Antimicrobial cationic peptide derivatives of bactenecin

Details Antimicrobial cationic peptide derivatives of bactenecin Antimicrobial cationic peptide derivatives of bactenecin

1998-05-20

2001-01-09

Borin, Michael (Department: 1631)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

15 to 23 amino acid residues in defined sequence

C530S317000, C530S327000, C514S009100, C514S014800

Reexamination Certificate

active

06172185

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to antimicrobial peptides and specifically to derivatives of the antimicrobial cationic peptide, bactenecin.
2. Description of Related Art
In 1981, the self-promoted uptake hypothesis was first proposed to explain the mechanism of action of polycationic antibiotics in
Pseudomonas aeruginosa
. According to this hypothesis, polycations interact with sites on the outer membranes of Gram-negative bacteria at which divalent cations cross-bridge adjacent lipopolysaccharide molecules. Due to their higher affinity for these sites, polycations displace the divalent cations and, since the polycations are bulkier than the divalent cations, cause structural perturbations in the outer membrane. These perturbations result in increased outer membrane permeability to compounds such as the &bgr;-lactam antibiotic nitrocefin, the eukaryotic non-specific defense protein lysozyme and to hydrophobic substances. By analogy, molecules accessing this pathway are proposed to promote their own uptake.
It has been clearly demonstrated that the outer membranes of Gram-negative bacteria are semipermeable molecular “sieves” which restrict access of antibiotics and host defense molecules to their targets within the bacterial cell. Thus, cations and polycations which access the self-promoted uptake system are, by virtue of their ability to interact with and break down the outer membrane permeability barrier, capable of increasing the susceptibility of Gram-negative pathogenic bacteria to antibiotics and host defense molecules. Hancock and Wong demonstrated that a broad range of such compounds could overcome the permeability barrier and coined the name “permeabilizers” to describe them (Hancock and Wong,
Antimicrob. Agents Chemother
., 26:48, 1984). While self-promoted uptake and permeabilizers were first described for
P. aeruginosa
, they have now been described for a variety of Gram-negative bacteria.
Over the past decade, non-specific defense molecules have been described in many animals, including insects and humans. One subset of these molecules have in common the following features: (a) they are small peptides, usually 15-35 amino acids in length, (b) they contain 4 or more positively charged amino acid residues, either lysines or arginines, and (c) they are found in high abundance in the organisms from which they derive. Several of these molecules have been isolated, amino acid sequenced and described in the patent literature (e.g., cecropins: WO 8900199, WO 8805826, WO 8604356, WO 8805826; defensins: EP 193351, EP 85250, EP 162161, U.S. Pat. No. 4,659,692, WO 8911291). However, only limited amounts of these peptides can be isolated from the host species. For example, Sawyer, et al., (
Infect. Immun
. 56:693, 1988) isolated 100-200 mg of rabbit neutrophil defensins 1 and 2 from 10
9
primed peritoneal neutrophils or lipopolysaccharide-elicited alveolar macrophages (i.e., the numbers present in a whole animal).
The gene for human defensin has been cloned and sequenced, but no successful expression has been demonstrated, as yet. Furthermore, production of these peptides using peptide synthesis technology produces peptides in limited amounts and is expensive when scaled up or when many variant peptides must be produced. Also, structural analysis is difficult without specific incorporation of
15
N and
13
C tagged amino acids which is prohibitively expensive using amino acid synthesis technology.
Recently, cationic peptides containing a disulphide bond forming a looped structure were identified (Morikawa et al.,
Biochim. Biophys. Res. Commun
. 189:184, 1992; Simmaco et al.,
FEBS
324:159, 1993; Clark et al.,
J. Biol. Chem
. 269:10849, 1994). One member of this group, bactenecin (i.e., dodecapeptide), is a twelve amino acid peptide isolated from bovine neutrophils (Romeo et al.,
J Biol. Chem
. 263:9573, 1988). Bactenecin is the smallest known cationic antimicrobial peptide. Two cysteine residues form a disulphide bond to make bactenecin a loop molecule. Bactenecin was previously found to be active against
Escherischia coli
and
Staphylococcus aureus
, and strongly cytotoxic for rat embryonic neurons, fetal rat astrocytes and human glioblastoma cells (Radermacher et al.,
J. Neuroscience Res
. 36:657, 1993).
There is a need to develop peptides having a broad range of potent antimicrobial activity against a plurality of microorganisms, including gram negative bacteria, gram positive bacteria, fungi, protozoa, viruses and the like. To that end, the small size and structure of bactenecin present an opportunity to identify derivatives of the polypeptide which are effective as therapeutics for microbial pathogens.
SUMMARY OF THE INVENTION
The present invention provides derivatives of the antimicrobial cationic peptide bactenecin, that have antimicrobial activity. In a first embodiment, the invention provides antimicrobial peptides derived from bactenecin. Exemplary peptides include: RLCRIVVIRVCR (SEQ ID NO:1); RLARIVVIRVAR NH
2
(SEQ ID NO:2); RLSRIVVIRVCR NH
2
(SEQ ID NO:3); RLSRIVVRVSR NH
2
(SEQ ID NO:4); RRCPIVVIRVCR NH
2
(SEQ ID NO:5); RICRIVVIRCIR (SEQ ID NO:6); RLCPRVRIRVCR NH
2
(SEQ ID NO:7); KKCPIVVIRVCK (SEQ ID NO:8); RRRCPIVVIRVCRR (SEQ ID NO:9); RRRLCPIVIRVCRR (SEQ ID NO:10); RRLCRIVVIRVCRR (SEQ ID NO:11); RLCRIVPVIRVCR (SEQ ID NO:12); RLCRIVWVIRVCR (SEQ ID NO:13); RRLCRIVWVIRVCRR (SEQ ID NO:14); RRCPIVWVIRVCR NH
2
(SEQ ID NO:15); RRCPIVWVIPVCRR NH
2
(SEQ ID NO:16); RLCRIVVIRVCR NH
2
(SEQ ID NO:17); RLCRIVVIRVCRIVIVIV (SEQ ID NO:18); RLSRIVVIRVSR (SEQ ID NO:19); and RRCPIVVIRVCR (SEQ ID NO: 20), and analogs, derivatives, amidated variations and conservative variations thereof.
The invention also provides a method of inhibiting the growth of bacteria including contacting the bacteria with an inhibiting effective amount of at least one peptide of the invention alone, or in combination with at least antibiotic. Classes of antibiotics that can be used in synergistic therapy with the peptides of the invention include aminoglycoside, penicillin, cephalosporine, fluoroquinolone, carbepenem, tetracycline and macrolide.
The invention further provides polynucleotides that encode the peptides of the invention. Exemplary polynucleotides encode peptides including: RLCRIVVIRVCR (SEQ ID NO:1); RLARIVVIRVAR NH
2
(SEQ ID NO:2); RLSRIVVIRVCR NH
2
(SEQ ID NO:3); RLSRIVVIRVSR NH
2
(SEQ ID NO:4); RRCPIVVIRVCR NH
2
(SEQ ID NO:5); RICRIVVIRCIR NH2 (SEQ ID NO:6); RLCPRVRIRVCR NH
2
(SEQ ID NO:7); KKCPIVVIRVCK (SEQ ID NO:8); RRRCPIVVIRVCRR (SEQ ID NO:9); RRRLCPIVIRVCRR (SEQ ID NO:10); RRLCRIVVIRVCRR (SEQ ID NO:11); RLCRIVPVIRVCR (SEQ ID NO:12); RLCRIVWVIRVCR (SEQ ID NO:13); RRLCRIVWVIRVCRR (SEQ ID NO:14); RRCPIVWVIRVCR NH
2
(SEQ ID NO:15); RRCPIVWVIPVCRR NH
2
(SEQ ID NO:16); RLCRIVVIRVCR NH
2
(SEQ ID NO:17); RLCRIVVIRVCRIVIVIV (SEQ ID NO:18); RLSRIVVIRVSR (SEQ ID NO:19); and RRCPIVVIRVCR (SEQ ID NO: 20), and analogs, derivatives and conservative variations thereof.
In another embodiment, the invention provides a method of inhibiting an endotoxemia or sepsis associated disorder in a subject having or at risk of having such a disorder, by administering to the subject a therapeutically effective amount of at least one peptide of the invention.
The invention also provides a method of inhibiting the growth of a eukaryotic cell. The method includes contacting the cell with an inhibiting effective amount of a peptide or combination thereof of the invention, alone, or in combination with an agent effective for inhibiting eukaryotic cell growth. Classes of such agents which can be used for synergistic therapy with the peptides of the invention include bleomycin, neocarcinostatin, suramin, doxorubicin, taxol, mitomycin C and cisplatin.
The invention further provides a method of inhibiting a cell proliferation-associated disorder in a subject having or at risk of having such a disorder. The method includes administering to the subject a therapeutically effective amount of at least one peptide of the invention, alone, or in comb

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