Parevins and tachytegrins

Details Parevins and tachytegrins Parevins and tachytegrins

1998-08-03

2001-10-23

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, C514S012200, C514S013800

Reexamination Certificate

active

06307016

ABSTRACT:

TECHNICAL FIELD
The invention relates to the field of antibiotic peptides. In particular, the invention concerns short peptides with unique patterns of cysteine type residues and conformations that have a wide range of antimicrobial activities.
BACKGROUND
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 of both plants and animals. PCT application WO 95/03325 published Feb. 2, 1995 contains a review of the literature on this subject. Such peptides include tachyplesins, which are 17-18 amino acid peptides containing four invariant cysteines, the defensins, &bgr;-defensins, and insect defensins, which are somewhat longer peptides characterized by six invariant cysteines, and antifungal and antibacterial peptides and proteins which have been found in plants.
The applications in the series of which WO 95/03325 is a part provide a new class of antimicrobial and antiviral peptides, designated “protegrins”, 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 some of the protegrin peptides was reported in a paper by Kokryakov, V. N. et al.
FEBS
(1993) 337:231-236 (July issue). A later publication described the presence of a new protegrin, whose sequence and that of its precursor were 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. 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. Additional publications related to protegrins are Harwig, S. S. L., et al.
J Peptide Sci
(1995) in press; Zhao, C., et al.
FEBS Lett
(1995) 376:130-134; Zhao, C. et al.
FEBS Lett
(1995) 368:197-202. See also, U.S. Pat. No. 5,464,823, U.S. Pat. No. 5,696,486, WO 95/03325, WO 96/37508 and WO 98/03192.
The protegrins 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. The protegrins are also effective in inhibiting the growth of organisms that are associated with sexually transmitted diseases such as
Chlamydia trachomatis
and
Neisseria gonorrhoeae.
The invention described below relates to peptide type compounds that are related to the protegrins described above, but reflect displacements of the protegrin cysteines at positions 6 and 15. The availability of these compounds, the preferred forms of which are designated parevins and tachytegrins, expands the repertoire of antimicrobial peptides and permits more exquisite matching of indications to antimicrobial formulations. Although at least one of C
4
, C
5
, C
16
or C
17
in the formula set forth below must be cysteine, the common name terminology of these components reflects particularly preferred situations wherein both of C
4
and C
17
are cysteine type residues (the tachytegrins) or where both C
5
and C
16
are cysteine type residues (the parevins).
DISCLOSURE OF THE INVENTION
The invention provides compounds which retain generally the antimicrobial activity of the protegrins discussed above, but differ in conformation due to the dislocation of the cysteine residues at positions 6 and/or 15 of these protegrins. Surprisingly, these modified compounds exhibit activity spectra which are analogous to those of the protegrins, but offer the opportunity to fine-tune the biological activity of antibiotics and antivirals. All of these peptides can be produced synthetically and those that contain only gene-encoded amino acids can also be produced recombinantly. These compounds are useful 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 or synthetically produced compound which contains the amino acid sequence
A
1
-A
2
-A
3
-C
4
-C
5
-C
6
-A
7
-C
8
-A
9
-A
10
-A
11
-A
12
-C
13
-A
14
-C
15
-C
16
-C
17
-A
18
  (1)
said compound containing 11-24 amino acid residues. The sequence shown as (1) can be extended at the N and/or C terminus with non-interfering amino acids or sequence.
The compounds also include the N-terminal acylated and/or C-terminal amidated or esterified forms and may be either in the, optionally—SH stabilized, linear or in a disulfide-bridged form.
In the amino acid sequence shown, each of A
1
-A
3
is independently present or not present, and if present each is independently a basic, hydrophobic, polar/large, or small amino acid;
each of C
4
, C
5
, C
6
, C
15
, C
16
and C
17
is independently cysteine, homocysteine or penicillamine or a basic, hydrophobic, polar/large, or small amino acid, and C
4
and/or C
17
may be present or not present; C
6
and/or C
15
may also be acidic;
each of C
8
and C
13
is independently cysteine, homocysteine or penicillamine;
each of A
7
and A
14
is independently a hydrophobic or a small amino acid;
A
9
-A
12
must be capable of effecting a or &bgr;-turn when contained in the compound and at least one of A
9
-A
12
must be a basic amino acid;
A
18
is present or not present, and if present, is a basic, hydrophobic, polar/large or small amino acid.
The compounds of the invention may, in the alternative, contain a modified form of formula (1) wherein one or both of C
8,
and C
13
is independently replaced by a basic, hydrophobic, polar/large, acidic, or small amino acid.
In all of the compounds of the invention at least about 15% and no more than about 50% of the amino acids must be basic amino acids, and the compounds must have a net charge of +1 at physiological pH;
with the proviso that at least one of C
4
, C
5
, C
16
and C
17
must be cysteine, homocysteine or penicillamine; and
only one of C
4
, C
5
, and C
6
, and only one of C
15
, C
16
and C
17
can be cysteine, homocysteine or penicillamine.
A particular advantage of some of the peptides of the invention, especially those which contain fewer amino acids, lies in their reduced size. As a result of this, they are less costly to produce, generally are expected to provide better distribution in tissue, and are less immunogenic. As they provide alternative structures, they are likely to have different pharmacokinetic and toxicological profiles.
In still other aspects, the invention is directed to recombinant materials useful for the production of the peptides of the invention as well as plants or animals modified to contain expression systems for the production of these peptides. The invention is also directed to pharmaceutical compositions and compositions for application to plants containing the peptides of the invention as active ingredients or compositions which contain expression systems for production of the peptides or for in situ expression of the nucleotide sequence encoding these peptides. The invention is also directed to methods to prepare the invention peptides synthetically, to antibodies specific for these peptides, and to the use of the peptides as preservatives.
In other aspects, the invention is directed to the use of the compounds of the invention as standards in antimicrobial assays. The compounds many also be used as antimicrobials in solutions useful in eye care, such as contact len

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