Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1996-08-23
2001-02-20
Housel, James C. (Department: 1641)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S324000, C530S326000, C530S327000, C514S012200, C514S013800
Reexamination Certificate
active
06191254
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to peptides that have antimicrobial activity.
BACKGROUND OF THE INVENTION
Systemic diseases that are associated with pathogenic microorganisms or their toxins in the blood (e.g., septicemia) are a leading cause of death among humans. Gram-negative bacteria are the organisms most commonly associated with such diseases, and pathogenesis has been related in many cases to the release of a toxic outer membrane component termed endotoxin. However, gram-positive bacteria are an increasing cause of fatal infections. In addition, antibiotic resistance is becoming a major problem for all classes of antibiotics, and novel antibiotics are urgently needed.
Cationic peptides having antimicrobial activity have been isolated from a wide variety of organisms. In nature, such peptides provide a defense mechanism against microorganisms such as bacteria and yeast. Generally, these cationic peptides are thought to exert their antimicrobial activity on bacteria by interacting with the cytoplasmic membrane to form channels or lesions. In gram-negative bacteria, they interact with surface lipopolysaccharide (LPS) to permeabilize the outer membrane, leading to self promoted uptake across the outer membrane and access to the cytoplasmic membrane. Examples of antimicrobial peptides include indolicidin, defensins, cecropins, and magainins.
SUMMARY OF THE INVENTION
The invention provides a novel class of isolated cationic peptides having antimicrobial activity. As a group, the peptides of the invention can be described by the following formulas:
X
1
X
1
PX
2
X
3
X
2
P(X
2
X
2
P)
n
X
2
X
3
(X
5
)
o
;
(SEQ ID NO: 23)
X
1
X
1
PX
2
X
3
X
3
X
4
(X
5
)
r
PX
2
X
3
X
3;
(SEQ ID NO: 24)
X
1
X
1
X
3
(PW)
u
X
3
X
2
X
5
X
2
X
2
X
5
X
2
(X
5
)
o
; and
(SEQ ID NO: 25)
X
1
X
1
X
3
X
3
X
2
P(X
2
X
2
P)
n
X
2
(X
5
)
m
;
(SEQ ID NO: 25)
wherein:
m is 1 to 5;
n is 1 or 2;
o is 2 to 5;
r is 0 to 8;
u is 0 or 1;
X
1
is Isoleucine, Leucine, Valine, Phenylalanine, Tyrosine, Tryptophan or Methionine;
X
2
represents Tryptophan or Phenylalanine;
X
3
represents Arginine or Lysine;
X
4
represents Tryptophan or Lysine; and
X
5
represents Phenylalanine, Tryptophan, Arginine, Lysine, or Proline.
Various derivatives, analogues, conservative variations, and variants of the peptides described herein are included within the invention. Preferably, the peptide is amidated or carboxymethylated. Isolated nucleic acids encoding the peptides of the invention also are included. Examples of preferred peptides include those having the following amino acid sequences, as defined using the one-letter amino acid code:
ILKKWPWWPWRRK (SEQ ID NO: 1);
ILKPWKWPWWPWRRKK (SEQ ID NO:2);
ILKPWKWPWWPWRR (SEQ ID NO:3);
ILPWKKWPWWRWRR (SEQ ID NO:4);
ILKKWPWWPWRR (SEQ ID NO:5);
ILPWKWPWWPWRKWR (SEQ ID NO:6);
ILPWKWPWWPWRRWR (SEQ ID NO:7);
ILPWKWPWWPWKKWK (SEQ ID NO:8);
PWKWPWWPWRR (SEQ ID NO:9);
ILPWKWPWRR (SEQ ID NO:10);
ILPWKWPWWPWWPWRR (SEQ ID NO:11);
ILPWKWPWWPWWKKPWRR (SEQ ID NO:12);
ILPWICPWRPSKAN (SEQ ID NO:13);
IVPWKWTLWPWRR (SEQ ID NO:14);
TLPCLWPWWPWSI (SEQ ID NO:15);
ILKKWPWWPWKRR (SEQ ID NO:17);
ILKKWPWWPWKWKK (SEQ ID NO: 18);
ILPWKWPWYVRR (SEQ ID NO: 19);
IKWPWYVWL (SEQ ID NO: 20);
ILPWKWFFPPWPWRR (SEQ ID NO: 21);
ILPWKWPPWPPWPWRR (SEQ ID NO: 22);
ILKKWPWWRWRR (SEQ ID NO: 27);
ILKKFPFFPFRRK (SEQ ID NO: 28);
ILKKFPFFPFKKK (SEQ ID NO: 29);
ILKKWAWWPWRRK (SEQ ID NO: 30);
ILKKWPWWAWRRK (SEQ ID NO: 31);
ILKKWPWWPWKKK (SEQ ID NO: 32);
ILRRWPWWPWRRR (SEQ ID NO: 33);
WWKKWPWWPWRRK (SEQ ID NO: 34);
FFKKWPWWPWRRK (SEQ ID NO: 35);
FFKKFPFFPFRRK (SEQ ID NO: 36);
FFKKFPFFPFKKK (SEQ ID NO: 37);
ILKKWPWWPWWPWRRK (SEQ ID NO: 38);
ILKKWPWWPWRWWRR (SEQ ID NO: 39);
ILKKWPWWPWRRWWK (SEQ ID NO: 40);
ILKKWPWWPWPPRRK (SEQ ID NO: 41);
ILKKWPWWPWPPFFRRK (SEQ ID NO: 42);
The invention also provides a method of inhibiting the growth of a bacterium (or bacteria) by contacting the bacterium with an inhibiting effective amount of one or more of the peptides of the invention, used simultaneously or sequentially. If desired, the peptide(s) can be used in combination with an antibiotic, simultaneously or sequentially. Such combination therapy may prove beneficial by a synergy occurring between the peptide and the antibiotic.
The invention also provides a method for inhibiting an endotoxemia or sepsis-associated disorder in a subject (e.g., a mammal such as a human) that has, or is at risk of having, such a disorder; the method entails administering to the subject a therapeutically effective amount of a peptide of the invention.
In addition, the invention provides a method for producing a cationic peptide variant having antimicrobial activity. This method entails: identifying the amino acid sequence of a reference cationic peptide having antimicrobial activity; producing an expression library encoding cationic peptide variants of the reference peptide (where a plurality of the variants each contain at least one substitution of an amino acid of the identified amino acid sequence); expressing the library in a plurality of host cells (thereby creating a plurality of clones); and isolating a clone that produces a cationic peptide variant having antimicrobial activity.
The invention provides several advantages. The peptides of the invention are compact and tend to have a unique polyproline type II extended helix structure that permits them to span the membrane with relatively few amino acids. The best peptides have very broad spectrum activity against antibiotic resistant bacteria, combined with activity against the medically important fungus,
Candida albicans.
These peptides also often possess the ability to work synergistically with antibiotics; in addition, they often possess anti-endotoxin activity. The peptides can be produced efficiently by recombinant DNA and protein chemical means. The invention also provides methods that permit both rationally designed and semi-random mutants of core structures (e.g., reference proteins) to be tested to permit variants within the formulas described herein to be produced and tested.
REFERENCES:
patent: 4810777 (1989-03-01), Zasloff
patent: 4822608 (1989-04-01), Benton et al.
patent: 5028530 (1991-07-01), Lai et al.
patent: 5073542 (1991-12-01), Zasloff
patent: 5166321 (1992-11-01), Lai et al.
patent: 5202420 (1993-04-01), Zasloff et al.
patent: 5206156 (1993-04-01), Lai et al.
patent: 5208220 (1993-05-01), Berkowitz
patent: 5217956 (1993-06-01), Zasloff et al.
patent: 5235038 (1993-08-01), Blondelle et al.
patent: 5254535 (1993-10-01), Zasloff et al.
patent: 5324716 (1994-06-01), Selsted et al.
patent: 5344765 (1994-09-01), Lai et al.
patent: 5357044 (1994-10-01), Lai et al.
patent: 5547939 (1996-08-01), Selsted
patent: 5635594 (1997-06-01), Lehrer et al.
patent: 12060510 (1991-10-01), None
patent: 0 356 409 (1990-02-01), None
patent: WO8911290 (1989-11-01), None
patent: WO9004407 (1990-05-01), None
patent: WO9004408 (1990-05-01), None
patent: WO9008552 (1990-08-01), None
patent: WO9112270 (1991-02-01), None
patent: WO9108758 (1991-06-01), None
patent: WO9112015 (1991-08-01), None
patent: WO9116918 (1991-11-01), None
patent: WO9117760 (1991-11-01), None
patent: WO9217197 (1992-10-01), None
patent: WO9217195 (1992-10-01), None
patent: WO9222317 (1992-12-01), None
patent: WO92/22308 (1992-12-01), None
patent: WO9301723 (1993-02-01), None
patent: WO9305802 (1993-04-01), None
patent: WO9311783 (1993-06-01), None
patent: WO9324138 (1993-12-01), None
patent: WO9406688 (1994-03-01), None
patent: WO 95/22338 (1995-08-01), None
“Biologically Active and Amidated Cecropin Produced in a Baculovirus Expression System From a Fusion Construct Containing the Antibody-Binding Part of Protein A” The Biochemical Journal 280, Part 1: 219-224, 1991.
Romeo et al., “Bovine Neutrophil Antibiotic Peptides and Their Precursors: Structure and Role Innate Immunity,” Croatica Chemica ACTA, vol. 68, No. 3, 1995 pp. 607-614.
Van Abel et al., “Synthesis and characterization of indolicidin, a tryptophan-rich antimicrobial peptide from bovine neutrophils”,Int. J. Peptide Res.;45, 1995, 401-409.
Uchida et al., “Antibacterial Acti
Falla Timothy J.
Gough Monisha
Hancock Robert E. W.
Devi S.
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Housel James C.
University of British Columbia
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