Compounds and uses thereof

Abstract

The invention relates to compounds that show antifungal and antibacterial properties in the context of treating plant and / or animal (e.g. human) diseases or infections. The invention relates to the use of a compound of formula (I) as an antifungal or antibacterial agent. The invention also relates to a compound of formula (I) for use in treating a fungal or bacterial disease or infection in a subject. Methods of producing a compound of formula (I) are also provided.

Description

[0001] COMPOUNDS AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The invention relates to compounds that show antifungal and antibacterial properties in the context of treating plant and / or animal (e.g. human) diseases or infections. The invention relates to the use of a compound of formula (I) as an antifungal agent or an antibacterial agent. The invention also relates to a compound of formula (I) for use in treating a fungal or bacterial disease or infection in a subject. Methods of producing a compound of formula (I) are also provided.

[0004] BACKGROUND

[0005] Antifungal compounds find applications across many medical and biotechnological fields. For example, they can be used to treat fungal infections in human and animal subjects, especially in subjects who are immunocompromised. They can also be used to treat or control a plant disease.

[0006] Wheat is one of the most important crops in feeding a growing world population. Out of over 200 diseases of wheat, 50 cause economic losses and are widely distributed. Each year, about 20% of wheat is lost due to diseases, with some diseases responsible for up to 50% yield loss. Some major wheat diseases are rusts, smut, tan spot, spot blotch, fusarium head blight, common root rot, septoria blotch, powdery mildew, blast, and several viral, nematode, and bacterial diseases. These diseases adversely impact the yield and cause mortality of the plants.

[0007] Reduced wheat yield leads to higher wheat prices for consumers and could lead to reduced regional food security. Thus, there is a need for a better disease control of plants, including wheat.

[0008] Fungal infections in human or animal subjects are especially prevalent in immunocompromised individuals. This includes individuals receiving immunosuppressive therapy for autoimmune or neoplastic disease, organ transplant recipients, and AIDS patients. As these individuals struggle to fight any type of infection, a need exists for an effective compound for treating a fungal disease.

[0009] Bacteria produce different compounds which might find therapeutic or industrial applications. For example, Bacillus species are known to produce compounds of antibacterial activity, such as Mersacidin or Pumilin. In addition, Streptomyces nodosus was found to produce amphotericin B, which has antifungal properties. Thus, bacteria can provide a source of new antibacterial and / or antifungal compounds.

[0010] SUMMARY OF THE INVENTION

[0011] The invention provides a compound of formula (I) comprising a peptide, a chromophore, and a side chain. The compound of formula (I) may be used to treat a fungal infection or disease. The fungal infection or disease may be affecting human or animal subjects, or it may be affecting plants. The compound of formula (I) may also be used to treat a bacterial infection or disease.

[0012] 1

[0013] 17211595 CXB CXB The inventors of the present application showed that compound of formula (I) is particularly effective in treating fungal diseases or infections. Specifically, compound of formula (I) is effective in treating infections or diseases caused by plant pathogens, such as Zymoseptoria tritici as well as human or animal pathogens, such as Aspergillus fumigatus. Compound of formula (I) also inhibits growth of Alternaria brassicicola, Botrytis cinerea, Microdochium nivale, Venturia inaequalis, Phytophthora cactorum, and Phytophthora infestans. The inventors of the present application showed that compound of formula (I) is also particularly effective in treating bacterial diseases or infections. Specifically, compound of formula (I) is effective in treating infections or diseases caused by different gram-positive and gram-negative bacteria, e.g. Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecalis. Thus, compound of formula (I) has broad applications in the medical and biotechnology fields. Compound of formula (I) is particularly suitable for use in therapy and for controlling plant diseases.

[0014] The invention provides a compound of formula (I) P-C-S (I), wherein:

[0015] P is a peptide chain comprising n amino acids, wherein n may be between 3-14; C is a chromophore; and S is a side chain.

[0016] The side chain may comprise:

[0017] Ri is OH, H, or NH2, and R2is OH, H or NH2.

[0018] 2

[0019] 17211595 CXB CXB Preferably, the compound of formula (I) is a compound of structure (la):

[0020] The compound of formula (la) may be in the following resonance forms:

[0021] 3

[0022] 17211595 CXB CXB

[0023]

[0024] Alternatively, preferably, the compound of formula (I) is a compound of structure (lb):

[0025] The compound of formula (lb) may be in the following resonance forms:

[0026] 4

[0027] 17211595 CXB CXB

[0028]

[0029] Compound of formula (I) may be used to treat plants, animals, and / or humans. Compound of formula (I) may be used to treat diseases caused by bacterium, fungus and / or yeast.

[0030] The invention provides a use of a compound of formula (I) as an antimicrobial agent. The invention provides a use of a compound of formula (I) as an antifungal agent. The invention provides a use of a compound of formula (I) as an antibacterial agent.

[0031] The invention provides a compound of formula (I) for use in therapy. The invention provides a compound of formula (I) for use as an antifungal agent. The invention provides a compound of formula (I) for use in treating a fungal disease or infection in a subject. The invention provides a compound of formula (I) for use as an antibacterial agent. The invention provides a compound of formula (I) for use in treating a bacterial disease or infection in a subject.

[0032] The invention provides an antifungal composition (or a composition of matter) comprising compound of formula (I) as an active ingredient and, optionally, an acceptable carrier (e.g. a pharmaceutically acceptable carrier). The invention also provides an antibacterial composition (or a composition of matter) comprising compound of formula (I) as an active ingredient and, optionally, an acceptable carrier (e.g. a pharmaceutically acceptable carrier).

[0033] The invention also provides a method for treating a plant to control a disease, the method comprising applying an effective amount of the compound of formula (I) as described herein or the antifungal or antibacterial composition as described herein to the plant, or a part of the plant.

[0034] 5

[0035] 17211595 CXB CXB P262959WQ00

[0036] The invention also provides a method for producing a compound of formula (I) comprising synthesising the compound of formula (I) from a biosynthetic gene cluster encoding at least 20, at least 30, at least 40, or all of the peptides of SEQ ID NOs: 1-54, or peptides of 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NOs: 1-54.

[0037] The invention also provides a biosynthetic gene cluster encoding at least 10 peptides of any one of SEQ ID NOs: 1-54, or peptides of 80%, 85%, 90%, 95%, 97%, or 99% identity to at least 10 peptides of any one of SEQ ID NOs: 1-54.

[0038] The invention provides a host cell comprising a plasmid encoding at least one, preferably at least 10, of the peptides of SEQ ID NOs: 1-54.

[0039] Other preferred embodiments of the compounds according to the invention appear throughout the specification and in the examples.

[0040] Without wishing to be bound by a particular theory, the antimicrobial (i.e. antifungal and / or antibacterial) properties of the compound of formula (I) might be the result of its ability to sequester iron into an iron-compound (I) complex, which depletes the iron pool. If the microbe lacks a transporter for compound of formula (I), the iron-compound (I) complex remains inaccessible, driving iron starvation and growth inhibition.

[0041] In the embodiments in which the compound of formula (I) forms (or is) a siderophore, the antimicrobial properties are especially prevalent when: (i) the compound of formula (I) has greater thermodynamic Fe(lll) affinity and sufficient kinetic stability compared with the pathogen’s native siderophore(s), (ii) environmental iron is limiting, and / or (iii) the pathogen cannot hijack the compound of formula (I) via compatible uptake receptors. Under conditions of limited iron in the environment, many pathogens depend on siderophores for Fe(lll) uptake. Thus, the compound of formula (I) might have a higher affinity for Fe(lll) than the native siderophores of the microbe. Native siderophores may be derived from the non-ribosomal peptide synthetase (NRPS)-dependent siderophore pathway or the NRPS- independent siderophore (NIS) synthetase. Examples of native siderophores include hydroxamates, such as desferrioxamine, and catecholates, such as enterobactin.

[0042] Other modes of action of the compound of formula (I) may include direct oxidative stress, cell membrane disruption, quorum sensing interference, induced systemic resistance in plants, biofilm inhibition and / or enzyme inhibition by metal removal.

[0043] The compound of formula (I) is effective against many pathogens. That is to say that the compound of formula (I) may, for example, inhibit the growth of many pathogens. Preferably, the compound of formula (I) is effective against microbes (e.g. fungi and / or bacteria) that rely on iron for survival and / or growth. Thus, the compound of formula (I) may be effective against microbes (e.g. fungi and / or

[0044] 6

[0045] 17211595 CXB CXB bacteria) that produce siderophores (i.e. small molecules that chelate iron to make it available for uptake). The compound of formula (I) may be effective against iron-scavenging microbes. The compound of formula (I) may be effective against iron-scavenging bacteria. The compound of formula (I) may be effective against iron-scavenging fungi. That is to say that the compound of formula (I) may be used to treat diseases caused by iron-scavenging microbes (e.g. bacteria or fungi). The compound of formula (I) may be used to inhibit the growth of iron-scavenging microbes (e.g. bacteria or fungi).

[0046] It will be understood that iron-scavenging microbes (i.e. bacteria and / or fungi) refers to microorganisms that utilise or produce siderophores for iron uptake, microorganisms that exhibit siderophore-mediated iron acquisition pathways, and / or microorganisms that depend on siderophores for growth and survival.

[0047] The compound of formula (I) may also be effective against microorganisms devoid of siderophore systems, but which are reliant on extracellular iron pools vulnerable to siderophore sequestration.

[0048] The present inventors have surprisingly discovered that compound of formula (I) is effective as an antifungal agent, for example against Zymoseptoria tritici, a wheat plant pathogen causing septoria leaf blotch. The present inventors have surprisingly discovered that compound of formula (I) is effective as an antifungal agent for treating fungal diseases or infections caused by fungi of the Aspergillus genus. This fungus may affect animal and / or human patients, for example, immunocompromised animal and / or human patients.

[0049] The inventors of the present application have also shown that compound of formula (I) has antimicrobial properties against Alternaria brassicicola, Botrytis cinerea, Microdochium nivale, Venturia inaequalis, Phytophthora cactorum, Phytophthora infestans. In addition, compound of formula (I) was shown to inhibit the growth of Cryptococcus neoformans, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecalis.

[0050] Each aspect or embodiment as defined herein may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0051] DETAILED DESCRIPTION

[0052] Unless otherwise defined herein, scientific, and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.

[0053] 7

[0054] 17211595 CXB CXB It should be understood that singular prepositions such as “a,” “an,” and “the,” are often used for convenience, however, all instances of the singular are intended to encompass the plural unless otherwise indicated either explicitly or from context. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Further, it should be understood that all references, including journal articles, books, patents, technical documents, and the like, mentioned in this disclosure are hereby incorporated by reference in their entirety and for all purposes.

[0055] The invention provides a compound of formula (I) comprising a peptide (denoted “P”), a chromophore (denoted “C”), and a side chain (denoted “S”).

[0056] The invention provides a compound of formula (I), or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof;

[0057] P-C-S (I), wherein:

[0058] P is a peptide chain comprising n amino acids, wherein n may be between 3-14;

[0059] C is a chromophore; and S is a side chain.

[0060] The invention provides a compound of formula (I), or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof;

[0061] P-C-S (I), wherein:

[0062] P is a peptide chain comprising n amino acids, wherein n may be between 3-14;

[0063] C is a chromophore; and

[0064] S is a side chain, wherein the side chain comprises or consists of:

[0065] Ri is OH, H, or NH2, and R2is OH, H or NH2.

[0066] Preferably, Ri in the side chain is NH2. Preferably, R2in the side chain is OH. The side chain may be linked to the chromophore’s 3-amino group.

[0067] The side chain may be bound at position C-3 of the chromophore. The side chain (S) may be or may comprise succinate, amide, malate, amide, a-ketoglutarate, or glutamate.

[0068] 8

[0069] 17211595 CXB CXB The term “chromophore” as used herein intends to encompass any molecule or chemical group that absorbs light at a specific wavelength and reflects colour. The chromophore may comprise or consist of a quinoline-derived fluorescent complex. The chromophore may comprise or consist of a quinoline or a quinazoline. The chromophore may comprise or consist of a dihydroxyquinoline. The chromophore may be a fluorescent chromophore. The chromophore may be derived from 2,3- diamino-6,7-dihydroxyquinoline.

[0070] The chromophore may comprise or consist of any of the following structures:

[0071] Preferably, the chromophore may comprise or consist of the following structure:

[0072] The chromophore may comprise or consist of any one of the following resonance forms:

[0073] Thus, preferably, the chromophore may comprise or consist of any of the following structures:

[0074] 9

[0075] 17211595 CXB CXB

[0076] Thus, in the structure of compound of formula (I), the chromophore may be selected from the group comprising:

[0077] Thus, preferably, in the structure of compound of formula (I), the chromophore may be selected from the group comprising:

[0078] In a preferred embodiment, the resonance forms are in equilibrium.

[0079] The peptide chain may be specific to a strain of bacteria from which the compound of formula (I) has been obtained.

[0080] The peptide chain may comprise n amino acids, wherein n may be between 3-14. The peptide chain may comprise n amino acids, wherein n may be between 6-14. The peptide chain may comprise n

[0081] 10

[0082] 17211595 CXB CXB amino acids, wherein n may be between 8-10. Preferably, the peptide chain comprises 9 amino acids. The peptide chain may be linear or cyclic (either entirely or partially).

[0083] The peptide chain may comprise at least one naturally occurring amino acid. The peptide chain may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten naturally occurring amino acids. The term “naturally occurring” is intended to encompass amino acids naturally produced by the human and / or animal body. For example, the naturally occurring amino acid may be glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, and / or glutamic acid.

[0084] The peptide chain may comprise at least one unnatural amino acid. The peptide chain may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten unnatural amino acids. The term “unnatural amino acid” is intended to encompass amino acids that are not found in natural polypeptide chains of animals and / or humans. The unnatural amino acids may be created synthetically or occur naturally in bacteria, fungi, plants, or marine organisms.

[0085] The unnatural amino acid may comprise or consist of hydroxyproline, beta-alanine, citrulline, ornithine, norleucine, 3-nitrotyrosine, nitroarginine, and / or pyroglutamic acid. The unnatural amino acid may be N-acetyl-N-hydroxy-ornithine. The unnatural amino acid may be N-hydroxy-cyclo ornithine. The peptide chain may comprise N-acetyl-N-hydroxy-ornithine and / or N-hydroxy-cyclo ornithine. The unnatural amino acid may comprise or consist of a D-isoform of a natural amino acid. The unnatural amino acid may be a naturally-occurring amino acid which comprises at least one modification in the side chain. For example, the unnatural amino acid may be a naturally-occurring amino acid which comprises a side chain that has been acetylated, hydroxylated, dehydrated, formylated, and / or chlorinated.

[0086] The peptide chain may comprise at least one, at least two, or at least three unnatural amino acids selected from:

[0087] 11

[0088] 17211595 CXB CXB

[0089] D-amino acids homo amino acids N-methyl amino acids alpha-methyl amino acid beta2amino acids beta3amino acids beta3homo amino acids AGHC peptoids wherein R is selected from a group of H, OH, COOH, NH2, CH3, or any side group of a naturally-occurring amino acid.

[0090] The peptide chain may comprise a sequence of X-[N-acetyl-N-hydroxy-ornithine]-glycine-threonine- threonine-glutamine-glycine-serine-[N-hydroxy-cyclo ornithine] (SEQ ID NO: 55), wherein X is any amino acid. Preferably, X is a hydrophobic amino acid. The hydrophobic amino acid may be alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, and / or valine. Preferably, X is alanine. X is preferably not serine.

[0091] The peptide chain may comprise a sequence of alanine-[N-acetyl-N-hydroxy-ornithine]-glycine- threonine-threonine-glutamine-glycine-serine-[N-hydroxy-cyclo ornithine] (SEQ ID NO: 56). Preferably, the peptide chain comprises alanine or other hydrophobic amino acid at the N-terminal end. The alanine may be an L-alanine or a D-alanine. Preferably, the first amino acid in the peptide chain (i.e. at the N-terminal end) is not serine. Preferably, the peptide is attached to the chromophore at the N-terminal end of the peptide.

[0092] The amino acid in the peptide chain may be a D-amino acid or an L-amino acid.

[0093] The side groups of amino acids in the peptide chain may comprise modification(s). The side groups of amino acids may comprise post-translational modifications, such as acetylation, phosphorylation, hydroxylation, ubiquitylation, and / or oxidation. The side groups of amino acids may comprise a functional group. The functional group may be a fatty acid chain, a flavin moiety, a heme C group, a palmitate group, an isoprenoid group, and / or a myristate group.

[0094] The peptide chain may comprise or consist of a sequence (C-terminus to N-terminus) of:

[0095] 12

[0096] 17211595 CXB CXB wherein Z is selected from a group comprising:

[0097] H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3.

[0098] Preferably, Z is CH3.

[0099] The peptide chain may comprise or consist of a sequence (C-terminus to N-terminus) of:

[0100] The peptide chain may comprise a sequence of SEQ ID NO: 55, or a sequence comprising at least one, or at least two amino acid substitutions in the sequence of SEQ ID NO: 56. The amino acid substitution(s) may be conservative amino acid substitution(s).

[0101] The peptide chain may be linked to the carboxyl group of the chromophore. The side chain may be linked to the NH2group of the chromophore. Preferably, the peptide chain is linked by its A / -terminus to the carboxyl group of the chromophore.

[0102] The compound of formula (I), or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may comprise:

[0103] (i) the peptide chain comprising: wherein Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0104] (ii) the chromophore comprising any one of:

[0105] 13

[0106] 17211595 CXB CXB

[0107] preferably

[0108] (iii) the side chain comprising: , wherein

[0109] Ri is OH, H, or NH2, and R2is OH, H or NH2.

[0110] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may comprise:

[0111] (i) the peptide chain comprising:

[0112] (ii) the chromophore comprising any one of:

[0113] 14

[0114] 17211595 CXB CXB

[0115] preferably

[0116] (iii) the side chain comprising:

[0117] R1is OH, H, or NH2, and R2is OH, H or NH2.

[0118] Preferably, the compound of formula (I) comprises:

[0119] (i) the peptide chain comprising:

[0120] (ii) the chromophore comprising any one of:

[0121] 15

[0122] 17211595 CXB CXB

[0123] preferably

[0124] (iii) the side chain comprising:

[0125] Preferably, the side chain is linked to the amine group of the chromophore and the peptide chain is linked to the carboxyl group of the chromophore.

[0126] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be: wherein:

[0127] 16

[0128] 17211595 CXB CXB Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0129] R1is OH, H, or NH2, and

[0130] R2is OH, H or NH2.

[0131] Preferably, R1is NH2, R2is OH and Z is CH3. Preferably, R1 is OH, R2is OH and Z is CH3.

[0132] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be: wherein:

[0133] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0134] R1is OH, H, or NH2, and

[0135] R2is OH, H or NH2.

[0136] Preferably, R1is NH2, R2is OH and Z is CH3. Preferably, R1is OH, R2is OH and Z is CH3.

[0137] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be: wherein:

[0138] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0139] 17

[0140] 17211595 CXB CXB R1is OH, H, or NH2, and

[0141] R2is OH, H or NH2.

[0142] Preferably, R1is NH2, R2is OH and Z is CH3. Preferably, R1is OH, R2is OH and Z is CH3.

[0143] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be:

[0144] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be:

[0145] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be:

[0146] 18

[0147] 17211595 CXB CXB

[0148]

[0149] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be:

[0150] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be:

[0151] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be:

[0152] 19

[0153] 17211595 CXB CXB

[0154]

[0155] The compound of formula (I) may be used in inhibiting the growth of fungi. The compound of formula (I) may be applied or administered to a plant or a subject in a form of a composition as described herein.

[0156] The compound of formula (I) may be of a hexdantate iron chelator. The compound of formula (I) may have a high iron-binding affinity. Without wishing to be bound by a particular theory, iron chelation may be mediated by the catechol group of the chromophore and the hydroxamate groups of the N- acyl N-hydroxy ornithine and N-hydroxy cyclo ornithine residues. This strong iron-binding interaction may induce iron starvation in a wide range of pathogenic microbes, resulting in growth inhibition.

[0157] The compound of formula (I) may have an IC50 value against a fungal pathogen, such as Z. tritici or A. fumigatus, of < 10pM, <5 pM, <3 pM, < 2 pM, or < 1 pM. The compound of formula (I) may have an IC50 value against a fungal pathogen, such as Z. tritici or A. fumigatus, of < 10 pg / mL, < 5 pg / mL, < 3 pg / mL, or < 2 pg / mL. The compound of formula (I) may have a minimum inhibitory concentration (MIC) value against a fungal pathogen, such as Z. tritici or A. fumigatus, of < 10pM, < 5 pM, < 3 pM, or < 2 pM. The compound of formula (I) may have a minimum inhibitory concentration (MIC) value against a fungal pathogen, such as Z. tritici or A. fumigatus, of < 10 pg / mL, < 5 pg / mL, < 3 pg / mL, or < 2 pg / mL.

[0158] As used herein, the term “antimicrobial” in the context of uses and / or agents is intended to encompass uses and agents that, upon administration or application to a plant or subject, result in the prevention or inhibition of microbial growth. The term “antimicrobial” relates to compounds (such as a compound of formula (I)) which are active against microbes. Microbes include microorganisms such as fungi, fungi-like microorganisms (e.g. Oomycota or water mold), mold and bacteria. The administration or application of antimicrobial agents may result in microbial death or growth inhibition. Preferably, antimicrobial agents are selective against fungal or fungal-like pathogens and / or bacterial pathogens. The subject may be an animal or human suffering from a microbial disease or infection.

[0159] Use as an antifungal or antibacterial agents to treat or control plant disease

[0160] 20

[0161] 17211595 CXB CXB The compound of formula (I) as described herein may be used to treat a fungal infection or disease. The fungal infection or disease may be affecting plants. The compound of formula (I) as described herein may be used to treat a bacterial infection or disease. The bacterial infection or disease may be affecting plants.

[0162] The invention provides a use of a compound of formula (I) as an antifungal agent. The invention provides a use of a compound of formula (I) as described herein as an antifungal agent. The invention also provides an antifungal use of a compound of formula (I) as described herein. The invention provides a use of a compound of formula (I) as an antibacterial agent. The invention provides a use of a compound of formula (I) as described herein as an antibacterial agent. The invention also provides an antibacterial use of a compound of formula (I) as described herein.

[0163] The invention provides a use of a compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof as an antifungal agent or an antibacterial agent, wherein the compound of formula (I) comprises:

[0164] (i) the peptide chain comprising: wherein Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0165] (ii) the chromophore comprising any one of: preferably

[0166] 21

[0167] 17211595 CXB CXB

[0168] (iii) the side chain comprising: , wherein

[0169] R1is OH, H, or NH2, and

[0170] R2is OH, H or NH2.

[0171] Preferably, R1is NH2, R2is OH and Z is CH3. Preferably, R1is OH, R2is OH and Z is CH3.

[0172] The invention provides a use of a compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof as an antifungal agent or an antibacterial agent, wherein the compound of formula (I) is wherein:

[0173] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0174] R1is OH, H, or NH2, and

[0175] R2is OH, H or NH2.

[0176] Preferably, R1is NH2, R2is OH and Z is CH3. Preferably, R1is OH, R2is OH and Z is CH3.

[0177] The invention provides a use of a compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof as an antifungal agent or an

[0178] 22

[0179] 17211595 CXB CXB antibacterial agent, wherein the compound of formula (I) is wherein:

[0180] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0181] R1is OH, H, or NH2, and

[0182] R2is OH, H or NH2.

[0183] Preferably, R1is NH2, R2is OH and Z is CH3. Preferably, R1is OH, R2is OH and Z is CH3.

[0184] The invention provides a use of a compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof as an antifungal agent or an antibacterial agent, wherein the compound of formula (I) is wherein:

[0185] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0186] R1is OH, H, or NH2, and

[0187] R2is OH, H or NH2.

[0188] Preferably, R1is NH2, R2is OH and Z is CH3. Preferably, R1is OH, R2is OH and Z is CH3.

[0189] The antifungal agent may be used to treat a disease of infection of a plant.

[0190] The term “antifungal” as used herein in the context of uses and / or agents is intended to encompass uses and agents that, upon administration or application to the plant, result in the prevention or

[0191] 23

[0192] 17211595 CXB CXB inhibition of fungal growth. The term “antifungal” relates to the application or administration of compounds (such as a compound of formula (I)) which are active against fungi (especially against fungal pathogens). The administration or application of antifungal agents may result in fungal death or growth inhibition. The antifungal agent may be selective for fungal pathogens. Compound of formula (I) may have specificity for fungal pathogens over, for example, microbial pathogens. The term “antibacterial” as used herein in the context of uses and / or agents is intended to encompass uses and agents that, upon administration or application to the plant, result in the prevention or inhibition of bacterial growth. The term “antibacterial” relates to the application or administration of compounds (such as a compound of formula (I)) which are active against bacteria (especially against bacterial pathogens). The administration or application of antibacterial agents may result in bacterial death or growth inhibition.

[0193] The term “fungi” or “fungal” is intended to cover fungi as well as fungi-like microorganisms such as Oomycota, Water mold and / or yeast. Preferably, fungi-like microorganisms include Peronoporales, more preferably, fungi-like microorganisms belong to the Peronosporaceae family, more preferably fungi-like microorganisms have the genus Phytophthora, more preferably the fungi-like microorganism is Phytophthora infestans and / or Phytophthora cactorum. The yeast may be Cryptococcus neoformans. Therefore, the term “antifungal” in the context of uses and / or agents is also intended to encompass uses and agents that, upon administration or application to the plant, result in the prevention or inhibition of the growth of fungi-like microorganisms.

[0194] The invention also provides a method for treating a plant to control a disease, the method comprising applying an effective amount of the compound of formula (I) as described herein to the plant, or a part of the plant.

[0195] The invention also provides a method for treating a plant to control a disease, the method comprising applying an effective amount of the antifungal composition or the antibacterial composition as described herein to the plant, or a part of the plant.

[0196] The expression “control a disease” as used herein (in the context of methods and uses of the invention) is intended to encompass inhibition of the progression of the disease and / or treatment of the disease. That is to say that compound of formula (I) may stop the disease from progressing or may reverse the effects of the disease. The disease may be anthracnose, leaf spot, rust, wilt, blight, coils, scab, gall, canker, damping-off, root rot, mildew, or dieback. The disease may be septoria leaf blotch.

[0197] The plant may be any plant suffering from a fungal or bacterial disease. Preferably, the plant is wheat.

[0198] In the context of treating a plant, the fungal disease may be anthracnose, leaf spot, rust, wilt, blight, coils, scab, gall, canker, damping-off, root rot, mildew, or dieback. The fungal disease may be septoria leaf blotch.

[0199] 24

[0200] 17211595 CXB CXB The term “effective amount” as used herein means the amount of the compound of formula (I) or the antifungal or antibacterial composition that, when administered to or applied to a plant for treating a disease, is sufficient to effect such treatment for the disease. The “effective amount” will vary depending on the disease and its severity, and / or, on the plant type, size and / or age.

[0201] The compound of formula (I) may be administered to or applied to a plant in the form of an antifungal composition (as described herein), or antibacterial composition (as described herein) or formulation. Such antifungal compositions or antibacterial compositions may be administered or applied to a plant by any acceptable route of administration including, but not limited to, spraying onto the plant or a part thereof, or addition to a water and / or nutrient solution provided to the plant.

[0202] Use as an antifungal agent or an antibacterial agent for therapy in a subject

[0203] Compound of formula (I) can be used to treat diseases caused by microbes. Microbes may be fungi, bacteria, yeast, and / or fungi-like pathogens (e.g. Phytophthora sp.). In addition to antifungal properties of a compound of formula (I) for plant treatment, the compound of formula (I) was also shown to have antifungal properties against fungal pathogens affecting (i.e. causing a disease or infection in) animal (especially human) subjects. The compound of formula (I) was also shown to have antibacterial properties against bacterial pathogens affecting (i.e. causing a disease or infection in) animal (especially human) subjects.

[0204] Thus, the invention provides a compound of formula (I) for use in therapy. The invention provides a compound of formula (I) for use as an antifungal agent in a subject. The invention provides a compound of formula (I) for use in treating a fungal disease in a subject. Thus, the invention provides a compound of formula (I) as described herein for use in therapy. The invention provides a compound of formula (I) as described herein for use as an antifungal agent in a subject. The invention provides a compound of formula (I) as described herein for use in treating a fungal disease or infection in a subject. The invention provides a compound of formula (I) for use as an antibacterial agent in a subject. The invention provides a compound of formula (I) for use in treating a bacterial disease or infection in a subject.

[0205] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may comprise:

[0206] (i) the peptide chain comprising:

[0207] 25

[0208] 17211595 CXB CXB wherein Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and

[0209] CH2CH(CH3)CH3;

[0210] (ii) the chromophore comprising any one of: preferably

[0211] (iii) the side chain comprising: , wherein

[0212] R1is OH, H, or NH2, and R2is OH, H or NH2.

[0213] Preferably, R1is NH2, R2is OH and Z is CH3. Preferably, R1is OH, R2is OH and Z is CH3.

[0214] 26

[0215] 17211595 CXB CXB The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be: wherein:

[0216] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0217] R1is OH, H, or NH2, and

[0218] R2is OH, H or NH2.

[0219] Preferably, R1is NH2, R2is OH and Z is CH3. Preferably, R1is OH, R2is OH and Z is CH3.

[0220] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be: (I), wherein:

[0221] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0222] R1is OH, H, or NH2, and

[0223] R2is OH, H or NH2.

[0224] Preferably, R1is NH2, R2is OH and Z is CH3. Preferably, R1is OH, R2is OH and Z is CH3.

[0225] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be:

[0226] 27

[0227] 17211595 CXB CXB

[0228] wherein:

[0229] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0230] R1is OH, H, or NH2, and

[0231] R2is OH, H or NH2.

[0232] Preferably, R1is NH2, R2is OH and Z is CH3. Preferably, R1is OH, R2is OH and Z is CH3.

[0233] The invention provides the compound of formula (I) as described herein for use as a medicament.

[0234] The invention also provides a method for treating a fungal disease or infection comprising administering to a subject an effective amount of the compound of formula (I) as described herein or the antifungal composition as described herein.

[0235] The subject may suffer from a fungal disease or infection. The subject may be immunocompromised (e.g. from a cancer therapy, after a transplant, or may be suffering from HIV / AIDS). The subject may be human or animal.

[0236] The fungal disease or infection may be a disease of subcutaneous tissue, such as porotrichosis, chromoblastomycosis, and eumycetoma. The fungal disease or infection may be a disease of a systemic nature, for example, histoplasmosis, cryptococcosis, coccidioidomycosis, blastomycosis, mucormycosis, aspergillosis, pneumocystis pneumonia and systemic candidiasis. The fungal disease or infection may be of superficial nature, for example, candidiasis or malassezia infections.

[0237] The invention provides a method for treating a fungal disease or infection in a subject (e.g. human), the method comprising applying an effective amount of the compound of formula (I) as described herein or the antifungal composition as described herein to the subject.

[0238] The invention also provides a method for treating a bacterial disease or infection comprising administering to a subject an effective amount of the compound of formula (I) as described herein or the antibacterial composition as described herein.

[0239] 28

[0240] 17211595 CXB CXB The subject may suffer from a bacterial disease or infection. The subject may be immunocompromised (e.g. from a cancer therapy, after a transplant, or may be suffering from HIV / AIDS). The subject may be human or animal.

[0241] The bacterial disease or infection may be a disease or infection caused by a gram-positive or gramnegative bacterium. The bacterial disease or infection may be a disease or infection caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and / or Enterococcus faecalis.

[0242] As used herein, the term “antibacterial” in the context of uses and / or agents is intended to encompass uses and agents that, upon administration or application to the subject, result in the prevention or inhibition of bacterial growth. The term “antibacterial” relates to the application or administration of compounds (such as a compound of formula (I)) which are active against bacteria (especially against pathogenic bacteria). The administration or application of antibacterial agents may result in bacterial death or growth inhibition. The antibacterial agent may be selective for bacterial pathogens, such as Klebsiella pneumoniae, Enterococcus faecalis, Escherichia coli, and / or Staphylococcus aureus. Compound of formula (I) may have specificity for bacterial pathogens over, for example, other microbial pathogens. The subject may be an animal or human. The subject may be an animal or human suffering from a bacterial disease or infection. Preferably, the subject is an individual with a compromised immune system, such as a subject suffering from HIV / AIDS, a hospital patient, a member of combat troops or an individual exposed to war or conflict zones.

[0243] The invention provides a method for treating a bacterial disease or infection in a subject (e.g. human), the method comprising applying an effective amount of the compound of formula (I) as described herein or the antibacterial composition as described herein to the subject.

[0244] The term “effective amount” as used herein means the amount of the compound of formula (I) or the antifungal or antibacterial composition that, when administered to a subject for treating a disease or infection, is sufficient to effect such treatment of the disease or infection. The “effective amount” will vary depending on the disease and its severity, and, if the subject is human or animal, the age and weight of the subject to be treated. The term “subject” includes, but is not limited to, animals such as, for example, mammals. Preferably, the subject is a human.

[0245] The expression “control a disease” is intended to encompass inhibition of the progression of the disease or infection and / or treatment of the disease or infection. That is to say that compound of formula (I) may stop the disease or infection from progressing or may reverse the effects of the disease or infection. The disease or infection may be a disease or infection of subcutaneous tissue, such as porotrichosis, chromoblastomycosis, and eumycetoma. The disease or infection may be a disease or infection of a systemic nature, for example, histoplasmosis, cryptococcosis, coccidioidomycosis, blastomycosis, mucormycosis, aspergillosis, pneumocystis pneumonia and

[0246] 29

[0247] 17211595 CXB CXB systemic candidiasis. The disease or infection may be of superficial nature, for example, candidiasis or malassezia infections. The disease or infection may be a disease or infection caused by a fungus of the Aspergillus or Candida genus.

[0248] The compound of formula (I) may be administered to a subject in the form of a pharmaceutical composition (e.g. the antifungal composition as described herein or the antibacterial composition as described herein) or formulation. Such pharmaceutical compositions may be administered to the subject by any acceptable route of administration including, but not limited to, oral, topical (including transdermal) and parenteral modes of administration.

[0249] The methods described herein may be in vitro methods or in vivo methods.

[0250] In the context of treating a subject, administration can be accomplished either by oral administration via tablets, pills, capsules, granules, powders, solutions, and the like, or parenteral administration, such as injections such as intra-articular, intravenous, and intramuscular injections, suppositories, ophthalmic solutions, eye ointments, or agents for external use, such as transdermal liquid preparations, ointments, transdermal patches, transmucosal liquid preparations, transmucosal patches, inhalers, and the like.

[0251] In oral administration, the daily dose is generally from about 0.0001 to 1000mg / kg per body weight, administered in one portion or in 2 to 4 separate portions. The dose is appropriately decided upon in response to the individual case by taking the symptoms, the age, and the gender, and the like into consideration.

[0252] Antifungal compositions and antibacterial compositions

[0253] The invention provides an antifungal composition comprising a compound of formula (I) as an active ingredient and, optionally, an acceptable carrier (e.g. a pharmaceutically acceptable carrier). The invention provides an antifungal composition comprising compound of formula (I) as described herein as an active ingredient and, optionally, an acceptable carrier (e.g. a pharmaceutically acceptable carrier). The antifungal composition as described herein is understood to refer to a composition of matter. The invention provides an antibacterial composition comprising a compound of formula (I) as an active ingredient and, optionally, an acceptable carrier (e.g. a pharmaceutically acceptable carrier). The invention provides an antibacterial composition comprising compound of formula (I) as described herein as an active ingredient and, optionally, an acceptable carrier (e.g. a pharmaceutically acceptable carrier). The antibacterial composition as described herein is understood to refer to a composition of matter.

[0254] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may comprise:

[0255] (i) the peptide chain comprising:

[0256] 30

[0257] 17211595 CXB CXB wherein Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0258] (ii) the chromophore comprising any one of: preferably

[0259] (iii) the side chain comprising: , wherein

[0260] R1is OH, H, or NH2, and

[0261] R2is OH, H or NH2.

[0262] Preferably, R1is NH2, R2is OH and Z is CH3. Alternatively, preferably, R1is OH, R2is OH and Z is CH3.

[0263] 31

[0264] 17211595 CXB CXB The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be: wherein:

[0265] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0266] R1is OH, H, or NH2, and

[0267] R2is OH, H or NH2.

[0268] Preferably, R1is NH2, R2is OH and Z is CH3. Alternatively, preferably, R1is OH, R2is OH and Z is CH3.

[0269] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be: (I), wherein:

[0270] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0271] R1is OH, H, or NH2, and

[0272] R2is OH, H or NH2.

[0273] Preferably, R1is NH2, R2is OH and Z is CH3. Alternatively, preferably, R1is OH, R2is OH and Z is CH3.

[0274] 32

[0275] 17211595 CXB CXB The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be: wherein:

[0276] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0277] R1is OH, H, or NH2, and

[0278] R2is OH, H or NH2.

[0279] Preferably, R1is NH2, R2is OH and Z is CH3. Alternatively, preferably, R1is OH, R2is OH and Z is CH3.

[0280] The compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof may be: wherein:

[0281] Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0282] R1is OH, H, or NH2, and

[0283] R2is OH, H or NH2.

[0284] Preferably, R1is NH2, R2is OH and Z is CH3. Alternatively, preferably, R1is OH, R2is OH and Z is CH3.

[0285] 33

[0286] 17211595 CXB CXB The invention provides an antifungal composition comprising compound (I) as an active ingredient and an acceptable carrier (e.g. pharmaceutically acceptable carrier), wherein compound (I) is described herein.

[0287] The expressions “antifungal composition” or “antibacterial composition” (or “composition of matter”) as used herein intend to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such term in relation to the antifungal or antibacterial composition, is intended to encompass a product comprising the compound of formula (I), and optionally the additional ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the antifungal or antibacterial compositions of the present invention encompass any composition comprising the compound of formula (I) as described herein, and optionally an acceptable carrier. By “acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0288] The antifungal or antibacterial composition (or the composition of matter) may comprise the compound of formula (I) in the salt form, or hydrate form. The compound of formula (I) may be in any isomeric form.

[0289] The pharmaceutical compositions (e.g. an antifungal composition or an antibacterial composition) or formulations are typically prepared by pharmaceutically acceptable carrier and, optionally, one or more optional ingredients. If necessary or desired, the resulting uniformly blended mixture can then be shaped or loaded into tablets, capsules, pills, canisters, cartridges, dispensers, and the like using conventional procedures and equipment.

[0290] In the context of treating a subject, when intended for oral administration in a solid dosage form (i.e., as capsules, tablets, pills and the like), the pharmaceutical composition will typically comprise a compound of formula (I) as the active ingredient. The pharmaceutical composition may comprise compound of formula (I) and no other ingredient. Alternatively, the pharmaceutical composition may comprise the compound (I) as the active ingredient and one or more pharmaceutically acceptable carriers. Suitable pharmaceutically acceptable carriers would be known by the person skilled in the art, for example, fats, water, physiological saline, alcohol (e.g., ethanol), glycerol, polyols, aqueous glucose solution, extending agent, disintegrating agent, binder, lubricant, wetting agent, stabilizer, emulsifier, dispersant, preservative, sweetener, colorant, seasoning agent or aromatizer, concentrating agent, diluent, buffer substance, solvent or solubilizing agent, chemical for achieving storage effect, salt for modifying osmotic pressure, coating agent or antioxidant, saccharides such as lactose or glucose; starch of corn, wheat or rice; fatty acids such as stearic acid; inorganic salts such

[0291] 34

[0292] 17211595 CXB CXB as magnesium metasilicate aluminate or anhydrous calcium phosphate; synthetic polymers such as polyvinylpyrrolidone or polyalkylene glycol; alcohols such as stearyl alcohol or benzyl alcohol; synthetic cellulose derivatives such as methylcellulose, carboxymethylcellulose, ethylcellulose or hydroxypropylmethylcellulose; and other conventionally used additives such as gelatin, talc, plant oil and gum arabic.

[0293] The pharmaceutical composition (e.g. an antifungal composition) comprising the compound of formula (I) can also be administered transdermally or transmucosally using known delivery systems and excipients. For example, the pharmaceutical composition can be admixed with permeation enhancers such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and the like, and incorporated into a patch or similar delivery system. Additional excipients including gelling agents, emulsifiers, and buffers, may also be used.

[0294] Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous solvents include, for example, distilled water for injection and / or physiological saline. Examples of non-aqueous solvents include alcohols such as ethanol.

[0295] The pharmaceutical composition (e.g. the antifungal composition or the antimicrobial composition) may comprise one or more further active agents. This combination therapy involves using the compound of formula (I) combined with one or more of the further active agents, either formulated together (for example, packaged together in a single formulation) or formulated separately (for example, packaged as separate unit dosage forms).

[0296] Biosynthetic gene cluster

[0297] The invention also provides a method for producing a compound of formula (I) comprising synthesising the compound of formula (I) from a biosynthetic gene cluster encoding the peptides of SEQ ID NOs: 1-54, or peptides of 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NOs: 1-54.

[0298] The invention provides the biosynthetic gene cluster encoding at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, or at least 50 peptides of any one of SEQ ID NOs: 1-54, or peptides of 80%, 85%, 90%, 95%, 97%, or 99% identity to at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, or at least 50 peptides of SEQ ID NOs: 1-54 The invention also provides the biosynthetic gene cluster encoding the sequences of SEQ ID NOs: 1-54, or sequences of 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NOs: 1-54. The biosynthetic gene cluster may be used to produce the compound of formula (I).

[0299] The at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, or at least 50 peptides of SEQ ID NOs: 1-54 may comprise peptide(s) of SEQ ID NOs: 17, 18, 19 and / or 50.

[0300] 35

[0301] 17211595 CXB CXB P262959WQ00

[0302] The invention provides a host cell comprising a plasmid encoding at least one of the peptides of SEQ ID NOs: 1-54. The host cell may comprise a plasmid encoding at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 of the peptides of SEQ ID NOs: 1-54. The host cell may comprise a plasmid encoding all of the peptides of SEQ ID NOs: 1-54. The host cell may comprise two or three plasmids. Each plasmid may encode at least one of the peptides of any one of SEQ ID NO:1-54. Each plasmid may encode at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 of the peptides of any one of SEQ ID NOs: 1-54.

[0303] The plasmid may comprise a promoter. Each peptide expressed by the plasmid may be under control of a different promoter or the same promoter.

[0304] The invention also provides a method of producing a compound of formula (I) as described herein, the method comprises culturing the host cell as described herein under conditions suitable for the production of proteins.

[0305] The invention provides the compound of formula (I) produced by the method as described herein.

[0306] These and other aspects of the invention will now be described with reference to the accompanying Figures, in which:

[0307] Figure 1 illustrates dose response curves for Compounds of formula (I) against A. fumigatus. Fig. 1A illustrates a dose response curve of Compound of formula (la) against A. fumigatus from a resazurin- based assay. Fig. 1 B illustrates a dose response curve of Compound of formula (lb) against A. fumigatus from a resazurin-based assay against a positive control, voriconazole (Vra).

[0308] Figure 2 illustrates dose response curves for compounds of formula (I) against Z. tritici from a fluorescence-based assay, where Fig. 2A shows dose response curves for Compound (la) and a positive control, amphotericin B (AmB) and Fig. 2B shows dose response curves for Compound (lb) and a positive control (AmB).

[0309] Figure 3 illustrates a dose response curve of compound of formula I against Z. tritici from an absorbance-based assay. Fig. 3A shows dose response curves for Compound (la) and a positive control (AmB) and Fig. 3B shows dose response curves for Compound (lb) and a positive control (AmB).

[0310] Figure 4 shows chromatograms obtained for compound of formula (la).

[0311] Figure 5 shows mass spectrum results for compound of formula (la).

[0312] Figure 6A shows 1 H NMR spectrum for compound of formula (la).

[0313] Figure 6B shows 13C NMR spectrum for compound of formula (la).

[0314] Figure 7 shows the chemical structure of compound of formula (la) after characterization in its different resonance forms. The double headed arrow ► is a “resonance arrow” used to depict the interconversion between different resonance forms.

[0315] 36

[0316] 17211595 CXB CXB Figure 8 shows mass spectrum results for compound of formula (lb).

[0317] Figure 9A shows 1 H NMR spectrum for compound of formula (lb).

[0318] Figure 9B shows 13C NMR spectrum for compound of formula (lb).

[0319] Figure 10 shows the chemical structure of compound of formula (lb) after characterization in its different resonance forms. The double headed arrow ► is a “resonance arrow” used to depict the interconversion between different resonance forms.

[0320] Figure 11 shows the architecture of the biosynthetic gene cluster.

[0321] Figure 12 shows a map of the plasmid (PL00377) used to produce a compound of formula (I) according to Example 4.

[0322] Figure 13 provides cloning confirmation of compound (la) according to formula (I) using PCR. Figure 14 shows LC-MS trace for [M-4H+Fe(lll)+2H]2+ion peak for compound (la) according to formula (I) obtained according to Examples 4 & 5 compared to material obtained from a nonengineered strain control.

[0323] Figure 15 shows a full UV spectrum of compound (la) after purification of compound (la) according to Example 4 showing a distinctive UV profile with maxima at 220 and 360 nm.

[0324] Figure 16 shows the effect of treatment with Compound (la) on disease caused by Zymoseptoria tritici on winter wheat plants.

[0325] Figure 17 provides a visual representation of the different levels of disease severity for wheat Zymoseptoria tritici based on the percentage of leaf area affected.

[0326] Figure 18 illustrates response curves for compound (la) and three different positive controls against three agricultural pathogens from an absorbance-based assay. Fig. 18a is a dose response curve for compound (la) against Alternaria brassicicola. Fig 18b is a dose response curve for compound (la) against Microdochium nivale. Fig 18c is a dose response curve for compound (la) against Phytophthora cactorum. Fig. 18d shows a dose response curve for positive control azoxystrobin (azoxy) against Alternaria brassicicola. Fig. 18e shows a dose response curve for positive control fluxapyroxad (flux) against Microdochium nivale. Fig 18f shows a dose response curve for mandipropamid (mandi) against Phytophthora cactorum.

[0327] Figure 19 illustrates response curves for compound (la) and two different positive controls against different strains of Zymoseptoria tritici from an absorbance-based assay. Fig. 19a is a dose response curve for compound (la) against a susceptible strain of Zymoseptoria tritici. Fig 19b is a dose response curve for compound (la) against a resistant strain of Zymoseptoria tritici, Haplotype H4 with reduced sensitivity to azoles. Fig 19c is a dose response curve for compound (la) against a succinate dehydrogenase inhibitor (SDHI) resistant strain of Zymoseptoria tritici. Fig 19d shows a dose response curve for positive control fluxapyroxad (flux) against a susceptible strain of Zymoseptoria tritici. Fig 19e shows a dose response curve for positive control prothioconazole-desthio (pro des) against a resistant strain of Zymoseptoria tritici, Haplotype H4 with reduced sensitivity to azoles. Fig. 19f shows a dose response curve for positive control fluxapyroxad (flux) against a succinate dehydrogenase inhibitor (SDHI) resistant strain of Zymoseptoria tritici.

[0328] Figure 20 shows the ratio of control as a function of the concentration of compound (la), and two control compounds: carbonyl cyanide 3-chlorophenylhydrazone, chlorpromazine to evaluate cellular

[0329] 37

[0330] 17211595 CXB CXB toxicity. The dashed lines represent the significant cut-off from vehicle control and was used to calculate the MEC (minimum effective concentration). The filled diamonds represent the mean data points for each concentration plus or minus standard deviation. The solid lines represent historical maximum and minimum responses which were used to calculate AC50.

[0331] Figure 21 shows the ratio of control as a function of the concentration of a) compound (la) in media containing glucose, b) compound (la) in media containing galactose, c) rotenone (control compound) in media containing glucose, d) rotenone (control compound) in media containing galactose, e) chlorpromazine (control compound) in media containing glucose, f) chlorpromazine (control compound) in media containing galactose to evaluate mitochondrial toxicity. The dashed lines represent the significant cut-off from vehicle control and was used to calculate the MEC (minimum effective concentration). The filled diamonds represent the mean data points for each concentration plus or minus standard deviation. The solid lines represent historical maximum and minimum responses which were used to calculate AC50.

[0332] The invention is further disclosed in the following clauses:

[0333] 1 . A compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof

[0334] P-C-S (I), wherein:

[0335] (i) P is a peptide chain comprising: wherein Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0336] (ii) C is a chromophore comprising any one of:

[0337] 38

[0338] 17211595 CXB CXB

[0339] preferably

[0340] (iii) S is a side chain comprising: , wherein

[0341] R1is OH, H, or NH2, and

[0342] R2is OH, H or NH2.

[0343] 2. The compound of clause 1 , wherein Z is CH3.

[0344] 3. The compound of clause 1 or 2, wherein R1is OH or NH2.

[0345] 4. The compound of any one of clauses 1-3, wherein R2is OH.

[0346] 5. The compound of any one of clauses 1-4, wherein the compound has a structure (la) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof:

[0347] 39

[0348] 17211595 CXB CXB

[0349]

[0350] 6. The compound of clause 5, wherein compound (la) is selected form the following resonance forms:

[0351] 7. The compound of any one of clauses 1-4, wherein the compound has a structure (lb) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof:

[0352] 40

[0353] 17211595 CXB CXB

[0354]

[0355] 8. The compound of clause 7, wherein compound (lb) comprises the following resonance forms:

[0356] 9. Use of a compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof:

[0357] P-C-S (I), wherein:

[0358] (i) P is a peptide chain comprising:

[0359] 41

[0360] 17211595 CXB CXB wherein Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and

[0361] CH2CH(CH3)CH3;

[0362] (ii) C is a chromophore comprising any one of:

[0363] Preferably

[0364] (iii) S is a side chain comprising: , wherein

[0365] R1is OH, H, or NH2, and

[0366] R2is OH, H or NH2, as an antifungal agent.

[0367] 10. A compound of formula (I) or a resonance form, tautomer, stereoisomeric form, pharmaceutically acceptable salt or solvate thereof:

[0368] P-C-S (I), wherein:

[0369] 42

[0370] 17211595 CXB CXB (i) P is a peptide chain comprising: wherein Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;

[0371] (ii) C is a chromophore comprising:

[0372] (iii) S is a side chain comprising: , wherein

[0373] R1is OH, H, or NH2, and

[0374] R2is OH, H or NH2, for use in treating a fungal disease or infection in a subject.

[0375] 11 . The use of clause 9, or the compound for use of clause 10, wherein Z is CH3.

[0376] 12. The use of clause 9 or 11 , or the compound for use of clause 10 or 11 , wherein R1is OH or NH2.

[0377] 43

[0378] 17211595 CXB CXB 13. The use of any one of clauses 9, 11 -12, or the compound for use of any one of clause 10-12, wherein R2is OH.

[0379] 14. The use of any one of clauses 9, 11 -13, or the compound for use of any one of clause 10-13, wherein the compound has a structure (la):

[0380] 15. The use or the compound for use of clause 14 wherein compound (la) comprises the following resonance forms:

[0381] 16. The use of any one of clauses 9, 11 -13, or the compound for use of any one of clause 10-13, wherein the compound has a structure (lb):

[0382] 44

[0383] 17211595 CXB CXB

[0384]

[0385] 17. The use or compound for use of clause 16 wherein compound (lb) comprises the following resonance forms:

[0386] 18. A method for treating a plant to control a disease, the method comprising applying an effective amount of the compound of formula (I) as described in any one of clauses 1-8 to the plant, or a part of the plant.

[0387] 19. The method of clause 18, wherein the disease is septoria leaf blotch.

[0388] 20. The method of clause 18 or 19 wherein the plant is wheat.

[0389] 21. An antifungal composition comprising the compound of formula (I) as described in any one of clauses 1-8 and an acceptable carrier.

[0390] 45

[0391] 17211595 CXB CXB 22. A method for producing a compound of formula (I) comprising synthesising the compound of formula (I) from a biosynthetic gene cluster encoding the peptides of SEQ ID NOs: 1-54, or peptides of 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NOs: 1-54.

[0392] 23. A biosynthetic gene cluster encoding at least 10 peptides of any one of SEQ ID NOs: 1-54, or peptides of 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NOs: 1-54.

[0393] 46

[0394] 17211595 CXB CXB EXAMPLES

[0395] The following non-limiting examples further illustrate the present invention.

[0396] Example 1 - Fungal Growth Assays

[0397] Aspergillus fumigatus

[0398] Spore stocks of A. fumigatus strain CEA10 (RS-AA23) (generated by Bactobio) (catalogue number: A1163) are prepared by spreading spores Potato Dextrose Agar. The plate is sealed and incubated at 37 °C for 2 days. Post incubation, spores are harvested by adding 25 mL of Tween-saline solution to the plate, physically disrupting mycelia to release spores, and transferring the spore suspension to a sterile Falcon tube. Spores are filtered through a 20 pm filter to remove filament debris and pelleted by centrifugation at 2250 rpm for 15 min. The pellet is resuspended in 25 mL of 10% glycerol-RPMI 1640 medium and spore concentration is quantified using a haemocytometer. Spore stock aliquots of 500 pL are prepared to a concentration of 106spores / mL in 10% glycerol-RPMI 1640 and stored at - 20°C. Bioassay inoculum is prepared by thawing aliquots for 10 min at room temperature and dilution to 104spores / mL with RPMI-1640 medium.

[0399] For bioassays, 12.5 pL of samples or the following controls are added to a sterile 384-well microtitre plate in triplicate at final concentrations of: 0.25% DMSO (negative control); 25 pg / mL cycloheximide (positive antifungal control) (Fisher Scientific (cat. no. 10661185)); uninoculated RPMI-1640 media (sterility control). Each test well is filled with 37.5 pL of the fungal inoculum. After inoculation the plate is sealed with a breathable membrane and incubated at 37 °C for 22 h at which point, 10 pL of 0.012% resazurin stock is added to each well and green fluorescence (excitation 485 nm, emission 535 nm, gain 40) measurements are taken at two time points (to, to) using a TECAN Spark® Multimode Microplate Reader.

[0400] Zymoseptoria tritici

[0401] The fluorescent Z. tritici strain (RS-AA10) (catalogue number: IMI 505230) (CABI genetic resource collection) was generated by transforming Z. tritici IPO323 with plasmid pBACTO4 (eAA69), which carries the ZTGFP_StrongKozak insert in a pCAMBIA0380 backbone. To create the bioassay inoculum, Z. tritici RS-AA10 is streaked out from glycerol stocks onto YMS agar plates containing 1 pL / mL of a 40 mg / mL carboxin stock solution. The plate is incubated in the dark at 18 °C for 1-2 weeks. Once colonies are visible, they are used to inoculate 10 mL of 25% YMS broth, which is incubated at 18 °C while shaking at 200 rpm for 3 days. 100 pL of this starter culture is transferred into fresh 25% YMS media, and incubated for another 24 h. Prior to the assay, the inoculum is adjusted to an ODeoo of 0.1 .

[0402] For bioassays 12.5 pL of samples or the following controls are added to a sterile 384-well microtitre plate in triplicate at final concentrations of: 0.25% DMSO (negative control); 15 pg / mL amphotericin B (positive antifungal control) (Fisher Scientific (cat. no. 15333671)); uninoculated 25% YMS media

[0403] 47

[0404] 17211595 CXB CXB (sterility control). Each test well is filled with 37.5 pL of the fungal inoculum. After inoculation, the plate is incubated in the dark at 18 °C for 48 h. ODeoo and green fluorescence (excitation 485 nm, emission 535 nm) are measured at two time points (to and t4s) using a TECAN Spark® Multimode Microplate Reader.

[0405] Bioassay Data Analysis

[0406] A. fumigatus

[0407] Percentage inhibition of each sample is calculated against A. fumigatus as follows:

[0408] 1. Each sample is time normalised to to: RFU(t0norm) = 7?FU(t2) - RFU(t0~)

[0409] 2. The mean ± standard deviation of the three independent replicates is calculated. The mean of the crude extracts of each growth media is used to control for full growth. The percentage inhibition of each crude extract is calculated using the following formula:

[0410] In cases where there are no crude extract controls, the negative control is used to represent 100% growth. Extracts which resulted in 50% inhibition relative to the culture media control are considered hits.

[0411] Figure 1A illustrates a dose response curve of compound of formula (la) against A. fumigatus from a resazurin-based assay. Figure 1 B illustrates a dose response curve of compound of formula (lb) against A. fumigatus from a resazurin-based assay. In this assay a Resazurin dye is used as an indicator for the presence of metabolic activity of A. fumigatus. The data indicates metabolic activity was maximally inhibited at a concentration of 0.78 pM and 0.39 pM for compound of formula (la) and (lb), respectively. IC50 values of 0.69 pM and 0.27 pM were derived from these respective results.

[0412] Z. tritici

[0413] Percentage inhibition of each sample is calculated against Z. tritici as follows:

[0414] 1 . Each sample is time normalised to to:

[0415] 2. The mean ± standard deviation of the three independent replicates is calculated. The mean of the negative control samples is used as control for full growth. Therefore, the percentage inhibition of each sample is calculated using the following formula:

[0416] The percentage inhibition is calculated separately for ODeoo and fluorescence.

[0417] Figure 2A illustrates a dose response curve of compound (la) against Z. tritici from a fluorescencebased assay. An IC50 of 0.89 pM was derived from these results. The data indicates metabolic activity was inhibited at a concentration of 1 .56 pM. Amphotericin B (AmB) was used as a control to

[0418] 48

[0419] 17211595 CXB CXB compare the results of compound (I) to an active antibiotic, showing similar inhibition values of 1 .12 pM.

[0420] Figure 2B illustrates a dose response curve of compound (lb) against Z. tritici from a fluorescencebased assay. An IC50 of 1 .64 pM was derived from these results. The data indicates metabolic activity was inhibited at a concentration of 3.12 pM. Amphotericin B (AmB) was used as a control to compare the results of compound (lb) to an active antibiotic.

[0421] Figure 3A illustrates a dose response curve of compound (la) against Z. tritici from an absorbancebased assay. An IC50 of 0.95 pM was derived from these results. The data indicates metabolic activity was inhibited at a concentration of 1 .56 pM. Amphotericin B (AmB) was used as a control to compare the results of compound (I) to an active antibiotic, showing similar inhibition values of 1 .23 pM.

[0422] Figure 3B illustrates a dose response curve of compound (lb) against Z. tritici from an absorbancebased assay. An IC50 of 1 .73 pM was derived from these results. The data metabolic activity was inhibited at a concentration of 3.12 pM. Amphotericin B (AmB) was used as a control to compare the results of compound (I) to an active antibiotic.

[0423] MIC and IC50 Calculations

[0424] The minimum inhibitory concentration (MIC) was defined as the minimum concentration of a compound of formula (I) required to inhibit the metabolic activity of a bacterial or fungal pathogen to a suitable extent, such as by 50 or 75%. The half maximal inhibitory concentration (IC50) was calculated in R using the ‘drm’ function from the ‘drc’ package (version 3.0.1). The values were determined using a four-parameter logistic model (Ritz, C., Baty, F., Streibig, J.C., and Gerhard, D. (2015). Dose-Response Analysis Using R. PLoS ONE, 10(12), e0146021).

[0425] The MIC and IC50 values for Compound (la) are shown in Table 1 .

[0426] Table 1 . The MIC and IC50 values for Compound (la) according to formula (I). *ND - not determined

[0427] The MIC and IC50 values for Compound (lb) are shown in Table 2.

[0428] Table 2. The MIC and IC50 values for Compound (lb) according to formula (I). *ND - not determined

[0429] 49

[0430] 17211595 CXB CXB Conclusion

[0431] As can be observed from the results set out in Tables 1 and 2, the MIC and IC50 values for Compound (lb) are comparable to those of Compound (la).

[0432] Example 2 - Production of compound of formula (I)

[0433] Construct Outline

[0434] Compound of formula (I) (specifically, la) is produced via a plasmid-borne biosynthetic gene cluster, maintained in the heterologous production host Pseudomonas putida (strain DSM 6125), using an antibiotic selection marker (aminoglycoside phosphotransferase, conferring resistance to Kanamycin).

[0435] The architecture of the biosynthetic gene cluster is shown in Figure 11 .

[0436] The sequence of proteins expressed by the biosynthetic gene cluster, their function and DNA size are shown in Table 3.

[0437] 50

[0438] 17211595 CXB CXB

[0439] 51

[0440] 17211595 CXBCXB

[0441]

[0442] 52

[0443] 17211595 CXBCXB

[0444]

[0445] 53

[0446] 17211595 CXBCXB

[0447] 54

[0448] 17211595 CXBCXB

[0449] 55

[0450] 17211595 CXBCXB

[0451] 56

[0452] 17211595 CXBCXB

[0453]

[0454] 57

[0455] 17211595 CXBCXB

[0456]

[0457] 58

[0458] 17211595 CXBCXB

[0459]

[0460] 59

[0461] 17211595 CXBCXB

[0462]

[0463] 60

[0464] 17211595 CXBCXB

[0465]

[0466] 61

[0467] 17211595 CXBCXB

[0468]

[0469] 62

[0470] 17211595 CXBCXB

[0471]

[0472] 63

[0473] 17211595 CXBCXB

[0474] 64

[0475] 17211595 CXBCXB

[0476]

[0477] 65

[0478] 17211595 CXBCXB

[0479]

[0480] Table 3. The sequence of proteins encoded by the biosynthetic gene cluster, their function and DNA size.

[0481] 66

[0482] 17211595 CXB CXB Heterologous Expression of compound of formula (I)

[0483] A swab of the glycerol stock, consisting of the heterologous host containing both the supplementary plasmid and the biosynthetic gene cluster of compound (l)-containing plasmid, is streaked out onto LB agar and incubated at 30°C for 16 hours. The agar plate contains 50 pg / mL kanamycin (Fisher Scientific: 10031553), selecting for the maintenance of the plasmid. A single colony is picked from the plate post-incubation for inoculation of a 5 mL LB broth pre-culture, also containing 50 pg / mL kanamycin. This culture is incubated for 16 hours at 30°C, shaking at 200 rpm. The pre-culture is used to inoculate a secondary pre-culture of 50 mL LB broth in a 250 mL baffled erlenmeyer flask (with 50 pg / mL kanamycin) with a 2% inoculum (1 mL) and is incubated for 16 hours at 30°C, shaking at 125 rpm.

[0484] Production cultures of compound of formula (I) are produced across two baffled 2 L erlenmeyer flasks containing 500 mL M9 minimal media (0.1 mM CaCh, 1 mM MgSC , 3 g / L KH2PO4, 0.5 g / L NaCI, 6.78 g / L Na2HPO4, 1 g / L NH4CI, 1% Glycerol, 50 pg / mL Kanamycin and 25 pg / mL Chloramphenicol) - for a total production culture volume of 1 L across the 2 flasks. The 50 mL pre-culture is used to inoculate the M9 media with a 2% inoculum (10 mL pre-culture added to each flask). Production cultures are incubated for 7 days at 30°C, shaking at 125 rpm, prior to harvesting and extraction.

[0485] Example 3 - Chemical characterization

[0486] Compound of formula (la) produced as in Example 2 was analysed structurally. Characterization of the compound of formula (lb) is also provided.

[0487] Liquid Chromatography Mass Spectrometry (LCMS)

[0488] Overview

[0489] LC-MS data are collected on an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6546 quadrupole time-of-flight (Q-TOF) mass spectrometer. Samples are prepared in H2O + 0.1% formic acid, at a crude concentration of < 5 mg / mL in 2 mL vials or 96-well plates. Visible precipitate is removed via filtration or centrifugation as required. For data collection, 2 pL of each sample of interest is injected on to a Luna Omega 3.0 pm Polar C18 column (50 x 2.1 mm, 100 A, Phenomenex), eluting with a linear gradient of 0 to 98% acetonitrile (ACN) over 6 min, with untargeted MS1 and MS2 positive mode data collected throughout.

[0490] Liquid Chromatography

[0491] Line A is H2O with 0.1 % formic Acid, Line B is ACN with 0.1 % formic acid. Solvent flow rate is 0.45 mL / min and column oven temperature 40 °C. Gradient is 0 % B 0.0 -1 .0 min, 0 - 98 % B 1 .0 - 7.0 min, 98 % B 7.0 - 8.0 min, with 1 .0 min of post-time for equilibration at 0% B.

[0492] Electrospray Ionisation Source Settings

[0493] 67

[0494] 17211595 CXB CXB All data are collected in positive ion mode. Gas temperature = 325 °C, gas flow = 11 (L / min), nebuliser = 50 (psig), sheath gas temperature = 325 °C, sheath gas flow = 12, capillary voltage = 4 kV, fragmentor voltage = 125 V, skimmer = 65, octopole RF = 650.

[0495] MS1 Data Collection

[0496] Data are collected in the range 100 - 1700 m / z with a scan rate of 4 spectra / s. Data storage threshold is set to 1000. The internal reference ions (121.05087 and 922.0098) are permanently infused to maintain mass accuracy throughout data collection.

[0497] MS2 Data Collection

[0498] Data are collected in a data dependent manner using a top 3 method in the range 50 - 1700 m / z with a scan rate of 8 spectra / s. Data storage threshold is set to 50. The PC is enabled for precursor selection with a minimum of height of 10,000 required to trigger fragmentation, precursors are sorted by abundance only. The system calibrant ions (121 .05087 and 922.0098) are excluded from selection with a ppm error of 20. Precursors are isolated with a narrow isolation width (~1 .3 amu). Collision energy for fragmentation is set using a formula of slope 3 with and offset of 10 V. Active exclusion is enabled with exclusion activated after collecting 1 spectrum and exclusion is released again after 0.05 min (3 s).

[0499] UV Data Collection

[0500] UV data are collected at wavelengths of 210, 254, 280 and 300 nm with a bandwidth of 4 nm.

[0501] Results

[0502] Compound (la)

[0503] Figure 4 shows obtained chromatograms. Figure 4 shows the retention time and purity of the isolated compound of formula (la) when Fe-bound. Purity was determined by m / z profile (total ion chromatogram, base peak chromatogram as well as UV profile at 210 and 254 nm). The extracted ion (EIC) chromatogram shows the presence of the targeted ion.

[0504] Figure 5 shows mass spectrum results. The mass profile shows the experimental [M]+and [M+H]2+ions for compound of formula (la), in agreement with predicted values.

[0505] Compound of formula (la) has the following characteristics:

[0506] Table 4. Characteristics of compound of formula (la).

[0507] The mass profile data for the experimental ions for Compound of formula (la) is listed in Table 5. The results show good experimental agreement between the proposed structure and the theoretical masses.

[0508] 68

[0509] 17211595 CXB CXB

[0510] Table 5. Mass profile data for the experimental ions of Compound (la) according to compound of formula (I).

[0511] Compound (lb)

[0512] Figure 8 shows mass spectrum results. The mass profile shows the experimental [M]+and [M+H]2+ions for compound of formula (lb), in agreement with predicted values.

[0513] Compound of formula (lb) has the following characteristics:

[0514] Table 6. Characteristics of compound (lb).

[0515] The mass profile data for the experimental ions for compound (lb) is listed in Table 7. The results show good experimental agreement between the proposed structure and the theoretical masses.

[0516] Table 7. Mass profile data for the experimental ions of compound (lb).

[0517] Nuclear Magnetic Resonance (NMR)

[0518] Overview

[0519] Structural characterisation is carried out by acquisition of one dimensional (1 D) and two dimensional (2D) NMR data and full assignment for elucidation. Chemical shifts are determined by 1 H and 13C (800 MHz and 201 MHz) NMR spectroscopy using a Bruker 800 MHz instrument. Data analysis and peak assignment is performed using MestReNova software.

[0520] Sample Preparation

[0521] Samples are dissolved in 0.560 pL of the deuterated solvent of choice based on solubility testing. If needed, the sample is sonicated for 5 minutes then transferred into a 5 mm NMR tube.

[0522] Preliminary1H data Acquisition

[0523] The NMR tubes containing the samples are placed in the autosampler and a preliminary 1 H data are acquired (ns = 1 , relaxation delay = 1 sec). Spectral width should be minimum from 14 to -2 ppm. Spectra are recorded at room temperature and chemical shifts are referenced to the solvent signal. The1H spectrum is inspected for concentration and purity to determine if quality and concentration are sufficient for further analysis.

[0524] 69

[0525] 17211595 CXB CXB13C data Acquisition

[0526] The minimum spectral width is 230 to -10 ppm, relaxation delay > 2 s. Number of scans is determined based on sample concentration, typically a minimum of 500 up to 4000 scans are required.

[0527] Secondary NMR Data Acquisition

[0528] Homonuclear correlations (1H-1H) are determined based on COSY, NOESY and ROESY experiments, (1H-13C) hetero-correlations are determined based on HMQC and HMBC experiments. A NOAH supersequence (BQCR or BOON) is used with 8 - 16 scans, with the acquisition spectral width the same as for 1 D data.

[0529] Results

[0530] Compound (la)

[0531] Figure 6A shows1H NMR spectrum for compound of formula (la).

[0532] Figure 6B shows13C NMR spectrum for compound of formula (la).

[0533] The NMR Peak assignments is shown in Table 8.

[0534] 70

[0535] 17211595 CXB CXB

[0536]

[0537] 71

[0538] 17211595 CXBCXB

[0539] Table 8. The NMR Peak assignments for compound (la).

[0540] Figure 7 shows a chemical structure of compound of formula (I) after characterization (i.e. structure la in its resonance forms).

[0541] Compound (lb)

[0542] Figure 9A shows1H NMR spectrum for compound of formula (lb).

[0543] Figure 9B shows13C NMR spectrum for compound of formula (lb).

[0544] The NMR Peak assignments is shown in Table 9.

[0545] 72

[0546] 17211595 CXB CXB

[0547]

[0548] Table 9. The NMR Peak assignments for compound (lb).113 values based on HMBC correlation.

[0549] 73

[0550] 17211595 CXB CXB Figure 10 shows a chemical structure of compound (lb), and its resonance forms, after characterization.

[0551] The structure of Compound (lb) differs from Compound (la) by substitution of a terminal amino group with a hydroxyl moiety. This modification is not expected to significantly alter the core bioactivity relative to Compound (la). Without wishing to be bound by theory, it is believed that the principal structural features responsible for iron coordination, and hence siderophore-like activity, include the catechol group of the chromophore and the hydroxamate groups of the N-acyl N-hydroxy ornithine and N-hydroxy cyclo ornithine residues and their spatial arrangement around the chelation centre. However, the replacement may influence other physicochemical properties of the molecule, including solubility, stability, membrane permeability and toxicity, thereby modulating its overall biological performance.

[0552] Example 4 - Production of compound of formula (I) with heterologous expression strain RS- AI49.

[0553] 4.1 - Construct Outline

[0554] P. putida DSM 6125 was transformed with plasmid PL00375 to yield the heterologous expression host. PL00375 harbours several genes which contribute to the successful activation of heterologously expressed BGCs, provide key precursors in compound synthesis, and facilitate extracellular transport of resulting compounds. To enable successful heterologous expression, multiple enzymes were inserted into a parent plasmid containing pBBR1 replication origin and apramycin resistance marker to generate PL00375. These include, a promiscuous phosphopantotheinyl transferase from Bacillus subtilis (sfp gene), enzymes for methyl malonyl coA production (pccB and accA2 from Streptomyces coelicolor A3) and a mutated enterobactin siderophore transporter (FepA L8T from Escherichia coli). No modifications were made to the DSM 6125 genomic background.

[0555] A further plasmid, (+PL00377; SEQ ID NO: 57) was provided with the biosynthetic gene cluster encoding the amino acid sequences as set out in Table 3 above. Figure 12 shows a map of plasmid PL00377. The plasmid contains 92,616 base pairs. The map illustrates the replication of origin DNA sequence (oriV), which serves as the starting point for DNA replication; the trfA gene, which encodes the replication initiator protein and KanR, which acts as an antibiotic resistance marker, along with the biosynthetic gene cluster encoding sequences as set out in Table 3.

[0556] P. putida DSM 6125 (+PL00375) heterologous expression host was further transformed with +PL00377 to yield the heterologous expression strain RS-AI49.

[0557] 4.2 - Heterologous Expression of compound of formula (I)

[0558] Strain RS-AI49 was streaked out onto LB (Lennox) agar plates containing 50 pg / mL Kanamycin and 50 pg / mL Apramycin and incubated in a standing incubator at 30°C overnight. A single colony was

[0559] 74

[0560] 17211595 CXB CXB used to inoculate 10 mL of LB (Lennox) media containing 50 pg / mL Kanamycin and 50 pg / mL Apramycin in a 50 mL Falcon tube, yielding the starter culture. This starter culture was incubated on an inclined rack in a shaking incubator at 30°C at 200 rpm, overnight. The starter culture was then used to inoculate 50 mL of M9 minimal media containing 50 pg / mL Kanamycin, 50 pg / mL Apramycin and 1 mM IPTG (isopropyl-p-D-thiogalactopyranoside) in a 250 mL shake-flask at 2% inoculum - yielding the production culture. This production culture was placed in a shaking incubator at 28°C, shaking at 150 rpm for a total of 14 days, with aliquots taken over several days to assess compound production via LC-MS analysis.

[0561] 4.2.1 - Cloning confirmation

[0562] Figure 13 provides cloning confirmation of a compound of formula (I) using PCR. Three sets of primers were designed; two to bind in regions at both junctions of the gene cluster and adaptor and one to bind in the middle of the gene cluster. Successful cloning was confirmed by presence of bands at the expected size as shown in the figure.

[0563] 4.3 - LC-MS data acquisition and analysis

[0564] LC-MS data was collected using the same method set out in Example 3.

[0565] 4.3.1- Results and identification via LC-MS

[0566] Figure 14 shows mass spectrum results obtained from the heterologous expression strain RS-AI49. The mass profile shows the experimental [M-4H+Fe(lll)+2H]2+ion for compound of formula (la), which is in agreement with the predicted value as shown in Table 5. Therefore, the results show good experimental agreement between the proposed structure and the theoretical masses and confirm the identify of compound (la).

[0567] This figure also provides a comparison with the LC-MS trace observed for a non-engineered control strain. The LC-MS trace for the engineered control strain does not show the presence of the [M- 4H+Fe(lll)+2H]2+ion and therefore it can be concluded that a compound of formula (I) was not obtained from the non-engineered control strain.

[0568] 4.4 - Conclusion

[0569] A compound of formula (I) was produced via heterologous expression in modified Pseudomonas putida (strain DSM 6125).

[0570] Example 5 - Purification of compound of formula (I)

[0571] 75

[0572] 17211595 CXB CXB Compound of formula (I) obtained in any of the previous examples was purified according to the following method.

[0573] 5.1 - Extraction

[0574] Bacterial culture was extracted with methanol (MeOH) as follows. The culture was concentrated to dryness and extracted with MeOH (1 / 3rdof the volume of the culture) with 15 min sonication in a water bath sonicator. This was repeated three times, and the combined extracts were subjected to centrifugation to remove particulates and then concentrated under vacuum, resulting in a dried crude extract.

[0575] 5.2 - Flash Column Chromatography

[0576] The crude extract was dissolved in 30 mL H2O + 0.1% formic acid (FA), and flash column chromatography was carried out with 3 x 10 mL injections,

[0577] The column used for this purpose was a Biotage Star C18 D - Duo 100 A 30 pm 60 g. The solvents were H2O for Line A and acetonitrile for Line B. Solvent flow rate was set at 50 mL / min and the following gradient was used:

[0578] For each run, 75 x 17 mL fractions were collected in 16 x 150 mm test tubes and then pooled as 2 x 17 mL to give 38 fractions. These were concentrated and subject to LCMS analysis. Compound (I) was observed in fraction 17, Further purification of the recovered compound was performed by preparative HPLC.

[0579] 5.3 - Preparative HPLC

[0580] Three runs of preparative HPLC were carried out as set out bellow,

[0581] Run 1

[0582] 1 .5 g of material collected in the previous step was dissolved in 5 mL of H2O + 0.1% FA and preparative HPLC was carried out with 6 x 990 pL injections. The column used for this purpose was a Luna Omega 5 pm Polar C18 100A, 250 x 21 .2 mm. The solvents were H2O+ 0.1% FA for Line A and acetonitrile + 0.1% FA for Line B. Solvent flow rate was set at 20 mL / min and the following gradient was used:

[0583] 76

[0584] 17211595 CXB CXB

[0585] For each run, 47 x 15 mL fractions were collected for 0 - 34 min, and tubes were pooled as 3 x 15 mL to give 17 fractions. These were concentrated and subject to LCMS analysis. Relevant fractions containing compound (I) were combined and dried.

[0586] Run 2

[0587] 700 mg of material collected in the previous run was dissolved in 2 mL of H2O + 0.1% FA and preparative HPLC was carried out with 2 x 1000 pL injections, with the following gradient:

[0588] For each run, 47 fractions were collected for 0 - 20 min, and tubes were pooled to give 20 fractions, which were concentrated and subject to LCMS analysis. Relevant fractions containing compound (I) were combined and dried.

[0589] Run 3

[0590] 187 mg of material collected from the previous run was dissolved in 0.5 mL of H2O + 0.1% FA and preparative HPLC was carried out with a 470 pL injection, with the following gradient:

[0591] 77

[0592] 17211595 CXB CXB

[0593] 48 fractions were collected for 0 - 24 min, and tubes were pooled to give 18 fractions, which were concentrated and subject to LCMS analysis. Relevant fractions containing compound (I) were combined and dried. Further purification of the recovered compound was performed by semipreparative HPLC.

[0594] 5.4 - Semi Preparative HPLC

[0595] 44 mg of material obtained from the previous step was dissolved in 0.2 mL of H2O + 0.1% FA and semi-preparative HPLC was carried out with 2 x 100 pL injection. The column used for this purpose was Luna Omega 5 pm Polar C18 100 A, 250 x 10 mm. The solvents were H2O+ 0.1% FA for Line A and acetonitrile + 0.1% FA for Line B. Solvent flow rate was set at 2 mL / min and the following gradient was used:

[0596] For each run, 96 fractions were collected for 0 - 44 min in a glass-coated 96-well plate. Fractions were concentrated and subject to LCMS analysis.

[0597] 5.5 -Results

[0598] 5.5.1 - Purity

[0599] Purity of compound (la) was confirmed by LCMS and NMR analysis.

[0600] Purity of compound (la) was assessed by analysis of LCMS chromatograms (Figure 4), whereby integration of the peaks observed demonstrated that compound (la) was present at >90% purity as determined by MS and UV (Figure 15). This was further confirmed by NMR analysis, whereby integration of the 1 H proton signals showed that compound (la) was >90% purity.

[0601] 5.6 - Conclusion

[0602] Compound (I) was purified via reverse-phase chromatography to achieve >90% purity.

[0603] 78

[0604] 17211595 CXB CXB Example 7 - Fungal Growth Study of Zymoseptoria tritici in planta

[0605] Preventative in planta activity testing of compound (la) against Zymoseptoria tritici in winter wheat plants were conducted.

[0606] Pots were sown using winter wheat (Triticum aestivum) cultivar JB Diego. Ten seeds were sown into 8 cm diameter pots, with three replicate pots sown per treatment. Once sown, pots were transferred to a controlled environment room and grown at 16°C for approximately 14 days by which point plants had reached BBCH 12 (two leaves unfolded on the main shoot). BBCH is a scale used to identify the phonological development stages of plants. Treatments were applied when wheat plants were at BBCH 12. Five treatments were applied in the screen: compound (la) at 4 different concentrations (300 ppm, 100 ppm, 50 ppm and 10 ppm), along with a positive control (Imtrex, 50 ppm) and a negative control (water). All treatments were applied using a handheld atomiser sprayer with 1 ml of spray solution applied per treatment. Following treatment pots were placed in a fully randomised block design.

[0607] Plants were inoculated one day after treatment with a spore suspension containing 7x106 conidia / ml. The spore suspension was made up in a 0.05% Tween 20 solution. Each replicate was inoculated to the point of run off (approximately 2 ml) using a handheld atomiser sprayer and then incubated at 18°C under conditions of high humidity for 72 hours, after which plants were grown at 18°C until symptoms expressed.

[0608] Disease assessment was performed 22 days after inoculation. Disease severity was assessed on five plants per replicate pot as a visual assessment of the percentage of the total area where pycnidia were present on the first leaf to emerge, following the visual key illustrated in Figure 17. Six examples of wheat leaves are provided with disease severities ranging from 1% to 75% (areas of disease are represented in black). Differences between treatments were determined through calculation of the least significant difference of the means using Genstat version 24, with analysis using ANOVA. Percentage values were transformed using an angular transformation before statistical analysis was carried out.

[0609] Figure 16 shows the effect of treatment with Compound (la) on disease caused by Zymoseptoria tritici on winter wheat plants. The compound showed dose-dependent disease control. When applied to plants at either 300, 100 or 50 pg / mL, Compound (la) produced a significant reduction (p=0.05) in Zymoseptoria tritici disease severity when compared to the water control. Over the dose range tested (10 pg / mL to 300 pg / mL) there was a near-linear increase in disease control as a function of dose between 10 pg / mL and 100 pg / mL, followed by a gradual-but-incomplete plateau in disease control above 100 ppm. Disease control likely reached a maximum at a concentration between 300-600 pg / mL. Under the conditions of this screen there was no observed phytotoxicity towards young (BBCH 12) winter wheat plants following treatment between 10 and 300 pg / mL. Plants were checked for phytotoxicity just prior to inoculation (24 h after treatment), 72 h after treatment and at the final assessment 23 days after treatment.

[0610] 79

[0611] 17211595 CXB CXB Conclusions

[0612] Compound (la) exhibited statistically significant and commercially applicable control of septoria tritici blotch caused by Z. tritici on wheat plants.

[0613] Example 8 - Bioassay Study of pathogens in vitro (agricultural fungal pathogens)

[0614] Compound (la) and standards were screened at 10 doses (130, 65, 32, 16, 8, 4, 2, 1 , 0.5 and 0.25 pg / mL) with each test combination replicated twice.

[0615] Compound (la) was initially prepared at a concentration of 65,000 pg / mL in 100% DMSO. Stock solutions (13,000 pg / mL) were then produced for both compound (la) and standards and from these a 1 :2 serial dilution using 100% DMSO created down to 25 pg / mL. From each serial dilution 2 pL was added to an appropriate well in a 96 well plate; an equivalent amount of DMSO was added to control wells. For each pathogen a spore suspension (containing 1 ,000 spores / mL) was produced in GPM broth and 198 pL of this added to the appropriate wells on the 96 well plate to give the required final well concentrations in 1% DMSO. Plate absorbance was read at 405 nm immediately after plate set up and then again after plates were incubated for 4 days at 18°C. IC50 values for each compound and pathogen were calculated based on a comparison of growth in the control and test wells. The dose-response curve was fit using a four-parameter logistic model 'LL.4' (Ritz, C., Baty, F., Streibig, J.C., and Gerhard, D. (2015). Dose-Response Analysis Using R. PLoS ONE, 10(12), e0146021). The IC50 was determined as the minimum concentration of test compound (i.e. compound of formula (I)) resulting in >50% inhibition when compared to the positive control.

[0616] Table 10 IC50 values, % growth inhibition plateau and rate at which plateau begins, across a range of agricultural fungal and fungal-like pathogens.

[0617] 80

[0618] 17211595 CXB CXB

[0619] Table 11 IC50 values, % growth inhibition plateau and rate at which plateau begins across susceptible and resistant Zymoseptoria tritici strains.

[0620] Table 12. Source information for pathogen samples screened.

[0621] Conclusions

[0622] As shown by the results obtained in Tables 10 and 11 , Compound (la) showed broad inhibition against panel of commercially relevant agricultural pathogens, indicating it has a mode of action that can be applied effectively in a broad number of key agricultural fungicide markets.

[0623] Without wishing to be bound by theory, it is believed that the activity of Compound (la) against Alternaria brassicicola, Botrytis cinerea, Microdochium nivale, Venturia inaequalis, Phytophthora cactorum, Phytophthora infestans and Zymoseptoria tritici may be associated with differences in iron- acquisition capacity and susceptibility to siderophore competition.

[0624] The strong inhibition observed for Compound (la) against Zymoseptoria tritici (IC50 value of 11 .2 pg / mL), Alternaria brassicicola (IC50 value of 8.1 pg / mL) and Microdochium nivale (IC50 value of 11 .9

[0625] 81

[0626] 17211595 CXB CXB pg / mL) suggests high sensitivity to iron limitation and supporting a siderophore-related mode of action. Therefore, it is believed that these fungal pathogens may possess limited or specialized siderophore biosynthetic capacity and reduced iron-uptake redundancy, rendering them more vulnerable to iron sequestration.

[0627] Venturia inaequalis and Phytophthora cactorum exhibit moderate inhibition plateaus (25 % at 33 pg / mL and 39 % at 32 pg / mL, respectively), consistent with partial Fe-starvation effects. Phytophthora infestans shows partial inhibition at lower concentrations (22 % at 4 pg / mL), possibly reflecting lower siderophore affinity but greater capacity for external iron uptake. In contrast, Botrytis cinerea displays only weak inhibition (18 % at 65 pg / mL), consistent with redundant iron-uptake mechanisms and moderate siderophore productivity.

[0628] The consistent IC50 values observed across Zymoseptoria tritici strains, including susceptible, SDHI- resistant and DMI-resistant isolates, indicates that Compound (la) acts via a mechanism distinct from succinate dehydrogenase or demethylation inhibition.

[0629] Figure 18 illustrates response curves for compound (la) and three different positive controls against three agricultural pathogens from an absorbance-based assay. Fig. 18a is a dose response curve for compound (la) against Alternaria brassicicola. Fig 18b is a dose response curve for compound (la) against Microdochium nivale. Fig 18c is a dose response curve for compound (la) against Phytophthora cactorum. Fig. 18d shows a dose response curve for positive control azoxystrobin (azoxy) against Alternaria brassicicola. Fig. 18e shows a dose response curve for positive control fluxapyroxad (flux) against Microdochium nivale. Fig 18f shows a dose response curve for mandipropamid (mandi) against Phytophthora cactorum.

[0630] Figure 19 illustrates dose response curves of compound (I) against three Zymoseptoria tritici strains from an absorbance-based assay: a susceptible strain, a succinate dehydrogenase inhibitor (SDH I)- resistant strain, and a demethylation inhibitor (DMI) -resistant strain. IC50 values are derivable from these results and are detailed in Table 11 . All three dose response curves plateau at approximately 80-85%, and have comparable IC50 values. The three control dose response curves (bottom row) behaved as expected.

[0631] Without being bound by theory, Compound (la) is believed to exert its activity primarily through a siderophore-related iron-starvation pathway and may additionally act via intracellular fungicidal effects following uptake (a “Trojan-horse” mechanism).

[0632] Example 9 - Bioassay Study of pathogens in vitro (human bacterial and fungal pathogens) Antibacterial testing was performed following CLSI guidelines (M07-Ed12) using broth microdilution methods. Compound (I) was prepared at 100x maximum testing concentration. These stocks were serially diluted 1 :1 in the relevant solvent to produce a 100x master plate. Ciprofloxacin or

[0633] 82

[0634] 17211595 CXB CXB meropenem were included as quality control compounds. Positive (growth) and negative (sterility) controls were also included. 100x master stocks were transferred into iron depleted cation-adjusted Mueller-Hinton broth in a flat-bottom 96-well plate to yield test articles at twice their required final test concentration. Each strain to be tested was grown on appropriate agar at 37°C overnight. A bacterial suspension was prepared from each strain equivalent to a 0.5 McFarland suspension. This suspension was diluted in relevant medium to produce a final inoculum level of 2-8 x 105 cfu / mL and added to the test plates. Plates were incubated at 37°C for 16-20 hours. After incubation, growth was assessed on a microplate reader (absorbance 600 nm). The concentration resulting in 50% growth inhibition was calculated from absorbance readings.

[0635] Antifungal susceptibility testing for yeast strains was performed following EUCAST guidelines (E.Def 7.4). C. krusei ATCC 6258 was included as a quality control strain. Amphotericin B was included as a comparator and quality control compound. Positive (growth) and negative (sterility) controls were also included. Compound (I) was prepared at 100x highest test concentration, serially diluted and added to 2x assay medium (Roswell Park Memorial Institute 1640 medium + 2% glucose + 0.165M MOPS) in a flat-bottom 96-well plate to result in test articles being at twice the required test concentration. Cryptococcus neoformans (HA99) was grown on Sabouraud dextrose agar at 35°C for 48 hours. A single colony suspension was prepared in sterile water, equivalent to a 0.5 McFarland standard. This suspension was further diluted in sterile water to produce a final inoculum level of between 1-5 x 105 cfu / mL. Adjusted inoculum was added to the test plates and plates were incubated at 35°C for 48 hours After incubation, a growth was assessed using a microplate reader (absorbance 530 nm). A “discrete IC50” was defined as the lowest tested concentration of Compound (I) that produced >50% inhibition relative to the positive control.

[0636] Table 13 Discrete IC50 values for Compound (la) across a range of human bacterial and fungal pathogens.

[0637] Conclusions

[0638] 83

[0639] 17211595 CXB CXB As shown by the results obtained in Table 13, Compound (la) exhibits broad inhibition against panel of commercially relevant fungal and bacterial human pathogens, indicating it has a mode of action that can be applied effectively in a broad number of key healthcare markets.

[0640] Without wishing to be bound by theory, it is believed that the strong bioactivity of Compound (la) to Cryptococcus neoformans (HA99), Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 43816, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212 may reflect the high siderophore susceptibility of these specific pathogens, which spans both fungal and bacterial pathogens.

[0641] Example 10 - Toxicity Studies

[0642] 10.1 - In vitro cellular toxicity (cytotoxicity)

[0643] HepG2 cells were plated on appropriate tissue culture treated plates. The cells were dosed with test compound for 72 hours at a range of concentrations as set out in Table 14. At the end of the incubation period, the cells were lysed to release ATP. The plates were then scanned using a plate reader.

[0644] Table 14. Summary of experimental parameters for cellular toxicity assay.

[0645] Cellular ATP is a direct readout of cell health and hence compound toxicity. Healthy cells are metabolically active and generate cellular ATP, therefore, a decrease in metabolically active cells is accompanied by a decrease in the level of cellular ATP.

[0646] 10.1.1 - Results

[0647] Table 15. Summary of cellular toxicity for compound (la) and two control compounds: carbonyl cyanide 3-chlorophenylhydrazone and chlorpromazine. MEC is the Minimum effective concentration that significantly crosses vehicle control threshold. AC50 is the concentration at which 50% maximum effect is observed for each cell health parameter. -(J. represents the direction of response: an upward

[0648] 84

[0649] 17211595 CXB CXB response may indicate either cell proliferation, metabolic stimulation or assay artefact; a downward response indicates lower ATP levels due to fewer metabolically active cells, and hence higher activity. NR stands for No response observed. MR stands for Maximum response (ratio of control), defined as the largest observed effect (i.e. change in ATP levels) caused by the compound in the assay relative to control conditions, and indicates the maximum level of toxicity observed at the concentrations tested. R2is a statistical measure of how well the regression model fits the data and is defined as 1 minus (sum of squared residuals (errors) / total sum of squared residuals). High R2values (i.e. above 0.90) indicate that the model explains most of the variability in the data and provides a strong, reliable fit to the observed trend.

[0650] As shown in Figure 20 Compound (la) maintains a ratio of control of approximately 1 across the entire concentration range tested, indicating no change in the number of metabolically active, healthy cells relative to the untreated control. Compound (la) therefore shows no cellular toxicity within the tested range (0-100 pM). In contrast, the positive control compounds carbonyl cyanide 3- chlorophenylhydrazone and chlorpromazine show reduced metabolic activity at higher concentrations, reflected by decreased ratios of control and consistent with known cellular toxicity.

[0651] 10.2 - In vitro mitochondrial toxicity

[0652] HepG2 cells were plated on appropriate tissue culture treated plates at a density of 6000 cells, 25pL per well. The cells were dosed with test compound at a range of concentrations as set out in Table 16 below. 4-6 hrs before dosing, the media was replaced with Dulbecco’s Modified Eagle’s Medium (DMEM) containing either galactose or glucose supplemented with 10% foetal bovine serum and antibiotics. At the end of the incubation period, the cells were loaded with the relevant dye / antibody for each cell health marker. The plates were then scanned using a microplate luminescence reader.

[0653] Table 16. Summary of experimental parameters for mitochondrial toxicity assay.

[0654] 10.2.1 - Determination of mitochondrial toxicity

[0655] Mitochondrial toxicity is determined from the relative sensitivity of cells grown in medium containing either galactose or glucose to the test compound.

[0656] The AC50 was calculated from a curve fit to determine percent survival of compound (la) and two control compounds. The fold change was calculated from AC50 curve fit data by comparison with cells dosed in glucose or galactose. A greater than 2-fold shift between glucose and galactose indicates the compound is considered to be a mitochondrial toxicant.

[0657] 85

[0658] 17211595 CXB CXB 10.2.2 - Results

[0659] Table 17. Summary of mitochondrial toxicity for compound (I) and two control compounds: carbonyl cyanide 3-chlorophenylhydrazone and chlorpromazine. AC50 is the concentration at which 50% maximum effect is observed for each cell health parameter, U indicates the direction of response: an upward response may indicate increased mitochondrial activity via mitochondrial uncoupling (i.e. increased ATP turnover) or a compensatory metabolic response indicative of mild stress; a downward response indicates lower mitochondrial ATP output or impaired mitochondrial function and hence mitochondrial toxicity. NR stands for “No Response” observed. R2is a statistical measure of how well the regression model fits the data and is defined as 1 minus (sum of squared residuals (errors) / total sum of squared residuals). High R2values (i.e. above 0.90) indicate that the model explains most of the variability in the data and provides a strong, reliable fit to the observed trend.

[0660] Figure 21 shows that Compound (la) maintains an average ratio of control of approximately 1 across the tested concentrations in both glucose- and galactose-containing media, indicating no significant perturbation of mitochondrial activity over the range of concentrations of Compound (la) tested. Accordingly, Compound (la) demonstrates no mitochondrial toxicity within the tested range (0-100 pM).

[0661] In contrast, the positive control compounds behaved as expected. Rotenone and chlorpromazine show significant decreases in mitochondrial activity at higher concentrations in the presence of glucose, indicating both are cellular toxicants. Only rotenone demonstrates true mitochondrial toxicity, as evidenced by a statistically significant 231 -fold shift in AC50 in the presence of galactose relative to glucose. Chlorpromazine, which does not show a substantial AC50 shift between glucose and galactose, is therefore not classified as a mitochondrial toxicant in this assay.

[0662] 10.3 - Conclusions

[0663] The horizontal lines shown in figures 19 and 20a), and b), for Compound (la) demonstrate that Compound (la) exhibits no human HepG2 cellular or mitochondrial toxicity up to 100 pM.

[0664] Selectivity index is a key parameter used to determine the therapeutic potential of a compound and is defined as AC50 (toxicity) / IC50 (efficacy). The lack of toxicity combined with the high efficacy of Compound (la) against a number of bacterial and fungal pathogens, as evidenced in Examples 1 , 2, 7 and 8, equates to a high selectivity index and thus good therapeutic potential. For example,

[0665] 86

[0666] 17211595 CXB CXB Compound (la) exhibits a selectivity index of greater than 140x for fungal healthcare pathogen A. fumigatus and of greater than 110x for fungal agricultural pathogen Z. tritici.

[0667] Example 11 - In silico toxicity screen

[0668] The toxicological assessment of compound (la) was performed using in silico Expert System DEREK Nexus, and two QSAR packages (T.E.S.T. and four VEGA models).

[0669] Compound (la) is predicted to be non-mutagenic and have no endocrine activity, overall suggesting a safe toxicology profile.

[0670] 87

[0671] 17211595 CXB CXB

Claims

CLAIMS1. Use of a compound of formula (I):P-C-S (I), wherein:(i) P is a peptide chain comprising:wherein Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, andCH2CH(CH3)CH3;(ii) C is a chromophore comprising any one of:; and(iii) S is a side chain comprising:, whereinR1is OH, H, or NH2, andR2is OH, H or NH2, as an antifungal agent.

2. The use of claim 1 , wherein compound of formula (I) has a structure:8817211595 CXB CXBwherein:Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;R1is OH, H, or NH2, andR2is OH, H or NH2.

3. The use of claim 1 or claim 2, wherein the compound of formula (I) has a structure:wherein:Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;R1is OH, H, or NH2, andR2is OH, H or NH2.8917211595 CXB CXB4. The use of claim 2 or claim 3, wherein R1is OH or NH2, R2is OH and / or Z is CH3.

5. A compound of formula (I):P-C-S (I), wherein:(i) P is a peptide chain comprising:wherein Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, andCH2CH(CH3)CH3;(ii) C is a chromophore comprising any one of:; and(iii) S is a side chain comprising:, whereinR1is OH, H, or NH2, andR2is OH, H or NH2, for use in treating a fungal or bacterial disease or infection in a subject.9017211595 CXB CXB6. The compound of formula (I) for use of claim 5, wherein the compound of formula (I) has a structure:wherein:Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, andCH2CH(CH3)CH3;R1is OH, H, or NH2, andR2 is OH, H or NH2.

7. The compound of formula (I) for use of claim 5 or claim 6, wherein the compound of formula(I) has a structure:wherein:Z is selected from a group comprising H, CH3, CH(CH3)CH3, CH(CH3)CH2CH3, and CH2CH(CH3)CH3;R1is OH, H, or NH2, and9117211595 CXB CXBR2is OH, H or NH2.

8. The compound of formula (I) for use of claim 6 or claim 7, wherein Ri is OH or NH2,2is OH and / or Z is CH3.

9. The compound of formula (I) for use of any one of claims 5-8, wherein the subject is human.

10. The compound of formula (I) for use of any one of claims 5-9, wherein the subject is: a) suffering from a fungal or bacterial disease or infection; and / or b) immunocompromised.11 . The compound of formula (I) for use of any one of claims 5-10, wherein the fungal disease or infection is a disease or infection of subcutaneous tissue, such as porotrichosis, chromoblastomycosis, and eumycetoma, or a disease or infection of a systemic nature, such as histoplasmosis, cryptococcosis, coccidioidomycosis, blastomycosis, mucormycosis, aspergillosis, pneumocystis pneumonia and systemic candidiasis, or a disease or infection of superficial nature, such as candidiasis or malassezia infections.

12. A method for treating a plant to control a disease, the method comprising applying an effective amount of the compound of formula (I) as described in any one of claims 1-6 to the plant, or a part of the plant.

13. An antifungal composition or an antibacterial composition comprising the compound of formula (I) as described in any one of claims 1-6 as an active ingredient and an acceptable carrier.

14. A method for producing the compound of formula (I) as described in any one of claims 1-6 comprising synthesising the compound of formula (I) from a biosynthetic gene cluster encoding the peptides of SEQ ID NOs: 1-54, or peptides of 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NOs: 1-54.

15. A biosynthetic gene cluster encoding at least 10 peptides of any one of SEQ ID NOs: 1-54, or peptides of 80%, 85%, 90%, 95%, 97%, or 99% identity to SEQ ID NOs: 1-54.

16. A compound selected from i) a compound of structure (la):9217211595 CXB CXBor ii) a compound of structure (lb):9317211595CXBCXB

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