New antibiotic that selectively kills gram-negative pathogens

Abstract

To provide a substance as an active pharmaceutical ingredient (API) in a medicament, having a different working mechanism, which is unhindered by the triple-defense-line of Gram-negative bacteria.SOLUTION: The present invention provides a specific bicyclic heptapeptide of formula I and a medical preparation comprising the substance for use in vertebrates or humans suffering from an infection caused by Gram-negative bacteria.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to bicyclic heptapeptide antibiotics against Gram-negative pathogens, their derivatives, and their manufacture (production) and use as active pharmaceutical ingredients (API) in medicine. [Background technology]

[0002] The current need for new antibiotics is particularly urgent with respect to drug-resistant Gram-negative pathogens. These microorganisms have highly restrictive permeability barriers that limit the penetration of most compounds. Consequently, the most recent classes of antibiotics active against Gram-negative bacteria were developed within the last 60 years of the last century.

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

[0004] Consequently, the complex barrier (inner membrane plus outer membrane) restricts all molecules, and nutrients enter through outer membrane porins and dedicated transporters. Drugs that leak through the barrier are extruded by trans-envelope multidrug resistance pumps (MDR), which recognize amphipathic compounds (as most drugs are) that pass through hydrophilic as well as hydrophobic regions of the cell (e.g., membranes). Thus, the state of the art is in great need of APIs with distinct mechanisms of action that are not hindered by the triple defense mechanism of Gram-negative bacteria (outer membrane, inner membrane, and MDR). Summary of the Invention

[0005] Darobactin A (DAR) is a ribosomally synthesized, post-translationally modified peptide (RiPP) antibiotic first identified in bacteria belonging to the genus Photorhabdus. Additionally, a corresponding biosynthetic gene cluster (BGC) was identified and subsequently detected in several bacterial genera. DAR represents a highly promising lead structure for the development of novel antibiotic therapeutics. DAR targets the outer membrane protein BamA and is therefore specific for Gram-negative bacteria. This, along with demonstrated in vivo activity in a mouse infection model, makes DAR a particularly promising candidate for future research. To improve compound availability for future DAR studies and enable the production of novel derivatives, it is highly desirable to establish an effective and versatile microbial production platform for these classes of RiPP antibiotics. Herein, we demonstrate the design and construction of a heterologous production and engineering platform for DAR and / or its derivatives, which ensures high production yields and facilitates structural modification approaches. The known useful Gram-negative bacteria Escherichia coli and Vibrio natriegens were tested as heterologous hosts. In addition, DAR-producing strains were generated, and the optimization of the expression constructs resulted in a DAR production titer 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, only two genes are required for heterologous production of RiPP (darobactin and / or its derivatives), and thus a minimal DAR BGC was identified and characterized.

[0006] Darobactin B 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. Other naturally occurring derivatives, such as darobactin B and brominated variants, are active 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). Experiments have demonstrated that DAR binds to BamA, a central component of the OM β-barrel assembly machinery. BamA assists in the folding and insertion of β-barrel proteins, such as porins, into the OM. Impairment of this chaperone-like function leads to disruption of OM formation. In addition to its favorable in vitro activity, DAR has shown promising efficacy in mouse sepsis and mouse thigh infection models without demonstrating cytotoxicity. Therefore, DAR has emerged as a promising drug lead.

[0007] The objective of the present invention is to provide novel substances that can be used as active pharmaceutical ingredients (APIs) for drugs that are effective against Gram-negative bacteria in a novel and unexpected way and therefore do not have resistance in Gram-negative bacteria. Surprisingly, bicyclic heptapeptides according to Formula I exhibit characteristics / interactions with cellular components to provide these positive effects: they act on the BamA chaperone and translocator, an attractive but highly unusual target. The BamA chaperone and translocator assist the folding and insertion of β-barrel proteins, such as porins, into the outer membrane. BamA itself is an outer membrane β-barrel protein. Drugs in general, and natural products in particular, typically target enzymes with this well-defined catalytic center rather than chaperones. Darobactin is a large molecule that is likely necessary to disrupt protein-protein binding between BamA and its substrate. While surface target location would solve the difficult problem of penetrating across the permeability barrier of Gram-negative bacteria, this is not necessary in the case of darobactin. Only two essential proteins, BamA and LptD, are exposed on the surface of the outer membrane. Thus, bicyclic heptapeptides targeting BamA effectively bypass the triple defense mechanisms of Gram-negative bacteria (outer membrane, inner membrane, and MDR) and offer a novel mode of action for antibiotic activity.

[0008] [ka]

[0009] Scheme 1: General formula I for bicyclic heptapeptides In Scheme I, the substituents and indices have the following values: R1, R2, R3, R4, independently of one another, are 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, (1H-imidazol-4-yl)-methyl halide, -CH(CH3)(C2H5), -CH2-CH(CH3)2, -CH2-CH2-CH2-CH2NH3, -CH2-CH2-S-CH3 , -CH2-CH5, halogenated -CH2-CH5, (1H-indol-3-yl)-methyl, halogenated (1H-indol-3-yl)-methyl, (4-hydroxyphenyl)-methyl, halogenated (4-hydroxyphenyl)-methyl, -CH-(CH3), 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, at any position of X, independently of any other position of X, is either O or S. R5 is selected from the list comprising methylsulfonyl, p-toluenesulfonyl; R6 is selected from the list comprising methylsulfonyl, p-toluenesulfonyl, -(C=NH)-NH2. Z1 and Z2 each represent a double bond or a single bond, provided that Z1 and Z2 are both single bonds, or only one of them is a single bond and the other is a double bond, Z1 is a single bond and Z2 is a double bond, or vice versa. Y1 is 3,7-indolylene or a halogenated 3,7-indolylene, Y2 is independently selected from the list of 3,6-indolylene, 1,4-phenoxylene, halogenated 3,6-indolylene, and halogenated 1,4-phenoxylene, and n is 1 or 2.

[0010] Darobactin is a modified heptapeptide with the amino acid sequence W1-N2-W3-S4-K5-S6-F7. NMR studies revealed two unusual macrocyclic bridges in darobactin: an unprecedented aromatic-aliphatic ether bond between the C7 indole of W1 and the β-carbon of W3, and a carbon-carbon bond between the C6 indole of W3 and the β-carbon of K5. A tryptophan-lysine bond is formed between the two unactivated carbons, which is unique for antibiotics. This bicyclic structure is characteristic and essential for darobactin A and all of the derivatives of the present invention.

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

[0012] The dar operon consists of a propeptide encoded by darA, a small relE-type ORF that may play a role in host resistance to compounds, darBCD, encoding an ABC-type transenvelope transporter, and darE, encoding a radical SAM enzyme. Radical SAM class enzymes catalyze free-radical-based reactions capable of linking unactivated carbons. This explains the formation of the tryptophan-lysine C-C bond in darobactin and its derivatives. Such a Trp-Lys C-C bond was recently reported in streptid, the peptide pheromone of Streptococcus thermophilus. While there is little overall homology between the two enzymes, DarE contains the SAM and SPASM domains characteristic of this group. The operon does not contain a separate enzyme for creating the ether bond in the first ring. The RiPP operon often encodes a protease that cleaves the active peptide, which was not present in the dar operon. Therefore, general proteolysis, autocleavage, or other proteases present in the producing strain may be involved in propeptide maturation. Surprisingly, the DarE radical SAM enzyme appears to catalyze the formation of both Trp-Lys C-C bonds and COC Trp-Trp ether bonds. The chemistries of these two reactions are quite distinct, and the mechanism of DarE catalysis clearly requires separate investigation. To link the putative BGC to darobactin production, we generated a markerless knockout mutant in which the entire BGC darABCDE was deleted from Photorhabdus khanii DSM3369 by double crossover. DAR production was abolished in the resulting mutant strain; no molecule with the corresponding molecular weight could be detected by MS (Figure 10). Importantly, darobactin was heterologously produced from the dar operon cloned into E. coli (Figure 10), demonstrating that the dar operon is sufficient to generate darobactin. We found that the dar operon is common in Photorhabdus, and we detected it in 16 different species for which genome sequences were available (Fig. 2a; Fig. 10).The dar operon was not present only in P. bodei. Synteny between the genome containing the dar locus and that of P. bodei helped to determine the boundaries of the operon (Figure 2a). We also tested darobactin production in several different Photorhabdus species and found that darobactin production was highest in P. khanii DSM 3369, a strain from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) culture collection. This indicates that strains with the BGC produce the compound.

[0013] We next expanded our search for dar-type operons in the bacterial genome sequence database (NCBI) using the propeptide and dar-encoding peptides as queries. These searches identified homologs of the dar operon that likely encode four darobactin analogs. Therefore, we proposed the names darobactin A for the first compound and darobactins B–E for the predicted analogs of this class of antibiotics. In Photorhabdus australis and Photorhabdus asymbiotica, sequence data suggest the production of darobactin B, which contains two amino acid changes at the N-terminus (SKSF→TKRF). In several Yersinia species, either the second amino acid (N→S) or the fifth amino acid (K→R), or both, are modified. We designated these analogs darobactins C, D, and E. Interestingly, the darobactin C sequence is present in Yersinia pestis, the causative agent of plague, and Y. frederiksenii from the human gut microbiome (respectively). The putative structures of darobactins B–E, deduced from their amino acid sequences, are shown in Figure 10. Of 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 are likely to emerge as more bacterial genomes are sequenced.

[0014] These experiments suggest that darobactin is a promising lead compound for developing therapeutic or pharmaceutical preparations against Gram-negative pathogens. The experimental results presented herein demonstrate that bicyclic heptapeptides derived from DAR are effective compounds (APIs) for pharmaceutical preparations against Gram-negative pathogens. Pharmaceutical preparations containing darobactin and / or bicyclic heptapeptides are effective in vertebrates, such as birds, fish, amphibians, reptiles, and mammals, as well as humans, suffering from infections caused by Gram-negative bacteria. The 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 invention and many other Gram-negative bacteria that belong to this group include, for example, 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 manufacture / produce bicyclic heptapeptides and derivatives, we selected several different heterologous hosts, cloned the respective DAR BGCs from different species, including the upstream regions of the BGCs, and finally generated DAR-resistant heterologous hosts to enhance DAR production. [Brief explanation of the drawings]

[0016] [Figure 1] Diagram of ribosomally synthesized darobactin (DAR) and post-translationally modified peptides (RiPPs) encoded by the dar operon. (a) DAR is a modified heptapeptide consisting of seven amino acids, W1-N2-W3-S4-K5-S6-F7, with an ether bond between W1 and W3 and a C-C bond between W3 and K5. (b) The dar BGC (total length 6.2 kb) consists of darA, encoding the precursor peptide; darBCD, encoding a subunit of the ABC transporter; and darE, encoding the radical S-adenosylmethionine enzyme (RaS). [Figure 2]Diagram of the darobactin biosynthetic gene cluster (BGC) in selected bacterial strains. (a) The BGC consists of the structural gene darA (blue), darBCD (a transporter-encoding gene, gray), and darE (encoding the radical SAM enzyme, orange). In addition, the ORF of a relE-like gene (black) can colocalize with the BGC at different positions. The BGC can be detected in a conserved gene region in most Photorhabdus strains. In addition, homologous BGCs (related genes show the same color code) exist in Yersinia, Vibrio, and Pseudoalteromonas strains. (b) Biosynthetic hypothesis. The propeptide encoded by darA consists of 58 amino acids. A bridge is introduced into the linear propeptide by DarE. In the next step, the leader and tail regions are cleaved, and darobactin is secreted by the ABC transporter DarBCD. (c) Amino acid sequences of the propeptides from selected bacterial strains are shown. The darobactin core peptide is highlighted in bold, and the amino acids involved in cross-linking are highlighted in bold red. Asterisks indicate stop codons. SEQ ID NO: 31 is darA from Photorhabdus temperata, SEQ ID NO: 32 is darA from Photorhabdus khanii, SEQ ID NO: 33 is darA from Photorhabdus australis, SEQ ID NO: 34 is darA from Photorhabdus laumondii, SEQ ID NO: 35 is darA from Yersinia frederiksenii, SEQ ID NO: 36 is darA from Vibrio tasmaniensis, and SEQ ID NO: 37 is darA from Pseudoalteromonas luteoviolacea. Boxes indicate consensus sequences from microorganisms that form a typical heptapeptide according to Formula I. [Figure 3]Diagram of the expression constructs generated in this study. The DAR BGC was cloned into the pRSFDuet™-1 vector under the control of the T7lac promoter. pZW-ADC3 contains a simplified DAR BGC derived from P. khanii HGB1456, with all intergenic regions removed, while pZW-ADC5 and pZW-ADC6 contain the native clusters derived from P. khanii HGB1456 and P. khanii DSM3369, respectively. A second copy of darA was added to pZW-ADC3 and pZW-ADC5, generating pZW-ADC3.2 and pZW-ADC5.2. Plasmid pZW-ADC7 contains the DAR BGC derived from P. khanii HGB1456 with an additional 200-bp upstream region of darA, and pZW-ADC8 contains the 605-bp upstream region. Plasmid pZW-ADC9 has the DAR BGC from P. khanii DSM3369 but lacks the darBCD; and pZW-YerA4 has the DAR BGC from Yersinia frederiksenii ATCC 33641. The black arrow indicates the T7lac promoter; the color codes for lacI, RSF, and the kanamycin resistance cassette (KanR) are kept constant. The lacI gene encodes the lac operon repressor, and RSF is the origin of replication derived from RSF1030, allowing the plasmid to be maintained at high copy number in cells. [Figure 4-1] FIG. 1 shows NMR chemical shifts (ppm) of darobactin B and additional NMR experimental data. [Figure 4-2] FIG. 1 shows NMR chemical shifts (ppm) of darobactin B and additional NMR experimental data. [Figure 4-3] FIG. 1 shows NMR chemical shifts (ppm) of darobactin B and additional NMR experimental data. [Figure 4-4] FIG. 1 shows NMR chemical shifts (ppm) of darobactin B and additional NMR experimental data. [Figure 4-5] FIG. 1 shows NMR chemical shifts (ppm) of darobactin B and additional NMR experimental data. [Figure 5A] NMR assignments of darobactin B. The structure of darobactin B is shown with the numbering of the NMR assignments. [Figure 5B] NMR assignments for darobactin B. Basic COSY and TOCSY correlations are shown (curved double-sided arrows indicate COSY correlations, thick bonds indicate TOCSY correlations). [Figure 5C] NMR assignments for darobactin B. Basic HMBC correlations (curved double-headed arrows indicate HMBC correlations, thick bonds indicate COSY / TOCSY correlations) are shown. [Figure 6] 1 is a diagram of additional exemplary bicyclic heptapeptides according to the present invention; these are not to be understood as limiting the scope of the invention (Bicyclic Heptapeptides III-VI). [Figure 7] 1 is a diagram of additional exemplary bicyclic heptapeptides according to the present invention; these are not to be understood as limiting the scope of the invention (bicyclic heptapeptides VII-X). [Figure 8] 1 is a diagram of additional exemplary bicyclic heptapeptides according to the present invention; these are not to be understood as limiting the scope of the invention (bicyclic heptapeptides XI-XIV). [Figure 9] 1 is a diagram of additional exemplary bicyclic heptapeptides according to the present invention; these are not to be construed as limiting the scope of the invention (bicyclic heptapeptides XV-XVIII). [Figure 10A] Figure 1. Diagram of the darobactin knockout strain and heterologous expression, as well as the predicted structure and production strains of darobactins A to E. Figure 2. Scheme of the double-crossover knockout vector pNB02 and the targeted genomic region. [Figure 10B] 1 is a scheme of the darobactin BGC expression plasmid. [Figure 10C]Diagram of test PCR of P. khanii DSM3369 ΔdarABCDE demonstrating loss of the darobactin BGC; left: amplification of darA (primers darA_f / r) yields a 177-bp fragment in WT and no fragment in mutant; right: when the BGC is deleted (primers DSMko_f / r), a 450-bp fragment is amplified after loss of pNB02 (indicated by sensitivity to Kan); positive controls: pNB03-darA–E and pNB02, respectively; positions of primers are shown in blue in scheme a. [Figure 10D] LC-MS extracted ion chromatograms (EICs) at m / z = 483.7089 ± 0.001. Yellow: P. khanii DSM3369 ΔdarABCDE+pNB03, Red: P. khanii DSM3369 ΔdarABCDE+pNB03-darA~E, Brown: E. coli BW25113+pNB03-darA~E, Blue: P. khanii DSM3369 WT. Inset: HRMS spectrum of the ion peak showing the doubly charged [M+2H]2+ ion corresponding to darobactin. [Figure 10E] This diagram depicts putative darobactin analogs B-E based on the amino acid sequence present in the darobactin BGC. The proposed producer was identified by a BLASTP search of the 7-amino acid sequence of darobactin A, confirming the presence of darBCDE downstream of the propeptide. Amino acid changes from darobactin A are highlighted in red. [Figure 10F] 1 is a table showing the propeptide sequences of various darobactin analogs. [Figure 11] 1 is a diagram of additional exemplary bicyclic heptapeptides according to the present invention; these are not to be understood as limiting the scope of the invention (Bicyclic Heptapeptides IXX-XXI). [Figure 12] 2 is a diagram of an additional exemplary bicyclic heptapeptide according to the present invention; these are not to be understood as limiting the scope of the invention (Bicyclic Heptapeptide XXII). DETAILED DESCRIPTION OF THE INVENTION

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

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

[0019] General description of the fermentation of Pseudoalteromonas strains for the production of bicyclic heptapeptides Pseudoalteromonas strains, such as Pseudoalteromonas luteoviolacea H33 and H33S, containing a biosynthetic gene cluster for expression of a bicyclic heptapeptide (e.g., eight genes encoding a propeptide, a modified radical SAM enzyme, and an FAD-dependent halogenase, one gene of unknown function, and four transporter genes) are cultured in a suitable medium, such as marine broth or a buffered artificial seawater formulation, a suitable carbon source, such as a sugar (e.g., glucose, rhamnose), a suitable nitrogen source, such as ammonium-containing salts (NH4Cl), or a complex carbon-nitrogen source, such as casitone or yeast extract. Cultures are incubated at a suitable temperature (4°C-40°C) in a volume of 20 mL-2 L, with or without shaking (or with / without shaking / mixing in a production-scale fermentor), for 1-7 days with or without a small molecule inducer, such as an N-acyl homoserine lactone derivative. The culture medium is separated from the cells, for example, by centrifugation, and the clarified medium is further processed for purification. Halogenated (e.g., Cl, Br, I, F) and non-halogenated bicyclic heptapeptides, as well as variants with or without an additional double bond (e.g., dehydrogenases), can be isolated from strains that possess a Photorhabdus BGC plus an FAD-dependent halogenase and one gene of unknown function. To produce halogenated derivatives, suitable enzymes can be used in vivo or in vitro, such as the FAD-dependent halogenase encoded in the BGC of Pseudoalteromonas luteoviolacea H33 and H33S. As known to those skilled in the art, the position of the halogen within the aromatic ring can be varied by using different halogenases catalyzing halogenation at specific positions.

[0020] Similarly, it is well known in the art that naturally occurring polyhalogenated (e.g., polybrominated, polychlorinated) compounds exist, for example, produced by marine organisms. Thus, the scope of the present invention expressly includes polyhalogenated bicyclic heptapeptides. Scheme 1 shows aromatic and heteroaromatic substructures of bicyclic heptapeptides, which can be monohalogenated or polyhalogenated.

[0021] [ka]

[0022] Scheme 2: Possible halogenated substructures IIa to IIf of bicyclic heptapeptides according to formula I, wherein the substituents R20, R21, R22, R23, R24, R25, R26, R27, R28, R29, R30, R31, R32, R33, R34, R35, R36, R37, R38 are, independently of one another, H, F, Cl, Br, I.

[0023] Substructure IIa represents a halogenated 3,7-indolylene as Y1 in formula I, substructure IIb represents a halogenated 3,6-indolylene as Y2 in formula I, substructure IIc represents a halogenated 1,4-phenoxylene as Y2 in formula I, and substructures IId, IIe, IIf and substructure IIc represent any halogenated aromatic or heteroaromatic substituent R1, R2, R3, R4 in formula I, in particular a (1H-imidazol-4-yl)-methyl halide, a -CH2-CH5 halide, a (1H-indol-3-yl)-methyl halide, a (4-hydroxyphenyl)-methyl halide, a benzyl halide.

[0024] Preparations containing FAD-dependent halogenases can be used for the halogenation of bicyclic heptapeptides. The enzymes can be heterologously overexpressed and purified by procedures well known to those skilled in the art. The purified enzyme (100 μL) was incubated with 10 μL of purified flavin reductase (which can be produced by procedures well known to those skilled in the art), 10 μM FAD, 2.4 mM NADH, 25 mM KBr, 0.15 mM substrate (e.g., darobactin A), and 10 mM potassium phosphate buffer (pH 7.2) in a total volume of 300 μL. The reaction was incubated at 18°C ​​for 24 hours and stopped by adding an equal volume of methanol prior to LC-MS analysis. The resulting product was brominated darobactin A.

[0025] General Method for Purification of Bicyclic Heptapeptides Method A: The cells are sedimented by centrifugation at 10,000 × g for 5 minutes, 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 minutes removes the insoluble portion. The crude extract is then subjected to reverse-phase flash chromatography (Interchim Puriflash 4125 chromatography system equipped with a Puriflash C18-HP 30 mm flash column, 1 hour gradient elution from 5% MeOH / H2O to 100% MeOH). The fraction containing DAR is further purified by HPLC. A C18 column (Macherey Nagel, EC 250 / 4.6 Nucleodur C18 Gravity-SB, 5 μm) is used in the following manner:

[0026] [Table 1]

[0027] Solvent A was 0.1% trifluoroacetic acid in water. The solvent was pumped into the HPLC system at a flow rate of 1 mL / min. The DAR was collected by a fraction collector from 15:90 to 17:10 min.

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

[0029] Those skilled in the art will appreciate that any type of peptide produced by the above-described fermentation process can be further chemically modified, if necessary, by using a suitable protecting group strategy, for example, by reacting them with different equivalents of Lawesson's reagent, O,O-diethylammonium phosphorodithioate salt, P4S10 / dimethicone, PSCl3 / HO / Et3N, or other reagents. By doing so, it is possible to obtain the replacement of one or even all amide bonds with thioamide bonds. Furthermore, conversion of an amino group to a sulfonamide is possible by treating (an optionally protected substrate) with mesyl chloride, tosyl chloride, or other suitable reagents. Therefore, bicyclic heptapeptides chemically modified in this manner or by other methods are also within the scope of the present invention.

[0030] Heterologous darobactin A expression and identification of the minimal biosynthetic gene cluster The scope of the present invention includes the generation of expression constructs by transferring the genes required for biosynthesis into any state-of-the-art expression vector, for example, by applying different promoters, ribosome binding sites, and additional elements for regulating expression, as will be apparent to those skilled in the art. Therefore, the following examples are not meant to limit the scope of the present invention.

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

[0032] It will be apparent to those skilled in the art that the scope of the present invention includes the use of any organism that has a biosynthetic gene cluster (BGC), amplifying or synthesizing the native or codon-optimized version of the BGC (in part or in whole), and cloning it into an expression vector. The vector is then transferred into an expression host. Therefore, the following examples are not meant 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_darobactin-halogenase codon-optimized. SEQ ID NO: 30 shows Pseudoalteromonas_luteoviolacea_protein_wo_homology_codon-optimized.

[0034] Plasmid pZW-ADC3 contains the DAR BGC genes without the intergenic region between darA and darB. Therefore, darA was amplified from Photorhabdus khanii HGB1456 using a primer pair; and darB-darE were amplified using their respective primers. All of these amplifications were performed 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 one-step isothermal DNA assembly protocol described by Gibson et al. was followed, with the minor modification of adding 1.2 μl of 10 U / μL T5 exonuclease instead of 0.64 μl. The final concentrations of the Gibson reaction mixture were as follows: 100 mM Tris-HCl pH 7.5, 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, and 0.02–0.5 pmol DNA fragment. The reaction mixture was then incubated at 50°C for 1 h. 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 the plasmid maintenance host by electroporation at 2.5 kV in a 0.2 cm electroporation cuvette using a Micropulser Electroporator (Bio-Rad, California, USA).

[0035] The second and third constructs 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 DNA was used as a template. Gibson assembly was carried out as described above.

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

[0037] Plasmids pZW-ADC7 and pZW-ADC8 contain the native DAR BGC from P. khanii HGB1456 with an additional 200 bp and 605 bp upstream region of darA, respectively. The BGC was 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 carries the DAR BGC from P. khanii DSM 3369 without the 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 carries the DAR BGC 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 propagation in E. coli TOP10 cells, all constructs were verified by test PCR and their restriction patterns.

[0041] A DAR-resistant strain was generated by introducing three point mutations, 1300A>G, 1334A>C, and 2113G>A, into the bamA gene. Previous studies have confirmed that these three point mutations result in a DAR-resistant phenotype, and DAR resistance increased to 128 μg / mL. This previous study is known to those skilled in the art. Mutations for creating resistant producer strains can be generated by means well known to those skilled in the art. The gene modified for this purpose can be, for example, bamA.

[0042] Expression constructs were transferred from the maintenance host, E. coli TOP10, to different expression hosts by electroporation. Transformation of the E. coli expression host was performed as described above for E. coli TOP10, except for V. natriegens Vmax™, which was transformed in a 0.1 cm electroporation cuvette at 900 V. After electroporation, strains were incubated for 1 hour in their respective growth medium and temperature. Cells were then plated onto LB(-ASW) plates containing kanamycin as the selection agent at the concentrations listed above, with chloramphenicol added when E. coli Rosetta™ (DE3) was used as the host. Individual single colonies were picked from the selection plates, and the presence of each expression plasmid was confirmed by PCR. Colonies carrying the correct construct were then inoculated into 3 mL of LB(-ASW) containing the required antibiotic 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, incubated at 37°C or 30°C until the OD600 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 with shaking at 180 rpm. Those skilled in the art will recognize that when an inducible promoter is used for expression, a non-resistant producer strain can be used without departing from the scope of the present invention, since expression can be induced when a certain cell density is reached. This ensures that the cells are not killed by antibiotic production.

[0043] DAR production was analyzed by UPLC-HRMS. A 1 mL aliquot was taken from the expression culture and centrifuged to separate the medium from the bacterial cells. The medium was lyophilized, 1 mL of methanol was added, and the mixture was sonicated for 30 minutes in a Bandelin Sonorex RK255 ultrasonic bath (Berlin, Germany) 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 a final 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 30 minutes of sonication. Next, 500 μL of deionized water was added, and sonication was continued for another 15 minutes. The solution was then 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 equipped with an Acquity UPLC BEH C18 1.7 μm (2.1 × 100 mm) column (Waters, Eschborn, Germany) and an Acquity UPLC BEH C18 1.7 μm VanGuard precolumn (2.1 × 5 mm; Waters, Eschborn, Germany) setup, coupled to a DAD detector and a micrOTOFQ II mass spectrometer (Bruker, Bremen, Germany). The LC portion was performed using a gradient (A: HO, 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. The column oven temperature was set to 45 °C. MS parameters were as follows: nebulizer gas 1.6 bar; gas temperature, 200 °C; gas flow rate, 8 L / min; capillary voltage, 4500 V; endplate offset, 500 V; and the measurement was performed in positive ion mode.

[0045] A DAR standard curve was generated by plotting the DAR peak area from extracted ion chromatograms (EICs) (relative to m / z values ​​of 483.7089 and 475.1956 ±0.01) against a series of DAR concentrations (2, 3, 4, 5, 10, 15, 20, 30, and 40 mg / L). DAR concentrations from heterologous expression cultures were quantified by calculating the peak area and interpolating it onto 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. A standard curve was measured for every batch analyzed by UPLC-HRMS to exclude technical differences between assays.

[0046] 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 to define a minimal DAR BGC, these genes were removed from the expression construct. Therefore, pZW-ADC9, a construct containing only darA and darE, was created. This experiment demonstrated that DAR could still be produced in the absence of darBCD. Furthermore, DAR was detected outside the cells.

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

[0048] (Heterologous expression of other bicyclic heptapeptides) Based on the expression construct exemplified above, amino acids of the heptapeptide can be replaced. This can be done specifically for individual amino acids, or in a randomized approach using primers to modify the sequence of the heptapeptide. Therefore, specific or degenerate primers (e.g., using triplets NNN or NNK for any proteinogenic amino acid to be 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 reaction contained the following: 34-34.5 μL water, 2.5 μL DMSO, 10 μL Q5 reaction buffer, 1 μL dNTPs, 0.5 μL forward primer (100 pmol / μL), 0.5 μL reverse primer (100 pmol / μL), 0.5 μL template DNA, and 0.5-1 μL Q5 DNA polymerase. 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; 30 cycles of steps 2-4; step 5, 72°C, 10 min; step 6, 4°C, infinity.

[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 a double bond according to formula I.

[0050] (Structure elucidation) All NMR data were recorded on a Bruker Avance III HD 600 MHz NMR spectrometer (Bruker BioSpin MRI GmbH, Ettlingen, Germany). Deuterium oxide (Deutero GmbH, Kastellaun, Germany) was used as the solvent for all NMR experiments. H NMR spectra were referenced to the literature solvent residual peaks. For C spectra, 3-trimethylsilyl-d4-propionic acid (TSPA) was used as an external standard, as known to those skilled in the art. Complete assignments were obtained using 2D experiments, including COSY (cosygpmfphpp), TOCSY (mlevetgp and mlevgpph19), H-C_HSQC (hsqcedetgpsisp2.3), and H-C_HMBC (hmbcetgpl3nd). To improve the resolution of the H-C HMBC spectra, additional experiments were performed using nonuniform sampling (NUS) and / or HO suppression. HO suppression was also applied to the recording of TOCSY spectra. NMR spectra were analyzed using the software TopSpin 3.6.0 (Bruker BioSpin MRI GmbH, Ettlingen, Germany).

[0051] (Identification of biosynthetic gene clusters) Direct screening of the core peptide sequence WNWSKSF was performed using the Basic Local Alignment Search Tool (BLAST) against all Photorhabdus genomes available in public databases. In P. temperata, the 7-amino acid sequence of darobactin is located near the C-terminus of an open reading frame encoding a 58-amino acid sequence 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 boundaries of the cluster were determined by comparison with the P. bodei genome, which does not contain an operon. Furthermore, the GC content of the dar cluster was significantly lower than the average GC content of the rest of the genome (32% vs. 45%). To identify other bacterial species potentially producing darobactin-like compounds, a search for homologous enzymes was performed in BLAST using the radical SAM protein sequence (DarE) as input. The genomic content of each hit was manually analyzed to confirm the presence of a DarA-like propeptide in the vicinity of the radical SAM protein. Additionally, searches using the propeptide DarA as input yielded identical hits.

[0052] (Generation and heterologous expression of darobactin deletion mutants) To delete the dar BGC (darABCDE) from the genome of the production strain Photorhabdus khanii DSM3369, a plasmid was constructed by assembly of five fragments that allow for markerless genome modification. Therefore, chromosomal DNA was isolated using the innuprep Bacteria DNA Kit (Analytik Jena, Jena, Germany).

[0053] (i) An upstream fragment and (ii) a downstream fragment of the BGC were amplified (approximately 1 kb in size) using the following primer pairs: 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), the origin of conjugal transfer (oriT) and the levansucrase gene sacB from Bacillus subtilis were amplified together from the 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 flanked by 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, which confers 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 using Q5 DNA polymerase (New England Biolabs, Ipswich, USA), gel-purified from 1% or 2% TAE agarose gel, and DNA was recovered using a Large Fragment DNA Recovery Kit (Zymo Research, Irvine, USA). All fragments were then fused by isothermal assembly to generate the plasmid pNB02. After assembly, E. coli WM3064 cells were transformed with pNB02 by electroporation, and correct assembly was confirmed by PCR and restriction analysis according to standard procedures. Conjugation between E. coli WM3064 and P. khanii DSM3369 was performed by growing both strains to an OD600 of approximately 0.6. After washing twice with LB medium, the cells were plated onto LB agar containing a 1:3 mixture of E. coli and P. khanii supplemented with diaminopimelic acid (0.3 mM) and incubated at 37°C for 3 hours, followed by overnight incubation at 0°C. The bacterial lawn was resuspended in LB medium and serially diluted and plated onto LB agar containing kanamycin (50 μg ml-1). Kanamycin-resistant single-crossover exconjugants were grown in LB medium to an OD600 of approximately 0.6. Expression of SacB was then induced by adding arabinose (0.2% w / v), followed by a 2-hour incubation.

[0055] The cultures were then plated onto LB agar supplemented with 0.2% (wt / vol) arabinose and 10% sucrose and incubated at 30°C for 48 hours. Individual single colonies were picked onto LBKan and LBAra / Suc agar. Sensitivity to kanamycin indicates loss of the plasmid, and thereby a successful double-crossover event. Clones were picked 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 called P. khanii DSM3369 ΔdarABCDE. The expression plasmid pNB03 was used for heterologous expression of the darobactin A BGC in E. coli and to complement P. khanii DSM3369 ΔdarABCDE. To avoid problems with the regulatory system between the propeptide and the modifying enzymes, all intergenic regions were removed, and the darA to darE genes were expressed in a simplified form under the control of the arabinose-inducible araB promoter.

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

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

[0059] To introduce the 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 SEQ ID NO:21: 5'-GCTCTAAGGAGGTTATAAAAATGCATAATACCTTAAATGAAACCGTTAAA-3', and SEQ ID NO:22: 5'-AATAGCATTCATTTATGGCTCTCCTTTTAAATTTCCTGGAAGCTTT-3' (iii) darB-darE was amplified using: SEQ ID NO:23: 5'-AAAGCTTCCAGGAAATTTAAAAGGAGAGCCATAAATGAATGCTATT-3', and SEQ ID NO:24: 5'-CGAAAACTCACGTTAAGGGATTACGCCGCGATGGTTTGTTTTATT-3'

[0060] All fragments were gel purified and assembled as described above. The resulting vector, pNB03-darABCDE, was transformed into E. coli TOP10 cells to confirm 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 harboring the expression plasmid, and E. coli ET pUB307 harboring the pUB307 conjugation helper plasmid was performed as described above (cell ratio 3:1:1). Because P. khanii DSM3369 is naturally resistant to carbenicillin and the kanamycin-resistant pUB307 lacks the bla promoter, final selection was performed on LB agar supplemented with kanamycin and carbenicillin. Kanamycin-resistant exconjugants were grown in LBKan, and the plasmid was isolated and its identity confirmed by PCR. For heterologous expression, the vector pNB03-darABCDE was electroporated into E. coli BW25113 (an arabinose non-utilizer). P. khanii DSM3369 WT, P. khanii DSM3369 ΔdarABCDE + pNB03, P. khanii DSM3369 ΔdarABCDE + pNB03-darABCDE, and E. coli + pNB03-darABCDE were then grown in LB or LBKan supplemented with 0.2% (wt / vol) arabinose for 5–7 days and analyzed by LCMS.

[0062] (Minimum inhibitory concentration (MIC)) MICs were determined by 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. baumannii ATCC19606, K. pneumoniae ATCC30104, and S. enterica ATCC13076 were adjusted to McFarland 1.0 and then diluted to 5 x 10 c.fu mL in MHIIB. Darobactin derivatives were screened in triplicate at 12 concentrations ranging from 64 to 0.03 μg mL. 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 by a colistin dilution series (16–0.007 μg mL).

[0063] Bacterial suspensions without standard antibiotics or darobactin were used as negative controls. After incubation (18 h, 180 rpm, 37 °C, 85% rH), cell growth was determined by measuring turbidity at 600 nm using a microplate spectrophotometer. The MIC was defined as the lowest concentration at which at least 85% growth inhibition was measured compared to the negative control.

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

[0065] Claim 1: A bicyclic heptapeptide of formula I: [ka] and / or a pharmaceutically acceptable salt, stereoisomer, tautomer or hydrate thereof, During the ceremony, R1, R2, R3, R4 are independently selected from the group 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, (1H-imidazol-4-yl)-methyl halide, -CH(CH3)(C2H5), -CH2-CH(CH3)2, -CH2-CH2-CH2-CH2NH3, -CH2-CH2-S-CH3, -CH2-C6H5, halide selected from the list comprising 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, benzyl halide, -CH2-CH2-CH2-NH-(C=NH)-NH2; - X, at any position of X, independently of any other position of X, is either O or S; - R5 is selected from the list comprising methylsulfonyl, p-toluenesulfonyl; - R6 is selected from the list comprising methylsulfonyl, p-toluenesulfonyl, -(C=NH)-NH2; - Z1 and Z2 are each a double bond or a single bond, provided a) Z1 and Z2 are both single bonds, or b) only one of them is a single bond and the other is a double bond, wherein: i) Z1 is a single bond and Z2 is a double bond, or ii) Z2 is a single bond and Z1 is a double bond; - Y1 is 3,7-indolylene or a halogenated 3,7-indolylene; Y2 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; Bicyclic heptapeptides.

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

[0067] Claim 3: A method for producing the bicyclic heptapeptide of claim 1, comprising: i) a fermentation step using a bicyclic heptapeptide-producing microorganism to provide a fermentation broth containing the bicyclic heptapeptide and microbial cells, wherein the microorganism is selected from the list of microorganisms comprising Photorhabdus spp., Photorhabdus laumondii, Photorhabdus khanii, Pseudoalteromonas spp., Pseudoalteromonas luteoviolacea, Pseudoalteromonas luteoviolacea, Yersinia spp., Escherichia spp., Vibrio spp.; ii) a separation step in which the fermentation broth is separated by sedimentation and / or centrifugation and / or filtration into an insoluble fraction containing microbial cells and / or microbial cell debris and a solution containing the bicyclic heptapeptide, wherein the microbial cells are or are not disrupted before applying the sedimentation and / or centrifugation and / or filtration to the fermentation broth; iii) a purification step of purifying the bicyclic heptapeptide contained within the solution obtained from the previous separation step ii) to provide a solution of purified bicyclic heptapeptide. A method comprising:

[0068] Claim 4: A method for producing the bicyclic heptapeptide of claim 3, wherein the purification step iii) of claim 3 comprises: a) drying the solution obtained from step ii) of claim 3 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, and dissolving the dried residue in deionized water to obtain a crude extract of the bicyclic heptapeptide; c) removing the insoluble portion of the crude extract of the previous step b) by sedimentation and / or filtration and / or centrifugation to obtain a solids-free crude extract; d) purifying the solid-free crude extract 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 the bicyclic heptapeptide of claim 3, wherein the purification step iii) of claim 3 comprises: a) contacting the solution obtained from step ii) of claim 3 with a 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 adsorbed bicyclic heptapeptide-loaded hydrophobic interaction material 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 organic solvent may or may not contain an acid in an amount between 0.001% and 1% by weight, d) concentrating the eluate obtained from step c) and removing the organic solvent by applying a vacuum and contacting the aqueous solution with a strong cation ion exchange material such that the bicyclic heptapeptide is adsorbed onto the cation ion exchange material; e) washing the bicyclic heptapeptide-loaded cation ion exchange material with an acid having a concentration between 0.001% and 1% by weight; f) eluting the bicyclic heptapeptide by applying an aqueous buffer solution having a pH value between pH 5 and pH 11 and collecting fractions containing the bicyclic heptapeptide; g) adsorbing the bicyclic heptapeptide obtained from the fractionation of step f) onto a hydrophobic interaction material in the same manner as described in step a); h) eluting the bicyclic heptapeptide with a gradient of HO and an organic solvent, the organic solvent being selected from the list comprising MeOH, MeCN, THF, acetone, ethanol, propanol; i) purifying the fraction containing the bicyclic heptapeptide obtained from step h) by applying HPLC using a gradient of HO and an organic solvent, the organic solvent being selected from the list comprising MeOH, MeCN, THF, acetone, ethanol, propanol. A method comprising:

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

[0071] Claim 7: The method for producing the bicyclic heptapeptide of claim 6, wherein the chemical modification is reacting 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 of claim 1, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

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

[0074] Claim 10: The pharmaceutical composition of any one of claims 8 to 9, which is administered topically, systemically, parenterally, subcutaneously, or transdermally, rectally, orally, intravaginally, intranasally, intrabronchially, intraocularly, intraaurally, intravenously, intramuscularly, or intraperitoneally.

[0075] Claim 11: The pharmaceutical composition of any one of claims 8 to 10, wherein the pharmaceutical composition is administered to a mammal suffering from a bacterial infection and for a sufficient period of time and with sufficient frequency to provide a beneficial effect to the mammal.

[0076] Claim 12: A pharmaceutical composition according to any one of claims 8 to 10, for administration 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] Level 13: Remove from the rest of the range, and leave Level 8 with 10 of them In particular, the phytochemical strain Pseudomonas Aettaginosa 、kleBLELA pneumoniae 、acaneisseria GONORRHOETHAE trachgella sonnei、salmonella Enterica Typhimurium lt2. Longum、bacteroides fragilis、Lactobacillus reuteri、Enterococcus faecalisおよびYersinia pestis、Pseudomonas、fluorescens、Pseudomonas acidovorans、Pseudomonas alkaligenes、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、Providence alcalifaciens、Providence rettgeri、Providence stuartii、Acinetobacter calcoaceticus、Acinetobacter haemolyticus、Yersinia enterocolitica、Yersiniapseudotuberculosis, 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 infections associated with the pharmaceutical composition.

[0078] 1. A pharmaceutical preparation comprising darobactin or a derivative thereof for use in vertebrates or humans suffering from infections caused by gram-negative bacteria.

[0079] 2. The pharmaceutical preparation of claim 1, wherein the Gram-negative bacteria are 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 not intended to limit the scope of the invention.

[0080] 3. The pharmaceutical preparation of claim 1 or 2, wherein the vertebrates include birds, fish, amphibians, reptiles, and mammals.

Claims

1. A bicyclic heptapeptide of formula I: 【Chemical 1】 and / or a pharmaceutically acceptable salt, stereoisomer, tautomer or hydrate thereof, During the ceremony, R1, R2, R3, R4 are each independently selected from the group 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, (1H-imidazol-4-yl)-methyl halide, -CH(CH3)(C2H5), -CH2-CH(CH3)2, -CH2-CH2-CH2-CH2NH3, -CH2-CH2-S-CH3, -CH2-C6H5, halide selected from the list comprising halogenated -CH2-C6H5, (1H-indol-3-yl)-methyl, halogenated (1H-indol-3-yl)-methyl, (4-hydroxyphenyl)-methyl, halogenated (4-hydroxyphenyl)-methyl, -CH-(CH3), 1-hydroxy-ethyl, hydroxy-methyl, sec-butyl, 1-acetamido, 1-thioacetamido, -CH2-CH2-NH-(C=NH)-NH2, benzyl, benzyl halide, -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; R5 is selected from the list comprising H, methylsulfonyl, p-toluenesulfonyl; R6 is selected from the list comprising H, methylsulfonyl, p-toluenesulfonyl, —(C═NH)—NH2; Z1 and Z2 are each a double bond or a single bond, provided a) Z1 and Z2 are both single bonds, or b) only one of them is a single bond and the other is a double bond, wherein: i) Z1 is a single bond and Z2 is a double bond, or ii) Z2 is a single bond and Z1 is a double bond; Y1 is 3,7-indolylene or a halogenated 3,7-indolylene; Y2 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, Bicyclic heptapeptides.

2. 2. The bicyclic heptapeptide of claim 1, which is darobactin.

3. 10. A method for producing the bicyclic heptapeptide of claim 1, comprising: i) a fermentation step using a bicyclic heptapeptide-producing microorganism to provide a fermentation broth containing the bicyclic heptapeptide and microbial cells, wherein the microorganism is selected from the list of microorganisms comprising Photorhabdus spp., Photorhabdus laumondii, Photorhabdus khanii, Pseudoalteromonas spp., Pseudoalteromonas luteoviolacea, Pseudoalteromonas luteoviolacea, Yersinia spp., Escherichia spp., Vibrio spp.; ii) a separation step in which the fermentation broth is separated by sedimentation and / or centrifugation and / or filtration into an insoluble fraction containing microbial cells and / or cell debris and a solution containing the bicyclic heptapeptide, wherein the microbial cells are or are not disrupted before applying sedimentation and / or centrifugation and / or filtration to the fermentation broth; iii) a purification step of purifying the bicyclic heptapeptide contained within said solution obtained from the previous separation step ii) to provide a solution of purified bicyclic heptapeptide. A method comprising:

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

5. 4. A method for producing the bicyclic heptapeptide of claim 3, wherein the purification step iii) of claim 3 comprises: a) contacting the solution obtained from step ii) of claim 3 with a 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 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 organic solvent may or may not contain an acid in an amount between 0.001% and 1% by weight, d) concentrating the eluate obtained from step c) and removing said organic solvent by applying a vacuum and contacting the aqueous solution with a strong cation ion exchange material such that the bicyclic heptapeptide is adsorbed onto the cation ion exchange material; e) washing the bicyclic heptapeptide-loaded cation ion exchange material with an acid having a concentration between 0.001% and 1% by weight; f) eluting the bicyclic heptapeptide by applying an aqueous buffer solution having a pH value between pH 5 and pH 11 and collecting fractions containing the bicyclic heptapeptide; g) adsorbing the bicyclic heptapeptide obtained from the fractionation of step f) onto a hydrophobic interaction material in the same manner as described in step a); h) eluting the bicyclic heptapeptide with a gradient of HO and an organic solvent, said organic solvent being selected from the list comprising MeOH, MeCN, THF, acetone, ethanol, propanol; i) purifying the fraction containing the bicyclic heptapeptide obtained from step h) by applying HPLC using a gradient of HO and an organic solvent, said organic solvent being selected from the list comprising MeOH, MeCN, THF, acetone, ethanol, propanol. A method comprising:

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

7. 7. The method for producing a bicyclic heptapeptide according to claim 6, wherein the chemical modification is by reacting the bicyclic heptapeptide with Lawesson's reagent or tosyl chloride or mesyl chloride.

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

9. 9. The pharmaceutical composition of claim 8, further comprising at least one pharmaceutically acceptable carrier, excipient, or diluent.

10. 10. The pharmaceutical composition of any one of claims 8 to 9, in the form of topical, systemic, parenteral, subcutaneous or transdermal, rectal, oral, vaginal, intranasal, intrabronchial, intraocular, intraaural, intravenous, intramuscular, or intraperitoneal administration.

11. administered to a mammal suffering from a bacterial infection, 11. The pharmaceutical composition of any one of claims 8 to 10, comprising administering said pharmaceutical composition to said mammal at a sufficient frequency and for a sufficient period of time to provide a beneficial effect to said mammal.

12. administered to a mammal suffering from a bacterial infection, 11. The pharmaceutical composition of any one of claims 8 to 10, wherein the bacterial infection is an infection involving at least one type / strain of Gram-negative bacteria.

13. administered to a mammal suffering from a bacterial infection, The bacterial infections 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 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, Bordetellapertussis 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, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron、Bacteroides uniformis、Bacteroides eggerthii、Bacteroides splanchnicus is a specimen of 11-year-old splanchnicus.

14. A pharmaceutical preparation comprising darobactin or a derivative thereof for use in vertebrates or humans suffering from infections caused by gram-negative bacteria.

15. 15. The pharmaceutical formulation of claim 14, 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.

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