Tuning the moenomycin pharmacophore to enable discovery of bacterial cell wall synthesis inhibitors

Article abstract of DOI:10.1021/ja4000933

New antibiotic drugs need to be identified to address rapidly developing resistance of bacterial pathogens to common antibiotics. The natural antibiotic moenomycin A is the prototype for compounds that bind to bacterial peptidoglycan glycosyltransferases

Full text of DOI:10.1021/ja4000933

Communication
pubs.acs.org/JACS
Tuning the Moenomycin Pharmacophore To Enable Discovery of
Bacterial Cell Wall Synthesis Inhibitors
Christian M. Gampe,† Hirokazu Tsukamoto,† Emma H. Doud,‡ Suzanne Walker,*,‡
and Daniel Kahne*,†,§
†Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
‡Department of Microbiology and Immunobiology and §Department of Biological Chemistry and Molecular Pharmacology, Harvard
Medical School, Boston, Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: New antibiotic drugs need to be identified
to address rapidly developing resistance of bacterial
pathogens to common antibiotics. The natural antibiotic
moenomycin A is the prototype for compounds that bind
to bacterial peptidoglycan glycosyltransferases (PGTs) and
inhibit cell wall biosynthesis, but it cannot be used as a
drug. Here we report the chemoenzymatic synthesis of a
fluorescently labeled, truncated analogue of moenomycin
based on the minimal pharmacophore. This probe, which
has optimized enzyme binding properties compared to
moenomycin, was designed to identify low-micromolar
inhibitors that bind to conserved features in PGT active
sites. We demonstrate its use in displacement assays using
PGTs from S. aureus, E. faecalis, and E. coli. 110,000
compounds were screened against S. aureus SgtB, and we
identified a non-carbohydrate based compound that binds
to all PGTs tested. We also show that the compound
inhibits in vitro formation of peptidoglycan chains by
several different PGTs. Thus, this assay enables the
identification of small molecules that target PGT active
sites, and may provide lead compounds for development of
new antibiotics.
Figure 1. Probe compound 2 was designed to identify compounds that
bind to the conserved features of the PGT active site. (a) The final stage
of the biosynthesis of peptidoglycan. (b) The moenomycin
he supporting structure of the bacterial cell wall is a layer of
polysaccharide strands containing peptide cross bridges,
pharmacophore is represented in black. Red arrows mark the functional
groups that form crucial hydrogen bonds to conserved active site amino
acid residues of PGTs. Parts of the molecule that do not contribute
significantly to protein binding are shown in gray. IC50 values are given
for in vitro PGT inhibition.3b,10
T
termed peptidoglycan (PG). This polymer protects the cell
membrane from rupture in harsh environments. The final stage
of the extracellular biosynthesis of PG proceeds in two steps: In
the transglycosylation step, the disaccharide phospholipid lipid II
is polymerized to form polysaccharide strands, and in the
subsequent transpeptidation step, these strands are cross-linked
(Figure 1a).1 These transformations are catalyzed by bifunctional
penicillin binding proteins (PBPs) that have both a glycosyl-
transferase (GT) and a transpeptidase active site.2 Additionally,
some bacteria possess monofunctional peptidoglycan glycosyl-
transferases (PGTs) that form polysaccharide strands, which are
then cross-linked by PBPs.3 All GT domains, whether found
within bifunctional PBPs or in monofunctional enzymes, contain
a set of invariant residues that both bind substrate and catalyze
the polymerization of lipid II.4 In bacteria, proper synthesis of PG
is required for cell viability, and inhibition of PG synthesis leads
to cell death. For decades, development of new antibiotics has
focused on targets involved in the cell wall synthesis and
remodeling.5 However, direct inhibition of PGT activity has so
far not been exploited for the development of antibiotics.6
The only known active site inhibitor of the PGTs is the natural
product moenomycin A (Figure 1b).6,7 Its desirable properties
include extraordinary potency without development of resist-
ance,8 but its clinical use is prevented due to physical properties
that result in poor oral bioavailability and long serum half-life.
However, moenomycin is potentially useful for discovering other
structural classes of molecules that target the same active site. In
one example of such a strategy, a fluorophore was directly
attached to the A-ring of the natural product, and displacement of
Received: January 4, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja4000933 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
this probe was monitored to discover new PGT inhibitors.9 The
beauty of this approach is its simplicity. However, because
moenomycin is a low-nanomolar inhibitor of PGTs, it cannot be
displaced by low-affinity binders found in typical compound or
fragment collections. Thus, we set out to design a probe based on
moenomycin that shows weaker binding affinity but retains the
structural features that determine the specificity for compounds
that bind to the active site of the PGTs, which is conserved across
all pathogens.
a
Scheme 1. Synthesis of 2
Four crystal structures of moenomycin bound to PGTs
showed that the E,F-disaccharide and the phosphoglycerate
engage in hydrogen bonds to several conserved, catalytically
essential amino acid residues in the active site (Figure 1b).11 It
was also reported that a lipid side chain of at least 10 carbon
atoms in length is required for enzyme inhibitory activity.7,11e,12
Thus, we needed to design a probe that retained these structural
features of moenomycin and contained a site that could be easily
derivatized for installation of a fluorophore. The crystal
structures indicated that a fluorescent label attached to the C-
ring N-acetyl group could be accommodated, since it points out
of the enzyme binding pocket. Thus, we targeted trisaccharide
phosphoglycerate 2 as a probe compound (Figure 1b) that would
allow us to identify low-affinity active site inhibitors.
The synthesis of probe 2 was achieved using a variety of
synthetic methods developed in our group (Scheme 1).12,13
Peracyl galactose was first converted into F-ring building block 3
in three steps (75% yield). Following carboxylation of the C3-
hydroxyl of 3, glycosylation with sulfoxide 4 and reductive
opening of the benzylacetal provided disaccharide 5. In a
sequence of six steps, the F-ring was equipped with the
moenomycin substituents crucial for contacting the active site.
First, the configuration at C4 was inverted (galacto to gluco
configuration) and the C3-carbamate as well as the C6-amide
were installed. Deprotection at the anomeric center then
provided disaccharide 6. Installation of phosphoglycerate 7
proceeded smoothly, and, following global deprotection, we
obtained the moenomycin pharmacophore 8. To our delight, we
noted that disaccharide 8 inhibits PG formation in vitro with IC50
values of 12 μM and 70 nM against E. coli PBP1b14 and S. aureus
SgtB,10b,15 respectively.3b,10
Next, we had to selectively attach the fluorescently labeled C-
ring to the unprotected disaccharide phosphoglycerate 8. To this
end, we took advantage of the bovine glycosyltransferase GalT
(Y289L), which was previously engineered to selectively transfer
a range of N-acetyl galactosamine derivatives, including N-
azidoacetyl galactosamine (GalNAz), to the C4 hydroxyl of N-
acetylglucosamine derivatives.16 Installation of galactosamine at
a model disaccharide proceeded smoothly;4 however, we found
that incubation of 8 and UDP-GalNAz with GalT (Y289L)
generated trisaccharide 9 only in yields below 10%. We
speculated that either 8 or product 9, being derivatives of
moenomycin A, might inhibit GalT. Gratifyingly, we found that
reducing the concentration of 8 by 10-fold while increasing the
amount of GalT to 5 mol% allowed the glycosyl transfer to
proceed in >90% yield after 60 h (>4 mg isolated). Treatment of
trisaccharide 9 with CuSO4/Na-ascorbate and fluorescein 6-
propargyl carboxamide in DMF provided 2 in 87% yield (>4 mg,
17 steps overall).
a
Reagents and conditions: (a) p-MeOC6H4OH, BF3·OEt2, DCM, rt,
24 h; (b) cat. NaOMe, MeOH, rt, 2 h; (c) PhCH(OMe)2, cat. PTSA,
CH3CN; (d) ClCO2Ph, pyridine, −40 °C, 2 h, 75% (over 3 steps); (e)
4, Tf2O, DTBMP, ADMB, mol. sieves 4 Å, DCM, −78 °C, 1.5 h, 56%;
(f) Et3SiH, TfOH, mol. sieves 4 Å, DCM, −78 °C; (g) Tf2O, pyridine,
DCM, −40 °C to rt, 2 h; (h) CsOAc, 18-crown-6, PhMe, rt, 14 h, 55%
(over 3 steps); (i) H2, 10% Pd/C (1 wt%), Cl3CCO2H/MeOH, rt, 45
min; (j) TEMPO, PhI(OAc)2, DCM/H2O (2/1), rt, 2 h; (k)
ClCO2iBu, N-methylmorpholine, THF, −40 °C, 5 min; then NH3,
iPrOH, rt, 24 h, 46% (over 3 steps); (l) CAN, MeCN/H2O (4/1), rt,
1.5 h; (m) 7, tetrazole, mol. sieves 3 Å, MeCN, 0 °C, 2 h; then
tBuO2H, 0 °C, 1 h; then P(OMe)3, rt, 0 °C, 47% (over 2 steps); (n)
Zn, Ac2O, AcOH, THF, rt, 10 h; (o) LiOH, THF/H2O2 (8/1), 0 °C, 2
h, 63% (over 2 steps); (p) 8 (0.1 mM); GalT (Y289L) (5 mol%);
UDP-GalNAz (1.3 mM), Tris (16 mM, pH 8.0), MnCl2 (16 mM),
CIP (0.2 U/μL), 37 °C, 60 h, >90%; (q) fluorescein 6-propargyl
carboxamide, CuSO4, Na-ascorbate, DMF, rt, 48 h, 87% (over 2 steps).
Abbreviations: PTSA, p-toluenesulfonic acid; ADMB, allyldimethoxy-
benzene; CIP, calf intestinal phosphatase; Troc, 2,2,2-trichloroethoxy-
carbonyl; CEO, cyanoethyl. IC50 values are given for in vitro
inhibition.3b,10
2 inhibits E. coli PBP1b and S. aureus SgtB with IC50 values of 600
nM and 31 nM, respectively. By comparison the parent natural
product moenomycin A shows low-nanomolar inhibition of both
enzymes. Second, we measured binding of 2 to PGTs based on
fluorescence polarization (FP) readout (Figure 2a) and obtained
KD values ranging from 0.15 to 0.38 μM (75 nM 2) for enzymes
from three different pathogens (E. coli, S. aureus, and E. faecalis;3i
Figure 2b). Lastly, we verified that 2 can be displaced from S.
aureus SgtB by moenomycin (1) and disaccharide 8, which is a
weak inhibitor of PGTs (Figure 2c). Similar behavior was seen
when E. coli PBP1b and E. faecalis PBP2a were used. In contrast,
no significant drop in FP was observed when the detergents
Tween-20 and dodecyl maltoside were used instead of
moenomycin. Likewise, using bovine serum albumin instead of
PBPs did not result in a significant change of FP.4
In order to assess the potential of trisaccharide phospho-
glycerate 2 to be used as a probe in the proposed displacement
assay, we examined its ability to bind to bacterial PGTs using
three different methods. First, using a well described biochemical
assay that monitors PGT formation in vitro,3b,10 we showed that
Taken together these results suggest that 2 binds to PGTs at
the same site as moenomycin A (1), i.e., the active site of PGTs.
Furthermore, we had successfully attenuated the affinity of the
B
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Journal of the American Chemical Society
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Figure 3. Hit compounds found in a screen against S. aureus SgtB also
inhibit PGTs of other pathogens. (a) Molecular structure of the
characterized hit compound 10 (b) Dose-dependent displacement of
probe 2 (75 nM) from PGTs of three pathogens by 10 was determined
by FP readout. (c) Inhibition of the polymerization of lipid II by PGTs
of different organisms. The following concentrations of enzymes were
chosen so that 30−40% conversion of lipid II (4 μM) was observed in
the uninhibited reaction: S. aureus SgtB, 50 nM; S. aureus PBP2, 1.2
μM;3d E. faecalis PBP2a, 50 nM; and E. coli PBP1b, 50 nM. (d)
Minimum inhibitory concentrations of 10 against S. aureus ATCC
29213 (MSSA), S. aureus USA 300 (MRSA), and B. anthracis ANR-1
were determined following the CLSI procedure.20
Figure 2. Probe 2 can be used in fluorescence polarization assays to
screen for PGT inhibitors. (a) Schematic representation of the
fluorescence polarization (FP) assay. (b) Probe 2 binds to PGTs as
observed by an increase of FP of 2 (75 nM) when exposed to PBP1b (E.
coli), PBP2a (E. faecalis), and SgtB (S. aureus); KD(E. coli PBP1b) = 0.15
μM; KD(E. faecalis PBP2a) = 0.38 μM; KD(S. aureus SgtB) = 0.18 μM.
(c) Probe 2 (75 nM) is displaced from S. aureus SgtB (0.2 μM) by
addition of either moenomycin (1) or the weaker PGT inhibitor 8, as
evidenced by reduction of FP. Ki(1) = 0.64 μM; Ki(8) = 3.17 μM. mP,
millipolarization; KD, dissociation constant; Ki, inhibitor constant.
showed similarly pan-selective inhibitory activity in an
established assay that monitors PGT activity in vitro.3b,10
Polymerization of lipid II19 by two PGTs of S. aureus, as well
as PGTs of E. faecalis and E. coli was inhibited with IC50 values in
the low micromolar range (Figure 3c). These findings indicate
that hit compound 10, identified in our screen using S. aureus
SgtB, is able to inhibit not only the enzymatic activity of S. aureus
PGTs but also the activity of PGTs from other pathogens.
In this paper we have described the development of a
displacement assay that uses a truncated moenomycin analogue
with attenuated binding affinity to enable the identification of
compounds that bind to PGTs. By using a natural product based
probe that contacts invariant residues in the active site, we hoped
to discover compounds that likewise contact conserved active
site features. We report a compound that binds to several
different PGTs and also inhibits their enzymatic activities. The
enzyme binding affinities do not allow accurate prediction of the
order of IC50 values for enzyme inhibition, but it is worth noting
that the conformations of PGTs in the process of polymerizing
lipid II may be different from those bound to moenomycin. This
phenomenon highlights the need for both enzyme inhibition and
binding assays to guide the development of PGT inhibitors.
Finally, we note that compound 10 has MICs of 4−16 μg/mL
against methicillin-sensitive and -resistant S. aureus strains as well
as B. anthracis,21 consistent with the PGT inhibitory activity
(Figure 3d). Further studies are underway to determine whether
the mechanism of cell killing is due to PGT inhibition.
probe compound to PGTs so that we could identify PGT
inhibitors with low micromolar potency in a FP-based displace-
ment assay (cf. 8, IC50(E. coli PBP1b) = 12 μM).17 At the same
time, probe 2 binds tightly enough to PGT enzymes so that the
amount of protein required for the assay is limited.
With the assay in hand, we set out to screen for new structural
classes of inhibitors using S. aureus SgtB. This enzyme is from a
relevant pathogen, accessible in sufficient quantities by
heterologous expression in E. coli (7 mg/L culture), and it can
be obtained as a well-behaved, stable monomer. We adjusted the
assay to a 1536 well plate format and screened 110,000
compounds of the ICCB library collection at Harvard Medical
School (Z′ = 0.78). Wells that showed 90% reduction of FP in
duplicate (as compared to controls) were scored as hits. The
initially obtained 186 hits (hit rate = 0.17%) were retested with
the same displacement assay, this time measuring a dose−
response curve rather than an end point. About 47% of primary
hits showed dose-dependent displacement of the probe and were
reconfirmed; 21% of the initial hits were found to be fluorescent
and were not further evaluated.
The PGT active site is conserved throughout prokaryotes, and
the assay is based on probe displacement from the active site.
Thus, hit compounds obtained in this screen, which uses S. aureus
SgtB, may not only show binding affinity to S. aureus PGTs but
also to PGTs of other organisms, provided they contact
conserved active site features. To examine their selectivity, hit
compounds were tested for their PGT inhibitory activity in two
orthogonal assays. In competitive binding studies, using PGTs
from the pathogens S. aureus, E. faecalis, and E. coli, we found that
hit compound 10 displaces probe 2 in a dose-dependent fashion
from all three enzymes. The corresponding inhibitor constants Ki
ranged from 2.6 μM to 94 nM (Figure 3).18 Compound 10
ASSOCIATED CONTENT
■
S
* Supporting Information
Rational probe design, PBP/PGT homology, and experimental
details. This material is available free of charge via the Internet at
http://pubs.acs.org.
C
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Journal of the American Chemical Society
Communication
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AUTHOR INFORMATION
■
Corresponding Author
suzanne_walker@hms.harvard.edu; kahne@chemistry.harvard.
edu
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
(R01GM066174 to D.K., R01GM076710 to S.W and D.K.,
1P01AI083214 to S.W.), a fellowship for C.M.G. within the
Postdoc-program of the German Academic Exchange Service
(DAAD), and a research fund of the New England Regional
Center for Excellence in Biodefense and Emerging Infectious
Disease Research (U54 AI057159). We are grateful to Drs.
Qasba and Ramakrishnan (NIH) for providing us with GalT. Dr.
B. Kraybill (Merck & Co., Inc.) is acknowledged for helpful
discussions. We thank the staff of the ICCB/NERCE BEID at
Harvard Medical School (Dr. S. Chiang, D. Flood, J. Nale, Dr. R.
Ross, B. Seiler, Dr. A. Onderdonk, C. Anderson) for their help
with setting up and carrying out the screening campaign. We
acknowledge Pfizer for their donation of the overexpression
plasmid for S. aureus SgtB.
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