Antibiotic Peptides

Abstract

This invention concerns novel antibiotic peptide and peptide derivates, especially for use in medicine. Further, the invention relates to compositions and methods for killing microbes, like bacteria or fungus, and methods to treat microbial infections. The invention further relates to a method for drug screening analysis. The peptides and peptide derivates have the general formula Sub 1 -X 1 N X 2 X 3 P V Y I P X 4 X 5 R P P H P-Sub 2 wherein X 1 is a neutral or positively charged moiety, X 2 is a polar or positively charged moiety, X 3 is a positively charged moiety, X 4 is a polar or positively charged moiety, X 5 is a proline or a proline derivate, Sub 1 being the free or modified N-terminus, and Sub 2 being the free or modified C-terminus. The peptides or peptide derivates according to the invention possess at least one of the following advantages compared to the natural occurring apidaecin peptides: (i) an increased half-live in mammalian serum due to a higher protease resistance and (ii) an increased antimicrobial activity against one or several bacterial strains, especially human pathogens, or fungus or other microbial infections (iii) show an enlarged spectrum of antimicrobial activity, (iv) do induce less resistance in microbes and (v) are not toxic to human cells including erythrocytes.

1 . Peptide or peptide derivate with at least 16 residues and the general formula
   Sub 1 -X 1  N X 2  X 3  P V Y I P X 4  X 5  R P P H P-Sub 2      wherein   X 1  is a neutral residue or a moiety having a net positive charge or a positively charged side chain under physiological conditions;   X 2  is a residue with a polar side chain or a moiety having a net positive charge or a positively charged side chain under physiological conditions;   X 3  is a moiety having a net positive charge or a positively charged side chain under physiological conditions;   X 4  is a neutral residue with a polar side chain, preferably not glutamine, or a moiety having a net positive charge or a positively charged side chain under physiological conditions;   X 5  is proline or a proline derivate;   Sub 1  being the free N-terminal amino group of the amino acid X 1  or a modification of the N-terminal amino group;   Sub 2  being the free C-terminal carboxyl group of the c-terminal amino acid or a modification of the C-terminal carboxyl group;   the native sequences according to SEQ ID No. 1 (GNNRPVYIPQPRPPHPRI-OH) and SEQ ID No. 2 (GNNRPVYIPQPRPPHPRL-OH) being excluded.   
     
     
         2 . Peptide or peptide derivate according to  claim 1 , incorporation at least one additional residue X 6  and/or X 7  in Sub 2 , whereas X 6  is selected from proline or a proline derivate or a moiety having a net positive charge or a positively charged side chain under physiological conditions and X 7  is selected from proline or proline derivates, a polar moiety or a hydrophobic moiety. 
     
     
         3 . Peptide or peptide derivate according to  claim 1  or  2 , whereas the residue X 1  is selected from the groups including citrulline, N-methylserine, N-methylglycine, dihydroxyphenylalanine, N-ethylasparagine, N-ethylglycine, homoserine, penicillamine, tetrahydropyranylglycine, allo-threonine, 3,5-dinitrotyrosine, N-methylleucine, N-methylisoleucine, tert.-butylglycine, β-alanine, norleucine, norvaline, N-methylvaline, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, aminovaleric acid, 2-aminopimelic acid, pipecolonic acid, iodo-tyrosine, 3,5-diiodo-tyrosine, 3,5-dibromo-tyrosine, β-cyclohexylalanine, p-aminobenzoic acid, ε-aminocaproic acid, 3,4-cis-methano-proline, phenylglycine, 3,4-dehydroproline, 4-amino-5-cyclohexyl-3-hydroxypentanoic acid, O-phosphotyrosine, O-sulfotyrosine, aminoethylpyrrolecarboxylic acid, 4-aminopiperidine-4-carboxylic acid, α-aminoadipic acid, homoproline, homophenylalanine, p-fluoro-phenylalanine, 3,4-dichlorophenylalanine, p-bromo-phenylalanine, p-iodo-phenylalanine, and p-nitro-phenylalanine as well as arginine, lysine, δ-hydroxylysine, homoarginine, D-arginine, methylarginine, nitroarginine, nitrosoarginine, arginal, guanidino propionic acid, 2,4-diaminobutyric acid, β-homoarginine, ε-N-methyllysine, allo-hydroxylysine, 2,3-diaminipropionic acid, 2,2′-diaminopimelic acid, ornithine, sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexynoic acid, histidine, 1-methyl-histidine, 3-methyl-histidine, 3-amino-tyrosine. 
     
     
         4 . Peptide or peptide derivate according to one of the  claims 1  to  3 , whereas the residue X 2  is selected from the groups including serine, threonine, homoserine, allo-threonine, and citrulline, as well as arginine, lysine, δ-hydroxylysine, homoarginine, D-arginine, methylarginine, nitrosoarginine, nitroarginine, arginal, guanidino propionic acid, 2,4-diaminobutyric acid, β-homoarginine, ε-N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2′-diaminopimelic acid, ornithine, sym-dimethylargininc, asym-dimethylarginine, 2,6-diaminohexynoic acid. 
     
     
         5 . Peptide or peptide derivate according to one of the  claims 1  to  4 , whereas the residue X 3  is selected from the group including arginine, lysine, δ-hydroxylysine, homoarginine, 2,4-diaminobutyric acid, β-homoarginine, ε-N-methyllysine, allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2′-diaminopimelic acid, lysine, arginine, ornithine, methylarginine, sym-dimethylarginine, asym-dimethylarginine, nitroarginine, nitrosoarginine, arginal, guanidino propionic acid, 2,6-diaminohexynoic acid, histidine, 1-methyl-histidine, 3-methyl-histidine, and 3-amino-tyrosine. 
     
     
         6 . Peptide or peptide derivate according to one of the  claims 1  to  5 , whereas the residue X 4  is selected from the groups including asparagines, N-methylserine, N-methylglycine, dihydroxyphenylalanine, N-ethylasparagine, N-ethylglycine, homoserine, penicillamine, tetrahydropyranylglycine, allo-threonine, 3,5-dinitrotyrosine, and citrulline, as well as arginine, lysine, δ-hydroxylysine, homoarginine, 2,4-diaminobutyric acid, β-homoarginine, ε-N-methyllysine, allo-hydroxylysine, 2,3-diaminipropionic acid, 2,2′-diaminopimelic acid, ornithine, methylarginine, sym-dimethylarginine, asym-dimethylarginine, nitroarginine, nitrosoarginine, arginal, guanidino propionic acid, 2,6-diaminohexynoic acid, histidine, 1-methyl-histidine, 3-methyl-histidine, and 3-amino-tyrosine. 
     
     
         7 . Peptide or peptide derivate according to one of the  claims 1  to  6 , whereas the residue X 5  is proline or a proline derivate, selected from 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 . Peptide or peptide derivate according to one of the  claims 2  to  7 , whereas the residue X 6  is selected from the groups including proline, cis-4-hydroxyproline, trans-4-hydroxyproline, cis-3-hydroxyproline, trans-3-hydroxyproline, β-cyclohexylalanine, 3,4-cis-methano-proline, 3,4-dehydroproline, homoproline or pseudoprolines, as well as arginine, preferably D-arginine, δ-hydroxylysine, homoarginine, 2,4-diaminobutyric acid, β-homoarginine, ε-N-methyllysine, allo-hydroxylysine, 2,3-diaminipropionic acid, 2,2′-diaminopimelic acid, lysine, ornithine, methylarginine, sym-dimethylarginine, asym-dimethylarginine, nitroarginine, nitrosoarginine, arginal, guanidino propionic acid, 2,6-diaminohexynoic acid, histidine, 1-methyl-histidine, 3-methyl-histidine, 3-amino-tyrosine. 
     
     
         9 . Peptide or peptide derivate according to one of the  claims 2  to  8 , whereas the residue X 7  is selected from the groups including proline, cis-4-hydroxyproline, trans-4-hydroxyproline, cis-3-hydroxyproline, trans-3-hydroxyproline, β-cyclohexylalanine, 3,4-cis-methanoproline, 3,4-dehydroproline, homoproline, pseudoprolines, serine, threonine, citrulline, N-methylserine, N-methylglycine, dihydroxyphenylalanine, N-ethylasparagine, N-ethylglycine, homoserine, penicillamine, tetrahydropyranylglycine, allo-threonine, 3,5-dinitrotyrosine, δ-hydroxylysine, as well as phenylalanine, leucine, isoleucine, valine, methionine, alanine, 1-amino-cylcohexyl carbonic acid, N-methylleucine, N-methylisoleucine, tert.-butylglycine, β-alanine, norleucine, norvaline, N-methylvaline, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, aminovaleric acid, 2-aminopimelic acid, pipccolonic acid, tryptophan, iodo-tyrosine, 3,5-diiodo-tyrosine, 3,5-dibromo-tyrosine, β-cyclohexylalanine, p-aminobenzoic acid, ε-aminocaproic acid, 3,4-cis-methanoproline, phenylglycine, 3,4-dehydroproline, 4-amino-5-cyclohexyl-3-hydroxypentanoic acid, O-phosphotyrosine, O-sulfotyrosine, aminoethylpyrrolecarboxylic acid, 4-aminopiperidine-4-carboxylic acid, α-aminoadipic acid, homoproline, homophenylalanine, p-fluoro-phenylalanine, 3,4-dichlorophenylalanine, p-bromo-phenylalanine, p-iodo-phenylalanine, p-nitro-phenylalanine or a short peptide sequence or a branched linker containing several peptide units. 
     
     
         10 . Peptide or peptide derivate according to one of the  claims 2  to  9 , selected from the sequences according to SEQ ID No. 3 to 160. 
     
     
         11 . Peptide or peptide derivate according to one of the  claims 2  to  10 , wherein at least one of the peptide bonds of the peptide backbone is replaced by a non-cleavable bond. 
     
     
         12 . Peptide or peptide derivate according to  claim 11  wherein the bond between X 6 -X 7  is a non-cleavable bond. 
     
     
         13 . Peptide or peptide derivate according to  claim 11  or  12 , wherein the non-cleavable bond is selected from the group consisting of a reduced amide bond, an alkylated amide bond, or a thioamide bond. 
     
     
         14 . Peptide or peptide derivate according to one of the  claims 1  to  13  obtained by chemical synthesis or by recombinant techniques. 
     
     
         15 . Peptide or peptide derivate according to one of the  claims 1  to  14 , being fused to a protein or coupled to a polymer. 
     
     
         16 . The peptide according to one of the  claims 1  to  15 , wherein said peptide is attached to a carrier. 
     
     
         17 . Multimer wherein at least two peptides or peptide derivates are coupled together, whereas at least one of the peptides or peptide derivates being a peptide or peptide derivate according to one of the  claims 1  to  16 . 
     
     
         18 . A pharmaceutical composition comprising at least one of the peptides or peptide derivates or a multimer according to one of the  claims 1  to  17 . 
     
     
         19 . A method of treating a microbial, bacterial or fungal infection in a mammal by administering to a mammal having said infection an amount of a peptide of  claims 1  to  17  or pharmaceutical composition according  claim 18 . 
     
     
         20 . Use of a peptide or peptide derivate or a multimer according to one of the  claims 1  to  17  as antibiotic, in a disinfecting or cleaning agent, as preserving agent or in a packaging material. 
     
     
         21 . Use according to  claim 20  to treat microbial, bacterial or fungal infections or contaminations. 
     
     
         22 . Use of a peptide or peptide derivate or a multimer according to one of the  claims 1  to  17  in biotechnical or pharmaceutical research or in a screening method. 
     
     
         23 . A method for identifying a compound, which has a potential antimicrobial, bactericidal or antifungal effect, comprising:
 (i) performing a competitive assay with:
 (a) a microorganism susceptible to a peptide or peptide derivate or multimer according to one of the  claims 1  to  17 ; 
 (b) a peptide or peptide derivate or multimer according to one of the  claims 1  to  17 ; and 
 (c) at least one compound to be tested; 
 by exposing (a) to (b) and (c); and 
   (ii) selecting a test compound which competitively displaces the binding of the peptide or peptide derivate or multimer to the microorganism.   
     
     
         24 . Method according to  claim 23 , wherein the selected compound is further screened for anti-bacterial or antifungal use. 
     
     
         25 . Method according to  claim 23  or  24 , wherein the microorganism is a species belonging to one of the genera selected from  Escherichia coli, Enterobacter cloacae, Erwinia amylovora, Klebsiella pneumoniae, Morganella morganii, Salmonella typhimurium, Salmonella typhi, Shigella dysenteriae, Yersinia enterocolitica, Acinetobacter calcoaceticus, Agrobacterium tumefaciens, Francisella tularensis, Legionella pneumophila, Pseudomonas syringae, Rhizobium meliloti  and  Haemophilus influenzae.    
     
     
         26 . Nucleic acid encoding a peptide or a multimer according to one of the  claims 1  to  17 . 
     
     
         27 . Host cell transfected or transformed with a nucleic acid according to  claim 26 .