Patent Application
20230348539
The invention relates to bicyclic heptapeptide antibiotics, derivatives thereof, and their manufacturing (production) and usage as active pharmaceutical ingredients (APIs) in medicaments against Gram-negative pathogens.
The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens. These microorganisms have a highly restrictive permeability barrier, which limits penetration of most compounds. As a result, the last class of antibiotics acting against Gram-negative bacteria was developed in the 60s years of the last century.
It is difficult to find compounds acting against Gram-negative bacteria. Gram-negative bacteria have an outer membrane to protect themselves from unwanted compounds. This membrane is decorated with an outer layer of negatively charged lipopolysaccharide (LPS), which serves as a barrier for large and hydrophobic compounds. The inner membrane limits the permeability by hydrophilic compounds.
As a result, the combined barrier (outer membrane plus inner membrane) restricts all molecules, and nutrients enter through outer membrane porins and specialized transporters. Drugs that leak through the barrier are extruded by trans-envelope multidrug resistance pumps (MDRs) that recognize amphiphilic compounds, which most drugs are in order to pass hydrophilic regions of cells as well as hydrophobic regions (e. g. membranes). Thus, there is great need within the state of the art for APIs with different working-mechanisms, being unhindered by the triple-defense-line of Gram-negative bacteria (outer membrane, inner membrane and MDRs).
Darobactin A (DAR) is a ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotic, which was initially 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 highly promising lead structure for the development of novel antibacterial therapeutic agents. It targets the outer membrane protein BamA and is therefore specific for Gram-negative bacteria. This, together with the convincing in vivo activities in mouse infection models, makes it a particular promising candidate for further research. To improve compound supply for further investigation of DAR and to enable production of novel derivatives, establishment of an efficient and versatile microbial production platform for these class of RiPP antibiotics is highly desirable. Herein design and construction of a heterologous production and engineering platform for DAR and/or its derivatives is revealed, which will ensure production yield and facilitates structure modification approaches. The known Gram-negative workhorses Escherichia coli and Vibrio natriegens were tested as heterologous hosts. In addition to that, DAR producer strains were generated and optimization of the expression constructs yielded production titers of DAR showing around 10-fold increase in concentration (titer) and 5-fold decrease in fermentation time compared to the product descriptions according to the state of the art. Also, the identification of the minimal DAR BGC is revealed, since only two genes are necessary for heterologous production of the RiPP (Darobactin and/or its derivatives).
DAR, the novel antibiotic that selectively kills Gram-negative pathogens, e.g. 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), was discovered in Photorhabdus khanii HGB1456. Other natural occurring derivatives, e.g. darobactin B, as well as brominated variants resp. derivatives show 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). Experimental proof was provided that DAR binds to BamA, which is the central component of the OM β-barrel assembly machinery. It helps to fold and insert β-barrel proteins such as porins into the OM. If this chaperone-like function is impaired, it will result in the disruption of OM formation. In addition to its good in vitro activity, DAR showed promising efficacy in mouse septicemia and thigh infection models without cytotoxic effects. Therefore, DAR has emerged as a promising drug lead.
An objective of the present invention is to provide new substances that can be used as pharmaceutical active ingredients (APIs) for medicaments being effective against Gram-negative bacteria in a new and unexpected way and against which there are therefore no resistances existing among Gram-negative bacteria. Surprisingly, bicyclic heptapeptides according to formula I are exerting characteristics/interactions with cellular components providing these positive effects: They act against an attractive, but also highly unusual target—the BamA chaperone and translocator. The BamA chaperone and translocator helps fold and insert p-barrel proteins such as porins into the outer membrane. BamA itself is an outer membrane p-barrel protein. Drugs in general, and natural products in particular, normally target enzymes with their well-defined catalytic centers, rather than chaperones. Darobactin is a large molecule, which is probably necessary to interfere with the protein-protein binding between BamA and its substrates. The location of the target on the surface resolves the intractable problem of penetration across the permeability barrier of Gram-negative bacteria, none is necessary in the case of darobactin. There are only two essential proteins exposed on the surface of the outer membrane—BamA; and LptD. Therefore, bicyclic heptapeptides that target BamA are bypassing the triple-defense-line of Gram-negative bacteria (outer membrane, inner membrane and MDRs) effectively and offer a new mode of action for antibiotic activity.
In Scheme I the substituents and indexes have the following values: R1, R2, R3, R4 are independently from 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-imidazole-4-yl)-methyl, halogenated (1H-imidazole-4-yl)methyl, —CH(CH3)(C2H5), —CH2—CH(CH3)2, —CH2—CH2—CH2—CH2NH3, —CH2—CH2—SCH3, —CH2—C6H5, halogenated —CH2—C6H5, (1H-indol-3-yl)-methyl, halogenated (1 Hindol-3-yl)-methyl, (4-hydroxyphenyl)-methyl, halogenated (4-hydroxyphenyl)-methyl, —CH—(CH3)2, 1-hydroxy-ethyl, hydroxy-methyl, sec-butyl, 1-acetamide, 1-thioacetamide, —CH2—CH2—NH—(C═NH)—NH2, benzyl, halogenated benzyl, —CH2—CH2—CH2—NH—(C═NH)—NH2. X is at any position of X independently from any other position of X 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, Z2 each are a double bond or a single bond in such a way that Z1 and Z2 both are single bonds or only one of them is a single bond and the other one is a double bond at the same time whereat Z1 is the single bond and Z2 is the double bond or vice versa. Y1 is 3,7-indolylene or halogenated 3,7-indolylene and Y2 is independently selected from the list 3,6-indolylene, 1,4-phenoxylene, halogenated 3,6-indolylene, halogenated 1,4-phenoxylene and n is 1 or 2.
Darobactin is a modified heptapeptide with an amino acid sequence W1—N2—W3—S4—K5—S6—F7. NMR studies revealed two unusual macrocycle cross linkages in darobactin: an unprecedented aromatic-aliphatic ether linkage 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. The tryptophan-lysine bond is made between two unactivated carbons, which is unique for an antibiotic. This bicyclic structure is characteristic and essential for darobactin A and all inventive derivatives.
Directly comparing the sequence of this 7 amino acid peptide (darobactin A) against the genome of P. temperata reveals a perfect match near the C-terminus of an open reading frame coding for a 58 amino acid long peptide. The ribosomal synthesis of darobactins suggests that the amino acid backbone is in L-configuration. The macrocycle cross linkages generate two chiral centers at the β-carbons of W3 and K5, which have R and S configurations, respectively, based on NOE correlations (
This dar operon consists of the propeptide encoded by darA, a small re/E-type ORF which may play a role in host resistance to the compound, darBCD coding for an ABC-type transenvelope exporter, and darE for a radical SAM enzyme. The radical SAM class of enzymes catalyze free radical-based reactions that can link unactivated carbons. This would explain 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 a peptide pheromone, streptide, from Streptococcus thermophilus. There is little overall homology between the two enzymes, but DarE contains the SAM and SPASM domains characteristic for this group. The operon does not contain a separate enzyme for making the ether bond in the first ring. RiPP operons often code for a protease that cleaves out the active peptide; this was not present in the dar operon. Hence, generic proteolysis, self-cleavage or other proteases present in producer strains can be involved in maturation of the propeptide. Surprisingly, it appears that the DarE radical SAM enzyme catalyzes 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 a separate investigation. To link the putative BGC with darobactin production, we generated a markerless knockout mutant in which the complete BGC darABCDE was deleted from Photorhabdus khanii DSM3369 by double crossover. DAR production was abolished in the resulting mutant strain; no molecule with a corresponding molecular weight could be detected by MS (
We then expanded the search for dar-type operons in databases of bacterial genome sequences (NCBI), using the propeptide and the dar encoding peptide as queries. The two searches identified homologues of the dar operon that appear to code for four darobactin analogs. We therefore propose the name darobactin A for the first compound, and darobactin B-E for the predicted analogs of this class of antibiotics. In Photorhabdus australis and Photorhabdus asymbiotica, the sequence data suggest production of darobactin B, which contains two amino acid changes on the N-terminus (SKSF→TKRF). In multiple Yersinia species either the second amino acid (N→S) or the fifth amino acid (K→R), or both, are modified. We named these analogs darobactin C, D, and E. Interestingly, darobactin C sequence is present in Yersinia pestis, the causative agent of plague, and by (resp. in) Y. frederiksenii from the human gut microbiome. The putative structures of darobactin B-E deduced from the amino acid sequence are shown in
These experiments suggest that darobactin is a promising lead compound for developing a therapeutic or a medical preparation against Gram-negative pathogens. The experimental results presented herein show that bicyclic heptapeptides derived from DAR are effective compounds (APIs) for medical preparations against Gram-negative pathogens. A medical preparation comprising darobactin and/or bicyclic heptapeptides is effective in vertebrates like birds, fishes, amphibians, reptiles and mammals and human suffering from an infection caused by Gram-negative bacteria. The Gram-negative bacteria are selected from the group 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 be restricting the scope of the invention—there are many other Gram-negative bacteria also belonging to this group, 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, 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 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.
For manufacturing/production of bicyclic heptapeptides and derivatives we choose several different heterologous hosts, cloned the respective DAR BGC from different species, including upstream regions of the BGC, and lastly created a DAR-resistant heterologous host to boost DAR production.
The following embodiments of the invention are examples of preferred embodiments which are nevertheless not meant to be confining the scope of the invention. It is obvious to the person skilled in the art, that similar microbial strains to the ones revealed herein can also be used without leaving the scope of the invention. The same is true for the chemical modifications revealed herein below, the steps of the manufacturing process and the presented pharmaceutical formulations and ways of application.
General Description of Fermentation of Photorhabdus Spp. for Production of Bicyclic Heptapeptides:
Photorhabdus sp. strains are inoculated in a suitable growth medium (e.g. 3 mL lysogeny broth: 10 g tryptone, 5 g yeast extract, 5 g NaCl) and incubated to promote growth. An aliquot of this pre-culture is used to inoculate the main culture (e.g. LB medium in shaking flasks and incubated to promote growth until harvesting.
General Description of Fermentation of Pseudoalteromonas Strains for Production of Bicyclic Heptapeptides:
Pseudoalteromonas strains such as Pseudoalteromonas luteoviolacea H33 and H33S containing the biosynthetic gene cluster corresponding to the expression of bicyclic heptapeptides (e.g., composed of 8 genes coding for the propeptide, the modifying radical SAM enzyme, an FAD-dependent halogenase, one gene of unknown function and four transporter genes), are cultivated in suited medium, such as Marine Broth or similar medium containing a buffered artificial seawater formulation, a suited carbon source, e.g. sugars (such as glucose, rhamnose), a suited nitrogen source, e.g. ammonium containing salts (such as NH4Cl) or a complex carbon and nitrogen source, e.g. casitone or yeast extract. Cultures are incubated at a suited temperature (4° C.-40° C.) in volumes from 20 mL to 2 L with or without shaking (or in a production-scale fermenter with or without stirring/mixing) and with or without addition of small molecule inducers such as N-acyl-homoserinelactone derivatives for 1 to 7 days. Culture medium is separated from the cells, e.g. by centrifugation, and cleared medium is further processed for purification. From strains carrying the BGC with an FAD-dependent halogenase and one gene of unknown function in addition to the Photorhabdus BGC, halogenated (e.g. Cl, Br, I, F) and non-halogenated bicyclic heptapeptides and variants either carrying or not carrying an additional double bond (e.g. dehydro-derivatives) can be isolated. For the generation of halogenated derivatives, suitable enzymes, as the FAD-dependent halogenase encoded in the BGCs of Pseudoalteromonas luteoviolacea H33 and H33S, can be used in vivo or in vitro. As it is known to the person skilled in the art, the position of the halogen within the aromatic ring can be altered by the usage of different halogenases, which are catalyzing halogenation at a specific position.
It is also well known within the state of the art that there are also naturally existing polyhalogenated (e. g. polybrominated, polychlorinated) compounds, e.g. produced by marine organisms. Therefore, the scope of the invention obviously also comprises polyhalogenated bicyclic heptapeptides. Scheme 1 shows the aromatic and heteroaromatic sub-structures of the bicyclic heptapeptides which are possibly mono- or poly-halogenated.
Scheme 2: possible halogenated sub-structures IIa-IIf of bicyclic heptapeptides according to formula I whereat substituents R20, R21, R22, R23, R24, R25, R26, R27, R28, R29, R30, R31, R32, R33, R34, R35, R36, R37, R38 are independently from each other H, F, Cl, Br, I.
Sub-structure IIa is describing halogenated 3,7-indolylene as Y1 in formula I, sub-structure IIb is describing halogenated 3,6-indolylene as Y2 in formula I, sub-structure IIc is describing halogenated 1,4-phenoxylene as Y2 in formula I and sub-structures IId, IIe, IIf as well as sub-structure IIc are describing any halogenated aromatic or heteroaromatic substituent R1, R2, R3, R4 in formula I, especially: halogenated (1H-imidazole-4-yl)-methyl, halogenated —CH2—C6H5, halogenated (1H-indol-3-yl)-methyl, halogenated (4-hydroxyphenyl)-methyl, halogenated benzyl.
Preparations containing the FAD-dependent halogenase can be used for halogenation of bicyclic heptapeptides. Enzyme can be heterologously overexpressed and purified by procedures well known by the person skilled in the art. Purified enzyme (100 μL) was incubated with 10 μL purified Flavin reductase (Enzyme can be generated by procedures well known by the person 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 24 h at 18° C. and stopped by adding an equal amount of methanol bevor LCMS analysis. The resulting product was brominated darobactin A.
General Methods for Purification of Bicyclic Heptapeptides:
Method A:
Cells are pelleted 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 second washing step, the methanol is removed completely and deionized water is added to dissolve the crude extract. Centrifugation step at 10,000×g for 5 minutes is done to remove insoluble part. Then, the crude extract is subjected to reverse phase flash chromatography (Interchim Puriflash 4125 chromatography system equipped with a Puriflash C18-HP30 mm Flash column, gradient elution of 5% MeOH/H2O to 100% MeOH over 1 h). 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, with the following method:
Solvent A is 0.1% trifluoroacetic acid in water. The solvents are pumped through the HPLC system with the flowrate of 1 mL/min. DAR is collected by fraction collector from 15.90-17.10 minutes.
Method B:
Cleared medium is applied to a hydrophobic interaction material (e.g. C18 silica, Amberlite XAD-16N), washed with H2O and eluted with a mixture of organic solvent (e.g. MeOH, MeCN) and H2O with or without 0.1% formic acid. Eluate is concentrated and organic solvent is removed in vacuo and the now aqueous elution is loaded to a strong cation ion exchange material (e.g. SP Sepharose XL). Subsequently, this material is washed with 0.1% formic acid and the material bound to the column is eluted using a suited buffer system such as NH4CH3COO with pH 5 to pH 11. Fractions are analysed by LCMS and fractions containing the compound of interest are pooled, bound to a hydrophobic interaction material (e.g. C18 silica, Amberlite XAD-16N) and eluted in a gradient with H2O and an organic solvent (e.g. MeCN). Peaks are collected based on UV absorption and collected fractions are analysed by LCMS. Peptides of interest containing fractions are separated by HPLC with a gradient of H2O and a suited organic solvent (e.g. MeOH, MeCN). Peaks are collected to yield the pure compounds of interest.
It is well known to the person skilled in the art that peptides of any type being produced by fermentation processes as described above can additionally be chemically modified, e.g. by reacting them with different equivalents of Lawson's Reagent, O,O-diethyl ammonium phosphorodithioate salt, P4S10/dimethicone, PSCl3/H2O/Et3N, or other reagents, optionally supported by a suitable protection group strategy. By doing so it is possible to acquire substitution of one or even all amide bonds by thioamide-bonds. Furthermore, conversion of amino groups to sulfonamides is possible by treatment (of the optionally protected substrates) with mesyl chloride, tosyl chloride or other suitable reagents. Bicyclic heptapeptides chemically modified in this or in other ways are therefore also comprised by the scope of the invention.
Heterologous Darobactin a Expression and Identification of the Minimal Biosynthetic Gene Cluster
The scope of the invention comprises the generation of expression constructs, applying e.g. different promoters, ribosomal binding sites, and further elements for regulation of expression, by transferring the genes needed for the biosynthesis into any state of the art expression vector, as is obvious to the person skilled in the art. Therefore, the following example is not meant to be confining the scope of the invention.
Exemplarily several constructs were generated during this project for heterologous expression of DAR (
It is obvious to the person skilled in the art that the scope of the invention comprises the use of any organism carrying the biosynthetic gene cluster (BGC) and amplifying or synthesizing a native or codon-optimized version of the BGC (parts or complete) and cloning it into an expression vector. The vector is then transferred into an expression host. Therefore, the following example is not meant to be confining the scope of the invention.
SeqID 25 shows codon-optimized darE from Photorhabdus_namnaonensis. SeqID 26 shows codon-optimized darA from Photorhabdus_namnaonensis. SeqID 27 shows codon-optimized darA from Pseudoalteromonas_luteoviolacea. SeqID 28 shows codon-optimized darE from Pseudoalteromonas_luteoviolacea. SeqID 29 shows Pseudoalteromonas_luteoviolacea_Darobactin-halogenase_codon-optimized. SeqID 30 shows Pseudoalteromonas_luteoviolacea_protein_wo_homology_codon-optimized.
Plasmid pZW-ADC3 carries the genes of the DAR BGC without the intergenic region between darA and darB. Therefore, darA was amplified from Photorhabdus khanii HGB1456 using a primer pair; and darB to darE was amplified using respective primers, all these amplifications being performed according to methods as are known to the person skilled in the art. pRSFDuet™-1 was linearized using NdeI and AvrII restriction enzymes (New England Biolabs, Ipswich, USA) to insert the two purified fragments 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 was followed, with the minor modification that 1.2 μl of 10 U/μL T5 exonuclease was added instead of 0.64 μL. Therefore, the final concentration of the Gibson reaction mix was the following: 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 fragments. This reaction mix was then incubated at 50° C. for one hour. After the isothermal assembly, the reaction was dialyzed using a 0.025 μm nitrocellulose membrane (Merck™ MF-Millipore™, Ireland), and subsequently transferred to E. coli TOP10 cells as a plasmid maintenance host by electroporation using Micropulser Electroporator (Bio-Rad, California, USA) in a 0.2 cm electroporation cuvette at a voltage of 2.5 kV.
The second and third construct, carries the native DAR BGC from Photorhabdus khanii HGB1456 (pZW-ADC5) and Photorhabdus khanii DSM3369 (pZW-ADC6), respectively. For both constructs, the respective BGC was amplified by PCR according to methods as are known to the person skilled in the art; therefore, the respective bacterial genomic DNA was used as template. Gibson Assembly was performed as described above.
Plasmids pZW-ADC3.2 and pZW-ADC5.2 were created by restriction-digest of pZW-ADC3 and pZW-ADC5. Each plasmid was restricted using NcoI and NotI (New England Biolabs, Ipswich, USA). Then, an additional codon optimized version of Photorhabdus sp. darA (SeqID 26) was inserted into the first multiple cloning site of the pRSFDuet™-1 based vectors by Gibson Assembly.
The plasmids pZW-ADC7 and pZW-ADC8 carry the native DAR BGC from P. khanii HGB1456 with an additional 200 bp and 605 bp upstream region of darA, respectively. The BGCs were amplified from the bacterial genomic DNA by PCR according to methods as are known to the person skilled in the art. The respective inserts were assembled to the NdeI-AvrII linearized pRSFDuet™-1 using Gibson Assembly.
The plasmid pZW-ADC9 carries the DAR BGC from P. khanii DSM3369 without the transporter genes, i.e. only darA and darE. Both fragments were amplified by PCR and assembled to the NdeI-AvrII linearized pRSFDuet™-1 using Gibson Assembly.
pZW-YerA4 carries the DAR BGC derived from Yersinia frederiksenii ATCC 33641 that was amplified by PCR. The amplificate was also assembled to the NdeI-AvrII linearized pRSFDuet™-1 by using Gibson assembly.
Following assembly and propagation in E. coli TOP10 cells, all constructs were checked by test PCR and by their restriction pattern.
The DAR resistant strain was generated by introduction of three point mutation into the bamA gene, which are 1300A>G, 1334A>C, and 2113G>A. That these three point mutations result in a DAR resistant phenotype was confirmed by a previous study, whereby DAR resistance increased to 128 μg/mL. This previous study is known to the person skilled in the art. Mutations to create a resistant producer strain can be generated by procedures well known by the person skilled in the art. The gene to be modified for this purpose can be—for example—bamA.
The expression constructs were transferred from the maintenance host, E. coli TOP10, into different expression hosts by electroporation. The transformation of E. coli expression hosts was performed as described above for E. coli TOP10, while the transformation of V. natriegens Vmax™ was done in a 0.1 cm electroporation cuvette at a voltage of 900 V. After the electroporation, the strains were incubated for 1 hour in their respective growth medium and temperature. Then, they were plated on LB(−ASW) plates containing kanamycin as the selective agent with the aforementioned concentration, with the addition of chloramphenicol when E. coli Rosetta™ (DE3) was used as the host. Single colonies were picked from the selective plates and the presence of the respective expression plasmid was confirmed by PCR. Colonies with correct constructs were then inoculated in 3 mL LB(−ASW) containing required antibiotics and incubated at 37° C. or 30° C. overnight. 500 μL of this pre-culture were used to inoculate 50 mL of fresh LB(−ASW) medium containing kanamycin, incubated at 37° C. or 30° C. until an OD600 of 0.4-0.6 was reached, and then induced with IPTG (final concentration of 0.5 mM). After IPTG induction, the cultures were incubated at 30° C. with 180 rpm shaking. The person skilled in the art knows that if an inducible promoter is used for expression one can use non-resistant producer strains without leaving the scope of the invention, because expression can be induced when a certain cell density is reached. Thereby the cells are not killed by the production of the antibiotic.
DAR production was analyzed by UPLC-HRMS. From the expression culture, a 1 mL aliquot was taken and centrifuged to separate the medium and the bacterial cell. The medium was lyophilized, 1 mL 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 deionized water. After a final centrifugation at 10,000 g for 5 minutes, the sample was ready to be injected to the UPLC-HRMS system. To prepare the sample from the cell pellet, 500 μL of methanol was added prior to sonication for 30 minutes. Then, 500 μL of deionized water was added, and the sonication was continued for another 15 minutes. Thereafter, the solution was centrifuged to pellet the insoluble part and the supernatant was injected to the UPLC-HRMS system.
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 Pre-Column (2.1×5 mm; Waters, Eschborn, Germany) setup coupled to a DAD detector and a micrOTOFQ II mass spectrometer (Bruker, Bremen, Germany). The LC part was run using a gradient (A: H2O, 0.1% FA; B: MeCN, 0.1% FA; Flow: 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 to 45° C. MS parameters were as follows: nebulizer gas 1.6 bar; gas temperature, 200° C.; gas flow, 8 L/min; capillary voltage, 4500 V; endplate offset, 500 V; measurement was done in positive ion mode.
A DAR standard curve was generated by plotting the peak area of DAR from the extracted ion chromatogram (EIC) (for the m/z of 483.7089 and 475.1956±0.01) to a series of DAR concentrations (2, 3, 4, 5, 10, 15, 20, 30, 40 mg/L). The DAR concentration from a heterologous expression culture was quantified by calculating the peak area and interpolating it to the DAR standard curve. The linear range for this quantification method was 3 μg/mL to 30 μg/mL. Therefore, the peak area below the border was not converted to concentration. The standard curve was measured with all batches that were analyzed by UPLC-HRMS to exclude technical differences between measurements.
The three genes darB, darC and darD are coding for subunits of an ABC transporter. To answer the question whether these transporter genes play an additional role to the biosynthesis of DAR and to define the minimum DAR BGC, these genes were removed from the expression construct. Therefore, pZW-ADC9, a construct that carries only darA and darE, was created. This experiment showed that without darBCD, DAR could still be produced. Furthermore, DAR was detected outside of the cell.
It was evaluated if the transporter-encoding genes darBCD are essential for the heterologous DAR expression or if darA, encoding the precursor peptide and darE, encoding the radical SAM modification enzyme, are sufficient. Deletion of darBCD did not abolish DAR production. However, the yield was lower (1.5-fold) than the one reached with the construct including transporter genes (pZW-ADC6). Most interestingly, DAR was also present in the medium, even without the transporter-encoding genes. On the one hand, this clearly defines the minimum DAR BGC, which consists of only darA and darE. On the other hand, it became clear that in E. coli DAR is present outside the cell even without the specific heterologous transporter genes darBCD.
Heterologous Expression of Other Bicyclic Heptapeptides
Based on expression constructs as exemplified above, the amino acids of the heptapeptide can be exchanged. 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 degenerated primers (e.g., using the triplet 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 example, a suitable PCR mix for a 50 μL scale reaction contained: Water: 34-34.5 μL, DMSO: 2.5 μL, Q5 reaction buffer: 10 μL, dNTPs: 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: step1 98° C., 10 min; step 2 98° C., 10 sec; step 3 65° C., 20 s; step 4 72° C., 7 min; 30 cycles step 2-4); step 5 72° C., 10 min; step 6 4° C., ∞.
In that way, all possible combinations of amino acids according to formula I can be incorporated into the heptapeptide. Furthermore, these derivatives can also be halogenized or modified by the presence of a double bond, according to formula I.
Structure Elucidation
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 solvent. 1H NMR spectra were referenced to the solvent residual peak according to the literature. For referencing of 13C spectra 3-trimethylsilyl-d4-propionic acid (TSPA) was employed as an external standard as is known to the person skilled in the art. Complete assignments were obtained using 2D experiments including COSY (cosygpmfphpp), TOCSY (mlevetgp and mlevgpph19), 1H-13C_HSQC (hsqcedetgpsisp2.3), and 1H-13C_HMBC (hmbcetgpl3nd). To improve resolution of 1H-13C_HMBC spectra, additional experiments were performed using non-uniform sampling (NUS) and/or H2O suppression. H2O suppression was also applied for the recording of TOCSY spectra. Analysis of NMR spectra was performed using the software TopSpin 3.6.0 (Bruker BioSpin MRI GmbH, Ettlingen, Germany).
Identification of Biosynthetic Gene Cluster
A direct screening for the core peptide sequence WNWSKSF was done on all Photorhabdus genomes available in public databases using Basic Local Alignment Search Tool (BLAST). In P. temperata the seven amino acid sequence of darobactin was located close to the C-terminus of an open reading frame coding for 58 amino acids, upstream of an ABC transporter and a radical SAM enzyme, suggesting a RiPP operon. This putative BGC was identified in the other darobactin producers, e.g. P. luminescens DSM-3368 and P. khanii DSM-3369. The boundaries of the cluster were determined by comparison with the P. bodei genome, which did not contain the operon. Furthermore, the GC content of the dar cluster was clearly lower than the rest of the average GC content in the genome (32% vs 45%). In order to identify other bacterial species that potentially produce darobactin-like compounds, homologous enzymes were searched using the radical SAM protein sequence (DarE) as input in BLAST. The genomic context of each hit was analyzed manually to confirm the presence of a DarA-like propeptide in the vicinity of the radical SAM protein. In addition, a search using the propeptide DarA as input was done, delivering the same hits.
Generation of a Darobactin Deletion Mutant and Heterologous Expression
To delete the dar BGC (darABCDE) from the genome of the producer strain Photorhabdus khanii DSM3369, a plasmid was constructed by assembly of 5 fragments, which enables marker less genome modification. Therefore, chromosomal DNA was isolated using the innuprepBacteria DNA Kit (AnalytikJena, Jena, Germany). Fragments (i) up- and (ii) downstream of the BGC were amplified (size ˜1 kb) using the primer pairs
(iii) The R6K origin of replication (ori), the origin of transfer (oriT) and the levan sucrase gene sacB from Bacillus subtilis were amplified in one piece from the vector pNPTS138 using the primers
The arabinose inducible expression system of pKD46 with the adjacent beta-lactamase (bla) promoter was amplified using the primers
SeqID No 7:
5′-ACTCTTCCTTTTTCAATATTATTGAAGCAT-3′ and
SeqID No 8:
5′-TGCATTTTTTATAACCTCCTTAGAGCTCGAATTCC-3′, and (v) the aph gene from pCAP03 conferring resistance to kanamycin, was amplified using the primers
All fragments were amplified by Q5 DNA polymerase (New England Biolabs, Ipswich, USA), gel purified with 1% or 2% TAE agarose gels and DNA was retrieved with the Large Fragment DNA Recovery Kit (Zymo Research, Irvine, USA). Subsequently all fragments were fused by isothermal assembly, generating the plasmid pNB02. After assembly, E. coli WM3064 cells were transformed with pNB02 by electroporation and correct assembly was corroborated by PCR and restriction analysis following standard procedures. Conjugation between E. coli WM3064 and P. khanii DSM3369 was performed by growing both strains to an OD600 of ˜0.6. After washing twice with LB medium, cells were mixed in 1:3 ratio of E. coli and P. khanii, plated out on LB agar supplemented with diaminopimelinic acid (0.3 mM) and incubated at 37° C. for 3 h, followed by overnight incubation at 30° C. The bacterial lawn was resuspended in LB medium and plated on LB agar with kanamycin (50 μg ml−1) in serial dilution. Kanamycin resistant single cross over transconjugants were grown in LB medium to an OD600 of ˜0.6. Then, expression of SacB was induced by adding arabinose (0.2% w/v), followed by 2 h incubation.
Subsequently, the culture was plated out on LB agar supplemented with 0.2% (w/v) arabinose and 10% sucrose and incubated at 30° C. for 48 h. Single colonies were picked on LBKan and LBAra/Suc agar. Sensitivity to kanamycin indicated plasmid loss and therewith a successful double crossover event. Clones were picked and analyzed for BGC loss by PCR using the primers
The knock out strain is called P. khanii DSM3369 ΔdarABCDE.
For heterologous expression of the darobactin A BGC in E. coli and to complement P. khanii DSM3369 ΔdarABCDE, the expression plasmid pNB03 was used. To avoid issues with the regulation system between the propeptide and the modifying enzymes, all intergenic regions were removed and the genes darA-darE were expressed streamlined under the control of the arabinose inducible araB promoter. pNB03 was created by amplification of (i) the p15A on from pACYC177 (primers SeqID No 13: 5′-GGTCGACGGATCCCCGGAATAGCGGAAATGGCTTACGAAC-3′ and SeqID No 14: 5′-CTCTAAGGAGGTTATAAAAAGCGGCCGCATCCCTTAACGTGAGTTTTC-3′), (ii) the arabinose expression system and kanamycin resistance of pNB02 (primers SeqID No 15: 5′-AAGCAGCTCCAGCCTACATCAGAAGAACTCGTCAAGAAGGCGA-3′ and SeqID No 16: 5′-TTTTTATAACCTCCTTAGAGCTCGAATTCC-3′), as well as (iii) the oriT and the aac(3) gene conferring resistance to apramycin from pIJ773 (primers SeqID No 17: 5′-ATTCCGGGGATCCGTCGACC-3′ and SeqID No 18: 5′-TGTAGGCTGGAGCTGCTT-3′).
Subsequently, all fragments were gel purified and assembled as described previously. E. coli TOP10 cells were transformed with the vector and correct assembly was corroborated.
To introduce the dar BGC into P. khanii DSM3369 ΔdarABCDE, (i) pNB03 was linearized using the primers SeqID No 19: 5′-TCCCTTAACGTGAGTTTTCG-3′ and SeqID No 20: 5′-TTTTATAACCTCCTTAGAGCTCGAA-3′,
(ii) darA was amplified using SeqID No 21: 5′-GCTCTAAGGAGGTTATAAAAATGCATAATACCTTAAATGAAACCGTTAAA-3′ and SeqID No 22: 5′-AATAGCATTCATTTATGGCTCTCCTTTTAAATTTCCTGGAAGCTTT-3′, (iii) darB-darE were amplified using SeqID No 23: 5′-AAAGCTTCCAGGAAATTTAAAAGGAGAGCCATAAATGAATGCTATT-3′ and SeqID No 24: 5′-CGAAAACTCACGTTAAGGGATTACGCCGCGATGGTTTGTTTTATT-3′.
All fragments were gel purified and assembled as described above. The resulting vector pNB03-darABCDE was transferred to E. coli TOP10 cells and correct assembly was corroborated.
The empty pNB03 as well as pNB03-darABCDE were transferred to P. khanii DSM3369 ΔdarABCDE by triparental conjugation. In brief, conjugation between P. khanii DSM3369 ΔdarABCDE, E. coli TOP10 carrying the expression plasmid and E. coli ET pUB307, harboring the pUB307 conjugation helper plasmid was carried out as before (cell ratio 3:1:1). Since P. khanii DSM3369 is naturally resistant to carbenicillin and the kanamycin resistance of pUB307 lacks the bla promoter, final selection took place on LB agar supplemented with kanamycin and carbenicillin. Kanamycin resistant transconjugants were grown in LBKan, the plasmid was isolated and the identity verified by PCR. For heterologous expression, the vector pNB03-darABCDE was transferred in E. coli BW25113 (arabinose non-utilizer) by electroporation. Subsequently, 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 LBKan supplemented with 0.2% (w/v) arabinose for 5-7 days and analyzed by LCMS.
Minimum Inhibitory Concentration (MIC)
The MICs were determined by microbroth dilution assays 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 MMG11 OXA-48, P. aeruginosa PAO 1, P. aeruginosa PAO 750, A. baumanii ATCC19606, K. pneumoniae ATCC30104 and S. enterica ATCC13076 were adjusted to McFarland 1.0 and subsequently diluted to 5×105 c.f.u. mL−1 in MHIIB. Darobactin derivatives were screened in 12 concentrations ranging from 64 to 0.03 μg mL−1 in triplicate. The same concentrations were tested for rifampicin, tetracycline and gentamycin as positive controls. For tetracycline resistant E. coli strains (NRZ14408, K0416 and MMG11) as well as for E. coli Survcare 052 tetracycline was substituted with a colistin dilution series (16-0.007 μg mL−1).
Bacteria suspension without supplemented standard antibiotics or darobactin was used as negative control. After incubation (18 h, 180 rpm, 37° C., 85% r.H.), cell growth was determined by measuring the turbidity with a microplate spectrophotometer at 600 nm. The MIC was defined as the minimum concentration where at least 85% growth inhibition relative to the negative control was measured.
The invention comprises several aspects, closely connected to each other. All-encompassing is the claim for bicyclic heptapeptides, their manufacturing and usage as APIs within medical preparations resp. pharmaceutical compositions. The expressions “medical preparation” and “pharmaceutical composition” are used synonymously herein. Thus, the invention also comprises:
Claim 1: Bicyclic heptapeptide of formula I,
and/or a pharmaceutically acceptable salt, stereoisomer, tautomer or hydrate thereof, wherein
Claim 2: Bicyclic heptapeptide according to claim 1, characterized in that it is darobactin.
Claim 3: Method for manufacturing the bicyclic heptapeptide according to claim 1, the method comprising
Claim 4: Method for manufacturing the bicyclic heptapeptide according to claim 3, characterized in that the purification step iii) of claim 3 comprises
Claim 5: Method for manufacturing the bicyclic heptapeptide according to claim 3, characterized in that the purification step iii) of claim 3 comprises
Claim 6: Method according to any one of claims 3 to 5, characterized in that the bicyclic heptapeptide according to claim 3 or the
Claim 7: Method for manufacturing the bicyclic heptapeptide according to claim 6, characterized in that the chemical modification is reacting the bicyclic heptapeptide with Lawson's Reagent or with tosyl chloride or with mesyl chloride.
Claim 8: A pharmaceutical composition for treating infections in a mammal caused by Gram-negative bacteria, comprising a therapeutically effective amount of the bicyclic heptapeptide according to claim 1 or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.
Claim 9: The pharmaceutical composition according to claim 8, further comprising at least one pharmaceutically acceptable carrier, excipient or diluent.
Claim 10: The pharmaceutical composition according to any one of claims 8 to 9, in a form of topical administration, systemic administration, parenteral administration, subcutaneous administration, or transdermal administration, rectal administration, oral administration, intravaginal administration, intranasal administration, intrabronchial administration, intraocular administration, intra-aural administration, intravenous administration, intramuscular administration, or intraperitoneal administration.
Claim 11: The pharmaceutical composition according to any one of claims 8 to 10 being administered to a mammal suffering of a bacterial infection, comprising administering the pharmaceutical composition to the mammal at a frequency and for a duration sufficient to provide a beneficial effect to the mammal.
Claim 12: The pharmaceutical composition according to any one of claims 8 to 10 being administered to a mammal suffering of a bacterial infection, wherein the bacterial infection is an infection involving at least one type/strain of Gram-negative bacteria.
Claim 13: The pharmaceutical composition according to any one of claims 8 to 10 being administered to a mammal suffering of a bacterial infection, wherein the bacterial infection is an infection involving 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, 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.
a, The BGC consists of the structural gene darA (colored in blue), darBCD (transporter encoding genes, in grey) and darE (encoding a radical SAM enzyme, in orange). In addition a relE-like gene (black) ORF can be co-located with the BGC at different positions. The BGC can be detected in most Photorhabdus strains in a conserved genetic region. In addition, homologous BGCs (related genes show the identical color code) are in Yersinia, Vibrio and Pseudoalteromonas strains. b, Biosynthetic hypothesis. The propeptide encoded by darA consists of 58 amino acids. The crosslinks are installed on the linear propeptide by DarE.
In a next step the leader and tail regions are cleaved off and darobactin is secreted by the ABC transporter DarBCD. c, Amino acid sequence of the propeptide from selected bacterial strains. The darobactin core peptide is highlighted in bold and the amino acids involved in the crosslinking in bold red. The star indicates the stop codon. SeqID 31 is darA from Photorhabdus temperata, SeqID 32 is darA from Photorhabdus khanii, SeqID 33 is darA from Photorhabdus australis, SeqID 34 is darA from Photorhabdus laumondii, SeqID 35 is darA from Yersinia frederiksenii, SeqID 36 is darA from Vibrio tasmaniensis, SeqID 37 is darA from Pseudoalteromonas luteoviolacea. The boxes are showing the consensus sequences from the microorganisms forming a typical heptapeptide according to formula I.
a, Scheme of the double cross-over knock out vector pNB02 and the targeted genomic region. b, Scheme of the darobactin BGC expression plasmid. c, Test PCRs on P. khanii DSM3369 ΔdarABCDE, proving the loss of the darobactin BGC; left: Amplification of darA (primers darA_f/r) resulting in a 177 bp fragment in the WT and in no fragment in the mutant; right: After loss of pNB02 (indicated by sensitivity to Kan) amplification of a 450 bp fragment if the BGC is deleted (primers DSMko_f/r); positive control: pNB03-darA-E and pNB02, respectively; primer positions indicated in blue in scheme a.
d, LC-MS extracted ion chromatogram (EIC) 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 double charged [M+2H]2+ ion corresponding to darobactin. e, Putative darobactin analogs B-E were drawn based on the amino acid sequence present in the darobactin BGC. The proposed producing organisms were identified by a BLASTP search of the 7 amino acid sequence of darobactin A, and confirming the presence of darBCDE downstream of the propeptide. Amino acid changes from darobactin A are highlighted in red. f, The table shows the propeptide sequence of the various darobactin analogs.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19210353.9 | Nov 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/025531 | 11/20/2020 | WO |
