The mechanism of action of ramoplanin and enduracidin
Xiao Fang,adKittichoat Tiyanont,bdYi Zhang,aJutta Wanner,cDale Bogercand Suzanne Walker*d
Received 28th October 2005, Accepted 16th November 2005
First published as an Advance Article on the web 29th November 2005
The lipoglycodepsipeptide antibiotic ramoplanin is proposed to inhibit bacterial cell wall
biosynthesis by binding to intermediates along the pathway to mature peptidoglycan, which
interferes with further enzymatic processing. Two sequential enzymatic steps can be blocked by
ramoplanin, but there is no definitive information about whether one step is inhibited
preferentially. Here we use inhibition kinetics and binding assays to assess whether ramoplanin
and the related compound enduracidin have an intrinsic preference for one step over the other.
Both ramoplanin and enduracidin preferentially inhibit the transglycosylation step of
peptidoglycan biosynthesis compared with the MurG step. The basis for stronger inhibition is a
greater affinity for the transglycosylase substrate Lipid II over the MurG substrate Lipid I. These
results provide compelling evidence that ramoplanin’s and enduracidin’s primary cellular target is
the transglycosylation step of peptidoglycan biosynthesis.
Peptidoglycan is a crosslinked carbohydrate polymer that
surrounds bacterial cells and prevents them from rupturing
under high internal osmotic pressures. Because peptidoglycan
is essential for survival and has no eukaryotic counterpart,
peptidoglycan biosynthesis (Fig. 1) is the target of a large
number of clinically used antibiotics, including the b-lactams,
cephalosporins, and glycopeptide antibiotics.1The emergence
of resistance to all these classes of antibiotics represents a
significant threat to public health and has stimulated efforts to
develop structurally novel antibacterial agents that inhibit the
peptidoglycan biosynthetic pathway. One molecule that has
received considerable attention in recent years is ramoplanin
(Fig. 2, 1), a lipoglycodepsipeptide antibiotic discovered in the
1980s in a screen for peptidoglycan synthesis inhibitors.2,3
Ramoplanin has good activity against a wide range of Gram-
positive organisms and is regarded as a promising candidate
for the treatment of many Gram-positive infections.4It is
currently in late stage clinical trials for two different
indications.5,6Due to hydrolytic instability and other issues,
however, neither of these indications involves systemic
administration of ramoplanin, and the full potential of this
compound has yet to be realized.4A better understanding of
the mechanism of action of ramoplanin may enable the
development of derivatives to treat systemic infections.
A mechanism of action for ramoplanin was first proposed in
1990 by Somner and Reynolds, who showed, using a cell-free,
particulate membrane assay, that the antibiotic blocks the
MurG-catalyzed conversion of Lipid I to Lipid II (Fig. 1) on
the biosynthetic pathway to peptidoglycan.7Although no
direct evidence for an interaction with Lipid I was presented,
these authors suggested that ramoplanin kills bacterial cells by
binding to this substrate, rendering it inaccessible to MurG.7A
decade later, also using a cell-free, particulate membrane
system, we showed that ramoplanin inhibits the transgly-
cosylase-catalyzed coupling of Lipid II molecules to form the
carbohydrate chains of peptidoglycan.8We established that
ramoplanin binds to synthetic Lipid II analogues, and so we
proposed that ramoplanin acts primarily by binding to Lipid II
and inhibiting the transglycosylation step of peptidoglycan
Our hypothesis that the primary mechanism of action of
ramoplanin involves binding to Lipid II and inhibition of
transglycosylation rather than binding to Lipid I and inhibition
of MurGrestedlargely onthe factthat Lipid IIistranslocated to
the external surface of the bacterial membrane as soon as it is
produced whereas Lipid I remains on the internal surface of the
membrane.10Ramoplanin is a large and highly water-soluble
molecule, and in the absence of a dedicated transport mecha-
nism, it seemed improbable that it could diffuse readily through
bacterial membranes to reach an intracellular target. In fact,
Somner and Reynolds made this point in their mechanistic
papers on ramoplanin, but when they did their studies it was not
known that Lipid I and MurG are intracellular.7,10
Comparative information on how well ramoplanin and
various analogues inhibit MurG and the bacterial trans-
glycosylases could provide more insight into the mechanism of
action of the molecule. We have developed synthetic routes to
Lipid I and Lipid II substrates and have developed assays to
study E. coli MurG and E. coli PBP1b, the major bacterial
transglycosylase in this organism.9,11,12These tools enable us
to carry out the studies required to assess the importance of the
different proposed targets of ramoplanin and structurally
related compounds. Below we report a comparative analysis of
aDepartment of Chemistry and Chemical Biology, Harvard University,
12 Oxford St., Cambridge, MA 02128, USA.
E-mail: firstname.lastname@example.org.; Tel: 617-432-5498
bDepartment of Chemistry, Princeton University, Princeton, NJ 08544,
cDepartment of Chemistry and The Skaggs Institute for Chemical
Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
dDepartment of Microbiology and Molecular Genetics, Harvard Medical
School, Harvard University, Boston, MA 02115, USA.
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other than the similarities in residues 3–8 may explain the
observation that ramoplanin and enduracidin behave similarly
with respect to ligand binding. For example, conformationally
defined backbone interactions requiring the full structure may
play the primary role in the recognition of Lipid I and Lipid
II.32Breukink and coworkers have reported that the lantibiotic
nisin, which also binds to Lipid II, uses primarily backbone
contacts in complexation, and they have identified a ‘‘pyro-
phosphate cage’’ as a key binding element.33Structural
studies of enduracidin and ramoplanin show that the back-
bones of these two molecules are very similar.24,25The
backbone conformations are determined by the number and
chirality of the amino acids in the macrocycles rather than by
the side chain identities, and it is quite possible that most of
the side chains are individually unimportant for binding.
Alanine scanning experiments on the ramoplanin structure
should shed more light on the importance of each particular
amino acid side chain in substrate binding and biological
activity, and changes in chirality can provide insight into the
importance of the backbone conformation in binding.
Having established a convergent synthetic route to the
ramoplanin aglycon analogues that enables such a detailed
examination of the importance of each residue,28,32,34–36and
with suitable assays in place to probe Lipid II vs. Lipid I
binding, transglycosylase vs. MurG inhibition, and biological
activity, we should be able to determine the essential
requirements for binding and biological activity, and we may
be able to identify analogues with more desirable properties to
use as antibiotics.
We compared the inhibitory behavior of ramoplanin and
two structurally related compounds, enduracidin and the
ramoplanin aglycon, with respect to two enzymes involved in
the late steps of peptidoglycan biosynthesis. We have shown
that the ramoplanin aglycon and enduracidin inhibit these two
enzymes, MurG and the bacterial transglycosylase PBP1b, by
binding to their substrates, Lipid I and Lipid II, respectively.
The inhibition kinetics shows that both compounds bind more
tightly to Lipid II than to Lipid I. We measured the Kds of
ramoplanin for fluorescent analogues of Lipid I and Lipid II
and found that Lipid II binds about ten-fold more tightly than
Lipid I. Based on our studies, we conclude that the primary
mechanism of action of both ramoplanin and enduracidin
involves inhibition of the transglycosylation step of peptido-
This work is supported by NIH grants AI50855 (to SW) and
CA41101 (to DLB). We thank Oscient Pharmaceuticals for a
generous gift of ramoplanin.
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