led to the identification of
compound 1 from fungal strain F717 (Fig. 1). Compound 1 was
isolated from the fermented whole medium of the fungal strain
F717, which was isolated from seashore slime collected at
Daechun beach, Chungcheongnam-do, Korea. The strain was
identified as Penicillium chrysogenum based on standard biological
and physiological tests and taxonomic determination. Seed
culture was conducted in a liquid culture medium containing
2% glucose, 0.2% yeast extract, 0.5% peptone, 0.05% MgSO4,
and 0.1% KH2PO4 (pH 5.7 before sterilization). A sample of the
strain from a mature plate culture was inoculated into a 500-mL
Erlenmeyer flask containing 80 mL of the above sterile seed
liquid medium and cultured on a rotary shaker (150 rpm) at
28uC for 3 days. Subsequently, 5 mL of the seed culture was
transferred into 500-mL Erlenmeyer flasks (54 flasks) containing
80 g of bran medium, which was cultivated for 7 days at 28uC to
produce the active compound. The culture solid state was
extracted with 80% acetone, and the extract was concentrated in
vacuo to an aqueous solution. The aqueous solution was then
extracted 3 times with an equal volume of ethyl acetate (EtOAc).
The EtOAc extract was concentrated in vacuo to dryness. The
crude extract was subjected to SiO2 (Merck Art No. 7734.9025)
column chromatography followed by stepwise elution with
CHCl3-MeOH (100:1, 50:1, and 10:1). The active fractions
eluted with CHCl3-MeOH (50:1) were pooled and concentrated
in vacuo to give an oily residue. The residue was applied again to
a Sephadex LH-20 and then eluted with CHCl3-MeOH (1:1).
The active fraction was dissolved in MeOH and was further
purified by reverse-phase high-performance liquid
chromatography (206150 mm; YMC C18) by using a photodiode array
detector. The column was eluted using MeOH: H2O (75:25) at a
flow rate of 5 mL/min to afford compound 1 with .99% purity
at a retention time of 19.4 min. The chemical structure of
compound 1 was determined to be meleagrin  by mass
spectroscopy (MS) and nuclear magnetic resonance (NMR)
spectra as follows: [a]D = 296.7u (c = 0.04, MeOH);
HRESIMS: m/z 434.18463 (M+H)+, C23H23N5O4 requires 434.18228;
1H-NMR (600 MHz, DMSO-d6): 8.30 (1H, s, NH-19), 8.17 (1H,
s, H-15), 7.77 (1H, s, H-20), 7.53 (1H, d, J = 7.5, H-4), 7.34 (1H,
s, H-18), 7.25 (1H, t, J = 7.5, H-6), 7.03 (1H, t, J = 7.5, H-5),
6.96 (1H, d, J = 7.5, H-7), 6.00 (1H, brs, H-22), 5.25 (1H, s,
H8), 5.01 (1H, d, J = 17.1, Ha-23), 4.98 (1H, d, J = 9.0, Hb-23),
3.66 (3H, s, 1-OCH3), 1.19 (6H, s, CH3-24 and 25), 13C-NMR
(150 MHz, DMSO-d6): 165.0 (C-13), 158.6 (C-10), 146.2 (C-7a),
143.3 (C-22), 142.7 (C-9), 137.6 (C-20), 134.1 (C-18), 127.8
(C6), 126.0 (C-3a), 125.9 (C-16), 124.7 (C-4), 123.7 (C-12), 123.1
(C-5), 112.8 (C-23), 111.6 (C-7), 109.2 (C-8), 106.7 (C-15), 101.5
(C-2), 64.8 (1-OCH3), 52.2 (C-3), 41.8 (C-21), and 23.0 (C-24
Preparation of derivatives of compound 1
Several derivatives of 1 were obtained by chemical
modification of functional groups such as hydroxyl and amine groups
(Fig. 1). Demethoxylation of compound 1 afforded glandicolin A
(2) together with compound 7 as a byproduct. Methylation of
compound 1 produced oxaline (3), N14-methylmeleagrin (4), and
O,N14-dimethylmeleagrin (5). O,N14-dimethylglandicolin (6) was
obtained by methylation of compound 2. Details regarding the
preparation procedures and spectral data of compounds 27 are
presented in Information S1.
FabI and FabK assay
S. aureus FabI and E. coli FabI enzymes were cloned,
overexpressed, and purified as described previously . The
wild-type fabK gene was amplified by PCR from genomic DNA
obtained from Streptococcus pneumoniae KCTC 5412 by using the
which contained NdeI and XhoI restriction sites, respectively. After
the DNA sequence was confirmed, the gene was cloned into the
pET22b vector (Novagen, Gibbstown, NJ, USA). The construct
was transformed into E. coli BL21 (DE3) for expression following
induction with isopropylthiogalactoside. The C-terminal
Histagged protein was purified as described previously . Assays
were conducted in half-area, 96-well microtiter plates. The
compounds were dissolved in DMSO and evaluated in 100-mL
assay mixtures containing components specific for each enzyme
(see below). Reduction of the trans-2-octenoyl N-acetylcysteamine
(t-o-NAC thioester) substrate analog was measured
spectrophotometrically following the utilization of NADH or NADPH at
340 nm at 30uC for the linear period of the assay. S. aureus FabI
assays contained 50 mM sodium acetate (pH 6.5), 200 mM
t-oNAC thioester, 200 mM NADPH, and 150 nM S. aureus FabI.
NADH was used as a cofactor rather than NADPH for the E.
coli FabI assay. Substrate concentrations used for the
LineweaverBurk plot were 100, 200, 300, and 400 mM, whereas the
concentrations of the cofactor were 100, 200, 400, and 600 mM.
The rate of decrease in the amount of NADPH in each reaction
was measured with a microtiter enzyme-linked immunosorbent
assay (ELISA) reader by using the SOFTmax PRO software
(Molecular Devices, Sunnyvale, CA, USA). The inhibitory
activity was calculated according to the following formula: %
of inhibition = 1006 [12 (rate in the presence of compound/
rate in the untreated control)]. IC50 values were calculated by
fitting the data to a sigmoid equation. An equal volume of
DMSO solvent was used for the untreated control. FabK assays
contained 100 mM sodium acetate (pH 6.5), 2% glycerol,
200 mM NH4Cl, 50 mM t-o-NAC thioester, 200 mM NADH,
and 150 nM S. pneumoniae FabK.
E. colib S. pneumoniaec
aS. aureus RN4220; bE. coli KCTC 1924; cS. pneumoniae KCTC 3932.
Figure 2. The mechanism of inhibition of Staphylococcus aureus FabI by meleagrin respective to t-o-NAC thioester (A) and NADPH (B), and
Ki determination of meleagrin (C).
Fluorescence quenching assay
Fluorescence spectra were measured using a SHIMADZU
fluorescence spectrophotometer (model RF-5310PC). S. aureus
FabI (15 ng/ml) was incubated with different concentrations of
triclosan (1, 2, 4, 8, and 16 nM in PBS buffer) and compounds 1,
5, or 7 (10, 20, 40, 80, and 160 nM in PBS buffer). Protein
quenching was monitored at 25uC by using 5-nm excitation and
5-nm emission wavelength. The excitation wavelength was
Determination of minimum inhibitory concentrations
Whole-cell antimicrobial activity was determined by broth
microdilution as described previously . The test strains except
for S. pneumoniae were grown to mid-log phase in MuellerHinton
broth and diluted 1,000-fold in the same medium. Cells (105/mL)
were inoculated into MuellerHinton broth and dispensed at
0.2 mL/well into a 96-well microtiter plate. S. pneumoniae was
grown in tryptic soy broth supplemented with 5% sheep blood.
MICs were determined in triplicate by serial 2-fold dilutions of test
compounds. The MIC was defined as the concentration of a test
compound that completely inhibited cell growth during a 24-h
incubation at 30uC. Bacterial growth was determined by
measuring the absorption at 650 nm by using a microtiter ELISA
Measurement of the inhibition of macromolecular
To monitor the effects of compound 1 on lipid, DNA, RNA,
protein, and cell wall biosynthesis, its effects on the incorporation
of [1-14C] acetate (50 mCi/mmol), [2-14C] thymidine (59.8 mCi/
mmol), [U-14C] uridine (539 mCi/mmol), L-[U-14C] leucine (306
mCi/mmol) or L-[U-14C] isoleucine (329 mCi/mmol), and
Nacetyl-D-[1-14C] glucosamine (58.1 mCi/mmol) into S. aureus and
S. pneumoniae were measured as described previously . S. aureus
was exponentially grown to an A650 of 0.2 in MuellerHinton
broth. S. pneumoniae was grown in tryptic soy broth supplemented
with 5% sheep blood. Each 1-mL culture was treated with drugs at
2 times the MIC for 10 min. An equal volume of DMSO solvent
was added to the untreated control. After incubation with the
radiolabeled precursors at 37uC for 1 h, followed by
centrifugation, the cell pellets were washed twice with PBS buffer. After
acetate incorporation, the total cellular lipids were extracted with
chloroform-methanol-water. The incorporated radioactivity in the
chloroform phase was measured by scintillation counting. For the
other precursors, incorporation was terminated by adding 10%
(w/v) TCA and cooling on ice for 20 min. The precipitated
material was collected on Whatman GF/C glass microfiber filters,
washed with TCA and ethanol, dried, and counted using a
scintillation counter. The total counts incorporated at 1 h of
incubation without inhibitors ranged from .7,000 for [U-14C]
uridine to ,13,000 for [1-14C] acetate. The inhibition of
radiolabeled precursor incorporation was calculated using the
following formula: % inhibition = 1006 [12 (radioactivity values
of the treated samples/control (no antibacterial) values)]. In all
experiments, known antibacterial agents were included as positive
Frequency of the spontaneously resistant mutant
The frequency of spontaneous resistance was determined for S.
aureus RN4220, S. aureus KCTC 1916, and E. coli KCTC 1942. E.
coli KCTC 1942 is highly sensitive to antibiotics. The organisms
were grown to log-phase by dilution of an overnight culture in
fresh media and re-incubation at 35uC until the cultures reached a
cell density of approximately 109 CFU/mL. A volume of 100 ml of
the bacterial suspension was then applied to solid media
containing 46 MIC of 1, 5, or triclosan. Inocula were determined
by applying 100 ml of 10-fold dilutions on solid media without
drug. Colony-forming units were counted after 48 h incubation at
35uC. The ratio of the number of colonies on drug-containing
plates to that on control plates was calculated as the in vitro
frequency of isolation of CFU.
An overexpression assay using S. aureus RN4220, S. aureus
RN4220 (pE194), and S. aureus RN4220 (pE194-fabI) was
conducted to perform target validation of FabI inhibitors as
described previously . Additionally, both fabI- and
fabKoverexpressing E. coli were constructed to test a multitarget effect
of the compounds. The wild-type fabI gene from the genomic
DNA of E. coli W3110 was amplified by PCR by using the primers
59TTTCAGTTCGAGTTCGTTCATT-39. The wild-type fabK
gene from the genomic DNA of S. pneumoniae KCTC 5412 was
amplified by PCR by using the primers
59-ATGAAAACGCGTATTACA-39 and 59-GTCATTTCTTAC
AACTCCTGTCCA39. The resulting products were cloned into the pBAD-TOPO TA
expression vector (Invitrogen, Carlsbad, CA, USA) to yield the
pBAD-fabI and pBAD-fabK recombinant plasmids, which placed
the expression of the genes fabI and fabK, respectively, under the
control of the arabinose promoter . Recombinant pBAD-fabI
and pBAD-fabK were then introduced into the pump-negative
(tolC) E. coli EW1b via electroporation to generate E. coli EW1b
(pBAD-fabI) and E. coli EW1b (pBAD-fabK), respectively.
Isolation of meleagrin as a new FabI inhibitor
A FabI inhibitor was isolated from Penicillium chrysogenum F717,
which is known as a penicillin-producing species. MS and NMR
spectral analyses of the inhibitor revealed that it was meleagrin (1)
(Fig. 1). Compound 1 inhibited both E. coli and S. aureus FabI with
S. aureus (pE194)
S. aureus (pE194-fabI)
S. aureus KCTC 1916
E. coli KCTC 1924
Frequency of resistance
IC50 values of 33.2 and 40.1 mM, respectively (Table 1). To
determine whether compound 1 selectively inhibited FabI, its
effect on FabK, which is the enoyl-ACP reductase of S. pneumoniae,
was examined. Compound 1 did not inhibit S. pneumoniae FabK
even at 200 mM, which indicates that it is selective for FabI.
Mode of FabI inhibition
The FabI reaction mechanism requires the nucleotide cofactors
NADH or NADPH as the first substrates . The FabI inhibitor
could bind to the free enzyme, the enzyme-substrate complex, or
both to prevent catalysis. In the first case, the inhibition pattern
with respect to the cofactor would be competitive; in the second,
the inhibition pattern would be non-competitive; and in the third
case, mixed-type inhibition would occur. Inhibition of S. aureus
FabI by compound 1 was mixed with respect to trans-2-octenoyl
Nacetylcysteamine, with a Ki value of 39.8 mM (Fig. 2A and 2C). In
addition, compound 1 exhibited mixed inhibition with respect to
NADPH, with a Ki value of 32.3 mM (Fig. 2B). Thus, compound 1
must bind to both the free enzyme and the FabI-NADPH complex
to prevent binding of the nucleotide cofactor and the substrate,
Effects of structural changes in compound 1 on FabI and
To determine whether structural changes in compound 1
influence its effects on FabI, compound 1 and its derivatives were
tested against S. aureus and E. coli FabI and bacterial growth
(Table 1). Compounds 5 and 6, which were modified at both the
9-OH and 14-NH groups, produced a significant increase in S.
aureus and E. coli FabI-inhibitory activity, and they enhanced
antibacterial activity against S. aureus and E. coli. In contrast,
compounds 2, 3, and 4, which were modified at the 1-NH, 9-OH,
and 14-NH groups, respectively, did not affect activity. Compound
7, which was brominated at the benzene ring of compound 2,
totally lost its activity.
Effects on fluorescence quenching of S. aureus FabI
We examined whether active compounds directly bind with
FabI by fluorescence quenching analysis. S. aureus FabI displayed
strong maximal fluorescence at 307 nm after excitation at 270 nm
(Fig. 3), whereas triclosan, kanamycin, 5, and 7 had no
fluorescence at this wavelength (data not shown). When S. aureus
FabI was incubated with increasing amounts of active compound
5, its fluorescence intensity decreased gradually (Fig. 3A), whereas
the inactive compound 7 did not exhibit such an effect (Fig. 3B).
Compound 1 showed the same pattern as compound 5 (data not
shown). As a positive control, triclosan binding resulted in
fluorescence quenching of S. aureus FabI (Fig. 3C), whereas
kanamycin as a negative control did not (Fig. 3D). These data
indicate that the active compounds 1 and 5 directly interact with
S. aureus FabI, whereas compound 7 does not, thus explaining their
effects on FabI.
Inhibition of cellular fatty acid synthesis
To evaluate whether the active compounds inhibit cellular fatty
acid synthesis, we determined whether the compounds inhibited
the incorporation of acetate into membrane fatty acids in vivo. We
measured their effects on the incorporation of [1-14C] acetate into
membrane fatty acids in S. aureus. In agreement with their
antibacterial activity and FabI-inhibitory activity, the more active
compounds 5 and 6 indeed blocked incorporation of
radioactivelylabeled acetate into chloroform/methanol-extractable
phospholipids in vivo in a concentration-dependent manner, with
approximately 2-fold higher activity than the less active compounds 1, 3,
and 4 (Table 1). The inactive compound 7 did not exhibit such
fatty acid synthesis inhibition even at 200 mM, as expected. As a
positive control, triclosan inhibited fatty acid synthesis in a
concentration-dependent manner (data not shown). In contrast,
Inhibition of precursor incorporation (%)
aS. aureus RN4220; bS. pneumoniae KCTC 3932. cReference antibacterials used for inhibition of acetate, thymidine, uridine, isoleucine, and N-acetyl-D-glucosamine
incorporation are triclosan, norfloxacin, rifampin, chlorampenicol, and vancomycin, respectively. dReference antibacterials in S. pneumoniae were the same as in S.
aureus, except cerulenin was used instead of triclosan for acetate inhibition.
E. coli EW1b(pBAD)
E. coli EW1b(pBAD-fabI)
E. coli EW1b(pBAD-fabK)
a3% arabinose was treated.
the incorporation of leucine into proteins was not inhibited by the
active compounds (Table 1), whereas the protein synthesis
inhibitor, chloramphenicol, inhibited incorporation (data not
Consistent with their FabI-inhibitory activity, compounds 5 and
6 showed 24 times higher antibacterial activity than compound 1
against S. aureus RN4220 and the highly sensitive strain E. coli
KCTC 1924 (Table 1), as expected. Interestingly, compounds that
were inactive against the FabK isoform exhibited antibacterial
activity against S. pneumoniae KCTC 3932, which contains only the
FabK isoform. This finding suggests that the compounds inhibit
not only FabI but also another target. Compounds 5 and 6 also
showed antibacterial activity against other gram-positive bacteria,
including S. aureus 503, S. aureus KCTC 1916, MRSA CCARM
3167, MRSA CCARM 3506, QRSA CCARM 3505, QRSA
CCARM 3519, Staphylococcus epidermis KCTC 3958, B. subtilis
KCTC 1021, and Micrococcus luteus KCTC 1056 with MIC values
of 816 mg/mL.
Effects on fabI-overexpressing S. aureus
The increase in the MIC for the fabI-overexpressing strain
relative to the wild type is indicative of FabI being the mode of
antibacterial action . The antibacterial activity of the active
compounds for the fabI-overexpressing strain was investigated to
determine whether overexpression of fabI shifted the MIC for S.
aureus. The MICs for the fabI-overexpressing strain S. aureus
RN4220 (pE194-fabI) were 48-fold higher than those of the
wildtype strain S. aureus RN4220, or the vector-containing strain S.
aureus RN4220 (pE194) (Table 2). The MIC for triclosan in the
fabI-overexpressing strain increased, which was used as a positive
control. Erythromycin, the selection marker for the vector pE194,
increased the MICs for both the fabI-overexpressing strain and the
vector-containing strain, which indicated that the engineered
constructs functioned as expected. Antibiotics with different modes
of action such as oxacillin and norfloxacin were applied as
negative controls and did not change the MICs of the 3 strains,
which indicates that altered expression of fabI does not alter the
sensitivity of cells to antibiotics in general. These results indicate
that the active compounds inhibited the growth of S. aureus by
inhibiting the fabI-encoded ENR.
Frequency of spontaneously resistant mutants
We isolated resistant mutants to determine which other gene or
genes were targeted by the active compounds (Table 3). As a
control, triclosan-resistant mutants were isolated at a frequency of
3.3060.1361028, 2.5860.0461029, and 9.0760.0861028 from
S. aureus RN4220, S. aureus KCTC 1916, and the
antibioticsensitive E. coli KCTC 1942, respectively. However, no mutants
resistant to compounds 1 and 5 were detected from the strains
tested. These results suggest that compounds 1 and 5 inhibit
Effects on macromolecular biosynthesis
To identify other pathways inhibited by compound 1, the effects
of compound 1 on the incorporation of radiolabeled precursors of
macromolecular synthesis in S. pneumoniae and in S. aureus were
investigated. All reference antibacterial agents selectively inhibited
the macromolecular synthesis pathway, which is consistent with
their known mechanism of action (Table 4). Compound 1
inhibited the incorporation of acetate into lipids in both S. aureus
and S. pneumoniae by 62% and 65%, respectively, whereas the
incorporation of thymidine, uridine, isoleucine, and
N-acetylglucosamine, into DNA, RNA, protein, and the cell wall, respectively,
was not inhibited. Because compound 1 is inactive against the
FabK isoform, these data suggest that compound 1 inhibits at least
one additional target in addition to FabI in the fatty acid pathway.
Effects on fabK-overexpressing E. coli
To demonstrate that active compounds 1 and 5 inhibit not only
FabI but also an additional target, we cloned fabK and fabI into an
arabinose-inducible expression system, vector pBAD TOPO, and
placed this plasmid in a TolC-negative E. coli host. Because FabK
is resistant to compounds 1 and 5, if the compounds inhibited only
FabI, expression of FabK in E. coli would lead to resistance to
compounds 1 and 5 because the expressed FabK can compensate
for the inhibited FabI. As expected, the MICs of compounds 1 and
5 for fabI-overexpressing E. coli EW1b (pBAD-fabI) were 4-fold
higher than those for wild-type E. coli EW1b and vector-containing
E. coli EW1b (pBAD) in the presence of arabinose (Table 5).
However, the MICs for the fabK-overexpressing E. coli EW1b
(pBAD-fabK) did not change. As a positive control, triclosan,
which does not inhibit FabK, showed inducer-dependent higher
MICs for fabK-overexpressing E. coli and fabI-overexpressing E.
coli. Therefore, S. pneumoniae FabK replaced E. coli FabI for fatty
acid synthesis, which, in turn, indicates that FabI is the only target
of triclosan in this system. Ampicillin, which is the selection marker
for the pBAD vector, increased the MICs for all vector-containing
strains, thereby demonstrating normal functioning of the
constructs. Actinonin, which is a PDF inhibitor applied as a negative
control, did not change the MICs of any of the tested strains. This
result clearly indicates that active compounds 1 and 5 inhibit an
additional target as well as FabI, unlike triclosan.
We screened 25,000 microbial extracts consisting of
actinomycetes and fungi to identify new FabI inhibitors. Meleagrin was
isolated from the solid-state fermentation of the fungal strain P.
chrysogenum F717. Meleagrin was previously isolated from P.
meleagrinum  and P. chrysogenum , but its biological activity,
including antimicrobial activity, has not been reported. Although
its activity was weak, meleagrin clearly showed inhibition selective
for S. aureus FabI over S. pneumoniae FabK. Importantly, the binding
of meleagrin with S. aureus FabI was demonstrated by the
fluorescence quenching assay. Furthermore, its inhibition of FabI
was supported by results obtained using its chemical derivatives,
the intracellular fatty acid synthesis assay, and the
fabI-overexpressing assay. Interestingly, meleagrin and its more active
derivatives showed antibacterial activity against S. pneumoniae, in
which FabK is the sole enoyl-ACP reductase, and it did not
produce spontaneously resistant mutants of S. aureus or E. coli, in
contrast to triclosan, which suggests that meleagrin inhibits
multiple targets. Meleagrin inhibited the incorporation of
radiolabeled acetate into lipids in S. pneumoniae and S. aureus, whereas
incorporation of thymidine (DNA), uridine (RNA), isoleucine
(protein), and N-acetylglucosamine (cell wall) was not inhibited,
which indicates that these compounds inhibit fatty acid synthesis
through one or more modes of action in addition to FabI
inhibition. The multitarget effect was confirmed by the
fabKoverexpression assay in E. coli. The multitarget effect is very
important from the point of view of drug development because a
single point mutation in one gene for a drug with a single target
renders the strain resistant and the drug useless. Thus, when
considering that one of the advantages of antibacterial agents
having multiple targets is the reduced development of drug
resistance , meleagrin and its derivatives hold promise for the
development of new antibiotics that can treat infections caused by
Several FabI inhibitors have been reported, and most were
derived from compound libraries and were synthetically developed
using structure-based approaches, including 1,4-disubstituted
imidazoles, aminopyridines, naphthyridinones, and thiopyridines
. Although synthetic inhibitors are potent, they have a
disadvantage, as resistant mutants occur at relatively high
frequency , A few natural FabI inhibitors have been reported,
such as vinaxanthone , cephalochromin , kalimantacin/
batumin , EGCG, and flavonoids . EGCG and flavonoids
inhibit several targets such as FabG, FabZ, and FabI. The mode of
action of vinaxanthone, cephalochromin, and kalimantacin/
batumin was demonstrated by FabI-overexpressing strains. To
our knowledge, this is the first study on a multitarget effect of FabI
In summary, meleagrin is a new class of FabI inhibitor with
antibacterial activity against multidrug-resistant bacteria such as
MRSA and QRSA. Meleagrin is structurally unique, and it
inhibits at least one more target in addition to FabI, thereby
resulting in a no resistance mutant; thus, meleagrin may have
potential as a useful lead compound for the development of a new
Preparation and spectral data of
comWe thank the Culture Collection of Antimicrobial Resistant Microbes of
Korea and the E. coli Genetic Stock Center of Yale University for providing
the bacterial strains used in this study.
Also we express our thanks to Korea Basic Science Institute for the
Conceived and designed the experiments: WGK. Performed the
experiments: CJZ MJS SL WGK. Analyzed the data: MJS WGK.
Contributed reagents/materials/analysis tools: CJZ MJS. Wrote the paper:
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