BG108399A - Antibacterial agent - Google Patents

Abstract

The invention relates to the use of substances, binding to the bacterial translation factor EF-Tu, to prevent the formation of a cytoskeleton in bacterial cells and for production of anti-bacterial agents. The invention further relates to anti-bacterial agents, comprising partial sections of the amino acid sequences of the domains 2 and 3 of a bacterial EF-Tu protein, preferably with a length of 4-20 aminoacids.

Description

The invention relates to an antibacterial agent. More specifically, it relates to the use of substances that bind to the bacterial translational factor EF-Tu to inhibit cytoskeletal formation in bacterial cells and to produce an antibacterial agent. The invention further relates to an antibacterial agent comprising fragments of the amino acid sequence of domains 2 and / or 3 of a bacterial EF-Tu protein of length, preferably of 4-20 amino acids.

BACKGROUND OF THE INVENTION

Currently, antibacterial agents include, among others, penicillin or other antibiotics that have a specific inhibitory effect on growing bacterial cells. This action is based on the fact that these antibiotics impede the necessary growth of the peptidoglycan skeleton of the growing cells. Through this destabilization of murine, the growing cells are decisively damaged. The bacteria in the stationary phase are not inhibited as there is no increase in the murine skeleton in this phase.

The bacterial protein EF-Tu contains domains 1, 2 and 3 (Song, H., Parsons, M. R., Rowell, S., Leonard, G., Phillips, E. V., J. Mol. Biol. 285, 12451256, 1999). The EF-Tu protein sequences and the coding genes have been published for Escherichia coli and a number of other eubacteria and are available in the database. It also describes that the EF-Tu domain 1 plays a role in protein synthesis.

The possible presence of a prokaryotic permanent cytoskeleton has been discussed in Naturwissensch. 85, 1998, 278-282 (Mayer et al.).

Ho, there is currently no known involvement of the bacterial protein EFTu in the construction of such a cytoskeleton.

So far described in the literature (cf. Schilstra, MJ, Slot, JW van der Meide, PH, Posthuma, G., Cremers, AF, Bosch, L .: Immunocytochemical localization of the elongation factor Tu in Escherichia coli cells., Biochem. Biophys. Acta 1291, (1996), 122-130) in view of the arrangement of EF-Tu in bacterial cells, that EF-Tu is virtually homogeneously distributed in the cytoplasm. To date, it has not been fully taken into account that artificially produced EF-Tu fibrils in vitro can be depolymerized by cooling.

It has surprisingly been found that there is a cytoskeleton in prokaryotic cells that can be stained with anti-EP-Ti antibodies. This cytoskeleton encompasses a network of protein fibrils that extends close to the surface of the cytoplasmic membranes and passes through the cytoplasm itself. The cytoplasmic membrane and the peripheral part of this network may be regarded as two concentrically arranged hollow tubes, wherein the cytoplasmic membrane is the outer of the two tubes and the peripheral part of the network (cytoskeleton) is the inner tube. Fibrils passing through the cytoplasm complete and stabilize the system and represent binding sites for ribosomes. Ribosomes can also be demonstrated in the periphery of the cytoskeleton, oriented in the direction of the cytoplasm.

The prokaryotic cytoskeleton is manifested in several variants:

• variants that confer special functions consisting of proteins that are close to the actin of the higher cells and determine the diameter and length of the cells in rod-shaped bacteria;

• variants composed of proteins that are close to the tubule of the higher cells, which take care of the proper cell division; and • a common variant (“major cytoskeleton”) occurring in all prokaryotes, consisting of a network of protofibrils of the EF-Tu protein (prolonging factor Ti), which serves as a structural element that stabilizes the cell shape, as well as binding structure for ribosomes and other complex molecular aggregates. The latter variant is also referred to here as the cytoskeletal network.

EF-Tu is a protein containing three domains, in which domain 1 is involved in translation. So far, no specific functions have been described for domains 2 and 3. It has now been found that the laterally exposed epitopes of domains 2 and 3 create alignment where one surface is concave and the other is convex. It is recognized that these combinations could induce the formation of EF-Tu-polymers, in particular linear, stacked fibrils, both in vitro and in vivo. These fibrils are elements of the network that acts as a cytoskeleton. Hence, it can be concluded that substances that bind to EF-Tu, especially in the domain of domains 2 and / or 3, can inhibit cytoskeletal formation in bacterial cells and thus serve to produce an antibacterial agent.

Therefore, the cytoskeletal network can be used as an attack target for a new class of antibiotics.

In particular, EF-Tu may serve as a target protein for novel bacterial agents that may occupy the blend sites of domains 2 and / or 3 and thereby impede the formation of EF-Tu polymers in cells that are at the core of bacterial cell structure.

This mode of action differs radically from the mode of action of other antibiotics acting on EF-TU (cf. eg Vogeley, L., Palm, GJ, Mesters, JR, Hilgenfeld, R .: Conformational change of elongation factor TU (EF-TU) induced by antibiotic binding. J. Biol. Chem. 276 (2001), 17149-17155). There, it has been shown that the previously known antibodies of the cyromycin (Kirromycin) type act in such a way that they prevent the reversibility of a conformational change in domain 1, whereby domain 1 bends in the direction of domain 2 when GTP is coupled. This mechanism is fundamentally different from the polymerization inhibition mechanism described herein, in which domains 2 and 3 are included.

EF-Tu covers 394 amino acids. Domain 1 belongs to amino acids 8-204, wherein amino acids 172-204 create a binding structure to domain 2. Domain 2 belongs to amino acids 205-298 and domain 3 belongs to amino acids 299-394.

Different secondary structures appear in domains 2 and 3. Of particular interest are amino acid sequences 317 to 328 and 343 to 354, which reside in domain 3 and form bands that extend freely in space and are candidate sequences for interaction with other amino acid sequences located in a correspondingly located a recess in the periphery of the domain

2, these sequences span amino acids 218 to 224.

According to the invention, it has surprisingly been found that in the bacterial cytoskeleton, cell damage can generally be achieved by inhibiting the polymerization of EF-TU. Such cell damage can also be achieved with normal bacterial cells that have a cell wall. The invention is directed specifically to eubacteria.

The substances used to inhibit cytoskeletal formation may be of a different nature as long as they are able to inhibit the interaction between domains 2 and 3 of two adjacent EF-Tumolecules. Suitable substances can be identified, for example, by a method including:

(a) contacting the substance to be tested with bacterial EF-Tu or a polymerizable fragment thereof, e.g. with a fragment comprising domains 2 and 3 and (b) determining whether this substance can inhibit the formation of EF-Tu-polymers.

This method can be performed both in vivo and in vitro. In the in vitro method, it is preferable to purified EF-Tu molecules, respectively. suitable fragments thereof to be incubated under conditions under which fibril formation occurs. The action of a test substance on the formation of fibrils can be determined by a simple method, e.g. by immunological staining with a labeled anti-EF-Tu antibody or by using EF-Tu-molecules having a labeled group, e.g. fluorescently labeled group. Of course, the method can also be carried out in vivo, where the action of adding the test substance to the fibril network in a cell can be determined by immunological methods, e.g. immunohistochemically with labeled anti-EF-Tu antibodies and microscopically evaluated.

Substances that inhibit the formation of EF-Tu polymers and which can be prepared as described above, as well as those resulting therefrom, e.g. by empirically preparing derivatives and / or by computer modeling, may be incorporated into the dosage form, optionally together with conventional pharmaceutical carriers, excipients and / or diluents.

The pharmaceutical composition can be in the form of a liquid or solid preparation, emulsion or dispersion. Administration may be, depending on the form, by injection, oral, rectal, nasal, topical, etc. The dosage will depend on the active ingredient, the route of administration, and the type and severity of the disease and will be selected to allow effective control of the bacterial infection.

The action of the antibacterial agent may be of a different nature. On the one hand, substances that can bind directly to the EF-Tu domains of domains 2 and / or 3 will be used. On the other hand, those that will bind to other parts of the EF-Tu molecule may be used, which will have an inhibitory effect on the alignment and thus interfere with the binding of the fibrils.

In a preferred embodiment of the invention peptide antibacterial agents are used. The peptide agents are based on oligopeptides that bind to EF-Tu, preferably in the region of the overlapping portions of domains 2 and / or 3. These oligopeptides may comprise fragments of amino acid sequences of domains 2 and / or 3, of length preferably from 4 to 20 amino acids, from 5 to 15 amino acids is preferred and from 6 to 12 amino acids is particularly preferred. These fragments are able to bind to complementary sequences of other domains, i. E. the sequences of domain 2 are able to bind to domain 3, and the sequences of domain 3 can bind to domain 2.

In another preferred embodiment of the invention, the substances that bind to EF-Tu contain fragments of amino acid sequences from domain 2 of at least 4 in length, in particular, at least 5 amino acids, and in particular domains 2 of amino acids 218 to 224, and at the same time, no fragment corresponding to the amino acid region 317 to 328 and / or the amino acid region 343 to 354 of EF-Tu domain 3. Accordingly, substances which contain fragments of amino acid sequences of domain 3 with a length of at least 4 amino acids, in particular at least 5 amino acids, are particularly preferred, especially those with at least 6 amino acids and which do not include fragments, corresponding to amino acids 218 to 224 of domain 2. Such fragments may be truncated EF-Tu consisting only of domain 3, without domains 1 and 2, or only domains 1 and 2 without domain 3. One such EF -Tu-segment competes in the cell with its own synthesized veins, natural EF-Tu-protein molecules, and tracks, during its formation, the chain of polymerization protofilament breaks, since it lacks the second necessary for chain extension. This way no more intact network is created. This is equivalent to a loss of bacterial cell viability. A disturbance in networking in a bacterial cell has a negative effect on the shape and behavior of this cell, which we have been able to experimentally establish. The negative effect on the shape and behavior of the cell is evidence of the expected cell death that occurs with the antibiotic administration of the invention.

An antibiotic may be used instead of the truncated "EF-Tu-sections" described, in which the polymerization of the EF-Tu protein molecules, i.e. circuit breakage can be achieved by other means, e.g. from the presence of fragments that will interfere with the attachment of other EF-Tu-protein molecules.

One particular advantage of the antibiotics of the present invention is that there is little risk of bacterial resistance to these new classes of antibiotics. Resistance will mean that the bacterium will break down a peptide that has penetrated inside the cell. If this happens, the bacterium will not be able to avoid its structurally identical peptide, which is an integral part of the cellular EF-Tu protein and which is of great importance for translation, to be destroyed together with it.

Antibacterial agents may include linear or cyclic peptide compounds or peptidomimetics. The peptide compounds may be composed of natural L-? -Amino acids but, on the other hand, may also be composed of other amino acids, e.g. of D-α-amino acids, azaamidic acids, β-amino acids, non-genetically encoded L- and / or D-α-amino acids, etc., and combinations thereof. The preparation of peptidomimetic compounds is described, e.g. in RIPKA, A.S., RICH, D.H. (1998) Peptidomimetic design, Curr. Op. Chem. Biol. 2, 441452.

In addition, peptide or peptidomimetic compounds may contain coupled hydrophobic groupings that facilitate transfer through cytoplasmic membranes or larger-filled groupings that will interfere with the attachment of other EF-Tu molecules and thus the formation of polymerization products. In addition, antibacterial agents may have groups with a protective function against degradation.

Antibacterial agents can be used against any prokaryotic organisms and archebacteria, especially against pathogens. Both Gram-positive, Gram-negative and mycoplasma bacteria have a cytoskeleton based on EF-Tu, which can be used to combat the agent of the invention. For example, antibacterial agents may be used against vancomycin-resistant embryos such as staphylococci.

For the new classes of antibiotics, this opens up a wide range of applications. These regions, which are responsible for the binding of monomers in protofilament formation, have been found to be very similar in all tested bacteria in terms of their amino acid sequence. EF-Tu is very well preserved in this area. Also, the distances between these regions in an EF-Tumolecule, i. the distances of the exposed regions of domains 2 and 3, expressed by the number of amino acids, are identical, with 126 amino acids always present between the preserved regions.

The antibiotics of the invention are highly specific and especially with very limited side effects. Large EFTu sequences are not found in human cells except in mitochondria. The mitochondrial EF-Tu-like sequences are protected against the antibiotic by the double membrane of the mitochondria.

The invention is further explained by the following examples and drawings.

In FIG. 1 shows the macromolecular structure of the bacterial protein EF-Tu, in which domains 1, 2 and 3 are more clearly indicated.

This bacterial protein, EF-Tu, can bind in periodically constructed fibrils upon polymerization, as shown in FIG. 2.

In FIG. 3 is a schematic representation of the polymerization in which (+) or (-) denotes the reactive constituent regions of domains 2 and 3.

FIG. 4 is an enlargement (magnification of about 1.5 million) of electron microscopic images of polymerized in vivo isolated fibrils of EF-Tu protein molecules. Above the dotted line are the domains 1, below them the stacked domains 2 and 3.

When the polymerization of the EF-Tu protein in the designations of FIG. 3 with (+) and (-) regions of attachment is suppressed by the addition of excess particles containing fragments of amino acid sequences of domains 2 or 3, which will render the cell affected unable to revive because the cellular structure disintegrates.

EXAMPLES FOR THE IMPLEMENTATION OF THE INVENTION

Example:

Studies have been conducted with the Gram-positive bacterium Thermoanaerobacterium thermosaccharolyticum EM1 (hereinafter referred to as EM1) and the non-bacterial bacterium Mycoplasma pneumoniae (hereinafter referred to as Mp) to show that these bacteria have a permanent cytoskeleton on the basis of EF There.

Studies cover the identification and cellular localization of candidate proteins for such a bacterial cytoskeleton using antiactin antibodies (obtained against actin of higher cells) whose more or less strong cross-activity with bacterial proteins is due to the fact that the bacteria have proteins belonging to the actin superfamily, without showing apparent sequential homology to the higher cell actin. Prokaryotes have no expressed actin genes.

In addition to immune electron microscopy with the antibodies mentioned above, ultra-thin sections of bacteria also used the Whole mount staining technique. Combining these techniques, it has been found that a network of protein fibrils, whose components cross-react with anti-actin antibodies, extends into the surface of the cytoplasmic membrane located close to the cytoplasm. The cytoplasmic membrane and the peripheral part of this network formally form two concentric hollow tubes, the cytoplasmic membrane being the outer of these tubes and the peripheral part of the network (cytoskeleton) the inner. Fibrils passing through the cytoplasm complete and stabilize the system and are the starting point for the capture of ribosomes. The ribosomes also attach to the peripheral part of the cytoskeleton, oriented in the direction of the cytoplasm.

Using a French press (French-Press), cells of EM1 are destroyed and the resulting material (soluble fraction, particle fraction) is performed by sodium dodecyl sulfate gel electrophoresis (SDSE) and Western blotting. The sodium dodecyl sulfate gel contained several defined zones, one of which (at about 43 kDa) could be stained with both anti-actin antibodies and anti-EF-Tu antibodies obtained against EF-Tu from Mr. This zone appears pronounced where, as a material for sodium dodecyl sulfate electrophoresis, the particulate fraction obtained by low-speed centrifugation is used. Anti-EF-Tu antibodies will be used because EF-Tu is usually found at 43 kDa (it represents up to 9% of the bacterial protein mass), because EF-Tu belongs to the actin superfamily and because EF-Tu is found in more in the prokaryotic cell.

The role of EF-Tu as a building block for the bacterial cytoskeleton is new. From this characteristic of EF-Tu, as a building block in a complex network, such as the cytoskeleton, it can be concluded that for this purpose the bacterial cell must contain a large amount of protein. A comparison between the structure of this bacterial cytoskeleton and that of the higher cells clearly shows that the bacterial cytoskeleton can also be made up of more proteins. EF-Tu is the main component. In higher cells, EF-Tu is not actually involved in the construction of the cytoskeleton (the higher cells do not contain EF-Tu), but they are known to contribute a large number of different proteins to the cytoskeleton.

We have been shown to show that the reacting components of the cytoskeleton network above with the anti-actin antibodies under section and the Whole mount method also react intensely with anti-EF-Tu antibodies.

This Mp reaction takes place on entire surfaces that have been exposed by treatment with Triton (removal of cytoplasmic membranes), now already exposed outward but which were initially covered. Controls obtained using cells that were not treated with newt but underwent all other treatments, i. E. who have not lost their cytoplasmic membranes show no marking. In this control experiment, the cytoplasmic membrane conceals potential binding sites for EF-Tu.

The surface revealed by removal of the cytoplasmic membrane is the peripheral part of the cytoskeleton of the cell. In this situation, the internal structures of the cell, e.g. ribosomes that have been shown to be binding sites during translation are also binding sites of the ancillary EFTu function (EF-Tu participates with its domain 1). From this fact, it was concluded that during translation, EF-Tu does not move to the ribosome, and conversely, the ribosome moves to EF-Tu, which, as a component of the cytoskeleton, is spatially fixed to the periphery of the cell and to cytoplasmic fibrils directly through the cytoplasm.

The presence of a permanent bacterial cytoskeleton could be further demonstrated in the eubacteria Escherichia coli, Bacillus sp., Ralstonia eutropha and Thermoanaerobacterium thermosulfurigens, as well as in the archaea Methanococcus jannaschii and Methanococcus voltae.

Claims (22)

1. Use of substances that bind to EF-Tu to inhibit cytoskeletal formation in bacterial cells. Use according to claim 1, characterized in that the substances bind in the region of domain 2 (amino acids 205-298) and / or domain 3 (amino acids 299 to 394) of EF-Tu. Use according to claim 1 or 2, characterized in that the substances bind in the region of amino acids 218 to 224 of EF-Tu domain 2. Use according to claim 1 or 2, characterized in that the substances bind in the region of amino acids 317 to 328 and / or 343 to 354 of EF-Tu domain 3. Use according to any one of claims 1 to 4, characterized in that the substances comprise fragments of amino acid sequences of domains 2 and / or 3 having a length of from 4 to 20 amino acids. Use according to claim 5, characterized in that the fragment is from 5 to 15 amino acids in length, in particular from 6 to 12 amino acids. Use according to any one of claims 1 to 6, characterized in that the substances are selected from linear or cyclic peptide compounds or peptidomimetics. Use according to claim 7, characterized in that the peptide compounds or peptidomimetics comprise coupled hydrophobic groups, larger groups and / or groups with a protective function against destruction. Use according to any one of claims 1 to 8 for the preparation of an antibacterial agent. Use according to claim 9, characterized in that the antibacterial agent is included in a pharmaceutical composition, optionally in admixture with conventional pharmaceutical carriers, diluents or auxiliary materials. Use according to claim 9 or 10 for the preparation of a Gram-positive or Gram-negative bacterial agent. Use according to claims 9 or 10 for the preparation of an antimycoplasma agent. An antibacterial agent comprising fragments of the amino acid sequences of domains 2 and / or 3 of the bacterial protein EFTu with a length of 4 to 20 amino acids. The antibacterial agent of claim 13, comprising fragments of 5 to 15 amino acids in length. The antibacterial agent of claim 13 or 14, comprising fragments of 6 to 12 amino acids in length. Antibacterial agent according to claims 13 to 15, characterized in that it contains fragment bound hydrophobic groups, larger groups and / or groups with a protective function against degradation. An antibacterial agent according to any one of claims 13 to 16, in the form of a pharmaceutical composition containing optionally the usual pharmaceutical carriers, diluents and / or excipients. 18. A method for identifying novel antibacterial active ingredients, comprising: (a) contacting one of the test substances with bacterial EF-Tu or with a polymerizable fragment thereof and (b) determining whether the substance can inhibit the formation of EFTu-polymers. A method according to claim 18, wherein the assay is performed in vitro. 20. The method of claim 18, wherein the assay is performed in vivo. A method according to any one of claims 18 to 20, characterized in that labeled antibodies and / or labeled EF-Tu proteins or fragments thereof can be used to determine the formation of EF-Tu polymers. are polymerized. A method according to any one of claims 18 to 20, characterized in that a pharmaceutical composition is obtained from a substance that inhibits the formation of EF-Tu polymers or from a derivative thereof, optionally by the addition of conventional pharmaceutical carriers , diluents and / or excipients. MODIFIED PATENT CLAIMS SUBMITTED TO THE INTERNATIONAL OFFICE ON 04.04.2003 Use of substances that bind to EF-Tu to produce a cytoskeleton-inhibiting agent in bacterial cells, wherein the substances bind to EF-Tu in domain 2 (amino acids 205-298) and / or of domain 3 (amino acids 299 to 394). Use according to claim 1, characterized in that the substances bind to EF-Tu in the region of amino acids 218 to 224 of domain 2. Use according to Claims 1 or 2, characterized in that the substances bind to EF-Tu in the region of amino acids 317 in to 328 and / or 343 to 354 of domain 3. Use according to any one of claims 1 to 3, characterized in that the substances contain fragments of amino acid sequences of domains 2 and / or 3 having a length of from 4 to 20 amino acids. Use according to claim 4, characterized in that the fragments are from 5 to 15 amino acids in length, in particular from 6 to 12 amino acids. Use according to any one of claims 1 to 5, characterized in that the substances are selected from linear or cyclic peptide compounds or peptidomimetics. Use according to claim 6, characterized in that the peptide compounds or peptidomimetics comprise coupled hydrophobic groups, larger groups and / or groups with a protective function against degradation. Use according to any one of claims 1 to 7 for the preparation of an antibacterial agent. Use according to claim 8, characterized in that the antibacterial agent is included in a pharmaceutical composition, optionally in admixture with conventional pharmaceutical carriers, diluents or auxiliary materials. Use according to claims 8 or 9 for the preparation of a Gram-positive or Gram-negative bacterial agent. Use according to claims 8 or 9 for the preparation of an antimycoplasma agent. An antibacterial agent comprising fragments of amino acid sequences of domains 2 and / or 3 of the bacterial protein EF-Tu with a length of from 4 to 20 amino acids. The antibacterial agent of claim 12, comprising fragments of 5 to 15 amino acids in length. The antibacterial agent of claim 12 or 13, comprising fragments of 6 to 12 amino acids in length. An antibacterial agent according to claims 12 to 14, characterized in that it contains fragment bound hydrophobic groups, larger groups and / or groups with a protective function against degradation. The antibacterial agent of any one of claims 12 to 15, in the form of a pharmaceutical composition, optionally comprising the usual pharmaceutical carriers, diluents and / or excipients. 17. A method for identifying novel antibacterial active ingredients, comprising: (a) contacting one of the test substances with bacterial EF-Tu or with a polymerizable fragment thereof and (b) determining whether the substance can inhibit the formation of EFTi-polymers. The method of claim 17, wherein the assay is performed in vitro. A method according to claim 18, wherein the assay is performed in vivo. A method according to any one of claims 17 to 19, characterized in that labeled antibodies and / or labeled EF-Tu proteins or fragments thereof can be used to determine the formation of EF-Tu polymers. are polymerized. 21. A method according to any one of claims 17 to 20, characterized in that a pharmaceutical composition, optionally containing conventional pharmaceutical carriers, diluents, is obtained from a substance that inhibits the formation of EF-Tu polymers or from a derivative thereof. and / or excipients.