Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Antibacterial Agent
Description
The invention concerns the use of substances which bind to the bacterial
translation
factor EF-Tu for inhibiting the formation of a cytoskeleton in bacterial cells
and for
producing antibacterial agents. The invention also concerns antibacterial
agents
which contain partial sections of the amino acid sequences of the domains 2
or/and 3
of a bacterial EF-Tu protein preferably having a length of 4-20 amino acids.
Penicillin or other antibiotics which have a specific inhibitory effect on
growing
bacterial cells have, among others, previously been used as antibacterial
agents. This
effect is based on an inhibition by these antibiotics of the extension of the
peptido-
glycan skeleton that is necessary for cell growth. Growing cells are
considerably
weakened by this destabilization of the murein. Bacteria in the stationary
phase are
not inhibited because the murein skeleton is not extended in this phase.
The bacterial protein EF-Tu contains the domains l, 2 and 3 (Song, H.,
Parsons,
M.R., Rowell, S., Leonard, G., Phillips, E.V., J. Mol. Biol. 285, 1245-1256,
1999).
The sequences of the protein EF-Tu and its encoding gene have been published
for
Escherichia coli and a number of other eubacteria and are accessible in
databases. It
has also been described that the domain 1 of EF-Tu plays a role in protein
synthesis.
The possible existence of a permanent prokaryotic cytoskeleton has been
discussed in
Naturwissensch. 85, 1998, 278-282 (Mayer et al.). However, an involvement of
the
bacterial protein EF-Tu in the formation of such a cytoskeleton was unknown.
With regard to the location of EF-Tu in bacterial cells, it has previously
been
assumed in the literature (cf. e.g. Schilstra, M.J., Slot., J.W. van tier
Meide, P.H.,
Posthuma, G., Cremers, A.F., Bosch, L.: Immunocytochemical localization of the
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elongation factor Tu in Escherichia coli cells., Biochem. Biophys. Acta 1291,
(1996),
122-130) that EF-Tu is distributed almost homogeneously in the cytoplasm.
However, previous experiments did not take into account the fact that
artificially
produced EF-Tu fibrils can be depolymerized in vitro by low temperatures.
It was surprisingly found that a cytoskeleton exists in prokaryotic cells
which can be
stained with anti-EF-Tu antibodies. This cytoskeleton comprises a network of
protein
fibrils which are located near to the surface of the cytoplasmic membrane that
faces
the cytoplasm and they extend through the cytoplasm. The cytoplasmic membrane
and the peripheral part of this network can be regarded as two concentric
hollow
tubes where the cytoplasmic membrane represents the outer of the two tubes and
the
peripheral part of the network (cytoskeleton) represents the inner tube.
Fibrils
running through the cytoplasm complement and stabilize the system and are
attachment sites for ribosomes. Ribosomes have also been detected in the
peripheral
part of the cytoskeleton oriented towards the cytoplasm.
Hence the prokaroytic cytoskeleton has several variants:
- Variants which mediate special functions consisting of proteins that are
similar to
the actin of higher cells and which, in the case of rod-shaped bacteria,
define the
length and diameter of the cell,
- variants which consist of proteins that are similar to the tubulin of higher
cells and ensure controlled cell division and
- a variant which occurs generally in all prokaryotes (basic cytoskeleton)
which
consists of a network of protofilaments of the protein EF-Tu (elongation
factor
Tu) which the cell uses as form-stabilizing structural elements and which act
as
an attachment structure for ribosomes and other complex molecular aggregates.
The last variant is also referred to herein as a cytoskeletal network.
EF-Tu is a protein which contains three domains of which domain 1 is involved
in
the translation process. Up to now no specific function has been described for
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domains 2 and 3. It has now been found that the laterally exposed epitopes of
domains 2 and 3 form a fit in which one surface is convex and one surface is
concave. It is assumed that these fits can result in the formation of EF-Tu
polymers
and especially a linear arrangement of fibrils in vitro as well as in vivo.
These fibrils
are the components of the network which act as a cytoskeleton. Hence
substances
which bind to EF-Tu especially in the region of domains 2 or/and 3 could be
used to
inhibit the formation of a cytoskeleton in bacterial cells and thus to produce
an
antibacterial agent.
Hence the cytoskeletal network could thus be used as a target for a new class
of
antibiotics.
In particular EF-Tu can be used as a target protein for new bacterial agents
which can
occupy the fitting sites of domains 2 or/and 3 and thus prevent the formation
of EF-
Tu polymers in the cell which are essential for the structure of the bacterial
cell.
This mode of action is fundamentally different from the mode of action of
other
antibiotics acting on EF-Tu (cf. e.g. Vogeley, L., Palm, G.J., Mesters, J.R.,
Hilgenfeld, R.: Conformational change of elongation factor Tu (EF-Tu) induced
by
antibiotic binding. J. Biol. Chem. 276 (2001), 17149-17155). This publication
shows
that the action of previously known antibiotics of the kirromycin type is due
to the
fact that they prevent the reversibility of a conformational change of domain
1
resulting in a bending of domain 1 towards domain 2 when GTP is bound. This
mechanism is fundamentally different from the mechanism of action described
herein
of an inhibition of polymerization in which domains 2 and 3 are involved.
EF-Tu comprises 394 amino acids. Amino acids 8-204 belong to domain 1 and
amino acids 172-204 form a linking structure to domain 2. Amino acids 205-298
belong to domain 2 and domain 3 comprises the amino acids 299-394.
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Different secondary structures occur within domains 2 and 3. In this context
the
amino acid sequences of 317 to 328 and of 343 to 354 which are located in
domain 3
are of particular interest since they form loops which protrude freely into
space and
are candidate sequences for an interaction with amino acid sequences which are
located in a depression on a corresponding position on the periphery of domain
2
where these sequences extend from amino acid 218 to 224.
It was surprisingly found according to the invention that in the case of the
bacterial
cytoskeleton it is basically possible to damage the cells by inhibiting the
polymerization of EF-Tu. In particular such cell damage is also achieved in
common
bacterial cells which have a cell wall. The invention is particularly
applicable to
eubacteria.
A wide variety of substances can be used to inhibit the formation of
cytoskeletons
provided they are able to inhibit the interaction between domain 2 and domain
3 of
two neighbouring EF-Tu molecules. Suitable substances can for example be
identified by a method comprising:
(a) contacting a substance to be tested with bacterial EF-Tu or with a partial
fragment thereof capable of polymerization such as a fragment which contains
the domains 2 and 3 and
(b) determining whether the substance can inhibit the formation of EF-Tu
polymers.
This method can be carried out in vitro as well as a vivo. In an in vitro
method
purified EF-Tu molecules or suitable partial fragments thereof are preferably
incubated under conditions in which fibril formation can take place. The
effect of a
test substance on fibril formation can be determined in a simple manner for
example
by means of immunological staining using labelled anti-EF-Tu antibodies or by
using
EF-Tu molecules which carry a marker group e.g. a fluorescent marker group. Of
course the method can also be carried out in vivo in which case the effect of
adding a
test substance on the fibril network in a cell can be determined by
immunological
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methods e.g. immunohistochemically using labelled anti-EF-Tu antibodies and
microscopic evaluation.
Substances which inhibit the formation of EF-Tu polymers and which can be
obtained by the method described above as well as substances derived therefrom
e.g.
by means of empirical derivatizadon orland by computer modelling, can be
formulated as a pharmaceutical composition optionally together with common
pharmaceutical vehicles, auxiliary substances or/and diluents.
The pharmaceutical composition can for example be present as a liquid
preparation,
solid preparation, emulsion or dispersion. Depending on the preparation it can
be
administered by injection or orally, rectally, nasally, topically etc. The
dosage is
selected depending on the active substance, the form of administration and the
type
and severity of the disease, such that it is possible to combat bacterial
infections.
The antibacterial agent can have a variety of effects. On the one hand
substances are
used which can bind directly to the fitting sites of domains 2 or/and 3 of EF-
Tu. On
the other hand it is also possible to use substances which bind to other
positions on
the EF Tu molecule but have an inhibitory effect on the fitting and thus
preventing
fibril formation.
Peptidic, antibacterial agents are used in a preferred embodiment of the
invention.
The peptidic agents are based on oligopeptides which bind to EF-'I~z
preferably in the
region of the sites of fit of domains 2 or/and 3. These oligopeptides may
contain
partial sections of the amino acid sequences of domains 2 or/and 3 having a
length of
preferably 4 to 20 amino acids, particularly preferably 5 to 15 amino acids
and
especially preferably having a length of b-12 amino acids. These partial
sections are
able to bind to complementary sequences of the other domain i.e. sequences
from
domain 2 are able to bind to domain 3 and sequences from domain 3 are able to
bind
to domain 2.
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In another preferred embodiment the substances which bind to EF-Tu contain a
partial section of the amino acid sequences from domain 2 having a length of
at least
4 and in particular of at least 5 amino acids, in particular partial sections
in the region
of domain 2 of amino acids 2I8 to 224 and at the same time no section which
corresponds to the region of amino acids 317 to 328 or/and the region of amino
acids
343 to 354 of domain 3 of EF-Tu. Alternatively substances are preferred which
contain partial sections of the amino acid sequences from domain 3 having a
length
of at least 4 amino acids, in particular of at least 5 amino acids and
particularly
preferably of at least 6 amino acids and no partial sections corresponding to
amino
acids 218 to 224 of domain 2. Such sections can for example be a truncated EF-
Tu
which is composed only of domain 3 without domains 1 and 2 or only of domains
1
and 2 without domain 3. Such an EF-Tu fragment competes in the cell with the
natural EF-Tu protein molecules synthesized by the cell and results in chain
termination when it is incorporated into the polymerizing protofilament since
in each
case the second domain required for chain extension is absent. As a result an
intact
network is no longer formed. This is synonymous with the loss of viability of
the
bacterial cell. A disorder in the development of the network in the bacterial
cell has
an adverse effect on the shape and behaviour of the bacterial cell as
demonstrated by
experiments. The adverse effect on the shape and behaviour or the cell
indicates the
expected cell death which occurs when the antibiotic according to the
invention is
used.
Instead of the described truncated EF-Tu fragment, it is also possible to use
an
antibiotic which prevents the polymerization of EF-Tu protein molecules i.e. a
chain
termination, by other means for example due to the presence of sections which
prevent binding of further EF-Tu protein molecules.
A particular advantage of the antibiotics according to the invention is that
there is
only a slight risk of the development of bacterial resistance to this new
class of
antibiotics. A resistance would mean that the bacterium would degrade the
peptide
that has been transferred to the inside of the cell. If this were to occur,
the bacterium
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would not be able to avoid also degrading its own structurally identical
peptide which
is a component of the cell's EF-Tu protein and which is of major importance
for
translation.
The antibacterial agents can comprise linear or cyclic peptidic compounds or
peptidomimetics. Peptidic compounds can be composed of natural L-a-amino
acids,
but also other amino acids e.g. D-a-amino acids, azaamino acids, ~3-amino
acids,
non-genetically coded L- or/and D-a-amino acids etc. or combinations thereof.
The
preparation of peptidomimetics is described for example in RIPKA, A.S., RICH,
D.H. (1998) Peptidomimetic design, Curr. Op. Chem. Biol. 2, 441-452.
In addition the pepddic compounds or peptidomimetics may contain bound
hydrophobic groups which facilitate transfer through the cytoplasmic membrane
or
very bulky groups which prevent the attachment of further EF-Tu molecules and
thus
prevent the formation of a polymerization product. The antibacterial agents
may also
carry groups which protect against degradation.
The antibacterial agents can be used against any prokaryotic organisms and
archaea
and especially pathogenic organisms. Gram-positive bacteria, Gram-negative
bacteria
and mycoplasma have a cytoskeleton based on EF-Tu and can therefore be
combated
by the agent according to the invention. For example antibacterial agents
against
vancomycin-resistant microorganisms e.g. staphylococci can be used.
Hence the new class of antibiotics has a wide spectrum of applications. It was
found
that the regions which are responsible for binding the monomers to form the
protofilaments, have a very similar amino acid sequence in all examined
bacteria.
EF-Tu is highly conserved in this region. Also the distances between these
regions in
a given EF-Tu molecule i.e. the distances between the exposed regions of
domains 2
and 3 are identical in terms of the number of amino acids and there are always
126
amino acids between the conserved regions.
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The antibiotics according to the invention are characterized by a high
specificity and
especially by very low side-effects. Large EF-Tu sequences do not occur in the
human cell apart from in mitochondria. The mitochondria) EF-Tu-like sequences
are
substantially protected from the antibiotic by the double membrane of the
mitochondria.
The invention is illustrated by the following figures and examples.
Figure 1 shows the macromolecular architecture of the bacterial protein EF-Tu
in
which domains l, 2 and 3 are described in more detail. This bacterial protein
EF-Tu
can associate during polymerization to form periodically structured fibrils as
shown
in figure 2.
Figure 3 shows a schematic representation of the polymerization at the
reactive
binding regions of domains 2 and 3 labelled with + or -.
Figure 4 shows an enlargement (enlargement: ca. 1.5 million) of an
electronmicrograph of an in vivo polymerized isolated fibril from EF-Tu
protein
molecules. The domains 1 are located above the dotted line and the juxtaposed
domains 2 and 3 are below.
If the polymerization of the EF-Tu protein is repressed at the binding regions
labelled
with + and - in figure 3 by adding an excess of particles containing partial
sections of
the amino acid sequence of domains 2 or 3, the affected bacterial cell is
unable to
survive because the cell structure breaks down.
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Example:
Experiments were carried out on the Gram-positive bacterium
Thermoanaerobacterium thermosaccharolyticum EM1 (abbreviated EMl in the
following) and the bacterium Mycoplasma pneumoniae (abbreviated Mp in the
following) which lacks a cell wall proving that these bacteria have a
permanent
cytoskeleton that is based on EF-Tu.
The experiments included the identification and cellular localization of
candidate
proteins for such a bacterial cytoskeleton by using anti-actin antibodies
(prepared
against actin of higher cells) which cross-react to a greater or lesser extent
with
bacterial proteins due to the fact that bacteria are known to have proteins
which
belong to the actin super family without having conspicuous sequence
homologies
with actin of higher cells. Prokaryotes do not have distinct actin genes.
In addition to immunoelectron microscopy with the aforementioned antibodies on
ultrathin sections through bacteria, whole mount techniques were also used. It
was
found by a combination of these techniques that a network of protein fibrils
is located
near to that surface of the cytoplasmic membrane which faces the cytoplasm and
extend through the cytoplasm. The components of these fibrils cross-react with
the
anti-actin antibodies. The cytoplasmic membrane and the peripheral part of
this
network form two concentric hollow tubes where the cytoplasmic membrane forms
the outer of the two tubes and the peripheral part of the network
(cytoskeleton) forms
the inner part. The fibrils extending through the cytoplasm complement and
stabilize
the system and are attachment sites for ribosomes. Ribosomes also sit on the
peripheral part of the cytoskeleton oriented towards the cytoplasm.
The cells of the EM1 cells were disrupted with the aid of a French press and
the
material obtained (soluble fraction, particulate fraction) was subjected to
SDS gel
electrophoresis and Western blotting. Several defined bands were obtained in
the
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SDS gel of which one band (at about 43 kDa) could be stained with anti-actin
antibodies as well as with anti-EF-Tu antibodies obtained against the EF-Tu of
Mp.
This band was particularly pronounced where the particulate fraction of cell
lysis
obtained by low-speed centrifugation had been used as the material for SDS gel
electrophoresis. Anti-EF-Tu antibodies were used because EF-Tu is usually
found at
43 kDa (comprises about 9 % of the protein mass of a bacterium), because EF-Tu
belongs to the actin superfamily and because EF-Tu occurs in large amounts in
a
prokaryotic cell.
The role of EF-Tu as a structural component of a bacterial cytoskeleton is
new. This
property of EF-Tu as a structural component of a complex network like that of
the
cytoskeleton infers that the bacterial cell has to use a large amount of
protein for this
purpose. A comparison of the structure of this bacterial cytoskeleton with
that of
higher cells makes it clear that the bacterial cytoskeleton must also be
composed of
several types of protein. EF-Tu is a major component. Although in higher cells
EF-
Tu is not involved in the formation of the cytoskeleton (the higher cell has
no EF-
Tu), it is, however, known that a large number of different proteins
contribute to the
formation of the cytoskeleton.
In the section and in the whole mount it was possible to show that components
of the
above-mentioned network of the cytoskeleton that react with anti-actin
antibodies
also intensively reacted with anti-EF-Tu antibodies.
This reaction occurred in the case of Mp with the entire originally covered
surface
that was exposed to the environment by Triton treatment (removal of the
cytoplasmic
membrane). A control that was prepared using cells which were not treated with
Triton but otherwise treated identically and thus had not lost their
cytoplasmic
membrane, exhibited no labelling. Hence in this control experiment the
cytoplasmic
membrane masked the potential binding sites for EF-Tu.
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The surface exposed by removing the cytoplasmic membrane is the peripheral
part of
the cytoskeleton of the cell. This, however, does not expose inner cell
components
such as ribosomes which have been shown to be the attachment sites for EF-Tu
which perform a helper function during translation (domain 1 of EF-Tu acts in
this
case). As a result it was concluded that during the course of translation EF-
Tu does
not go to the ribosome but the ribosome goes to EF-Tu which, due to its
property as a
component of the cytoskeleton, is spatially fixed on the cell periphery and on
fibrils
that run crosswise through the cytoplasm.
The occurrence of a permanent bacterial cytoskeleton was also detected in the
eubacteria Escherichia coli, Bacillus sp., Ralstonia eutropha and
Thermoanaero8acterium thermosulfurigenes and in Archaea Methanococcus
jannaschii and Methanococcus voltae.
