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Antimicrobial Agents and Chemotherapy, May 2004, p. 1541-1547, Vol. 48, No. 5
0066-4804/04/$08.00+0 DOI: 10.1128/AAC.48.5.1541-1547.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Genome Therapeutics Corporation, Waltham, Massachusetts 02453,1 ArQule, Inc., Woburn, Massachusetts 018012
Received 3 July 2003/ Returned for modification 26 September 2003/ Accepted 1 February 2004
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ABSTRACT |
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INTRODUCTION |
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While the antibacterial activities of triclosan and isoniazid establish the suitability of the target for drug intervention, the compounds themselves are limited in their utility. Isoniazid has been a frontline drug for the treatment of tuberculosis since 1950 but is ineffective against most other species. Triclosan has found wide use in consumer products but has never been used for systemic therapeutic purposes due to toxicity. Consequently, broader-acting, more selective inhibitors are needed. Recently, one research group described two new series of ENR inhibitors (11, 19, 21, 23). Here, we report a new chemically distinct series, and we show that these 2-(alkylthio)-4,6-diphenylpyridine-3-carbonitriles ("thiopyridines") inhibit bacterial growth through a mechanism that includes ENR inhibition.
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MATERIALS AND METHODS |
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rfaC::Kanr), E. coli WO153 (AB1157 [EGSC]; recJ asmB1 tolC::Kanr), B. subtilis NO8 (BD170 [Bacillus Genetic Stock Center]; bmr::CAT), vancomycin-resistant Enterococcus faecium ATCC 700221, methicillin-resistant S. aureus ATCC 700699, and methicillin-sensitive S. aureus CYL316 (17). Purification of E. coli ENR. The E. coli fabI gene (AAC74370) was cloned into the pET30a expression vector (Novagen, Inc., Madison, Wis.) and expressed in E. coli BL21(DE3) cells. The purification procedure utilized chromatography with Q-Sepharose, blue resin, and hemagglutinin resin as follows. Cell pellets were suspended in lysis buffer (50 mM KH2PO4 [pH 8.0], 100 mM NaCl, 2 mM EGTA, and 10% glycerol), and cells were broken by passage through a Microfluidics cell disrupter. Lysates were centrifuged, and the supernatant was applied to a Q-Sepharose column preequilibrated in buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], 100 mM NaCl, 10% glycerol, 0.1% ß-mercaptoethanol, and 0.02% Brij 35). ENR was eluted with a NaCl gradient (0.1 to 1 M) in the equilibration buffer. The major peak fractions were pooled and concentrated, dialyzed (10 mM Tris-HCl [pH 7.5], 0.1 mM EGTA, 0.1 mM PMSF, 10% glycerol, 0.1% ß-mercaptoethanol, and 0.02% Brij 35), and centrifuged. The supernatant was loaded on a preequilibrated blue resin column (10 mM Tris-HCl [pH 7.5], 0.1 mM EGTA, 1 mM PMSF, 50 mM NaCl, 10% glycerol, 0.1% ß-mercaptoethanol, and 0.02% Brij 35). ENR was eluted with the equilibration buffer containing NaCl (gradient of 50 to 1,000 mM), dialyzed in equilibration buffer, and further purified on a hydroxyapatite column (20 mM KH2PO4 [pH 8.0], 0.1 mM EGTA, 0.1 mM PMSF, 10% glycerol, 0.1% ß-mercaptoethanol, and 0.02% Brij 35). ENR was eluted with a gradient of KH2PO4 up to 500 mM. The peak fractions were pooled and dialyzed in storage buffer (10 mM morpholinepropanesulfonic acid [MOPS; pH 7.0], 150 mM NaCl, 0.1 mM EGTA, 50% glycerol, 0.02% Brij 35) and then stored at –20°C.
Endpoint assay of ENR and high-throughput screen.
An ENR endpoint assay was engineered utilizing crotonoyl coenzyme A (CoA) as a substrate and measuring the overall decrease in NADH by fluorescence according to the following reaction: crotonoyl-CoA + NADH 
butyryl-CoA + NAD+. By comparing sample readings to those of negative (absence of compound) and positive (absence of enzyme) controls, the percent inhibition of enzymatic activity by each compound was determined. The assay was performed in 96-well half-area black plates (Corning). The final concentration of each component in the 50-µl reaction mixture was as follows: sodium phosphate, 100 mM (pH 7.5); NADH, 100 µM; dithiothreitol, 1 mM; FabI, 1.67 µg/ml; crotonoyl-CoA, 0.4 mM (Sigma; C-4146). The assay was initialized by adding all components except substrate to plates containing compounds. After 20 min, the crotonoyl-CoA substrate was added and plates were incubated for 35 min and then read on a Victor2V plate reader (PE Lifescience) with the fluorometry option set at the following parameters: umbelliferone (0.1 s); CW-lamp filter, F355; emission filter, F460. The signal/background ratio of the resulting screening assay was 3 to 4, with a Z' factor of 0.69 (26). Throughput of the screen with a screening platform constructed in-house was 80 plates/day. Hits were confirmed by using a kinetic assay.
Kinetic assay for ENR and IC50 measurements. A kinetic assay in microtiter wells was used to confirm the activity of compounds identified by the endpoint assay screen and to measure 50% inhibitory concentrations (IC50s). The final concentrations of each component in each 50-µl reaction mixture are as follows: sodium phosphate, pH 7.5, 100 mM; NADH, 200 µM; dithiothreitol, 1 mM; ENR, 3 µg/ml; crotonoyl-CoA, 0.8 mM. For IC50 measurements, compounds were tested in serial dilution. The reaction was initiated by addition of the substrate, crotonoyl-CoA, and monitored continuously for 5.5 min on a spectrophotometer by measuring absorption at 340 nm. The rate of decrease in the amount of NADH in each reaction well was converted to percentage of inhibition by using SOFTmax PRO software (Molecular Devices, Sunnyvale, Calif.) and the following formula: % of inhibition = 100 x [(rate in the presence of compound – rate of negative control)/(rate of positive control – rate of negative control)]. Reaction in the presence of 2% dimethyl sulfoxide but without inhibitory compounds served as the positive control, and reaction in the presence of all components except ENR served as the negative control.
Synthesis of ENR inhibitors. Analogs were prepared, from readily synthesized chalcones, in two additional steps. Treatment of the chalcones with 2-cyanoethanethioamide in methanolic sodium methoxide provided the intermediate 1,2-dihydro-4,6-diaryl-2-thioxopyridine-3-carbonitriles (15). Alkylation on sulfur with cesium carbonate in dimethyl formamide and an organic bromide gave the desired 2-(alkylthio)-4,6-diphenylpyridine-3-carbonitriles.
Antimicrobial activity assay. Assays to determine the MICs of compounds were performed according to NCCLS recommendations (20) in a 100-µl volume in 96-well format, with serial twofold dilutions of the drug concentration over a twofold dilution range of 0.125 to 64 µg/ml unless noted otherwise.
Mode of action studies. (i) Measurement of inhibition of macromolecular biosynthesis. Macromolecular biosynthesis in S. aureus CYL316 was measured essentially as described previously (12) except that radioactive precursors (acetate, N-acetylglucosamine, uridine, thymidine, or a mixture of amino acids) were 14C labeled and obtained from ICN Biochemicals Inc. Total counts incorporated at 45 min of incubation without inhibitors ranged from 3,000 for acetate, thymidine, and amino acids to >8,000 for N-acetylglucosamine and >11,000 for uridine. In these experiments, the GTC-004061 MIC for S. aureus CYL316 was 8 µg/ml and the triclosan MIC was 0.08 µg/ml.
(ii) Staphylococcus fabI underexpression hypersensitivity assay.
The endogenous S. aureus fabI gene was replaced with a drug resistance marker (Ermr) in a two-step allele-replacement procedure (4). About 1 kb of DNA sequence flanking each side of the S. aureus fabI gene (16) was PCR amplified from genomic DNA, ligated to an erythromycin resistance cassette by crossover PCR (16), and cloned into an allele-replacement suicide vector carrying a kanamycin resistance marker (Kanr) and the sacB gene encoding levansucrase (2). This allele-replacement plasmid was introduced by electroporation into S. aureus host strain CYL316 containing the multicopy plasmid pCL11219 with an int gene which constitutively expresses integrase (17). Selection for Kanr yielded cells containing the entire plasmid by means of a single crossover recombination. Next, a Staphylococcus fabI gene (AAO04309) was cloned by PCR and placed under control of the tetracycline-regulated promoter (xyl/tet) (6) on an integration vector carrying a chloramphenicol resistance marker (Cmr) and an attP site. This plasmid was introduced into the S. aureus host strain CYL316 containing the single crossover allele-replacement construct. Selection for chloramphenicol-resistant, lipase-negative clones resulted in the isolation of strains in which the plasmid had integrated at the attB/L54a site within the lipase gene geh. Insertion at this site inactivated the lipase gene. Propagation in the absence of selection resulted in the loss of the multicopy plasmid pCL11219. Finally, growth in sucrose and in the presence of anhydrotetracycline, the tet promoter inducer, selected for cells that had completed a second crossover event to eliminate the sacB gene and yield a total replacement of the endogenous fabI gene with the Ermr cassette.
This strain with a single copy of the Staphylococcus fabI gene under the regulation of the xyl/tet promoter at the geh locus grew poorly in the absence of the inducer, anhydrotetracycline, indicating that cells are dependent on fabI expression from the xyl/tet promoter for robust growth and that the tet promoter retains some activity even in the absence of inducer. For underexpression studies, cells were streaked and propagated overnight on Mueller-Hinton broth supplemented with 50 µg of anhydrotetracycline/ml. Several similar-sized colonies were resuspended in Trypticase soy broth and added to the MIC microtiter dish wells at a final cell concentration producing an optical density at 600 nm (OD600) of 0.005. Cell growth was measured as OD600 after an overnight incubation (about 20 h).
(iii) Staphylococcus fabI overexpression rescue assay. The lac repressor regulated by the penicillinase promoter (PPCN) and T7 RNA polymerase regulated by the spac-I promoter (25) were cloned onto an integration plasmid containing the attP site and introduced into the geh locus of S. aureus CYL316 as described above. The Staphylococcus fabI gene (AAO04309) was cloned as a PCR product and placed under the control of the T7 promoter on a replicating shuttle vector, which also contains PPCN driving lacI to ensure adequate repression of T7 RNA polymerase in the absence of isopropyl-ß-D-thiogalactopyranoside (IPTG). In the presence of inducer IPTG, excess ENR is produced. For overexpression studies, the cells were grown overnight in the absence of inducer. Several similar-sized colonies were resuspended in Trypticase soy broth and inoculated into the MIC plates at a final cell concentration equivalent to an OD600 of 0.001, with and without 1 mM IPTG. Cell growth was measured as OD600 after an overnight incubation (about 20 h).
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RESULTS |
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Three compounds, GTC-004061, GTC-330346, and GTC-341772, inhibited B. subtilis strain NO8 with MICs of 64, 8, and 16 µg/ml, respectively. While B. subtilis contains both a FabI and a FabL ENR, the two exhibit sufficient similarity to suggest that a compound could inhibit both (9). None of the compounds inhibited E. faecium. Compound GTC-004061 was tested against E. faecalis, but no inhibition of growth was observed in studies up to a concentration of 64 µg/ml. This is consistent with the known presence of both a FabI and an unrelated FabK ENR in E. faecalis (8).
Cells that underexpress fabI are hypersensitive to compounds in the thiopyridine series. Since the correlation between the MICs and the IC50s of this set of thiopyridine analogs indicates that these compounds are inhibiting cell growth by means of their activity on the ENR target, we investigated the cellular mode of action (MOA) of these compounds further. Previous studies have established that bacterial strains underproducing and overproducing ENR are more and less susceptible, respectively, to the ENR inhibitor triclosan than is the parental wild-type strain (3, 21). Accordingly, strains of S. aureus that underexpress fabI under the control of a tet-regulated promoter (1) and that overexpress fabI under the control of a T7 promoter were constructed and tested for sensitivity to thiopyridine compounds as well as to positive- and negative-control compounds.
The growth of cells underexpressing fabI is about fourfold more sensitive to the thiopyridine compound GTC-004061 than is the growth of the overexpressing strain or the wild-type parent (Fig. 2A). The growth of the same fabI-underexpressing strain is also about fourfold more sensitive to the known ENR inhibitor triclosan, while the fabI-overexpressing strain is about threefold less sensitive to triclosan, in comparison to growth of the parental strain in the presence of triclosan (Fig. 2B). These results are consistent with previous reports regarding changes in the triclosan susceptibilities of cells under- and overexpressing the fabI gene (3, 21) and indicate that the engineered constructs are functioning as expected. The growth of fabI-overexpressing and -underexpressing cells in the presence of novobiocin, which acts on a different target, was identical in all three strains (Fig. 2C), indicating that altered expression of fabI does not change the sensitivity of cells to antibiotics in general. The growth of fabI-underexpressing cells in the presence of a second thiopyridine analog, GTC-330346, was also reduced compared to growth of the wild-type strain in the presence of the compound; however, fabI-overexpressing cells did not exhibit reduced sensitivity to GTC-330346 (data not shown). These results indicate that compounds in the thiopyridine series inhibit growth of the underexpressing strain through inhibition of the fabI-encoded ENR.
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DISCUSSION |
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Spectrum. While these thiopyridine compounds were initially identified in a high-throughput screen for E. coli ENR inhibitors and they do exhibit potent IC50s for the E. coli enzyme, they do not show activity against E. coli cells. Several of the compounds arrest the growth of S. aureus strains and Bacillus subtilis, but they have no detectable effect on the growth of either wild-type E. coli cells or a strain carrying a deletion of the tolC gene along with the asmB1 allele of lpxC, which enhances the permeability of E. coli to some compounds (13). We examined the susceptibility of the tolC deletion strain because TolC is the outer membrane factor for the acrAB efflux pump (5), and tolC mutants show increased sensitivity to a large number of antibacterial compounds (5, 24). However, mutational analysis has revealed that genes encoding two additional putative outer membrane factors, yjcP and yohG, play roles in drug resistance in E. coli (24). Therefore, the thiopyridine compounds described here may not enter the cell, or they may be pumped out by a tolC-independent mechanism. The activity of these compounds on gram-positive species lacking a fabK gene, such as S. aureus and B. subtilis, is consistent with better accessibility in these cells, which lack an outer membrane. While B. subtilis carries both a fabI gene and a fabL gene, the two gene products share considerable sequence identity, and triclosan is known to inhibit both, although with different potencies (8). It is possible that compounds in the thiopyridine series can be engineered to gain activity on E. coli cells by focusing on structural changes that could contribute to outer membrane permeability.
Target specificity. Inhibition of cell growth by compounds in the thiopyridine series appears to involve inhibition of ENR as well as an additional target(s). In wild-type cells, direct measurement of the incorporation of radiolabeled precursors into macromolecules indicates that GTC-004061 inhibits fatty acid biosynthesis more potently than it inhibits protein or peptidoglycan synthesis. However, the action of GTC-004061 is not specific for fatty acid biosynthesis in wild-type cells, since it also inhibits DNA and RNA synthesis even more effectively. By contrast, in cells underexpressing the fabI gene, the two thiopyridines, GTC-004061 and GTC-330346, as well as triclosan all exhibit enhanced potency, consistent with specific hypersensitivity of these cells to ENR inhibitors such as the thiopyridines and triclosan but not to novobiocin. Based on these data, we propose that the thiopyridine compounds inhibit both ENR and one or more secondary targets in S. aureus and that reduction in the amount of ENR in fabI-underexpressing cells causes ENR inhibition to become the dominant mechanism of inhibition. This is consistent not only with the macromolecular biosynthesis results but also with the fact that cells overexpressing the fabI gene do not exhibit reduced sensitivity to GTC-004061 and GTC-330346. According to this hypothesis, underexpression of a target is a more sensitive method for evaluating target specificity than is overexpression because it permits detection of target-specific activity even in the presence of some degree of secondary target activity. This capability of the underexpression method to decouple dual target activities, together with the fact that it permits the use of less compound and the use of compounds with moderate MICs, makes it a valuable resource for compound evaluation.
While the thiopyridines appear to inhibit more than one target in S. aureus, considerable evidence indicates that the compounds in this series are not acting as membrane-perturbing agents or general biocides. First, we have tested GTC-004061 in a membrane permeability assay using the dye DiSC35 and including a known membrane-perturbing salicylamide compound as a positive control (Ling et al., 43rd ICAAC). While the salicylamide caused significant leakage of the dye from the cells, GTC-004061 did not (data not shown). Second, the same salicylamide compound inhibited all five macromolecular biosynthetic pathways potently, in contrast to the more selective pattern of inhibition exhibited by GTC-004061 (Fig. 3). Third, GTC-004061 does not significantly inhibit any of 10 other enzymes, including other reductases. Fourth, three compounds in the thiopyridine series are active against both S. aureus and B. subtilis, but they are inactive against E. coli and E. faecium. Such selective growth inhibition properties are not typical of general biocides.
Two classes of ENR inhibitors with antibacterial activity have been described recently by other groups. Heerding et al. (11) reported on 1,4-disubstituted imidazoles with IC50s for E. coli ENR of about 4 µM and MICs for S. aureus of 8 µg/ml. The thiopyridine series described here exhibits very similar potency. Seefeld et al. (23) and Payne et al. (21) reported another chemical series of compounds which exhibit IC50s for E. coli ENR of about 4 µM and MICs for S. aureus of 0.5 µg/ml. These compounds are similar in potency to the thiopyridines against the E. coli enzyme but display slightly higher MICs for S. aureus. In total, this group of inhibitors from three quite different chemotypes, two from previous efforts and the new chemotype reported here, suggests that ENR is a feasible target and that antibacterials directed against this target can be developed.
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ACKNOWLEDGMENTS |
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This work was supported by Genome Therapeutics Corp.
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FOOTNOTES |
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REFERENCES |
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