DE102024117130A1 - Compounds and compositions derived from Pseudomonas syringae with antimicrobial properties - Google Patents

Abstract

The present invention relates to compounds derived from the species Pseudomonas syringae and their use in medicine and plant protection, e.g., against amoebae and/or fungi. Furthermore, pharmaceutical compositions containing the identified compound(s) are provided.

Description

The present invention relates to compounds derived from the species Pseudomonas syringae and their use in medicine and plant protection, e.g., against amoebae and/or fungi. Furthermore, pharmaceutical compositions containing the identified compound(s) are provided.

Background of the invention

Bacteria of the species Pseudomonas syringae are widespread and ubiquitous plant pathogens (1,2) known for their rich secondary metabolism (3,4). The genome of this soil-dwelling bacterium contains a large number of biosynthetic gene clusters (BGCs) encoding enzymes responsible for the biosynthesis of structurally and functionally diverse natural products. These compounds include biosurfactants (5), siderophores (6), toxins (7,8), and signaling molecules (9), enabling the bacterium to adapt to different habitats and infect a wide variety of plant hosts (10,11).

While some of these metabolites exhibit a broad spectrum of activity, many have highly specialized functions shaped by very specific evolutionary selection pressures. Consequently, much of the structural diversity remains undiscovered, as it eludes detection by conventional screens that test for generic antibiotic, antifungal, or anticancer activity.

WO 2013/130680A1 Disclosing this document relates to compounds and compositions derived from Pseudomonas sp., in particular from Pseudomonas fluorescens or Pseudomonas protegens, and especially from strains with the identifying characteristics of Pseudomonas ATCC 55799, which exhibit antimicrobial properties and, in particular, antibacterial properties. However, such compounds derived from Pseudomonas species are disclosed as having the characteristics of being obtainable from a Pseudomonas species, in particular a Pseudomonas species that produces at least one compound that controls zebra, quagga, and golden mussels, modulates one or more species of phytopathogenic microorganisms, and is distinguished by molecular weight and HPLC retention time.

Mehmood N, et al. (in: Multifaceted Impacts of Plant-Beneficial Pseudomonas spp. in Managing Various Plant Diseases and Crop Yield Improvement. ACS Omega. 2023 Jun 16;8(25):22296-22315. doi: 10.1021/acsomega.3c00870. PMID: 37396244; PMCID: PMC10308577 ) highlights the need to explore the potential of native Pseudomonas spp. as biological control agents and to use them in the development of biopesticides to support sustainable agriculture.

Helfrich et al. (in: Evolution of combinatorial diversity in trans-acyltransferase polyketide synthase assembly lines across bacteria. Nat Commun. 2021 Mar 3;12(1):1422. doi: 10.1038/s41467-021-21163-x. PMID: 33658492; PMCID: PMC7930024 ) describe a new polyketide, which was named Secimide (here Secimide A) according to the following formula.

The core structure of Secimid A resembles a truncated migrastin-like or an elongated cycloheximide-like polyketide.

Previous research by the inventors also suggested that many natural products produced by Pseudomonas species might be useful as defenses against bacteria-eating predators such as amoebas or nematodes (12-17).

New strategies for the treatment and control of microbial diseases are desirable. It is therefore an objective of the present invention to provide such strategies, particularly against amoebae. and mushrooms. Further goals and benefits will become readily apparent to the expert from the following more detailed description and examples.

In a first aspect of the present invention, the problem of the present invention is solved by providing a syringamide lipopeptide according to the following general formula I. $${\text{R}}^1-\text{L}-\text{Leu}-\text{D}-\text{Ser}-\text{L}-\text{homo}-\text{Ser}-\text{L}-\text{Val}-\mathrm{\beta }-\text{Ala}-\text{L}-\text{Ser}$$
where
R ¹ is selected from a saturated or unsaturated fatty acid unit with a chain length between C12 and C14, wherein at least one amino acid may be a modified amino acid and optionally comprises a macrolactone ring formed between D-Ser and L-Ser, or a pharmaceutically acceptable salt thereof.

In a second aspect of the present invention, the problem of the present invention is solved by providing a secimide compound according to general formula II, wherein R ² indicates a chemically possible halogenation of the pyridine ring, or a pharmaceutically acceptable salt thereof.

Within the scope of the present invention, the inventors performed a comprehensive pangenomic analysis of 18 taxonomically distinct P. syringae strains, leading to the identification of 231 biosynthetic gene clusters (BGCs). Among these, nonribosomal peptide synthetases (NRPS) were particularly abundant, indicating their potential importance in this ecological context. Furthermore, the bioinformatic analysis enabled the identification of a novel halogenase, Halo5, which is essential for the chlorination of secimide B. The inventors identified and elucidated the structures of two new classes of bioactive compounds: the syringamides and chlorosecimides. These syringamides and secimides, and especially their mixtures, exhibited amoebicidal activity. Moreover, the secimides showed selective activity against fungi that share the same habitat as P. syringae. Furthermore, bioinformatic analysis enabled the identification of a novel halogenase, Halo5, which is essential for the chlorination of Secimide B. These results provide valuable insights into the potential for biotechnological applications and the therapeutic development of P. syringae.

In a third aspect of the present invention, the problem of the present invention is solved by providing a process for the production of Secimid B according to the present invention, comprising the enzymatic halogenation of Secimid A to Secimid B, wherein a suitable halogenase, in particular Pseudomonas syringae halo5, is used.

In a fourth aspect of the present invention, the problem of the present invention is solved by providing a pharmaceutical or plant protection composition comprising at least one compound according to the present invention together with a pharmaceutically or plant protection-compatible diluent or carrier. Preferably, the pharmaceutical or plant protection composition according to the present invention comprises a combination of at least two compounds according to the present invention.

In a fifth aspect of the present invention, the problem of the present invention is solved by making the compound according to the present invention or the pharmaceutical composition according to the present invention available for use in the prevention or treatment of amoebic or fungal infections.

In a sixth aspect of the present invention, the problem of the present invention is solved by providing a method for preventing or treating amoebic or fungal infections in a plant, wherein the compound according to the present invention or the plant protection composition according to the present invention is applied to the plant and/or the soil of the plant.

In a seventh aspect of the present invention, the problem of the present invention is solved by providing the use of the compound or the medicament or plant protection product according to the invention for the prevention or treatment of amoeba or fungal growth.

An eighth aspect of the present invention provides for the use of Pseudomonas syringae halo5 for the halogenation of Secimid A to Secimid B.

As already mentioned, the object of the present invention is achieved in a first aspect of the present invention by providing a syringamide lipopeptide compound according to the following general formula I. $${\text{R}}^1-\text{L}-\text{Leu}-\text{D}-\text{Ser}-\text{L}-\text{homo}-\text{Ser}-\text{L}-\text{Val}-\mathrm{\beta }-\text{Ala}-\text{L}-\text{Ser}\left(\text{SEQ ID NO}:1\right)$$ wherein R ¹ is selected from a saturated or unsaturated fatty acid unit having a chain length between C12 and C14, and wherein at least one amino acid may be a modified amino acid, and which optionally comprises a macrolactone ring formed between D-Ser and L-Ser, or a stereoisomer or a pharmaceutically acceptable salt thereof.

Within the scope of the present invention, the term “modified amino acid” refers to the amino acids of the compound, leucine, serine, homoserine, valine, and alanine, which have undergone chemical modifications. Generally, modifications can be made to improve the pharmacokinetic properties of the molecule, such as stability or solubility, or to enhance its biological or pharmaceutical activities. Common modifications include hydroxylation, acylation, or the incorporation of unnatural amino acids. See, for example, Baslé E et al. (in: Protein chemical modification on endogenous amino acids. Chem Biol. 2010 Mar 26;17(3):213-27. doi: 10.1016/j.chembiol.2010.02.008. PMID: 20338513 ), and other references to the state of the art.

A syringamide lipopeptide compound according to the present invention is preferred, wherein ^R1 is selected from a saturated or unsaturated fatty acid unit with a chain length of C12 or C14 or a pharmaceutically acceptable salt thereof. Even more preferred is that the syringamide lipopeptide compound according to the invention contains a double bond in the fatty acid, for example at C-5'/C-6' or C-7'/C-8', or a pharmaceutically acceptable salt thereof.

Even more preferred is a syringamide lipopeptide compound according to the present invention, which is a cyclic lipopeptide, or a pharmaceutically acceptable salt thereof. The molecule may comprise a ring, in particular a macrolactone ring, formed between the D-serine at position 2 of the amino acid chain and the L-serine at position 6 of the amino acid chain, preferably by esterification between the hydroxyl group of serine 2 and the C-terminus of serine 6.

Particularly preferred is a syringamide lipopeptide compound according to the present invention, which is selected from syringamide A, B, C and a pharmaceutically acceptable salt thereof. The chemical structures of these compounds are as follows: , or or a stereoisomer or pharmaceutically acceptable salts thereof.

Another aspect of the invention provides a secimide compound according to general formula II, (Formula II), where ^R2 denotes a chemically possible halogenation of the pyridine ring, i.e., a halo group. Any haloalkane or alkane containing a halogen atom, such as chlorine, bromine, or fluorine, is considered a halogen group. The functional halogen group contains a carbon-halogen bond to one of the ring members, or a stereoisomer or pharmaceutically acceptable salt thereof.

Further analysis of the secondary metabolites of P. syringae DSM 1242 revealed a prominent natural product with the molecular formula _C16H22O6NCl , which _was identified as _either undescribed chlorosecimide A or secimide B. Secimide A showed no amoebicidal activity, while secimide B exhibited toxicity against amoebae. This activity appears to be related to the halo group introduced by the enzyme Halo 5 (see below).

Therefore, the Secimid B according to the invention is preferred, as shown in the following formula III.

(Formula III), or a stereoisomer or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention relates to a process for the preparation of Secimid B according to the present invention, comprising the enzymatic halogenation of Secimid A to Secimid B using a suitable halogenase, in particular Pseudomonas syringae halo5. This process can be carried out in vitro or in vivo. Further details are given in the examples and discussion of Halo 5 below.

Another aspect of the present invention relates to a method for producing a pharmaceutical composition, comprising mixing a compound according to the present invention with at least one pharmaceutical diluent or carrier (see also below).

Another aspect of the present invention relates to a pharmaceutical or plant protection composition comprising at least one compound according to the present invention together with at least one pharmaceutically or plant protection-acceptable carrier. Preferably, a pharmaceutical or plant protection composition according to the present invention comprises a combination of at least two compounds according to the present invention. The combination may be contained in the same or separate containers and used together or separately, provided that the compounds can interact at the desired site of action.

The inventors then tested combinations of compounds that reflected the production ratios observed in SM/5 media. A preferred mixture of the syringamides AC (1:1:1, wt/wt/wt) exhibited a synergistic _IC50 (D. discoideum) value of 1.1 µg ^mL⁻¹ .

A preferred mixture of the secimides A and B (4:1, wt/wt) showed increased (synergistic) toxicity compared to the individual components, with a combined _IC50 (D. discoideum) value of 27.1 µg ^mL⁻¹ .

A preferred combination of all five compounds (1:1:1:4:1, wt/wt/wt/wt/wt, syringamides A, B, C or secimide A, B) resulted in a total _IC50 value (D. discoideum) of 5.5 µg ^mL⁻¹ .

Furthermore, the two preferred secimides A and B showed selective activity against the fungal competitor Sporobolomyces salmonicolor (MIC = 25 µg/mL)

Preferably, the composition consists of an aqueous formulation. The term "pharmaceutical composition" refers to a preparation that is in such a form that the biological activity of an active ingredient (in this case, the at least one compound) contained therein is effective, and that does not contain any additional components that are unacceptably toxic to a subject to whom the composition is administered.

A pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. As those skilled in the art know, the route and/or method of administration varies depending on the desired results. Pharmaceutically acceptable diluents include saline solution and aqueous buffer solutions. A "pharmaceutically acceptable diluent or carrier" refers to a component of a pharmaceutical formulation that is not an active ingredient and is non-toxic to the patient. Pharmaceutically acceptable carriers include all solvents, dispersants, coatings, antibacterial and antifungal agents, isotonic agents, absorption retardants, and the like, which are physiologically acceptable. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). The pharmaceutical composition for use according to the present invention is preferred, wherein the composition is intended for injection, oral and/or nasal application.

The pharmaceutical compositions according to the invention may also contain excipients such as preservatives, wetting agents, emulsifiers, and dispersants. The prevention of microorganisms can be ensured both by sterilization processes and by the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be achieved by adding absorption-delaying agents, such as aluminum monostearate and gelatin.

The medicinal products according to the invention can be in liquid, dry or semi-solid form, such as tablets, coated tablets, effervescent tablets, capsules, powders, granules, lozenges, pills, ampoules, drops, suppositories, emulsions, ointments, gels, tinctures, pastes, creams, moist compresses, gargles, plant juices, nasal preparations, inhalation mixtures, aerosols, mouthwashes, oral sprays, nasal sprays or room sprays. An injectable composition is preferred.

In some embodiments, the composition comprises the active ingredient, such as the compounds, in a concentration ranging from about 0.1 mg/ml to about 1 mg/ml to about 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, or 20 mg/ml. In some embodiments, the composition also comprises one or more additional therapeutic agents, e.g., a second, third, or fourth therapeutic agent, selected, for example, from antimicrobial agents.

Similar considerations to those above apply to the plant protection product, which contains a diluent or carrier that is acceptable for the plant protection product. Here, the additional considerations relate more to the stability and toxicity of the carrier, which is applied to the soil or the plants to be protected.

Another aspect concerns the compound or pharmaceutical composition according to the invention for use in the prevention or treatment of diseases, preferably for use against amoebic or fungal infections and/or infestations in a patient.

The compound or pharmaceutical composition according to the invention is preferably used in accordance with the present invention, wherein the amoeba is Dictyostelium discoideum and the fungus is a fungal competitor, such as Sporobolomyces salmonicolor, and the prevention or treatment preferably takes place in combination and has a synergistic effect.

In the context of the present invention, the term "subject" refers to a mammal, an animal or an individual, such as a human being or a patient, who is subjected to prevention or treatment according to the invention against an infectious disorder or disease.

In the context of the present invention, the term “treatment” or “therapy” means the (attempted) remedy of a health problem as disclosed herein, i.e., the patient’s infection.

In the context of the present invention, the term “synergistic effect” means the result of the interaction of two or more compounds or processes as disclosed herein. are, in order to achieve an effect that is greater than the cumulative effect that these methods achieve when used individually.

Another aspect of the present invention relates to a method for preventing or treating amoebic or fungal infections in a plant, in which the compound or plant protection composition according to the invention is applied to the plant and/or the soil of the plant.

The method according to the present invention is preferred, wherein the amoeba is Dictyostelium discoideum and the fungus is a fungal competitor, such as Sporobolomyces salmonicolor, and preferably the prevention or treatment is carried out in combination and has a synergistic effect.

Another aspect of the present invention relates to the use of the compound or the pharmaceutical or plant-protecting composition according to the invention for the prevention or treatment of amoeba or fungal growth, as disclosed herein.

Another aspect of the present invention relates to the use of the enzyme Pseudomonas syringae halo5 for the halogenation of a pyridine ring, preferably for the halogenation of Secimide A to Secimide B, as described herein.

In the context of the present invention, the term “Halo 5” refers to the bacterial protein α-KG-dependent 2,4-dichlorophenoxyacetate dioxygenase, as disclosed in (43). A preferred amino acid sequence of the Halo5 protein, as used according to the present invention, comprises an amino acid sequence that is at least 90%, preferably at least 95%, and particularly preferably at least 99% or even 100% identical to the amino acids of the bacterial Halo5 protein. Orthologs of the Halo5 amino acid sequence according to the present invention can be selected from other bacterial and fungal strains, and Halo5 can be produced by recombinant biotechnological processes.

In the context of the present invention, the term "approximately" means a deviation of +/- 10% of the stated value, unless otherwise specified.

The ability of P. syringae to thrive in its ecological niche relies on its dual capacity to both infect plant hosts and resist antagonistic interactions with other soil microorganisms, including amoebic predators (16). These requirements exert strong evolutionary selection pressure on its infection and defense mechanisms, which are highly dependent on the synthesis of bioactive natural products (10). As a result, P. syringae has evolved a rich secondary metabolome of natural products with diverse and often highly context-dependent functions. Although members of the species P. syringae occupy similar ecological niches, the species exhibits remarkable strain-specific variability in its bioactive metabolites (BGCs). Thus, the genome of P. syringae DSM 50255 has a 3.5 times higher number of BGCs than that of P. syringae DSM 50272, and the genome of P. syringae DSM 50256 has 16 NRPS-BGCs, while the genome of P. syringae DSM 50293 has only 3. Some of these biosynthetic genes are integral to the nuclear genome, such as those required for the production of pyoverdin siderophores (54, 55), highlighting their universal importance across species. Conversely, many BGCs are located in the accessory genome or exist as singletons with minimal prevalence across species. These accessory or singular genes often confer specific ecological advantages and facilitate niche-specific diversification (56-58).

A pangenomic analysis of 18 taxonomically distinct P. syringae strains enabled the identification of accessory biosynthesis genes with minimal similarity to known P. syringae BGCs. The inventors isolated and structurally elucidated the corresponding natural products, syringamides A-C, which are cyclic lipopeptides with six amino acids and fully reduced C12 or C14 fatty acid moieties at the C3 position. Furthermore, the inventors identified a novel chlorinated congener of secimide A, derived from trans-AT type I PKS, and subsequently discovered a previously undescribed halogenase, initially classified as a dioxygenase for the hydroxylation of the aspartic acid residue (59,60), which converts secimide A to secimide B via a late halogenation. The syringamides and secimides show amoebic activity both individually and in combination, and the secimides show selective inhibitory activity against S. salmonicolor.

• Gegenstand 1. Syringamid-Lipopeptid gemäß der folgenden allgemeinen Formel R¹-L-Leu-D-Ser-L-homo-Ser-L-Val-β-Ala-L-Ser (Formel I
• worin R¹ ausgewählt ist aus einer gesättigten oder ungesättigten Fettsäureeinheit mit einer Kettenlänge zwischen C12 und C14, und worin mindestens eine Aminosäure eine modifizierte Aminosäure sein kann,
• und gegebenenfalls einen zwischen D-Ser und L-Ser gebildeten Makrolactonring umfasst, oder ein Stereoisomer oder ein pharmazeutisch akzeptables Salz davon.

The present invention relates to the following items:

• Item 1. Syringamide lipopeptide according to the following general formula R ¹ -L-Leu-D-Ser-L-homo-Ser-L-Val-β-Ala-L-Ser (Formula I
• wherein R ¹ is selected from a saturated or unsaturated fatty acid unit with a chain length between C12 and C14, and wherein at least one amino acid may be a modified amino acid,
• and optionally includes a macrolactone ring formed between D-Ser and L-Ser, or a stereoisomer or a pharmaceutically acceptable salt thereof.

Item 2. The syringamide lipopeptide according to Item 1, wherein R ¹ is selected from a fatty acid unit having a chain length of C12 or C14 comprising a double bond at C-5'/C-6' or C-7'/C-8', or a stereoisomer or a pharmaceutically acceptable salt thereof.

Item 3. The syringamide lipopeptide according to Item 1 or 2, which is a cyclic lipopeptide,
or a stereoisomer or a pharmaceutically acceptable salt thereof.

Item 4. Secimide compound according to general formula II, wherein R ² signifies a chemically possible halogenation of the pyridine ring, or a stereoisomer or a pharmaceutically acceptable salt thereof.

Item 5. Secimid B according to the following formula III (Formula III), or a stereoisomer or a pharmaceutically acceptable salt thereof.

Item 6. Process for the production of Secimide B according to Item 5, comprising the enzymatic halogenation of Secimide A to Secimide B, comprising the use of a suitable halogenase, in particular Pseudomonas syringae halo5.

Item 7. Pharmaceutical or plant protection composition comprising at least one compound according to any of items 1 to 5 together with a pharmaceutically or plant protection-acceptable diluent or carrier.

Item 8. Medicinal products or plant protection products according to Item 7, containing a combination of at least two compounds according to any one of Items 1 to 5.

Item 9. The compound according to any of items 1 to 5 or the pharmaceutical composition according to item 7 or 8 for use in the prevention or treatment of diseases, preferably for use against amoebic or fungal infections.

Item 10. The compound according to any of Items 1 to 5 or the pharmaceutical composition according to Item 7 or 8 for use according to Item 9, wherein the amoeba is Dictyostelium discoideum and the fungus is a fungal competitor, such as Sporobolomyces salmonicolor.

Item 11. Method for preventing or treating amoebic or fungal infections in a plant, comprising applying the compound according to any of Items 1 to 5 or the plant protection products according to Item 7 or 8 to the plant and/or the soil of the plant.

Item 12. Method according to Item 11, wherein the amoeba is Dictyostelium discoideum and the fungus is a fungal competitor, such as Sporobolomyces salmonicolor.

Item 13. Use of the compound according to any of items 1 to 5 or of the medicinal product or plant protection product according to item 7 or 8 for the prevention or treatment of amoeba or fungal growth.

Subject 14. Use of the enzyme Pseudomonas syringae halo5 for the halogenation of a pyridine ring, preferably for the halogenation of Secimide A to Secimide B.

• zeigt die Domänenorganisation des sym BGC auf der Grundlage der Vorhersagen von antiSMASH und NaPDoS. Die vorhergesagten Substrate der A-Domäne und die Spezifitäten der C-Domäne sind angegeben. C_S: Starter-C-Domäne; ^LC_L: C-Domänen, die zwei L-Aminosäuren kondensieren; ^DC_L: C-Domänen, die eine L-Aminosäure und eine D-Aminosäure kondensieren.
• zeigt die metabolische Analyse von P. syringae-Stämmen. (A) Extraktions-Ionen-Chromatogramme (EIC) von Kulturextrakten aus verschiedenen P. syringae-Stämmen, die zeigen, dass DSM 1242 der produktivste Produzent von Syringamiden (1-3) und Secimiden (4-5) ist. (B) HPLC-Profil von Rohextrakten aus DSM 1242-Wildtyp und Gen-Deletionsmutanten, das das Fehlen von Syringamiden in der DSM 1242Δsym-Mutante, das Fehlen von Secimiden in der DSM 1242Δsem-Mutante und das Fehlen beider Moleküle in der Doppelknockout-Mutante zeigt. Darüber hinaus fehlt Secimid B (4) in der DSM 1242Δhalo5-Mutante, und es kommt zu einer Anreicherung von Secimid A (5) (nachgewiesen bei λ = 210 nm).
• zeigt die Strukturen von Syringamiden, Secimiden und Scandium(III)-Triflat-vermittelter Peptidhydrolyse. (A) Chemische Strukturen der Syringamide A-C (1-3) und der Secimide A (5a-5d) und B (4). Die wichtigsten ¹H-¹H-, COSY-, HMBC- und NOESY-Korrelationen sind angegeben. (B) Die Scandiumtriflat-vermittelte Peptidspaltung ergibt das Fragment F1, das nur D-Serin enthält.
• zeigt das Schema der Halo5-katalysierten Chlorierung von Secimid A.

The invention will now be described in more detail in the following examples with reference to, but without being limited to, the accompanying figures. For the purposes of the present invention, all references as cited are incorporated by reference in their entirety.

• This figure shows the domain organization of the sym BGC based on the predictions of antiSMASH and NaPDoS. The predicted substrates of the A domain and the specificities of the C domain are indicated. C S : starter C domain; L C L : C domains condensing two L-amino acids; D C L : C domains condensing one L-amino acid and one D-amino acid.
• Metabolic analysis of P. syringae strains is shown. (A) Extraction ion chromatograms (EIC) of culture extracts from different P. syringae strains, showing that DSM 1242 is the most productive producer of syringamides (1-3) and secimides (4-5). (B) HPLC profile of crude extracts from DSM 1242 wild type and gene deletion mutants, showing the absence of syringamides in the DSM 1242Δsym mutant, the absence of secimides in the DSM 1242Δsem mutant, and the absence of both molecules in the double knockout mutant. Furthermore, Secimid B (4) is missing in the DSM 1242Δhalo5 mutant, and there is an enrichment of Secimid A (5) (detected at λ = 210 nm).
• Figure 1 shows the structures of syringamides, secimides, and scandium(III) triflate-mediated peptide hydrolysis. (A) Chemical structures of syringamides AC (1-3) and secimides A (5a-5d) and B (4). The main 1H - 1H , COSY, HMBC, and NOESY correlations are shown. (B) Scandium triflate-mediated peptide cleavage yields fragment F1, which contains only D-serine.
• shows the scheme of the Halo5-catalyzed chlorination of Secimide A.

SEQ ID NO: 1 shows the amino acid chain of the syringamides according to the present invention LShSVAS, where hS stands for homoserine.

Examples

Data and materials

All data are available in the main text or in the supplementary materials. The genome sequences of all 12 newly sequenced P. syringae strains are accessible via the National Center for Biotechnology Information (NCBI): JBEDVB000000000-JBEDVM000000000. Known genomes can also be found in the NCBI under the following accession numbers: GCF_000988485.1, GCF_000597765.1, GCF_000498595.1, GCF_000007805.1, GCF_001482725.1, and GCF_009495975.1 (database status as of June 15, 2024).

An atlas of biosynthetic gene clusters (BGCs) in P. syringae

Eighteen taxonomically distinct P. syringae genomes were analyzed for natural product BGCs using antiSMASH 7.0 (18). A total of 231 BGCs were identified and classified into five categories, averaging 13 BGCs per species, which is about 2–8 times higher than the average number of BGCs in other Enterobacteriaceae (19). Trans-acyltransferase type I polyketides (trans-AT type I PKSs), responsible for the production of secimide (20), were enriched and widespread. In contrast, ribosomally synthesized and post-translationally modified peptides (RiPP) and terpene BGCs are rare in P. syringae.

Pangenome analysis of P. syringae

Pangenomic analysis (22,23) of P. syringae led to the identification of a unique NRPS BGC with six modules, each comprising condensation (C), adenylation (A), and thiolation (T) domains. Furthermore, the starter C domain indicated an N-terminal fatty acid unit (32), suggesting that this NRPS can produce a six-amino-acid lipopeptide ( Furthermore, this BGC was found in 6 of the 18 P. syringae strains examined.

Identification, isolation and structural elucidation of syringamides.

To identify the natural substances associated with the unique NRPS BGC ( Since the inventors initially cultured all six P. syringae strains containing this BGC in different liquid media, the analysis of the SM/5 medium culture extracts showed that only DSM 1242 produced three different compounds with molecular masses of m/z 739.45, 741.45, and 767.45. The inventors then inactivated the respective BGC by generating an in-frame deletion of the gene fragment encoding the C _starter domain (Δsym). Comparison of the HPLC profiles of culture extracts from DSM 1242 and the deletion mutant showed that the three peaks present in the wild type were absent in the deletion mutant, suggesting that these peaks can be attributed to the products of this NRPS-BGC ( To isolate sufficient quantities of these natural products, the high-producing strain P. syringae DSM 1242 was cultivated on an 8-liter scale in SM/5 medium. Extraction of the supernatant with ethyl acetate and chromatographic purification of the extract led to the isolation of three related compounds, which the inventors named syringamides (sym). High-resolution mass spectrometry (HRMS) measurements revealed pseudomolecular ion peaks with m/z 739.4574 [M + H] ⁺ for syringamide A (1), m/z 741.4727 [M + H] ⁺ for syringamide B (2) and m/z 767.4886 [M + H] ⁺ for syringamide C (3), which correspond to the molecular formulas C ₃₆ H ₆₂ N ₁₀ O ₆ , C ₃₆ H ₆₄ N ₁₀ O ₆ and C ₃₈ H ₆₆ N ₁₀ O ₆ respectively.

To fully elucidate the structure of the syringamides, the inventors began with an in silico prediction of the specificities of each A domain of the sym NRPS. This revealed the amino acid sequence of the syringamides. The presence of a C _starter domain indicated the presence of an N-terminal fatty acid moiety (32), which was consistent with nuclear magnetic resonance (NMR), tandem mass spectrometry (MS ² ), and chemical degradation studies. Furthermore, NMR spectroscopy revealed that all three compounds contain a fatty acid residue with chain lengths of C12 (compounds 1 and 2) or C14 (compound 3) that features a double bond at C-5'/C-6' (1) or C-7'/C-8' (3). Derivatization of the hydrolyzed amino acids with Marfey's reagent confirmed the presence of 1 × L-Ser, 1 × D-Ser, 1 × L-Leu, 1 × L-Val, 1 × L-Hse, and 1 × β-Ala. However, these combined analyses did not allow us to determine the specific positions of L- and D-serine ( .

Scandium(III) triflate-mediated peptide hydrolysis (12,33) selectively cleaves amide bonds near serine or threonine residues. Using this method, the inventors obtained a shorter fragment of syringamide (F1), which was completely hydrolyzed and derivatized with the Marfey reagent. Analysis of F1, which contains 1 × L-leucine, 1 × D-serine, and 1 × L-homoserine, allowed us to assign the D-configuration of serine at position 2 and the L-configuration of serine at position 6 ( .

The predicted molecular mass for a linear peptide, based on the amino acids and fatty acid identified by MS ² experiments, differed by 18 mass units from the observed mass, suggesting that syringamide could be a macrocycle. Further analysis of the NMR data revealed an HMBC correlation between the hydrogens H-11 (1: δ _H 4.42; 2: δ _H 4.43; 3: δ _H 4.43) and C-30 (1: δ _C 170.04; 2: δ _C 170.01; 3: δ _C 170.01), indicating that syringamides are cyclic lipopeptides with macrolactones formed by esterification between the hydroxyl group of serine 2 and the C-terminus of serine 6. Interestingly, the syringamides do not belong to any of the six established groups (34) of cyclic lipopeptides derived from Pseudomonas (35,36). Instead, they form a new group characterized by six amino acids, including an unusual β-alanine and a fatty acid moiety lacking the 3-OH group ( .

Identification, isolation and structural elucidation of secimides

Further analysis of the secondary metabolites of P. syringae DSM 1242 revealed two prominent natural products exhibiting pseudomolecular ion peaks at m/z 360.1198 [M + H] ⁺ and 326.1585 [M + H] ⁺ , corresponding to the molecular formulas C ₁₆ H ₂₂ O ₆ NCl and C ₁₆ H ₂₃ O ₆ N, respectively. The compound with the molecular formula C ₁₆ H ₂₃ O ₆ N was identified as Secimide A (20), a known product of trans-AT type I PKS, while the other was identified as undescribed Chlorosecimide A (Secimide B). This finding is consistent with the results of the anvi'o platform, which identified six PKS gene hits in the accessory gene group, suggesting that these PKS hits are aggregated in the BGC of secimid (sem). The BGC for this class of molecules is present in 7 of 18 P. syringae strains, with DSM 1242 appearing as the optimal producer ( Deletion of the AT domain (Δsem) suppressed the production of both molecules, reinforcing the link between the identified trans-AT PKS BGC and its biosynthesis ( After purification of these compounds by HPLC, it was found that the peak at m/z 360.1198 represented a single compound designated Secimid B (4). In contrast, the peak at m/z 326.1585 consisted of a mixture of four isomers in a 1:1:1:1 ratio, designated Secimid A (5a-5d) ( .

To fully elucidate the structures of the secimides, a combination of extensive NMR analyses and computational predictions was employed. These investigations revealed that secimide B exhibits a 3-chloro substitution compared to 5a, and its absolute configuration was determined to be 3S,4S,8S,11R. Regarding the isomeric mixture of 5a–5d, the individual isomers were separated by HPLC using a C18 column. However, subsequent NMR analysis showed that the isomers had transformed into a mixture. The structural differences between 5a and 5b, as well as 5c and 5d, were attributed to variations in the chiral centers at C-11 and C-13, respectively. Specifically, 5a and 5b possess a double bond at C-12/C-13, while 5c and 5d have a double bond at C-11/C-12. It is noteworthy that the double bond can easily switch between these positions, allowing for a rapid conversion between the four. (see also .

Identification of the enzyme that chlorinates Secimide A

Since Secimid B is an unknown 3-chloro congener of Secimid A, the inventors investigated the underlying chlorination mechanism. A detailed examination of the trans-AT PKS BGC revealed no candidate genes encoding a halogenase enzyme. This suggests that Secimid A is chlorinated after assembly via a halogenase that may not be encoded in the sem BGC. Using genes from all four known halogenase groups as a query for a BLAST search, the inventors identified four potential halogenase genes in the genome of P. syringae DSM 1242 (halo1–halo4) belonging to three different groups. Based on their PFAM analysis (Table S8), halo1–halo4 encode either Fe(II)/α-KG-dependent or flavin-dependent halogenases (39–41). Halo1, Halo3, and Halo4 were the most likely candidates; however, the mutants Δhalo1, Δhalo2, Δhalo3, Δhalo4, Δhalo3Δhalo4, and Δhalo1Δhalo3Δhalo4 were still able to produce chlorinated secimide B. Given that Fe(II)/α-KG-dependent halogenases belong to the oxygenase family and are very similar to related enzymes that catalyze hydroxylation reactions (42), a gene, Halo5, located near the sem BGC and annotated as α-KG-dependent 2,4-dichlorophenoxyacetate dioxygenase (43), attracted the inventor's attention. In fact, the Δhalo5 mutant was unable to produce Secimid B, but instead accumulated Secimid A. Subsequent heterologous expression of halo1, halo3, and halo5 in Escherichia coli showed that only the strain containing halo5 was able to convert Secimid A to Secimid B. Thus, the results confirmed that halo5, although annotated as a dioxygenase-encoding gene, encodes a functional halogenase that enables the chlorination of secimide A in the final step of biosynthesis ( It is conceivable that Halo5 is a novel halogenase that differs from all previously known halogenases, but belongs to the Fe(II)/α-KG-dependent halogenase family.

Biological activity and synergy of syringamides and secimides

Since P. syringae and social amoebae share the same ecological niche (44), the inventors investigated the susceptibility of 18 P. syringae strains to amoebic feeding using a plaque test. For this purpose, vegetative Dictyostelium discoideum AX2 cells were placed on a solid medium containing a lawn of the respective bacterial strain. The formation of a grazing plaque, which eventually develops into amoebic fruiting bodies, indicates that the amoebae can feed on the bacteria. In contrast, the absence of grazing plaques or fruiting bodies indicates bacterial resistance to amoebic feeding (16). Of the 18 P. syringae strains tested, only P. syringae DSM 1242 showed resistance to amoebic feeding. Given that the production of amoebicidal secondary metabolites enables bacteria to efficiently kill amoebic predators, the inventors investigated the toxicity of syringamides and secimides to amoebae. This ecological function of bacterial secondary metabolites appeared particularly relevant because only P. syringae DSM 1242 was capable of producing both syringamides and secimides in significant quantities. Syringamide AC exhibited strong amoebicidal activity with _IC50 (D. discoideum) values of 13.5, 5.5, and 2.1 µg ^mL⁻¹ , respectively. In contrast, secimide A showed no amoebicidal activity, while secimide B exhibited moderate toxicity with an _IC50 (D. discoideum) value of 49.2 µg ^mL⁻¹ . The inventors then tested combinations of compounds that reflected the production ratios observed in SM/5 media. A mixture of syringamides AC (1:1:1, wt/wt/wt) exhibited an _IC50 value (D. discoideum) of 1.1 µg ^mL⁻¹ . A mixture of secimides A and B (4:1, wt/wt) showed increased toxicity compared to the individual components, with a combined _IC50 value (D. discoideum) of 27.1 µg ^mL⁻¹ . A combination of all five compounds (1:1:1:4:1, wt/wt/wt/wt/wt; syringamide AC and secimide A, B) yielded a total _IC50 value (D. discoideum) of 5.5 µg ^mL⁻¹ . Furthermore, both secimides A and B showed selective activity against the fungal competitor Sporobolomyces salmonicolor (MIC = 25 µg/mL), which uses the same habitat (45). These results underscore the important biological role of bacterially produced cyclic lipopeptides and trans-AT PKS (46,47), particularly in the context of microbial interactions (12,13,15,17,48,49). Importantly, modification of secimide A by a halogenase alters its biological function (50-53).

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QUOTES INCLUDED IN THE DESCRIPTION

This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions.

Cited patent literature

• WO 2013/130680A1 [0004]

Cited non-patent literature

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• Helfrich et al. (in: Evolution of combinatorial diversity in trans-acyltransferase polyketide synthase assembly lines across bacteria. Nat Commun. 2021 Mar 3;12(1):1422. doi: 10.1038/s41467-021-21163-x. PMID: 33658492; PMCID: PMC7930024 [0006]
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• Young, J.M. Taxonomy of Pseudomonas syringae. J. Plant Pathol. 2010, S5-S14 [0080]
• Strange, R. N. Phytotoxins produced by microbial plant pathogens. Nat. Prod. Rep. 2007, 24 (1), 127-144 [0080]
• Götze, S.; Stallforth, P. Structure, properties, and biological functions of nonribosomal lipopeptides from pseudomonads. Nat. Prod. Rep. 2020, 37 (1), 29-54 [0080]
• Burch, A.Y.; Zeisler, V.; Yokota, K.; Schreiber, L.; Lindow, S. E. The hygroscopic biosurfactant syringafactin produced by Pseudomonas syringae enhances fitness on leaf surfaces during fluctuating humidity. Environ. Microbiol. 2014, 16 (7), 2086-2098 [0080]
• Taguchi, F.; Suzuki, T.; Inagaki, Y.; Toyoda, K.; Shiraishi, T.; Ichinose, Y. The siderophore pyoverdine of Pseudomonas syringae pv. tabaci 6605 is an intrinsic virulence factor in host tobacco infection. J. Bacteriol. 2010, 192 (1), 117-126 [0080]
• Mittal, S.; Davis, K. R. Role of the phytotoxin coronatine in the infection of Arabidopsis thaliana by Pseudomonas syringae pv. tomato. Mol. Plant Microbe Interact. 1995, 8 (1), 165-171 [0080]
• Hutchison, M.L.; Tester, M.A.; Gross, D. C. Role of biosurfactant and ion channel-forming activities of syringomycin in transmembrane ion flux: a model for the mechanism of action in the plant-pathogen interaction. Mol. Plant. Microbe Interact. 1995, 8 (4), 610-620 [0080]
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Claims (14)

Syringamide lipopeptide according to the following general formula I $${\text{R}}^1-\text{L}-\text{Leu}-\text{D}-\text{Ser}-\text{L}-\text{homo}-\text{Ser}-\text{L}-\text{Val}-\mathrm{\beta }-\text{Ala}-\text{L}-\text{Ser}$$ wherein R ¹ is selected from a saturated or unsaturated fatty acid unit with a chain length between C12 and C14, at least one amino acid may be a modified amino acid, and optionally comprises a macrolactone ring formed between D-Ser and L-Ser, or a stereoisomer or a pharmaceutically acceptable salt thereof. Syringamide lipopeptide after Claim 1 , where R ¹ is selected from a fatty acid unit with a chain length of C12 or C14 having a double bond at C-5'/C-6' or C-7'/C-8', or a stereoisomer or a pharmaceutically acceptable salt thereof. Syringamide lipopeptide after Claim 1 or 2 , which is a cyclic lipopeptide, or a stereoisomer or a pharmaceutically acceptable salt thereof. Secimide compound according to general formula II, where R ² indicates a chemically possible halogenation of the pyridine ring, or a stereoisomer or a pharmaceutically acceptable salt thereof. Secimid B according to the following formula III or a stereoisomer or a pharmaceutically acceptable salt thereof. Method for the production of Secimid B according to Claim 5 , comprising the enzymatic halogenation of Secimid A to Secimid B using a suitable halogenase, in particular Pseudomonas syringae halo5. Pharmaceutical or plant protection composition comprising at least one compound according to one of the Claims 1 until 5 , together with a pharmaceutically or plant-protecting compatible diluent or carrier. pharmaceuticals or plant protection products Claim 7 , containing a combination of at least two compounds according to one of the Claims 1 until 5 . The connection after one of the Claims 1 until 5 or the pharmaceutical composition according to Claim 7 or 8 For use in the prevention or treatment of diseases, preferably for use against amoebic or fungal infections. The connection after one of the Claims 1 until 5 or the pharmaceutical composition according to Claim 7 or 8 for use after Claim 9 , where the amoeba is Dictyostelium discoideum and the fungus is a fungal competitor, such as Sporobolomyces salmonicolor. Methods for preventing or treating amoebic or fungal infections in a plant, comprising applying the compound after one of the Claims 1 until 5 or the pesticides Claim 7 or 8 on the plant and/or the soil around the plant. Procedure according to Claim 11 , where the amoeba is Dictyostelium discoideum and the fungus is a fungal competitor, such as Sporobolomyces salmonicolor. Use of the connection according to one of the Claims 1 until 5 or of the medicinal product or plant protection product after Claim 7 or 8 for the prevention or treatment of amoeba or fungal growth. Use of the enzyme Pseudomonas syringae halo5 for the halogenation of a pyridine ring, preferably for the halogenation of Secimide A to Secimide B.