WO2004015416A2

WO2004015416A2 - Methods for the identification of agents with anti-microbial action

Info

Publication number: WO2004015416A2
Application number: PCT/EP2003/008145
Authority: WO
Inventors: Gerald Kleymann; Hans-Otto Werling
Original assignee: Bayer Healthcare Ag
Priority date: 2002-08-06
Filing date: 2003-07-24
Publication date: 2004-02-19
Also published as: DE10235967A1; DE10235967B4; AU2003255282A1; WO2004015416A3

Abstract

The invention relates to a novel method and the use thereof for the discovery of novel anti-microbial agents, characterised in that potential agents are simultaneously incubated with eukaryotic cells and non-viral microbes in a test composition, the viability of the eukaryotic cells is determined and subsequently those agents are selected for which the signal for the corresponding test composition is significantly larger than the mean value of the infection control at at least one concentration of the agent.

Description

Method of identifying substances with anti-microbial activity

A goal of inventions in the pharmaceutical industry is to provide the healthcare system with compatible medication and therapy options for treating patients.

This invention describes a method for the identification of anti-infectively active substances for the treatment of intra- or extracellular, non-viral microbial infections. The method is based on a combined analysis of the anti-microbial effect and the tolerance of the agent in viti-o. In particular, this assay allows the characterization of an anti-microbial agent with respect to the anti-microbial effect, the inhibition of the cytopatic effect which the pathogen exerts on the infected culture, a possible cytotoxicity and protective effects on the infected cell culture.

In the field of anti-infective research, many anti-microbial agents have been identified using a sensitivity test in which potentially active compounds are tested for growth inhibition against pathogens (Principles and Practice of Infectious Diseases, GL Mandall, RG Douglas, JE Bennett ed., Churchill Livingstone Inc ., 1995). Historically, natural products have often been the basis for numerous developments in the field of anti-microbial agents. The identified compounds are usually chemically optimized and tested in secondary test systems with regard to compatibility, the so-called selectivity index in vitro. The compounds are then tested in animal models and active substances with a suitable pharmacokinetic and pharmacodynamic profile, which also have a corresponding therapeutic index, are selected for clinical studies in humans, provided that toxicological studies do not find any prohibitive results.

With the publication of numerous bacterial genome sequences, new ones

On the way to discovering anti-microbial drugs, it began so-called era of genome research. (Novel approaches to the discovery of anti-microbial agents, Schmid MB, Current Opinion In Chemical Biology, 2, 4, 529-534, 1998). Strategies for the efficient use of genomic information were developed to identify and characterize targets, to develop test systems and to evaluate substances. Theoretically, the number is antibacterial

Targets for drug discovery have skyrocketed, but even though these targets and assay systems are well characterized, the identified compounds must continue to meet the criteria listed above that are essential for successful medication.

The NCCLS committee (National Committee for Clinical Laboratory Standards, USA) publishes standardized guidelines for the MIC test (minimum inhibitory concentration, or MIC test, minimum inhibitory concentration), which is based on solid or liquid media.

This standard sensitivity test is exactly in "NCCLS, Villanova, PA, 1997. Methods for dilution anti-microbial susceptibility tests for bacteria that grow aerobically 5 ^' ed. Approved Standard. NCCLS Document M7-A5 (Vol. 20 No.2), M11-A4 (Vol 17 No 22) ". In principle, the multiplication of the pathogen, in this case bacteria, on solid media (growth of the culture ) or in liquid media (turbidity in the medium) in the presence or absence of potential active substances after a defined period of time and the sensitivity of the pathogen to potential active substances is determined At this point, however, note that only a fraction of the compounds identified in this way have the desired selectivity index or the necessary compatibility in downstream, additional test systems.

Bedard J et al. (Antiviral Research; 41; 1; 35-43; 1999 Feb; 9910) describes a colorimetric cell proliferation test to identify compounds with Activity against human cytomegahevirus (HCMV), in which the cell, virus and compound are incubated simultaneously. This method is not readily transferable to non-viral systems because other microbes may have mitochondria or enzyme complexes by means of which the dye used WST (tetrazolium salt WST-1) can be converted.

The aim of this invention is to provide a method which makes it possible to find alternative or more active non-viral, anti-microbial compounds which have a better selectivity index and / or tolerance and / or protective effects on the infected eukaryotic cell culture.

Another object of this invention is to provide a robust, inexpensive, sensitive and efficient method by which alternative, new or more potent anti-microbial substances can be identified.

Another object of this invention is to provide a method that is compatible with high throughput test methods.

Another aspect of the present invention is to provide a method by which substances can be identified which have a broad-spectrum activity or an activity against therapy-resistant pathogens.

The present invention solves the problems listed above by providing a method for finding active substances against non-viral microbes, characterized in that, in a test batch, potential active substances are simultaneously incubated together with eukaryotic cells and with non-viral microbes, the vitality of the eukaryotic cells are determined and then those active substances are selected in which the signal of the corresponding test batch is significantly larger than the mean value of the infection control at at least one concentration of the active substance. For the first time, this method allows a combined assessment of both the antimicrobial activity of the compound and, at the same time, its tolerance (selectivity index) and its cytoprotective effects on an infected cell culture. From a different perspective, it is precisely this in vitro assay

Configuration that simulates the natural process of infection very well, while the widespread MIC test only tests the sensitivity of the microbes to substances. Consequently, the infectiological assay according to the invention allows an efficient screening of substance libraries and the subsequent optimization of the identified anti-microbial active substances, because a reproducible and

Dose-effect-dependent test, which identifies anti-microbial activities of substances with predominantly suitable tolerability, ultimately represents the simplest stage in vitro of a sensible evaluation of substances in relation to their anti-microbial potential.

A further advantage of this invention is that this infectiological in vitro method not only simultaneously tests all of the pathogen's targets that are essential for microbial growth and infection, but also the targets of the eukaryotic host cells that are relevant for possible therapeutic intervention in the almost natural infection process without, for example like with

Genome approach to preselect individual targets based on current knowledge based on more or less scientifically correct criteria. The infectiological assay therefore provides answers to the question of whether there are structures that can be optimized, are tolerable and that are anti-microbial and that inhibit the occurrence of infection. Once a suitable substance has been identified, the underlying mechanism of action or the target can be quickly clarified using the available methods. With this procedure, the previously necessary identification, evaluation and testing of numerous individual targets can become redundant sequentially or in parallel. The assay clearly differs from all publications published to date. Numerous assays have been described in various modifications to identify bacteriostatic or bactericidal compounds, but these test systems are basically all based on the growth inhibition of the pathogen in the presence of the active ingredient or the test substance. Other tests test the inhibition or activation of a single target of the pathogen in question, which may be selected, criticized and expressed based on theoretical considerations of genome research, possibly purified in the case of a protein and analyzed in an enzyme test or binding test. But these strategies do not simultaneously provide the selectivity index or do not evaluate the tolerability of the active ingredients in vitro.

Precisely because these tests only check the reduction in bacterial growth or the change in the binding behavior or the enzymatic activity of a single target in the assay system, these tests identify not only the desired anti-microbial compounds with respect to the development of a drug, but to a large extent cytotoxic and cytostatic active substances, which require numerous downstream test systems in order to filter out compatible active substances from the undesired cell-toxic substances.

The term microbe is a collective term and stands for a wide variety of microorganisms including bacteria, viruses, protozoa and fungi etc.

The term signal describes any form of tapping information from the test batch which is suitable for evaluating the anti-microbial activity and tolerance of the potential active compounds, e.g. a signal can be referred to as a signal, which corresponds to the vitality of the cells and thus indicates the anti-microbial activity of the test substance and its tolerance.

The term prokaryote describes an organism that, unlike eukaryotic cells, has no nucleus. In this context, it also includes genetically engineered prokaryotes, such as those that produce a reporter gene product. The term eukaryotic cell describes an organism that, unlike prokaryotes, has a nucleus. In this context, it also includes genetically engineered eukaryotes, such as those that produce a reporter gene product.

The abbreviation MIC is a synonym for the German name MHK (Minimum Inhibitory Concentration) stands for "Minimum Inhibition Concentration" and describes the lowest concentration of a compound that is still able to completely inhibit bacterial growth, for example, with reference to visual turbidity the

Incubation media (described in National Committee for Clinical Laboratory Standards, Villanova, PA, 1997. Methods for dilution anti-microbial susceptibility tests for bacteria that grow aerobically 4 ed. Approved Standard. NCCLS Document M7-A4).

The terms Gram-positive and Gram-negative describe the result of the so-called Gram staining, which is only one of a large number of published staining methods for microorganisms to examine samples from organisms. (Principles and Practice of Infectious Diseases, GL Mandall, RG Douglas, JE Bennett ed., Churchill Livingstone Inc., 1995)

The abbreviation moi stands for "multiplicity of infection" and is the quotient of the cell number of the pathogen divided by the cell number of the cells to be infected. (MOI = cell number of the pathogen / number of cells that are infected).

The term selectivity index (SI) refers to the quotient of the concentrations at which the cell vitality is reduced to 50% compared to the cell control, divided by the concentration at which the anti-microbial effect reaches at least 50% of a maximum possible inhibition of the pathogen multiplication, ie SI = (CC ₅₀ / IC ₅₀ ). The larger this number, the greater the compatibility of the connection. Compounds with SI values greater than 10 are preferred. The term IC ₅₀ or EC ₅₀ describes the concentration or amount of active ingredient that is required to achieve 50% inhibition in a given test system or the effective concentration that is required to achieve a half-maximum signal in an assay.

The term simultaneous with reference to the infectious assay refers to a situation in which the active substance, microbe and eukaryotic cells are simultaneously incubated in one solution at a time; however, the sequence of adding the components is variable.

The term therapeutic index (TI; tolerance) refers to a quotient of the lethal dose (LD; lethal dose) at which 50% of the test animals die due to the substance effects and the effective dose (ED; effective dose) at 50% of the test animals Infection survive TI = (LDso / ED ₅ o).

The term inhibit, when used in connection with the infectious assay, generally describes the inhibition of microbial growth by approximately 50% at a concentration of IC ₅₀ = 100 μM or less and preferably a concentration that is as low as possible to to achieve the MIC value.

The term vital dyes refers to dyes that allow a distinction to be made between vital and non-vital cells, e.g. by only penetrate into non-vital cells (tryp blue) or are only converted by intact cells (e.g. fluorescein diacetate). Fluorescent dyes are preferred.

The term significance expresses the probability with which you can be sure that the positive test result did not come about by accident.

In one embodiment, the method is characterized in that

Active substances are selected for which the signal of the corresponding test rate is greater than 2 times, particularly greater than 4 times, very particularly greater than 10 times the mean value of the infection control and in particular greater than 50% of the cell control.

In a further embodiment, the method is characterized in that the non-viral microbes are prokaryotic cells. The prokaryotic cells are preferably bacteria, in particular intracellular and or therapy-resistant microbes.

Another preferred embodiment of the invention is characterized in that vital dyes are used. Fluorescent dyes, in particular fluorescein diacetate, fluorescein dibutyrate, 5-carboxyfluorescein diacetate, acetoxymethyl ester (5-CFDA, AM) and 4,5,6,7-tetrafluorofluorescein diacetate (TFFDA) are preferred.

A preferred embodiment of the invention is characterized in that eukaryotic cells are used which have a so-called reporter gene for generating the test signal.

A preferred embodiment of the invention is characterized in that the non-viral microbes, such as genetically engineered organisms, have non-naturally occurring properties. For example, For example, microbes can be used which have a changed adhesion behavior on cell surfaces or which produce special toxins.

A preferred embodiment of the invention is characterized in that the eukaryotic cells are Vero (african green monkey kidney cells) or CHO cells (chinese hamster ovary cells).

Another preferred embodiment of the invention is characterized in that a mixture of different microbial strains is used. So can Test substances are tested directly for possible anti-microbial broadband activity.

In a further preferred embodiment, the method is characterized in that a mixture of various eukaryotic cell lines is used.

In this way, in addition to the anti-microbial activity of active substances, their compatibility can be checked simultaneously against various eukaryotic cells. It is also possible to use different eukaryotic cells that carry the same or different so-called reporter genes, in order to draw conclusions about the target itself, in addition to the compatibility aspect.

In a further embodiment, the invention relates to compounds or active ingredients which have been identified by means of the above-mentioned method, to pharmaceutical compositions which contain a compound found in this way, which

Use of these substances for the manufacture of medicaments for the treatment and prevention of microbial infections and for the treatment of microbial infections in mammals by administering a therapeutically effective dose of the pharmaceutical composition to the mammal in need of such therapy.

In a further preferred embodiment, the method is characterized in that the compounds are selected which have a selectivity index of greater than or equal to 10 (selectivity index = CC ₅₀ / IC ₅ Q> = 10).

In a further preferred embodiment, the method is carried out as high-throughput screening (HTS) at a concentration of the active substance in the concentration range from 100 nM to 100 μM.

In a further embodiment, compounds are preferred which have a so-called broad-spectrum spectrum anti-microbial effect. Particularly preferred are active substances whose mechanism of action is based on a target of the pathogen or pathogen.

In a further preferred embodiment, the method is characterized in that those active substances are selected which have the property of inhibiting microbial growth in cell culture at a concentration of approximately 100 μM and less by at least 50%.

The present invention therefore relates to a method for identifying active substances, characterized in that

a) test substances or control antibiotics are presented at a suitable substance concentration; b) appropriate eukaryotic cells are added; c) microbes are added; d) the assay is incubated; e) a read out or a so-called test signal is generated.

This will be explained in more detail below, without thereby restricting the invention:

Step a)

If only a concentration of the test substance is used in carrying out the assay, such as in a high-throughput test, any concentration is suitable at which there is anti-microbial activity and a certain tolerance to the eukaryotic cells. A concentration of about 100 μM or less or at least in the range of the MIC concentration can be used, a concentration of about 10 μM is preferred. If a concentration gradient is used to show a dose dependency of the antimicrobial effect and a tolerance to eukaryotic cells over a wide concentration range, concentrations of 250 μM to less than 1/10 (one tenth) of the MIC concentration or less are usually used.

Step b)

Eukaryotic cells which optionally carry a reporter gene or mixtures of the corresponding cells or cell lines can be used. Any one

The number of cells per area can be sown provided that a reasonable quotient of the signals from the cell control and the infection control is achieved. The number of cells sown should not exceed an amount which leads to so-called contact inhibition of the cells during the incubation period; furthermore, cell vitality should not be impaired by the resulting cell metabolism products or nutrient deficiency. Suitable cell numbers allow a clear differentiation between possible cytostatic or cytotoxic effects of the test substances and the contact inhibition or nutrient deficiency as described above.

For example, 20,000 eukaryotic cells per well of a 96-well MTP are preferred.

Step c)

Microbes that may have been genetically manipulated, as well as intracellularly or extra-cellularly growing microbial cells or mixtures thereof, are added to the test in the desired multiplicity of infection (moi). It is necessary to have a microbial cell count that exerts a cytopathic effect on the eukaryotic cells directly or indirectly during the incubation period, so that a suitable measurement signal is generated after the corresponding incubation period. A moi of 10 to 0.001 is preferred. Step d)

A microtiter plate, which consists of a mixture of the assay components, namely the test substance, the eukaryotic cells and the microbes, is incubated under the optimal conditions for the growth of eukaryotic cells for a corresponding period of time in order to generate a suitable measurement signal.

Steps)

The cell culture supernatant is optionally removed, the remaining eukaryotic

If necessary, cells are washed and a suitable dye or the appropriate reagent for generating the signal in the case of the reporter gene-expressing eukaryotic cells is added to generate the measurement signal, which is recorded with the aid of a suitable measuring device or camera system at the corresponding wavelengths.

It must be emphasized that work steps a) b) and c) are interchangeable and can even be carried out simultaneously if necessary. Ideally, suitable dyes are added in step e) without first removing the cell culture supernatant and or carrying out additional washing steps, for example this is possible if defined cell culture media are used which do not interfere with the generation of the measurement signal. In one embodiment, this can also be achieved by using reporter gene-expressing, eukaryotic cells that allow a suitable measurement signal to be recorded after the corresponding incubation time. In principle, the vitality of the cells can be measured continuously (on-line) from the time of incubation by providing the cells with a suitable reporter system (eg GFP). The infectious assay

In principle, any eukaryotic cell (including those that express a so-called reporter gene) or a mixture of the named eukaryotic cells that are able to generate a suitable test signal and the measurement. of the corresponding selectivity index allow to be used in this assay. The eukaryotic cells can be sown long before the test incubation; preference is given to adding the eukaryotic cells to a microtiter plate suitable for cell culture, which already contains an active ingredient or test substance of a suitable concentration. Then these eukaryotic cells are then infected with the corresponding inoculum (these are microbes, including those that carry a so-called reporter gene, or hybrids of the microbes that have a cytophatic effect on the infected eukaryotic cells), with a suitable ratio of the multiplicity of infection (moi) allows a significant difference in the test signal (read out) between the eukaryotic cell control or the therapy control (antibiotic control) compared to the infection control. It is clear that all three components can be mixed and dispensed on microtiter plates, but a short pre-incubation period of the eukaryotic cells before infection may allow the identification of compounds that may adhere or colonize the microbes on the surface of the eukaryotic Prevent cells and or the invasion of the microbes into the eukaryotic cells.

The test substances can be present in any concentration, ideally in an area that is not toxic to the eukaryotic cells, so that an anti-microbial effect as a result generates a positive test signal. The microbes, whether they are Gram-positive or Gram-negative bacteria or intra- or extracellularly growing microbes, are added in an infection dose of (moi about 0.001 to 10). It is possible to vary the moi over a wide range and yet a reasonable test signal can be generated, but a change in the infection dose or the moi can also affect the IC ₅₀ and or the maximum test Increase signal accordingly or shift to lower values. The assay is then incubated for a suitable period of time under the conditions necessary for cell culture, such as temperature, atmosphere, etc. Following the incubation, the microtiter plates are washed and the test signal or read out is generated. A large number of fluorescent dyes can be used for various cell lines, while cell lines with reporter genes such as luciferase, green fluorescent protein (GFP), alkaline phosphatase etc. have to be generated or acquired in individual cases; However, such embodiments can provide better test signal / background ratios or make various steps such as washing steps superfluous.

Ausftihrungsbeispiel

The assay is for Vero cells (growth in medium 199 with Earle's salts supplemented with 5% fetal calf serum (FCS) and 2 mM L-glutamine) and CHO cells (growth in RPMI-1640 medium supplemented with 2 mM L-glutamine and 10%

FCS) and also shown for the corresponding cells with reporter gene. The cells are available from ATCC (American Tissue Cell Culture) or the DSMZ (German Collection for Microorganisms and Cell Cultures). The cells constitutively expressing the reporter gene were produced using standard methods. The reporter-bearing plasmids for luciferase, aequorin and eGFP (green fluorescent protein) as well as the transfection and detection reagents of the reporter genes are available from various commercial suppliers (e.g. Clontech).

An appropriate amount of test substance is placed in an MTP, so that the final concentration for a high-throughput test is 10 μM or for a gradient

(for example 250 μM in the highest concentration and then diluted 1: 2 to 1/10 of the IC _{50 of} the test substance). The gradient test is particularly suitable for determining the selectivity index. If the substances are dissolved in dimethyl sulfoxide, the solvent should not exceed final concentrations of 1% or better than 0.5%. Then the eukaryotic cells in the corresponding

Medium was added (e.g. 20,000 cells per well of a 96 well MTP, or 5,000 cells per well of a 384 well MTP and so on and so on for 1,536 well MTP or other cell culture vessels). For infection, the microbes are added at a moi of 0.025. The assay with a total volume of 200 μl is then carried out for 2 to 3 days, in the case of Vero or CHO cells at 37 ° C. and 5%

CO ₂ , incubated. After the incubation period, the MTPs are washed once with 200 μl PBS (phosphate buffer saline) and the read out or test signal is generated by adding 200 μl 10 μg / ml fluorescein diacetate in PBS or the corresponding detection reagents for luciferase or aequorin. In the case of fluorescein diacetate, the MTP are incubated for 15-45 min at room temperature until a visually visible discoloration of the cell control and the resulting fluorescence signal is measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm in a suitable measuring device (eg Fluoroskan Ascent from Labsystems). The other test signals are generated according to the manufacturer's instructions for the detection reagents.

Table 1 shows typical standardized data of the test described above.

Table 2 shows the evaluation of the standardized data of a 96-well microtiter plate from a high-throughput screening. Two hits are marked in bold (B8 and C4), the connections of which meet the selection criteria and are examined further.

Selection criteria: The percentage value of the test batch is significantly larger than the mean value of the infection control. It is preferred if the significance value is at least 99.95%.

Tables 3 and 4 are examples of a simultaneous dose effect and tolerance test over a broad concentration gradient for determining the IC ₅₀ values and the SI.

Table 1

Normalized data of cell control = 100%, bacterial control, infection control, and therapy control are listed above.

The percentage signal = (signal of the therapy or drug test approach) / ^' (signal of the cell control) * 100

Table 3 Determination of the SI for various antibiotics

assay parameters

Germ: E. coli-Neumann; moi = 0.00125

Cells: 40,000 CHO cells

Incubation: 3 days

Read out: Fluorescein diacetate 10 μg / ml in PBS

IC ₅₀ [µg / ml] SI

Antibiotics [µg / ml] 4 2 1 0.5 0.25 0.125 0.0625 0.031 0.016 0.008

Ciprofloxacin 119% 113% 112% 117% 97% 99% 118% 108% 116% 86% <0.008> 500

Moxifloxacin 111% 105% 107% 112% 98% 95% 87% 92% 105% 1% 0.012> 333

Trovafloxacin 39% 54% 77% 105% 109% 102% 109% 83% 101% 1% 0.012 166

Cell control 97% 102% 101% 99% 102% 97% 99% 100% 101% 100%

Infection control 2.1% 1.7% 1.3% 1.9% 1.8% 1.5% 1.6% 2.0% 1.7% 1.9%

Table 4 Bestirr imunc i of the SI for various antibiotics

assay parameters

Germ: Staphylococcus aureus 133; moi = 0.00125

Cells: 40,000 CHO cells

Incubation: 3 days

Read out: Fluorescein diacetate 10 μg / ml in PBS

IC ₅₀ [µg / ml] SI

Antibiotics [µg / ml] 4 2 1 0.5 0.25 0.125 0.0625 0.031 0.016 0.008

Ciprofloxacin 110% 109% 116% 118% 110% 2% 1% 2% 1% 1% 0.2> 20

Moxifloxacin 97% 98% 106% 1 14% 104% 101% 96% 106% 1% 1% 0.02> 200

Trovafloxacin 23% 30% 71% 101% 105% 106% 107% 97% 2% 1% 0.02 ^♦ 75

Cell control 98% 101% 100% 98% 99% 98% 102% 102% 100% 101%

Infection control 2.3% 1, 5% 1, 5% 1, 3% 1, 7% 1, 6% 1.9% 2.3% 2.1% 1, 8%

Claims

Patentansprtiche

1. A method for finding active substances against non-viral microbes, characterized in that potentials are simultaneously present in a test batch

5 active substances together with eukaryotic cells and with non-viral ones

Microbes are incubated, the vitality of the eukaryotic cells is determined and then those active substances are selected in which the signal of the corresponding test batch is significantly greater than the mean value of the infection control at at least one concentration of the active substance. Fri.

2. The method according to claim 1, characterized in that active substances are selected in which the signal is greater than twice the mean value of the infection control.

15

3. The method according to claim 1 or 2, characterized in that the non-viral microbes are prokaryotes.

4. The method according to claim 3, characterized in that the 20 prokaryotes are bacteria.

5. The method according to claim 1, 2, 3 or 4, characterized in that the non-viral microbes are intracellular microbes or therapy-resistant microbes.

25

6. The method according to claim 1, 2, 3, 4 or 5, characterized in that vital dyes are used to determine the vitality of the eukaryotic cells.

30 7. The method according to claim 6, characterized in that the vital dye

Is fluorescent dye.

8. The method according to any one of claims 1 to 7, characterized in that the eukaryotic cells have a reporter gene which can be used to generate the test signal.

9. The method according to any one of claims 1 to 8, characterized in that the non-viral microbes have non-naturally occurring properties, such as e.g. genetically engineered microorganisms.

10. The method according to any one of claims 1 to 9, characterized in that CHO cells or Vero cells are used as eukaryotic cells.

11. The method according to any one of claims 1 to 10, characterized in that a mixture of different bacterial strains is used.

12. The method of any one of claims 1 to 11, characterized in that a mixture of different eukaryotic cells is used.

13. The method according to any one of claims 1 to 12, characterized in that those substances are selected which have a selectivity index = CCso / ICs _ö of at least 10.

14. The method according to any one of claims 1 to 13, characterized in that the method is carried out as high-throughput screening at a concentration of the active ingredient of between 100 nm and 100 μm.

15. Compounds found by the method according to any one of claims 1 to 15.

16. A pharmaceutical composition containing a compound according to claim 15.

17. Use of a compound according to claim 16 for the manufacture of medicaments for the treatment and prevention of bacterial infections.