ANTIBIOTIC PEPTIDES

• US 20120021975A1
• Filed: 01/29/2010
• Published: 01/26/2012
• Est. Priority Date: 01/29/2009
• Status: Active Grant

• Current Assignee: AMP-Therapeutics GmbH (EnBiotix, Inc.)
• Original Assignee: AMP-Therapeutics GmbH & Co. KG
• Inventors: Hoffmann, Ralf, Knappe, Daniel, Hansen, Anna Klara Brigitte
• Granted Patent: US 8,686,113 B2
• Time in Patent Office: Days
• US Class Current: 514/2.4
• CPC Class Codes: A01N 47/44 Guanidine; Derivatives thereof
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/10 Peptides having 12 to 20 amino acids A61K38/043 - A61K38/046 take precedence
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: C07K 14/43563 from insects
• CPC Class Codes: C07K 7/08 having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to a peptide or peptide derivative having the general formula: Sub₁-X₁-D₂K₃-P₄-P₅-Y₆-L₇-P₈-R₉-P₁₀-X₂-P₁₂-P₁₃-R₁₄-X₃-I₁₆-P₁₇/Y₁₇-N₁₈-N₁₉-X₄-Sub₂, wherein X₁ is a non-polar, hydrophobic group or a positively charged group, D₂ is asparagine or glutamine, K₃, X₂, and X₄ are positively charged groups, X₃ is a positively charged group, proline, or a proline derivative; L₇ and I₁₆ are non-polar, hydrophobic groups, Y₆ and Y₁₇ are tyrosine, R₉ and R₁₄ are arginine, N₁₈ and N₁₉ are asparagine or glutamine, P₄, P₅, P₈, P₁₀, P₁₂, P₁₃, and P₁₇ are proline, hydroxyproline, or derivatives thereof, wherein possibly one or two of the groups selected from D₂, P₄, P₅, P₈, P₁₀, P₁₂, P₁₃, P₁₇, and Y₁₇ are replaced by an arbitrary group and/or P₁₃ and R₁₄ are exchanged, Sub₁ is the free or modified N-terminus, and Sub₂ is the free or modified C-terminus. The invention further relates to the use of the peptides and peptide derivatives in medicine, as an antibiotic, in a disinfectant or cleaning agent, as a preservative or in a packaging material, in pharmaceutical research, or in a screening method.

Claims

1. A peptide or peptide derivative, which contains one of the following sequences:

Sub₁-X₁-D₂-K₃-P₄-P₅-Y₆-L₇-P₈-R₉-P₁₀-X₂-P₁₂-P₁₃-R₁₄-X₃-I₁₆-P₁₇/Y₁₇-N₁₈-N₁₉-X₄-Sub₂

X₁ is a residue with a nonpolar, hydrophobic side chain or with a positive net charge or a side chain that is positively charged under physiological conditions;

D₂ is an aspartic acid or glutamic acid residue,

K₃ is a residue with a positive net charge or a side chain that is positively charged under physiological conditions, preferably lysine or arginine,

X₂ and X₄ are selected independently of one another from residues with a positive net charge or a side chain that is positively charged under physiological conditions;

X₃ is a residue with a positive net charge or a side chain that is positively charged under physiological conditions or proline or a proline derivative;

L₇ is selected from residues with a nonpolar, hydrophobic side chain, preferably leucine, isoleucine and value,

I₁₆ is selected from residues with a nonpolar, hydrophobic side chain, preferably leucine, isoleucine, tert-butylglycine and valine,

Y₆ and Y₁₇ are in each case tyrosine, R₉ and R₁₄ are in each case arginine, N₁₈ is asparagine or glutamine, N₁₉ is asparagine or glutamine or is absent, P₄, P₅, P₈, P₁₀, P₁₂, P₁₃ and P₁₇ are selected independently of one another from proline and proline derivatives or hydroxyproline and hydroxyproline derivatives,

Sub₁ is the free N-terminus of the amino acid X₁ or a modified N-terminal amino group;

Sub₂ is the free C-terminal carboxyl group of the C-terminal amino acid (—

COOH) or a modified C-terminal carboxyl group.

2. The peptide or peptide derivative of claim 1, wherein P₁₃ and R₁₄ are exchanged, and/or one or two of the residues selected from D₂, P₄, P₅, P₈, P₁₀, P₁₂, P₁₃, P₁₇ and Y₁₇ are replaced with any residue.

3. The peptide or peptide derivative of claim 1, wherein the N-terminus is linked to another peptide directly or via a linker.

4. The peptide or peptide derivative of claim 1 containing at least one additional residue X₅ and/or X₆ in Sub₂, wherein X₅ is selected from the group comprising proline, a proline derivative and a building block, which has a positive net charge or bears a side chain that is positively charged under physiological conditions and in that X₆ is selected from proline, a proline derivative, a polar building block or a hydrophobic building block.

5. The peptide or peptide derivative of claim 1, wherein the residue X₁ is selected from arginine, lysine, δ

-hydroxylysine, homoarginine, 2,4-diaminobutyric acid, β

-homoarginine, D-arginine, arginal, 2-amino-3-guanidinopropionic acid, nitroarginine, N-methylarginine, ε

-N-methyllysine, allot hydroxylysine, 2,3-diaminopropionic acid, 2,2′

-diaminopimelic acid, ornithine, dimethylarginine, asym-dimethylarginine, 2,6-diaminohexinic acid, p-aminobenzoic acid, 3-aminotyrosine, valine, isoleucine, leucine and methionine, alanine, phenylalanine, N-methylleucine, tert-butylglycine, cyclohexylalanine, β

-alanine, 1-aminocyclohexylcarboxylic acid, N-methylisoleucine, norleucine, norvaline and N-methylvaline.

6. The peptide or peptide derivative of claim 1, wherein the residue X₂ and/or the residue X₄ are selected independently of one another from arginine, lysine, δ

-hydroxylysine, homoarginine, β

-homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl-arginine, ε

-N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2′

-diaminopimelic acid, ornithine, sym-dimethylarginine, asym dimethylarginine, 2,6-diaminohexinic acid, p-aminobenzoic acid and 3-aminotyrosine.

7. The peptide or peptide derivative of claim 1, wherein the residue X₃ is selected from arginine, lysine, δ

-hydroxylysine, homoarginine, β

-homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid, β

-homoarginine, 2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine, N-methyl-arginine, ε

-N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2′

-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexinic acid, p-aminobenzoic acid, 3-aminotyrosine, proline, cis-4-hydroxyproline, trans-4-hydroxyproline, cis-3-hydroxyproline, trans-3-hydroxyproline, β

-cyclohexylalanine, 3,4-cis-methanoproline, 3,4-dehydroproline, homoproline and pseudoproline.

8. The peptide or peptide derivative of claim 1, selected from the group of sequences comprising SEQ ID NO. 5 to 9, 14 to 26, 29, 30, 32, 33, 36, 38, 40, 41, 44 to 46, 49, 50, 53 to 59, 61 to 85, 93, 94, 101, 102 and 107 to 112.

13. The peptide or peptide derivative of claim 1 selected from the group comprising SEQ ID NO. 95 to 102 and 106.

14. The peptide or peptide derivative of claim 1, wherein at least one of the peptide bonds of the peptide backbone is chemically modified.

15. The peptide or peptide derivative of claim 14, characterized in that the bond between X₆-X₇ is a chemical bond that cannot be cleaved under physiological conditions.

16. The peptide or peptide derivative of claim 14, wherein the chemically modified bond is selected from a reduced amide bond, an alkylated amide bond or a thioamide bond.

17. The peptide or peptide derivative of claim 1, which is joined to a protein or coupled to a polymer or bound to a carrier.

18. A multimer, in which at least two peptides or peptide derivatives are joined together, characterized in that at least one of the peptides or peptide derivatives is a peptide or peptide derivative of claim 1.

19. A pharmaceutical composition, characterized in that it contains at least one peptide, peptide derivative or multimer of claim 1.

20. A method of production of a peptide or peptide derivative or multimer of claim 1, characterized in that the peptide or peptide derivative or multimer is produced by chemical synthesis or by means of recombinant methods.

21. The use of as peptide, peptide derivative or multimer of claim 1 for the production of a medicinal product.

22. The use of a peptide, peptide derivative or multimer of claim 1 as a disinfectant and/or cleaning agent, as a preservative and/or in a packaging material.

23. The peptide, peptide derivative and/or multimer of claim 1 for use in the treatment of microbial, bacterial or fungal infections.

24. The use of a peptide, peptide derivative and/or multimer of claim 1 for removing contamination by microbes, bacteria or fungi.

25. The use of a peptide, peptide derivative or multimer of claim 1 in pharmaceutical research or in a screening process.

26. A method for identifying a substance with antibacterial or antimycotic action, comprising the following steps:

(i) carrying out a competitive assay with;

(a) a microorganism that is sensitive to a peptide, peptide derivative or multimer of claim 1;

(b) a peptide, peptide derivative or multimer as claimed in claim 1; and

(c) at least one substance to be tested by bringing (a) in contact with (b) and (c); and

(ii) selecting a test substance, which competitively displaces the binding of the peptide, peptide derivative or multimer on the microorganisms.

27. The method of claim 26, wherein the microorganism is a species selected from the group of genera comprising Escherichia coli, Enterobacter cloacae, Erwinia amylovora, Klebsiella pneumoniae, Morganella morganii, Salmonella typhimurium, Salmonella typhi, Shigeo dysenteriae, Yersinia enterocolitica, Acinetobacter calcoaceticus, Acinetobacter baumannii, Agrobacterium tumefaciens, Francisella tularensis, Legionella pneumophila, Pseudomonas syringae, Rhizobium meliloti, Pseudomonas aeruginosa, Proteus vulgaris, Proteus mirabilis, Stenotrophomonas maltophilia and Haemophilus influenzae.

28. A nucleic acid coding for a peptide or multimer of claim 1.

29. A host cell, comprising one or more nucleic acids claim 28.

9. A peptide or peptide derivative containing an antibacterial peptide and a cell-penetrating peptide.

10. The peptide or peptide derivative of claim 9, wherein said antibacterial peptide is selected from the group comprising apidaecin, drosocin, formaecin pyrrhocoricin and metalnikowin 1.

11. The peptide or peptide derivative of claim 9, wherein said antibacterial peptide comprises a peptide or peptide derivative, which contains one of the following sequences:

Sub₁-X₁-D₂-K₃-P₄-P₅-Y₆-L₇-P₈-R₉-P₁₀-X₂-P₁₂-P₁₃-R₁₄-X₃-I₁₆-P₁₇/Y₁₇-N₁₈-N₁₉-X₄-Sub₂

X₁ is a residue with a nonpolar, hydrophobic side chain or with a positive net charge or a side chain that is positively charged under physiological conditions;

D₂ is an aspartic acid or glutamic acid residue,

K₃ is a residue with a positive net charge or a side chain that is positively charged under physiological conditions, preferably lysine or arginine,

X₂ and X₄ are selected independently of one another from residues with a positive net charge or a side chain that is positively charged under physiological conditions;

X₃ is a residue with a positive net charge or a side chain that is positively charged under physiological conditions or proline or a proline derivative;

L₇ is selected from residues with a nonpolar, hydrophobic side chain, preferably leucine, isoleucine and valine,

I₁₆ is selected from residues with a nonpolar, hydrophobic side chain, preferably leucine, isoleucine, tert-butylglycine and valine,

Y₆ and Y₁₇ are in each case tyrosine, R₉ and R₁₄ are in each case arginine, N₁₈ is asparagine or glutamine, N₁₉ is asparagine or glutamine or is absent, P₄, P₅, P₈, P₁₀, P₁₂, P₁₃ and P₁₇ are selected independently of one another from proline and proline derivatives or hydroxyproline and hydroxyproline derivatives,

Sub₁ is the free N-terminus of the amino acid X₁ or a modified N-terminal amino group;

Sub₂ is the free C-terminal carboxyl group of the C-terminal amino acid (—

COOH) or a modified C-terminal carboxyl group.

12. The peptide or peptide derivative of claim 9, wherein said cell-penetrating peptide is selected from the group comprising penetratin, Tat peptides, model amphipathic peptides, transportan, SynB and cis-γ

-amino-1-proline-containing peptides.