Protegrins

Details Protegrins Protegrins

1999-08-30

2003-11-25

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

25 or more amino acid residues in defined sequence

C530S325000, C435S069100, C435S252300, C435S320100, C536S023100, C514S012200, C514S013800, C424S049000, C424S401000

Reexamination Certificate

active

06653442

ABSTRACT:

TECHNICAL FIELD
The invention relates to the field of antibiotic peptides. In particular, the invention concerns short peptides, some of which are isolated from porcine leukocytes, that have a wide range of antimicrobial activities.
BACKGROUND ART
One of the defense mechanisms against infection by both animals and plants is the production of peptides that have antimicrobial and antiviral activity. Various classes of these peptides have been isolated from tissues both of plants and animals. One well known class of such peptides is the tachyplesins which were first isolated from the hemocytes of the horseshoe crab as described by Nakamura, T. et al.
J Biol Chem
(1988) 263:16709-16713. This article described the initial tachyplesin isolated, Tachyplesin I, from the Japanese species. Tachyplesin I is a 17-amino acid amidated peptide containing four cysteine residues providing two intramolecular cystine bonds. A later article by this group, Miyata, T. et al.
J Biochem
(1989) 106:663-668, reports the isolation of a second tachyplesin, Tachyplesin II, consisting of 17 residues amidated at the C-terminus, also containing four cysteine residues and two intramolecular disulfide bonds. Two additional 18-mers, called polyphemusins, highly homologous to Tachyplesin II and containing the same positions for the four cysteine residues, were also isolated from the American horseshoe crab. Polyphemusin I and Polyphemusin II differ from each other only in the replacement of one arginine residue by a lysine. All of the peptides were described as having antifungal and antibacterial activity. A later article by Murakami, T. et al.
Chemotherapy
(1991) 37:327-334, describes the antiviral activity of the tachyplesins with respect to vesicular stomatitis virus; Herpes Simplex Virus I & II, Adenovirus I, Reovirus II and Poliovirus I were resistant to inactivation by Tachyplesin I. Morimoto, M. et al.
Chemotherapy
(1991) 37:206-211, found that Tachyplesin I was inhibitory to Human Immunodeficiency Virus. This anti-HIV activity was found also to be possessed by a synthetic analog of Polyphemusin II as described by Nakashima, H. et al.
Antimicrobial Agents and Chemotherapy
(1992) 1249-1255. Antiviral peptides have also been found in rabbit leukocytes as reported by Lehrer, R. I. et al.
J Virol
(1985) 54:467-472.
Other important classes of cysteine-containing antimicrobial peptides include the defensins, &bgr;-defensins and insect defensins. The defensins are somewhat longer peptides characterized by six invariant cysteines and three intramolecular cystine disulfide bonds. Defensins were described by Lehrer, R. I. et al.
Cell
(1991) 64:229-230; Lehrer, R. I. et al.
Ann Rev Immunol
(1993) 11:105-128. A review of mammalian-derived defensins by Lehrer, R. I. et al. is found in
Annual Review Immunol
(1993) 11:105-128; three patents have issued on the defensins: U.S. Pat. No. 4,705,777; U.S. Pat. No. 4,659,692; and U.S. Pat. No. 4,543,252. Defensins have been found in the polymorphonucleated neutrophils (PMN) of humans and of several other animals, as well as in rabbit pulmonary alveolar macrophages, and in murine small intestinal epithelial (Paneth) cells and in corresponding cells in humans.
&bgr;-Defensins are found in bovine respiratory epithelial cells, bovine granulocytes and avian leukocytes. See Selsted, M. E. et al.
J Biol Chem
(1993) 288:6641-6648 and Diamond, G. et al.
Proc Natl Acad Sci
(USA) (1991) 88:3952-3958. Insect defensins have been reported by Lambert, J. et al.
Proc Natl Acad Sci
(USA) (1989) 88:262-265.
Antifungal and antibacterial peptides and proteins have also been found in plants (Broekaert, W. F. et al.
Biochemistry
(1992) 31:4308-4314) as reviewed by Cornelissen, B. J. C. et al.
Plant Physiol
(1993) 101:709-712. Expression systems for the production of such peptides have been used to transform plants to protect the plants against such infection as described, for example, by Haln, R. et al.
Nature
(1993) 361:153-156.
The present invention provides a new class of antimicrobial and antiviral peptides, designated “protegrins” herein, representative members of which have been isolated from porcine leukocytes. These peptides are useful as antibacterial antiviral and antifungal agents in both plants and animals.
The isolation of the protegrin peptides of the invention was reported by the present applicants in a paper by Kokryakov, V. N. et al.
FEBS
(1993) 337:231-236 (July issue). A later publication of this group described the presence of a new protegrin, whose sequence, and that of its precursor, was deduced from its isolated cDNA clone. Zhao, C et al,
FEBS Letters
(1994) 346:285-288. An additional paper disclosing cationic peptides from porcine neutrophils was published by Mirgorodskaya, O. A. et al.
FEBS
(1993) 330:339-342 (September issue). Storici, P. et al.
Biochem Biophys Res Comm
(1993) 196:1363-1367, report the recovery of a DNA sequence which encodes a pig leukocyte antimicrobial peptide with a cathelin-like prosequence. The peptide is reported to be one of the protegrins disclosed hereinbelow. Additional publications related to protegrins are Harwig, S. S. L., et al.
J. Peptide Sci
. (1995) in press; and Zhao, C., et al. FEBS-MS MB-283 (1995) in press.
The protegrins of the invention have also been found to bind to endotoxins—i.e., the lipopolysaccharide (LPS) compositions derived from gram-negative bacteria which are believed responsible for gram-negative sepsis. This type of sepsis is an extremely common condition and is often fatal. Others have attempted to design and study proteins which bind LPS/endotoxin, and illustrative reports of these attempts appear in Rustici, A. et al.
Science
(1993) 259:361-364; Matsuzaki, K. et al.
Biochemistry
(1993) 32:11704-11710; Hoess, A. et al.
EMBO J
(1993) 12:3351-3356; and Elsbach, P. et al.
Current Opinion in Immunology
(1993) 5:103-107. The protegrins of the present invention provide additional compounds which are capable of inactivating of LPS and ameliorating its effects.
In addition to the foregoing, the protegrins of the invention are effective in inhibiting the growth of organisms that are associated with sexually transmitted diseases. It is estimated that 14 million people world-wide are infected with HIV and that millions of women sustain pelvic inflammatory disease each year.
Chlamydia trachomatis
and
Neisseria gonorrhoeae
cause over half of this inflammatory disease although
E. coli, Mycoplasma hominis
and other infectious microorganisms can also be responsible. Pathogens include viral, bacterial, fungal and protozoan pathogens. It is especially important that the antibiotics used to combat these infections be effective under physiological conditions. The protegrins of the present invention offer these properties.
DISCLOSURE OF THE INVENTION
In one embodiment, the invention is directed to peptides of 16-18 amino acid residues characterized by four invariant cysteines and either by a characteristic pattern of basic and hydrophobic amino acids and/or being isolatable from animal leukocytes using the method of the invention. In a second embodiment, the invention is directed to the above peptides wherein 1-4 of these cysteines is replaced by a hydrophobic or small amino acid. All of these peptides can be produced synthetically and some can be produced recombinantly or can be isolated from their native sources and purified for use as preservatives or in pharmaceutical compositions in treating or preventing infection in animals. Alternatively, the peptides can be formulated into compositions which-can be applied to plants to protect them against viral or microbial infection. In still another approach, the DNA encoding the peptides can be expressed in situ, in animals or preferably in plants, to combat infections. The peptides are also useful as standards in antimicrobial assays and in binding endotoxins.
Accordingly, in one aspect, the invention is directed to a purified and isolated or recombinantly produced compound of the formula
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