Antimicrobial peptides

Details Antimicrobial peptides Antimicrobial peptides

2000-10-13

2003-10-28

Russel, Jeffrey E. (Department: 1654)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Tablets, lozenges, or pills

C514S012200, C514S013800, C514S014800, C530S324000, C530S325000, C530S326000, C530S327000

Reexamination Certificate

active

06638531

ABSTRACT:

The present invention relates to new peptides with an antimicrobial activity. The antimicrobial activity is particularly aimed at bacteria, fungi and yeasts.
The use of the known antibiotics is in an increasing number of cases no longer sufficient for the treatment of infections. Many bacteria strains have developed resistance to the known classes of antibiotic and in the last thirty years no new classes of antibiotic have been discovered. In view of the above, a new class of antimicrobial agents is very desirable. Alkaline peptides and proteins are found in saliva which have a bactericidal and fungicidal activity in vitro. Histatins form a known family of such salivary peptides. However, in order to be clinically applicable as well it is desirable that the antimicrobial activity is even higher. A higher activity compensates the proteolytic degradation of the agent which always occurs to a greater or lesser degree. Furthermore, a reduced proteolytic degradation relative to the naturally occurring peptides is desirable. Finally, from an economic point of view in respect of the production of the peptides, it is recommended that antimicrobial agents are relatively small.
It is the object of the present invention to provide new antibacterial and antifungal agents which do not have the above stated drawbacks and which comply as far as possible with the recommended requirements.
This is achieved with the invention by peptides consisting of an amino acid chain which contains a domain of 10 to 25 amino acids, wherein the majority of the amino acids of the one half of the domain are positively charged amino acids and the majority of the other half of the domain are uncharged amino acids.
The structure of these peptides has a number of variations. Firstly, the domain can form an &agr;-helix, of which at least a majority of the positions 1, 2, 5, 6, 9 (12, 13, 16, 19, 20, 23 and 24) contains a positively charged amino acid, position 8 is a positive or an uncharged amino acid and at least a majority of the positions 3, 4, 7, 10, (11, 14, 15, 17, 18, 21, 22, 25) contains an uncharged amino acid. These peptides have a lateral amphipathicity, i.e. a maximum hydrophobic moment at 100°. Stated simply, these peptides are hydrophobic on the left side and hydrophilic on the right side or vice versa. These peptides are referred to herein as “type I”.
The domain can further form an &agr;-helix, of which at least a majority of the positions 1, 2, 5, 6, 9 (12, 13, 16, 19, 20, 23 and 24) contains an uncharged amino acid, position 8 is a positive or an uncharged amino acid and at least a majority of the positions 3, 4, 7, 10, (11, 14, 15, 17, 18, 21, 22, 25) contains a positively charged amino acid. These peptides have a lateral amphipathicity, i.e. a maximum hydrophobic moment at 100°. Stated simply, these peptides are hydrophobic on the right side and hydrophilic on the left side or vice versa. These peptides are designated “type II” herein and are in principle mirror-symmetrical to type I peptides.
In addition, the domain can form an &agr;-helix, wherein at least a majority of the positions 1 to 6 (or 7 or 8 or 9 or 10 or 11 or 12) contains an uncharged amino acid and a positively charged amino acid is found at position 7 (or 8 or 9 or 10 or 11 or 12 or 13) to 25. These peptides have a longitudinal amphipathicity, i.e. a minimum hydrophobic moment at 100°. These peptides are hydrophobic on their “top” and hydrophilic on their “bottom”. Such peptides are designated “type III”.
Conversely, the domain can form an &agr;-helix, wherein at least a majority of the positions 1 to 6 (or 7 or 8 or 9 or 10 or 11 or 12) contains a positively charged amino acid and an uncharged amino acid is found at position 7 (or 8 or 9 or 10 or 11 or 12 or 13) to 25. These peptides likewise have a longitudinal amphipathicity and therefore a minimum hydrophobic moment at 100°. These peptides are hydrophobic on their “bottom” and hydrophilic on their “top”. Such peptides are designated “type IV”.
Finally, the domain can form a so-called &bgr;-strand and contain a positively charged amino acid on at least a majority of the positions 1, 3, 5, 7, 9 (11, 13, 15, 17, 19, 21, 23 and 25) and an uncharged amino acid on at least a majority of the positions 2, 4, 6, 8, 10, (12, 14, 16, 18, 20, 22, 24). Such a &bgr;-strand is laterally amphipathic and has a maximum hydrophobic moment at 180°. The &bgr;-strand structure is flatter than the &agr;-helix and, stated simply, is hydrophobic on the left and hydrophilic on the right or vice versa. These are “type V” peptides.
The positively charged amino acids are preferably chosen from the group consisting of ornithine (O), lysine (K), arginine (R) and histidine (H), while the uncharged amino acids are preferably chosen from the group consisting of the aliphatic amino acids glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), the amino acids with a dipolar side chain methionine (M), asparagine (N), glutamine (Q), serine (S), threonine (T), the amino acids with an aromatic side chain phenylalahine (F), tyrosine (Y), tryptophan (W). Amino acids on the border between hydrophilic and hydrophobic can be chosen from both groups or from the remaining amino acids.
Hardly any difference in activity can in principle be detected when one of the positive amino acids and/or one of the uncharged amino acids is replaced by a random amino acid. The majority of the positively charged amino acids is therefore preferably the total number of positively charged amino acids minus 1 and the majority of the uncharged amino acids is preferably the total number of uncharged amino acids minus 1.
The domain can be a part of a larger peptide but can itself also make up the entire peptide. When the domain forms part of a larger peptide, the C-terminal and/or N-terminal amino acids which are then additionally present can be random amino acids.
The following peptides of the type I are particularly recommended:
KRLFKELKFSLRKY
(peptide 3)
(SEQ ID NO: 1)
KRLFKELLFSLRKY
(peptide 4)
(SEQ ID NO: 2)
KRLFKELKKSLRKY
(peptide 5)
(SEQ ID NO: 3)
KRLFKELLKSLRKY
(peptide 6)
(SEQ ID NO: 4)
OOLFOELOOSLOOY
(peptide 7)
(SEQ ID NO: 5)
OOLFOELLOSLOOY
(peptide 8)
(SEQ ID NO: 6)
KRLFKKLKFSLRKY
(peptide 9)
(SEQ ID NO: 7)
KRLFKKLLFSLRKY
(peptide 10)
(SEQ ID NO: 8)
A preferred peptide of the type Ill has the following amino acid sequence:
LLLFLLKKRKKRKY (peptide 11) (SEQ ID NO: 9)
The peptides according to the invention can also contain further modifications. These modifications are for instance an N-termninal amide ring, for instance with acetic acid anhydride, or an alternative cleavage of the synthesis resin by which the C-terminus is modified. For this latter a replacement of the C-terminal carboxylic acid group by an amide, ester, ketone, aldehyde or alcohol group can be envisaged. Peptides with such a modification are for instance:
KRLFKELKFSLRKY-amide (peptide 12) (SEQ ID NO: 10)
KRLFKELLFSLRKY-amide (peptide 13) (SEQ ID NO: 11)
In addition to single peptides, oligomers can also be made. These are preferentially linear oligomers of the peptides according to the invention. The coupling can be head-to-head and tail-to-tail as well as head-to-tail, either by direct synthesis or post-synthetic enzymatic coupling. For a trans-membrane pore formation a minimum peptide length is required. Oligomers of the peptides according to the invention are double length and are thereby better able in principle to span the whole phospholipid double layer of the bacterial cell membrane at one time. The activity of the peptide could hereby improve even further. In addition, extension of the peptides provides stabilisation of the helix conformation. A spacer must usually be inserted. In direct synthesis of head-to-tail coupled oligomers a spacer can be inserted to size by the use of a chain of unnatural amino acids of the correct length, for instance &bgr;-alanine, &ggr;-amino butyric acid, &egr;-amino caproic acid, etc. Heterodifuctional coupling reagents, such as are commercially available for coupling peptide antigens to carrier proteins (for instan

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