The bacterial strains used in antibacterial activity assays were obtained from the American Type Culture Collection, the E. coli
Genetic Stock Center (EGSC; Yale University), the Bacillus
Genetic Stock Center (Ohio State University), or the Genome Therapeutics (GENE) collection, as noted: E. coli
ATCC 35218, E. coli
WO159 (AB1157 [EGSC]; recJ asmB1
), E. coli
WO153 (AB1157 [EGSC]; recJ asmB1
), B. subtilis
NO8 (BD170 [Bacillus
Genetic Stock Center]; bmr
), vancomycin-resistant Enterococcus faecium
ATCC 700221, methicillin-resistant S. aureus
ATCC 700699, and methicillin-sensitive S. aureus
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% β-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% β-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% β-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% β-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 Victor2
V 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 × [(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 14
C 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
) 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 pCL112Δ19 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
) 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 pCL112Δ19. 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
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.
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
to ensure adequate repression of T7 RNA polymerase in the absence of isopropyl-β-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).