![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, November 2001, p. 3182-3188, Vol. 45, No. 11
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.11.3182-3188.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification and Characterization of New
Inhibitors of the Escherichia coli MurA
Enzyme
Ellen Z.
Baum,*
Deborah A.
Montenegro,
Lisa
Licata,
Ignatius
Turchi,
Glenda C.
Webb,
Barbara D.
Foleno, and
Karen
Bush
The R. W. Johnson Pharmaceutical
Research Institute, Raritan, New Jersey 08869
Received 13 April 2001/Returned for modification 11 June
2001/Accepted 2 August 2001
 |
ABSTRACT |
The bacterial enzyme MurA catalyzes the transfer of enolpyruvate
from phosphoenolpyruvate (PEP) to uridine
diphospho-N-acetylglucosamine (UNAG), which is the first
committed step of bacterial cell wall biosynthesis. From
high-throughput screening of a chemical library, three novel inhibitors
of the Escherichia coli MurA enzyme were identified: the
cyclic disulfide RWJ-3981, the purine analog RWJ-140998, and the
pyrazolopyrimidine RWJ-110192. When MurA was preincubated with
inhibitor, followed by addition of UNAG and PEP, the 50% inhibitory
concentrations (IC50s) were 0.2 to 0.9 µM, compared to
8.8 µM for the known MurA inhibitor, fosfomycin. The three compounds
exhibited MICs of 4 to 32 µg/ml against Staphylococcus aureus; however, the inhibition of DNA, RNA, and protein
synthesis in addition to peptidoglycan synthesis by all three
inhibitors indicated that antibacterial activity was not due
specifically to MurA inhibition. The presence of UNAG during the MurA
and inhibitor preincubation lowered the IC50 at least
fivefold, suggesting that, like fosfomycin, the three compounds may
interact with the enzyme in a specific fashion that is enhanced by
UNAG. Ultrafiltration and mass spectrometry experiments suggested that
the compounds were tightly, but not covalently, associated with MurA.
Molecular modeling studies demonstrated that the compounds could fit
into the site occupied by fosfomycin; exposure of MurA to each compound reduced the labeling of MurA by tritiated fosfomycin. Taken together, the evidence indicates that these inhibitors may bind noncovalently to
the MurA enzyme, at or near the site where fosfomycin binds.
| |
INTRODUCTION |
Both gram-positive and
gram-negative bacteria are surrounded by a cell wall which protects the
cell from destruction by osmotic pressure. It is well established that
interference with cell wall biosynthesis is an excellent mechanism for
bacterial killing; for example, penicillin and vancomycin specifically
interact with the cell wall at different stages of its formation. A
major component of the cell wall is a layer of peptidoglycan (murein),
which is a polymer of the sugars N-acetylglucosamine
(NAG) and N-acetylmuramic acid, and various amino acids. The
bacterial MurA enzyme (UDP-NAG enolpyruvyl transferase), in the first
committed step of peptidoglycan biosynthesis, catalyzes the transfer of
enolpyruvate from phosphoenolpyruvate (PEP) to UDP-NAG (UNAG),
releasing inorganic phosphate (6).
MurA is conserved across both gram-positive and gram-negative bacterial
species; gram-negative bacteria have one copy of the murA
gene (5), and gram-positive bacteria have two copies
(9). MurA is an essential enzyme in that its deletion from
Escherichia coli and Streptococcus pneumoniae
(both copies) is lethal (5, 9), and it has no mammalian
homolog. One marketed antibacterial drug, fosfomycin, is a natural
product that is a specific inhibitor of the MurA enzyme
(13). MurA is thus an attractive target for antibiotic discovery.
Inhibition of MurA by fosfomycin is well characterized. Fosfomycin is a
PEP surrogate which forms a covalent adduct with Cys115 of MurA
(16). Resistance to fosfomycin can be achieved by several different mechanisms, including enzymatic modification of the antibiotic (3), decreased uptake of the antibiotic
(1), and overexpression of MurA (12). In
addition, MurA containing a Cys115Asp mutation was enzymatically active
but was resistant to fosfomycin (15).
Despite the availability since 1992 of both recombinant MurA and a
straightforward assay method to identify inhibitors of the enzyme
(17), fosfomycin has remained the only MurA inhibitor of
record in the literature to date. In this study, we report the
identification of three new inhibitors of the E. coli MurA enzyme. Each compound apparently has a mechanism distinct from that of
fosfomycin, in that a noncovalent enzyme-inhibitor complex appears to
be formed. The characterization of these inhibitors is presented.
(This study was presented in part at the 40th Interscience Conference
on Antimicrobial Agents and Chemotherapy [D. A. Montenegro, L. Licata, J. Melton, I. Turchi, G. C. Webb, B. D. Foleno, E. Wira, W. Jones, J. Masucci, K. Bush, and E. Z. Baum, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2021, 2000].)
| |
MATERIALS AND METHODS |
Compounds.
Fosfomycin (disodium salt, catalog no. P-5396)
was purchased from Sigma (St. Louis, Mo.) and was dissolved in
distilled H20 at 40 mM. Compounds RWJ-3981,
RWJ-110192, and RWJ-140998 and their analogs were obtained from The
R. W. Johnson Pharmaceutical Research Institute (Raritan, N.J.)
and were dissolved in 100% dimethyl sulfoxide at 10 mg/ml for assay purposes.
Expression of MurA.
E. coli MurA (GenBank
accession number AE000399) was expressed as a fusion to the carboxy
terminus of the maltose binding protein (MBP) as follows. The entire
MurA coding region (nucleotides 6508 to 7768) was amplified from
E. coli OC3340 DNA (strain K12) using the primers
5'-AATTCCCGATGGATAAATTTCGTGTTCAG-3' and
5'-TGCAGATTATTCGCCTTTCACACG-3' and Pfu polymerase
(Stratagene, La Jolla, Calif.). The resultant PCR fragment was flanked
by EcoRI and PstI sites which allowed its cloning
in frame into the corresponding restriction sites of the pMalC2
polylinker (New England Biolabs, Beverly, Mass.). The 89-kDa MBP-MurA
fusion protein (hereafter designated MurA) was expressed and purified
by chromatography on amylose resin columns (New England Biolabs) by
standard protocols. For mass spectrometry experiments, murA
was amplified from E. coli OC3340 DNA using the primers
5'-TATGGATAAATTTCGTGTTCAG-3' and
5'-TCGAGTTCGCCTTTCACACGCTC-3' and cloned into the
NdeI and XhoI sites of the pET23a(+) polylinker (Novagen). This construct encodes a 46-kDa protein of MurA with LeuGluHis6 at its carboxy terminus (MurA-His).
The His-tagged protein was purified on a nickel-nitrilotriacetic acid
agarose column (Qiagen, Valencia, Calif.) as recommended by the supplier.
Assay of MurA activity.
MurA (100 nM, 10 µg/ml) was
incubated with 20 µM PEP and 75 µM UNAG in 25 mM Tris-HCl (pH 7.8)
for 30 min, and inorganic phosphate (Pi) was
monitored using malachite green as described previously (17). All MurA incubations were at room temperature. For
inhibition studies, MurA and inhibitor were incubated together for 10 min unless otherwise noted, prior to addition of the PEP and UNAG substrates. The IC50 (concentration of inhibitor
at which 50% inhibition of MurA is achieved) was determined
graphically. In the enzymatic assay, the specific activity of the
MurA-His protein and its inhibition by fosfomycin and the compounds
were similar to those of the MBP-MurA protein.
Mass spectrometry.
To examine covalent adduct formation,
MurA-His (100 µM) was incubated with 1 mM UNAG in 100 µl of 15 mM
Tris-HCl (pH 7.8) for 20 min followed by addition of 1 mM inhibitor and
continued incubation for 1 h. Samples were desalted, and free
compound was removed using MicroSpin G-50 columns (Amersham Pharmacia
Biotech, Parsippany, N.J.). Samples were digested with trypsin as
described previously (16) except that Triton X-100 was
omitted, and peptide masses were determined by matrix-assisted laser
desorption ionization mass spectrometry.
Reversibility of inhibition.
MurA (800 nM) and UNAG (1 mM)
were incubated alone, or with 63 µM RWJ-3981, RWJ-110192, or
RWJ-140998, or 1 mM fosfomycin in 480 µl of 10 mM ammonium acetate
(pH 6.8) for 30 min. An aliquot was then set aside for testing as the
before-filtration sample. To separate free inhibitor from enzyme-bound
complexes, the remainder of each reaction mixture was applied to
Ultrafree 0.5-ml centrifugal filtration devices (10K NMWL; Millipore,
Bedford, Mass.) that were used as per the manufacturer's instructions.
The filtrate should contain free compound and other low-molecular-mass
material (<10 kDa); the retentate, containing MurA and MurA inhibitor
complexes, becomes concentrated (to 5 to 20 µl) during each
centrifugation step. The retentate was washed three times with 0.3 ml
of 10 mM ammonium acetate (pH 6.8). The amounts of protein recovered in the washed retentates were determined by bicinchinonic acid protein assay (Pierce, Rockford, Ill.) and were similar for all of the samples
(
50%). Samples were then assayed for MurA activity, with the
before-filtration and after-washing samples separately normalized to
their respective MurA controls (MurA sample without added inhibitor, 0% inhibition; reaction containing no MurA, 100% inhibition).
Competition of each inhibitor with [3H]fosfomycin
for binding to MurA.
MurA (13 µM) and UNAG (225 µM) were
incubated in 100 µl of 10 mM Tris-HCl (pH 7.8) with 1 mM RWJ-3981,
RWJ-110192, or RWJ-140998 or 4 mM fosfomycin for 10 min.
[3H]fosfomycin (1 µCi of >0.5 Ci/mmol, 20 µM final concentration; SibTech, Newington, Conn.) was added, and the
incubation was continued. Aliquots (20 µl in duplicate) were removed
at 10 and 35 min and added to 100 µl of 0.5-mg/ml bovine serum
albumin used as carrier. Cold 10% trichloroacetic acid (TCA; 1 ml) was
added to each sample, and labeling of MurA by
[3H]fosfomycin was determined by precipitation
onto Whatman GF/A filters and scintillation counting as described
earlier (11). The extent of labeling of MurA with
[3H]fosfomycin in the absence of added
inhibitor is defined as the 100% control; counts per minute retained
on the filter in the absence of MurA is defined as background (0% control).
MIC determinations.
Broth microdilution MICs were determined
according to the National Committee for Clinical Laboratory Standards
(18). The MIC is the lowest concentration of compound that
inhibited bacterial growth.
Macromolecular synthesis and membrane damage assays.
DNA,
RNA, and protein synthesis were monitored by the incorporation of
tritiated thymidine, uridine, or amino acids, respectively, into
TCA-precipitable material as described previously (11) using S. aureus ATCC 29213. Peptidoglycan synthesis was
monitored in a similar fashion by incorporation of
[3H]NAG (Amersham catalog no. T-2238,
8.3 Ci/mmol) into TCA-precipitable material, using 0.25 µCi of
[3H]NAG per ml of S. aureus culture.
Compounds were used at four times the MIC and were administered 10 min
before the addition of radiolabel. Membrane damage was assessed by the
BacLight assay (Molecular Probes, Eugene, Oreg.), which was performed
as described previously (11) on S. aureus 29213 cells at the MICs and at four times the MICs of fosfomycin, RWJ-3981,
RWJ-110192, and RWJ-140998.
Modeling studies.
The X-ray crystal structure of MurA with
bound fosfomycin was used in modeling studies (23). The
docking programs FlexX (19) and Dockvision
(10) were used to dock RWJ-3981, RWJ-110192, and
RWJ-140998 into the catalytic site of the MurA enzyme. Nonprotein atoms
were removed prior to the docking runs. The poses from both programs
showed similar orientations for all three inhibitors. The program Hint
(14) was used to analyze the more important interactions
of the inhibitors with the residues in the catalytic site of the
enzyme. The AM1 Hamiltonian (8) was used to calculate the
lowest unfilled molecular orbital (LUMO) energies of the analogs of
RWJ-3981.
| |
RESULTS AND DISCUSSION |
Identification of three inhibitors of MurA.
By screening a
chemical library, compounds RWJ-3981, RWJ-110192, and RWJ-140998 were
identified as inhibitors of the E. coli MurA enzyme (Fig.
1). All three compounds exhibited lower
MurA IC50s (0.2 to 0.9 µM) than did the known
MurA inhibitor, fosfomycin (IC50 = 8.8 µM)
(Table 1). The three compounds have no
apparent structural similarity to fosfomycin. Compound RWJ-3981 is a
cyclic disulfide. Compound RWJ-110192 is a pyrazolopyrimidine.
RWJ-140998 is a purine and contains the 2,4-dioxopyrimidine ring which
is also found in the uracil portion of the UNAG substrate. Inhibition of MurA by RWJ-3981, RWJ-110192, or RWJ-140998 was prevented by dithiothreitol (10 mM) added either before or after the
enzyme-inhibitor preincubation. In contrast, inhibition of MurA by
fosfomycin was unaffected by the presence of dithiothreitol.
Presence of UNAG during MurA inhibitor preincubation lowers the
observed IC50.
Inhibition of MurA by the PEP surrogate
fosfomycin requires the presence of the substrate UNAG, which effects a
conformation change in the enzyme (16, 22). From
structural studies, it has been determined that MurA assumes an open
conformation in the absence of ligand (21) and a closed
conformation in the presence of UNAG and fosfomycin (23).
The binding of UNAG creates an induced fit which renders the enzyme
catalytically competent (20, 22). Furthermore, MurA is
isolated from E. coli with PEP covalently bound to Cys115;
addition of UNAG turns over the bound PEP to product, purging the
enzyme (4). Since the new MurA inhibitors might bind to
the blocked PEP site and/or require UNAG for binding to the enzyme, the
effects of the order of addition of reaction components on
IC50s were examined (Table 1).
The presence of PEP during preincubation of MurA with each inhibitor
did not change the IC50 observed with
preincubation of only MurA and inhibitor. In contrast, the presence of
UNAG during the preincubation of MurA with inhibitor decreased the
IC50 22-fold for fosfomycin, from 8.8 to 0.4 µM. Similarly, when UNAG was present during the preincubation, each
of the RWJ inhibitors exhibited a decrease in its
IC50, as follows: 5-fold for RWJ-3981, 7.5-fold for RWJ-110192, and 13-fold for RWJ-140998. The decrease in the IC50s suggests that the presence of UNAG enhances
the interaction of the three inhibitors with MurA, possibly in a manner
similar to that of the binding of fosfomycin. It is possible that the binding of each compound requires the enzymatically active conformation of MurA, which is achieved in the presence of UNAG (20, 22, 23). In addition, if the binding site of a particular compound overlaps with the PEP binding site, the effect of UNAG may be to purge
MurA of PEP and provide access to the binding site.
Since MurA was present in the assay at 100 nM, the
IC50s for the compounds (40 to 70 nM) in the
presence of UNAG are close to the theoretical lower limit of 50 nM
(half the enzyme concentration) for a 1:1 stoichiometric addition of
inhibitor to enzyme. These data suggested that the possibility of
formation of a covalent or tightly bound complex between MurA and each
inhibitor should be investigated.
Lack of covalent adduct formation between RWJ-3981, RWJ-110192, or
RWJ-140998 and MurA.
The mechanism of inhibition of MurA by
fosfomycin is well established. Fosfomycin forms a covalent adduct with
the active site nucleophile Cys115 of MurA (15, 16, 23).
Using conditions that detected the covalent adduct between fosfomycin
and MurA-Cys115 in mass spectrometry of tryptic digests, none of the
three RWJ inhibitors appeared to form a covalent adduct with MurA,
either at Cys115 or elsewhere on the MurA protein (data not shown).
Determination of reversibility of inhibition.
To determine
whether the inhibition by the compounds was reversible, each compound
was incubated with MurA and UNAG, followed by filtration and washing to
separate free compound and other low-molecular-weight components from
free enzyme and from enzyme-inhibitor complex. An irreversible complex
is expected to remain inhibited after washing.
Incubation of MurA with fosfomycin, RWJ-3981, RWJ-110192, or
RWJ-140998 prior to filtration and washing substantially inhibited each
sample (77 to 116% inhibition) (Table
2). After washing, the fosfomycin sample,
which is expected to covalently and irreversibly modify the MurA
protein, remained inhibited. Similarly, the RWJ-3981 and RWJ-140998
samples remained inhibited after washing. In contrast, the RWJ-110192
sample, which was fully inhibited before washing, recovered 64% of its
activity, displaying only a 36% inhibition after washing.
Based on these data, inhibition of MurA by RWJ-140998 and RWJ-3981
appeared to be irreversible; inhibition by RWJ-110192 appeared to be
reversible. The lack of evidence for covalent adducts between MurA and
each compound from the tryptic peptide mass spectrometry experiment
discussed above would suggest that the basis for inhibition is complex
formation via strong noncovalent binding of compound to MurA. The
binding of RWJ-110192 would appear to be weaker than that of RWJ-3981
or RWJ-140998 since inhibited enzyme recovered partial activity during washing.
Competition with [3H]fosfomycin for binding to
MurA. To determine whether interaction with each inhibitor
prevented the binding of fosfomycin to MurA, MurA (with UNAG) was
incubated with each inhibitor, followed by incubation with
[3H]fosfomycin and determination of
radioactivity bound to MurA. As expected (Table
3), the presence of unlabeled fosfomycin
reduced the labeling of MurA to background levels.
Each of the three compounds (RWJ-3981, RWJ-110192, and RWJ-140998)
reduced the labeling of MurA by [3H]fosfomycin
compared to that of the MurA control, suggesting that these compounds
may bind at or near the active site of the enzyme and prevent access of
fosfomycin. Compared to RWJ-3981 and RWJ-140998, RWJ-110192 was less
effective at preventing labeling of MurA with
[3H]fosfomycin. Whereas labeling of MurA by
[3H]fosfomycin was essentially abolished by the
presence of either RWJ-3981 or RWJ-140998, labeling in the presence of
RWJ-110192 was 29% at 10 min and 49% at 35 min, suggesting either
weaker interactions with MurA than fosfomycin or less overlap in the binding sites of RWJ-110192 and fosfomycin. Moreover, the increase in
labeling of MurA at 35 min compared to 10 min suggested that the
MurA-RWJ-110192 interaction was reversible, consistent with the partial
recovery of MurA activity observed after filtration and washing of MurA
with RWJ-110192 (Table 2).
Thus, all three inhibitors reduced the labeling of MurA by
[3H]fosfomycin, suggesting that the compounds
may bind at or near the active site. However, we cannot exclude the
possibility that the binding of the compounds may cause a conformation
change in MurA which prevents fosfomycin from binding.
Modeling studies: docking of inhibitors into the active site of
MurA.
The crystal structure of the MurA enzyme complexed with the
substrate UNAG and with fosfomycin, a PEP surrogate which binds at the
PEP site, consists of two globular domains with the active site located
between them (23). This structure was used as the starting
point for modeling studies, to dock each of the compounds into the
active site of MurA. The predicted binding of each compound is shown in
Fig. 2.
View larger version (191K):
[in this window]
[in a new window]
|
FIG. 2.
DockVision poses of the crystal structure of MurA with
bound fosfomycin (A), RWJ-3981 (B), RWJ-110192 (C), and RWJ-140998 (D).
The carbons of the inhibitors are shown in cyan.
|
|
For RWJ-3981 (Fig. 2B), the centroid of the sulfur-containing ring was
predicted to bind 7.1 Å from the sulfur of the catalytic Cys115. The
carbonyl oxygen of the inhibitor may form strong hydrogen bonds with
Arg120 and Arg397. The sulfur atoms can make hydrophobic contacts with
the side chain carbon atoms of Met90 and Arg91.
For RWJ-110192 (Fig. 2C), the centroid of the rings was predicted to
bind farther from Cys115 than for RWJ-3981. The centroid of the
bicyclic ring system was 8.8 Å from the sulfur of Cys115. This
inhibitor may overlap with the NAG of UNAG. The carbonyl oxygen can
form a strong hydrogen bond with Arg91. Also, there may be a relatively
strong electrostatic attraction between the guanidinium group of Arg91
and the negatively charged pyrazolone ring of RWJ-110192.
RWJ-140998 (Fig. 2D) was also predicted to bind farther away than
RWJ-3981 from the catalytic Cys115. The centroid of the rings was 8.7 Å from the sulfur of Cys115. Although RWJ-140998 contains the
2,4-dioxopyrimidine ring also found in the uracil portion of the UNAG
substrate, the proposed binding of the dioxopyrimidine of RWJ-140998
was relatively far (>10 Å) from the UNAG site. The NH of the
pyrimidindione ring can form a strong hydrogen bond to Asp305, while
the carbonyl oxygen may form a hydrogen bond to Asn23. Arg120 may form
a hydrogen bond with one of the nitrogen atoms of the imidazole. The
phenyl ring of Phe328 can form a hydrophobic contact with the sulfur
atom of the thiazocine ring, while the side chains of Thr304 and Val163
may form hydrophobic contacts with the methylene carbons of this ring.
Confirmation of these hypotheses about the interactions between MurA
and RWJ-3981, RWJ-110192, and RWJ-140998 would require cocrystallization studies of MurA with each inhibitor.
SARs of RWJ-3981 and RWJ-140998.
To investigate the
structure-activity relationships (SARs) of RWJ-3981 and RWJ-140998,
several structural analogs of these two compounds were tested for
inhibitory activity in the MurA assay. The RWJ-140998 analog,
1-methylxanthine (Fig. 3), did not inhibit the MurA enzyme at a concentration of 25 µM. Thus, some features of the thiazocine ring appear to be necessary for inhibitory activity, consistent with the prediction of interactions between MurA
and this moiety in the modeling study (Fig. 2).
For the RWJ-3981 analogs (Table 4), the
presence of the chlorine on the ring containing the disulfide appeared
to be essential for activity, as Compound 3, which lacked the chlorine,
was inactive (IC50 > 25 µM). Replacing the
chlorine with a dimethylamino group (Compound 2) decreased
activity about 100-fold. Replacing it with 4-methoxyanilino,
2-hydroxyethylthio, or N-morpholino (Compounds 4, 5, and 6, respectively) decreased activity >100-fold. Compound 1, with a
dichlorinated phenyl ring, was approximately as active as RWJ-3981.
The SAR of the dithiolanones (Table 4) demonstrated that the chlorine
atom is necessary for activity, yet Hint calculations showed no
significant interaction of the chlorine with any of the neighboring
residues in the active site of MurA in the modeling studies. The LUMO
energies of the dithiolane derivatives were calculated (Table 4). The
LUMO is centered on the disulfide moiety in the ring, suggesting that
attack of a nucleophile (i.e., Cys115) would occur at one of the sulfur
atoms. Replacing the chlorine at position 5 of the dithiolanone ring
lowered the LUMO energy of RWJ-3981 by 3.7 kcal/mole relative to
Compound 3, which had no substitutions. All of the other substitutions
(dimethylamino, 4-methoxyanilino, 2-hydroxyethylthiol, or
N-morpholino [Compounds 2, 4, 5, and 6, respectively])
increased the LUMO energy relative to that of RWJ-3981. These results
suggest that RWJ-3981 should be more reactive than the other analogs
toward nucleophilic attack, thus rationalizing the observed SAR.
The presence of a disulfide in RWJ-3981 does indeed raise the
possibility that Cys115 of MurA could perform nucleophilic attack, with
the opening of the ring of RWJ-3981 and concomitant formation of a
disulfide bond between the compound and Cys115. This mechanism would be
analogous to that observed for fosfomycin, with nucleophilic attack by
Cys115 at C-2 of the antibiotic, opening of the ring of the
epoxide, and formation of a covalent bond between C-2 and Cys115
(13, 16). However, repeated attempts to detect a putative covalent adduct between MurA and RWJ-3981 by mass spectrometry were
unsuccessful, under conditions that did detect the adduct between MurA
and fosfomycin. If the covalent adduct did form in the case of
RWJ-3981, it did not survive the conditions of mass spectrometry. The
docking programs used in the modeling study cannot address covalent
binding between proteins and ligands. Thus, it is possible that the
irreversible complex detected between MurA and RWJ-3981 (Table 2) was
the result of either tight binding or covalent adduct formation.
Antibacterial activity of the MurA inhibitors.
Each of the
MurA inhibitors was tested for antibacterial activity using both
gram-negative and gram-positive bacteria (Table 5). Each of the compounds had modest
gram-positive antibacterial activity, with MICs of 4 to 32 µg/ml
against the strains of Staphylococcus aureus,
Enterococcus faecalis, and Enterococcus
faecium tested. Like fosfomycin, RWJ-3981 inhibited a
lipopolysaccharide-deficient E. coli strain
approximately equally as well as it inhibited S. aureus.
Gram-positive bacteria contain two copies of murA, encoding MurA1 and MurA2. Both MurA1 and MurA2 from S. pneumoniae
were inhibited by fosfomycin in enzyme assays (9). Our
enzyme assays used E. coli MurA, and it is not known whether
RWJ-3981, RWJ-110192, and RWJ-140998 can inhibit either of the MurA
enzymes from gram-positive bacteria.
Because fosfomycin uptake is enhanced by the presence of
glucose-6-phosphate due to the induction of the hexose phosphate transporter system (2, 7, 13), broth microdilution MICs were also determined in the presence of this sugar (25 µg/ml) (data
not shown). The MICs of fosfomycin did decline as much as eightfold for
S. aureus 29213, OC2878, and the hypersensitive E. coli but were unchanged for the other strains tested. However, for
RWJ-3981, RWJ-110192, and RWJ-140998, glucose-6-phosphate had no effect
on MICs for any of the strains, consistent with the lack of structural
similarity of the compounds to either fosfomycin or to hexose phosphate.
To determine whether the three compounds inhibited peptidoglycan
synthesis in S. aureus, the incorporation of
[3H]NAG was examined (Fig.
4A). In addition, the effects of these compounds on DNA, RNA, and protein synthesis were determined (Fig. 4B,
C, and D). All three compounds inhibited peptidoglycan synthesis in
S. aureus, as determined by reduced incorporation of
[3H]NAG compared to that of the control (Fig.
4A). However, all three compounds also inhibited DNA, RNA, and protein
synthesis within 10 min at four times the MIC (Fig. 4B, C, and D show
[3H]thymidine incorporation,
[3H]uridine incorporation, and
[3H]-amino acid incorporation, respectively).
The rapid inhibition of multiple bacterial functions suggested that the
antibacterial activities of these compounds against S. aureus in the MIC assay was not due to specific inhibition of
MurA. In contrast, inhibition by vancomycin was specific to
peptidoglycan synthesis, without inhibiting DNA, RNA, or protein
synthesis (Fig. 4B, C, and D). In an attempt to ascertain whether cell
wall synthesis might be specifically inhibited at a lower concentration
of the compounds, the labeling experiment was repeated at the MIC (data
not shown). RWJ-3981 and RWJ-110192 inhibited DNA, RNA, protein, and
cell wall synthesis from 78 to 99%, whereas RWJ-140998 did not inhibit any of these processes by more than 22% under these conditions. Thus,
for all three compounds, it was not possible to detect specific inhibition of cell wall biosynthesis.
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 4.
Effects of MurA inhibitors on peptidoglycan (A), DNA
(B), RNA (C), and protein synthesis (D) in S. aureus
29213 cells. Vancomycin, levofloxacin, rifampin, and tetracycline were
positive controls for inhibition of peptidoglycan, DNA, RNA, and
protein synthesis, respectively. NSB, nonspecific binding (cpm bound to
filter in the absence of cells).
|
|
Because of our previous experience with membrane-damaging agents which
inhibited bacterial DNA, RNA, and protein synthesis within 10 min of
exposure, these compounds were tested in a propidium iodide uptake
assay to measure cellular integrity (11). None of the
compounds appeared to damage the bacterial cell membrane (data not
shown). RWJ-3981 did not protect mice from death in an S. aureus lethal infection model (data not shown), nor did it cause
overt toxicity when dosed at 80 mg/kg of body weight.
In summary, we have identified and characterized three new inhibitors
of the MurA enzyme which are chemically different from fosfomycin and
appear to bind to the enzyme. These compounds represent three new
scaffolds available for further chemical modification to develop MurA
inhibitors with increased specificity and antibacterial activity.
| |
ACKNOWLEDGMENTS |
We thank Raul Goldschmidt and Yuan Chang for cloning of the MurA
enzymes, Jeffrey Fernandez and Haiyong Jin for enzyme purification and
characterization, and John Masucci and Bill Jones for mass spectrometry
experiments. We thank John Melton, Mike Loeloff, and Ellyn Wira for
assistance with screening, and Jamese Hilliard for performing the
BacLight and some macromolecular synthesis assays. We thank Raul
Goldschmidt, Anne Marie Queenan, and Mark Macielag for critical reading
of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: The R.W. Johnson
Pharmaceutical Research Institute, 1000 Route 202, Raritan, NJ 08869. Phone: (908) 704-4320. Fax: (908) 526-3047. E-mail:
ebaum{at}prius.jnj.com.
| |
REFERENCES |
| 1.
|
Arca, P.,
G. Reguera, and C. Hardisson.
1997.
Plasmid-encoded fosfomycin resistance in bacteria isolated from the urinary tract in a multicenter survey.
J. Antimicrob. Chemother.
40:393-399[Abstract/Free Full Text].
|
| 2.
|
Barry, A. L., and P. C. Fuchs.
1991.
In vitro susceptibility testing procedures for fosfomycin tromethamine.
Antimicrob. Agents Chemother.
35:1235-1238[Abstract/Free Full Text].
|
| 3.
|
Bernat, B. A.,
L. T. Laughlin, and R. N. Armstrong.
1997.
Fosfomycin resistance protein (fosA) is a manganese metalloglutathione transferase related to glyoxalase I and the extradiol dioxygenases.
Biochemistry
36:3050-3055[CrossRef][Medline].
|
| 4.
|
Brown, E. D.,
J. L. Marquardt,
J. P. Lee,
C. T. Walsh, and K. S. Anderson.
1994.
Detection and characterization of a phospholactoyl-enzyme adduct in the reaction catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase, MurZ.
Biochemistry
33:10638-10645[CrossRef][Medline].
|
| 5.
|
Brown, E. D.,
E. I. Vivas,
C. T. Walsh, and R. Kolter.
1995.
MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli.
J. Bacteriol.
177:4194-4197[Abstract/Free Full Text].
|
| 6.
|
Bugg, T. D. H., and C. T. Walsh.
1992.
Intracellular steps of bacterial cell wall peptidoglycan biosynthesis: enzymology, antibiotics, and antibiotic resistance.
Nat. Prod. Rep.
9:199-215[CrossRef][Medline].
|
| 7.
|
Dette, G. A.,
H. Knothe,
B. Schoenenbach, and G. Plage.
1983.
Comparative study of fosfomycin activity in Mueller-Hinton media and in tissues.
J. Antimicrob. Chemother.
11:517-524[Abstract/Free Full Text].
|
| 8.
|
Dewar, M. J. S.,
E. G. Zoebisch,
E. F. Healy, and J. J. P. Stewart.
1985.
Development and use of quantum mechanical molecular models. 76. AM1: a new general purpose quantum mechanical molecular model.
J. Am. Chem. Soc.
107:3902-3909[CrossRef].
|
| 9.
|
Du, W.,
J. R. Brown,
D. R. Sylvester,
J. Huang,
A. F. Chalker,
C. Y. So,
D. J. Holmes,
D. J. Payne, and N. G. Wallis.
2000.
Two active forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria.
J. Bacteriol.
182:4146-4152[Abstract/Free Full Text].
|
| 10.
|
Hart, T. N.,
S. R. Ness, and R. J. Read.
1997.
Critical evaluation of the research docking program for the CASP2 challenge.
Proteins Suppl.
1:205-209.
|
| 11.
|
Hilliard, J. J.,
R. M. Goldschmidt,
L. Licata,
E. Z. Baum, and K. Bush.
1999.
Multiple mechanisms of action for inhibitors of histidine protein kinases from bacterial two-component systems.
Antimicrob. Agents Chemother.
43:1693-1699[Abstract/Free Full Text].
|
| 12.
|
Horii, T.,
T. Kimura,
K. Sato,
K. Shibayama, and M. Ohta.
1999.
Emergence of fosfomycin-resistant isolates of Shiga-like toxin-producing Escherichia coli O26.
Antimicrob. Agents Chemother.
43:789-793[Abstract/Free Full Text].
|
| 13.
|
Kahan, F. M.,
J. S. Kahan,
P. J. Cassidy, and H. Kropp.
1974.
Mechanism of action of fosfomycin (phosphonomycin).
Ann. N. Y. Acad. Sci.
235:364-386[Medline].
|
| 14.
|
Kellogg, G. E.,
G. S. Joshi, and D. J. Abraham.
1991.
New tools for modeling and understanding hydrophobicity and hydrophobic interactions.
Med. Chem. Res.
1:444-453.
|
| 15.
|
Kim, D. H.,
W. J. Lees,
K. E. Kempsell,
W. S. Lane,
K. Duncan, and C. T. Walsh.
1996.
Characterization of a Cys115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-GlcNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin.
Biochemistry
35:4923-4928[CrossRef][Medline].
|
| 16.
|
Marquardt, J. L.,
E. D. Brown,
W. S. Lane,
T. M. Haley,
Y. Ichikawa,
C. H. Wong, and C. T. Walsh.
1994.
Kinetics, stoichiometry, and identification of the reactive thiolate in the inactivation of UDP-GlcNAc enolpyruvoyl transferase by the antibiotic fosfomycin.
Biochemistry
33:10646-10651[CrossRef][Medline].
|
| 17.
|
Marquardt, J. L.,
D. A. Siegele,
R. Kolter, and C. T. Walsh.
1992.
Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N-acetylglucosamine enolpyruvyl transferase.
J. Bacteriol.
174:5748-5752[Abstract/Free Full Text].
|
| 18.
|
National Committee for Clinical Laboratory Standards.
1997.
Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard M7-A4.
National Committee for Clinical Laboratory Standards, Wayne, Pa.
|
| 19.
|
Rarey, M.,
B. Kramer,
T. Lengauer, and G. Klebe.
1996.
A fast flexible docking method using an incremental construction algorithm.
J. Mol. Biol.
261:470-489[CrossRef][Medline].
|
| 20.
|
Schonbrunn, E.,
S. Eschenburg,
F. Krekel,
K. Luger, and N. Amrhein.
2000.
Role of the loop containing residue 115 in the induced-fit mechanism of the bacterial cell wall biosynthetic enzyme MurA.
Biochemistry
39:2164-2173[CrossRef][Medline].
|
| 21.
|
Schonbrunn, E.,
S. Sack,
S. Eschenburg,
A. Perrakis,
F. Krekel,
N. Amrhein, and E. Mandelkow.
1996.
Crystal structure of UDP-N-acetylglucosamine enolpyruvyltransferase, the target of the antibiotic fosfomycin.
Structure (London)
4:1065-1075[Medline].
|
| 22.
|
Schonbrunn, E.,
D. I. Svergun,
N. Amrhein, and M. H. J. Koch.
1998.
Studies on the conformational changes in the bacterial cell wall biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyltransferase (MurA).
Eur. J. Biochem.
253:406-412[Medline].
|
| 23.
|
Skarzynski, T.,
A. Mistry,
A. Wonacott,
S. E. Hutchinson,
V. A. Kelly, and K. Duncan.
1996.
Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, and enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin.
Structure (London)
4:1465-1474[Medline].
|
Antimicrobial Agents and Chemotherapy, November 2001, p. 3182-3188, Vol. 45, No. 11
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.11.3182-3188.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Eschenburg, S., Priestman, M. A., Abdul-Latif, F. A., Delachaume, C., Fassy, F., Schonbrunn, E.
(2005). A Novel Inhibitor That Suspends the Induced Fit Mechanism of UDP-N-acetylglucosamine Enolpyruvyl Transferase (MurA). J. Biol. Chem.
280: 14070-14075
[Abstract]
[Full Text]
-
He, X., Reeve, A. M., Desai, U. R., Kellogg, G. E., Reynolds, K. A.
(2004). 1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH). Antimicrob. Agents Chemother.
48: 3093-3102
[Abstract]
[Full Text]
-
El Zoeiby, A., Sanschagrin, F., Darveau, A., Brisson, J.-R., Levesque, R. C.
(2003). Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries. J Antimicrob Chemother
51: 531-543
[Abstract]
[Full Text]
-
He, X., Reynolds, K. A.
(2002). Purification, Characterization, and Identification of Novel Inhibitors of the {beta}-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) from Staphylococcus aureus. Antimicrob. Agents Chemother.
46: 1310-1318
[Abstract]
[Full Text]
Top
Abstract
Introduction
