X.-M. Wang, H.-L. Zhu, et al.
MED
4–6 h. The precipitates were separated by filtration, recrystallized
from methanol, washed with methanol three times, and dried in
a vacuum desiccator containing anhydrous CaCl2 to give oxime de-
rivatives 5–41 in yields of 81–89%.
pET19; FabH in pET28). All proteins were expressed in E. coli strain
BL21 (DE3). Transformed cells were grown on Luria–Bertani (LB)
agar plates supplemented with kanamycin (30 mgmLÀ1). SDS-PAGE
analysis was used to screen colonies for overexpression of proteins.
One such positive colony was used to inoculate 10 mL LB medium
with 30 mgmLÀ1 kanamycin; this was grown overnight at 378C,
1 mL of which was used to inoculate 100 mL LB medium supple-
mented with 30 mgmLÀ1 kanamycin. The culture was shaken for
4 h at 378C, and then induced with 0.5 mm isopropyl b-d-thioga-
lactopyranoside (IPTG). The culture was grown for 4 h, and harvest-
ed by centrifugation (30 min at 21000 g).
Terephthalaldehydes (0.25 mmol) with two aldehyde groups and
the substituted O-benzylhydroxylamines 1–4 hydrochloride with
one amino group were dissolved in methanol (10 mL) and stirred
at room temperature for 4–6 h. The precipitates were separated by
filtration, recrystallized from methanol, washed with methanol
three times, and dried in a vacuum desiccator containing anhy-
drous CaCl2 to give oxime derivatives 42–47 in yields of 78–83%.
Harvested cells containing His-tagged ACP, ACPS, and FabH were
lysed by sonication in 20 mm Tris (pH 7.6), 5 mm imidazole, and
0.5m NaCl and centrifuged at 37100 g for 30 min. The supernatant
was applied to a Ni–NTA agarose column, washed, and eluted
using a gradient of 5–500 mm imidazole over 20 column volumes.
Eluted protein was dialyzed against 20 mm Tris (pH 7.6), 1 mm DTT,
and 100 mm NaCl. Purified FabH was concentrated to 2 mgmLÀ1
and stored at À808C in 20 mm Tris (pH 7.6), 100 mm NaCl, 1 mm
DTT, and 20% glycerol for enzyme assays.
General method for the synthesis of compounds 48 and 49:
Compounds 48 and 49 were prepared according to the procedure
used for oxime derivatives. The O-benzylhydroxylamine was re-
placed by the corresponding benzylamine and 2-phenylethylamine,
and yields were 80–82%.
Biology
Antibacterial activity: The antibacterial activities of the synthe-
sized compounds were tested against three Gram-negative bacteri-
al strains: E. coli ATCC 25922, P. aeruginosa ATCC 27853, and P. fluo-
rescens P13, along with three Gram-positive strains: B. subtilis ATCC
530, S. aureus ATCC 25923, and E. faecalis ATCC 29212 using meth-
ods recommended by the Clinical Laboratory Standards Institute
(CLSI), formerly the National Committee for Clinical Laboratory
Standards (NCCLS).[36]
Purified ACP contains the apo form that needs to be converted
into the holo form. The conversion reaction is catalyzed by ACP
synthase (ACPS). In a final volume of 50 mL, 50 mg ACP, 50 mm
Tris, 2 mm DTT, 10 mm MgCl2, 600 mm CoA, and 0.2 mm ACPS were
incubated for 1 h at 378C. The reaction was then adjusted to pH~
7.0 using 1m potassium phosphate. Holo-ACP was purified by frac-
tionation of the reaction mixture by Source Q-15 ion-exchange
chromatography using a 0–500 mm NaCl gradient over 25 column
volumes.
In vitro activities of the compounds were tested in nutrient broth
(NB) for bacteria by the twofold serial dilution method. Seeded
broth (broth containing microbial spores) was prepared in NB from
24-hour-old bacterial cultures on nutrient agar (Hi-media) at 37Æ
18C. The bacterial suspension was adjusted with sterile saline to
a concentration of 1ꢁ104–105 CFUmLÀ1. The tested compounds
and reference drugs were prepared by twofold serial dilution to
obtain the required concentrations of 100, 50, 25, 12.5, 6.25,
3.13 mgmLÀ1, and even lower concentrations. The tubes were incu-
bated in BOD incubators at 37Æ18C for bacteria. The MICs were
recorded by visual observations after 24 h incubation (for bacteria).
Fleroxacin and DDCP were used as standards.
In a final 20 mL reaction, 20 mm Na2HPO4/NaH2PO4 (pH 7.0), 0.5 mm
DTT, 0.25 mm MgCl2, and 2.5 mm holo-ACP were mixed with 1 nm
FabH, and H2O was added to 15 mL. After 1 min incubation, a 2 mL
mixture of 25 mm acetyl-CoA and 0.75 mCi [3H]acetyl-CoA was
added for FabH reaction for 25 min. The reaction was stopped by
adding 20 mL ice-cold 50% TCA, incubating for 5 min on ice, and
centrifuging (16000 g, 10 min) to pellet the protein. The pellet was
washed with 10% ice-cold TCA and resuspended in 5 mL 0.5m
3
NaOH. Incorporation of the H signal in the final product was de-
termined by liquid scintillation. For IC50 determination, inhibitors
were added from a concentrated DMSO stock such that the final
concentration of DMSO did not exceed 2%.
Cytotoxicity assays: The cytotoxicity of compound 44 was evalu-
ated against normal Vero, BHK-21, and Raw 264.7 cell lines. Target
tumor cell lines were grown to log phase in RPMI 1640 medium
supplemented with 10% fetal bovine serum. After diluting to 2ꢁ
104 cellsmLÀ1 with the complete medium, 100 mL of the obtained
cell suspension was added to each well of 96-well culture plates.
The subsequent incubation was carried out at 378C under an at-
mosphere containing 5% CO2 for 24 h before cytotoxicity assess-
ments. Tested samples at pre-set concentrations were added to six
wells. After an exposure period of 48 h, PBS (40 mL) containing
Docking simulations
Molecular docking of compound 44 into the 3D X-ray structure of
E. coli FabH (PDB code: 1HNJ) was carried out with Discovery
Studio software (version 3.1) as implemented through the graphi-
cal user interface DiscoveryStudio CDOCKER protocol.
2.5 mgmLÀ1
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
The 3D structures of the aforementioned compounds were con-
structed using ChemBio 3D Ultra 11.0 software [Chemical Structure
Drawing Standard, Cambridge Soft Corp., USA (2008)]; they were
then energy-minimized by using MMFF94 with 5000 iterations and
a minimum RMS gradient of 0.10. The crystal structures of E. coli
FabH were retrieved from the RCSB Protein Data Bank (http://
were removed from the protein, and polar hydrogen atoms were
added. The entire 1HNJ structure was defined as a receptor and
the site sphere was selected based on the active center of 1HNJ
according to a previous report,[28] then compound 44 was placed
in during the molecular docking procedure. The types of interac-
bromide) (MTT) was added to each well. Four hours later, 100 mL
extraction solution (10% SDS, 5% isobutyl alcohol, 0.01m HCl) was
added. After an overnight incubation at 378C, the optical density
was measured at l 570 nm on an ELISA microplate reader. In all ex-
periments three replicate wells were used for each drug concentra-
tion. Each assay was carried out at least three times. The results
are summarized in Table 3.
E. coli FabH purification and activity assay: Full-length E. coli
acyl-carrier protein (ACP), acyl-carrier-protein synthase (ACPS), and
b-ketoacyl-ACP synthase III (FabH) were individually cloned into
pET expression vectors with an N-terminal His tag (ACP, ACPS in
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ChemMedChem 0000, 00, 1 – 8
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These are not the final page numbers!
Luo, Yin
