Antimicrobial cyclophane-containing peptides

Abstract

The present disclosure relates to a polypeptide represented by Formula (I): XA1-XA2-XA3- Xn-XB1-XB2-XB3; wherein XA1-XA2-XA3 forms a first 3-residue motif and XB1-XB2-XB3 forms a second 3-residue motif; wherein XA1 and XB1 are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2 and XB2 are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein XA1 and XA3 are connected to form a first cyclophane moiety, and XB1 and XB3 are connected to form a second cyclophane moiety; wherein Xn is an amide bond or 1 to 3 amino acid residue; and wherein the polypeptide optionally comprises a further 3-residue motif.

Description

[0001] Antimicrobial Cyclophane-Containing Peptides

[0002] Technical Field

[0003] The present invention relates, in general terms, to peptides having at least one cyclophane moiety and the methods of synthesising the peptides thereof. The peptides have antimicrobial properties.

[0004] Background

[0005] The CDC and WHO classify Carbapenem-resistant Enterobacteriaceae (CRE) which include the Gram-negative bacteria Klebsiella pneumoniae and Escherichia coli as two of the highest priority pathogens for which new antibiotics are urgently needed. CRE are an immediate threat because of their resistance to any carbapenem and their 50% increase over the last 5 years. Extended-spectrum p-lactamase-producing Enterobacterales (ESBL-E) account for a greater number of cases and more deaths compared to CRE but may still be treated with selected carbapenem antibiotics. The increased use of carbapenems, along with transmission of various resistance mechanisms have likely contributed to the rise in CRE. Both CRE and ESBL-E can lead to severe and deadly infections in hospital and nursing home patients via pneumonia, bloodstream infections, urinary tract infections, wound infections, and meningitis. New antibiotics able to treat both types of infections would reduce the mortality rate and decrease the spread of resistance mechanisms.

[0006] Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a rapidly growing family of natural products with potential antibiotic activities against a broad range of pathogens. RiPPs may be biosynthesized from a ribosomally synthesized precursor, posttranslationally modified, cleaved, then exported to give the mature RiPP. For example, RiPP pathways involving radical S-adenosylmethionine (rSAM) enzymes in their biosynthesis are of particular interest due to their ability to catalyze distinct chemically-demanding reactions leading to unique and bioactive RiPP natural products. The structural diversity and antibiotic activities are demonstrated by several RiPP families including lasso peptides, plantazolicins, lanthipeptides, thiopeptides, and sactipeptides. RiPP biosynthetic gene clusters (BGCs) are attractive for genome mining and synthetic biology due to their compact size and ease of genetic manipulation. For chemically-guided discovery, RJPP pathways are particularly appealing because a single posttranslational modifying enzyme can create unique, structurally complex, and bioactive peptides. Since RiPP biosynthesis is determined by a logic rather than genetically tractable features, their true number and diversity remains enigmatic and a promising source for new peptide scaffolds and antibiotics.

[0007] It would be desirable to overcome or ameliorate at least one of the above-described problems.

[0008] Summary

[0009] The present invention provides a polypeptide represented by Formula (I) : XA1-XA2-XA3-Xn-XB1-XB2-XB3 (I) wherein XAI-XAZ-XAS forms a first 3-residue motif and Xsi-XB2-Xs3 forms a second 3- residue motif; wherein XAI and XBI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2 and XB2 are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein XAI and XA3 are connected to form a first cyclophane moiety, and XBI and XB3 are connected to form a second cyclophane moiety; wherein Xnis an amide bond or 1 to 3 amino acid residue; and wherein the polypeptide optionally comprises a further 3-residue motif.

[0010] In some embodiments, XA3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof.

[0011] In some embodiments, XA2 and XB2 are each independently an amino acid residue, the amino acid independently selected from leucine (L), isoleucine (I), valine (V), alanine (A), proline (P), serine (S), lysine (K), asparagine (N), phenylalanine (F), aspartic acid (D) or a derivative thereof. In some embodiments, XA2 and XB2 are each independently an amino acid residue, the amino acid independently selected from valine (V), alanine (A), or a derivative thereof.

[0012] In some embodiments, XA2 and XB2 are both a valine (V) amino acid residue or a derivative thereof, or both an alanine (A) amino acid residue or a derivative thereof.

[0013] In some embodiments, the polypeptide is represented by Formula (la) :

[0014] XA1-XA2-XA3-Xna-XB1-XB2-XB3-Xnt>-Xci-Xc2-Xc3 (la) wherein XAI-XA2-XA3 forms a first 3-residue motif, XBI-XB2-XB3 forms a second 3-residue motif, and Xci-Xc2-Xc3 forms a third 3-residue motif; wherein XAI, XBI and Xci are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2, XB2 and Xc2 are each independently any amino acid residue or a derivative thereof; wherein XA3 and Xc3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein XAI and XA3 are connected to form a first cyclophane moiety, XBI and XBS are connected to form a second cyclophane moiety, and Xci and Xcs are connected to form a third cyclophane moiety; and wherein Xnaand Xnb are independently an amide bond or 1 to 3 amino acid residue.

[0015] In some embodiments, Xna is 1 to 3 amino acid residue and Xnb is an amide bond.

[0016] In some embodiments, the polypeptide is represented by Formula (lb) :

[0017] XA1-XA2-XA3-Xna-XB1-XB2-XB3-Xnb-Xci-Xc2-Xc3-Xnc-XD1-XD2-XD3 (lb) wherein XAI-XA2-XA3 forms a first 3-residue motif, XBI-XB2-XB3 forms a second 3-residue motif, Xci-Xc2-Xc3 forms a third 3-residue motif, and XDI-XD2-XD3 forms a fourth 3- residue motif; wherein XAI, XBI, XCI and XDI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2, XB2, XC2 and Xo2 are each independently any amino acid residue or a derivative thereof; wherein XA3, XC3 and XDS is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein XAI and XA3 are connected to form a first cyclophane moiety, XBI and XB3 are connected to form a second cyclophane moiety, Xci and Xcs are connected to form a third cyclophane moiety, and XDI and XD3 are connected to form a fourth cyclophane moiety; and wherein Xna, Xnt and Xnc are independently an amide bond or 1 to 3 amino acid residue.

[0018] In some embodiments, Xna is 1 to 3 amino acid residue and Xnb and Xnc are both an amide bond.

[0019] In some embodiments, XAZ, XB2, XCZ and XD2 are each connected to XA3, XBS, XC3 and XD3 respectively via a 3,6 or 3,7 substituted indolylene moiety. It was found that the 3,6 or 3,7 substitution is advantageous for providing an antibacterial effect.

[0020] In some embodiments, the polypeptide further comprises at least two C-terminus residues.

[0021] In some embodiments, at least one of the two C-terminus residues is a polar and / or basic residue.

[0022] In some embodiments, at least one of the two C-terminus residues is an aromatic residue.

[0023] In some embodiments, the first and second three residue motifs are separated by 1 to 3 amino acid residue.

[0024] In some embodiments, the cyclophane moieties are not fused.

[0025] In some embodiments, the polypeptide is represented by an amino acid sequence selected from:

[0026] RGEGWVKAYWVKRF (SEQ ID 1)

[0027] RGEGWVKAYWAKRF (SEQ ID 2) RGEGWAKAYWAKRF (SEQ ID 3)

[0028] RGEGWIKAYWIKRF (SEQ ID 4)

[0029] RGEGWLKAYWLKRF (SEQ ID 5)

[0030] RGEGWMKAYWMKRF (SEQ ID 6)

[0031] RGEGWWKAYWWKRF (SEQ ID 7)

[0032] RGEGWFKAYWFKRF (SEQ ID 8)

[0033] RGEGWHKAYWHKRF (SEQ ID 9)

[0034] RGEGWYKAYWYKRF (SEQ ID 10)

[0035] RGEGWIRAYWIRRF (SEQ ID 11)

[0036] RGEGWLRAYWLRRF (SEQ ID 12)

[0037] RGEGWMRAYWMRRF (SEQ ID 13)

[0038] RGEGWWRAYWWRRF (SEQ ID 14)

[0039] RGEGWFRAYWFRRF (SEQ ID 15)

[0040] RGEGWHRAYWHRRF (SEQ ID 16)

[0041] RGEGWYRAYWYRRF (SEQ ID 17).

[0042] In some embodiments, the polypeptide is selected from: In some embodiments, the polypeptide is an isolated polypeptide.

[0043] In some embodiments, the polypeptide is characterised by an antibacterial activity. In some embodiments, the polypeptide is characterised by an antibacterial activity against Gram-negative bacteria. In some embodiments, the polypeptide is characterised by an antibacterial activity against drug-resistant bacteria.

[0044] In some embodiments, the polypeptide is characterised by a minimal inhibitory concentration (MIC) of about 2 pg / mL to about 10 pg / mL.

[0045] The present invention also provides a composition comprising a polypeptide as disclosed herein.

[0046] The present invention also provides a method of producing a polypeptide in a host cell, the method comprising : a) introducing to the host cell one or more nucleic acid molecules, the nucleic acid molecules configured to express a precursor polypeptide (A), a rSAM / SPASM maturase (B), a protease (C), a transporter (D) and a protease / transporter (E); wherein the precursor polypeptide is represented by Formula (I):

[0047] XA1-XA2-XA3-Xn-XB1-XB2-XB3 (I) wherein XAI-XA2-XA3 forms a first 3-residue motif and XBI-XB2-XB3 forms a second 3- residue motif; wherein XAI and XBI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2 and XB2 are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein Xn is an amide bond or 1 to 3 amino acid residue; and wherein the polypeptide optionally comprises a third 3-residue motif; wherein the rSAM / SPASM maturase (B) is capable of modifying the precursor polypeptide (A) in the host cell to form a modified precursor polypeptide with a cyclophane moiety connecting XAI and XAS to form a first cyclophane moiety, and XBI and XBS to form a second cyclophane moiety; wherein the protease (C), transporter (D) and protease / transporter (E) are capable of cleaving the modified precursor polypeptide from the rSAM / SPASM maturase (A) to form a cleaved modified polypeptide and exporting the cleaved modified polypeptide out from the host cell.

[0048] In some embodiments, at least the nucleic acid molecule configured to express A is derived from a Xye maturase system.

[0049] In some embodiments, the nucleic acid molecules configured to express A and B are from one Xye species and the nucleic acid molecules configured to express C, D and E are from another Xye species.

[0050] In some embodiments, at least the nucleic acid molecules configured to express C, D and E are fused.

[0051] In some embodiments, the nucleic acid molecules configured to express A and B are fused.

[0052] In some embodiments, the nucleic acid molecules configured to express B, C, D and E are fused.

[0053] In some embodiments, the nucleic acid molecules configured to express A, B, C, D and E are fused.

[0054] In some embodiments, the nucleic acid molecule configured to express A is at least 70% identical to and derived from a bacterial species selected from Serratia marcescens (smc), Erwinia toletana (etc), Photorhabdus australis (pac), Xenorhabdus nematophila (xnc), Xenorhabdus griffiniae VH1 (xgc), Pandoraea sp. PE-S2R-1 (psc), Pandoraea oxalativorans DSM 23570 (poc), Photorhabdus heterorhabditis Q614 (phc), Kosakonia cowanii pasteuri (kcc2 and keel), Bordetella bronchialis AU17976 (bbc) and Photorhabdus laumondii BOJ-47 (pic). In some embodiments, the nucleic acid molecules configured to express C, D and E are at least 70% identical to and derived from Xenorhabdus nematophila (xnc).

[0055] The present invention also provides a method of treating a bacterial infection, comprising administering an effective amount of a polypeptide as disclosed herein to subject in need thereof.

[0056] In some embodiments, the bacterial infection is a Gram-negative bacterial infection. In some embodiments, the bacterial infection is characterised by a drug-resistance.

[0057] In some embodiments, the bacterial infection is caused by a Gram-negative bacteria selected from Escherichiacoli, Pseudomonas aeruginosa, Candidates Liberibacter, Agrobacterium tumefaciens, Acinetobactor baumannii, Moraxella catarrhalis, Citrobacterdi versus, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Salmonella typhimurium, Neisseria meningitidis, Serratia marcescens, Shigella sonnei, Shigella boydii, Neisseria gonorrhoeae, Acinetobacter baumannii, Salmonella enteriditis, Fusobacterium nucleatum, Veillonella parvula, Actinobacillus actinomycetemcomitans, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Helicobacter pylori, Francisella tularensis, Yersinia pestis, Vibrio cholera, Morganella morganii, Edwardsiella tarda, Campylobacter jejuni, Haemophilus influenza, Enterobacter cloacae, or a combination thereof.

[0058] Brief description of the drawings

[0059] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0060] Figure 1. Biosynthesis and types of Xenorceptides.

[0061] Figure 2. (a) BGC of sec cluster, the sequence of SecA is given at right side, (b) LC-MS analysis of SecAB and SecAB+SecDE full-length precursors, the truncated leader only existed in full-cluster expression, (c) LC-MS analysis of SPE elute fraction of SecAB and SecAB+SecDE, with peak 34 indicating sec end product.

[0062] Figure 3. (a) BGC of tvc cluster, the sequence of NHise-SUMO-TvcA is given at right side, (b) Strategies for obtaining tvc products, (c) LC-MS analysis of trypsin-digested samples of NHise-SUMO-TvcA and NHise-SUMO-TvcA+TvcB, peak 35 indicating tvc product only exists in the NHiss-SUMO-TvcA+TvcB sample, (d) MS / MS analysis of 35 shows that it has two mass shifts of -2 Da that is localized to the FAN and FSK motifs.

[0063] Figure 4. (a) BGC of kcc2 cluster, displaying the sequence of Kcc2A and its derivatives on the right side, (b) Strategies employed for acquiring xenorceptide DI derivatives, (c) LC-MS analysis of Kcc2A derivatives coexpressed with Kcc2BDE, peak 36 represents the end product of Kcc2A_WVKAYWVK, peak 37 and 38 indicate the doubly modified (-4D) and singly modified (-2D) products of Kcc2A_WVKAYWAK, peak 39 signifies the end product of Kcc2A_F10W.

[0064] Figure 5. (a) BGC of kcc2 cluster, displaying the sequence of Kcc2A on the right side, (b) Strategies employed for acquiring xenorceptide DI ring product, (c) LC-MS analysis results after using aminopeptidase and GluC on NHise-Kcc2A co-expressed with Kcc2B, with peak 40 represents the presence of the modified ring product.

[0065] Figure 6. Summary of Xye Type B and Type D biosynthetic gene clusters and the corresponding sequence of the precursor.

[0066] Detailed description

[0067] The term "cyclophane group" or "cyclophane" may be used interchangeably to refer to a macrocycle or ring consisting of an aromatic unit (aryl or heteroaryl) and an optionally substituted aliphatic chain that forms a bridge between two non-adjacent positions of the aromatic ring. For example, the "cyclophane group" or "cyclophane" can refer to a macrocycle or ring formed when an aromatic unit in an aromatic amino acid Xi (such as W, F, Y or H) in a peptide comprising a 3 residue motif X1-X2-X3 is joined to a CP in X3 via a carbon to carbon bond.

[0068] The terms "polypeptide", "peptides" and "protein" are used interchangeably and include any polymer of amino acids (dipeptide or greater) linked through peptide bonds or modified peptide bonds, whether produced naturally or synthetically. The polypeptides of the invention may comprise non-peptidic components, such as carbohydrate or fatty acid groups.

[0069] The term "amino acid" refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, by way of example, an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (by way of example, norleucine) or may have modified peptide backbones, while still retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. The amino acid as referred to herein may be a D or L amino acid. The amino acid may also be a p-amino acid. The term "amino acid" can include D-amino acids, a,a-disubstituted amino acids, N-alkyl amino acids, homo-amino acids, dehydroamino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan), and ortho-, meta- or paraaminobenzoic acid, non-conventional amino acids such as compounds which have an amine and carboxyl functional group separated in a 1,3 or larger substitution pattern, such as p-alanine, y-amino butyric acid, Freidinger lactam, the bicyclic dipeptide (BTD) , amino- methyl benzoic acid and others well known in the art. Statine-like isosteres, hydroxyethylene isosteres, reduced amide bond isosteres, thioamide isosteres, urea isosteres, carbamate isosteres, thioether isosteres, vinyl isosteres and other amide bond isosteres known to the art are also included.

[0070] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:

[0071] Table 1 : Amino Acid Subclassification

[0072]

[0073] Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in Table 2 under the heading of exemplary and preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity. Table 2: Exemplary and Preferred Amino Acid Substitutions

[0074] Unnatural amino acids may include amino acids which are not in the L conformation. These can include non-a amino acids such as p amino acids and D amino acids. Unnatural amino acids incorporated into peptides may include 1) a ketone reactive group (as found in para or meta acetyl-phenylalanine) that can be specifically reacted with hydrazines, hydroxylamines and their derivatives (Addition of the keto reactive group to the genetic code of Escherichia coli. Wang L, Zhang Z, Brock A, Schultz P G. Proc Natl Acad Sci USA. 2003 Jan. 7; 100(1) : 56-61; Bioorg Med Chem Lett. 2006 Oct. 15; 16(20) : 5356-9. Genetic introduction of a diketone-containing amino acid into proteins. Zeng H, Xie J, Schultz P G), 2) azides (as found in p-azido-phenylalanine) that can be reacted with alkynes via copper catalysed "click chemistry" or strain promoted (3+2) cyloadditions to form the corresponding triazoles (Addition of p-azido-L- phenylalanine to the genetic code of Escherichia coli. Chin J W, Santoro S W, Martin A B, King D S, Wang L, Schultz P G. J Am Chem Soc. 2002 Aug. 7; 124(31) :9026-7; Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae. Deiters A, Cropp T A, Mukherji M, Chin J W, Anderson J C, Schultz P G. J Am Chem Soc. 2003 Oct. 1; 125(39) : 11782-3), or azides that can be reacted with aryl phosphines, via a Staudinger ligation (Selective Staudinger modification of proteins containing p-azidophenylalanine. Tsao M L, Tian F, Schultz P G. Chembiochem. 2005 December; 6(12) :2147-9), to form the corresponding amides, 4) Alkynes that can be reacted with azides to form the corresponding triazole (In vivo incorporation of an alkyne into proteins in Escherichia coli. Deiters A, Schultz P G. Bioorg Med Chem Lett. 2005 Mar. 1; 15(5) : 1521-4), 5) Boronic acids (boronates) than can be specifically reacted with compounds containing more than one appropriately spaced hydroxyl group or undergo palladium mediated coupling with halogenated compounds (Angew Chem Int Ed Engl. 2008; 47(43) :8220-3. A genetically encoded boronate-containing amino acid, Brustad E, Bushey M L, Lee J W, Groff D, Liu W, Schultz P G), 6) Metal chelating amino acids, including those bearing bipyridyls, that can specifically co-ordinate a metal ion (Angew Chem Int Ed Engl. 2007; 46(48):9239-42. A genetically encoded bidentate, metal-binding amino acid. Xie J, Liu W, Schultz P G).

[0075] The majority of strains on the WHOs Priority Pathogens List for R&D of new antibiotics belong to the family Enterobactericiae and include Klebsiella pneumoniae, Escherichia coli, Enterobacter spp., Serratia spp., Proteus spp., Providencia spp., and Morganella spp. These strains are multi-drug resistant and lead to severe and deadly infections in hospitals and nursing homes. The discovery of new antibiotics with the ability to treat these infections will have significant impact in the clinic and can save thousands of lives annually.

[0076] The present invention is predicated on the understanding that RiPP cyclophane- containing natural products may be a source of antibiotics against Gram-negative pathogens. For example, Darobactin was isolated from Photorhabdus khanii in efforts targeting animal associated symbionts as a promising source of new antibiotics. The structure of darobactin is composed of two fused three-residue cyclophanes and an ether linkage. Homologues of the maturase DarE, have also been characterized to install an ether which is a characteristic feature for this class of maturases and products. Dynobactin was recently reported by a research group by expanding on this class of natural products bioinformatically and optimizing the purification protocol by testing of purified fractions. Dynobactin contains one four-residue and one three-residue cyclophane with the latter incorporating an imidazole via Ne2 linkage. Sequence comparison of DynA precursors shows the 4-residue cyclophane is likely conserved while the second cyclophane appears to be formed between two aromatic residues.

[0077] In an alternative approach to natural products drug discovery, the inventors pursued identification of a new RiPP family prior to knowledge of the bioactivity of the natural products. The rationale was that new RiPP families will contain new products for screening platforms and biosynthetic enzymes that could be applied for making druglike molecules. To do this the inventors systematically characterized three unique TIGRFAMs annotated as rSAM / SPASM maturases (Xye, TIGR04996: Grr, TIGR04261; and Fxs, TIGR04269) and found they are unified in their ability to catalyze 3-residue cyclophane formation. Cyclophane formation occurs via a C(sp2)-C0(sp3) bond between an aromatic ring and -position on 3-residue Q1-X2-X3 motifs where all aromatic residues (Phe, Trp, Tyr, and His) appear at the QI position (Figure 10b). Collectively, the maturases is referred to as 3-residue cyclophane forming enzymes (3-CyFEs). 3- CyFEs can be differentiated from DarE, DynA, and other radical SAM / SPASM maturases by the lack of Cys residues that bind auxiliary cluster 1 of the SPASM domain (Figure 10c). BGCs that contain at least one 3-CyFE define a new family of RiPPs are termed as triceptides. 3-CyFEs were localized within a region of rSAM / SPASM sequence-function space and analysis of this biosynthetic landscape allowed the identification of ~4000 triceptide precursors which are broadly distributed in bacteria. With a new RiPP family identified the inventors focused on a specific maturase system for antibiotic discovery.

[0078] As the activity and function for triceptides was unknown, the Xye maturase systems (GenPropl090) as a source of potential antibiotics for several reasons. First, xye BGCs are reminiscent of Class I bacteriocins, a well-known source of antibacterial peptides. Shared biosynthetic features include precursors encoding a Gly-Gly motif that separates the leader and core peptide, and protease / transporter proteins that cleave and export the mature RiPP (Figure 10a and la). Second, most xye BGC-containing bacteria are isolated from human or animal microbiomes. Since these end products are likely secreted and act in a biological environment similar to that experienced by clinically used antibiotics, the inventors hypothesize that these molecules would have evolved ideal drug-like features. Third, the inventors previously demonstrated production of xenorceptide Al, as a representative from the Xye maturase system. To their knowledge, xenorceptide Al is the first characterized triceptide natural product. The inventors collectively refer to the triceptides derived from the Xye maturase systems as xenorceptides. Although xenorceptide Al was not active when tested against several bacterial strains, the inventors believed that the production of xenorceptide Al provided an entry point to produce and study this subfamily further. The inventors hypothesized that the diversity in bacterial and core sequences within XyeA precursors had the potential to generate peptide antibiotics.

[0079] Further, it was found that particular peptides are more stable to heat, proteolytic degradation, and low pH. This may be advantageous as only a limited number of antibiotics have been approved that selectively target Gram-negative bacteria. Additionally, particular peptides were found to be more potent (at least having a lower MIC values) than xenorceptide Bl.

[0080] The Xye natural products are encoded by a 5-gene cassette containing precursor, radical SAM enzyme (XyeB), protease, transporter, fused protease transporter (XyeE). The radical SAM enzyme (XyeB) introduces the 3 rings and the protease-transporter (XyeE) cleaves the modified precursor. Since XyeE cleaves the modified precursor, the peptide may be more efficiently produced. In comparison, Darobactin, which is the most comparable antibiotic is produced from by the dar gene cluster. The dar gene cluster contains 5 genes (precursor, radical SAM enzyme, and 3 x transporters). The radical SAM enzyme (DarE) is responsible for the 2-rings in the natural product. The protease responsible for cleavage has not been identified. To obtain the darobactin, an undefined protease in E coli is used.

[0081] The present invention provides a polypeptide represented by Formula (I) : XA1-XA2-XA3-Xn-XB1-XB2-XB3 (I) wherein XAI-XA2-XA3 forms a first 3-residue motif and XBI-XB2-XB3 forms a second 3- residue motif; wherein X I and XBI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein X 2 and XB2 are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein XAI and XA3 are connected to form a first cyclophane moiety, and XBI and XB3 are connected to form a second cyclophane moiety; wherein Xnis an amide bond or 1 to 3 amino acid residue; and wherein the polypeptide optionally comprises a further 3-residue motif.

[0082] A cyclophane is a hydrocarbon consisting of an aromatic unit and a chain that forms a bridge between two non-adjacent positions of the aromatic ring.

[0083] When the polypeptide comprises two three residue motifs, the two three residue motifs may be referred to as a first three residue motif (from the N-terminus) and a second three residue motif (following the first motif).

[0084] The three residue motif may be each represented by X1-X2-X3, with the alphabet denoting the sequence order of the three residue motif from the N-terminus. For example, the first three amino acid motif is denoted "A" and the second three amino acid motif is denoted "B".

[0085] The polypeptide is modified such that Xi and X3 in each motif are linked. The linkage is via W, to form indolylene-bridged cyclophanes. The modified polypeptide may, for example, display restricted rotation of the aromatic ring and induce planar chirality in the asymmetric indole bridge. In some embodiments, Xi and X3 are connected via indolylene to form a cyclophane moiety. The connection may be via a 3,6 or 3,7 substituted indolylene moiety. It was found that the 3,6 or 3,7 substitution is advantageous for providing an antibacterial effect. This may for example be represented pictorially as follows:

[0086] In some embodiments, Xi is an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof. In some embodiments, X3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof. In some embodiments, X3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof. In some embodiments, XA3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof.

[0087] In some embodiments, X2 is an amino acid residue, the amino acid selected from leucine (L), isoleucine (I), valine (V), alanine (A), proline (P), serine (S), lysine (K), asparagine (N), phenylalanine (F), aspartic acid (D), methionine (M), tryptophan (W), histidine (H), tyrosine (Y), or a derivative thereof. In some embodiments, the amino acid is selected from I, L, M, W, F, H, Y, V, A, or a derivative thereof. In some embodiments, X2 is an amino acid residue, the amino acid selected from valine (V), alanine (A), or a derivative thereof.

[0088] In some embodiments, XA2 and Xe2 are each independently an amino acid residue, the amino acid independently selected from leucine (L), isoleucine (I), valine (V), alanine (A), proline (P), serine (S), lysine (K), asparagine (N), phenylalanine (F), aspartic acid (D), methionine (M), tryptophan (W), histidine (H), tyrosine (Y), or a derivative thereof. In some embodiments, XA2 and XB2 are each independently an amino acid residue, the amino acid independently selected from I, L, M, W, F, H, Y, V, A, or a derivative thereof. In some embodiments, XA2 and Xs2 are each independently an amino acid residue, the amino acid independently selected from valine (V), alanine (A), or a derivative thereof. In some embodiments, XA2 and XB2 are the same amino acid residue, the amino acid selected from I, L, M, W, F, H, Y, V, A, or a derivative thereof. In some embodiments, XA2 and XB2 are both a valine (V) amino acid residue or a derivative thereof, or both an alanine (A) amino acid residue or a derivative thereof.

[0089] In some embodiments, the polypeptide is characterised by a common three residue motif. In this regard, the first three residue motif (X I-X 2-X S) is the same as the second three residue motif (XBI -XB2-XB3) . In some embodiments, the three residue motif is WVK. In other embodiments, the three residue motif is WAK.

[0090] In some embodiments, the first and second three residue motifs are separated by 0 amino acid residue. In some embodiments, the first and second three residue motifs are separated by 1 to 3 amino acid residue. In some embodiments, the two three residue motifs are separated by 1 to 2 amino acid residue. In some embodiments, the two three residue motifs is separated by 1, 2 or 3 amino acid residue. In some embodiments, the two three residue motifs is separated by 2 amino acid residue. In this regard, Xnis 2 amino acid residue.

[0091] The first and second three residue motifs may be separated by any type of amino acid residue, natural or non-natural. In some embodiments, the two three residue motifs is separated by a residue selected from A, V, Y, F, T, Q, G, L, D, or S. In some embodiments, the two three residue motifs is separated by A. In some embodiments, the two three residue motifs is separated A and Y.

[0092] In some embodiments, the first three residue motif is not fused with the second three residue motif other than via 1-3 amino acid residues or an amide bond. In other embodiments, the cyclophane moiety in the first three residue motif is not fused to the cyclophane moiety in the second three residue motif. In some embodiments, the cyclophane moieties connecting Xi and X3 in each motif are not fused to each other. In this regard, in contrast to darobactin for example, the polypeptide of the present invention does not comprise linked three-residue cyclophanes. The polypeptide of the present invention also does not comprise an ether linkage between the three-residue cyclophanes motifs.

[0093] In some embodiments, the polypeptide further comprises at least two C-terminus residues. These residues do not form part of the three residue motif. In some embodiments, the C-terminus comprises at least three residues, or at least four residues. In other embodiments, the C-terminus comprises 2 to 8 residues, 2 to 7 residues, 2 to 6 residues, 2 to 5 residues, or 2 to 4 residues. In some embodiments, the C-terminus comprises at least three residues.

[0094] At least one of the two C-terminus residues is an aromatic residue. For example, at least one of the C-terminus residue may be tryptophan, tyrosine, phenylalanine, or histidine. In some embodiments, at least one of the two C-terminus residues is a polar and / or basic residue. In some embodiments, the C-terminus comprises an aromatic residue and a polar and / or basic residue. In some embodiments, the C-terminus comprises R and F. It was found that having at least an aromatic residue at the C-terminus improves the anti-bacterial property of the polypeptide.

[0095] In one embodiment, the polypeptide is a linear polypeptide. The polypeptide may be of any sequence length, having any number of residues at the N-terminus or C-terminus as long as it comprises at least two three residue motif optionally separated by 1 to 3 amino acid residue and at least two C-terminus residues.

[0096] The polypeptide may further comprise additional three residue motifs. In this regard, the polypeptide may comprise motif "C", "D", and so forth. The additional three residue motifs may be positioned anywhere in the polypeptide, as long as the first and second three residue motifs are maintained. In some embodiments, the polypeptide comprises at least three three residue motifs. In this regard, the three three residue motifs may be referred to as a first motif (from the N-terminus), a second motif (following the first motif), and a third motif (following the second motif and in proximity to the C-terminus). In this example, if the three residue motifs are each abbreivated as "A", "B" and "C", the polypeptide may be represented as: A-B-C, A-C-B, C-A-B.

[0097] In some embodiments, the polypeptide is represented by Formula (la) : XA1-XA2-XA3-Xna-XB1-XB2-XB3-Xr1t>-Xci-Xc2-Xc3 (la) wherein XAI-XA2-XAS forms a first 3-residue motif, XBI-XB2-XB3 forms a second 3-residue motif, and Xci-Xc2-Xc3 forms a third 3-residue motif; wherein XAI, XBI and Xci are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2, XB2 and Xc2 are each independently any amino acid residue or a derivative thereof; wherein XA3 and Xc3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein XAI and XA3 are connected to form a first cyclophane moiety, XBI and XB3 are connected to form a second cyclophane moiety, and Xci and Xc3 are connected to form a third cyclophane moiety; and wherein Xnaand Xnb are independently an amide bond or 1 to 3 amino acid residue. The third three amino acid motif is denoted "C".

[0098] In some embodiments, Xc2 is an amino acid residue, the amino acid selected from leucine (L), isoleucine (I), valine (V), alanine (A), proline (P), serine (S), lysine (K), asparagine (N), phenylalanine (F), aspartic acid (D), methionine (M), tryptophan (W), histidine (H), tyrosine (Y), or a derivative thereof. In some embodiments, Xc2 is an amino acid residue, the amino acid independently selected from I, L, M, W, F, H, Y, V, A, or a derivative thereof. In some embodiments, Xc2 is an amino acid residue, the amino acid selected from valine (V), alanine (A), or a derivative thereof.

[0099] In some embodiments, XA2, Xs2 and Xc2 are each independently an amino acid residue, the amino acid independently selected from leucine (L), isoleucine (I), valine (V), alanine (A), proline (P), serine (S), lysine (K), asparagine (N), phenylalanine (F), aspartic acid (D), methionine (M), tryptophan (W), histidine (H), tyrosine (Y), or a derivative thereof. In some embodiments, XA2, XB2 and Xc2 are each independently an amino acid residue, the amino acid independently selected from I, L, M, W, F, H, Y, V, A, or a derivative thereof. In some embodiments, XA2, XB2 and Xc2 are each independently an amino acid residue, the amino acid independently selected from valine (V), alanine (A), or a derivative thereof. In some embodiments, XA2, Xs2 and Xc2 are the same amino acid residue or the same derivative thereof. The amino acid may be I, L, M, W, F, H, Y, V, A, or a derivative thereof, or preferably valine (V) or alanine (A).

[0100] In some embodiments, Xc3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof.

[0101] Xn refers to the amino acid separating the three amino acid residue motifs. When more than two three residue motifs are present, an alphabet is added to denote the sequence order of Xn. For example, the first three residue motif "A" is separated from the second three residue motif "B" by by Xra, and the second three residue motif "B" is separated from the third three residue motif "C" by by Xnt>.

[0102] In some embodiments, Xnb is amide bond. Accordingly, the second three residue motif and the third three residue motif are not separated by any residue. In some embodiments, the polypeptide comprises at least four three residue motifs. In this regard, the four three residue motifs may be referred to as a first motif (from the N-terminus), a second motif (following the first motif), a third motif (following the second motif), and a fourth motif (following the third motif and in proximity to the C- terminus).

[0103] In some embodiments, the polypeptide comprises four three residue motifs. If the three residue motifs are each abbreivated as "A", "B", "C" and "D", the polypeptide may be represented as: A-B-C-D, A-C-B-D, C-A-B-D, A-C-D-B, C-A-D-B.

[0104] In some embodiments, the polypeptide is represented by Formula (lb) :

[0105] XA1-XA2-XA3-Xna-XB1-XB2-XB3-Xnb-Xci-Xc2-Xc3-Xnc-XD1-XD2-XD3 (lb) wherein XAI, XBI, XCI and XDI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2, XB2, XC2 and XD2 are each independently any amino acid residue or a derivative thereof; wherein XA3, XC3 and XDS is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein XAI and XA3 are connected to form a first cyclophane moiety, XBI and XB3 are connected to form a second cyclophane moiety, Xci and Xc3 are connected to form a third cyclophane moiety, and XDI and XD3 are connected to form a fourth cyclophane moiety; and wherein Xna, Xnb and Xncare independently an amide bond or 1 to 3 amino acid residue.

[0106] The fourth three amino acid motif is denoted "D".

[0107] In some embodiments, XD2 is an amino acid residue, the amino acid selected from leucine (L), isoleucine (I), valine (V), alanine (A), proline (P), serine (S), lysine (K), asparagine (N), phenylalanine (F), aspartic acid (D), methionine (M), tryptophan (W), histidine (H), tyrosine (Y), or a derivative thereof. In some embodiments, XD2 is an amino acid residue, the amino acid independently selected from I, L, M, W, F, H, Y, V, A, or a derivative thereof. In some embodiments, XD2 is an amino acid residue, the amino acid selected from valine (V), alanine (A), or a derivative thereof. In some embodiments, XA2, XB2, XC2 and XD2 are each independently an amino acid residue, the amino acid independently selected from leucine (L), isoleucine (I), valine (V), alanine (A), proline (P), serine (S), lysine (K), asparagine (N), phenylalanine (F), aspartic acid (D), methionine (M), tryptophan (W), histidine (H), tyrosine (Y), or a derivative thereof. In some embodiments, XA2, XB2, Xcz and Xo2 are each independently an amino acid residue, the amino acid independently selected from I, L, M, W, F, H, Y, V, A, or a derivative thereof. In some embodiments, XA2, XB2, XC2 and XD2 are each independently an amino acid residue, the amino acid independently selected from valine (V), alanine (A), or a derivative thereof. In some embodiments, X 2, XB2, Xc2 and Xo2 are the same amino acid residue or the same derivative thereof. The amino acid may be I, L, M, W, F, H, Y, V, A, or a derivative thereof, or preferably valine (V) or alanine (A).

[0108] In some embodiments, XD3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof.

[0109] In some embodiments, Xnc is amide bond. Accordingly, the second three residue motif and the third three residue motif are not separated by any residue.

[0110] In some embodiments, Xnb and Xncare amide bonds. Such polypeptides may be Type D peptides.

[0111] In some embodiments, the polypeptide is a xenorceptide Type D peptide. The polypeptide may be derive from Kosakonia cowanii pasteuri. The polypeptide may be selected from Table 3:

[0112] aBold residues indicate aromatic amino acids predicted to be in cyclophane

[0113] In some embodiments, the polypeptide is selected from: In some embodiments, the polypeptide is characterised by an antibacterial activity. In some embodiments, the polypeptide is characterised by an antibacterial activity against Gram-negative bacteria. The Gram-negative bacteria may be of the Enterobacteriaceae family. In some embodiments, the polypeptide is characterised by an antibacterial activity against drug-resistant bacteria. In some embodiments, the polypeptide shows antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Morganella morganii, Pseudomonas aeruginosa, Acinetobacter baumanii, Enterobacter cloacae, Salmonella typhimurium, Salmonella entereditis, Shigella flexneri, or a combination thereof. In some embodiments, the polypeptide shows antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Salmonella typhimurium, Salmonella entereditis, Shigella flexneri, or a combination thereof.

[0114] It is believed that the varying activities of the peptides is due to different affinities to target proteins.

[0115] In some embodiments, the polypeptide is characterised by a minimal inhibitory concentration (MIC) of about 2 pg / mL to about 10 pg / mL. In other embodiments, the MIC is less than about 90 pg / mL, about 80 pg / mL, about 70 pg / mL, about 60 pg / mL, about 50 pg / mL, or about 40 pg / mL.

[0116] In some embodiments, the polypeptide is an isolated polypeptide. "Isolated polypeptide" refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The polypeptide may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. The polypeptide is then separated from its native medium in order to form the isolated polypeptide.

[0117] In some embodiments, the polypeptide is synthetically produced. In this regard, the polypeptide can be formed via recombinant methods, phage systems, biological systems and / or via chemical synthesis. For example, solid-phase peptide synthesis can be used. The polypeptide may be synthesised by providing the corresponding nucleic acid sequence to a host cell and the polypeptide produced and modified in vivo. The present invention also provides a method of producing a polypeptide in a host cell, the method comprising : a) introducing to the host cell one or more nucleic acid molecules, the nucleic acid molecules configured to express a precursor polypeptide (A), a rSAM / SPASM maturase (B), a protease (C), a transporter (D) and a protease / transporter (E); wherein the precursor polypeptide is represented by Formula (I):

[0118] XA1-XA2-XA3-Xn-XB1-XB2-XB3 (I) wherein XAI and XBI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein X 2 and XB2 are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein Xnis an amide bond or 1 to 3 amino acid residue; wherein the rSAM / SPASM maturase (B) is capable of modifying the precursor polypeptide (A) in the host cell to form a modified precursor polypeptide with a cyclophane moiety connecting X I and X 3 to form a first cyclophane moiety, and XBI and XBS to form a second cyclophane moiety; wherein the protease (C), transporter (D) and protease / transporter (E) are capable of cleaving the modified precursor polypeptide from the rSAM / SPASM maturase (A) to form a cleaved modified polypeptide and exporting the cleaved modified polypeptide out from the host cell.

[0119] The nucleic acid molecule is a polynucleotide. In some embodiments, at least the nucleic acid molecule configured to express the precursor polypeptide (A) is derived from a Xye species. In some embodiments, at least the nucleic acid molecule configured to express the precursor polypeptide (A) and the nucleic acid molecule configured to express the rSAM / SPASM maturase (B) is derived from a Xye species.

[0120] In some embodiments, the nucleic acid molecule configured to express the precursor polypeptide (A) is from one Xye species while the nucleic acid molecules configured to express the rSAM / SPASM maturase (B), the protease (C), the transporter (D) and the protease / transporter (E) are from another Xye species. In some embodiments, the nucleic acid molecule configured to express the rSAM / SPASM maturase (B) is from one Xye species while the nucleic acid molecules configured to express the precursor polypeptide (A), the protease (C), the transporter (D) and the protease / transporter (E) are from another Xye species. In some embodiments, the nucleic acid molecule configured to express the protease (C) is from one Xye species while the nucleic acid molecules configured to express the precursor polypeptide (A), the rSAM / SPASM maturase (B), the transporter (D) and the protease / transporter (E) are from another Xye species. In some embodiments, the nucleic acid molecules configured to express the transporter (D) is from one Xye species while the nucleic acid molecules configured to express the precursor polypeptide (A), the rSAM / SPASM maturase (B), the protease (C), and the protease / transporter (E) are from another Xye species. In some embodiments, the nucleic acid molecules configured to express the protease / transporter (E) is from one Xye species while the nucleic acid molecules configured to express the precursor polypeptide (A), the rSAM / SPASM maturase (B), the protease (C), and the transporter (D) are from another Xye species. In some embodiments, the nucleic acid molecules configured to express the precursor polypeptide (A) and the rSAM / SPASM maturase (B) are from one Xye species while the nucleic acid molecules configured to express the protease (C), the transporter (D) and the protease / transporter (E) are from another Xye species. In some embodiments, the nucleic acid molecules configured to express the precursor polypeptide (A), the rSAM / SPASM maturase (B), the protease (C), the transporter (D) and the protease / transporter (E) are from one Xye species.

[0121] In some embodiments, the nucleic acid molecule is derived from a Xenorhabdus, Yersinia and Erwinia (Xye) maturase system. The Xye maturase system is named after three bacterial genera where it is commonly found : Xenorhabdus, Yersinia, and Erwinia, but also includes other bacterial genus where it may also be found, such as Serratia and Photorhabdus. In some embodiments, the nucleic acid molecule configured to express the precursor polypeptide is derived from a bacterial species selected from Serratia marcescens (smc), Erwinia toletana (etc), Photorhabdus australis (pac) or Xenorhabdus nematophila (xnc). In some embodiments, the nucleic acid molecule configured to express the rSAM / SPASM maturase is derived from a bacterial species selected from Serratia marcescens (smc), Erwinia toletana (etc), Photorhabdus australis (pac) or Xenorhabdus nematophila (xnc). In some embodiments, the nucleic acid molecule configured to express the protease, transporter and protease / transporter are derived from Xenorhabdus nematophila (xnc). In some embodiments, the nucleic acid molecules configured to express the precursor polypeptide is derived from a bacterial species selected from Xenorhabdus griffiniae VH1 (xgc), Pandoraea sp. PE-S2R-1 (psc), Pandoraea oxalativorans DSM 23570 (poc), Photorhabdus heterorhabditis Q614 (phc), Kosakonia cowanii pasteuri (kcc2 and keel keel), Bordetella bronchialis AU17976 (bbc) and Photorhabdus laumondii BOJ-47 (pic).

[0122] In some embodiments, the precursor polypeptide is:

[0123] Accordingly, the precursor polypeptide sequence for the other polypeptide may be correspondingly obtained by modifying the relevant amino acid(s).

[0124] In some embodiments, only the nucleic acid molecules configured to express protease, transporter and protease / transporter are derived from Xenorhabdus Spp.

[0125] The nucleic acid molecules may each individually express a precursor polypeptide, a rSAM / SPASM maturase, a protease, a transporter and a protease / transporter. Alternatively, the nucleic acid molecules may be fused. In other words, the nucleic acid molecules are operably linked to a first promoter; i.e. the nucleic acid molecules are part of one expression unit. In some embodiments, at least the nucleic acid molecule expressing the protease, the nucleic acid molecule expressing the transporter and the nucleic acid molecule expressing the protease / transporter are fused. In some embodiments, the nucleic acid molecule expressing the precursor polypeptide and the nucleic acid molecule expressing the rSAM / SPASM maturase are fused. In some embodiments, the nucleic acid molecule expressing the rSAM / SPASM maturase, the nucleic acid molecule expressing the protease, the nucleic acid molecule expressing the transporter and the nucleic acid molecule expressing the protease / transporter are fused. In some embodiments, the nucleic acid molecule expressing the precursor polypeptide, the nucleic acid molecule expressing the rSAM / SPASM maturase, the nucleic acid molecule expressing the protease, the nucleic acid molecule expressing the transporter and the nucleic acid molecule expressing the protease / transporter are fused.

[0126] In some embodiments, the nucleic acid molecule expressing the precursor polypeptide and the nucleic acid molecule expressing the rSAM / SPASM maturase are fused or operably linked to a first promoter, and the nucleic acid molecule expressing the protease, the nucleic acid molecule expressing the transporter and the nucleic acid molecule expressing the protease / transporter are fused or operably linked to a second promoter.

[0127] In some embodiments, the nucleic acid molecule expressing the precursor polypeptide is operably linked to a first promoter, and the nucleic acid molecule expressing the rSAM / SPASM maturase, the nucleic acid molecule expressing the protease, the nucleic acid molecule expressing the transporter and the nucleic acid molecule expressing the protease / transporter are fused or operably linked to a second promoter.

[0128] When the nucleic acid molecules are fused or linked, they may be fused in any order. For example, the nucleic acid molecule expressing the precursor polypeptide (A), the nucleic acid molecule expressing the rSAM / SPASM maturase (B), the nucleic acid molecule expressing the protease (C), the nucleic acid molecule expressing the transporter (D) and the nucleic acid molecule expressing the protease / transporter (E) may be fused as BACDE, BADEC, BAECD, BADCE, BACED, BAEDC, ABCDE, ABDEC, ABECD, ABDCE, ABCED, or ABEDC. When C, D and E are fused, they may be fused as CDE, DEC, ECD, DCE, CED, or EDC. When A and B are fused, they may be fused as AB or BA.

[0129] The present invention also provides a method of producing a polypeptide in a host cell, the method comprising : a) introducing to the host cell one or more nucleic acid molecules, the nucleic acid molecules configured to express a precursor polypeptide, a rSAM / SPASM maturase, a protease, a transporter and a protease / transporter; wherein the precursor polypeptide is represented by Formula (I):

[0130] XA1-XA2-XA3-Xn-XB1-XB2-XB3 (I) wherein X I and XBI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein X 2 and XB2 are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein Xn is an amide bond or 1 to 3 amino acid residue; wherein the rSAM / SPASM maturase (B) is capable of modifying the precursor polypeptide (A) in the host cell to form a modified precursor polypeptide with a cyclophane moiety connecting X I and XA3 to form a first cyclophane moiety, and XBI and XB3 to form a second cyclophane moiety; wherein only the protease, transporter and protease / transporter are derived from Xenorhabdus Spp; wherein the protease, transporter and protease / transporter are capable of cleaving the modified precursor polypeptide from the rSAM / SPASM maturase to form a cleaved modified polypeptide and exporting the cleaved modified polypeptide out from the host cell.

[0131] The terms "host", "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A host cell is any type of cellular system that can be used to synthesis a modified polypeptide of the present invention. Host cells include cultured cells, e.g., mammalian cultured cells, such as CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue.

[0132] In some embodiments, the method further comprises a step of culturing the host cell under conditions suitable for the production of the polypeptide.

[0133] The precursor polypeptide may be of any sequence length, as long as it comprises at least two of the three residue motif optionally separated by 1 to 3 amino acid residue and at least two C-terminus residues. The precursor polypeptide, which does not comprise a cyclophane, is then modified by the rSAM / SPASM maturase to form a cyclophane containing modified precursor polypeptide. The modified precursor polypeptide may then be cleaved and transported out from the host cell by the protease, transporter and protease / transporter.

[0134] In some embodiments, the precursor polypeptide or the nucleic acid molecule configured to express the precursor polypeptide is derived from a bacterial strain. In some embodiments, the precursor polypeptide or the nucleic acid molecule configured to express the precursor polypeptide is derived from Serratia marcescens (smc), Erwinia toletana (etc), Photorhabdus australis (pac), Xenorhabdus nematophila (xnc), Xenorhabdus griffiniae VH1 (xgc), Pandoraea sp. PE-S2R-1 (psc), Pandoraea oxalativorans DSM 23570 (poc), Photorhabdus heterorhabditis Q614 (phc), Kosakonia cowanii pasteuri (kcc2 and keel ), Bordetella bronchialis AU17976 (bbc) or Photorhabdus laumondii BOJ-47 (pic).

[0135] The precursor polypeptide and the rSAM / SPASM maturase (or the nucleic acid molecule configured to express the precursor polypeptide and rSAM / SPASM maturase) may be derived from the same bacterial strain, or may be of different bacterial strains. In some embodiments, the precursor polypeptide is fused to the rSAM / SPASM maturase. In some embodiments, the precursor polypeptide are transcribed and translated separately from the rSAM / SPASM maturase.

[0136] The amino acid sequence of the precursor polypeptide may be at least 70% identical to the amino acid sequence of of SEQ ID NO: [XyeA] (see Table 4 below). The amino acid sequence of the precursor polypeptide may be at least 70% identical to the amino acid sequence of SEQ ID NO: [SmcA], SEQ ID NO: [EtcA], SEQ ID NO: [PacA], SEQ ID NO: [XgcA], SEQ ID NO: [PscA], SEQ ID NO: [PocA], SEQ ID NO: [PhcA], SEQ ID NO: [Kcc2A] SEQ ID NO: KcclA, SEQ ID NO: [BbcA] or SEQ ID NO: [PIcAJ.

[0137] The term "rSAM" refers to radical S-adenosylmethionine. The rSAM enzyme may be an rSAM enzyme of the Xenorhabdus, Yersinia and Erwinia (XYE) maturase system (Xye, TIGR04496, IPR030989), Glycine-rich repeat (Grr) maturase system (GrrM, TIGR04261, IPR026357) or the Fxs maturase system (FxsB, TIGR04269, IPR026335). In some embodiments, the rSAM / SPASM maturase is from a Xenorhabdus, Yersinia and Erwinia (XYE) maturase system.

[0138] The rSAM enzyme may also be an enzymatically active fragment of an rSAM enzyme of the Xenorhabdus, Yersinia and Erwinia (XYE) maturase system (XyeB, TIGR04496, IPR030989), Glycine-rich repeat (Grr) maturase system (GrrM, TIGR04261, IPR026357) or the Fxs maturase system (FxsB, TIGR04269, IPR026335). In some embodiments, the rSAM / SPASM maturase is an enzymatically active fragment from a Xenorhabdus, Yersinia and Erwinia (XYE) maturase system.

[0139] The rSAM enzyme may have an amino acid sequence that is at least 70% (or 75%, 80%, 85%, 90% or 95%) identical to the following sequences:

[0140] XncB (Xenorhabdus nematophila) : MTTSKSEKIKHLEIILKISERCNINCSYCYVFNMGNSLATDSPPVISLDNVLALRGFFERSAAENEI EVIQVDFHGGEPLMMKKDRFDQMCDILRQGDYSGSRLELALQTNGILIDDEWISLFEKHKVHASI SIDGPKHINDRYRLDRKGKSTYEGTIHGLRMLQNAWKQGRLPGEPGILSVANPTANGAEIYHHFA NVLKCQHFDFLIPDAHHDDDIDGIGIGRFMNEALDAWFADGRSEIFVRIFNTYLGTMLSNQFYRV IGMSANVESAYAFTVTADGLLRIDDTLRSTSDEIFNAIGHLSELSLSGVLNSPNVKEYLSLNSELPS DCADCVWNKICHGGRLVNRFSRANRFNNKTVFCSSMRLFLSRAASHLITAGIDEETIMKNIQK (SEQ ID NO: 22)

[0141] YkcB (Yersinia kristensenii .

[0142] MEVITGSEGRVMLNLLIEKNIRHLEIILKISERCNINCDYCYVFNKGNSAADDSPARLSNKNIHHLV CFLQRACQEYKIGTVQIDFHGGEPLLMKKENFTDMCIQLISGNYCGSNIRLALQTNATLIDNEWIA IFEKYSVNVSISIDGPKHINDRHRLDTKGRSTYESTVRGLRILQNAYQQGRLPSDPGILCVTNAQA NGAEIYRHFVDELGVYSFDFLIPDDSYKDAHPDAVGIGRFLNEALDEWVKDNNAKIFVRLFQTHIA SLLGQKNSGVLGHTPNITGVYALTVSSDGFVRVDDTLRSTSDRMFNPIGHLSEVNLSNVFASPQF QEYSSIGQSLPTECEGCIWENICAGGRIVNRFSTEDRFKHKSIYCYSMRTFLSRSSAHLLNMGIKE ERIMAAIRA

[0143] (SEQ ID NO: 23)

[0144] EtcB (Erwinia toletana):

[0145] MTQLKGEKIKHLEIILKISERCNINCTYCYVFNMGNTLATDSTPVISLDNVYALRGFFERSAAENDI EVIQVDFHGGEPLMMKKDRFDRMCQILLQGNYRSSKFELALQTNGILIDDEWIALFEKHQVHASI SVDGPKHINDRHRLDRKGKSTYEGTITGLRLLQNAWQQGRLPGEPGILSVANANANGAEIYRHF ADTLQCQRFDFLIPDDHHDDSPDGEGVGRFLNEALDAWFADGRPEIFIRIFNTYLGTMLNSQFNR VLGMSANVESAYAFTVTADGMLRIDDTLRSTSDEIFNAVGHVSELSLARVLETSCVKEYLALSSNL PTVCAECVWNNICHGGRLVNRFSRTNRFNNKTVFCKSMRLFLSRAASHLMASGVDEKEIMKNIQ K

[0146] (SEQ ID NO: 24)

[0147] In one embodiment, the rSAM enzyme is an enzymatically active fragment of any one of the above sequences. In one embodiment, the enzymatically active fragment is one that comprises the rSAM and SPASM domains (such as CNINCSYC (SEQ ID NO: 25) and CADCVWNKIC (SEQ ID NO: 26) in XncB). In one embodiment, the enzymatically active fragment is from YkcB, wherein the rSAM domain is CNINCDYCYVFNK (SEQ ID NO: 27) and the SPASM domain is CEGCIWENIC (SEQ ID NO: 28). In one embodiment, the enzymatically active fragment is from EtcB, wherein the rSAM domain is CNINCTYC (SEQ ID NO: 29), and the SPASM domain is CAECVWNNIC (SEQ ID NO: 30). In one embodiment, the enzymatically active fragment is from MscB, wherein the rSAM domain is CDLACDHC (SEQ ID NO: 31), and the SPASM domain is CRRCPVVDQC (SEQ ID NO: 32). In one embodiment, the enzymatically active fragment is from OscB, wherein the rSAM domain is CNLNCDYC (SEQ ID NO: 33), and the SPASM domain is CRETCEYFGVC (SEQ ID NO: 34). In one embodiment, the enzymatically active fragment is from LscB, wherein the rSAM domain is CNLNCDYC (SEQ ID NO: 35), and the SPASM domain is CRQSCEYFGLC (SEQ ID NO: 36). In one embodiment, the enzymatically active fragment is from GscB, wherein the rSAM domain is CNLDCDYC (SEQ ID NO: 37), and the SPASM domain is CSDNCSYFGIC (SEQ ID NO: 38).

[0148] In one embodiment, the rSAM enzyme or enzymatically active fragment has two Cys- rich domains that are critical or essential for activity. The two Cys-rich domains may include the rSAM binding domain in the N-terminus (CXXXCXXC) and the SPASM domain in the C-terminus (CXXXCXXXXXC) or CXXCXXXXXC, where X may be any amino acid).

[0149] The term "domain", as used herein, refers to a part of a molecule or structure that shares common physicochemical features, such as, but not limited to, hydrophobic, polar, globular and helical domains or properties such as ligand-binding, membrane fusion, signal transduction, cell penetration and the like. Often, a domain has a folded protein structure which has the ability to retain its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and / or of the domain. Domains may be co-extensive with regions or portions thereof; domains may also include distinct, non-contiguous regions of a molecule.

[0150] The rSAM enzyme may be a recombinant enzyme or is isolated from bacteria.

[0151] The term "recombinant" when used with reference to, e.g., polypeptide, enzyme, nucleic acid or cell refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0152] In some embodiments, the nucleic acid sequence which encodes a rSAM / SPASM maturase comprises Xye, Grr or Fxs. In other embodiments, the nucleic acid sequence comprises Xye.

[0153] In one embodiment, the maturase is an enzyme from the XYE maturase system. The enzyme may be a XyeB SPASM protein (e.g. xncB, ykcB or etcB) or an enzymatically active fragment of the enzyme. The polypeptide may be a polypeptide having at least 80% identity to a XyeA precursor peptide (e.g. xncA, ykcA and etcA). In one embodiment, the enzyme is an enzyme from the GRR maturase system. The enzyme may be an GrrM SPASM protein (e.g. oscB, IscB or gscB) or an enzymatically active fragment of the enzyme. The enzyme may, for example, act on a peptide having at least 80% identity to an GrrA precursor peptide (e.g. oscA, IscA and gscA).

[0154] In one embodiment, the enzyme is an enzyme from the FXS maturase system. The enzyme may be an FxsB SPASM protein (e.g. mscB) or an enzymatically active fragment of the enzyme. The enzyme may, for example, act on a peptide having at least 80% identity to an FxsA precursor peptide (e.g. mscA).

[0155] The terms "Percentage of sequence identity" and "percentage identity" are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sei. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally. Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.. Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89: 10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), using default parameters provided.

[0156] The term "nucleic acid" includes a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. The terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence" and polynucleotide etc. are used interchangeably herein unless the context indicates otherwise.

[0157] As used herein, the terms "encode", "encoding" and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to "encode" a polypeptide if it can be transcribed and / or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and / or translated to produce the polypeptide. Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms "encode", "encoding" and the like include a RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of a RNA molecule, a protein resulting from transcription of a DNA molecule to form a RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide a RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.

[0158] The term "construct" refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources. Thus, constructs are chimeric molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule and include any construct that contains (1) nucleic acid sequences, including regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative constructs include any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably linked. Constructs of the present invention will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct, such as, for example, a target nucleic acid sequence or a modulator nucleic acid sequence. Such elements may include control elements such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors. An "expression construct" generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell. For the practice of the present invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning : A Laboratory Manual, 3rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0159] By "control element" or "control sequence" is meant nucleic acid sequences (e.g., DNA) necessary for expression of an operably linked coding sequence in a particular host cell. The control sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a cis-acting sequence such as an operator sequence and a ribosome binding site. Control sequences that are suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.

[0160] In some embodiments, the precursor polypeptide and the rSAM enzyme are selected from the following Table 4.

[0161] Table 4. Combination of precursor polypeptide sequence and rSAM sequence. aC-terminal residues after the GG motif.

[0162] ‘’Molecular weight of the fully modified core peptide.

[0163] Topology of xyeCDE genes in the biosynthetic gene cluster.dProtein ID and sequence for a representative pair of precursor and rSAM are shown.

[0164] The protease, transporter and protease / transporter may be fused or may be separately expressed. In some embodiments, the protease, transporter and the protease / transporter are encoded by the same nucleic acid molecule. In some embodiments, the protease, transporter and protease / transporter are derived from Xenorhabdus nematophila (Xnc).

[0165] In some embodiments, an amino acid sequence of the protease is at least 70% identical to the amino acid sequence of SEQ ID NO: [XncC]. In some embodiments, an amino acid sequence of the transporter is at least 70% identical to the amino acid sequence of, SEQ ID NO: [XncD], In some embodiments, an amino acid sequence of the protease / transporter is at least 70% identical to the amino acid sequence of SEQ ID NO: [XncE],

[0166] In some embodiments, the protease and / or the protease / transporter is capable of cleaving the modified precursor polypeptide to form the polypeptide. In some embodiments, the protease and / or the protease / transporter is capable of cleaving the modified precursor polypeptide at a Gly-Gly motif.

[0167] In some embodiments, the transporter and / or the protease / transporter is capable of transporting the polypeptide out from of a host cell.

[0168] In some embodiments, the nucleic acid sequence is provided to the host cell via a phage.

[0169] In some embodiments, the method comprises b) isolating the cleaved modified polypeptides that are exported out from the host cell. In some embodiments, the method comprises isolating the polypeptide from the culture medium.

[0170] The method may be performed under anaerobic or oxygen-free conditions.

[0171] Table 5 shows a list of precursor polypeptide and rSAM sequences, and protease, transporter and protease / transporter sequences that may be used.

[0172] Table 5. Precursor polypeptide, rSAM, protease, transporter and protease / transporter sequences

[0173]

[0174]

[0175]

[0176] In some embodiments, the nucleic acid molecules are introduced into the host cell via a pET28a(+) vector and / or pCDFduet-1 vector. In some embodiments, the nucleic acid molecules are introduced into the host cell via a pET28a(+) vector, pCDFduet-1 vector, pACYCDuet-1 vector, pETDuet-1 vector, pCOLADuet-1 vector, pRSFDuet-1 vector, pBAD vector, or a combination thereof.

[0177] In some embodiments, the host cell is E. coll NiCo21(DE3) cell. In some embodiments, the host cell is E. coll NICo21(DE3), BL21(DE3), BL21-AI, BL21 Star™ (DE3) pLysS, Rosetta™(DE3), or a combination thereof.

[0178] Through the method described above, the polypeptides obtained may be distinct from each other. These polypeptides are then tested for the desired properties. In this way, resources can be preserved as polypeptides having the same chemical structure is not tested .

[0179] The present invention also provides a method of producing a polypeptide, the method comprising : a) expressing a precursor polypeptide and a rSAM / SPASM maturase; wherein the precursor polypeptide is represented by Formula (I): XA1-XA2-XA3-Xn-XB1-XB2-XB3 (I) wherein XAI-XAZ-XAS forms a first 3-residue motif and Xsi-XB2-Xs3 forms a second 3- residue motif; wherein XAI and XBI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2 and XB2 are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein Xnis an amide bond or 1 to 3 amino acid residue; wherein the polypeptide optionally comprises a third 3-residue motif; wherein the rSAM / SPASM maturase is capable of modifying the precursor polypeptide to form a modified precursor polypeptide with a cyclophane moiety connecting XAI and XA3 to form a first cyclophane moiety, and XBiand XB3 to form a second cyclophane moiety.

[0180] In some embodiments, the method further comprises contacting the polypeptide of step a) with a protease.

[0181] The present invention also provides a method of producing a polypeptide, the method comprising : a) expressing a precursor polypeptide and a rSAM / SPASM maturase in order to form a modified precursor polypeptide; and b) cleaving the modified precursor polypeptide from the rSAM / SPASM maturase using a protease to form a cleaved modified polypeptide; wherein the precursor polypeptide is represented by Formula (I):

[0182] XA1-XA2-XA3-Xn-XB1-XB2-XB3 (I) wherein XAI-XA2-XA3 forms a first 3-residue motif and XBI-XB2-XB3 forms a second 3- residue motif; wherein XAI and XBI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2 and XB2 are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein Xnis an amide bond or 1 to 3 amino acid residue; wherein the polypeptide optionally comprises a third 3-residue motif; wherein the rSAM / SPASM maturase is capable of modifying the precursor polypeptide to form a modified precursor polypeptide with a cyclophane moiety connecting X I and XA3 to form a first cyclophane moiety, and XBI and XB3 to form a second cyclophane moiety.

[0183] This allows the method to be more versatile as a commercial protease can be used to cleave the modified precursor polypeptide in vitro.

[0184] In some embodiments, the protease is derived from Xenorhabdus Spp. In some embodiments, only the protease is derived from Xenorhabdus Spp.

[0185] The present invention also provides a method of synthesising a polypeptide as disclosed herein, the method comprising :

[0186] (a) coupling a pre-sequence peptide to a support, wherein said pre-sequence peptide comprises amino acid residues having side chain functionalities which are, if necessary, protected during the synthesis;

[0187] (b) coupling one or more N-protected amino acids to the N-terminus of the presequence peptide to form a precursor polypeptide, wherein each coupling is performed in stepwise fashion and under conditions in which each of the amino acids of the target peptide is coupled and subsequently N -deprotected; c) cleaving said precursor polypeptide from the support; and d) synthetically or enzymatically connecting the Xi and X3 in each motif to form a cyclophane moiety.

[0188] The step of d) connecting the Xi and X3 in each motif to form a cyclophane moiety can occur before the cleaving step c). In this regard, the modification of the precursor polypeptide can occur on the support. The step of d) may be performed synthetically. For example, the precursor peptide may comprise an alkyne moiety and an ortho-iodoaniline moiety. A Larock indole synthesis may be performed to form an indolyene containing cyclophane. Alternatively, the precursor peptide may comprise a halophenyl moiety such that a halo substitution may be performed to form a phenylene containing cyclophane.

[0189] The support may be a solid phase material or resin (for example, low cross-linked polystyrene beads) which may form a covalent bond between the carbonyl group and the resin, most often an amido or an ester bond. Alternatively, the synthetic method may be performed without the use of a support.

[0190] Accordingly, the method may comprise:

[0191] (a) synthesising a precursor polypeptide, wherein the precursor polypeptide is represented by Formula (I) :

[0192] XA1-XA2-XA3-Xn-XB1-XB2-XB3 (I) wherein XAI-XA2-XA3 forms a first 3-residue motif and XBI-XB2-XB3 forms a second 3- residue motif; wherein XAI and XBI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2 and XB2 are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein Xnis an amide bond or 1 to 3 amino acid residue; wherein the polypeptide optionally comprises a third 3-residue motif; b) synthetically or enzymatically connecting the Xi and X3 in each motif to form a cyclophane moiety.

[0193] The present invention also provides a method of modifying a precursor polypeptide, the precursor polypeptide is represented by Formula (I) :

[0194] XA1-XA2-XA3-Xn-XB1-XB2-XB3 (I) wherein XAI-XA?-XA3 forms a first 3-residue motif and XBI-XB2-XB3 forms a second 3- residue motif; wherein XAI and XBI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2 and XBZ are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein Xnis an amide bond or 1 to 3 amino acid residue; wherein the polypeptide optionally comprises a third 3-residue motif; the method comprising : enzymatically connecting the Xi and X3 residues in each motif to form a cyclophane moiety.

[0195] In some embodiments, the enzyme is rSAM / SPASM maturase.

[0196] The present invention also concerns polypeptides derived from biosynthetic gene clusters and their methods of synthesis thereof. The gene clusters may be are from the strain Salmonella enterica (sec), Trinickia violacea DHOD12 (tvc), Burkholderia sp. Nafp2 / 4-lb (bcs), Trinickia sp. DHG64 (tsc) and Providencia alcalifaciens (pfc).

[0197] In some embodiments, the nucleic acid molecules configured to express the precursor polypeptide is derived from a bacterial species selected from Salmonella enterica, Trinickia violacea DHOD12, Burkholderia sp. Nafp2 / 4-lb, Trinickia sp. DHG64, and Providencia alcalifaciens. In some embodiments, the nucleic acid molecules configured to express the precursor polypeptide is derived from Salmonella enterica.

[0198] In some embodiments, the precursor polypeptide are:

[0199] For sec and pfc cluster, His6-tagged precursor and the corresponding rSAM genes constructs were synthesized and inserted into pRSFDuet-1 vector. For tvc, bcs and tsc clusters, His6-SUMO-tagged precursor were synthesized and inserted into pET-28a (+) vector while rSAM genes were on pCDFDuet-1 vector.

[0200] Among the five clusters, only SecB and TvcB had relatively good conversion rate. Interestingly, it was discovered that the C gene may not be required for full-cluster expression. Therefore, we focused on using AB and DE for "full-cluster expression." We synthesized SecDE and inserted them into the pCDFDuet-1 vector to co-express with SecAB. In full-length LC-MS samples, significantly truncated precursors were detected in the SecAB-i- SecDE sample and the cleavage site is the GG motifs (Figure 2, b). LC- MS data from SPE experiment revealed that full-cluster expression of sec led to the detection of the end product (34), as compared to only His6-XyeAB expression. As demonstrated in Figure 2c, the products obtained from the SecAB+ SecDE construct included a double-charged fragment at m / z 845.4520, corresponding to -4 Da mass loss from the C-terminal core region of SecA (RGSGWVRATWRKSF, m / z 845.4553 [M + 2H]2+).

[0201] To scale up production, we conducted a large-scale fermentation of 12 L, followed by SPE and preparative reversed-phase HPLC, resulting in approximately 2.6 mg of compound 34 for further bioactivity evaluation.

[0202] For tvc, we opted to co-express NHis6-SUMO-TvcA+TvcB and subjected it to trypsin digestion to isolate the product containing 1X2X3 before trying to determine its cleavage site and obtain the true natural product. This is because the predicted end product is large and it contains R, which is the cutting site of trypsin. SUMO-tag was added to the precursor to increase its yield, and the co-expression results were shown in Figure 3. The desired modified product AWFANASFSKRF (m / z 1427.7148 [M+H]+) only exists in the NHis6-SUMO-TvcA and TvcB coexpression sample.

[0203] Following the large-scale fermentation of 18 L of NHis6-SUMO-TvcA+TvcB, nickel affinity chromatography was used for purification, followed by semi-preparative HPLC to obtain a certain amount of compound 35.

[0204] Accordingly, the present invention provides a polypeptide comprising : a) a first three residue motif (from a N-terminus) and a second three residue motif, the first and second three residue motif optionally separated by 1 to 3 amino acid residue; and b) at least two C-terminus residues; wherein the three residue motif is each represented by X1-X2-X3; wherein each Xi is a residue independently selected from tryptophan, phenylalanine, tyrosine, histidine, an unnatural aromatic amino acid residue or a derivative thereof; wherein each X2 and X3 are independently any amino acid residue; wherein Xi and X3 in each motif are connected to form a cyclophane moiety; wherein at least one of the two C-terminus residues is an aromatic residue.

[0205] The polypeptide is modified such that Xi and X3 in each motif are linked. The linkage may be via W, F, Y or H to form imidazolylene, indolylene or phenylene-bridged cyclophanes. The modified polypeptide may, for example, display restricted rotation of the aromatic ring and induce planar chirality in the asymmetric indole bridge. In some embodiments, Xi and X3 are connected via phenylene or indolylene to form a cyclophane moiety. In some embodiments, Xi and X3 in the second motif are connected via phenylene to form a cyclophane moiety.

[0206] X2 and X3 may each independently be any amino acid. In some embodiments, X2 is I, G, E, Y, V, L, A, D, S, T, N or Q. X3 may be a non-aromatic amino acid. In some embodiments, X3 is an amino acid that is not W, F, Y or H. In some embodiments, X3 is N, R, S, D, Q or K. In somne embodiment, X3 is N, R or K.

[0207] In some embodiments, X2 is I, G, E, Y, V, L, A, D, S, T, N or Q, and X3 is N, R, S, D or K. In some embodiments, X2 is I, G, E, Y, V, L, A, D, S, T, N or Q, and X3 is N, R or K.

[0208] The first and second three residue motifs may be separated by any type of amino acid residue, natural or non-natural. In some embodiments, the two three residue motifs is separated by a residue selected from A, V, Y, F, T, Q, G, L, D, or S. In some embodiments, the two three residue motifs is separated by A. In some embodiments, the two three residue motifs is separated by A and another amino acid residue.

[0209] In some embodiments, the first three residue motif is not fused with the second three residue motif other than via 1-3 amino acid residues or an amide bond. In other embodiments, the cyclophane moiety in the first three residue motif is not fused to the cyclophane moiety in the second three residue motif. In some embodiments, the cyclophane moieties connecting Xi and X3 in each motif are not fused to each other. In this regard, in contrast to darobactin for example, the polypeptide of the present invention does not comprise linked three-residue cyclophanes. The polypeptide of the present invention also does not comprise an ether linkage between the three-residue cyclophanes motifs.

[0210] In some embodiments, the polypeptide is selected from:

[0211] The present invention also provides a method of producing a polypeptide in a host cell, the method comprising : a) introducing to the host cell one or more nucleic acid molecules, the nucleic acid molecules configured to express a precursor polypeptide (A), a rSAM / SPASM maturase (B), a transporter (D) and a protease / transporter (E); wherein the precursor polypeptide comprises a first three residue motif (from a N- terminus) and a second three residue motif, the first and second three residue motif optionally separated by 1 to 3 amino acid residue, and at least two C-terminus residues; wherein the three residue motif is each represented by X1-X2-X3; wherein each Xi is a residue independently selected from tryptophan, phenylalanine, tyrosine, histidine, an unnatural aromatic amino acid residue or a derivative thereof; wherein each X2 and X3 are independently any amino acid residue; wherein at least one of the two C-terminus residues is an aromatic residue; wherein the rSAM / SPASM maturase is capable of modifying the precursor polypeptide to form a polypeptide with a cyclophane moiety connecting the Xi and X3 residues in each motif; and wherein the transporter (D) and protease / transporter (E) are capable of cleaving the modified precursor polypeptide from the rSAM / SPASM maturase (A) to form a cleaved modified polypeptide and exporting the cleaved modified polypeptide out from the host cell.

[0212] In this method, in addition to the above, the nucleic acid molecule configured to express the protease (C) is not used. Accordingly, when the nucleic acid molecules are fused or linked, they may for example be fused as AB and DE.

[0213] The present invention also provides a composition comprising a polypeptide as disclosed herein.

[0214] In one embodiment, there is provided a pharmaceutical composition comprising a polypeptide as defined herein. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like. Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives {e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient(s), its use in the pharmaceutical compositions is contemplated.

[0215] The present invention also provides a use and / or method of treating a disease. In one embodiment, there is provided a method of treating a disease in a subject, comprising administering an effective amount of a polypeptide or composition as defined herein to the subject in need thereof. Provided herein is also a modified polypeptide or composition as defined herein for use in treating a disease. Also provided herein is the use of the modified polypeptide or composition in the manufacture of a medicament for the treatment in a subject. The disease may, for example, an infectious disease. The disease may be caused by a bacteria, or a bacterial infection.

[0216] The term "treating" as used herein may refer to (1) preventing or delaying the appearance of one or more symptoms of the disorder; (2) inhibiting the development of the disorder or one or more symptoms of the disorder; (3) relieving the disorder, i.e., causing regression of the disorder or at least one or more symptoms of the disorder; and / or (4) causing a decrease in the severity of one or more symptoms of the disorder.

[0217] The term "subject" as used throughout the specification is to be understood to mean a human or may be a domestic or companion animal. While it is particularly contemplated that the methods of the invention are for treatment of humans, they are also applicable to veterinary treatments, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as primates, felids, canids, bovids, and ungulates. The "subject" may include a person, a patient or individual, and may be of any age or gender. The term "administering" refers to contacting, applying, injecting, transfusing or providing a composition of the present invention to a subject.

[0218] In some embodiments, the bacterial infection is caused by a Gram-negative bacteria. In other embodiments, the Gram-negative bacteria is selected from Escherichiacoli, Pseudomonas aeruginosa, Candidates Liberibacter, Agrobacterium tumefaciens, Acinetobactor baumannii, Moraxella catarrhalis, Citrobacter di versus, Enterobacter aerogenes, Klebsiella pneumoniaee, Proteus mirabilis, Salmonella typhimurium, Neisseria meningitidis, Serratia marcescens, Shigella sonnei, Shigella boydii, Neisseria gonorrhoeae, Acinetobacter baumannii. Salmonella enteriditis, Fusobacterium nucleatum, Veillonella parvula, Actinobacillus actinomycetemcomitans, Aggregatibacter actinomycetemcomitans, Porphyromonas gingiva Ils, Helicobacter pylori, Francisella tularensis, Yersinia pestis, Vibrio cholera, Morganella morganii, Edwardsiella tarda, Campylobacter jejuni, Haemophilus influenza, Enterobacter cloacae, or a combination thereof. The present disclsoure also concerns a method of killing and / or inhibiting proliferation of bacteria, comprising contacting the bacteria with an effective amount of a polypeptide as disclosed herein.

[0219] The present disclsoure also concerns a method of disinfecting a surface, comprising contacting the surface with an effective amount of a polypeptide as disclosed herein. The surface may be a medical device or implant.

[0220] In the embodiments that follows, the invention is described in relation to some conditions for consistency to showcase the present invention. However, the skilled person would understand that the invention is not limited to such.

[0221] Examples

[0222] Heterologous expression of xenorceptides in E. coli.

[0223] For production of xenorceptides, we used two different expression systems that allowed systematic production of xenorceptides from different bacterial genera. We first established that WINAFGNWERAFH (1) can be produced in E. coli by expressing the xnc BGC split into two vectors: His6-xncAB in pET28a(+) and xncCDE in pCDFDuet-1. The xncA gene was expressed with as an N-terminal His x 6 tag (His6) so that the precursor could be purified, and the modifications were detected. This two-vector system allows testing of His6-xyeAB expressions first to ensure maturation by the rSAM / SPASM enzyme then xyeCDE in a second vector can be expressed in a subsequent expression to facilitate cleavage and export.

[0224] To initiate heterologous expression, native AB constructs were synthesized and inserted into pET28a(+) vector. The three constructs containing His6-A+B were coexpressed in E. coli NiCo21(DE3) cells. The precursors were purified by Ni-affinity chromatography, digested with trypsin and subjected to LC-MS. The digest obtained from the smcAB construct included a double-charged fragment at m / z 1389.6797, corresponding to -6 Da mass loss from the C-terminal region of SmcA (ELVDSLLDTVSGGWVNAFARWSKSF, m / z 1392.7032 [M+2H]2+). Expressions of etcAB and pacAB constructs also resulted in detecting similar modified fragments. These experiments showed efficient modification by rSAM enzymes in E. coli and we proceeded with full cluster expression. The remaining genes (CDE) for each cluster were synthesized and inserted into pCDFduet-1. Native His6-A+B constructs were coexpressed with native XyeCDE constructs in E. coli Nico21(DE3). Both the cell biomass and the medium were analyzed separately by two methods. First, the cell pellet was processed as above to detect whether the precursor peptide was cleaved. Purified His6-SmcA, His6-EtcA, and His6- PacA were detected as truncated leaders losing C-terminal residues after the GG motif, implying the protease (C or E) are functioning. The products were extracted and purified from the culture medium by solid-phase extraction using a reversed-phase polymeric resin. The desired end products from smc, etc, and pac clusters were either undetectable or detectable in trace amounts. This result suggested D or E transporters are not functioning efficiently for native His6-AB+CDE expressions. To increase the yields of end products, we tested nonnative combinations of His6-AB +CDE; i.e. AB is from one species and CDE is from another species. As shown in Figure 3c, Smc, Etc, and Pac products could be efficiently produced using combinations of native His6-XyeAB + XncCDE. In this case, XyeAB are selected from SmcAB, EtcAB and PacAB. Tandem mass spectrometry (MSMS) analysis of these products confirmed the primary amino acid sequence and localized -2 Da losses to each of the three 1-X2-X3 motifs. Using these combinations, we proceeded with production of the Smc, Etc, and Pac products by larger scale fermentation, solid-phase extraction (polymeric resin), and preparative reversed phase HPLC which provided sufficient material for biological testing.

[0225] The second approach used to produce xenorceptides was expression of chimeric leadercore hybrids with the Xnc maturation and export machinery. These constructs were composed of His6-XncA leader (His6-XncAL) fused to the XyeA core of the target natural product inserted in pET28a(+). This precursor construct was coexpressed with XncBCDE encoded in pCDFDuet-1. This combination of genetic components allows a small gene fragment for the precursor to be synthesized and avoids the costly synthesis of the transport machinery. Using these constructs we pursued production of the products from different bacterial genera including: Yersinia kristensenii (y c), Xenorhabdus sp. (xec), Sodalis sp. (soc), Aeromonas jandaei (ajc), Provedencia huaxiensis phc), and Vibrio sagamiensis (vsc). Upon fermentation and extraction all of these products could be detected and analyzed -2 Da mass losses localized to the expected motifs.

[0226] Heterologous expression of xenorceptides Al l (11), A12-1 (12) and A12-2 (13) in E. coli. For the production of xenorceptides All (11), A12-1 (12) and A12-2 (13), they were produced in E. coli by expressing the Smc2A / pET28a(+), Smc3A-l / pET28a(+) or Smc3A-2 / pET28a(+) + Smc3B-XncCDE / pCDFDuet-l. The Smc2A, Smc3A-l or Smc3A- 2 gene was expressed as an N-terminal His x 6 tag (Hiss) so that the precursor could be purified, and the modifications detected. This two-vector system allows Hiss-xyeA precursor peptides modified by the rSAM / SPASM enzyme xyeB followed by xncCDE to cleave and export that is in a similar manner as above mentioned xenorceptides.

[0227] The Hiss-Smc2A / pET28a(+), Hiss-Smc3A-l / pET28a(+) or Hiss-Smc3A-2 / pET28a( + ) construct was co-expressed with Smc3B-XncCDE / pCDFDuet-l construct in E. coli. The cell medium was analyzed by extraction of the culture medium using solid-phase extraction (SPE). The desired end products, xenorceptide All (11), xenorceptide A12- 1 (12) and xenorceptide A12-2 (13) from Smc2A, Smc3A-l and Smc3A-2 precursors, respectively were detected from LCMS and confirmed by MSMS analysis to localized -2 Da losses to each of the three fil-X2-X3 motifs (Figures 14-16). To sufficiently produce the end products 11-13 for antimicrobial assays, large scale culture was carried out. Total 10 liter of Smc2A, 6 liter of Smc3A-l and 8 liter of Smc3A-2 were cultured, SPE extracted and HPLC purified to yield 11 (8.5 mg, 0.85 mg per liter), 11 (3.6 mg, 0.60 mg per liter) and 11 (5.5 mg, 0.68 mg per liter). Xenorceptide Al l (11), xenorceptide A12-1 (12) and xenorceptide A12-2 (13) were tested against a panel of clinical drugresistant isolates. These results are summarized in Table 15.

[0228] Full cluster expression of type B and type D xenorceptides

[0229] The Xye maturase system (GenPropl090) is derived from the names of three bacterial genera where it is commonly found : Xenorhabdus, Yersinia, and Erwinia. The substrate precursors are collectively referred to as XyeA, the rSAM proteins as XyeB, the proteases as XyeC, the transporters as XyeD, and the proteases / transporters as XyeE. Type B XyeA precursors containing xx xxxx (n = 2) and type D precursors containing QxxxxQxxxx (n = 16) through homology searches of rSAM / SPASM XyeB maturases in the RefSeq database. Subsequently, we screened the function of all the rSAM through co-expression of the precursor-rSAM pairs in E. coli. Based on these screening results, we have selected certain type B and type D family BGCs for full-gene cluster expression, specifically xgc, psc, poc, phc, kcc2, bbc, keel and pic (as shown in Figure 17). These three-letter short name to the gene clusters were given from the strain Xenorhabdus griffiniae VHl (xgc), Pandoraea sp. PE-S2R-1 (psc), Pandoraea oxalativorans DSM 23570 (pol), Photorhabdus heterorhabditis Q614 (phc), Kosakonia cowanii pasteuri (kcc2 and keel), Bordetella bronchialis AU 17976 (bbc) and Photorhabdus laumondii BOJ-47 (pic). For the xgc cluster, which contains two precursor genes, we named these two precursors XgcAl and XgcA2. Additionally, the kcc2 and keel clusters share the same protease and transporter, so both kcc2AB and kcclAB were coexpressed with the protease and transporter genes labeled kcc2CDE.

[0230] To investigate whether XyeCDE can function on corresponding Xye precursor in E. coll, type B and type D family His6-tagged precursor and rSAM genes constructs were synthesized and inserted into pRSFDuet-1 vector, along with the relevant protease, transporter genes were cloned onto pCDFDuet-1 vector. These pairs of plasmids were then transformed into E. coll NiCo (DE3) host cells. The two-vector system enables testing of His6-xyeAB expression to ensure proper maturation by the rSAM enzyme, followed by expression of xyeCDE in a second vector to facilitate cleavage and export.

[0231] Each gene cluster was fermented in a small scale of 200 mL in LB media firstly, then the truncated leader and modified full-length peptides were purified using Nickel-affinity chromatography and digested with trypsin; the end products were purified by solid phase extraction (SPE) from culture media. The full-length peptides, truncated precursors, trypsin digested fragments and end products were then detected through LC-MS analysis.

[0232] Similarly, genes of each cluster's His6-tagged precursor and rSAM enzyme were cloned into pRSFDuet-1 plasmid, while the relevant protease, transporter genes were cloned into pCDFDuet-1 plasmid. These pairs of plasmids were then transformed into E. coli NiCo21 host cells. The two-vector system enables testing of His6-xyeAB expression to ensure proper maturation by the rSAM / SPASM enzyme, followed by expression of xyeCDE in a second vector to facilitate cleavage and export. Each gene cluster was fermented in a small scale of 200 mL, then the full-length precursors were purified by nickel affinity chromatography, digested with trypsin and subjected to LCMS, the end products were purified by SPE form culture media.

[0233] Table 1. Summary of Xye Type B and Type D full-cluster expression screening

[0234] BGC Core sequence SEQ ID Detection by LC-MS Truncated

[0235] Modified Core

[0236] Leader xgcAl ASTAETWFKLDWKKSF 44 Yes Yes xgc / 12 SSDDDGIFFKTTWDRR 45 Yes Yes kcc2 RGEGWVRAYWAKRF 46 Yes Yes keel DGRWLQWIKNH 47 Yes Yes sec RGSGWVRATWRKSF 48 Yes Yes phc KPGEGWVNFTWNKSF 49 Yes Yes pic GDRWLKWIKNH 50 Yes No poc NVFVNATWSRAM 51 No No psc GNAFVNATWSRAM 52 No No bbc FANATWSKSF 53 No No tve ADSQPKARAWFANASFSKRF 54

[0237] The clear peaks of truncated leaders from LC-MS data suggested that protease from xgc, phc, kcc2 and phc clusters can work well in E. coll for their corresponding precursors, and the cleavage site of these cluster are the GG motif as predicted. In the precursors XgcAl, XgcA2 and PhcA, there is an arginine located at the C-terminal immediately adjacent to Gly-Gly, which serves as the cleavage site of trypsin. Therefore, only full-length data for these three precursors are presented. (Figure 18) Taking XgcAl as an example, the LC-MS data shows that both singly modified (-2D) and doubly modified (-4D) full-length precursors can be detected in both XgcAlB and XgcAlB + XgcDEC expression systems. However, the truncated leader that cleaves at the GG motif is only present in the full-cluster expression system. This suggests that the presence of protease is necessary for the successful cleavage of the XgcAl precursor at the Gly-Gly motif. (Figure 18)

[0238] In the case of kcc2 and keel, truncated leader is detectable in full-length, but in small quantities, so only the relatively clear digested fragment is shown. The characteristic fragment "AAHVANLLDNVQGG" ([M+H]+, m / z 1378.3395) is only detectable in Kcc2AB + Kcc2CDE expression, and similarly characteristic fragment "FSQSLLDDVQGG" ([M+H]+, m / z 1151.5164) is only detectable in keel full-cluster expression. Observations have revealed that the pic precursor contains three consecutive Gly motifs at its C-terminal. (Figure 19a) In full-length LCMS samples, significantly truncated precursors were detected from the first two GG motifs, (Figure 19b, c) and similarly, trypsin-digested samples also showed clear evidence of cleavage at the first two GG motifs in the Pic precursors, supporting that these motifs act as a cleavage site. However, no product was detected in the supernatant, which suggests that the pic protease can function in E. coll, but the transporter is not operational in this organism. (Figure 19). The other three clusters psc, bbc and poc, we attempted to use various combinations of proteases and transporters, but no desired compound was detected. Alternative strategy would be utilized on these clusters.

[0239] LC-MS data from small-scale SPE experiments revealed that full gene cluster expression of kcc2, keel, phe, xgc (Al and A2) led to the detection of their respective end products, as compared to only His6-XyeAB expression. As demonstrated in Figure 21, the products obtained from the kcc2AB + kcc2CDE construct included a double-charged fragment at m / z 889.4837, corresponding to -4 Da mass loss from the C-terminal core region of Kcc2A (RGEGWVRAYWAKRF, m / z 891.4710 [M+2H]2+), as well as a double-charged fragment at m / z 890.4916, corresponding to -2 Da mass loss of the core fragment, and an unmodified fragment at m / z 891.4988. Similarly, expression of keel constructs resulted in the detection of -4 Da and -2 Da mass losses modified and unmodified core peptide fragments, which were displayed using an extracted ion chromatogram (EIC) in Figure 10c because they were trace amounts. Tandem mass spectrometry (MS / MS) was conducted to locate the modifications to specific residues. MSMS analysis localized the -2 Da modifications to the first Q1X2X3 motif for Kcc2A core peptide and the second 1X2X3 motif for -2 Da Keel product. For phe and xgc (Aland A2), only fully modified end products were detected. In comparing the precursor Al and A2 of Xgc, the efficiency of the Xgc transporter for XgcAl is higher than that for XgcA2, evidenced by the significantly larger amount of XgcAl end product detected in the supernatant compared to XgcA2. These results are summarized in Table 14 and illustrated in Figure 20-22.

[0240] Large scale fermentation followed by SPE and preparative reversed phase HPLC was carried out for xgc(Al), phe and kcc2 clusters based on their good yield in small-scale experiments, to obtain a sufficient amount of compound from xgcAl, kcc2, keel, phe, pic. However, the yields of compounds from xgcA2, poc, psc and bbc were relatively low, making it difficult to obtain sufficient quantities for biological evaluation by SPE. Therefore, we designed several variants and utilize alternative strategies for go42 and keel, as well those clusters that failed in full cluster expression.

[0241] In vitro cleavage of leader peptide from modified precursors

[0242] For the precursors that cannot be produced using the full-cluster expression strategy, we designed G-to-K / R / E variants in an attempt to obtain the predicted natural products via peptidase digestion. The core peptides are composed of 10-16 amino acids, which we have labelled with positive numbers starting from the first residue of the predicted core sequence. We were initially interested in the bbc cluster due to the presence of two Gly-Gly motifs at the C-terminal region (Figure 17), with the GG closer to the C-terminal adjacent to the first , which is a unique feature of type A Xye precursors. However, it was found that the rSAM BbcB can only catalyze the formation of one ring, which different from previous screening results. To determine which GG motif is the boundary between leader and core peptide and investigate the possibility of using another rSAM to form two rings, we designed a fusion precursor consisting of the BbcA leader and Kcc2A core and co-expressed it with BbcB. The purified product was trypsin-digested and analyzed via LCMS, revealing that only the longer leader helped to produce -2D modification in the Kcc2A core. These results suggest that the boundary between the precursor and core is located at the second GG motif.

[0243] We investigated whether PocB rSAM could assist BbcA in forming two rings, as PocB has a high conversion rate to modify PocA, and the PocA core peptide is similar to the BbcA core. We also designed the Gly(-l) to Lys variant of PocA leader to generate the expected BbcA core peptide after trypsin cleavage. The results showed that PocB could indeed assist in the production of -4D and -2D modified BbcA core peptides, labelled compound 30 and 31, respectively. (Figure 23c) We also designed variants of XgcA2(G- 1K), KcclA(G-lE), and PocA(G-lR) to co-express their corresponding rSAM and then digested with appropriate peptidases to produce the predicted natural products. Figure 23 a, b, d shows that the yield of these targeted fragments was good. The core peptides of PIcA and PscA have similarities with KcclA and PocA, respectively.

[0244] After the large-scale fermentation of 14-18 L of each variant, nickel affinity chromatography was used for purification, followed by semi-preparative HPLC to obtain a certain amount of compound 22, 27, 28, 30 and 31. Table 13. Xye Type B and Type D core peptides

[0245] * Bold residues refer to Xi of the three-amino acid motif, where a cyclophane is formed between Xi and X3.

[0246] Antibacterial activity

[0247] To assess the antibacterial activity of the compounds under investigation and determine their minimum inhibitory concentration (MIC), we purchased linear core peptides as internal standards and employed a spectroscopic method to quantify the samples for preliminary screening. Promising compounds will be produced in larger quantities and subjected to a more accurate MIC measurement. Our panel for testing consisted of E. coli, K. pneumoniae, E. cloacae, A. baumannii, E. faecalis and S. aureus (Table 14). MIC values were obtained for the compounds 21-29 and 30, 31, using broth microdilution assays. XgcAl (21), XgcA2 (22), and both -4D and -2D Bbc products (30 and 31) showed no activity against all the strains that we tested. But we were encouraged by Kcc2 (24-25), Phc (23) and Keel (27), 27 only had selective activity against K. pneumoniae with MIC value 8 pg / mL, 23 had some activity against E. coli, E. cloacae, A. baumannii and K. pneumoniae, with MIC value range from 8-32 pg / mL. Notably, fully modified kcc2 core peptide (24) showed reasonable activity against Gram-negative strains E. coli, E. cloacae, A. baumannii, and K. pneumoniae with MIC value range from 1-4 |jg / mL. From this result, it seems that the antibacterial activity of 24 is stronger but more narrow-spectrum than Darobactin, and selectively kills Gram-negative bacteria. Secondly, 25, which is single modified Kcc2 product, was also active against these test bacteria, but weaker than 24 that is fully modified, the unmodified product 26 was not active against any of the test bacteria, which confirms that the cyclophane rings are critical to the bioactivity of the Xye peptides.

[0248] Structure elucidation

[0249] Compound 24 has the strongest and broadest spectrum of anti-microbial activity among all the type A, type B and type D xenorceptides we have obtained so far, so we decided to prioritize the production of sufficient amounts of 24 for structure analysis. Concentrated SPE elute fraction from 40 L culture of Kcc2AB coexpressed with Kcc2CDE was subjected to reverse phase preparative HPLC using a C18 column followed by a Luna PFP column to get ~6.8 mg of pure product.

[0250] Compound 24 is composed of 14 amino acids, which we have labelled with positive numbers starting from the first residue of the predicted core sequence (Figure 24). Sequential assignment of backbone NHs and their corresponding spin systems was performed using MS / MS and 2D NMR analysis, which confirmed the N-terminal (RGEG) and C-terminal (RF) sequences were unmodified. MS / MS of compound 24 showed -2 Da mass shifts localized to each of the WVR and WAK motifs within the predicted core peptide fragmentation, indicating that cyclization may have occurred within the two motifs.

[0251] Chemical shifts of side chain protons were assigned using COSY and TOSCY spectra. COSY and TOCSY correlations were observed between Ha and methyl group (Ala8 and Alall) and through the spin system of iso-propyl side chain of Val6. The chemical shifts of HP / CP of Arg 7 (6 2.82 ppm / 46.38 ppm) and Lysl2 (6 2.70 ppm / 49.60 ppm) were assigned by TOCSY, COSY, and HSQC correlations starting from NH signals. 1H and 13C chemical shifts of the Trp5 and TrplO were assigned starting from Arg7 Hp / Cp and Lysl2 HP / CP respectively.

[0252] For the first macrocyclic ring, 2D NMR analysis indicated that Trp5 was now substituted at Trp5-C6, based on the following observations: Trp5-H4 (6 7.15 ppm) and Trp5-H5 (6 6.72 ppm) were assigned adjacent based on 3JHH coupling. The location of Trp5-H5 was supported by HMBC correlations to Arg7C0 and a NOESY correlation to Arg7H0, 1H signals of Trp5-H5 appeared as a doublet. Trp5-H7 (6 7.14 ppm) was assigned based on HMBC correlations to Arg7Cp, a NOESY correlation to Arg7Hp, Arg7Hy (3 2.13 ppm) and Trp5-indole NH (6 10.74 ppm). The assignment of Trp5-H2 (6 7.14 ppm) was supported by 3JHH coupling with Trp5-indole NH and a NOESY correlation to Trp5Hg (6 2.94 ppm). The indole NH gave correlations to C2, C3, C7, C7a. The protons for Hl, H2, H4, H5, and H7 of TrplO could be assigned while H6 was not observed. Collectively, these observations supported a new C-C bond between Trp5C6 and Arg7Cg. Determination of the newly formed bond in the WAK motif was carried out in a similar fashion. Figure 25 revealed key correlations that allowed assignment of the newly formed bonds.

[0253] Figure 46-51 shows the NMR spectra used to derive the structure of xenorceptide DI (24). Table 21 shows the summarised NMR data for xenorceptide DI (24).

[0254] Xenorceptide DI derivatives

[0255] Due to the good bioactivity of xenorceptide DI, we further enhance its efficacy by structurally modifying it to generate more potent derivatives. According to the biosynthetic pathway of kcc2, we chose to manipulate precursor amino acids, altering their core sequences, and co-expressed these variations with Kcc2BDE genes to produce the intended end products. Initially, we designed four derivatives (Figure 4a), where the first three introduced alterations to X2 and X3, while the fourth substituted the final amino acid, changing it from F to W.

[0256] LCMS results showed that the co-expression of Kcc2A_WVKAYWVK and Kcc2BDE yielded fully modified end product 36. Conversely, co-expression of Kcc2A_WVKAYWAK and Kcc2BDE resulted in both doubly and singly modified products (37 and 38). While the co-expression of Kcc2A_WAKAYWAK and Kcc2BDE failed to produce the desired product. So, we co-expressed Kcc2A_WAKAYWAK with Kcc2B only, revealing an exceptionally low conversion rate of rSAM for this variant. This low efficiency might stem from the critical role of valine in the initial fil-X2-X3 sequence for rSAM activity. Within the LCMS data from the co-expression of the Kcc2A_F10W and Kcc2BDE, peak 39 emerged as the desired end product. Compared to Darobactin, Dynobactin, or Xenorceptide A, one distinguishing features of Xenorceptide D is the presence of several amino acids preceding the initial cyclophane. We were intrigued by the potential impact of these amino acids on its activity. Our approach aimed to isolate the product containing solely WVRAYWAKRF. To achieve this, we employed a strategy involving the co-expression of NHise-Kcc2A and Kcc2B. Subsequently, we utilized aminopeptidase to digest the modified full-length precursor over 24 hours. Following this, GlucC was used for a 4-hour digestion process to attain the desired target product, as illustrated in Figure 5b. The LC-MS results presented in Figure 5c suggest the successful generation of the "ring product" WVRAYWAKRF, showcasing our ability to isolate and produce this specific sequence.

[0257] For these derivatives, we first chose to scale up fermentation and obtain compounds 36, 39 and 40.

[0258] The bioactivitv of the newly obtained compounds and five test strains were added for compounds 21-30

[0259] We added four new Gram-negative test strains Salmonella enterica ATCC 14028, Salmonella typhimurium NCTC 8391, Salmonella enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 9027 and one new Gram-positive test strain Staphylococcus aureus ATCC 43300 (MRSA) to the previous activity tests of four Gram-negative bacteria and two Gram-positive bacteria.

[0260] Table 2 compiles the activities of previous compounds 21-30 alongside the newly acquired 34 and 35 against a total of 8 Gram-negative bacteria and 3 Gram-positive bacteria. Notably, compound 34 demonstrates good activity against all 8 Gramnegative bacteria, displaying efficacy within the range of 1-16 ug / mL. Meanwhile, compound 35, despite being an unconfirmed natural product, exhibits considerable antibacterial activity against the same 8 Gram-negative bacteria, with efficacy ranging between 4-16 ug / mL. Compound 35 is the first compound where both cyclophanes are FXX, and the activity of compound 35 further confirms the potential of type D xye triceptides as antibiotics.

[0261] Table 2. Antimicrobial activity MD

[0262] Table 3 provides a comprehensive summary of the antibacterial activities of xenorceptide DI derivatives, inclusive of xenorceptide DI for comparative purposes. Upon comparison, compound 36 which has two WVK motifs displayed a 2-4 times heightened activity against all eight Gram-negative bacteria in contrast to xenorceptide DI. However, the anticipated increase in activity with the F10W product did not materialize; instead, it led to a reduction in activity across most tested bacteria, ranging from 2-4 times, with a notable exception of a fourfold increase against A. baumanii ATCC 19606.

[0263] Moreover, the activity of the ring products exhibited a significant decrease, ranging from 4-32 times compared to xenorceptide DI. This stark decline strongly suggests the essentiality of the chain amino acid preceding the first QI for the activity within this compound class. Importantly, this distinction sets this compound apart from Darobactin, Dynobactin, or xenorceptide A, underlining the critical role played by this specific amino acid chain for the compound's antibacterial efficacy.

[0264] Table 3. Antimicrobial activity of xenorceptide DI derivatives

[0265] Conclusions

[0266] This work identified five new type D xye gene clusters. Products from two gene clusters were successfully obtained and tested for activity, showcasing promising efficacy against Gram-negative bacteria.

[0267] Among the four xenorceptide DI derivatives acquired, noteworthy observations were made. The compound 36 featuring two WVK motifs exhibited increased activity by 2-4 times compared to the natural product. The activity of the ring product displayed significantly diminished activity, indicating the significance of the chain amino acid preceding the first QI.

[0268] Materials, equipment, and general experimental procedures. Chemicals and reagents were purchased from the following suppliers: Acetonitrile from Tedia (USA); Isopropanol and methanol from Thermo Fisher Scientific (USA); Kanamycin and spectinomycin from GoldBio; Isopropyl g-D-l-thiogalactopyranoside (IPTG) from Combi-Blocks; and Strata-X® Polymeric Solid Phase Extraction (SPE) Sorbent (33 pm) from Phenomenex (USA); NMR solvent DMSO-de from Cambridge Isotope Labs (USA). Other chemicals and reagents were purchased from either Sigma (USA) or Bio Basic (Canada). Synthetic genes inserted into expression vectors were purchased from Twist Bioscience (USA). Escherichia coli NiCo21(DE3) cells were purchased from New England Biolabs (USA). Electroporation was carried out using mode p2 (2.5 kV, 5.6 ms) on a MicroPulser Electroporator (Bio-Rad, USA). Ultrasonication was carried out using an Ultrasonic Cleaner 142-0307 (VWR, USA). Centrifugation was carried out using either an Eppendorf® Centrifuge 5424R or 5810R (Germany), or an Avanti JXN-26 Ultracentrifuge (Beckman Coulter, USA). SPE was performed using either 12-Position Vacuum Manifold Set (Phenomenex, USA) or Vac-Man® Vacuum Manifold (Promega, USA). Sample solutions were concentrated using either a rotary evaporator (Rotavapor® R-210, Buchi, Switzerland), centrifugal evaporator (Genevac EZ-2 Elite, SP Scientific, UK), or freeze dryer (ScanVac CoolSafe, LaboGene, Denmark). LC-MS experiments were performed on a Waters Acquity UPLC System coupled to Xevo G1 QToF Mass Spectrometer (USA) and data was analyzed using MassLynx v.4.1. Preparative HPLC was carried out on a Shimadzu Nexera Prep System. NMR spectra were acquired at 298 K using a Bruker 400 MHz Avance Neo Nanobay NMR Spectrometer (USA) with a Bruker iProbe 5 mm SmartProbe or a Bruker 800 MHz Avance Neo NMR Spectrometer (USA) with a Bruker 5 mm CPTXI Cryoprobe and data was analyzed using Bruker Topspin v3.6.

[0269] Transformation of plasmids into E. coli cells. Plasmids containing precursor (xyeA) and rSAM (xyeB) genes or those containing peptidase and transporter (xyeCDE genes were synthesized by Twist Bioscience. The plasmids were reconstituted in autoclaved Milli-Q grade 1 water to a final concentration of 10 ng / pL. For full-length gene cluster expression,! pL of plasmid DNA was added to 70 pL of E. coli electrocompetent cells and transformed in a 2 mm electroporation cuvette. For coexpression, 1 pL of each plasmid DNA containing the appropriate genes was added to 70 pL of E. coli electrocompetent cells and transformed in a 2 mm electroporation cuvette. 1 mL of lysogeny broth (LB) was subsequently added to the transformed cells in an Eppendorf tube and incubated in the shaker at 37 °C, 200 rpm for 1 h. Following this, the bacteria cells were centrifuged at 4,000 rpm for 10 min at 25 °C and the cell pellet obtained by disposing the supernatant. The cell pellet was then resuspended with the residual supernatant and streaked on LB agar supplemented with appropriate antibiotics to be grown overnight at 37 °C.

[0270] Expression and purification of Hise-precursors. An overnight culture of the transformant was inoculated into LB medium in an Ultra Yield® flask (Thomson) at a ratio of 1 : 100 v / v with appropriate antibiotics. The flask was shaken at 250 rpm and 37 °C until ODeoo reaches 1.5-3.0. The culture was cooled in an ice bath for 30 min. Protein expression was induced in the presence of 1 mM IPTG at 16 °C and shaken at 250 rpm for 16 to 24 h. The cells harvested by centrifugation were reconstituted in denaturing lysis buffer (100 mM NaH2PC>4, 10 mM Tris, 9 M urea, 10 mM imidazole, pH 8.0) and then lysed by ultrasonication. The Hise-precursor in the supernatant was captured on HisPur Ni-NTA resin (Thermo Scientific, 625 mL per 20 mL supernatant) and purified according to the instructions provided by the manufacturer. The protein was eluted using NPI-250 (50 mM NaH2PC>4, 300 mM NaCI, 250 mM imidazole, pH 8.0) and the buffer was exchanged into 50 mM Tris-HCI (pH 7.5) using a PD Minitrap G-10 column (GE Healthcare). When XyeAB were expressed, the purified protein was digested by trypsin (10 pg per 1 mL eluate) at 37 °C for 16 h, or by GluC (10 pg per 1 mL eluate) at 25 °C for 16 h. Digested precursors were analyzed by LC-MS using the following conditions: column = Phenomenex Kinetex XB-C18, 5 pm, 150 x 4.6 mm; mobile phase / gradient = solvent A: HzO (+0.1% formic acid, FA), solvent B: CH3CN (+0.1% FA), isocratic 4% B for 2 min, followed by a linear gradient to 60% B over 10 min; flow rate = 0.5 ml / min; column temp. = 50 °C. When XyeAB and XyeCDE were coexpressed, the purified protein was directly analyzed by LC-MS using the following conditions: column = Phenomenex Aeris WIDEPORE C4, 3.6 pm, 150 x 4.6 mm; mobile phase / gradient = solvent A: H2O (+0.1% formic acid, FA), solvent B: 1 : 1 CHsCN / f-PrOH (+0.1% FA), isocratic 4% B for 2 min, followed by a linear gradient to 60% B over 12 min; flow rate = 0.5 mL / min; column temp. = 50 °C.

[0271] Expression and purification of tvc product

[0272] A 50 mL falcon tube containing 10 mL LB medium supplemented with appropriate antibiotics was inoculated with a colony from the transformation above. The overnight culture was inoculated into LB medium in an Ultra Yield® flask (Thomson) at a ratio of 1 : 100 v / v with appropriate antibiotics. The flask was shaken at 250 rpm and 37 °C until ODsoo reaches 1.5-3.0. The culture was cooled in an ice bath for 40 min. Protein expression was induced in the presence of 1 mM IPTG at 16 °C and shaken at 250 rpm for 16 to 24 h.

[0273] The cells harvested by centrifugation were reconstituted in denaturing lysis buffer (100 mM NaFhPC , 10 mM Tris, 9 M urea, 10 mM imidazole, pH 8.0) and then lysed by microfluidizer at 17,000 psi with the Microfluidics LM20 Microfluidizer. The homogenised suspension was then centrifuged at 4°C, 17,000 ref, and the supernatant incubated with HisPur Ni-NTA resin to capture the Hise precursor. 630pL of Ni-NTA resin were used per 20 mL of supernatant. The mixture was then incubated in ice on a countertop shaker for an hour, and then purified according to the manufacturer's instructions. The protein was eluted using NPI-250 (50 mM NaH2PC>4, 300 mM NaCI, 250 mM imidazole, pH 8.0) and the buffer was exchanged into 50 mM Tris-HCI (pH 8.0) using a PD Minitrap G-10 column (GE Healthcare). When XyeAB were expressed, the purified protein was digested by trypsin (10 pg per 1 mL eluate) at 37 °C for 16 h, or by GluC (10 pg per 1 mL eluate) at 25 °C for 16 h.

[0274] Purification of full-gene cluster expression by SPE and preparative HPLC After the overnight protein expression by IPTG, cells were removed by centrifugation at 4,000 rpm for 15 min at 4 °C. 1 L supernatant was combined with 5.5 g of free-standing Strata-X® resin in a 2 L conical flask and shaken at 16 °C, 160 rpm to allow binding of the core peptide to the resin. Peptide-bound resin was then washed twice with 60% methanol (55 mL), 100% methanol (55 mL), and finally eluted with 60% CH3CN with 0.1% FA (55 mL). The elution fraction was concentrated in vacuo, reconstituted in 20% CH3CN with 0.1% FA, and subjected to purification by preparative HPLC at the following conditions: solvent A: H2O (+0.1% TFA), solvent B: CH3CN (+0.1% TFA) Kinetex XB- C18, 5 pm, 250 x 21.2 mm; isocratic 4% B for 1 min, followed by a linear gradient to 30% B over 22 min; flow rate = 20 mL / min; UV detection = 280 nm; column temp. = room temperature.

[0275] Purification of xenorceptides. After the overnight protein expression by IPTG, cells were removed by centrifugation at 4,000 rpm for 15 min at 4 °C. 1 L supernatant was combined with 5.5 g of free-standing Strata-X® resin in a 2 L conical flask and shaken at 16 °C, 160 rpm to allow binding of the core peptide to the resin. Peptide-bound resin was then washed twice with 60% methanol (55 mL), 100% methanol (55 mL), and finally eluted with 60% acetonitrile with 0.1% FA (55 mL). The elution fraction was concentrated in vacuo, reconstituted in 20% acetonitrile with 0.1% FA, and subjected to purification by preparative HPLC at the following conditions: column = Imtakt, Cadenza 5CD-C18, 5 pm, 250 x 20 mm; mobile phase / gradient = solvent A: H2O (+0.1% FA), solvent B: CH3CN (+0.1% FA), isocratic 5% B for 1 min, followed by a linear gradient to 25% B over 17 min; flow rate = 21.2 mL / min; UV detection = 220 nm; column temp. = room temperature.

[0276] Yields of xenorceptides. Xenorceptide Al (1) was obtained with yield of 5.0 mg / L of culture as a white powder. Xenorceptide A2 (2) was obtained with yield of 4.6 mg / L of culture as a white powder. Xenorceptide A3 (3) was obtained with yield of 1 mg / L of culture as a slightly yellow powder. Xenorceptide A4 (4) was obtained with yield of 3.3 mg / L of culture as slightly yellow powder.

[0277] Minimum inhibitory concentration (MIC) determination. MIC screening of the peptides against a panel of ATCC and clinical strains was performed using broth microdilution method.1Briefly, peptides stock solutions in DMSO (0.1% TFA) were diluted into Mueller Hinton Broth (MHB), followed by two-fold serial dilution in a 96-well plate. Bacteria culture in mid-log phase was diluted into MHB to yield 106 colonyforming units (CFU) / mL. Equal volume of the starting inoculum was added to the peptide samples, then incubated for 18-20 h (37 °C, 120 rpm). ODeoo of the samples was then measured using Tecan Infinite M200 (TECAN, Mannedorf, Switzerland). MIC is defined as the lowest peptide concentration to achieve more than 90% reduction in ODeoo relative to the drug-free control. The experiments were repeated three times. Colistinresistant clinical isolates are a kind gift from Dr. Jeanette Koh (National University Hospital, Singapore). Multidrug-resistant clinical isolates are a kind gift from Dr. Lakshminarayanan Rajamani (Singapore Eye Research Institute, Singapore).

[0278] Killing kinetics determination. Peptides stock solutions were diluted into MHB to desired concentrations. Bacteria culture in mid-log phase was diluted into MHB to yield 106CFU / mL. The mixture was incubated at 37 °C with shaking. At each time point, 10 pL of the sample was drawn out and subjected to ten-fold serial dilution. 20 pL of relevant dilutions was dropped onto MHA plate using the drop plate method. The plate was incubated for 18-20 h at 37 °C. Colony number was counted, and used for calculating the CFU / mL according to the equation : CFU / mL = Colony count x 50 x dilution factor

[0279] Field-emission scanning electron microscopy (FE-SEM) microscopy. E. coli M6 culture at mid-log phase was diluted to an ODeoo of 0.1. After incubating the bacteria with the peptide at 8xMIC for 1 h, 2 h, or 4 h at 37 °C with shaking, the samples were washed thrice in PBS. After overnight fixation with 2.5% glutaraldehyde (in PBS) at 4 °C, the samples were washed twice in PBS, and then re-suspended in 500 pL of PBS. Sample was dropped onto cover slips pre-treated with poly-l-lysine. After 30 min, unbound cells were washed away with PBS. Following post-fixation with 1% OsO4 for 30 min, 0s04 was removed, and the cover slips were washed twice with distilled water. Samples were dehydrated using a series of ethanol solutions (50%, 75%, 95%, 3 x 100%). They were then subjected to critical point drying using Leica EM CPD300 (Wetzlar, Germany), followed by sputter gold coating using Leica EM ACE200 (Wetzlar, Germany). Viewing of the samples was performed using JEOL JSM-6701F (Tokyo, Japan). Images were processed using Image! (National Institutes of Health, Bethesda, MD). Serial passage. Resistance development of E. coli M6 against xenorceptide A2 was assessed by serial passaging of the bacteria in broth containing subinhibitory concentrations of the peptide. In brief, bacteria culture at mid-log phase was diluted to 105-10sCFU / mL in MHB containing 0.25x, 0.5x, lx, 2x, and 4x MIC of the peptide. After 24h of incubation (37 °C, 120 rpm shaking), the new visually observed MIC value was recorded, and the culture at highest peptide concentration showing visible growth was diluted to 105-106CFU / mL in MHB. A new set of peptide concentration range was added to the cultures based on the latest MIC. This process was repeated over 14 days for three independent starting cultures.

[0280] In vivo efficacy in peritonitis model.

[0281] All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at National University of Singapore (Singapore). Female C57BL / 6NTac mice aged 6-8 weeks were acquired from InVivos Pte Ltd (Singapore, Singapore). Solutions for injections were prepared fresh in pharmaceutical grade saline and filter-sterilized. Murine peritonitis model was established according to literature. Briefly, healthy mice were rendered neutropenic by administering i.p. injection (0.5 mL) of cyclophosphamide on day -4 (150 mg / kg) and day -1 (100 mg / kg). On day 0, mice were infected with E. coli M6 (109 CFU / mL) through i.p. injection (0.1 mL). At 30 min post-inoculation, mice were given i.p. injection (0.5 mL) of a single dose of Smc (5 or 50 mg / kg), colistin (5 mg / kg), or saline control (n = 5 mice per treatment group). At 2 h post-treatment, mice were humanely euthanized by carbon dioxide asphyxiation and cervical dislocation. Sterile PBS (3 mL) was injected into the peritoneal cavity, followed by abdominal massage and collection of peritoneal fluid (1-2 mL). Blood (0.3-0.5 mL) was collected through cardiac puncture. Liver, spleen, and kidney were surgically removed and stored in 0.1% Triton X-100 (in PBS). Tissue homogenization was performed using gentleMACS dissociator (Miltenyi Biotec, Germany) by following a published protocol. Cell aggregates were removed using a 30 pm mesh MACS Smartstrainer (Miltenyi Biotec). Blood, peritoneal fluid, and tissue homogenates were plated on LB agar and incubated overnight for colony counting.

[0282] LC-MS experiments

[0283] Mobile phases used are as follows: (Al) H2O + 0.1% formic acid; (Bl) CH3CN + 0.1% formic acid; (B2) 1 : 1 CH3CN / isopropanol + 0.1% formic acid. Details of conditions used for various samples are listed below: For full-length precursors analyses, 10 pL of sample was injected into the system and left to run with the Phenomenex® Aeris Widepore 3.6 pm C4 column (150 x 4.6 mm) as stationary phase and mobile phases of Al and B2 were used at a flow rate of 0.5 mL / min for 20 minutes and 10-75% B2 gradient over 12.5 minutes.

[0284] For digested fragment analyses, 40 pL of sample was injected into the system and left to run with Phenomenex Kinetex XB-C18, 5 pm, 150 x 4.6 mm column (150 x 4.6 mm) as stationary phase and mobile phases of Al and Bl were used at a flow rate of 0.5 mL / min for 25 minutes and 4-60% Bl gradient over 17 minutes.

[0285] For SPE fractions, 40 pL of sample was injected into the system and left to run with Phenomenex Kinetex XB-C18, 5 pm, 150 x 4.6 mm column (150 x 4.6 mm) as stationary phase and mobile phases of Al and Bl were used at a flow rate of 0.5 mL / min for 15 minutes and 4-32% Bl gradient over 7 minutes.

[0286] For subsequent MS / MS of fragmentation of selected ions, a collision energy of 30-45 eV was used. MassLynx v.4.1 was finally used to analyze the data collected.

[0287] Antimicrobial Assays

[0288] MIC values for compounds (21-36, 39, 40) were assessed using 96-well plate format with Mueller Hinton (MH) broth, using the two-fold dilution method, previously reported in standard methods provided by Clinical and Laboratory Standards S8 Institute (CLSI). Kanamycin and ampicillin were used as antibacterial control agents. According to the reference, the compounds (34-36, 39, 40) were first dissolved in DMSO + 0.1%TFA at a concentration of 3.2 mg / mL and 4 pL was serially diluted in 96 pL of MH broth. Then, sequential 2-fold serial dilutions of the mix were diluted in 50 pL MH broth and 50 pL cell cultures were added to wells. After incubation at 37 °C for 18 h, the lowest concentrations that completely inhibited the growth of bacteria in microdilution wells were detected by microplate reader for each tested compound, the values were recorded in Table 2 and 3. All assays were carried out in triplicate.

[0289] General cyclophane synthetic protocol

[0290] Precursor peptide containing alkyne moiety and 2-bromoacetanilide moiety (1.00 g, 1.04 mmol, 1.0 equiv) and Pd(PtBua)2 (180 mg, 0.347 mmol, 0.3 equiv) were added to a flame-dried round bottom flask. The flask was evacuated and backfilled with argon (3X). Dry dioxane (100 mL) and DIPEA (0.99 mL, 5.20 mmol, 5.0 equiv) were added and the mixture was heated to 85 °C. After 1.5 h, the reaction solution was cooled to ambient temperature then evaporated under vacuum. The crude solid may be purified via flash column chromatography using a gradient of 30% to 90% EtOAc in DCM.

[0291] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0292] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0293] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0294] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

Claims1. A polypeptide represented by Formula (I):XA1-XA2-XA3-Xn-XB1-XB2-XB3 (I) wherein XAI-XA2-XA3 forms a first 3-residue motif and XBI-XB2-XB3 forms a second 3- residue motif; wherein X I and XBI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein X 2 and XB2 are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein XAI and XA3 are connected to form a first cyclophane moiety, and XBI and XB3 are connected to form a second cyclophane moiety; wherein Xn is an amide bond or 1 to 3 amino acid residue; and wherein the polypeptide optionally comprises a further 3-residue motif.

2. The polypeptide according to claim 1, wherein XA3 is an amino acid residue, the amino acid is lysine (K) or a derivative thereof.

3. The polypeptide according to claim 1 or 2, wherein XA2 and XB2 are each independently an amino acid residue, the amino acid independently selected from leucine (L), isoleucine (I), valine (V), alanine (A), proline (P), serine (S), lysine (K), asparagine (N), phenylalanine (F), aspartic acid (D) or a derivative thereof.

4. The polypeptide according to any one of claims 1 to 3, wherein XA2 and XB2 are each independently an amino acid residue, the amino acid independently selected from valine (V), alanine (A), or a derivative thereof.

5. The polypeptide according to any one of claims 1 to 4, wherein XA2 and XB2 are both a valine (V) amino acid residue or a derivative thereof, or both an alanine (A) amino acid residue or a derivative thereof.

6. The polypeptide according to any one of claims 1 to 5, wherein the polypeptide is represented by FormulaXA1-XAwherein XAI-XA2-XA3forms a first 3-residue motif, XBI-XB2-XB3 forms a second 3-residue motif, and Xci-Xc2-Xc3forms a third 3-residue motif; wherein XAI, XBI and Xci are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2, XB2 and Xc2 are each independently any amino acid residue or a derivative thereof; wherein XA3 and Xcs is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein XAI and XA3 are connected to form a first cyclophane moiety, XBI and XBS are connected to form a second cyclophane moiety, and Xci and Xc3 are connected to form a third cyclophane moiety; and wherein Xna and Xnb are independently an amide bond or 1 to 3 amino acid residue.

7. The polypeptide according to claim 6, wherein Xnais 1 to 3 amino acid residue and Xnb is an amide bond.

8. The polypeptide according to any one of claims 1 to 7, wherein the polypeptide is represented by Formula (lb) :XA1-XA2-XA3-Xna-XB1-XB2-XB3-Xnb-Xci-Xc2-Xc3-Xnc-XD1-XD2-XD3 (lb) wherein XAI-XA2-XA3forms a first 3-residue motif, XBI-XB2-XB3 forms a second 3-residue motif, Xci-Xc2-Xc3forms a third 3-residue motif, and XDI-XD2-XD3forms a fourth 3- residue motif; wherein XAI, XBI, XCI and XDI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2, XB2, XC2 and XD2 are each independently any amino acid residue or a derivative thereof; wherein XA3, XCS and XD3is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof;wherein XAI and XA3 are connected to form a first cyclophane moiety, XBI and XB3 are connected to form a second cyclophane moiety, Xci and Xcs are connected to form a third cyclophane moiety, and XDI and XDS are connected to form a fourth cyclophane moiety; and wherein Xna, Xnb and Xnc are independently an amide bond or 1 to 3 amino acid residue.

9. The polypeptide according to claim 8, wherein Xna is 1 to 3 amino acid residue and Xnb and Xncare both an amide bond.

10. The polypeptide according to claim 8 or 9, wherein XA2, XB2, XC2 and XD2 are each connected to XA3, XB3, XC3 and XD3 respectively via a 3,6 or 3,7 substituted indolylene moiety.

11. The polypeptide according to any one of claims 1 to 10, wherein the polypeptide further comprises at least two C-terminus residues.

12. The polypeptide according to claim 11, wherein at least one of the two C- terminus residues is a polar and / or basic residue.

13. The polypeptide according to claim 10 or 11, wherein at least one of the two C- terminus residues is an aromatic residue.

14. The polypeptide according to any one of claims 1 to 13, wherein the first and second three residue motifs are separated by 1 to 3 amino acid residue.

15. The polypeptide according to any one of claims 1 to 14, wherein the cyclophane moieties are not fused.

16. The polypeptide according to any one of claims 1 to 15, wherein the polypeptide is represented by an amino acid sequence selected from:RGEGWVKAYWVKRF (SEQ ID 1)RGEGWVKAYWAKRF (SEQ ID 2)RGEGWAKAYWAKRF (SEQ ID 3)RGEGWIKAYWIKRF (SEQ ID 4)RGEGWLKAYWLKRF (SEQ ID 5)RGEGWMKAYWMKRF (SEQ ID 6)RGEGWWKAYWWKRF (SEQ ID 7)RGEGWFKAYWFKRF (SEQ ID 8)RGEGWHKAYWHKRF (SEQ ID 9)RGEGWYKAYWYKRF (SEQ ID 10)RGEGWIRAYWIRRF (SEQ ID 11)RGEGWLRAYWLRRF (SEQ ID 12)RGEGWMRAYWMRRF (SEQ ID 13)RGEGWWRAYWWRRF (SEQ ID 14)RGEGWFRAYWFRRF (SEQ ID 15)RGEGWHRAYWHRRF (SEQ ID 16)RGEGWYRAYWYRRF (SEQ ID 17).

17. The polypeptide according to any one of claims 1 to 16, wherein the polypeptide is selected from:

18. The polypeptide according to any one of claims 1 to 17, wherein the polypeptide is an isolated polypeptide.

19. The polypeptide according to any one of claims 1 to 18, wherein the polypeptide is characterised by an antibacterial activity against Gram-negative bacteria and / or a drug-resistant bacteria.

20. The polypeptide according to any one of claims 1 to 19, wherein the polypeptide is characterised by a minimal inhibitory concentration (MIC) of about 2 pg / mL to about 10 pg / mL.

21. A composition comprising a polypeptide according to any one of claims 1 to 20.

22. A method of producing a polypeptide in a host cell, the method comprising : a) introducing to the host cell one or more nucleic acid molecules, the nucleic acid molecules configured to express a precursor polypeptide (A), a rSAM / SPASM maturase (B), a protease (C), a transporter (D) and a protease / transporter (E); wherein the precursor polypeptide is represented by Formula (I):XA1-XA2-XA3-Xn-XB1-XB2-XB3 (I) wherein XAI-XA2-XA3 forms a first 3-residue motif and XBI-XB2-XB3 forms a second 3- residue motif; wherein XAI and XBI are each independently an amino acid residue, the amino acid selected from tryptophan (W) or a derivative thereof; wherein XA2 and XB2 are each independently any amino acid residue or a derivative thereof; wherein XA3 is an amino acid residue, the amino acid selected from arginine (R), lysine (K) or a derivative thereof; wherein XB3 is an amino acid residue, the amino acid selected from lysine (K) or a derivative thereof; wherein Xnis an amide bond or 1 to 3 amino acid residue; and wherein the polypeptide optionally comprises a third 3-residue motif; wherein the rSAM / SPASM maturase (B) is capable of modifying the precursor polypeptide (A) in the host cell to form a modified precursor polypeptide with a cyclophane moiety connecting XAI and XAS to form a first cyclophane moiety, and XBI and XBS to form a second cyclophane moiety;wherein the protease (C), transporter (D) and protease / transporter (E) are capable of cleaving the modified precursor polypeptide from the rSAM / SPASM maturase (A) to form a cleaved modified polypeptide and exporting the cleaved modified polypeptide out from the host cell.

23. The method according to claim 22, wherein at least the nucleic acid molecule configured to express A is derived from a Xye maturase system.

24. The method according to claim 22 or 23, wherein the nucleic acid molecules configured to express A and B are from one Xye species and the nucleic acid molecules configured to express C, D and E are from another Xye species.

25. The method according to any one of claims 22 to 24, wherein at least the nucleic acid molecules configured to express C, D and E are fused.

26. The method according to any one of claims 22 to 25, wherein the nucleic acid molecules configured to express A and B are fused.

27. The method according to any one of claims 22 to 26, wherein the nucleic acid molecules configured to express B, C, D and E are fused.

28. The method according to any one of claims 22 to 27 , wherein the nucleic acid molecules configured to express A, B, C, D and E are fused.

29. The method according to any one of claims 22 to 28, wherein the nucleic acid molecule configured to express A is at least 70% identical to and derived from a bacterial species selected from Serratia marcescens (smc), Erwinia toletana (etc), Photorhabdus australis (pac), Xenorhabdus nematophila (xnc), Xenorhabdus griffiniae VH1 (xgc), Pandoraea sp. PE-S2R-1 (psc), Pandoraea oxalativorans DSM 23570 (poc), Photorhabdus heterorhabditis Q614 (phc), Kosakonia cowanii pasteuri (kcc2 and keel), Bordetella bronchialis AU17976 (bbc) and Photorhabdus laumondii BOJ-47 (pic).

30. The method according to any one of claims 22 to 29, wherein the nucleic acid molecules configured to express C, D and E are at least 70% identical to and derived from Xenorhabdus nematophila (xnc).

31. A method of treating a bacterial infection, comprising administering an effective amount of a polypeptide according to any one of claims 1 to 20 to subject in need thereof.

32. The method according to claim 31, wherein the bacterial infection is a Gramnegative bacterial infection and / or is characterised by a drug-resistance.

33. The method according to claim 31 or 32, wherein the bacterial infection is caused by a Gram-negative bacteria selected from Escherichiacoli, Pseudomonas aeruginosa, Candidatus Liberibacter, Agrobacterium tumefaciens, Acinetobactor baumannii, Moraxella catarrhalis, Citrobacter di versus, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Salmonella typhimurium, Neisseria meningitidis, Serratia marcescens, Shigella sonnei, Shigella boydii, Neisseria gonorrhoeae, Acinetobacter baumannii, Salmonella enteriditis, Fusobacterium nucleatum, Veillonella parvula, Actinobacillus actinomycetemcomitans, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Helicobacter pylori, Francisella tularensis, Yersinia pestis, Vibrio cholera, Morganella morganii, Edwardsiella tarda, Campylobacter jejuni, Haemophilus influenza, Enterobacter cloacae, or a combination thereof.

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