Meleagrin, a New FabI Inhibitor from Penicillium chryosogenum with at Least One Additional Mode of Action

PLOS ONE, Nov 2013

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.

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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 the manuscript. 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 [35]. 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 [10], 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 [1315]. In addition, rapid mutation development has been often reported in synthetic FabI inhibitors [16]. Thus, it has been recently emphasized that ideal antibiotics should bind to multiple targets [17]. 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 [18]. 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 [19]. A few FabI inhibitors have been reported from microorganisms [2022], and most of these are phenolic compounds. Therefore, more unique FabI inhibitors need to be obtained from microorganisms. 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 Bacterial strains 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 University. 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 [23] 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 and 25). 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 [24]. The wild-type fabK gene was amplified by PCR from genomic DNA obtained from Streptococcus pneumoniae KCTC 5412 by using the primers 59-GGAAACCATATGAAAACGCGTATTACGAA-39 and 59-CCGCTCGAGGTCATTTCTTACAACTCCTGT-39, 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 [24]. 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. doi:10.1371/journal.pone.0078922.t001 [14C] acetate incorporation 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). doi:10.1371/journal.pone.0078922.g002 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 (MICs) Whole-cell antimicrobial activity was determined by broth microdilution as described previously [21]. 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 reader. Measurement of the inhibition of macromolecular biosynthesis 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 [21]. 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 controls. 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. Overexpression assay 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 [21]. 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 59-ATGGGTTTTCTTTCCGGTAAGCGCA-39 and 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 [25]. 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 3.3060.1361028 2.5860.0461029 9.0760.0861028 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 [26]. 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, respectively. Effects of structural changes in compound 1 on FabI and related activity 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 (%) S. pneumoniaeb Reference antibacteriald Reference antibacterialc N-Acetyl-D-[1-14C] Glucosamine 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. doi:10.1371/journal.pone.0078922.t004 E. coli EW1b(pBAD) E. coli EW1b(pBAD-fabI) E. coli EW1b(pBAD-fabK) a3% arabinose was treated. doi:10.1371/journal.pone.0078922.t005 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 shown). Antibacterial activity 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 [27]. 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 multiple targets. 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 [28] and P. chrysogenum [29], 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 [10], meleagrin and its derivatives hold promise for the development of new antibiotics that can treat infections caused by multidrug-resistant pathogens. 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 [30]. Although synthetic inhibitors are potent, they have a disadvantage, as resistant mutants occur at relatively high frequency [16], A few natural FabI inhibitors have been reported, such as vinaxanthone [21], cephalochromin [31], kalimantacin/ batumin [22], EGCG, and flavonoids [32]. 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 inhibitors. In summary, meleagrin is a new class of FabI inhibitor with antibacterial activity against multidrug-resistant bacteria such as MRSA and QRSA. 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Chang Ji Zheng, Mi-Jin Sohn, Sangku Lee, Won-Gon Kim. Meleagrin, a New FabI Inhibitor from Penicillium chryosogenum with at Least One Additional Mode of Action, PLOS ONE, 2013, DOI: 10.1371/journal.pone.0078922