Dual inhibitor of MurD and MurE ligases from escherichia coli and staphylococcus aureus

Article abstract of DOI:10.1021/ml300047h

MurD and MurE ligases, consecutive enzymes participating in the intracellular steps of bacterial peptidoglycan biosynthesis, are important targets for antibacterial drug discovery. We have designed, synthesized, and evaluated the first d-glutamic acid-containing dual inhibitor of MurD and MurE ligases from Escherichia coli and Staphylococcus aureus (IC₅₀ values between 6.4 and 180 μM) possessing antibacterial activity against Gram-positive S. aureus and its methicillin-resistant strain (MRSA) with minimal inhibitory concentration (MIC) values of 8 μg/mL. The inhibitor was also found to be noncytotoxic for human HepG2 cells at concentrations below 200 μM.

Full text of DOI:10.1021/ml300047h

Letter
pubs.acs.org/acsmedchemlett
Dual Inhibitor of MurD and MurE Ligases from Escherichia coli and
Staphylococcus aureus
†
†
†
†
‡,§,∥
̌
Tihomir Tomasic, Roman Sink, Nace Zidar, Anja Fic, Carlos Contreras-Martel,
̌
́
Andrea Dessen,‡,§,∥ Delphine Patin,⊥,# Didier Blanot,⊥,# Manica Muller-Premru,∇ Stanislav Gobec,†
́
̈
Anamarija Zega,† Danijel Kikelj,† and Lucija Peterlin Masic*,†
̌
̌
†Faculty of Pharmacy, University of Ljubljana, Ask
‡Institut de Biologie Structurale, Bacterial Pathogenesis Group, Universite
§Commissariat a
l'Energie Atomique (CEA), IBS, 38027 Grenoble, France
∥Centre National de la Recherche Scientifique (CNRS), IBS, 41 rue Jules Horowitz, 38027 Grenoble, France
⊥Univ Paris-Sud, Laboratoire des Enveloppes Bacter
́
iennes et Antibiotiques, Institut de Biochimie et Biophysique Moleculaire et
̌
erce
̌
va 7, 1000 Ljubljana, Slovenia
Grenoble I, 38027 Grenoble, France
́
̀
́
Cellulaire, UMR 8619, 91405 Orsay, France
#CNRS, 91405 Orsay, France
∇Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, 1105 Ljubljana, Slovenia
S
* Supporting Information
ABSTRACT: MurD and MurE ligases, consecutive enzymes
participating in the intracellular steps of bacterial peptidogly-
can biosynthesis, are important targets for antibacterial drug
discovery. We have designed, synthesized, and evaluated the
first D-glutamic acid-containing dual inhibitor of MurD and
MurE ligases from Escherichia coli and Staphylococcus aureus
(IC50 values between 6.4 and 180 μM) possessing antibacterial
activity against Gram-positive S. aureus and its methicillin-
resistant strain (MRSA) with minimal inhibitory concentration
(MIC) values of 8 μg/mL. The inhibitor was also found to be
noncytotoxic for human HepG2 cells at concentrations below 200 μM.
KEYWORDS: antibacterial agent, inhibitor, Mur ligase, peptidoglycan, 2-thioxothiazolidin-4-one
Ala (MurC), D-Glu [UDP-N-acetylmuramoyl-L-Ala:D-Glu ligase
he increasing emergence of bacterial strains resistant to
Tcurrently available antibiotics has created unmet medical (MurD)], L-Lys or meso-diaminopimelic acid [UDP-N-
acetylmuramoyl-L-Ala-D-Glu:meso-diaminopimelate(or L-Lys)
ligase (MurE)], and D-Ala-D-Ala dipeptide (MurF) to the
starting MurC substrate UDP-MurNAc.3
needs in antibacterial therapy. Development of novel effective
antibacterial drugs directed toward previously unexploited
targets is therefore essential to combat bacterial drug resistance
in the future.1 Peptidoglycan, an essential cell wall polymer
unique to prokaryotic cells, is thus an important target for
antibacterial drug discovery.2 The biosynthesis of peptidoglycan
is a multistep process comprising intracellular assembly of the
uridine 5′-diphosphate (UDP)-MurNAc-pentapeptide (Park's
nucleotide), which is subsequently translocated through the
cytoplasmic membrane and incorporated into the growing
peptidoglycan layer.3 While the majority of currently approved
antibiotics that target peptidoglycan biosynthesis act on the
extracellular transpeptidation steps,4 there has been increased
interest in exploiting the enzymes involved in the early
intracellular steps of peptidoglycan precursor biosynthesis.5
This is in particular the case for ATP-dependent Mur ligases
[UDP-N-acetylmuramate:L-Ala ligase (MurC) to UDP-N-
acetylmuramoyl-L-Ala-γ-D-Glu-meso-diaminopimelate(or L-
Lys):D-Ala-D-Ala ligase (MurF)], which catalyze a series of
reactions leading to Park's nucleotide by sequentially adding L-
Mur ligases catalyze the formation of a peptide or amide
bond between the UDP-substrate and the condensing amino
acid. They operate by similar chemical mechanisms6,7 and, as
shown for MurC and MurF, by an ordered kinetic
mechanism.8,9 The enzymatic reaction is initiated by the
binding of ATP to the free enzyme, followed by binding of the
corresponding UDP substrate. The terminal carboxyl group of
the UDP substrate is then activated by ATP-promoted
phosphorylation, resulting in the formation of an acylphosphate
intermediate that is attacked by the amino group of the
incoming amino acid residue or dipeptide after its binding to
the enzyme. The resulting tetrahedral high-energy intermediate
collapses with elimination of inorganic phosphate and
Received: February 22, 2012
Accepted: June 27, 2012
Published: June 27, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
concomitant formation of a peptide or amide bond.6−9 The
crystal structures of Mur ligases from different bacterial strains
show the similar three-domain topology, with the N-terminal
and central domains binding UDP precursor and ATP,
respectively, while the C-terminal domain binds the condensing
amino acid or dipeptide residue. While the topologies of the
central and C-terminal domains are similar among the Mur
ligases, those of the N-terminal domains show differences, with
MurC and MurD more closely related to each other than to
MurE and MurF. These differences are related to the lengths of
the UDP precursor substrates.10
Recently, we have designed a series of thiazolidin-4-one-
based inhibitors of Mur ligases that act either as multitarget11,12
or MurD-selective inhibitors.13−16 Multitarget inhibitors of
bacterial enzymes constitute a promising strategy to combat
bacterial resistance because target-mediated resistance to such
compounds is less likely to evolve, since mutations conferring
resistance would have to occur in at least two different target
genes during a single generation.17 Although sequence
alignment of Mur ligase orthologues and paralogues revealed
relatively low overall homology, there is quite high homology of
residues present especially in the ATP-binding site.18−21 The
common catalytic mechanism and the conserved active sites
make Mur ligases attractive targets for the design of multitarget
inhibitors.11,12,22,23 Because MurD and MurE ligases are
consecutive enzymes in the biosynthesis of Park's nucleotide
(Figure S1 in the Supporting Information), it is reasonable to
assume that it would be possible to inhibit both enzymes with a
single molecule that would act as MurD product and MurE
substrate analogue. Moreover, superposition of MurD and
MurE crystal structures revealed that residues important for
binding of D-Glu in the MurD enzyme have their corresponding
counterparts in the MurE enzyme, which gives further support
to our hypothesis.22 Indeed, glutamic acid-based MurD
inhibitors, designed as transition-state analogues, were reported
to inhibit Staphylococcus aureus MurE to some extent.24,25
Similarly, compound I (Figure 1) and its analogous Escherichia
observations provide the rationale for evaluating MurD
inhibitors for MurE inhibition.
Analysis of the binding mode of previously reported
thiazolidine-2,4-dione-based inhibitor I (Figure 1) in the E.
coli MurD active site revealed that the D-Glu moiety and the
thiazolidine-2,4-dione ring form the majority of hydrogen
bonds with E. coli MurD active site residues, while the linker
between them is involved mainly in hydrophobic interactions
and hydrogen bonds through water molecules.14,15 Compound
I inhibited E. coli MurD ligase with an IC50 of 35 μM14 and also
showed weak inhibition of S. aureus MurE ligase (RA = 64% at
500 μM, Table 1). Using structure-based design, through
several structure optimization cycles, we improved E. coli MurD
ligase inhibitory potency of I down to an IC50 value of 3 μM.15
Here, we report further structural modifications of the E. coli
MurD inhibitor I by variation of the linker connecting the two
phenyl rings and replacement of the thiazolidine-2,4-dione ring
by the rhodanine moiety (Figure 1), while leaving other parts of
the molecule unchanged, given their important interactions
with E. coli MurD active site residues. The synthesis of target
compounds 9 and 10 is outlined in Scheme 1 and described in
detail in the Supporting Information.
a
Scheme 1. Synthesis of Target Compounds 9 and 10
a
Reagents and conditions: (a) Ethylene glycol, p-toluenesulfonic acid,
benzene, reflux, 24 h. (b) 3-Aminobenzoic acid, NaCNBH3, MeOH, rt,
16 h. (c) Pyridinium tosylate, THF/H2O, rt, 18 h. (d) Compound 5:
rhodanine, piperidine, AcOH, EtOH, reflux, 48 h; compound 6:
thiazolidine-2,4-dione, piperidine, AcOH, EtOH, microwave, 140 °C,
30 min. (e) H-D-Glu(OMe)-OMe·HCl, O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), Et3N,
CH2Cl2, rt, 5 h. (f) Compound 9: 2 M LiOH, MeOH/H2O, rt, 16
h; compound 10: 1 M NaOH, MeOH/H2O, rt, 3 h.
Figure 1. Structural modification of E. coli MurD inhibitor I.
Considering a possibility of dual MurD and MurE inhibition,
the target compounds 9 and 10 were tested against MurD and
MurE ligases from E. coli and S. aureus using the radioactivity
assays (Table 1). The compounds were tested in the presence
of detergent Tween-20 to avoid nonspecific inhibition due to
aggregate formation.26 Compound 9 inhibited MurD ligases
coli MurD inhibitors showed weak affinity for S. aureus MurE
with residual activities (RA) between 35 and 76% at 500 μM
compound (Table S1 in the Supporting Information). Although
reported MurE inhibitions are in general moderate, these
Table 1. Inhibitory Activity of Compounds 9 and 10 against MurD and MurE Ligases and Their Antibacterial Activity
IC50 (μM)
MIC (μg/mL)
9 10
enzyme
I
9
10
bacterial strain
E. coli MurD ligase
S. aureus MurD ligase
E. coli MurE ligase
S. aureus MurE ligase
35
8.2 0.6
6.4 1.0
180 60
17 1.5
36
5
S. aureus ATCC 29213
MRSA ATCC 43300
E. faecalis ATCC 29212
E. coli ATCC 25922
8
8
>128
>128
>128
>128
>128
ND
64%
ND
100
5
a
b
∼200
>200
64
>128
>128
c
P. aeruginosa ATCC 27853
a
b
c
Residual activity (RA) at 500 μM compound. Residual activity at 200 μM compound: 51 2%. No inhibitory activity at 200 μM compound; ND,
not determined.
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ACS Medicinal Chemistry Letters
Letter
from E. coli and S. aureus with IC50 values of 8.2 and 6.4 μM,
respectively, and additionally showed inhibitory activity against
MurE ligases from E. coli and S. aureus with IC50 values of 180
and 17 μM, respectively, thus acting as a dual inhibitor of the
intracellular steps of peptidoglycan biosynthesis. Dual inhib-
ition of MurD and MurE from S. aureus was well balanced,
which was not the case for MurD and MurE from E. coli.
Thiazolidine-2,4-dione 10 was found to be weaker inhibitor of
all four Mur ligases than its rhodanine counterpart 9, which has
already been observed previously in the case of E. coli MurD for
similar compounds.13−16 Compound 10 inhibited MurD ligases
from E. coli and S. aureus with IC50 values of 36 and 100 μM,
respectively, while it was found to be inactive against S. aureus
MurE and showed comparable E. coli MurE inhibition as 9.
Following the promising inhibition of MurD and MurE
ligases from the two bacterial strains, inhibitors 9 and 10 were
also tested for their antibacterial activity against three Gram-
positive [S. aureus ATCC 29213, methicillin-resistant S. aureus
(MRSA) ATCC 43300, and Enterococcus faecalis ATCC 29212]
and two Gram-negative (E. coli ATCC 25922 and Pseudomonas
aeruginosa ATCC 27853) bacteria (Table 1). Although
compound 9 was found to be inactive against both Gram-
negative bacteria, presumably because of its inability to cross
the outer membrane, it inhibited the growth of E. faecalis with
MIC of 64 μg/mL and showed more potent activity against S.
aureus and MRSA with MIC values of 8 μg/mL. Compound 10,
a weaker inhibitor of Mur ligases than 9, was devoid of
antibacterial activity. Compound 9 represents, to the best of
our knowledge, the first D-Glu-based inhibitor of Mur ligases
possessing antibacterial activity; however, its mode of action
still remains to be determined. To evaluate the preliminary
safety profile of compound 9, it was tested for its cytotoxicity
on human HepG2 cells and found to be noncytotoxic at
concentrations at least up to 200 μM (Figure S2 in the
Supporting Information).
resolution of 1.5 Å, the inhibitor is seen to occupy the binding
site of MurD product UDP-MurNAc-L-Ala-D-Glu (UMAG).
The D-Glu moiety of the inhibitor is positioned at the same site
as that previously observed for the D-Glu residue of UMAG6
and other MurD inhibitors containing the D-Glu moi-
ety,14,15,27,28 while the rhodanine moiety occupies the uracil-
binding pocket, where it forms a hydrogen bond with Oγ of
Thr36. However, there are important differences in the position
of α-carboxylate group of D-Glu moiety (Figures 2 and S4 in
the Supporting Information). The α-carboxylate group of the D-
Glu moiety of UMAG and of the previously reported
thiazolidin-4-one inhibitor I forms a charge-based interaction
with Nξ of Lys348 and is additionally hydrogen-bonded
through a conserved water molecule to Oγ of Thr321 and the
carboxyl group of Asp182. In the case of E. coli MurD−9
complex, the α-carboxylate group of D-Glu is rotated by
approximately 75°, which results in a different hydrogen-
bonding network. Because of a different overall conformation,
the α-carboxylate group loses direct contact with the Nξ of
Lys348 and forms hydrogen bonds with three water molecules
(W19, W92, and W102), which form a hydrogen bond network
with residues Lys115, Glu157, Asp182, Thr321, and Lys348
(for more details, see Figure S3 and the text in the Supporting
Information). The described linker modification of I to give 9
results in the different conformation of the middle part of the
inhibitors (Figure S4 in the Supporting Information). The
benzylidene moiety of 9 is held in place by the hydrophobic
interaction of the phenyl ring with Gly73, while the phenyl ring
of the phenylamino moiety contributes to recognition of the
inhibitor within the active site, with hydrophobic interactions to
Leu416 and possible π−π interactions to Phe161. In addition to
these hydrophobic interactions, the most crucial interactions
between the inhibitor 9 and E. coli MurD active site residues are
hydrogen bonds formed between the carboxylates of D-Glu
moiety and Thr321, Lys348, Ser415, and Phe422, since they
contribute to the recognition of the D-Glu moiety in the D-Glu-
binding site, as well as hydrogen bond between rhodanine NH
group and Thr36. On the other hand, it is hard to estimate the
importance of the water-mediated interactions, since it was
shown by high-resolution NMR and molecular dynamics that
the bound ligands exhibit conformational flexibility in the E. coli
MurD active site.29 Therefore, water-mediated interactions in
the solution are very different from those observed in the
crystal structure.
The binding mode of inhibitor 9 in the E. coli MurD active
site was determined by solving the crystal structure of E. coli
MurD−9 complex, the structure of E. coli apo-MurD being
employed as a search model in a molecular replacement
experiment (Figures 2 and S3 in the Supporting Information).
In the crystal structure of E. coli MurD−9 complex, solved to a
In summary, structural modification of our recently disclosed
thiazolidin-4-one-based MurD inhibitors led to the discovery of
compound 9 that inhibited E. coli and S. aureus MurD ligase
with IC50 values of 8.2 and 6.4 μM, respectively. It also
inhibited E. coli and S. aureus MurE ligases with respective IC50
values of 180 and 17 μM. Compound 9 is, to the best of our
knowledge, the first D-Glu-based dual inhibitor of Mur ligases
that possesses antibacterial activity against Gram-positive S.
aureus and MRSA with MIC values of 8 μg/mL; however, its
mode of action still remains to be determined. Moreover,
compound 9 was found to be noncytotoxic on human HepG2
cells at concentrations at least up to 200 μM. These results
make compound 9 a promising inhibitor of two consecutive
steps of peptidoglycan biosynthesis and highlight it as an
interesting candidate for further optimization toward com-
pounds with improved antibacterial activity.
Figure 2. X-ray binding mode of inhibitor 9 (in magenta) in the active
site of E. coli MurD ligase (PDB code: 2Y1O). Hydrogen bonds
between 9 and the E. coli MurD active site residues (in yellow) and
crystal water molecules (in red) are presented as dashed green lines.
Comparison of the binding modes of inhibitors I and 9 in the E. coli
MurD active site is shown in Figure S4 in the Supporting Information.
628
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ACS Medicinal Chemistry Letters
Letter
alanyl-D-alanine-adding enzyme: Use of a glutathione S-transferase
fusion. Biochemistry 1996, 35, 16264−16269.
ASSOCIATED CONTENT
* Supporting Information
■
S
(10) Smith, C. A. Structure, function and dynamics in the mur family
of bacterial cell wall ligases. J. Mol. Biol. 2006, 362, 640−655.
Experimental procedures and characterization of intermediate
and target compounds, description of crystallization, prepara-
tion of the inhibitor complex, data collection, crystal structure
solution, and model refinement, description of enzyme assays,
and determination of antibacterial activity and cytotoxicity
assay. This material is available free of charge via the Internet at
http://pubs.acs.org.
(11) Tomasi
̌
c,
́
T.; Zidar, N.; Kovac,
̌
A.; Turk, S.; Simci
M.; Grdadolnik, S. G.; Zega, A.;
Anderluh, M.; Gobec, S.; Kikelj, D.; Peterlin Masi , L. 5-
̌ ̌
c, M.; Blanot,
D.; Muller-Premru, M.; Filipic,
̌
̈
̌ ̌
c
Benzylidenethiazolidin-4-ones as multitarget inhibitors of bacterial
Mur ligases. ChemMedChem 2010, 5, 286−295.
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T.; Kovac,
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bacterial MurC and MurD ligases. J. Mol. Model. 2012, 18, 1063−1072.
(13) Tomasic, T.; Zidar, N.; Rupnik, V.; Kovac, A.; Blanot, D.;
Gobec, S.; Kikelj, D.; Peterlin Masic, L. Synthesis and biological
̌
A.; Klebe, G.; Blanot, D.; Gobec, S.; Kikelj,
̌ ̌
D.; Peterlin Masi L. Virtual screening for potential inhibitors of
Accession Codes
The PDB code for the E. coli MurD−9 complex is 2Y1O.
̌
́
̌
̌
̌
AUTHOR INFORMATION
Corresponding Author
*Tel: +386-1-4769635. Fax: +386-1-4258031. E-mail: lucija.
peterlin@ffa.uni-lj.si.
evaluation of new glutamic acid-based inhibitors of MurD ligase.
■
Bioorg. Med. Chem. Lett. 2009, 19, 153−157.
̌
̌ ́ ̌
(14) Zidar, N.; Tomasic, T.; Sink, R.; Rupnik, V.; Kovac, A.; Turk, S.;
Patin, D.; Blanot, D.; Martel, C. C.; Dessen, A.; Muller-Premru, M.;
̈
̌ ̌
Zega, A.; Gobec, S.; Peterlin Masic, L.; Kikelj, D. Discovery of novel 5-
Funding
benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhib-
This work was supported by the EU FP6 Integrated Project
EUR-INTAFAR (Project No. LSHM-CT-2004-512138), by the
Slovenian Research Agency (Grant No. P1-0208), and by the
World Federation of Scientists.
itors of MurD ligase. J. Med. Chem. 2010, 53, 6584−6594.
̌
̌ ́ ̌
(15) Tomasic, T.; Zidar, N.; Sink, R.; Kovac, A.; Blanot, D.;
Contreras-Martel, C.; Dessen, A.; Muller-Premru, M.; Zega, A.; Gobec,
̈
̌ ̌
S.; Kikelj, D.; Peterlin Masic, L. Structure-based design of a new series
of D-glutamic acid based inhibitors of bacterial UDP-N-acetylmur-
amoyl-L-alanine:D-glutamate ligase (MurD). J. Med. Chem. 2011, 54,
4600−4610.
Notes
The authors declare no competing financial interest.
(16) Tomasi
̌
c,
́
T.; Kovac,
̌
A.; Simci
̌
c,
̌
M.; Blanot, D.; Grdadolnik, S.
c, L. Novel 2-thioxothiazolidin-
ACKNOWLEDGMENTS
We thank Professor Roger Pain for critical reading of the
manuscript.
■
G.; Gobec, S.; Kikelj, D.; Peterlin Masi
̌
̌
4-one inhibitors of bacterial MurD ligase targeting D-Glu- and
diphosphate-binding sites. Eur. J. Med. Chem. 2011, 46, 3964−3975.
(17) Wong, K. K.; Kuo, D. W.; Chabin, R. M.; Fournier, C.; Gegnas,
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13528.
ABBREVIATIONS
■
UDP, uridine 5′-diphosphate; UMA, UDP-N-acetylmuramoyl-
L-Ala; UMAG, UDP-N-acetylmuramoyl-L-Ala-D-Glu; MurC,
UDP-N-acetylmuramate:L-Ala ligase; MurD, UDP-N-acetyl-
muramoyl-L-Ala:D-Glu ligase; MurE, UDP-N-acetylmuramoyl-
L-Ala-D-Glu:meso-diaminopimelate(or L-Lys) ligase; MurF,
UDP-N-acetylmuramoyl-L-Ala-γ-D-Glu-meso-diaminopimelate-
(or L-Lys):D-Ala-D-Ala ligase
(18) Ikeda, M.; Wachi, M.; Jung, H. K.; Ishino, F.; Matsuhashi, M.
Homology among MurC, MurD, MurE and MurF proteins in
Escherichia coli and that between Escherichia coli MurG and a possible
MurG protein in Bacillus subtilis. J. Gen. Appl. Microbiol. 1990, 36,
179−187.
(19) Bouhss, A.; MenginLecreulx, D.; Blanot, D.; van Heijenoort, J.;
Parquet, C. Invariant amino acids in the mur peptide synthetases of
bacterial peptidoglycan synthesis and their modification by site-
directed mutagenesis in the UDP-MurNAc:L-alanine ligase from
Escherichia coli. Biochemistry 1997, 36, 11556−11563.
(20) Eveland, S. S.; Pompliano, D. L.; Anderson, M. S. Conditionally
lethal Escherichia coli murein mutants contain point defects that map to
regions conserved among murein and folyl poly-gamma-glutamate
ligases: Identification of a ligase superfamily. Biochemistry 1997, 36,
6223−6229.
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