GB2505448A

GB2505448A - Anti-infective peptides comprising a C terminal amine group

Info

Publication number: GB2505448A
Application number: GB1215431.6A
Authority: GB
Inventors: Helge B Bodo; Daniela Reimer; Anna Venneri; Friederike Nollmann
Original assignee: Goethe Universitaet Frankfurt am Main
Current assignee: Goethe Universitaet Frankfurt am Main
Priority date: 2012-08-30
Filing date: 2012-08-30
Publication date: 2014-03-05
Also published as: GB201215431D0

Abstract

The invention relates to non-ribosomal peptides isolated from ontamopathogenic bacteria which have antibiotic activities against protozoan parasites wherein said peptides comprise a C terminal amine group. This group may be as a phenylethylamine, agmatine or tryptamne amino acid. Furthermore, the invention provides novel gene clusters which encode for the non-ribosomal peptide synthetases (NRPS) that mediate the biochemical synthesis of the inventive peptides. The peptides provided by the invention fall under the categories Xenortides, Rhabdopeptides and Mevalagmapeptides. Their use in medicine, specifically in the treatment of a disease caused by a protozoan parasite, such as malaria or sleeping sickness; and/or as insecticidals, for example in plant protection agents is disclosed. Furthermore provided are methods for the production of the peptides of the invention, and the enzymes and. nucleic acids useful for their synthesis in a host cell.

Description

Novel Anti-Parasitic Peptides from Entomopathogenic Bacteria The present invention pertains to novel non-ribosomal peptides isolated from entomopatho-genic bacteria which have antibiotic activities against protozoan parasites. Furthermore, the invention provides novel gene clusters which encode for the non-ribosomal peptide syntheta-ses (NRPS) that mediate the biochemical synthesis of the inventive peptides. The peptides provided by the invention fall under the categories Xenortides, Rhabdopeptides and Me-valagmapeptides. They are particularly useful as medicine, specifically in the treatment of a disease caused by a protozoan parasite, such as malaria or sleeping sickness; and/or as insec-ticidals, for cxamplc in plant protection agents. Furthermore provided arc methods for the production of the peptides of the invention, and the enzymes and nucleic acids useful for their synthesis in a host cell.

Background

Natural products and compounds isolated from microorganisms provide a rich source for new therapeutically active molecules like for example novel antibiotics. Bacterial symbionts of insect pathogenic nematodes are interesting host organisms to screen for such new com-pounds with antibiotic or insecticidal properties. The Gram-negative bacterial strains of the genus Xenorhabdus and Photorhabdus are known to be symbiotically associated with soil dwelling nematodes of the Steinernernatidae and HetereorhabditLs family, respectively. After entering the insect larvae via natural opcnings, ncmatodes release bacteria from their intestine to the host's hemocoel. Bacteria are involved in killing the insect host by producing insecti-cidal proteins and inhibitors of the insect immune system. The bacteria proliferate in the kil led host and favour the reproduction of the nematode by degrading the insect biomass and by producing antibiotics that inhibits the development of the other microorganisms present in the insect corpse (bacteria, fungi).

Xenorhabdus produces different classes of secondary metabolites identified in different strains. For example secondary metabolites isolated from Xenorhahdus comprise Xenocou-macin 1 which has antibacterial and antifungal activity and xenocoumacin 2 which has weakly antibacterial effects (Mclncrney et al., 1991b; Rcimer et al., 2009). Nematophin is an active indole derivative against clinically relevant Staphi'lococcus aureus strains (Li et al., 1997a). Indole derivatives in general exhibit antibacterial and antithngal effects (Li et al., I 997b; Li et a]., 1998). Xenematide is a cyclic peptide with antibiotic and insecticidal activity (Lang ct al., 2008) and phcnylethylamidcs havc cytotoxic activity (Proschak ct al., 2011).

Furthermore, there are xenofuranones with weakly cytotoxic effects (Brachmann et al., 2006), iodinine as a pigment (Bode, 2009) and xenorhabdins as well as xenorxides have antibacte- rial, antifungal and insecticidal effects (Mclnerney et al., 199 Ia). Szentiamide is a depsipep-tide that shows activity against the malaria-causing parasite P. .talci2anim (Ohlendorf et al., 2011, Nollmann et al., 2012). PAX (peptide-antimicrobial-Xenorhabdus) are lysine-rich cyclopeptides exhibiting antifungal and antibacterial activity (Gaultieri et al., 2009; Fuchs et al., 2011).

Non-ribosomal peptide synthetases (NRPSs) are large muhienzyme complexes that assemble amino acids into non-ribosomal peptides (NRPs). NRPSs consist of several modules and each is responsible for the incorporation of one specific amino acid into the final peptide product.

Each modulc is subdividcd into domains with charactcristic and highly conscrvcd scqucncc motifs (Strieker et al., 2010; Sieber and Marahiel, 2005).

Parasitic infcctions, chiefly malaria but also Icishmaniasis, trypanosomiasis and amocbiasis, constitute one of the most important causes of disease and mortality in the world. Research in this field is of primary interest not only for endemic, mostly tropical countries but also for other areas in which some formerly indigenous infections are presently extinct or eatly re-duced in incidence, but are steadily imported by travelers from other lands.

One particular prominent example of a disease caused by a protozoan parasite is malaria. Ma-laria is a major cause of morbidity and mortality in many parts of the word. Almost one third of the world's population is exposed to the risk of infection. There are an estimated 150 mil- lion cases of malaria per year and in Africa alone the aimual mortality, mainly among chil- dren, is about I million. Accordingly malaria constitutes a major constraint on economic pro-grcss in tropical and subtropical arcas of thc world.

Another example are trypanosomes, which are flagellated protozoa of the genus Trypano- soma. Various species of T;panosoina cause numerous diseases in humans and other mam-mals. For example, Trypanosotna brucel gambiense and Trjpanosorna brucie rhodesiense cause trypanosomiasis, also referred to as African sleeping sickness, transmitted by tsetse flies of the genus Gloss/na.

In South America, a different trypanosomc, Ti;panosotna cruzi, causes Chaga's disease, which affects the nervous system and heart. This parasite is not transmitted by tsetse flies but by a different type of blood-sucking arthropod, Triatoina injèstans, also referred to as the tria-tomine kissing bug. Other species, restricted in distribution to Africa and Asia, cause diseases of horses and cattle. Agents for treating trypanosomiasis are still not satisfactory, and all of the agents require hospital admission and a long period administration. These agents are ex-pensive, and often have haimful side-effects WO 98/50427 dcscribcs thc production, isolation and charactcrization of Xenorhabdus pro-teins, which are toxic to insects upon exposure. The proteins are of a higher molecular weight (above lOOkD) and can be applied to insect larval foods or to plants for controlling insect pests.

WO 03/08964 1 describes domains of non-ribosomalpeptide synthetases (NRPSs that exhibit dual condensation and epimerization activities, which allow for the incorporation of non- protcinogcnic substrates, such as D-amino acids, into pcptidc products. These dual condensa- tioniepimerization NRPS domains may further be used to modify the stereochemistry of syn-thesized peptides at selected amino acid sites.

In view of the above, there is an ongoing interest to identifi new secondary metabolites with antibiotic properties, specifically against protozoan parasites, which can bc used as mcdicincs or plant protection agents in agriculture. Since non-ribosomal peptides from bacterial symbi-onts are known to possess such activities and constitute a class of compounds comprising a wide variety of different substances with assorted functions, which are for the most part still unidentified or insufficiently characterized, it is also a key interest of the present invention to unravel the biochemical pathways of non-ribosomal peptide synthesis and the identification of the essential enzymes and functions of this process.

The above objective is solved in a first aspect by a peptide of the general formula X1...

wherein each x is an amino acid residue independently selected from any D-or L-amino acid, preferably an L-amino acid, n is the number of total amino acid residues x, and y is an amine.

In the above formula the amino acid residues x are interconnected by a peptide bond. The peptide is however in a preferred embodiment a linear peptide, and not a circular peptide. The number n shall denote the number of total amino acid residues within the peptide of the inven-tion. For example for n = 3, the peptide has the general formula Xi -X2 --y, wherein each of Xi, X2 and X3 can be the same or different amino acids. For the above formula n is never 0, and n = 1 shall refer to a peptide with the general formula Xi -y, whereas n = 2 shall rcfcr to a pcptidc with thc gcncral formula Xi -X2 -y.

In the context of the present invention the amino acid residue x can be selected from any amino acid known to the person of skill in the art, such as standard L-or D amino acids, or modified amino acids, such as N-methylated amino acids. While in the context of the present invcntion thc twenty-two amino acids arc prcfcrrcd which arc naturally incorporatcd into polypeptides, so called proteinogenic or natural amino acids, also non-standard amino acids obtainable by chemical modification of the standard amino acids are included; preferably in- cludcd arc N-mcthylatcd amino acids which carry a mcthyl group at thc pcptidc chain nitro-gen.

In the above formula the amine y is bound to the last amino acid residue x on the C-terminal end of the peptide backbone via the amino acid's carboxyl group.

In one first embodiment the peptide according to the invention is preferred wherein n is a number between I and 8, and more preferably wherein n is a number between 2 and 7.

On the other hand in other embodiments of the invention the peptide carries a y which is an amine selected from the amines of formulas a, f3, x or 6

NH x

Preferred peptides of the invention include such peptides which in the above formula have an x which is independently selectcd from any amino acid or N-methyl amino acid, prefcrably wherein thc amino acid or N-methyl amino acid is s&ected from phcnylalaninc, tyrosine, valine or leucine, or the respective N-methyl variants thereof In one embodiment of the invention a peptide is preferred wherein at least one amino acid residue x is a N-methyl amino acid.

In yet another preferred embodiment the peptide of the invention has the general formula xl -x2 -y, wherein xl and x2 are N-methyl amino acids, and y is an amine of formulas CL or preferably wherein x2 is N-methyl phenylalanine. Such peptides are generally referred to as Xenortides.

Preferred Xenortides of the invention are selected from the peptides of formulas A to D In one embodiment Xenortides A to D are composed of only L-amino acids, or of only D-amino acids.

Specifically preferred is in one embodiment Xenortide D. Xenortide D can in a further em-bodiment also be used for medicinal purposes as described herein below.

In another embodiment a peptide selected from Xcnortide A to C is preferred fbr use in medi-cine as described herein below. Preferably Xenortide A or B are used for treating a disease caused by a parasite selected from Trypanosoma brucel rhodesiense (STIB900), Trypano- sotna cruzt (Tulahuen C4), Leishinania donovani (MHOM-ET-67/L82), and Piastnodiuinfai-cz7arum (NFS4).

Other embodiments of the invention relate to peptides wherein each x is independently se-lected from valine, leucine, N-methyl valine or N-methyl leucine, n is number between 3 and 7, and y is an amine of formulas a or 3. Those pcptidcs arc referred to as Rhabdopcptidcs.

In preferred embodiments of the invention Rhapdopeptides are provided selected from formu-las Ito 26 H OKH OvH E uH -cN 1 (1 2 o o 0 0 A 3 H °H °TH :x)*i5±nfnnc

H

4 N (i 4 i $4 K..NK. -. JK$N * r N 13 14 15 1 4 1 II I i -H 24.. -K. KF4 *& -L..--..----. H..N.N..,K. -N*--..-.

N H Nç r NH( H) j H

C -16 17

I

H H

U -... N --. N 4 1 J It 1.. JH iJJ: C 21 u H H H I H [ [ I N N N I N 14 -N --N -K N -yxj: i1fl ii -( )T 22 23 o Y C

II -

C

H

In one preferred embodiment Rhabdopeptides 24 to 26 are used for the treatment of a para-sitic disease caused by a parasite selected from Tr;panosonza bruce! rhodesiense (STIB900), Tnpanoso;na cruzi (Tulahuen C4), Leis/unania donovani (M HOM-ET-67!L82), and Plasma-cliumfalciparum (NF54).

In yet another embodiment of the invention a peptide is provided, wherein in the above for-mula each x is independently selected from valine or N-methyl valine, n is a number between 4 and 8, preferably 5, andy is an amine of formulas x or. These peptides are referred to as Mevalagmapeptides.

In a further embodiment of the invention a Mevalagmapeptide is selected from formula I or II

N 0 0 1 H H

N NH

S H: .H

I

N N N

H I 0 0

II

Further preferred peptides of the present invention are the peptides shown in Figures 8 and 9.

Independent of the above, the peptides as described herein arc preferably non-ribosomal syn-thesized peptides. This includes specifically peptides, which can be synthesized by one or more specialized non-ribosomal peptide-synthetase (NRPS) enzymes. The NRPS genes for a certain peptide are usually organized in one operon in bacteria. The enzymes are organized in modules that are responsible for the introduction of one additional amino acid. Each module consists of several domains with defined functions, separated by short spacer regions of about 14-36 amino acids. Those modules are used iteratively in the biosynthesis process.

The peptides according to the present invention are preferably for use in a method for treat-ment of the human or animal body by surgery or therapy, or for diagnostic methods practised on the human or animal body. Thus it is preferred that the peptide is for use in the treatment of a human or animal disease.

In preferred embodiments of the present invention the peptides have a low cytotoxicity.

in the context of the present invention the disease is preferably a disease caused by a proto- zoan parasite, in particular wherein the disease is selected from Malaria, Amoebiasis, Giardi-asis, Toxoplasmosis, Cryptosporidiosis, Trichomoniasis, Chaga's disease, Leishmaniasis, Sleeping Sickness or Dysentery leishmaniasis.

The above problem of the invention is also solved by the use of a peptide according to the above embodiments as an insecticide, and/or as a plant protection agent.

In a further aspect, the objective of the invention is solved by a method for the production of a peptide according to the above embodiments, comprising culturing a host cell that expresses a non ribosomal peptide synthetase (NRPS), wherein said NRPS comprises at least one of each of the following domains (or activities): (i) an adenylation (A) domain, (ii) a peptidyl carrier protein (PCP) domain (also referred to as thiolation (T) domain), and (iii) a condensation (C) domain; wherein said host cell is cultured under conditions suitable for the synthesis of non-ribosomal peptides.

For an NRPS, the minimal elongation module consists of the following domains: The approx.

550 amino acid long adenylation (A) domain is responsible for the activation of one specific amino acid by formation of an aminoacyl adenylate intermediate at the expense of adenosine- 5'-triphosphate (ATP) and release of pyrophosphate (PPi). Moreover, it facilitates the nucleo-philic attack of the free thiol group of the phosphopantetheinyl cofactor of the peptidyl carrier protein to the aminoacyl adenylate.

The peptidyl carrier protein (PCP) domain, also referred to as thiolation (T) domain is the only domain without catalytic activity. It is located downstream the A domain and contains approx. 100 amino acids. Attachment of the phosphopantetheinyl group of coenzyme A eova-lently to the high conserved specific serine residue converts the inactive apo-PCP into the active holo-PCP. Holo-PCP is able to transport the covalently bound intermediates to the cata-lytic centers of the other domains.

The condensation (C) domain comprises approx. 450 amino acids and catalyzes the nucleo-philie attack of the downstream PCP-bound acceptor amino acid on the activated thioester of the upstream PCP-bound donor amino acid (Strieker et al., 2010; Sieber and Marahiel 2005).

Said NRPS further comprises one or more additional domains (or activities) selected from the group of (iv) thioesterase (TE) domain, (v) an epimerization (E) domain, (vi) heterocycliza-tion (Cy) domain, (vii) an N-methylation (N-MT or MT) domain, (viii) oxidation (0) domain or (ix) reduction (R) domain.

For the release of the product, usually an approx. 280 amino acid long thioesterase (TE) do-main is present at the C-terminus catalyzing the transfer of the intermediate from the PCP domain to the serine in the catalytic center of the TE domain. The intermediate can then either -10-be released by hydrolysis as a linear acid or by an intramolecular reaction as a cyclic peptide (Strieker et al., 2010; Sieber and Marahiel, 2005).

Atcrnatively, a C-terminal condensation domain can release thc peptide by attack of an amine.

Furthermore, there are several modification domains such as the 450 amino acid long epim-erization (E) domain, that catalyzes the epimerization of L-amino acids to D-amino acids, the hererocyclization (Cy) domain that introduces a thiazoline or oxazoline ring into the peptide on amino acid substrates possessing a f3-heteroatom such as cysteine, senile and threonine (Kelly et al., 2005) and is also responsible for peptide elongation, the approx. 420 amino acid long N-methylation (N-MT or MT) domain which is inserted into the accompanying A do-main and transfers thc S-methyl group of S-adcnosylmethionine (SAM) to thc amino group of the thioesterified amino acid and the approx. 250 amino acid long oxidation (Ox) or reduction (Red) domains that influence the oxidative state of the building blocks (Strieker et al., 2010; Sieber and Marahiel, 2005).

The term "building blocks" shall in the context of the present invention refer to the individu-ally introduced amino acid residues x of the peptides according to the present invention.

In one frirther embodiment of the invention the above method for the production of a peptide according to the invention is preferred, wherein at least one, preferably more, most preferably all of said domains (or activities) (i) to (ix) are encoded by a fragment of a nucleic acid se-quence according to SEQ ID No. I to II, wherein said fragment encodes for a domain (or activity) (i) to (ix) as defined above.

Non-ribosomal peptide synthetases are not limited to proteinogenic amino acids and can also include fatty acids, sugars and other substrates, thus resulting in a very large chemical diver-sity of the final peptides of the invention.

In certain embodiments of the invention said NRPS is encoded by a nucleic acid sequence according to SEQ ID No. I to II, or a nucleic acid sequence that is at least 50%, preferably 75%, more preferably 90% or 95 % identical to a sequence according to SEQ ID No. ito 11; or wherein said NRPS comprises an amino acid sequence according to SEQ ID No: 12 to 41, -11-or an amino acid sequence that is at lease 50%, preferably 75%, more preferably 90% or 95 % identical to a sequence according to SEQ ID No. 12 to 41.

In preferred embodiments of the present invention a method for the production of an inventive peptide is provided, wherein said host cell is a Xenorhabdus or Photorhabdus cell. Alterna- tively the NRPS can be produced in other host organisms, which are suitable for the produc- tion of non-ribosomal peptides, specifically which are suitable for the production of the pep-tides according to the present invention.

Preferred is that the Rhabdopeptides and Xenortides of the present invention are produced with the above method wherein the host cell is a Xenorhahdus nernatophila cell, preferably a cell of thc strain ATC 19061 (HGBO81), or wherein thc host cell is a Xenorhahdus cahanil-las/i cell, preferably of the strain DSM 17905 (for Rhabdopeptides 24 and 25). Also preferred is a method for the production of the Mevalagmapeptides of the invention, preferably of for-mulas I or II, wherein the host cell is a Photorhabdus luininescens cell, such as a TTOI cell.

Other preferred host cells are indicated in the figures 2 to 7.

In the above method for the production of an inventive peptide said NRPS is ectopically ex- pressed, preferably under the control of a strong constitutive promoter, such as the rpsM pro-moter of the ribosomal protein S13 of Photorhabdus lutninescens. However, in general any promoter can be used which is active in the host cell of choice. Of course such promoters are preferred which allow for a strong expression of the peptide in said host cell. Other promoters that can be used in context of the present invention arc promoters of cipB (Crystal inclusion protein B) and the arabinose inducible PB system.

In the above method for the production of an inventive peptide said culture conditions com- prise the additional supplementation with amino acids and/or amines, preferably the addi-tional supplementation ofphenyethylamine and/or tryptamine.

In one embodiment the method of the invention includes a flirthcr step wherein the pcptidcs are isolated from said culture using adsorptive beads, such as Amberlite XAD 16® beads.

-12 -The above objective is in another aspect solved by a genetic construct comprising a sequence encoding for an NRPS, wherein said NRPS comprises at least one of each of the following domains (or activities): (i) an adenylation (A) domain, (ii) a peptidyl carrier protein (PCP) domain (also referred to as thiolation (T) domain), and (iii) a condensation (C) domain; and optionally one or more additional domains (or activities) selected from the gronp of(iv) thio-esterase (TE) domain, (v) an epimerization (E) domain, (vi) heterocyclization (Cy) domain, (vii) an N-methylation (N-MT or MT) domain, (viii) oxidation (Ox) do-main or (ix) reduction (Red) domain.

In another aspect the present invention pertains to an non-ribosomal peptide synthetase (NRPS) module, comprising at least one of each of the following domains (or activities): (i) an adcnylation (A) domain, (ii) a pcptidyl carrier protcin (PCP) domain (also rcfcrrcd to as thiolation (T) domain), and (iii) a condensation (C) domain; and optionally one or more addi-tional domains (or activities) selected from the group of (iv) thioesterase (TE) domain, (v) an epimerization (E) domain, (vi) heterocyclization (Cy) domain, (vii) an V-methylation (N-MT or MT) domain, (viii) oxidation (0) domain or (ix) reduction (R) domain.

One embodiment of this aspect is a NRPS module as described before, comprising an amino acid sequence according to SEQ ID No. 12 to 41, or an amino acid sequence that is at least 50, preferably 75, more preferably 90, 95 or 100% identical to an amino acid sequence ac-cording to SEQ ID No. 12 to 41.

A further aspect relates to an NRPS complex comprising at least two NRPS modules accord-ing to the invention. Preferably the NRPS complcx comprises such NRPS modulcs that arc encoded by the same gene cluster according to Figures 2 to 7.

The NRPS complexes or modules as described before, for use in the production of non-ribosomal peptides.

In a preferred embodiment the NRPS of the invention is a NRPS from Xenorhabdus. The pre-ferred NRPS consists either of four modules RdpA, B, C and D (Figure 10), or -in the casc of Xenortides -of the two modules XndA and XndB (Figure 10). Each module is responsible for the incorporation of one amino acid into the final product. Since the length of the rhabdopep- tides does not correspond to the number of the modules some of the modules are used itera-tively during the rhabdopeptide biosynthesis.

In certain embodiments of the invention the provided genetic construct is usable for the ex-pression of the NRPS in a method for the production of an inventive peptide as described herein above.

In a preferred embodiment of the invention, the genetic construct comprises a nucleic acid sequence according to SEQ ID No. Ito 11, or a nucleic acid sequence that is at lease 50%, preferably 75%, more preferably 90% or 95 % identical to a sequence according to SEQ ID No. I to 11; or coi"p' ising a nucleic acid sequence encoding for an NRPS having an amino acid scqucncc according to SEQ ID No: 12 to 41, or an amino acid scqucncc that is at lease 50%, preferably 75%, more preferably 90% or 95 % identical to a sequence according to SEQ IDNo. 12to41.

One additional aspect of the invention pertains to a pharmaceutical composition couiptising as active ingredient one or more peptides as described herein, or salts or derivatives thereof; and optionally pharmaceutically acceptable stabilizers and/or exipients.

In a preferred embodiment of the invention the pharmaceutical composition is for use in the treatment of a disease, specifically wherein the disease is caused by a protozoan parasite, such as Malaria, Amoebiasis, (3iardiasis, Toxoplasmosis, Cryptosporidiosis, Trichomoniasis, Cha-gas disease, Leishmaniasis, Sleeping Sickness or Dysentery leishmaniasis.

Other embodiments of the invention pertain to the pharmaceutical composition, which further comprises at least one additional therapeutically active ingredient and/or at least one pharma-cokinetic booster.

The products and compounds of the present invention can be used in preferred embodiments as medicines or insecticides.

Also provided as one aspect of the invention is a method for the production of a peptide as described herein, wherein said method is a chemical synthesis.

-14 -The present invention will now be explained in the ibliowing examples with reference to the accompanying Figures, without being limited thereto. For the purposes of the present inven- tion, a!! references as cited herein are incorporated by reference in their entireties. In the Fig-ures, Figure 1: Structures of Xenortides A (1), B (2). C (3) and D (4), Rhabdopeptides pro- duced by X. nematophila HGBO81 A (5), B (6), C (7), 1) (8), F (9) and F (10) and Rhab-dopeptides G (11) and H (12) produced byX cahanillasil DSM 17905.

Figure 2: Biosynthesis gene cluster of Xenorhahdus nenatophi1a (K neinatophila) ATCC 19061. Legend: Arrows genes, circles domains, C (dark) condensation domain, A = adcnyiation domain, PCP = peptidyl carricr protcin, MT = N-mcthyitra.nsfcrasc domain, C (light): terminal condensation domain. Top gene cluster: produce rhabdopeptides 1-i2 and 13-23 (numbering according to claims); Bottom gene cluster: produces xenortides Al) (num-bering according to claims) Figure 3: Biosynthesis gene cluster of further organisms. Legend as in Figure 2. Photor-habtius luminescens TTOI gene cluster produces Mevalagmapeptides I and 11.

Figure 4: Biosynthesis gene chister of fhrther organisms. Legend as in Figure 2.

Figure 5: Biosynthesis gene cluster of further organisms. Legend as in Figure 2.

Figure 6: Biosynthcsis gene cluster of further organisms. Legend as in Figure 2.

Figure 7: Biosynthesis gene cluster of further organisms. Legend as in Figure 2.

Figure 8: Peptides produced by the flirther organisms of Figures 3 to 7. Legend: MeVal: N-methyl-vahne, Val: va!ine, MePhe: N-methvl-phenvia!anine, TRA: tryptamine, PEA: phenylcthylaminc, AGM: agmatine. Top four compounds are only produced by Xenorhahdus iniraniensis DSM 17902. Bottom seven compounds are only produced by Xenorhabdus innexi DSM 16336: -15 -Figure 9: Peptides produced by the fbrther organisms of Figures 3 to 7. Legend as above.

Two check marks indicate two con-mounds with the same mass but two different retention times. (RI) suggesting different sequence of huflding blocks. 668 and 682 are mevalag-mapeptides.

Figure 10: Domain organization of the biosynthetic gene clusters colTesponding for the production of the rhabdopeptides (a) and xenorlides (b) in Xènorhabdus nematophila. In the case of the xenortides, enzyme bound intermediates of 1 and a possible termination mecha-nism catalyzed by the terminal C-domain are shown. C: condensation domain, A: adenylation domain, MT: methyitransferase domain, PEP: peptidyl carrier protein domain.

Figure 11: HPLC MS analysis of xcnortidcs and rhabdopcptides produced in a rhabdopcp-tide overproducer strain of Xenorhabdus nematophila. MS analysis of I and 5 are shown. De picted are (a) base peak chromatograns (BPC) of the rhabdopeptide overproducer strain rpsM-Rhab and (b) BPC of the HGBO8I wild-type. Production after 3 days of incubation was compared in strains cultivated in LB medium (continuous lines). inLB medium*suppIemented with 3 mM of tryptaniine (dashed lines) and in LB medium supplemented with 3 miVi of phenylethylamine (dotted lines). All chrornatograms per figure are scaled in the same inteti-sity.

SEQ ID No, 1 --11: Nucleic acid sequences of newly identified biosynthesis gene clusters SEQ ID No. 12-41: Amino acid sequences of NRPS proteins encoded by the corresponding gene clusters in SEQ ID Nos. I -Ii.

The gene clusters and proteins indicated in the Figures 2 to 7 of the herein disclosed NRPS correspond to the sequence identifiers according to the following table: Nucleotide Sequence Amino Acid Sequence Figure Strain ORF/Protein SEQ ID No.1 SEQ ID No.12 2 ATCCI9O6I RdpA SEQ ID No.1 SEQ IDNo. 13 2 ATCCI9O6I RdpB SEQ ID No.1 SEQ ID No.14 2 ATCCI9O6I RdpC SEQ ID No.1 SEQ IDNo. 15 2 ATCC19O61 RdpD SEQIDNo.2 SEQIDN0.16 2 ATCC19O61 XndA SEQIDNo.2 SEQIDNo.17 2 ATCC19O61 XndB SEQ ID No.3 SEQ IDNo. 18 3 TTO1 Plu0897 SEQ ID No.3 SEQ IDNo. 19 3 TTO1 P1u0898 -16-SEQ ID No. 3 SEQ ID No. 20 3 TTO1 P1u0899 SEQ ID No.4 SEQ ID No.21 3 DSM15199 011153 SEQ ID No.4 SEQ IDNo. 22 3 DSM15199 0RF54 SEQ ID No.4 SEQ IDNo. 23 3 DSM15199 0RF55 SEQ ID No. 5 SEQ ID No. 24 3 DSM16342 0RE21 SEQ ID No. 5 SEQ ID No. 25 3 DSM16342 0RF22 SEQ ID No. 5 SEQ ID No. 26 3 DSM16342 011123 SEQ ID No. 6 SEQ ID No. 27 4 DSM17904 ORF1 SEQ ID No. 6 SEQ ID No. 28 4 D5M17904 ORF3 SEQ ID No. 6 SEQ ID No. 29 4 D5M17904 ORF4 SEQ ID No. 7 SEQ ID No. 30 5 DSM17902 0RF123 SEQ ID No.7 SEQ ID No.31 5 DSM17902 0RF124 SEQ ID No. 8 SEQ ID No. 32 6 DSM16338 ORF6 SEQ ID No.8 SEQ ID No.33 6 DSM16338 ORF8 SEQ ID No. 9 SEQ ID No. 34 7 DSM16336 ORF1 SEQ ID No.9 SEQ ID No.35 7 DSM16336 ORF2 SEQ ID No. 9 SEQ ID No. 36 7 D5M16336 ORF4 SEQ ID No. 10 SEQ ID No. 37 6 DSM17382 0RE66 SEQ ID No. 10 SEQ ID No. 38 6 D5M17382 0RF67 SEQ ID No.10 SEQ ID No.39 6 DSM17382 0RF68 SEQ ID No. 11 SEQ ID No.40 6 DSM 17382 vorORFi SEQ ID No. 11 SEQ ID No. 41 6 DSM17382 ORF1 -17-

Examples

Material and Methods: Bacterial strains and culture condifions E. coli strains were grown on solid Luria-Bertani (LB. pH 7.0) medium at 37 °C and on liquid LB medium at 30 °C and 180 rpm at a rotary shaker. For plasmid selection in E. coil, chloramphenicol (34 jig mt-i) or ampicillin (100 jig mL-i) were added, respectively. All Xenorhabdus strains were cuhivated on solid and liquid LB medium at 30 °C. Xenorhabdus mutants were selected on LB containing rifampicin (40 jig mL-1) and chloramphenieol (34 jig mL-l) at 30 oC All strains were initially grown overnight in S ml LB medium and antibiotic, respectively and inoculated with 0.1% (v!v) of the preculture in 10 mL of the same medium and incubated for 72 h. Construction of rhabdopeptide promoter exchanQe mutant For the construction of the Rhabdopeptide promoter exchange mutant a fragment of 890 bp initiating from the start codon of xncl 2228 was amplified with primers Rhabd NheI for and Rhab Mliii rev, subcloned into pietl.2/blunt and cloned via the restriction sites Nhel and MIul into the conjugatable plasmid pCK_rpsM harboring the native promoter region of the very strong constitutive rpslI promoter of the ribosomal protein S13 of the 30S ribosomal subunit of Photorhabtius iuininescens TTO1. The resulting plasmid was introduced into X nematophlla by biparental conjugation. The mutant was confirmed by phenotypie analysis of the produced compounds.

Compound isolation For the isolation of compound 11 and 12 X. cahanillasil DSM 17905 was cultivated in four 5 L Erlenmeyer flasks at 30 °C on a rotary shaker (160 rpm). Every flask contained I L of LB medium and 2% (v/v) of Amberlite XAD-16 (Sigma-Aldrich). 0.1% (v/v) of a 24h preculture cultivated in the same medium without XAD-l 6 was added. The beads were harvested after three days of cultivation, and separated from cells by sieving. XAD beads were extracted with MeOH (600 mL), and the solvent was removed at reduced pressure on a rotary evaporator, yielding a dark brown oily residue. The crude extract was redissolved in MeOH and prefrac-tionated by column chromatography using a hexane-chloroform-methanol gradient. The 8:2 and 7:3 fractions were used for further purification by preparative RP HPLC MS (Waters 515 -18-HPLC Pump, Waters 2545 Binary Gradient Module, Waters 2998 Photodiode Array Detector, Waters SF0 System Fluidics Organizer, Waters Selector Value, Waters 2767 Sample Man-ager coupled to a Waters 3100 mass detector; XBridge C 18 5 jim RP column, Waters) using a 32 mm gradient from 20-95% MeOH, yielding pure rhabdopeptide G (11; Rt = 7.8 mm; 23.4 mg L-1) and pure rhabdopeptide H (12; Rt = 9.3 mm; 13.6mg L-1). The fractions were dried and analyzed by HPLC MS prior to NMR analysis.

Biological activity testing Hemocyte cytotoxictiy assays with last instar Galleria inellonella larvae were carried out as described previously (Proschak, A. et al. 2011). Bioactivity against the four protozoan para-sites P. falctparum NF54, I cruziTulahuen C4, T. b. rhodesiense STIB900, and L. donovan! MHOM-ET-67!L82, and against rat skeletal myoblasts (L-6 cells) for cytotoxicity assessment was determined as previously described (Orhan,I., et al. 2010).

Example I: Identification of the biosynthesis gene cluster of rhabdopeptkles and xenor-tides, and structure elucidation of the new compounds To get an insight in the produced compounds in Xenorhabdus, the inventors decided to con-struct mutants in the corresponding gene cluster and additional in other similar NRPS gene clusters to identi' the produced compounds. In Xenorhabdus nematophila the NRPS gene cluster is composed of the genes xncl 2228 (it/pA), xnc] 2229 (it/pB), xncl_2230 (rdpC) and xncl 2233 (rdpD).

In the following the compounds referred to in the examples are denoted as in Figure 1. Analy-sis of a plasmid insertion mutant of xncl 2228 (rdpA) and deletion mutants of xncl 2230 (rdpC) and xncl 2233 (rdpD) led to the identification of the six rhabdopeptides A -F (5 - 10) not produced anymore in the mutant strains. Furthermore, an insertion mutant of xncl 2300 (xndA) allowed the identification of the NRPS biosynthesis gene cluster responsi-ble for the production of the already known xenortides A -C (1 -3) and a new derivative xenortide D (4).

According to the herein newly identified NRPS gene cluster in Xenorhahdus neniatophila rdpABCD (SEQ ID No. 1) and xndAB (SEQ ID No. 2), the corresponding NRPS gene clusters in other micro organisms were identifies: P. lutninescens TTOI (SEQ ID No. 3), P. teinperata -19-thracensis DSM 15199 (SEQ ID No. 4), X budapestensis DSM 16342 (SEQ ID No. 5), X stock/ac DSM17904 (SEQ ID No. 6), X in/ran/ens/s DSM17902 (SEQ ID No.7), , X szen-t/nna// DSM16338 (SEQ ID No. 8), X innexi DSM 16336 (SEQ ID No. 9) and X. inc//ca DSM 17382 (SEQ ID Nos. 10 and 11). The corresponding protein sequences arc depicted in SEQ ID Nos. 12 to 41. A schematic representation of the composition of the gene clusters is given in Figures 2 to 7.

Structure elucidation of rhabdopeptides and a new xenortide derivative As the new rhabdopeptides were only produced in small amounts in X neinatophila HGB08 1 cultivated in LB medium under standard lab condition and an isolation of these compounds via preparative RP HPLC MS was not possible therefore and due overlapping with other compounds produced, the inventors decided to elucidate the structure of all compounds using a method for rapid structure elucidation of non-ribosomal peptides using feeding and inverse feeding experiments combining detailed ESI HPLC MS and HR MS analysis without com- pound isolation and structure elucidation via NMR analysis. Feeding experiments with L-methionine-methyl-d3, L-[2,3,4,4,4,5,5,5-D8]valine and L-[2,3,3,4,5,5,5,6,6,6-D10]leucinc to LB medium and for an inverse feeding approach with L-leucine, L-valine, L-phenylalanine and L-phenylethylamine to the wild-type strain X. netnatophila HGBO8 1 and cultivated in [U- 13C]medium revealed the rhabdopeptides as five to seven amino acids large non-ribosomal peptides containing valine, leucine and phenylethylamine differing in N-methylations occur-ring in two different series of building block composition (compound 5, 7, 9 and compound 6, 8, 10). 5 and 6 are composed of phenylethylamine with N-methylleucine and two N-methylvaline and one unmethylated valine or three N-methylated valines, respectively (Figure 1). 7, 8 and 9, 10 showing the same incorporation pattern extended with one or two more N-methylated valines for each, respectively (Figure 1). High-resolution mass spectrometry (HR MS) analysis of the identified compounds indicated as molecular formulas for 5 -10 C2H56N5O4 (5: [M+H] in/z 574.4313, eale. 574.4327), C3H58N5O4 (6: [M+Hj tn/z 588.4469, calc. 588.4483), C33H57N505, (7: [M+H] in/z 687.5158, cale. 687.5167), C9H59N5O5, (8: [M+H] ,n/z 701.5312, calc. 701.5324), C44H78N705 (9: [M+H] tn/z 800.6000, calc. 800.6008) and C45H80N,06 (10: [M+W] in/z 814.6154, calc. 814.6154), re- spectively (Table I). As all predicted molecular formulas were conform to the identified in-corporated building blocks the sequential order of the building blocks in the non-ribosomal peptides were assigned by analysis of the MS2 fragmentation pattern of the rhabdopeptides.

Based on the MS2 fragmentation pattern the rhabdopeptides could be elucidated as linear pep- -20 -tides and analysis of the MS1 fragmentation pattern revealed the correct order of valine and leucine as depicted in Figure 1.

Table 1. High-resolution MS analysis of xenortides A -D (1 -4) and rhabdopeptides A -F (5 -10) produced inXenorhahdus nematophila and rhabdopeptides U -H (11 -12) produced in Xenorha bus cahanillasil.

compound sum formula [He] miz calc. [M +Ht] m/z det. [M +H'] zppm Xenortide A (1) C25H36N302 410.2802 410.2790 2.959 Xenortide B (2) C27HN4O2 449.2911 449.2902 2.076 Xenortide C (3) C24H34N3Q2 396.2646 396.2638 1.928 Xenortide D (4) C26H35N402 435.2755 435.2747 1.821 Rhabdopeptide A (5) C32H56N504 574.4327 574.4313 2.458 Rhabdopeptide B (6) C33H58N504 588.4483 588.4469 2.433 Rhabdopeptide C (7) C38H67N605 687.5167 687.5158 1.361 Rhabdopeptide D (8) C39H69N605 701.5324 701.5312 1.719 Rhabdopeptide F (9) C44H73N706 800.6008 800.6000 0.961 Rhabdopeptide F (10) C45H80N706 814.6165 814.6154 1.276 RhabdopeptideG (11) C32HN6O4 585.4123 585.4111 2.068 Rhabdopeptide H (12) C33HN6O4 599.4279 599.4267 2.036 Additional to the rhabdopeptides produced in X. nematophila I-10B081, in X. cabanillasil DSM 17905 two related rhabdopeptides 13-Fl (11 and 12) (Figure 1) consisting of two valine and two N-mcthylatcd valincs or one valinc, one icucinc and two N-mcthylatcd valincs in combination with tryptamine as the terminal amine could be identified, isolated and character-ized via feeding experiments and NMR analysis. II was identified with a mass of [M+H] tn/z 585.4111, calc. 585.4123 (C2I-l53N6O4) and 12 of [M+I-(] ,n/z 599.4267, calc. 599.4279 (CH55N6O4) (Table 1, Table 2).

Table 2. NMR data of rhabdopeptide U (11) and H (12) fromx cabanillasli. Me (methyl), TRA (tryptamine).

11 (isolated) 12 (isolated) subunit position 6H, mult. (J in Hz) 6 6H, mull. (J in Hz) N-Me-L-Va11 1 170.9 172.7 2 68.7 2.91, d (4.79) 70.1 2.73, t (5.77) 3 30.4 1.85, m 31.0 1.78, m 4 18.7a 0.88, m 17.8a 0.77, m 19.28 0.87, mb 18.38 0.78, mb HN-CH3 38.8 2.25, s 35.0 2.19, S -21 -L-LeuI L-VaV 1 172.1 171.2 2 50.3 4.48, d (8.91) 56.8 4.45, d (8.46) 3 41.0 1.39, m 31.0 1.90, m 4 23.8 1.55, m 18.4a 0.80, m' 23.r 0.89, m 19.la 0.82, m' 6 19.2 0.87, m NH 8.13, d (8.41) 7.86, d (9.14) L-VaI 1 172.6 172.7 2 53.9 4.47, d (8.91) 54.2 4.46, d (8.66) 3 30.0 1.96, m 30.6 1.95, m 4 18.7a 0.81, mb 18.5a 0.84, m' 18.sa 0.79, mb 19.2a 0.86, mb NH 8.20, d 8.19, d (8.36) N-Me-L-Va14 1 170.8 169.7 2 61.0 4.60,d(11.0) 61.4 4.59,d(11.01) 3 25.9 2.10,m 26.1 2.11,m 4 21.1 0.82, m 18.7 0.68, d (6.55) 18.0 0.67, d (6.55) 19.3a 0.86, mb N-CH3 30.2 3.06, s 30.7 3.08, s TRAb 1 136.7 136.7 2 118.1 7.32, d (8.07) 118.6 7.51, d (7.87) 3 118.2 6.97, t (7.04) 118.5 6.96, t (7.48) 4 120.8 7.06, t (7.19) 121.5 7.06, t (7.54) 111.2 7.52, d (7.83) 111.5 7.32, d (7.97) 6 127.2 127.5 7 111.5 111.2 8 122.4 7.11,s 122.8 7.11,s NH (md) 10.78 10.77 9 25.0 2.77, d (7.48) 25.4 2.78, d (6.56) 38.7 3.32, m 39.3 3.33, m NH 8.07, t (5.62) 8.07, t (5.33) °: ambiguous 1C protons h: ambiguous H protons Using the same approach for structure elucidation of the xenortides a fourth up to now un-known xenortide derivative could be identified (Figure 1). Xenortide D (4) ([M+H'] in/z 435.2747, calc. 435.2755, C26H35N402) consists of N-methylated valine as in the case of xenortide C (3), N-methylated phenylalanine and tryptamine as in xenortide B (2) (Table 1,

Table 3).

Table 3. Structure elucidation of xenortides A -D (1 -4) resulting from (a) feeding experi-ments in HOBOS1 (b) following ESI MS2 fragmentation analysis for the verification of the new Xenortide D (4). a)

labeling experiment corn-m/z Sum formula corn-m/z Sum formula pound [M + WI [Hi pound [M + H] [WI IZC 1 410.2 C25H36N302 3 396.2 C24H34N302 -22 - + methyi-2H3-met 416.3 C25H302H6N302 402.2 C24H282H6N302 + 2Ho-ieu 419.2 C25H272H9N302 396.2 C24HN3O2 ± 2H8-vai 410.2 C25H36N302 403.3 C24H272H7N302 ± 2H8-phe 417.2 C25H292H7N302 403.3 C24H272H7N302 424.3 C25H222H14N302 410.3 C24H202H14N302 435.3 420.2 + ieu 429.3 420.2 + lie 435.3 420.3 1CHNO + vai 435.3 415.3 C51C19HN3O2 + phe 426.3 C913C16H36N302 411.2 C91'C15HN3O2 418.2 403.2 C171C7HN3O2 ± phenyiethyia-427.3 C813C17H36N302 412.3 C313C16HN3O2 mine + tryptamine n.d. n.d. n.d. nd.

413.3 C25H3615N302 399.3 C24H15N3O2 IZC 2 449.2 C27H37N402 4 435.2 C26H35N402 ± methyi-2H3-met 455.3 C27H312H6N402 441.2 C26H292H6N402 + 2H10-ieu 458.2 C27H252H9N402 435.2 C25H35N402 + 2H3-vai 449.2 C27H37N402 442.3 C26H232H7N402 + 2H8-phe 456.2 C27H302H7N402 442.2 C26H282H7N402 476.4 461.4 + eu n.d. n.d. n.d. nd.

+ iie n.d. n.d. n.d. nd.

+ vai 476.3 nd. nd.

± phe n.d. n.d. n.d. nd.

± phenyiethyia-n.d. n.d. n.d. nd. mine

+ tryptamine 466.3 C1013C17H37N402 451.4 C101C16H35N4O2 453.3 C27H3715N402 439.3 C26H3515N402 n.d. (not detected) b) compound fragmentation pattern incooperated amino acid 1 410.2 -289.1 phenyiethyiamine 289.1 -162.2 methyileucine 162.2 methyiphenyiaianine 2 449.2 -289.1 tryptamine 289.1 -162.2 methyileucine 162.2 methyiphenyiaianine 3 396.2-275.2 phenyiethyiamine 275.2-162.2 methyivahne 162.2 methyiphenyiaianine 4 435.2-275.2 tryptamine 275,2-162.2 methyivahne 162.2 methyiphenyiaianine Further novel peptides were elucidated using a similar approach P. luniinescens TTO 1, P. reniperasa thracensis DSM 15199, X buclapestensis DSM 16342, X stock/ac DSM17904, X iniraniensis DSM17902, X szentir;naii DSM16338, X innexi DSM 16336 and X inc//ca DSM 17382. The composition of the peptides is given in Figures 8 and 9.

-23 -Example II: Biosynthesis of rhabdopeptides and xenortides by iterative NRPS module usage Detailed bioinformatic analysis of the postulated biosynthesis gene cluster corresponding to the production of the rhabdopeptides led to a four module NRPS system (RdpABCD). Each module is standalone and responsible for the loading and incorporation of valine or N-methylated valine and N-methylated leucine. RdpA and RdpB contained C-A-MT-T extender modules each and RdpC (C-A-MT-T-C) an extender module with a terminal C-domain, which might be involved in the condensation of the amine with the peptide during the release mechanism (Figure 10 a)). Interestingly, RdpD might act as an additional termination module (C-A-T-C) for alternative rhabdopcptides consisting a C-terminal tryptaminc instead of the phenylethylaminc (Figure 10 a), Table 4).

Table 4. Additional rhabdopeptide derivates identified in the rhabdopeptide overproducer strain rpsM-Rhab or in Galleria niellonella infected with X. nematophila FIGBO8I 14 d post infection. Tryptamine rhabdopeptides (13 -18), derivates of 5 -10, could be identified in the wild type strain in trace amounts and in the insect model. Furthermore, 15 new phenylethyl-amine derivates (19 -33) could be identified in the overprodueer strain. For 11 derivates a possible structure and sum formula could be elucidated via feeding experiments and analysis of the fragmentation patterns (data not shown).

compound m/z sum for-identified in proposed structure [M+H] mula > -C) 0 W = 0 -=

S

C C

13 613.3 C34HN6O4 / / * 14 627.8 C35HsaN6O4 / / X 726.6 C40H67N705 / / * 16 740.5 C41H69N,05 / / x -24 -3N7 0NV 17 839.5 C46H78N806 I I x 18 853.4 C47H80N806 I I x

H o

19 433.2 C24H40N403 x x I oi 447.2 C25H42N403 x (1) 1 oi 21 461.3 C26H44N403 x x H H ° H 22 532.3 C29H49N504 x x I o o 23 546.3 C30H51N504 x (1) 1 o o -L 24 560.3 C3i H53N504 1 1 1

H

602.2 C34H59N504 x I I I o'K o 26 645.2 C35H60N605 x (I) I I ° ° 27 659.3 C35H52N605 x (1) I 28 673.4 C37H64N605 I j a 29 715.4 C40H70N605 x I I a 0H 772.4 C42H73N706 x (I) I -25 - 31 786.4 C43H75N706 x.7 a 32 899.5 C49HN8O7 x (.1) a 33 913.5 C50H53N307 (/) 1 1 a (ambiguous; rhabdopeptide fragmentation pattern identified, but no structure elucidation is possible) The biosynthesis gene cluster for the xenortides consists of two NRPS genes. XndA consists of C-A-MT-T and might be responsible in loading N-methylvaline oder N-methyleucine.

XndB (C-A-MT-T-C) is responsible for the elongation with N-methylphenylalanine and also carrying a terminal C-domain. As for the rhabdopeptides this C-domain might catalyze the termination of the enzyme bound peptide intermediate by condensation with the decarboxy-lated amino acid during the release mechanism (Figure 10 b).

All adenylation (A) domains of RdpABC and XndAB harboring an additional S-adenosylmethionine-dependent N-methyhransferase, which is nested between the A-domain motifs A1-8 and A9-l0. All methyltransferases harbor the highly conserved GxGxG amino acid sequence, like it is invariably available in motif I of N-methyltransferases. In contrast, RdpD contains an A-domain without additional methylation. Prediction of the amino acid specificity for the xenortide A-domains XndA Al and XndB A2 using the 10 amino acid code of Stachelhaus revealed phenylalanine as most likely incorporated amino acid. But only, for XndB_A2 this specificity could be detected. For RdpA_A1, RdpB_A2 and RdpC_A3 a loading of L-2,3-diaminopropionic acid (LDAP) were predicted and the 10 amino acid code show no invariation for one of these domains as the inventors would expect due the different incorporation of leucine or valine in the rhabdopeptides (Table 5).

Table 5:

-26 -Adenylation Gene: Small-cluster or Single amino ada Stacheihaus domain locus tag large cluster predIction: or code nearest rle:iQhhor XndAAIMTI AntI 2228 Phe.Tro. FhqTw.Bh Phe DALLMGAVCK XndB_A2_MT2.rnei_2229 Phe, Tp Phe DAFTVAGVCK RdpAAl_MTi XflCi 2230 Phe.Trp.PhgjyrBht WAP DALYLAYSIK RdpB_A2_MT2 wet 2233 Phe Tru.PhgTvr.Bht LOAF DALVLAVSK RdpC_A3_M13 xncl 2300 Phe,TrpFhqTyi.Bhl LOAF DALVLAVSFK RdpD_A4 xnci_2299 n/a Fhe VAIMLGASM- n/a not avuar4e). LOAP (L-2<3-daminoDrop[.nnic Pha:(ohenyl-ycie. Bht ibeta-iydroxy-tyione) Example III: Identification of additional rhabdopeptide derivatives in rhabdopeptide overexpression mutant Encouraged by the idea of an iterative usage of the NRPS modules in the rhabdopeptide bio-synthesis, a rhabdopeptide overproducer strain using the strong constitutive rpsM promoter of the ribosomal protein S13 of the 30S ribosomal subunit of Photorhabdus iwninescens TTOI was constructed. Afier cultivation for three days an overwhelming amount of new rhabdopep- tide derivatives could be observed (Figure 11). Additional supplementation with phenyla-lanine and tryptamine, respectively, enforced the production especially in the phenylalanine supply.

MS analysis of this extract allowed identifying 15 new phenylethylamine derivatives (19-33, Table 4). All these compounds exhibit the typical rhabdopeptide fragmentation pattern includ-ing the mass difference of m/z 121 for the incorporation ofphenylethylamine and;n/z 99, 113, 127 for valine, methylvaline/leucine, and methylleucine, respectively.

Example IV: Biological Activity of Rhabdopeptides and Xenortides In order to determine the biological activities of the peptides of the present invention 1C50 values of Xenortides A and B, as well as Rhabdopeptides 24 to 26 (see below) against four different protozoan parasites were determined. As control, aLso the 1C50 values of known anti parasitic substances were included as well the cytotoxicity values of the tested peptides (table XY).

-27 -

Table 6.

--z::

U --J o

0-50 0-50 IC-SO IC-SO IC-SO [pg/mL] (fig/mLI [pg/rnLI [pg/rnL] [pg/mU -MelarsoproF 0.004 6enzndazoIe 0.498 MiItefosne 0.174 Chloroquine 0.003 -Podophyliotoxin 0.009 1 XenortidA(LL 181 35.2 68.7 10.5 47.9 2 XenortidA(D,D 9.22 19.0 51.0 7.85 47.3 3 Xenortid B(L.L)S.N 0.731 5.24 49.9 0.343 6.14 4 Xenortid B (D,D 0.729 7.09 48.9 0.046 8.1 Rhabdopepd 24 (all L)N 6.79 18.4 37.4 4.53 45.3 Rhabdopepd 25 (oIl L) 4.88 15.5 17.1 3.32 40.2 7 Rhabdopepd26(aIILI 0.202 21.4 86.6 0,367 7.62 In the Table 6 the biologic activity of isolated non-ribosomal peptides is shown. L, or D de- notes the stereochemistry of the amino acid building blocks of the compounds. S, and N de-note whether the compound was chemically synthesized (S) or is an isolated natural product (N). Rhabdopeptid 24 (5) and 25 (6) show also activity against Galleria mellon ella hemocytes (EC-50 of 19.8 and 7.6 gIml) The following structures correspond to the compounds used in the above experiments 1 to 7

(Table 6):

-28 -

--

I

H

H H

--

7 AN-Cr\\

H H

Example 1: Chemical Synthesis of Rhabdopeptides Rhabdopeptide Synthesis Rhabdopeptides differ in their amino acid composition or order, C-terminal amine and their methylation pattern or degree. According to their methylation patter the inventors divided them in three different groups; partly methylated, highly methylated and permethylated Rhab-dopeptides.

Synthesis of N-mcthyl-L-valinc-L-valinc-phenylethylaminc (shod partly methylated Rhab dopeptide) -29 - N-(tert-Butoxyearbonvl)-L-valine-phenylethylamine. 1.0 eq of N-(tert-Butoxycarbonyl)-L-valine was dissolved in 2 mL/mmol dry dichioromethane and cooled to 0°C while stirring, then 1. 0 eq 1 -Hydroxybenzotriazole hydrate and 1.1 eq N-Ethyl-N'-(3-dimethyl-aminopropyl)carbodiimide were added to the reaction mixture, followed by the addition of 1.5 eq phenylethylamine. The reaction mixture was stirred at 0°C and warmed to room tem-perature within 18 h. The reaction's progress was monitored using thin layer chromatography (0.2 mm silica gel with fluorescent indicator pre-coated polyester sheets; eluent: 90% chloro-form, 10% methanol). Then the reaction mixture was diluted with dichloromethane and washed once with water, satured NaF1CO solution and I M NaFISO4 solution. Subsequently, the organic layer was dried with Na2SO4 and the solvent removed under reduced pressure.

The product was obtained as a white powder (yield: 89%) and submitted to NMR speetros-copy.

L-valine-phenylethylamine. To remove the N-terminal protecting group the peptide was dissolved in 6 mlL/mmol dichloromethane and incubated while stirring at 0°C with 3 mL/mmol triflouroacetic acid for 60 mm. The reaction's progress was monitored using thin layer chromatography (0.2mm silica gel with fluorescent indicator pre-coated polyester sheets; eluent: 90% chloroform, 10% methanol). After all of the N-(tert-Butoxycarbonyl)-peptide was consumed, the solvent was removed under reduced pressure.

N-methyl-L-valine-L-valine-phenylethylaniine. 1.1 eq of L-valine-phenylethylamine was dissolved in 10 mL!mmol dry dichioromethane and cooled to -10°C, then 1.1 eq 2-Bromo-I -ethyl-pyridinium tetrafluoroborate and 3.2 eq iYN-diisopropylethylamine together with 1.0 eq N-(iert-Butoxycarbonyl)-N-methyl-L-valine. The reaction mixture was stirred at -10°C for mm and then slowly warmed to room temperature within 18 h. The reaction's progress was monitored using thin layer chromatography (0.2 mm silica gel with fluorescent indicator pre-coated polyester sheets; eluent: 90% chloroform, 10% methanol). Then the organic solvent was removed under reduced pressure and the N-terminal protecting group cleaved according -30 - to standard protocol (see L-valine-phenylethylamine), followed by a HPLC-ESI-MS purifica-tion.

Following the synthesis of N-methyl-L-valine-L-valine-phenylethylamine all other short pep-tide sequences (up to three amino acids) were synthesized in solution.

Synthcsis of N-mcthyl-L-valinc-L-valinc-L-valinc-L-valine-phcnylethylaminc (longer partly methylated Rhabdopeptide) N-(tert-Butoxycarbonyl)-N-methyl-L-valine-L-valine-L-valine. The peptide sequence was assembled with standard 9-flourenylmethoxycarbonyfltert-Butyl chemistry on a preloaded 2-Chiorotrityl chloride resin. The amino acids (6.0 eq, 0.2 mM in N,N-dimcthylformamide) with exception of the methylated building block (and the successive amino acid) were activated in situ with O-(Benzotriazol-1 -yl)-NivcNcN'-tetramethyluronium hexafluorophosphate (5.0 eq, 0.5 mM in N,N-dimethylformamide) in the presence of N,N-diisopropylethylamine (10.0 eq.

2.0 mM in NN-dimcthylformamidc) assisted by microwave irradiation (20W, 70°C, 10 mlii).

In the case of the methylated N-9-flourenylmethoxycarbonyl amino acid and the adjacent amino acid (3.0 eq. 0.5 mM in N-Methyl-2-pyrrolidone) the coupling was conducted with 0- (7-Azabenzotriazol-I -yl)-JV,AçNçN'-tetramethyluronium hexafluorophosphate (3.0 eq, 0.5 mM in V-Methyl-2-pyrrolidonc) and 1-Hydroxy-7-azabcnzotriazole (3.0 eq. 0.5 mM in iV-Mcthyl- 2-pyrrolidone) in the presence of 4-Methylmorpholine (3.3 eq) still assisted with microwave irradiation but at lower temperatures (20W, 50°C, 20 mm). The completion of each coupling was monitored using the Kaiser-or Chloranil-tcst, respectively. The i\T_terminal 9 flourenylmethoxycarbonyl protecting group was cleaved according to standard protocol (20% piperidine in NN-dimethylformamide; 25W, 70°C, 30s/3 min/3 mm). The cleavage of fully protected peptide fragments was achieved by incubation for 10 mm at room temperature with 20% 1,1,1,3,3,3-Flexafluoro-2-propanol in dichloromethane.

N-methyl-L-valine-L-valine-L-valine-L-valine-phenylethylamine. The amine condensa-tion, the cleavage of the N-terminal protecting group and the purification followed standard protocol (see N-methyl-L-valine-L-valine-phenylethylamine).

Following the synthesis of N-methyl-L-valine-L-valine-L-valine-L-valine-phenylethylamine all other longer peptide sequences with only a few methylated amino acids were synthesized prior on resin followed by the amine condensation in solution.

Synthesis of N-methyl-L-valine-N-methyl-L-leucine-N-methyl-L-valine-methyl-L-valine-phe nylethylamine (permethylated Rhabdopeptide N-methyl-L-valine-N-inethyl-L-Ieucine-N-inethyl-L-valine-methyl-L-valine-p henyl-ethylamine. The assembly of the peptide sequence and the cleavage of the fully protected peptide fragment were conducted following standard protocols (see N-(tert-Butoxycarbonyl)-N-methyl-L-valine-L-valine-L-valine). Then the peptide was permethylated with 10.0 eq iodomethane and 6.3 eq sodium hydride in a mixture of 0.4 mlL/mmol tetrahy-drofuran and 0.2 mL/mmol YN-dimethylformamide at 0°C while stirring and warmed to room temperature within I Sh. The reaction's progress was monitored using thin layer chro-matography (0.2 mm silica gel with fluorescent indicator pre-coated polyester sheets; eluent: 90% chloroform, 10% methanol). After completion of the reaction the solvents were removed under reduced pressure. The residue was dissolved in water, washed with n-hexane, acidified to pH 1 with I M hydrochloric acid, diluted with satured brine and extracted with ethyl ace-tate. The organic phases were combined, dried with Na2504 and the solvents removed under reduced pressure. This was followed by an ester saponification with lithium hydroxide solu- tion (6.0 eq. 0.4 M in water) in a mixture of 5 mL/mm& methanol and IS mL!mm& tetrahy-drofuran. After completion of the reaction the reaction mixture was dissolved with saturated NaHCO3 and extracted with diethyl ether. The organic phases were combined, dried with Na2504 and the solvents removed under reduced pressure. The amine condensation, the -32 -cleavage of the N-terminal protecting group and the purification followed standard protocol (see N-methyl-L-valine-L-valine-phenylethylamine).

Following the synthesis of N-methyl-L-valine-N-mcthyl-L-lcucine-N-methyl-L-valine-methyl-L-valine-phe nylethylamine other permethylated rhabdopeptides were synthesized prior on resin followed by permethylation and amine condensation in solu-tion.

Synthesis of N-methyl-L-leucine-N-methyl-L-valine-L-valine-N-methyl-L-valine-yhenyl ethylamine (hiihly methylated Rhabdopeptide) N-(tert-Butoxycarbonyl)-N-methyl-L-leucine-N-methyl-L-valine-L-valine. The synthesis was conducted following standard 9-flourenylmethoxycarbonyl/tert-Butyl chemistry on a preloaded 2-Chlorotrityl chloride resin (see N-(tert-butoxycarbonyl)-N-methyl-L-valine-L-valine-L-valine) using methylated building blocks or via amino acid specific methylation according to literature. The cleavage of the fully pro- tected peptide fragment was carried out following standard protocols (see N-(tert-butoxycarbonyl)-N-methyl-L-val me-L-val me-L-val I ne).

N-methyl-L-valine-phenylethylamine. The reaction of the amino acid with the phenylethyl-amine was carried out in solution followed by the cleavage of the N-terminal protecting group according to standard protocols (see N-methyl-L-valine-L-valine-phenylethylamine).

N-methyl-L-Ieucine-N-methyl-L-valine-L-valine-N-methyl-L-valine-phenylethy lamine.

Condensation of the two building blocks and cleavage of the N-terminal protecting group as well as purification followed standard protocols (see N-methyl-L-valine-L-valine-phenylethylamine).

-33 - The synthesis of other highly methylated Rhabdopeptides differed dependent on theft methy- lation pattern, but all of the building blocks were synthesized and coupled following the stan-dard synthesis pmtocols described prior to this.

Claims

-34 -Claims 1. A peptide of the general formula xi-...-x1I-y, wherein each x is an amino acid residue independently selected from any D-or L-amino acid, n is the number of total amino acid residues x, andy is an amine.
2. The peptide according to claim 1, wherein n is a number between 0 and 8, preferably wherein n is a number between 2 and 7.
3. The peptide according to claim 1 or 2, wherein y is an amine selected from the amines of formulas a, 13, x or 6H NH x 5
4. The peptide according to any one of daims I to 3, wherein x is independently selected from any amino acid or N-methyl amino acid, preferably wherein the amino acid or N-methyl amino acid is selected from phenylalanine, tyrosine, valine or leucine, or the respective N-methyl variants thereof.
5. The peptide according to any one of claims Ito 4, wherein at least one amino acid residue x is a N-methyl amino acid.
6. The peptide according to any one of claims Ito 5, of the general formula x1 --y, wherein x1 and x2 are N-methyl amino acids, and y is an amine of formulas a or 13, preferably wherein x2 is N-methyl phenylalanine.-35 -
7. The peptide according to claim 6, wherein the peptide is a Xenortide selected from the peptides of formulas A to D

OKO
8. The peptide according to anyone of claims I to 5, wherein each x is independently selected from valine, leucine, N-methyl valine or N-methyl leucine, n is number between 3 and?, andy is an amine of formulas a or
9. The peptide according to claim 8, wherein the peptide is a Rhabdopeptide selected from formulas I to 26 o -/ xH-5 <ic o ---------0 0 0H -I IX t) rN 13 4 O" 0 2-t(1)±fU fffto 2: 5 (I Ji / C N = : --4 :; : / zr 71.I0J) (\ .-< N \/t ( zi /_) / / / -/ /\ .. / \/ -,2 -. -& a \ r / I / ,-, ax / / aS j /\ / IN. _2 -2 Co a 1. 0 K fl::::/ C::: T N.. I --/ ,2i -/ -.( K) / / C) / -.-, .___Zf -Zr...,-J N / / N 7 S..2 K. \ ai ( \ XI o /o _/ N. - / ø_:: -H. /t/\ / C frk.__z I ç1J <NiU-37 -
10. The peptide according to any one of claims Ito 5, wherein each xis independently selected from valine or N-methyl valine, n is a number between 4 and 8, preferably 5, andy is an amine of formulas xor 6.
11. The peptide according to claim 10, wherein the peptide is a Mevalagmapeptide selected from formula I or II H Pt 9 HN N NHIHN t Nk NH H H:CII12. The peptide according to any one of claims Ito 11, wherein the peptide is non-ribosomal synthesized peptide.13. A peptide according to any one of claims I to 12 for use in a method for treatment of the human or animal body by surgery or therapy, or for diagnostic methods practised on the human or animal body.14. The peptide according to claim 13, for use in the treatment of a human or animal disease.15. The peptide according to claim 14, wherein the disease is a disease caused by a protozoan parasite, in particular wherein the disease is selected from Malaria, Amoebiasis, Giardiasis, Toxoplasmosis, Cryptosporidiosis, Trichomoniasis, Chagas disease, Leishmaniasis, Sleeping Sickness or Dysentery leishmaniasis.16. Use ofa peptide according to any one of claims I to 12 as an insecticide, and/or as a plant protection agent.17. A method for the production of a peptide according to any one of claims Ito 15, comprising culturing a host cell that expresses a non-ribosomal peptide synthetase (NRPS), wherein said NRPS comprises at least one of each of the following domains (or activities): (i) an adenylation (A) domain, (ii) a peptidyl carrier protein -38-mains (or activities): (i) an adenylation (A) domain, (ii) a peptidyl carrier protein PCP) domain (also referred to as thiolation (T) domain), and (iii) a condensation (C) domain; wherein said host cell is cultured under conditions suitable for the synthesis of non-ribosomal pcptidcs.18. The method according to claim 17, wherein said NRPS frirther comprises one or more additional domains (or activities) selected from the group of(iv) thioesterase (TE) domain, (v) an epimerization (E) domain, (vi) heterocyclization (Cy domain, (vii) an N-methylation (N-MT or MT) domain, (viii) oxidation (0) domain or (ix) reduction (R) domain.19. The mcthod according to claim 17 or 18, whcrcin said NRPS is cncodcd by a nucleic acid sequence according to SEQ ID No. Ito 11, or a nucleic acid sequence that is at lease 50%, preferably 75%, more preferably 90% or 95 % identical to a sequence according to SEQ ID No. I to 11; or wherein said NRPS comprises an amino acid sequence according to SEQ ID No: 12 to 41, or an amino acid sequence that is at lease 50%, preferably 75%, more preferably 90% or 95 % identical to a sequence according to SEQ ID No. 12 to 41.20. The method according to any one of claims 17 to 19, wherein said host cell is a Xenorhabdus or Photorhabdus cell.21. The method according to any one of claims 17 to 20, wherein said NRPS is cctopically cxpresscd, preferably undcr the control of a strong constitutivc promoter, such as the rpsi%I promoter of the ribosomal protein S13 of Pijotorliabdus luininescens.22. The method according to any one of claims 17 to 21, wherein said culture conditions comprise the additional supplementation with amino acids andlor amincs, preferably thc additional supplcmentation of phcnylethylaminc and/or ryptam inc.23. The method according to any one of claims 17 to 20, wherein in a further step the peptides are isolated from said culture using adsorptive beads, such as Amberlite XAD I 6 beads.-39 - 24. A genetic construct comprising a sequence encoding for an NRPS, wherein said NRPS comprises at least one of each of the following domains (or activities): (i) an adenylation (A) domain, (ii) a peptidyl carrier protein (PCP) domain (also referred to as thiolation (T) domain), and (iiO a condensation (C) domain; and optionally one or more additional domains (or activities) selected from the group of (iv) thioesterase (TE) domain, (v) an epimerization (B) domain, (vi) heterocyclization (Cy) domain, (vii) an N-methylation (N-MT or MT) domain, (viii) oxidation (0) domain or (ix) reduction (R) domain.25. The genetic construct according to claim 24, usable for the expression of the NRPS in a mcthod according to anyonc of claims 17 to 23.26. The genetic construct according to claim 24 or 25, comprising a nucleic acid sequence according to SEQ ID No. I to 11, or a nucleic acid sequence that is at lease 50%, preferably 75%, more preferably 90% or 95 % identical to a sequence according to SEQ ID No. 1 to 11; or comprising a nucleic acid sequence encoding for an NRPS having an amino acid sequence according to SEQ ID No: 12 to 41, or an amino acid sequence that is at lease 50%, preferably 75%, more preferably 90% or 95 % identical to a sequence according to SEQ ID No. 12 to 41.27. A pharmaceutical composition comprising as active ingredient one or more peptides according to anyone of claims I to 15, or salts or derivatives thereof, and optionally pharmaceutically acceptable stabilizers and/or exipicnts.28. The pharmaceutical composition according to claim 27 for use in the treatment of a disease.29. The pharmaceutical composition according to claim 28, wherein the disease is caused by a protozoan parasite, such as Malaria, Amoebiasis, Giardiasis, Toxoplasmosis, Cryptosporidiosis, Triehomoniasis, Chagas disease, Leishmaniasis, Sleeping Sickness or Dysentery leishmaniasis.-40 - 30. The pharmaceutical composition according to any one of claims 27 to 29, further comprising at least one additional therapeutically active ingredient and/or at least one pharmacokinetic booster.31. A method for the production of a peptide according to any one of claims Ito 15, wherein said method is a chemical synthesis.32. An non-ribosomal peptide synthetase (NRPS) module, comprising at least one of each of the following domains (or activities): (i) an adenylation (A) domain, (ii) a peptidyl carrier protein (PCP) domain (also referred to as thiolation (T) domain), and (iii) a condensation (C) domain; and optionally one or more additional domains (or activities) selected from the group of (iv) thioesterase (TE) domain, (v) an epimerization (E) domain, (vi) heterocyelization (Cy) domain, (vii) an N-methylation (N-MT or MT) domain, (viii) oxidation (0) domain or (ix) reduction (R) domain.33. An NRPS complex comprising at least two NRPS modules according to claim 32.34. The NRPS module of claim 32 or the NRPS complex of claim 33 for use in the production of an non-ribosomal peptide, preferably a peptide according to any one of claims 1 to 15, or for use in medicine, preferably in the treatment of a parasitic disease.