Novel Antibiotics That Selectively Kill Gram-Negative Pathogenic Bacteria

Abstract

The present invention relates to pharmaceutical formulations comprising bicyclic heptapeptides for use in vertebrates or humans suffering from infections caused by Gram-negative bacteria, such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Neisseria gonorrhoeae, Chlamydia trachomatis, Shigella sonnei, Salmonella enterica Typhimurium LT2, Enterobacter spp., Bifidobacterium longum, Bacteroides fragilis, Lactobacillus reuteri, Enterococcus spp., Yersinia pestis, and many other Gram-negative bacteria.

Description

Technical Field

[0001] The present invention relates to bicyclic heptapeptide antibiotics against Gram-negative pathogenic bacteria, their derivatives, and their production (generation), as well as their use as active pharmaceutical ingredients (APIs) in medicine.

Background Art

[0002] The current need for new antibiotics is particularly urgent with respect to drug-resistant Gram-negative pathogenic bacteria. These microorganisms have a highly restrictive permeability barrier that limits the penetration of most compounds. As a result, even the most recent classes of antibiotics that act against Gram-negative bacteria were developed during the past six decades of the previous century.

[0003] It is difficult to discover compounds that act against Gram-negative bacteria. Gram-negative bacteria have an outer membrane to protect themselves from unwanted compounds. This membrane is covered by an outer membrane of negatively charged lipopolysaccharide (LPS), which serves as a barrier to large and hydrophobic compounds. The inner membrane limits the permeability by hydrophobic compounds.

[0004] As a result, the complex barrier (in addition to the outer membrane, the inner membrane) restricts all molecules, and nutrients enter through the outer membrane porins and dedicated transporters. Drugs that leak through the barrier are extruded by the transmembrane multidrug resistance pump (MDR), which recognizes amphiphilic compounds (as most drugs are) that pass through both the hydrophilic and hydrophobic regions (e.g., membranes) of the cell. Thus, there is a great need for APIs with different mechanisms of action that are not hindered by the triple defense mechanism (outer membrane, inner membrane, and MDR) of Gram-negative bacteria at the state of the art.

Summary of the Invention

[0005] Darbactin A (DAR) is a ribosomally synthesized and post-translationally modified peptide (RiPP)-type antibiotic, which was first identified from bacteria belonging to the genus Photorhabdus. In addition, the corresponding biosynthetic gene cluster (BGC) was identified and subsequently detected in several bacterial genera. DAR represents a very promising lead structure for the development of novel antibiotic therapeutics. DAR targets the outer membrane protein BamA and is thus specific for Gram-negative bacteria. Along with the demonstration of in vivo activity in a mouse infection model, this makes DAR a particularly promising candidate for future research. To improve the compound supply for future studies of DAR and to enable the production of novel derivatives, it is highly desirable to establish an efficient and versatile microbial production platform for these classes of RiPP antibiotics. Herein, the design and construction of a heterologous production and engineering platform for DAR and / or its derivatives are disclosed, which ensures production yields and facilitates structure modification approaches. The well-known useful Gram-negative bacteria Escherichia coli and Vibrio natriegens were tested as heterologous hosts. In addition, DAR-producing strains were generated, and by optimizing the expression constructs, DAR production titers were obtained that showed an approximately 10-fold increase in concentration (titer) and a 5-fold reduction in fermentation time compared to state-of-the-art product descriptions. Similarly, since only two genes are required for the heterologous production of RiPPs (darbactin and / or its derivatives), the minimal DAR BGC is identified and elucidated.

[0006] DAR is a novel antibiotic that selectively kills Gram-negative pathogens such as Acinetobacter baumannii (MIC, 8 μg / ml), Pseudomonas aeruginosa PAO1 (MIC, 2 μg / ml), Escherichia coli wild-type and MDR strains (MIC, 2 - 4 μg / ml), Klebsiella pneumoniae (MIC, 2 - 4 μg / ml), and Salmonella enteritidis (MIC, 4 μg / ml), and was discovered in Photorhabdus khanii HGB1456. Each of the other naturally occurring derivatives, such as darobactin B, and brominated variants shows activity against E. coli wild-type and MDR strains (MIC, 0.5 - 1 μg / ml), Klebsiella pneumoniae (MIC, 1 μg / ml), and Salmonella enteritidis (MIC, 1 μg / ml). It has been experimentally proven that DAR binds to BamA, a central component of the OM β-barrel assembly machinery. BamA helps with the folding and insertion of β-barrel proteins such as porins into the OM. Disruption of this chaperone-like function leads to disruption of OM formation. In addition to its good in vitro activity, DAR has shown promising efficacy in mouse sepsis and mouse thigh infection models without showing cytotoxic effects. Therefore, DAR is emerging as a promising drug lead.

[0007] The object of the present invention is to provide a medicament that is effective against Gram-negative bacteria in a novel and unexpected way and, therefore, a novel substance that can be used as a pharmaceutical active ingredient (API) of a medicament for which resistance does not exist in Gram-negative bacteria. Surprisingly, the bicyclic heptapeptides according to formula I show an interaction with a feature / cellular component and provide these positive effects: they act against the BamA chaperone and translocator, which are attractive but very rare targets. The BamA chaperone and translocator assist in the folding and insertion into the outer membrane of β-barrel proteins such as porins. BamA itself is an outer membrane β-barrel protein. Drugs in general, especially natural products, usually target enzymes with this well-defined catalytic center rather than chaperones. Darobactin is a large molecule that is probably necessary to interfere with the protein-protein binding between BamA and this substrate. The fact that the target is located on the surface solves the difficult problem of penetrating through the permeability barrier of Gram-negative bacteria, which is not necessary in the case of darobactin. There are only two essential proteins exposed on the surface of the outer membrane, BamA and LptD. Therefore, the bicyclic heptapeptides targeting BamA effectively bypass the triple defense mechanism (outer membrane, inner membrane and MDR) of Gram-negative bacteria and provide a novel mode of action regarding the activity of antibiotics.

[0008]

Chemical formula

[0009] Scheme 1: General formula I of bicyclic heptapeptides In Scheme I, the substituents and indices have the following values: R 1 , R 2 , R 3 , R 4is independently selected from the list consisting of H, -CH3, -CH2-CH2-CH2-NH-C(NH)(NH2), -CH2-CO-NH2, -CH2-CO2H, -CH2-SH, -CH2-CH2-CO2H, -CH2-CH2-CO-NH2, (1H-imidazol-4-yl)-methyl, halogenated (1H-imidazol-4-yl)-methyl, -CH(CH3)(C2H5), -CH2-CH(CH3)2, -CH2-CH2-CH2-CH2NH3, -CH2-CH2-S-CH3, -CH2-C6H5, halogenated -CH2-C6H5, (1H-indol-3-yl)-methyl, halogenated (1H-indol-3-yl)-methyl, (4-hydroxyphenyl)-methyl, halogenated (4-hydroxyphenyl)-methyl, -CH-(CH3)2, 1-hydroxy-ethyl, hydroxy-methyl, sec-butyl, 1-acetamido, 1-thioacetamido, -CH2-CH2-NH-(C=NH)-NH2, benzyl, halogenated benzyl, -CH2-CH2-CH2-NH-(C=NH)-NH2. X is, at any position of X, independently of any other position of X, either O or S. R 5 is selected from the list consisting of methylsulfonyl, p-toluenesulfonyl; R 6 is selected from the list consisting of methylsulfonyl, p-toluenesulfonyl, -(C=NH)-NH2. Z 1 Z 2 are each a double bond or a single bond, provided that Z 1 and Z 2 are both single bonds, or only one of them is a single bond while the other is a double bond, and Z 1 is a single bond and Z 2 is a double bond, or vice versa. Y 1 is 3,7-indolylene or halogenated 3,7-indolylene, and Y 2 is independently selected from the list of 3,6-indolylene, 1,4-phenoxylene, halogenated 3,6-indolylene, halogenated 1,4-phenoxylene, and n is 1 or 2.

[0010] Darobactin is a modified heptapeptide having the amino acid sequence W 1 -N 2 -W 3 -S 4 -K 5 -S 6 -F 7 NMR tests revealed two rare macrocyclic crosslinks in darobactin: an unprecedented aromatic-aliphatic ether bond between the C7 indole of W 1 and the β-carbon of W 3 and a carbon-carbon bond between the C6 indole of W 3 and the β-carbon of K 5 . The tryptophan-lysine bond is formed between two non-activated carbons, which is unique to antibiotics. This bicyclic structure is characteristic and essential for darobactin A and all derivatives of the present invention.

[0011] Direct comparison of the sequence of this 7-amino acid peptide (darobactin A) with the genome of P. temperata reveals a perfect match near the C-terminus of an open reading frame encoding a 58-amino acid long peptide. The ribosomal synthesis of darobactin suggests that the amino acid backbone has an L-configuration. The macrocyclic crosslinks generate two chiral centers at the β-carbons of W3 and K5, having R and S configurations respectively, based on NOE correlations (Figure 4). The putative operon encoding darobactin biosynthesis (Figure 2) is typical of RiPPs that encode diverse ribosomally produced natural products, including the food preservative antibiotic nisin and thiostrepton.

[0012] This dar operon consists of a propeptide encoded by darA, a small relE-type ORF that may play a role in the host's resistance to compounds, darBCD encoding an ABC-type transenvelope transporter, and darE encoding a radical SAM enzyme. Enzymes of the radical SAM class catalyze reactions based on free radicals that can ligate non-activated carbons. This accounts for the formation of the tryptophan-lysine C-C bond in darobactin and its derivatives. Such Trp-Lys C-C bonds have recently been reported in streptopeptide, a peptide pheromone of Streptococcus thermophilus. There is little overall homology between the two enzymes, but DarE contains SAM and the SPASM domain characteristic of this group. The operon does not contain an individual enzyme for creating ether bonds in the first ring. RiPP operons often encode proteases that cleave the active peptide, but this was not present in the dar operon. Thus, general proteolysis, self-cleavage, or other proteases present in the production strain may be involved in the maturation of the propeptide. Surprisingly, the DarE radical SAM enzyme appears to catalyze the formation of both the Trp-Lys C-C bond and the C-O-C Trp-Trp ether bond. The chemistries of these two reactions are quite different, and the mechanism of DarE catalysis clearly requires individual investigation. To link the putative BGC to darobactin production, the inventors generated a markerless knockout mutant in which the complete BGC darABCDE was deleted from Photorhabdus khanii DSM3369 by double crossover. DAR production was lost in the resulting mutant strain; molecules with the corresponding molecular weight could not be detected by MS (Figure 10). Importantly, darobactin was heterologously produced from the dar operon cloned into E. coli (Figure 10). This indicates that the dar operon is sufficient to produce darobactin. The inventors discovered that the dar operon is common in Photorhabdus, and they detected it in 16 different species with available genomic sequences (Figure 2a; Figure 10).The dar operon was not present only in P. bodei. Synteny between the genome containing the dar locus and the genome of P. bodei was useful for determining the operon boundaries (Figure 2a). We also tested the production of darobactin in several different Photorhabdus and found that production was highest in the strain of P. khanii DSM 3369 from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) culture collection. This indicates that the strain with the BGC is producing the compound.

[0013] The inventors next used the propeptide and the dar-coded peptide as queries to expand the search for dar-type operons in the bacterial genomic sequence database (NCBI). Two searches identified homologs of the dar operon that are likely to encode four darobactin analogs. Accordingly, the inventors proposed the name darobactin A for the first compound and darobactins B - E for the expected analogs of this class of antibiotics. In Photorhabdus australis and Photorhabdus asymbiotica, the sequence data suggest the production of darobactin B containing two amino acid changes (SKSF→TKRF) at the N-terminus. In multiple Yersinia species, either the second amino acid (N→S) or the fifth amino acid (K→R), or both, are modified. The inventors named these analogs darobactins C, D, and E. Interestingly, the darobactin C sequence is present (respectively) in Yersinia pestis, the causative agent of plague, and Y. frederiksenii from the human gut microbiome. The putative structures of darobactins B - E predicted from the amino acid sequences are shown in FIG. 10. Among the five compounds, darobactin A is the most common, and the corresponding propeptide sequence is present in nine sequenced Photorhabdus species, seven Yersinia species, Vibrio crassostreae, and Pseudoalteromonas luteoviolacea, all of which are γ-proteobacteria. Additional members of this class of antibiotics may emerge as more bacterial genomes are sequenced.

[0014] These experiments suggest that darobactin is a promising lead compound for the development of therapeutic or pharmaceutical agents against Gram-negative pathogenic bacteria. The experimental results presented herein indicate that the bicyclic heptapeptide derived from DAR is an effective compound (API) for pharmaceutical agents against Gram-negative pathogenic bacteria. Pharmaceutical agents containing darobactin and / or the bicyclic heptapeptide are effective in vertebrates such as birds, fish, amphibians, reptiles and mammals, as well as humans, suffering from infectious diseases caused by Gram-negative bacteria. Gram-negative bacteria are selected from the group of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Neisseria gonorrhoeae, Chlamydia trachomatis, Shigella sonnei, Salmonella enterica Typhimurium LT2, Enterobacter cloacae, Bifidobacterium longum, Bacteroides fragilis, Lactobacillus reuteri, Enterococcus faecalis, and Yersinia pestis. This group is not meant to limit the scope of the present invention, and many other Gram-negative bacteria belonging to this group, such as Pseudomonas, fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Enterobacter aerogenes, Enterobacter spp., Klebsiella oxytoca, SerratiaSerratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus also exist.

[0015] To produce and generate bicyclic heptapeptides and derivatives, the inventors selected several different heterologous hosts, cloned each DAR BGC from different species containing the upstream region of the BGC, and finally created DAR-resistant heterologous hosts to enhance DAR production.

Brief Description of the Drawings

[0016]

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Mode for Carrying Out the Invention

[0017] The following embodiments of the present invention are examples of preferred embodiments and are not meant to limit the scope of the present invention in any way. It will be apparent to those skilled in the art that microbial strains similar to the strains disclosed herein can also be used without departing from the scope of the present invention. The same applies to the chemical modifications, steps of the manufacturing process, and pharmaceutical formulations and methods of application presented in the following specification.

[0018] (General description of the fermentation of Photorhabdus species for the production of bicyclic heptapeptides) Inoculate a strain of Photorhabdus species into a suitable growth medium (e.g., 3 mL of lysogeny broth: 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl) and incubate to promote growth. Use an aliquot of this preculture to inoculate the main culture (e.g., LB medium) in an Erlenmeyer flask and incubate to promote growth until harvest.

[0019] (General description of the fermentation of Pseudoalteromonas strains for the production of bicyclic heptapeptides) Cultivate a Pseudoalteromonas strain containing a biosynthetic gene cluster corresponding to the expression of a bicyclic heptapeptide (e.g., composed of 8 genes encoding a propeptide, a modified radical SAM enzyme, and an FAD-dependent halogenase, 1 gene of unknown function, and 4 transporter genes), such as Pseudoalteromonas luteoviolacea H33 and H33S, in a suitable medium. Suitable media include, for example, marine broth, or a buffered artificial seawater formulation, a suitable carbon source such as sugars (e.g., glucose, rhamnose), a suitable nitrogen source such as ammonium-containing salts (NH4Cl), or a similar medium containing a complex carbon-nitrogen source such as casitone or yeast extract. Incubate the culture for 1 - 7 days at a suitable temperature (4°C - 40°C) in a volume of 20 mL - 2 L, with or without shaking (or with or without shaking / mixing in a production-scale fermenter), and with or without the addition of a small molecule inducer such as an N-acyl homoserine lactone derivative. Separate the culture medium from the cells, for example, by centrifugation, and further process the clarified medium for purification. From BGCs having an FAD-dependent halogenase in addition to the Photorhabdus BGC and strains having 1 gene of unknown function, halogenated (e.g., Cl, Br, I, F) and non-halogenated bicyclic heptapeptides, and variants with or without additional double bonds (e.g., dehydro derivatives) can be isolated. To generate halogenated derivatives, a suitable enzyme can be used in vivo or in vitro as the FAD-dependent halogenase encoded in the BGC of Pseudoalteromonas luteoviolacea H33 and H33S. As is known to those skilled in the art, the position of the halogen within the aromatic ring can be varied by using different halogenases that catalyze halogenation at specific positions.

[0020] Similarly, it is also well known in the art that there are also naturally occurring polyhalogenated (e.g., polybrominated, polychlorinated) compounds, such as those produced by marine organisms. Accordingly, the scope of the present invention clearly includes polyhalogenated bicyclic heptapeptides. Scheme 1 shows the aromatic and heteroaromatic partial structures of bicyclic heptapeptides that can be mono- or polyhalogenated.

[0021]

Chemical formula

[0022] Scheme 2: Possible halogenated partial structures IIa - IIf of the bicyclic heptapeptide according to formula I, wherein the substituents R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 are, independently of one another, H, F, Cl, Br, I.

[0023] Partial structure IIa represents halogenated 3,7-indolylene as Y 1 in formula I, partial structure IIb represents halogenated 3,6-indolylene as Y 2 in formula I, partial structure IIc represents halogenated 1,4-phenoxylene as Y 2 in formula I, and partial structures IId, IIe, IIf and partial structure IIc are any halogenated aromatic or heteroaromatic substituents R 1 , R 2 , R 3 , R 4represents, in particular, halogenated (1H-imidazol-4-yl)-methyl, halogenated -CH2-C6H5, halogenated (1H-indol-3-yl)-methyl, halogenated (4-hydroxyphenyl)-methyl, and halogenated benzyl.

[0024] Formulations containing FAD-dependent halogenase can be used for the halogenation of bicyclic heptapeptides. The enzyme can be heterologously overexpressed and purified by procedures well known to those skilled in the art. Purified enzyme (100 μL) was incubated in a total volume of 300 μL with 10 μL of purified flavin reductase (the enzyme can be produced by procedures well known to those skilled in the art), 10 μM FAD, 2.4 mM NADH, 2.5 mM KBr, 0.15 mM substrate (e.g., darobactin A), and 10 mM potassium phosphate buffer (pH 7.2). The reaction was incubated at 18 °C for 24 h and stopped by adding an equal volume of methanol prior to LC-MS analysis. The product obtained was brominated darobactin A.

[0025] (General method for the purification of bicyclic heptapeptides) Method A: Cells are sedimented by centrifugation at 10,000×g for 5 min, and the supernatant is collected in a round-bottom flask for lyophilization. The dried supernatant is washed twice with methanol. After the two washing steps, the methanol is completely removed, and deionized water is added to dissolve the crude extract. A centrifugation step at 10,000×g for 5 min is performed to remove the insoluble fraction. Next, the crude extract is subjected to reverse-phase flash chromatography (Interchim Puriflash 4125 chromatography system equipped with a Puriflash C18-HP 30 mm flash column, gradient elution of 5% MeOH / H2O to 100% MeOH over 1 h). Fractions containing DAR are further purified by HPLC. A C18 column (Macherey Nagel, EC 250 / 4.6 Nucleodur C18 Gravity-SB, 5 μm) is used as follows:

[0026]

Table 1

[0027] Solvent A is an aqueous solution of 0.1% trifluoroacetic acid. The solvent is pumped into the HPLC system at a flow rate of 1 mL / min. The DAR is collected by a fraction collector at 15.90 minutes to 17.10 minutes.

[0028] Method B: The clarified medium was applied to a hydrophobic interaction material (e.g., C18 silica, Amberlite XAD-16N), washed with H2O, and eluted with a mixture of an organic solvent (e.g., MeOH, MeCN) and H2O with or without 0.1% formic acid. The eluate was concentrated, the organic solvent was removed under vacuum, and the aqueous eluate was loaded onto a strong cation exchange material (e.g., SP Sepharose XL). The material was then washed with 0.1% formic acid, and the material bound to the column was eluted at pH 5 to pH 11 using a suitable buffer system, e.g., NH4CH3COO. The fractions were analyzed by LCMS, and the fractions containing the target compound were pooled, bound to a hydrophobic interaction material (e.g., C18 silica, Amberlite XAD-16N), and eluted with a gradient of H2O and an organic solvent (e.g., MeCN). The peaks were collected based on UV absorption, and the collected fractions were analyzed by LCMS. The fractions containing the target peptide were separated by HPLC using a gradient of H2O and a suitable organic solvent (e.g., MeOH, MeCN). The peaks were collected to obtain the pure compound of interest.

[0029] Any type of peptide produced by the above fermentation process can be, for example, reacted with different equivalents of Lawesson's reagent, O,O-diethylammonium phosphorodithioate salt, P4S 10It is well known to those skilled in the art that further chemical modification can be carried out by reacting with dimethicone, PSCl3 / H2O / Et3N, or other reagents, optionally with a suitable protecting group strategy. By doing so, it is possible to obtain substitution of one or even all amide bonds with thioamide bonds. Furthermore, conversion of the amino group to a sulfonamide is possible by treatment with mesyl chloride, tosyl chloride or other suitable reagents (on a substrate protected as necessary). Thus, bicyclic heptapeptides chemically modified by this or other methods are also included within the scope of the present invention.

[0030] (Heterologous darobactin A expression and identification of the minimal biosynthetic gene cluster) As will be apparent to those skilled in the art, the scope of the present invention includes the generation of expression constructs by transferring the genes required for biosynthesis into expression vectors at any level of technology, for example applying different promoters, ribosome binding sites, and additional elements for the regulation of expression. Thus, the following examples are meant not to limit the scope of the present invention.

[0031] In this project regarding the heterologous expression of DAR, several exemplary constructs were generated (Figure 3). In this example, the vector background used for the expression construct was pRSFDuet™-1 (Merck KGaA, Darmstadt, Germany). The chromosomal DNA used as a template for amplifying the DAR BGC was isolated using the innuPREP Bacteria DNA Kit (Analytik Jena AG, Jena, Germany). Generally, the fragments were amplified using Q5 DNA polymerase (New England Biolabs, Ipswich, USA) and purified from the agarose gel using the Large Fragment DNA Recovery Kit (Zymo Research, Irvine, USA). The polymerase chain reaction (PCR) was performed in a Biometra TRIO thermocycler (Analytik Jena AG, Jena, Germany) using the following program. 2 minutes at 95°C; 45 seconds at 95°C, 45 seconds at 60 - 70°C (the annealing temperature applied depends on the primer sequence), 30 seconds / kb at 72°C (the extension time varies according to the length of the amplified fragment) for 34 cycles, followed by a final extension step of 5 minutes at 72°C.

[0032] It will be apparent to those skilled in the art that the scope of the present invention includes the use of any organism having a biosynthetic gene cluster (BGC), amplifying or synthesizing the native or codon-optimized form (partially or entirely) of the BGC, and cloning this into an expression vector. The vector is then transferred into an expression host. Accordingly, the following examples are meant not to limit the scope of the present invention.

[0033] SEQ ID NO: 25 shows codon-optimized darE from Photorhabdus namnaonensis. SEQ ID NO: 26 shows codon-optimized darA from Photorhabdus namnaonensis. SEQ ID NO: 27 shows codon-optimized darA from Pseudoalteromonas luteoviolacea. SEQ ID NO: 28 shows codon-optimized darE from Pseudoalteromonas luteoviolacea. SEQ ID NO: 29 shows Pseudoalteromonas luteoviolacea dhalobactine-halogenase codon optimization. SEQ ID NO: 30 shows Pseudoalteromonas luteoviolacea protein w / o homology codon optimization.

[0034] Plasmid pZW-ADC3 has genes of a DAR BGC that has no intergenic region between darA and darB. Thus, darA was amplified from Photorhabdus khanii HGB1456 using a primer pair; and darB-darE were amplified using their respective primers, and all these amplifications were carried out according to methods known to those skilled in the art. pRSFDuet™-1 was linearized using NdeI and AvrII restriction enzymes (New England Biolabs, Ipswich, USA), and the two purified fragments were inserted into the second multiple cloning site of the vector under the control of the T7lac promoter. To do this, the 1-step isothermal DNA assembly protocol described by Gibson was followed, with a minor modification that 1.2 μl instead of 0.64 μl of 10 U / μL of T5 exonuclease was added. Thus, the final concentrations of the Gibson reaction mixture were as follows: 100 mM Tris-HCl pH7.5, 10 mM MgCl2, 0.2 mM each dNTP, 10 mM DTT, 5% PEG-8000, 1 mM NAD, 7.5 U / mL T5 exonuclease, 25 U / mL Phusion polymerase, 4 U / μL Taq DNA ligase, 0.02-0.5 pmol DNA fragment. Next, this reaction mixture was incubated at 50 °C for 1 hour. After isothermal assembly, the reaction was dialyzed using a 0.025 μm nitrocellulose membrane (Merck™ MF-Millipore™, Ireland), and then transferred into E. coli TOP10 cells as a plasmid maintenance host by electroporation at a voltage of 2.5 kV in a 0.2 cm electroporation cuvette using a Micropulser Electroporator (Bio-Rad, California, USA).

[0035] The second and third constructs each have the native DAR BGC from Photorhabdus khanii HGB1456(pZW-ADC5) and Photorhabdus khanii DSM3369(pZW-ADC6), respectively. For both constructs, the respective BGCs were amplified by PCR according to methods known to those skilled in the art; thus, the respective bacterial genomic DNAs were used as templates. Gibson assembly was performed as described above.

[0036] Plasmids pZW-ADC3.2 and pZW-ADC5.2 were generated by restriction enzyme digestion of pZW-ADC3 and pZW-ADC5, respectively. Each plasmid was restriction digested using NcoI and NotI (New England Biolabs, Ipswich, USA). Next, an additional codon-optimized form of Photorhabdus sp. darA (SEQ ID NO: 26) was inserted into the first multiple cloning site of the pRSFDuet™-1-based vector by Gibson assembly.

[0037] Plasmids pZW-ADC7 and pZW-ADC8 each have the native DAR BGC from P. khanii HGB1456 with additional 200 bp and 605 bp upstream regions of darA, respectively. The BGCs were amplified from bacterial genomic DNA by PCR according to methods known to those skilled in the art. Each insert was assembled into NdeI-AvrII linearized pRSFDuet™-1 using Gibson assembly.

[0038] Plasmid pZW-ADC9 has the DAR BGC from P. khanii DSM3369 that does not have transporter genes, i.e., only darA and darE. Both fragments were amplified by PCR and assembled into NdeI-AvrII linearized pRSFDuet™-1 using Gibson assembly.

[0039] pZW-YerA4 has the DAR BGC derived from Yersinia frederiksenii ATCC 33641, which was amplified by PCR. The amplicon was also assembled into NdeI-AvrII linearized pRSFDuet™-1 using Gibson assembly.

[0040] After assembly and growth in E. coli TOP10 cells, all constructs were confirmed by test PCR and their restriction patterns.

[0041] DAR-resistant strains were generated by introducing three point mutations 1300A>G, 1334A>C, and 2113G>A into the bamA gene. That these three point mutations result in the DAR-resistant phenotype has been confirmed by past tests, and the DAR resistance increased to 128 μg / mL. This past test is known to those skilled in the art. Mutations for generating resistant strains can be generated by means well known to those skilled in the art. The gene to be modified for this purpose can be, for example, bamA.

[0042] The expression construct was transferred by electroporation from the maintenance host E. coli TOP10 to different expression hosts. Transformation of the E. coli expression host was carried out as described above for E. coli TOP10, while transformation of V. natriegens Vmax™ was carried out at a voltage of 900 V in a 0.1 cm electroporation cuvette. After electroporation, the strains were incubated for 1 hour in their respective growth media and temperatures. Next, the cells were plated on LB(-ASW) plates containing kanamycin as the selective agent at the above concentrations, and chloramphenicol was added when using E. coli Rosetta™(DE3) as the host. Individual single colonies were picked from the selection plates, and the presence of each expression plasmid was confirmed by PCR. Next, colonies with the correct construct were inoculated into 3 mL of LB(-ASW) containing the necessary antibiotics and incubated overnight at 37 °C or 30 °C. 500 μL of this preculture was used to inoculate 50 mL of fresh LB(-ASW) medium containing kanamycin, and OD600 It was incubated at 37°C or 30°C until it reached 0.4 - 0.6, and then induced with IPTG (final concentration 0.5 mM). After IPTG induction, the culture was incubated at 30°C while shaking at 180 rpm. Those skilled in the art are aware that when using an inducible promoter for expression, expression can be induced when a certain cell density is reached, and thus non-resistant producing strains can be used without departing from the scope of the present invention. By doing so, the cells are not killed by the production of antibiotics.

[0043] DAR production was analyzed by UPLC - HRMS. From the expression culture, a 1 mL aliquot was taken and centrifuged to separate the medium and bacterial cells. The medium was lyophilized, 1 mL of methanol was added, the mixture was sonicated in a Bandelin Sonorex RK255 ultrasonic bath (Berlin, Germany) for 30 minutes, and centrifuged at 10,000 g for 5 minutes. The methanol was removed, and the pellet was resuspended in 1 mL of deionized water. After the last centrifugation at 10,000 g for 5 minutes, the sample was ready for injection into the UPLC - HRMS system. To prepare the sample from the cell pellet, 500 μL of methanol was added followed by sonication for 30 minutes. Next, 500 μL of deionized water was added and sonication was continued for an additional 15 minutes. Then, the solution was centrifuged to sediment the insoluble portion, and the supernatant was injected into the UPLC - HRMS system.

[0044] The UPLC-HRMS system was an Agilent Infinity 1290 UPLC system, which was equipped with an Acquity UPLC BEH C18 1.7μm (2.1×100mm) column (Waters, Eschborn, Germany) and an Acquity UPLC BEH C18 1.7μm VanGuard pre-column (2.1×5mm; Waters, Eschborn, Germany) setup and was connected to a DAD detector and a micrOTOFQ II mass spectrometer (Bruker, Bremen, Germany). The LC part was carried out using a gradient (A: H2O, 0.1% FA; B: MeCN, 0.1% FA; flow rate: 600 μL / min): 0 min: 95% A; 0.80 min: 95% A; 18.70 min: 4.75% A; 18.80 min: 0% A; 23.00 min: 0% A; 23.10 min: 95% A; 25.00 min: 95% A, and the column oven temperature was set at 45°C. The MS parameters were as follows: nebulizer gas 1.6 bar; gas temperature, 200°C; gas flow rate, 8 L / min; capillary voltage, 4500 V; end plate offset, 500 V; the measurement was carried out in positive ion mode.

[0045] The DAR standard curve was prepared by plotting the peak area of DAR from the extracted ion chromatogram (EIC) (for m / z ±0.01 of 483.7089 and 475.1956) against a series of DAR concentrations (2, 3, 4, 5, 10, 15, 20, 30, 40 mg / L). The DAR concentration from the heterologous expression cultures was quantified by calculating the peak area and interpolating this into the DAR standard curve. The linear range of this quantification method was 3 μg / mL to 30 μg / mL. Therefore, peak areas below the limit were not converted to concentrations. The standard curve was measured for all batches analyzed by UPLC-HRMS to exclude technical differences between measurements.

[0046] The three genes darB, darC, and darD encode subunits of an ABC transporter. To answer the question of whether these transporter genes play an additional role in DAR biosynthesis and, furthermore, to define the minimal DAR BGC, these genes were removed from the expression construct. Thus, the construct pZW-ADC9, which has only darA and darE, was generated. This experiment showed that DAR could still be produced even in the absence of darBCD. Furthermore, DAR was detected outside the cells.

[0047] We evaluated whether the transporter-encoding genes darBCD are essential for heterologous DAR expression or whether darA, which encodes the precursor peptide, and darE, which encodes the radical SAM modifying enzyme, are sufficient. The deletion of darBCD did not abolish DAR production. However, the yield was lower (1.5-fold) than that achieved by the construct containing the transporter genes (pZW-ADC6). Most interestingly, DAR was also present in the medium in the absence of the transporter-encoding genes. On the one hand, this clearly defines the minimal DAR BGC consisting only of darA and darE. On the other hand, it was revealed that in E. coli, DAR is present outside the cells even in the absence of the specific heterologous transporter gene darBCD.

[0048] (Heterologous expression of other bicyclic heptapeptides) Based on the expression constructs exemplified above, the amino acids of the heptapeptide can be exchanged. This can be done specifically for individual amino acids or in a randomization approach using primers to modify the sequence of the heptapeptide. Thus, specific or degenerate primers (e.g., using the triplets NNN or NNK for any proteinogenic amino acid incorporated at a given position) were designed to modify one or more amino acids of the core (= hepta) peptide. As an example, a PCR mixture suitable for a 50 μL scale reaction contained the following: water: 34 - 34.5 μL, DMSO: 2.5 μL, Q5 reaction buffer: 10 μL, dNTP: 1 μL, forward primer (100 pmol / μL): 0.5 μL, reverse primer (100 pmol / μL): 0.5 μL, template DNA: 0.5 μL, Q5 DNA polymerase: 0.5 - 1 μL. The PCR program can be as follows: Step 1, 98 °C, 10 min; Step 2, 98 °C, 10 s; Step 3, 65 °C, 20 s; Step 4, 72 °C, 7 min; Steps 2 - 4 for 30 cycles; Step 5, 72 °C, 10 min; Step 6, 4 °C, ∞.

[0049] In this way, all possible combinations of amino acids according to formula I can be incorporated into the heptapeptide. Furthermore, these derivatives can also be halogenated or modified by the presence of double bonds according to formula I.

[0050] (Elucidation of Structure) All NMR data were recorded on a Bruker Avance III HD 600 MHz NMR spectrometer (Bruker BioSpin MRI GmbH, Ettlingen, Germany). For all NMR experiments, deuterium oxide (Deutero GmbH, Kastellaun, Germany) was used as the solvent. 1 The 1H NMR spectra were referenced to the solvent residual peaks according to the literature. 13For the reference of the C spectrum, 3-trimethylsilyl-d4-propionic acid (TSPA) was used as an external standard as known to those skilled in the art. 2D experiments including COSY (cosygpmfphpp), TOCSY (mlevetgp and mlevgpph19), 1 H- 13 C_HSQC (hsqcedetgpsisp2.3), and 1 H- 13 C_HMBC (hmbcetgpl3nd) were used to obtain complete assignments. 1 H- 13 To improve the resolution of the H-

[0051] (Identification of Biosynthetic Gene Clusters) The direct screening of the core peptide sequence WNWSKSF was performed on all Photorhabdus genomes available in the public database using the Basic Local Alignment Search Tool (BLAST). In P. temperata, the 7 - amino acid sequence of darobactin is located near the C - terminus of an open reading frame encoding 58 amino acids upstream of an ABC transporter and a radical SAM enzyme, suggesting a RiPP operon. This putative BGC was also identified in other darobactin - producing strains, such as P. luminescens DSM - 3368 and P. khanii DSM - 3369. The cluster boundaries were determined by comparison with the P. bodei genome, which does not contain the operon. Furthermore, the GC content of the dar cluster was clearly lower than the other average GC content in the genome (32% vs 45%). To identify other bacterial species potentially producing darobactin - like compounds, the radical SAM protein sequence (DarE) was used as an input in BLAST to search for homologous enzymes. The genomic content of each hit was manually analyzed to confirm the presence of a DarA - like pre - peptide in the vicinity of the radical SAM protein. In addition, a search was performed using the pre - peptide DarA as an input, which resulted in the same hits.

[0052] (Generation and heterologous expression of darobactin deletion mutants) To delete the dar BGC (darABCDE) from the genome of the producing strain Photorhabdus khanii DSM3369, a plasmid was constructed by the assembly of five fragments enabling marker - less genome modification. Thus, chromosomal DNA was isolated using the innuprepBacteria DNA Kit (AnalytikJena, Jena, Germany).

[0053] The (i) upstream and (ii) downstream fragments of the BGC were amplified using the following primer pairs (size approximately 1 kb). SEQ ID NO: 1: 5’-TTTGACGTTGGAGTCCACGTGTTATGGACGTGGCAAACGCGGTTCTTGAC-3’, and SEQ ID NO:2: 5’-TTGAAATATCAGGATAGCATTGCGCTCGCTCACCCCGGTCACATAGTTCG-3’, and SEQ ID NO:3: 5’-ATGCTATCCTGATATTTCAAATGCAAGTAAAATGTTTCATCATAATAACC-3’, and SEQ ID NO:4: 5’-TTCTTGACGAGTTCTTCTGAGATGGGTTGATATCCACTGATATAAATCTC-3’ (iii) The R6K origin of replication (ori), origin of conjugative transfer (oriT), and levansucrase gene sacB from Bacillus subtilis were amplified as a unit from vector pNPTS138 using the following primers. SEQ ID NO:5: 5’-TCGAGCTCTAAGGAGGTTATAAAAAATGAACATCAAAAAGTTTGCAAAACAAGCA-3’, and SEQ ID NO:6: 5’-ACGTGGACTCCAACGTCAAA-3’ (iv) The arabinose-inducible expression system of pKD46 adjacent to the beta-lactamase (bla) promoter was amplified using the following primers. SEQ ID NO:7: 5’-ACTCTTCCTTTTTCAATATTATTGAAGCAT-3’, and SEQ ID NO:8: 5’-TGCATTTTTTATAACCTCCTTAGAGCTCGAATTCC-3’ (v) The aph gene from pCAP03 conferring resistance to kanamycin was amplified using the following primers. SEQ ID NO:9: 5’-TCAGAAGAACTCGTCAAGAAGGCGA-3’, and SEQ ID NO:10: 5’-TCAATAATATTGAAAAAGGAAGAGTATGATTGAACAAGATGGATTGCACG-3’

[0054] All fragments were amplified by Q5 DNA polymerase (New England Biolabs, Ipswich, USA), gel purified by 1% or 2% TAE agarose gel, and the DNA was recovered using the Large Fragment DNA Recovery Kit (Zymo Research, Irvine, USA). Then, all fragments were fused by isothermal assembly to generate plasmid pNB02. After assembly, E. coli WM3064 cells were transformed with pNB02 by electroporation, and the correct assembly was confirmed by PCR and restriction analysis following standard procedures. The conjugation between E. coli WM3064 and P. khanii DSM3369 was performed by growing both strains until the OD 600 was approximately 0.6. After washing twice with LB medium, the cells were mixed with E. coli and P. khanii in a ratio of 1:3 and plated on LB agar supplemented with diaminopimelic acid (0.3 mM), incubated at 37 °C for 3 h, and then at 0 °C overnight. The bacterial colonies were resuspended in LB medium and serially diluted and plated on LB agar containing kanamycin (50 μg ml -1 ). The kanamycin-resistant single crossover conjugation products were grown in LB medium until the OD 600 was approximately 0.6. Next, the expression of SacB was induced by adding arabinose (0.2 wt / vol%) and subsequently incubating for 2 h.

[0055] Then, the culture was plated on LB agar supplemented with 0.2% (wt / vol) arabinose and 10% sucrose and incubated at 30 °C for 48 h. Individual single colonies were grown in LB Kan and LB Ara / SucIt was collected on agar. Susceptibility to kanamycin indicated plasmid loss, thereby indicating the success of the double crossover event. Clones were collected and analyzed for BGC loss by PCR using the following primers. SEQ ID NO: 11: 5’-ATCTCCATCAAAGCGCTACC-3’, and SEQ ID NO: 12: 5’-CCGCGCTGCAACTCGAAATC-3’

[0056] The knockout strain is designated as P. khanii DSM3369 ΔdarABCDE. For heterologous expression of the darobactin A BGC in E. coli and also for complementing P. khanii DSM3369 ΔdarABCDE, the expression plasmid pNB03 was used. To avoid problems with the control system between the propeptide and the modifying enzymes, all intergenic regions were removed and the genes darA - darE were expressed under the control of the arabinose-inducible araB promoter in a simplified form.

[0057] pNB03 contains (i) the p15A ori from pACYC177 (primers SEQ ID NO: 13: 5’-GGTCGACGGATCCCCGGAATAGCGGAAATGGCTTACGAAC-3’, and SEQ ID NO: 14: 5’-CTCTAAGGAGGTTATAAAAAGCGGCCGCATCCCTTAACGTGAGTTTTC-3’), (ii) the arabinose expression system and kanamycin resistance of pNB02 (primers SEQ ID NO: 15: 5’-AAGCAGCTCCAGCCTACATCAGAAGAACTCGTCAAGAAGGCGA-3’, and SEQ ID NO: 16: 5’-TTTTTATAACCTCCTTAGAGCTCGAATTCC-3’), and (iii) The aac(3) gene conferring resistance to apramycin derived from oriT and pIJ773 (Primer SEQ ID NO: 17: 5’-ATTCCGGGGATCCGTCGACC-3’, and SEQ ID NO: 18: 5’-TGTAGGCTGGAGCTGCTT-3’) was prepared by amplification.

[0058] All fragments were then gel-purified and assembled as described above. E. coli TOP10 cells were transformed with the vector to confirm accurate assembly.

[0059] To introduce dar BGC into P. khanii DSM3369 ΔdarABCDE, (i) pNB03 was linearized using the following primers, SEQ ID NO: 19: 5’-TCCCTTAACGTGAGTTTTCG-3’, and SEQ ID NO: 20: 5’-TTTTATAACCTCCTTAGAGCTCGAA-3’ (ii) darA was amplified using the following, SEQ ID NO: 21: 5’-GCTCTAAGGAGGTTATAAAAATGCATAATACCTTAAATGAAACCGTTAAA-3’, and SEQ ID NO: 22: 5’-AATAGCATTCATTTATGGCTCTCCTTTTAAATTTCCTGGAAGCTTT-3’ (iii) darB-darE was amplified using the following. SEQ ID NO: 23: 5’-AAAGCTTCCAGGAAATTTAAAAGGAGAGCCATAAATGAATGCTATT-3’, and SEQ ID NO: 24: 5’-CGAAAACTCACGTTAAGGGATTACGCCGCGATGGTTTGTTTTATT-3’

[0060] All the fragments were gel-purified and assembled as described above. The resulting vector pNB03-darABCDE was transferred into E. coli TOP10 cells to confirm the correct assembly.

[0061] Empty pNB03 and pNB03-darABCDE were transferred into P. khanii DSM3369 ΔdarABCDE by triparental conjugation. Briefly, conjugation between P. khanii DSM3369 ΔdarABCDE, E. coli TOP10 with the expression plasmid, and E. coli ET pUB307 with the pUB307 conjugation helper plasmid was performed as described above (cell ratio 3:1:1). Since P. khanii DSM3369 is originally resistant to carbenicillin and the kanamycin-resistant pUB307 lacks the bla promoter, the final selection was carried out on LB agar supplemented with kanamycin and carbenicillin. The kanamycin-resistant conjugation products were grown in LB Kan and the plasmids were isolated and confirmed for identity by PCR. For heterologous expression, the vector pNB03-darABCDE was transferred into E. coli BW25113 (non-arabinose-utilizing strain) by electroporation. Then, P. khanii DSM3369 WT, P. khanii DSM3369 ΔdarABCDE + pNB03, P. khanii DSM3369 ΔdarABCDE + pNB03-darABCDE, and E. coli + pNB03-darABCDE were grown in LB or LB supplemented with 0.2% (weight / volume) arabinose for 5 - 7 days and analyzed by LCMS. Kan

[0062] (minimum inhibitory concentration (MIC)) The MIC was determined by the microbroth dilution assay in round-bottom 96-well plates. Overnight cultures of E. coli ATCC35218, E. coli NRZ14408 KPC-2, E. coli K0416 VIM-1, E. coli Survcare 052 NDM-5, E. coli MMGI1 OXA-48, P. aeruginosa PAO 1, P. aeruginosa PAO 750, A. baumanii ATCC19606, K. pneumoniae ATCC30104, and S. enterica ATCC13076 were adjusted to MacFarland 1.0 and then diluted in MHIIB to 5×10 5 c.f.u. / mL -1 . Darobactin derivatives were screened in triplicate at 12 concentrations in the range of 64 to 0.03 μg / mL -1 . The same concentrations were tested for rifampicin, tetracycline, and gentamicin as positive controls. For tetracycline-resistant E. coli strains (NRZ14408, K0416, and MMGI1) and E. coli Survcare 052, tetracycline was replaced with a colistin dilution series (16 to 0.007 μg / mL -1 ).

[0063] A bacterial suspension without the addition of standard antibiotics or darobactin was used as a negative control. After incubation (18 h, 180 rpm, 37 °C, 85% r.H.), cell growth was determined by measuring the turbidity at 600 nm using a microplate spectrophotometer. The MIC was defined as the minimum concentration at which at least 85% growth inhibition was measured compared to the negative control.

[0064] (Claims of the invention) The present invention includes several aspects that are closely related to each other. The claims encompass bicyclic heptapeptides, their production, pharmaceutical formulations, and their use as APIs in respective pharmaceutical compositions. The expressions "pharmaceutical formulation" and "pharmaceutical composition" are used synonymously herein. Thus, the present invention also includes the following:

[0065] Claim 1: A bicyclic heptapeptide of formula I, [Chemical formula] and / or a pharmaceutically acceptable salt, stereoisomer, tautomer or hydrate thereof, wherein, - R 1 , R 2 , R 3 , R 4 are, independently of each other, selected from the list comprising H, -CH3, -CH2-CH2-CH2-NH-C(NH)(NH2), -CH2-CO-NH2, -CH2-CO2H, -CH2-SH, -CH2-CH2-CO2H, -CH2-CH2-CO-NH2, (1H-imidazol-4-yl)-methyl, halogenated (1H-imidazol-4-yl)-methyl, -CH(CH3)(C2H5), -CH2-CH(CH3)2, -CH2-CH2-CH2-CH2NH3, -CH2-CH2-S-CH3, -CH2-C6H5, halogenated -CH2-C6H5, (1H-indol-3-yl)-methyl, halogenated (1H-indol-3-yl)-methyl, (4-hydroxyphenyl)-methyl, halogenated (4-hydroxyphenyl)-methyl, -CH-(CH3)2, 1-hydroxy-ethyl, hydroxy-methyl, sec-butyl, 1-acetamido, 1-thioacetamido, -CH2-CH2-NH-(C=NH)-NH2, benzyl, halogenated benzyl, -CH2-CH2-CH2-NH-(C=NH)-NH2; - X is, at any position of X, independently of any other position of X, either O or S; - R 5 is selected from the list comprising methylsulfonyl, p-toluenesulfonyl; - R 6 is selected from the list including methylsulfonyl, p-toluenesulfonyl, -(C=NH)-NH2; - Z 1 and Z 2 are each a double bond or a single bond, provided that a) Z 1 and Z 2 are both single bonds, or b) only one of them is a single bond while the other is a double bond, where i) Z 1 is a single bond and Z 2 is a double bond, or ii) Z 2 is a single bond and Z 1 is a double bond; - Y 1 is 3,7-indolylene or halogenated 3,7-indolylene; - Y 2 is independently selected from the list of 3,6-indolylene, 1,4-phenoxylene, halogenated 3,6-indolylene, halogenated 1,4-phenoxylene; - n is 1 or 2, a bicyclic heptapeptide.

[0066] Claim 2: The bicyclic heptapeptide according to claim 1, which is darobactin.

[0067] Claim 3: A method for producing the bicyclic heptapeptide according to claim 1, comprising i) A fermentation step of using a microorganism that produces a bicyclic heptapeptide to provide a fermentation broth containing the bicyclic heptapeptide and microbial cells, wherein the microorganism is selected from the list of microorganisms including Photorhabdus spp., Photorhabdus laumondii, Photorhabdus khanii, Pseudoalteromonas spp., Pseudoalteromonas luteoviolacea, Pseudoalteromonas luteoviolacea, Yersinia spp., Escherichia spp., Vibrio spp; ii) A separation step of separating the fermentation broth into an insoluble part containing microbial cells and / or fragments of microbial cells and a solution containing the bicyclic heptapeptide by sedimentation and / or centrifugation and / or filtration, wherein the microbial cells are either disrupted or not disrupted before sedimentation and / or centrifugation and / or filtration is applied to the fermentation broth; iii) A purification step of purifying the bicyclic heptapeptide contained in the solution obtained from the previous separation step ii) to provide a solution of the purified bicyclic heptapeptide A method comprising the above steps.

[0068] Claim 4: A method for producing the bicyclic heptapeptide according to claim 3, wherein the purification step iii) of claim 3 comprises a) A step of drying the solution obtained from step ii) of claim 3 by freeze-drying and / or distillation under reduced pressure to provide a residue; b) A step of washing the residue from the previous step a) with alcohol, drying the washed residue, dissolving the dried residue in deionized water to provide a crude extract of the bicyclic heptapeptide; c) A step of removing the insoluble part of the crude extract from the previous step b) by sedimentation and / or filtration and / or centrifugation to provide a solid-free crude extract; d) Purifying the crude extract free of the solid obtained from the previous step c) by chromatography, thereby providing a pure solution of the bicyclic heptapeptide A method comprising

[0069] Claim 5: A method for producing a bicyclic heptapeptide according to claim 3, wherein the purification step iii) of claim 3 is a) Contacting the solution obtained from step ii) of claim 3 with the hydrophobic interaction material such that the bicyclic heptapeptide is adsorbed onto the hydrophobic interaction material b) Separating the remaining clear solution from the hydrophobic interaction material and washing the hydrophobic interaction material loaded with the adsorbed bicyclic heptapeptide with water c) Eluting the bicyclic heptapeptide from the hydrophobic interaction material with a mixture of water and an organic solvent, wherein the organic solvent is selected from the list comprising MeOH, MeCN, THF, acetone, ethanol, propanol, and the mixture of water and the organic solvent may or may not contain an acid in an amount between 0.001 wt% and 1 wt% d) Concentrating the eluate obtained from step c), removing the organic solvent by applying a vacuum, and contacting the aqueous solution with a strong cation exchange material such that the bicyclic heptapeptide is adsorbed onto the cation exchange material e) Washing the cation exchange material loaded with the bicyclic heptapeptide with an acid having a concentration between 0.001 wt% and 1 wt% f) Eluting the bicyclic heptapeptide by applying an aqueous buffer solution having a pH value of pH 5 to pH 11 and collecting the fraction containing the bicyclic heptapeptide g) Adsorbing the bicyclic heptapeptide obtained from the fraction of step f) onto the hydrophobic interaction material in the same manner as described in step a) h) A step of eluting the bicyclic heptapeptide with a gradient of H2O and an organic solvent, wherein the organic solvent is selected from the list including MeOH, MeCN, THF, acetone, ethanol, and propanol, i) A step of purifying the fraction containing the bicyclic heptapeptide obtained from step h) by applying HPLC using a gradient of H2O and an organic solvent, wherein the organic solvent is selected from the list including MeOH, MeCN, THF, acetone, ethanol, and propanol, A method comprising the above steps.

[0070] Claim 6: The method according to any one of claims 3 to 5, wherein the bicyclic heptapeptide according to claim 3 or the bicyclic heptapeptide according to claim 4 or the bicyclic heptapeptide according to claim 5 is further processed by chemical modification.

[0071] Claim 7: The method for producing a bicyclic heptapeptide according to claim 6, wherein the chemical modification is to react the bicyclic heptapeptide with Lawesson's reagent or tosyl chloride or mesyl chloride.

[0072] Claim 8: A pharmaceutical composition for treating an infectious disease in a mammal caused by a Gram-negative bacterium, comprising a therapeutically effective amount of the bicyclic heptapeptide according to claim 1, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof.

[0073] Claim 9: The pharmaceutical composition according to claim 8, further comprising at least one pharmaceutically acceptable carrier, excipient, or diluent.

[0074] Claim 10: The pharmaceutical composition according to any one of claims 8 to 9, which is in the form of topical administration, systemic administration, parenteral administration, subcutaneous administration, transdermal administration, rectal administration, oral administration, intravaginal administration, intranasal administration, intratracheal administration, intraocular administration, intra-aural administration, intravenous administration, intramuscular administration, or intraperitoneal administration.

[0075] Claim 11: The pharmaceutical composition according to any one of claims 8 to 10, comprising administering a pharmaceutical composition to a mammal suffering from a bacterial infection over a sufficient period of time at a sufficient frequency to provide a beneficial effect to the mammal.

[0076] Claim 12: The pharmaceutical composition according to any one of claims 8 to 10, administered to a mammal suffering from a bacterial infection, wherein the bacterial infection is an infection involving at least one type / strain of Gram-negative bacteria.

[0077] Claim 13: A pharmaceutical composition according to any one of claims 8 to 10, which is administered to a mammal suffering from a bacterial infection, wherein the bacterial infection is caused by Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Neisseria gonorrhoeae, Chlamydia trachomatis, Shigella sonnei, Salmonella enterica Typhimurium LT2, Enterobacter cloacae, Bifidobacterium longum, Bacteroides fragilis, Lactobacillus reuteri, Enterococcus faecalis and Yersinia pestis, Pseudomonas, fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Enterobacter aerogenes, Enterobacter spp., Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, YersiniaA pharmaceutical composition for the treatment of infectious diseases associated with Pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, Bacteroides splanchnicus.

[0078] 1. A pharmaceutical preparation containing darobactin or a derivative thereof for use in vertebrates or humans suffering from an infectious disease caused by Gram-negative bacteria.

[0079] 2. The pharmaceutical preparation according to claim 1, wherein the Gram-negative bacterium is selected from the group consisting of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Neisseria gonorrhoeae, Chlamydia trachomatis, Shigella sonnei, Salmonella enterica Typhimurium LT2, Enterobacter cloacae, Bifidobacterium longum, Bacteroides fragilis, Lactobacillus reuteri, Enterococcus faecalis, and Yersinia pestis. This list is meant not to limit the scope of the present invention.

[0080] 3. The pharmaceutical preparation according to claim 1 or 2, wherein the vertebrate includes birds, fish, amphibians, reptiles, and mammals.

Claims

1. Darbactam B having the following structure, 【Chemical Formula 4】 or a pharmaceutically acceptable salt, stereoisomer, tautomer or hydrate thereof, a bicyclic heptapeptide.

2. The bicyclic heptapeptide according to Claim 1, which is Darbactam B having the following structure. [Chemical Formula 5]

3. Darbactam C having the following structure, 【Chemical Formula 6】 or a pharmaceutically acceptable salt, stereoisomer, tautomer or hydrate thereof, a bicyclic heptapeptide.

4. The bicyclic heptapeptide according to Claim 3, which is Darbactam C having the following structure. [Chemical Formula 7]

5. Darbactam D having the following structure, 【Chemical 8】 or a pharmaceutically acceptable salt, stereoisomer, tautomer or hydrate thereof, a bicyclic heptapeptide.

6. The bicyclic heptapeptide according to Claim 5, which is Darbactam D having the following structure. 【Chemical Formula 9】

7. Darbactam E having the following structure, 【Chemical Formula 10】 or a pharmaceutically acceptable salt, stereoisomer, tautomer or hydrate thereof, a bicyclic heptapeptide.

8. The bicyclic heptapeptide according to Claim 7, which is Darbactam E having the following structure. 【Chemical 11】

9. A method for producing the bicyclic heptapeptide according to any one of Claims 1 to 8, i) a fermentation step of using a microorganism that produces the bicyclic heptapeptide, wherein the microorganism is selected from Photorhabdus spp., Escherichia spp., and Vibrio spp., which provides a fermentation broth containing the bicyclic heptapeptide and microbial cells, the fermentation step; ii) a separation step of separating the fermentation broth into an insoluble portion containing microbial cells and / or fragments of the microbial cells and a solution containing the bicyclic heptapeptide by sedimentation and / or centrifugation and / or filtration, wherein the microbial cells are either disrupted or not disrupted before applying sedimentation and / or centrifugation and / or filtration to the fermentation broth, the separation step; iii) a purification step of purifying the bicyclic heptapeptide contained in the solution obtained from the previous separation step ii) to provide a solution of the purified bicyclic heptapeptide comprising the method.

10. A method for producing the bicyclic heptapeptide according to claim 9, wherein the purification step iii) of claim 9 is a) drying the solution obtained from step ii) of claim 9 by freeze-drying and / or distillation under reduced pressure to provide a residue; b) washing the residue from the previous step a) with alcohol, drying the washed residue, dissolving the dried residue in deionized water to provide a crude extract of the bicyclic heptapeptide; c) removing the insoluble portion of the crude extract from the previous step b) by sedimentation and / or filtration and / or centrifugation to provide a solid-free crude extract; d) purifying the solid-free crude extract obtained from the previous step c) by chromatography to thereby provide a pure solution of the bicyclic heptapeptide comprising the method.

11. A method for producing the bicyclic heptapeptide according to claim 9, wherein the purification step iii) of claim 9 is a) contacting the solution obtained from step ii) of claim 9 with the hydrophobic interaction material such that the bicyclic heptapeptide is adsorbed to the hydrophobic interaction material; b) separating the remaining clear solution from the hydrophobic interaction material and washing the hydrophobic interaction material loaded with the adsorbed bicyclic heptapeptide with water; c) eluting the bicyclic heptapeptide from the hydrophobic interaction material with a mixture of water and an organic solvent, wherein the organic solvent is selected from the list comprising MeOH and MeCN; d) concentrating the eluate obtained from step c), removing the organic solvent by applying a vacuum, and contacting the aqueous solution with the strong cation exchange material such that the bicyclic heptapeptide is adsorbed to the cation exchange material; e) washing the cation exchange material loaded with the bicyclic heptapeptide with formic acid having a concentration of 0.1% by weight; f) eluting the bicyclic heptapeptide by applying an aqueous buffer solution having a pH value of pH 5 to pH 11 and collecting the fraction containing the bicyclic heptapeptide; g) adsorbing the bicyclic heptapeptide obtained from the fraction of step f) to the hydrophobic interaction material in the same manner as described in step a) h) eluting the bicyclic heptapeptide with a gradient of H ２ O and an organic solvent, wherein the organic solvent is selected from the list comprising MeOH and MeCN, i) H ２ A step of purifying the fraction containing the bicyclic heptapeptide obtained from step h) by applying HPLC using a gradient of H₂O and an organic solvent, wherein the organic solvent is selected from the list containing MeOH and MeCN, step comprising the method.

12. A pharmaceutical composition for treating infections in mammals caused by Gram-negative bacteria, comprising a bicyclic heptapeptide according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, in a therapeutically effective amount.

13. The pharmaceutical composition according to claim 12, further comprising at least one pharmaceutically acceptable carrier, excipient, or diluent.

14. The pharmaceutical composition according to claim 12 or claim 13, which is in the form of topical administration, systemic administration, parenteral administration, subcutaneous administration, transdermal administration, rectal administration, oral administration, intravaginal administration, intranasal administration, intratracheal administration, intraocular administration, intra-aural administration, intravenous administration, intramuscular administration, or intraperitoneal administration.

15. Administered to a mammal suffering from a bacterial infection, The pharmaceutical composition according to any one of claims 12 to 14, comprising administering the pharmaceutical composition to the mammal at a sufficient frequency for a sufficient period to provide a beneficial effect to the mammal.

16. Administered to a mammal suffering from a bacterial infection, The pharmaceutical composition according to any one of claims 12 to 14, wherein the bacterial infection is an infection associated with at least one type / strain of Gram-negative bacteria.

17. Administered to a mammal suffering from a bacterial infection, The bacterial infectious diseases are Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Neisseria gonorrhoeae, Chlamydia trachomatis, Shigella sonnei, Salmonella enterica Typhimurium LT2, Enterobacter cloacae, Bifidobacterium longum, Bacteroides fragilis, Lactobacillus reuteri, Enterococcus faecalis, and Yersinia pestis, Pseudomonas, fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneriShigella flexneri, Enterobacter aerogenes, Enterobacter spp., Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhaliscatarrhalis), Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides Infections associated with Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides Bacteroides vulgatus, Bacteroides Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides Bacteroides eggerthii, Bacteroides The pharmaceutical composition according to any one of claims 12 to 14, which is an infection associated with Bacteroides splanchnicus.

18. A pharmaceutical preparation comprising the bicyclic heptapeptide according to any one of Claims 1 to 8 for use in a vertebrate or human suffering from an infectious disease caused by a Gram-negative bacterium selected from the group consisting of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Neisseria gonorrhoeae, Chlamydia trachomatis, Shigella sonnei, Salmonella enterica Typhimurium LT2, Enterobacter cloacae, Bifidobacterium longum, Bacteroides fragilis, Lactobacillus reuteri, Enterococcus faecalis, and Yersinia pestis.

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