U.S. patent application number 09/865943 was filed with the patent office on 2002-10-10 for parevins and tachytegrins.
This patent application is currently assigned to IntraBiotics Pharmaceuticals, Inc.. Invention is credited to Chang, Conway C., Gu, Chee L., Harwig, Sylvia S.L., Lehrer, Robert I..
Application Number | 20020147301 09/865943 |
Document ID | / |
Family ID | 26668280 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020147301 |
Kind Code |
A1 |
Lehrer, Robert I. ; et
al. |
October 10, 2002 |
Parevins and tachytegrins
Abstract
The present invention provides a new class of broad-spectrum
antimicrobial peptides effective against a wide variety of
microbes, including bacteria, viruses, retroviruses, fungi, yeast
and protozoa.
Inventors: |
Lehrer, Robert I.; (Santa
Monica, CA) ; Harwig, Sylvia S.L.; (Woodland Hills,
CA) ; Chang, Conway C.; (San Francisco, CA) ;
Gu, Chee L.; (Saratoga, CA) |
Correspondence
Address: |
PENNIE & EDMONDS LLP
1155 Avenue of the Americas
New York
NY
10036-2711
US
|
Assignee: |
IntraBiotics Pharmaceuticals,
Inc.
|
Family ID: |
26668280 |
Appl. No.: |
09/865943 |
Filed: |
May 24, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09865943 |
May 24, 2001 |
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09128344 |
Aug 3, 1998 |
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6307016 |
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09128344 |
Aug 3, 1998 |
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08674622 |
Jul 3, 1996 |
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60000898 |
Jul 6, 1995 |
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Current U.S.
Class: |
530/324 ;
530/325; 530/326; 530/327 |
Current CPC
Class: |
Y02A 50/473 20180101;
C07K 7/08 20130101; A61L 12/14 20130101; A01N 37/46 20130101; Y02A
50/30 20180101; A61K 38/00 20130101 |
Class at
Publication: |
530/324 ;
530/325; 530/326; 530/327 |
International
Class: |
C07K 007/06; C07K
007/08; C07K 014/00 |
Goverment Interests
[0002] This invention was made with funding from NIH Grant No.
A122839. The U.S. Government has certain rights in this invention.
Claims
What is claimed is:
1. A compound composed of 11-24 amino acid residues comprising the
amino acid sequence:
9
A.sub.1-A.sub.2-A.sub.3-C.sub.4-C.sub.5-C.sub.6-A.sub.7-C.sub.8-A-
.sub.9-A.sub.10-A.sub.11-A.sub.12-C.sub.13-A.sub.14-C.sub.15-C.sub.16-C.su-
b.17-A.sub.18
or a pharmaceutically acceptable salt or an N-terminal acylated or
C-terminal amidated or esterified form thereof, said compound being
either in a linear or in a disulfide-bridged form, wherein: each of
A.sub.1-A.sub.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.sub.4 and C.sub.17 is independently
present or not present, and if present each is independently
selected from the group consisting of cysteine, homocysteine,
penicillamine, a basic amino acid, a hydrophobic amino acid, a
polar/large amino acid and a small amino acid; C.sub.5 is selected
from the group consisting of cysteine, homocysteine, penicillamine,
a basic amino acid, a hydrophobic amino acid, a polar/large amino
acid and a small amino acid; each of C.sub.6, C.sub.8, C.sub.13 and
C.sub.15 is independently selected from the group consisting of
cysteine, homocysteine, penicillamine, a basic amino acid, a
hydrophobic amino acid, a polar/large amino acid, a small amino
acid and an acidic amino acid; C.sub.16 is selected from the group
consisting of cysteine, homocysteine, penicillamine, a hydrophobic
amino acid or a small amino acid; each of A.sub.7 and A.sub.14 is
independently a hydrophobic or a small amino acid; A.sub.9-A.sub.12
taken together are capable of effecting a .beta.-turn when
contained in the compound and at least one of A.sub.9-A.sub.12 is a
basic amino acid; A.sub.18 is present or not present, and if
present, is a basic, hydrophobic, polar/large, or small amino acid;
at least about 15% to about 50% of the amino acid residues
composing said compound are basic amino acids; and said compound
has a net positive charge of at least +1 at physiological pH; with
the provisos that: (i) when one of C.sub.4, C.sub.5 or C.sub.6 is
cysteine, homocysteine or penicillamine, the other two are other
than cysteine, homocysteine and penicillamine; (ii) when one of
C.sub.15, C.sub.16 or C.sub.17 is cysteine, homocysteine or
penicillamine, the other two are other than cysteine, homocysteine
and penicillamine; and (iii) at least one of C.sub.4, C.sub.5,
C.sub.16 or C.sub.17 is cysteine, homocysteine or
penicillamine.
2. The compound of claim 1 which comprises two disulfide
bridges.
3. The compound of claim 2, wherein one of said disulfide bridges
links C.sub.5-C.sub.16 and the other links C.sub.8-C.sub.13.
4. The compound of claim 3 which is selected from the group
consisting of: 14and the C-terminal amidated forms thereof, wherein
X is Har, x is D-Har, lower case letters represent D-amino acids
and lines between C or c residues represent disulfide linkages.
5. The compound of claim 2, wherein one of said disulfide bridges
links C.sub.5-C.sub.8 and the other links C.sub.13-C.sub.16.
6. The compound of claim 5 which is selected from the group
consisting of: 15and the C-terminal amidated forms thereof, wherein
X is Har, x is D-Har, lower case letters represent D-amino acids
and lines between C and c residues represent disulfide
linkages.
7. The compound of claim 2, wherein one of said disulfide bridges
links C.sub.4-C.sub.17 and the other links C.sub.8-C.sub.13.
8. The compound of claim 7 which is selected from the group
consisting of: 16and the C-terminal amidated forms thereof, wherein
X is Har, x is D-Har, lower case letters represent D-amino acids
and lines between C or c residues represent disulfide linkages.
9. The compound of claim 1 which comprises one disulfide
bridge.
10. The compound of claim 9 in which said disulfide bridge links
C.sub.4-C.sub.17.
11. The compound of claim 10 which is selected from the group
consisting of: 17and the C-terminal amidated forms thereof, wherein
X is Har, Z is MeGly and lines between C residues represent
disulfide linkages.
12. The compound of claim 9 in which said disulfide bridge links
C.sub.5-C.sub.16.
13. The compound of claim 12 which is selected from the group
consisting of: 18and the C-terminal amidated forms thereof, wherein
X is Har, x is D-Har, lower case letters represent D-amino acids
and lines between C residues represent disulfide linkages.
14. The compound of claim 9 in which the disulfide bridge links
C.sub.8 and C.sub.13.
15. The compound of claim 1 which is in the linear form.
16. The compound of claim 1 in which at least one of A.sub.1,
A.sub.2 or A.sub.3 is not present.
17. The compound claim 1 in which A.sub.1, A.sub.2 and A.sub.3 are
not present.
18. The compound of claim 1 in which at least one of A.sub.1,
A.sub.2 or A.sub.3 is a hydrophobic amino acid.
19. The compound of claim 1 in which each of C.sub.5 and C.sub.16
is independently selected from the group consisting of cysteine,
homocysteine, penicillamine, I, V, L, NLe, W, Y, F, A, S, G and
T.
20. The compound of claim 1 in which each of C.sub.4 and C.sub.17
is independently selected from the group consisting of cysteine,
homocysteine, penicillamine, I, V, L, NLe, W, Y, F, A, S, G and
T.
21. The compound of claim 1 in which each of A.sub.7and A.sub.14 is
independently selected from the group consisting of I, V, L, NLe,
W, Y, F, A, S, G and T.
22. The compound of claim 1 in which one of A.sub.9 or A.sub.12 is
R, K, Har, Orn or H and the other is I, V, L, NLe, W, Y, F, A, S, G
or T.
23. The compound of claim 1 in which all amino acids are in the
D-configuration.
24. The compound of claim 1 in which A.sub.7 and A.sub.14 are each
independently a hydrophobic amino acid.
25. The compound of claim 1 in which A.sub.9 or A.sub.12 is a
hydrophobic amino acid or a small amino acid.
26. The compound of claim 1 in which A.sub.10 and A.sub.11 are each
independently selected from the group consisting of proline, a
basic amino acid, a hydrophobic amino acid and a small amino
acid.
27. The compound of claim 1 in which each of C.sub.8 and C.sub.13
is independently cysteine, homocysteine or penicillamine.
28. The compound of claim 1 in which A.sub.9-A.sub.10-A.sub.12 is
selected from the group consisting of: R-R-R-F, R-G-W-I, R-P-R-F,
X-R-R-F, R-X-RF, R-K-K-W, R-X-R-Y, R-R-K-W, r-R-R-F, R-x-R-F,
R-G-R-F, C-R-G-R, Y-C-G-R, V-P-R-R-F, K-P-K-F, V-G-R-F, R-P-R-I and
R-Z-R-F, where X is Har, x is D-Har, Z is MeGly and r is D-Arg.
29. The compound of claim 1 which is in the linear or
disulfide-bridged form and which is selected from the group
consisting of: +M1 !,4 RGGRCLYCRRRFCVVCGR? ,19 (SEQ ID NO:11);? !
!RGGCRLYCRRRFCVVGCR? (SEQ ID NO:12);? ! !RGGRCLYCRRRFCIVCG? (SEQ ID
NO:13);? ! !RGGCRLYCRRRFCIVGC? (SEQ ID NO:14);? !
!RGGGCLYCRRRFCVVCGR? (SEQ ID NO:15);? ! !RGGCGLYCRRRFCVVGCR? (SEQ
ID NO:16);? ! !RGGRCLYCRGWICFVCGR? (SEQ ID NO:17);? !
!RGGCRLYCRGWICFVGCR? (SEQ ID NO:18);? ! !RGGRCLYCRPRFCVVCGR? (SEQ
ID NO:19);? ! !RGGCRLYCRPRFCVVGCR? (SEQ ID NO:20);? !
!RGGRCVYCRRRFCVVCG? (SEQ ID NO:21);? ! !RGGCRVYCRRRFCVIGC? (SEQ ID
NO:22);? ! !KGGRCLYCRRRFCVVCG? (SEQ ID NO:23);? !
!KGGCRIYCRRRFCVIGC? (SEQ ID NO:24);? ! !RGGXCLYCRRRFCVVC? (SEQ ID
NO:25);? ! !RGGCXLYCRRRFCVIC? (SEQ ID NO:26);? !
!RGGXCLYCXRRFCVVCGR? (SEQ ID NO:27);? ! !RGGCXLYCXRRFCVIGCR? (SEQ
ID NO:28);? ! !RGGRCVYCRXRFCVVCGR? (SEQ ID NO:29);? !
!RGGCRVYCRXRFCVVGCR? (SEQ ID NO:30);? ! !RGGRCLYCRKKWCVVCGR? (SEQ
ID NO:31);? ! !RGGCRLYCRKKWCVVGCR? (SEQ ID NO:32);? !
!RGGRCLYCRXRYCVVCGR? (SEQ ID NO:33);? ! !RGGCRLYCRXRYCVVACR? (SEQ
ID NO:34);? ! !RGSGCLYCRRKWCVVCGR? (SEQ ID NO:35);? !
!RGSCGLYCRRKWCVVGCR? (SEQ ID NO:36);? ! !RATRCIFCRRRFCVVCGR? (SEQ
ID NO:37);? ! !RATCRIFCRRRFCVIGCR? (SEQ ID NO:38);? !
!RGGKCVYCRXRFCVVCGR? (SEQ ID NO:39);? ! !RGGCKVYCRXRFCVIGCR? (SEQ
ID NO:40);? ! !RATRCIFCrRRFCVVCGr? (SEQ ID NO:41);? !
!RATCRIFCrRRFCVVGCr? (SEQ ID NO:42);? ! !RGGKCVYCRxRFCVVCGR? (SEQ
ID NO:43);? ! !RGGCKVYCRxRFCVVGCR? (SEQ ID NO:44);? !
!rggrclycrrrfcvvcgr? (SEQ ID NO:45);? ! !rggcrlycrrrfcvvgcr? (SEQ
ID NO:46);? ! !rggrclycrrrfcivcg? (SEQ ID NO:47);? !
!rggcrlycrrrfcivgc? (SEQ ID NO:48);? ! !rgggclycrrrfcvvcgr? (SEQ ID
NO:49);? ! !rggcglycrrrfcvvgcr? (SEQ ID NO:50);? !
!rggrclycrgwicfvcgr? (SEQ ID NO:51);? ! !rggcrlycrgwicfvgcr? (SEQ
ID NO:52);? ! !RGGCLRYCRPRFCVRVCR? (SEQ ID NO:53);? !
!RGGCRLYCRRRFCVVGCR? (SEQ ID NO:54);? ! !RGVCLRYCRGRFCVRLCR? (SEQ
ID NO:55);? ! !RGRVCLRYCRGRFCVRLCFR? (SEQ ID NO:56);? !
!RWRVCLRYCRGRFCVRLCLR? (SEQ ID NO:57);? ! !RGWRVCLKYCRGRFCVKLCLR?
(SEQ ID NO:58);? ! !RGGRVCLRYCRGKFCVRLCLR? (SEQ ID NO:59);? !
!RGGRCLYARRRFAVVCGR? (SEQ ID NO:6O);? ! !RGGRCLYARRRFSIVC? (SEQ ID
NO:61);? ! !RGGGCLYSRRRFAVVCGR? (SEQ ID NO:62);? !
!RGGRCLYARRRFGVVC? (SEQ ID NO:63);? ! !KGGRCLYVRRRFIVVC? (SEQ ID
NO:64);? ! !RGGXCLYARRRFVGCV? (SEQ ID NO:65);? !
!RGGXCLYAXRRFSVVCR? (SEQ ID NO:66);? ! !RGGCXLYAXRRFSVVGCR? (SEQ ID
NO:67);? ! !RGGRCVYVRXRFLVCVGR? (SEQ ID NO:68);? !
!RGGRCLYSRKKWAVSCGR? (SEQ ID NO:69);? ! !RGGRCLYSRXRYSVICGR? (SEQ
ID NO:70);? ! !RGSGCIYCRRKWGVVGCR? (SEQ ID NO:71);? !
!RATRCIFSRRRFSVVCGR? (SEQ ID NO:72);? ! !RGGKCVYGRXRFSVVCGR? (SEQ
ID NO:73);? ! !RATRCIFGrRRFGVVCGr? (SEQ ID NO:74);? !
!RGGKCVYLRXRFLVVCGR? (SEQ ID NO:75);? ! !RGGRCVFLRPRIGVVCGR? (SEQ
ID NO:76);? ! !RGGCLRYAVPRFAVRVCR? (SEQ ID NO:77);? !
!RGGCLRYTKPKFTVRVCR? (SEQ ID NO:78);? ! !RGGCLRYAVGRFAVRVCR? (SEQ
ID NO:79);? ! !RGGCLRYARZRFAVRVCR? (SEQ ID NO:80);? !
!RGFCLRYTVPRFTVRFCVR? (SEQ ID NO:81);? ! !RGFCLRYKVGRFKVRFCVR? (SEQ
ID NO:82);? ! !RGFCLRYZVGRFZVRFCVR? (SEQ ID NO:83);? !
!RGGCLRYARZRFAVRVCR? (SEQ ID NO:84);? ! !RGGCLRYAVGRFAVRVCR? (SEQ
ID NO:85);? ! !RGGRCLYCRRRFCVVGCR? (SEQ ID NO:86);? !
!RGGCRLYCRRRFCVCVGR? (SEQ ID NO:87);? ! !RGGRCLYCRRRFCVCVGR? (SEQ
ID NO:88);? ! !RGGCRLYCRRRFCVCVGR? (SEQ ID NO:89);? !
!RGGRLCYCRRRFCVVCGR? (SEQ ID NO:90);? ! !RGGRLCYCRRRFCVVGCR? (SEQ
ID NO:91);? ! !RGGCRLYCRRRFCVVGC? (SEQ ID NO:92);? !
!RGGRCLYCRRRFCVVGC? (SEQ ID NO:93);? ! !RGGCRLYCRRRFCVVCG? (SEQ ID
NO:94);? ! !RGGRCLYCRRRFCVCVG? (SEQ ID NO:95);? !
!RGGCRLYCRRRFCVCVG? (SEQ ID NO:96);? ! !RGGRLCYCRRRFCVVCG? (SEQ ID
NO:97);? ! !RGGRLCYCRRRFCVVGC? (SEQ ID NO:98);? !
!RGGGCLYCRRRFCVVGCR? (SEQ ID NO:99);? ! !RGGGCLYCRRRFCVCVGR? (SEQ
ID NO:100);? ! !RGGCGLYCRRRFCVCVGR? (SEQ ID NO:101);? !
!RGGGLCYCRRRFCVVCGR? (SEQ ID NO:102);? ! !RGGGLCYCRRRFCVVGCR? (SEQ
ID NO:103);? !!and the C-terminal amidated and N-terminal acylated
forms thereof, wherein X is Har, x is D-Har, Z is MeGly and lower
case letters represent D-amino acids.
30. A pharmaceutical composition comprising a compound according to
claim 1 and a pharmaceutically acceptable excipient.
31. A method of inhibiting the growth of a microbe or the
replication of a virus which comprises the step of contacting said
virus or said microbe with an amount of a compound according to
claim 1 effective to inhibit said growth or said replication.
32. The method of claim 31 in which the microbe is a bacteria.
33. The method of claim 32 in which the bacteria is selected from
the group consisting of E. coli, L. monocytogenes, B. subtilis, S.
typhimurium, S. aureus and P. aeruginosa.
34. The method of claim 31 in which the microbe or virus is a
sexually-transmitted microbe or virus.
35. The method of claim 34 in which the sexually-transmitted
microbe or virus is selected from the group consisting of HIV-1, C.
trachomatis, T. pallidum, N. gonorrhoeae, T. vaginalis, HSV-1,
HSV-2, H. ducreyi and human papilloma virus.
36. The method of claim 31 in which the microbe or virus is
HIV.
37. The method of claim 31 in which the microbe or virus is
methicillin-resistant S. aureus (MRSA) or vancomycin-resistant E.
faecalis (VREF).
38. A method to inactivate the endotoxin of gram-negative bacteria,
which method comprises contacting said endotoxin with an amount of
a compound according to claim 1 effective to inactivate said
endotoxin.
39. A method to treat or prevent a microbial or viral infection in
a subject, which method comprises administering to a subject in
need of such treatment an amount of a compound according to claim 1
effective to ameliorate said infection in the subject.
40. The method of claim 39 in which the infection is a bacterial
infection.
41. The method of claim 40 in which the bacteria is selected from
the group consisting of E. Coli, L. monocytogenes, B. subtilis, S.
typhimurium, S. aureus and P. aeruginosa.
42. The method of claim 39 in which the infection is caused by a
sexually-transmitted pathogen.
43. The method of claim 42 in which the sexually-transmitted
pathogen is selected from the group consisting of HIV-1, C.
trachomatis, T. pallidum, N. gonorrhoeae, T. vaginalis, HSV-1,
HSV-2, H. ducreyi and human papilloma virus.
44. The method of claim 39 in which the infection is an HIV
infection.
45. The method of claim 39 in which the infection is a
methicillin-resistant S. aureus (MRSA) or vancomycin-resistant E.
faecalis (VREF) infection.
46. The method of claim 39 in which the compound is administered
topically.
47. The method of claim 39 in which the compound is administered
prophylactically.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 08/674,622, filed Jul. 3, 1996, which is a
continuation-in-part of provisional application Ser. No.
60/000,898, filed Jul. 6, 1995. The contents of these applications
are hereby incorporated herein by references in their
entireties.
TECHNICAL FIELD
[0003] 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 ART
[0004] 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,
.beta.-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.
[0005] 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.
[0006] 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.
[0007] 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.sub.4, C.sub.5, C.sub.16 or C.sub.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.sub.4 and C.sub.17 are cysteine type residues (the
tachytegrins) or where both C.sub.5 and C.sub.16 are cysteine type
residues (the parevins).
DISCLOSURE OF THE INVENTION
[0008] 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.
[0009] 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
1
A.sub.1-A.sub.2-A.sub.3-C.sub.4-C.sub.5-C.sub.6-A.sub.7-C.sub.8-A-
.sub.9-A.sub.10-A.sub.11-A.sub.12-C.sub.13-A.sub.14-C.sub.15-C.sub.16-C.su-
b.17-A.sub.18 (1)
[0010] 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.
[0011] 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.
[0012] In the amino acid sequence shown, each of A.sub.1-A.sub.3 is
independently present or not present, and if present each is
independently a basic, hydrophobic, polar/large, or small amino
acid;
[0013] each of C.sub.4, C.sub.5, C.sub.6, C.sub.15, C.sub.16 and
C.sub.17 is independently cysteine, homocysteine or penicillamine
or a basic, hydrophobic, polar/large, or small amino acid, and
C.sub.4 and/or C.sub.17 may be present or not present; C.sub.6
and/or C.sub.15 may also be acidic;
[0014] each of C.sub.8 and C.sub.13 is independently cysteine,
homocysteine or penicillamine;
[0015] each of A.sub.7 and A.sub.14 is independently a hydrophobic
or a small amino acid;
[0016] A.sub.9-A.sub.12 must be capable of effecting a or
.beta.-turn when contained in the compound and at least one of
A.sub.9-A.sub.12 must be a basic amino acid;
[0017] A.sub.18 is present or not present, and if present, is a
basic, hydrophobic, polar/large or small amino acid.
[0018] The compounds of the invention may, in the alternative,
contain a modified form of formula (1) wherein one or both of
C.sub.8, and C.sub.13 is independently replaced by a basic,
hydrophobic, polar/large, acidic, or small amino acid.
[0019] 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;
[0020] with the proviso that at least one of C.sub.4, C.sub.5,
C.sub.16 and C.sub.17 must be cysteine, homocysteine or
penicillamine; and
[0021] only one of C.sub.4, C.sub.5, and C.sub.6, and only one of
C.sub.15, C.sub.16 and C.sub.17 can be cysteine, homocysteine or
penicillamine.
[0022] 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.
[0023] 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.
[0024] 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 lens solutions, and
in topical or other pharmaceutical compositions for treatment of
sexually transmitted diseases (STDs). The invention is also
directed to use of the invention compounds as preservatives for
foods or other perishables. As the invention peptides can
inactivate endotoxin, the invention is also directed to a method to
inactivate endotoxins using the compounds of the invention and to
treat gram-negative sepsis by taking advantage of this
property.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIGS. 1A and 1B show antibacterial activity of two of the
parevins against E. coli ML-35p;
[0026] FIGS. 2A and 2B show antibacterial activity of two of the
parevins against Listeria monocytogenes;
[0027] FIGS. 3A and 3B shows antifungal activity of two of the
parevins against Candida albicans;
[0028] FIGS. 4A and 4B shows antibacterial activity of a
tachytegrin against E. coli ML-35p;
[0029] FIGS. 5A and 5B shows antibacterial activity of a
tachytegrin against B. subtilis; and
[0030] FIGS. 6A and 6B shows antibacterial activity of a
tachytegrin against S. typhimurium 14028s.
MODES OF CARRYING OUT THE INVENTION
[0031] The peptides of the invention are characterized by the amino
acid sequence:
2 (1)
A.sub.1-A.sub.2-A.sub.3-C.sub.4-C.sub.5-C.sub.6-A.sub.7-C.sub-
.8-A.sub.9-A.sub.10-A.sub.11-A.sub.12-C.sub.13-A.sub.14-C.sub.15-C.sub.16--
C.sub.17-A.sub.18
[0032] and its defined modified forms. Any of these peptides which
may coincidentally occur in nature must be in purified and isolated
form or prepared recombinantly or synthetically.
[0033] The designation A.sub.n in each case represents an amino
acid at the specified position in the peptide. As defined,
A.sub.1-A.sub.3, C.sub.4, C.sub.17 and/or A.sub.18 may or may not
be present. However, the peptides of the invention contain 11-24
amino acids. Thus, the sequence shown as (1) can be extended at the
N and/or C terminus with non-interfering amino acids or sequence.
The positions of the cysteine, homocysteine or penicillamine
residues, shown as C in formula (1), are invariant in one
embodiment of the peptides of the invention; however, in the
modified forms of the peptides containing the sequence of formula
(1), also included within the scope of the invention, one or more
of these cysteines may be replaced by a small, basic acidic or
hydrophobic amino acid. However, at least one of C.sub.4, C.sub.5,
C.sub.16 and C.sub.17 must be cysteine, homocysteine
penicillamine.
[0034] All of the peptides of the invention, however, have a net
positive charge of at least +1 at physiological pH; approximately
15%-50% of the amino acid residues included in the sequence should
be basic. For embodiments having as few as 11 amino acids, there
may be only one basic amino acid residue; however, at least two
basic residues, even in this short-chain residue, are preferred. If
the peptide contains as many as 15 amino acid residues, two basic
residues are required. It is preferred that at least 20% of the
amino acids in the sequence be basic, more preferably 30%, but not
more than 50%.
[0035] The active peptides also preferably contain a .beta.-turn
bracketed by two strands that form a .beta.-sheet. While not
intending to be bound by any theory, applicants believe that
antimicrobial activity of the compounds containing the sequence of
formula (1) is associated with such a .beta.-turn bracketed by two
strands that form a .beta.-sheet structure. The amino acids
A.sub.9-A.sub.12 must be capable of effecting a .beta.-turn, which
can be encouraged by the cystine bond between C.sub.8 and C.sub.13
as well as by hydrogen bonding between A.sub.9 and A.sub.12. The
presence of proline at A.sub.10 and/or A.sub.11 does not interfere
with the .beta.-turn stabilized by the presence of a hydrophobic
amino acid at positions A.sub.9 or A.sub.12.
[0036] As used herein, ".beta.-turn" refers to a recognized
sub-class of reverse-turns. Typically, a ".beta.-turn" is a four
amino acid residue peptide segment that reverses the direction of a
polypeptide chain so as to allow a single polypeptide chain to
adopt an anti-parallel .beta.-sheet secondary structure. Generally,
the two internal amino acid residues of the .beta.-turn are not
involved in the hydrogen-bonding of the .beta.-sheet; the two amino
acid residues on either side of the internal residues are included
in the hydrogen-bonding of the J-sheet. The term ".beta.-turn"
expressly includes all types of peptide .beta.-turns commonly known
in the art including, but not limited to, type-I, type-II,
type-III, type-I', type-II', and type-III' .beta.-turns (see, Rose
et al., 1985, Adv. Protein Chem. 37:1-109; Wilmer-White et al.,
1987, Trends Biochem. Sci. 12:189-192; Wilmot et al., 1988, J. Mol.
Biol. 206:759-777; Tramontano et al., 1989, Proteins; Struct.
Funct. Genet. 6:382-394).
[0037] The presence of the four invariant cysteines of the
protegrins or of the C.sub.8 and C.sub.13 cysteines, homocysteine
or penicillamine of the compounds of the present invention is
helpful in effecting the .beta.-turn conformation; however, by
properly choosing the substitutions, one or both of the cysteine,
homocysteine or penicillamine residues at C.sub.8 or C.sub.13 can
be replaced without substantially disturbing the three-dimensional
shape of the molecule.
[0038] The .beta. sheets are believed to be effected by the
sequences surrounding C.sub.8 and C.sub.13 and are inclusive of
these residues. Thus, in the unmodified forms of the compound,
A.sub.7 and A.sub.14 are preferably hydrophobic amino acids. The
cysteine residues may also, then, be replaced by other residues
which do not affect the maintenance of the .beta. sheet formation;
these substitutions would include acidic, basic, hydrophobic polar
or small amino acids.
[0039] The amino terminus of the peptide may be in the free amino
form or may be acylated by a group of the formula RCO--, wherein R
represents a hydrocarbyl group of 1-6C. The hydrocarbyl group is
saturated or unsaturated and is typically, for example, methyl,
ethyl, i-propyl, t-butyl, n-pentyl, cyclohexyl, cyclohexene-2-yl,
hexene-3-yl, hexyne-4-yl, and the like.
[0040] The C-terminus of the peptides of the invention may be in
the form of the underivatized carboxyl group, either as the free
acid or an acceptable salt, such as the potassium, sodium, calcium,
magnesium, or other salt of an inorganic ion or of an organic ion
such as caffeine. In some embodiments, it is difficult to make
salts since the remainder of the molecule bears a positive charge
which may repel the relevant cation. The carboxyl terminus may also
be derivatized by formation of an ester with an alcohol of the
formula ROH, or may be amidated by an amine of the formula
NH.sub.3, or RNH.sub.2, or R.sub.2NH, wherein each R is
independently hydrocarbyl of 1-6C as defined above. Amidated forms
of the peptides wherein the C-terminus has the formula CONH.sub.2
are preferred.
[0041] As the peptides of the invention contain substantial numbers
of basic amino acids, the peptides of the invention may be supplied
in the form of the acid addition salts. Typical acid addition salts
include those of inorganic ions such as chloride, bromide, iodide,
fluoride or the like, sulfate, nitrate, or phosphate, or may be
salts of organic anions such as acetate, formate, benzoate and the
like. The acceptability of each of such salts is dependent on the
intended use, as is commonly understood.
[0042] The peptides of the invention that contain at least two
cysteines, homocysteine or penicillamine may be in straight-chain
or cyclic form. The straight-chain forms are convertible to the
cyclic forms, and vice versa. Methods for forming disulfide bonds
to create the cyclic peptides are well known in the art, as are
methods to reduce disulfides to form the linear compounds. The
linear compounds can be stabilized by addition of a suitable
alkylating agent such as iodoacetamide.
[0043] The cyclic forms are the result of the formation of
disulfide linkages among all or some of the four cysteine,
homocysteine or penicillamine residues that may be present. Cyclic
forms of the invention include all possible permutations of
disulfide bond formation; if the --SH containing amino acids are
numbered in order of their occurrence starting at the N-terminus as
C.sub.4, C.sub.5, C.sub.6, C.sub.8, C.sub.13, C.sub.16, C.sub.17 or
C.sub.18, these permutations include, when two disulfides are
present:
[0044] a) C.sub.4-C.sub.17 and C.sub.8-C.sub.13;
[0045] b) C.sub.4-C.sub.16 and C.sub.8-C.sub.13;
[0046] c) C.sub.4-C.sub.15 and C.sub.8-C.sub.13;
[0047] d) C.sub.5-C.sub.17 and C.sub.8-C.sub.13;
[0048] e) C.sub.5-C.sub.16 and C.sub.8-C.sub.13;
[0049] f) C.sub.5-C.sub.15 and C.sub.8-C.sub.13;
[0050] g) C.sub.6-C.sub.17 and C.sub.8-C.sub.13;
[0051] h) C.sub.6-C.sub.16 and C.sub.8-C.sub.13;
[0052] i ) C.sub.4-C.sub.8 and C.sub.13-C.sub.17;
[0053] j) C.sub.4-C.sub.8 and C.sub.13-C.sub.16;
[0054] k) C.sub.5-C.sub.8 and C.sub.13-C.sub.17; and
[0055] l) C.sub.5-C.sub.8 and C.sub.13-C.sub.16;
[0056] When one disulfide is present, these permutations
include:
[0057] C.sub.4-C.sub.7;
[0058] C.sub.4-C.sub.16;
[0059] C.sub.4-C.sub.15;
[0060] C.sub.5-C.sub.17;
[0061] C.sub.5-C.sub.16;
[0062] C.sub.6-C.sub.17;
[0063] C.sub.6-C.sub.16;
[0064] C.sub.8-C.sub.13;
[0065] C.sub.4-C.sub.8;
[0066] C.sub.13-C.sub.17; and
[0067] C.sub.13-C.sub.16.
[0068] In the modified forms of the peptides, where 1 or 2
cysteines, homocysteine or penicillamine are replaced, similar
permutations are available as in when 2-3 cysteines, homocysteine
or penicillamine are present.
[0069] The linearalized forms of the native cyclic peptides have
valuable activities, even when chemically stabilized to preserve
the sulfhydryl form of cysteine, homocysteine or penicillamine for
example, by reaction with iodoacetamide. The compounds of the
invention also include linearalized forms which are stabilized with
suitable reagents. As defined herein, "SH-stabilized" forms of the
peptides of the invention contain sulfhydryl groups reacted with
standard reagents to prevent reformation into disulfide
linkages.
[0070] An alternative approach to providing linear forms of the
invention compounds comprises use of the modified form of the
peptides where residues at C.sub.8 and/or C.sub.13 are replaced by
amino acids which do not form cystine linkages, in combination with
stabilization of any cysteine, homocysteine or penicillamine
residues at C.sub.4, C.sub.5, or C.sub.6 and/or C.sub.15, C.sub.16,
C.sub.17.
[0071] Forms of the invention compounds which have only one
disulfide bond are conveniently obtained by replacing the cysteine,
homocysteine or penicillamine residues at C.sub.8 and/or C.sub.13,
preferably both, with amino acids which do not form disulfide
linkages.
[0072] The amino acids denoted by A.sub.n may be those encoded by
the gene or analogs thereof, and may also be the D-isomers thereof.
One preferred embodiment of the peptides of the invention is that
form wherein all of the residues are in the D-configuration thus
conferring resistance to protease activity while retaining
antimicrobial or antiviral properties. The resulting peptides are
enantiomers of the native L-amino acid-containing forms.
[0073] In one class of peptides described herein, either one or
both of the residues found at C.sub.5 and/or C.sub.16 is a basic
amino acid and/or at least one of A.sub.1-A.sub.3 and C.sub.4 is
hydrophobic and/or at least one, and preferably all four of these
amino acids are deleted. By suitable manipulation of these and
other features, the range of conditions under which the class of
peptides of the present invention are effective can be varied.
Furthermore, the spectrum of microbes against which they are
effective can also be modified. This is further described
hereinbelow.
[0074] The amino acid notations used herein are conventional and
are as follows:
3 One-Letter Three-Letter Amino Acid Symbol Symbol Alanine A Ala
Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys
Glutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H His
Isoleucine I Ile Leucine L Leu Lysine K Lys Methioninie M Met
Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr
Tryptophan W Trp Tyrosine Y Tyr Valine V Val
[0075] The amino acids not encoded genetically are abbreviated as
indicated in the discussion below.
[0076] In the specific peptides shown in the present application,
the L-form of any amino acid residue having an optical isomer is
intended unless the D-form is expressly indicated by a dagger
superscript (.dagger.).
[0077] The compounds of the invention are peptides which are
partially defined in terms of amino acid residues of designated
classes. Amino acid residues can be generally subclassified into
major subclasses as follows:
[0078] Acidic: The residue has a negative charge due to loss of H
ion at physiological pH and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH.
[0079] Basic: The residue has a positive charge due to association
with H ion at physiological pH and the residue is attracted by
aqueous solution so as to seek the surface positions in the
conformation of a peptide in which it is contained when the peptide
is in aqueous medium at physiological pH.
[0080] Hydrophobic: The residues are not charged at physiological
pH and the residue is repelled by aqueous solution so as to seek
the inner positions in the conformation of a peptide in which it is
contained when the peptide is in aqueous medium.
[0081] Polar/large: The residues are not charged at physiological
pH, but the residue is not sufficiently repelled by aqueous
solutions so that it would seek inner positions in the conformation
of a peptide in which it is contained when the peptide is in
aqueous medium.
[0082] This description also characterizes certain neutral amino
acids as "small" since their side chains are not sufficiently
large, even if polar groups are lacking, to confer hydrophobicity.
"Small" amino acids are those with four carbons or less when at
least one polar group is on the side chain and three carbons or
less when not.
[0083] It is understood, of course, that in a statistical
collection of individual residue molecules some molecules will be
charged, and some not, and there will be an attraction for or
repulsion from an aqueous medium to a greater or lesser extent. To
fit the definition of "charged," a significant percentage (at least
approximately 25%) of the individual molecules are charged at
physiological pH. The degree of attraction or repulsion required
for classification as polar or nonpolar is arbitrary and,
therefore, amino acids specifically contemplated by the invention
have been classified as one or the other. Most amino acids not
specifically named can be classified on the basis of known
behavior.
[0084] Amino acid residues can be further subclassified as cyclic
or noncyclic, and aromatic or nonaromatic, self-explanatory
classifications with respect to the side-chain substituent groups
of the residues, and as small or large. The residue is considered
small if it contains a total of four carbon atoms or less,
inclusive of the carboxyl carbon, provided an additional polar
substituent is present; three or less if not. Small residues are,
of course, always nonaromatic.
[0085] For the naturally occurring protein amino acids,
subclassification according to the foregoing scheme is as
follows.
4 Acidic Aspartic acid and Glutamic acid Basic Noncyclic: Arginine,
Lysine Cyclic: Histidine Small Glycine, Serine, Alanine, Threonine
Polar/large Asparagine, Glutamine Hydrophobic Tyrosine, Valine,
Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan
[0086] The gene-encoded secondary amino acid proline is a special
case due to its known effects on the secondary conformation of
peptide chains, and is not, therefore, included in a group.
Cysteine and other --SH containing amino acid residues are also not
included in these classifications since their capacity to form
disulfide bonds to provide secondary structure is critical in the
compounds of the present invention.
[0087] Certain commonly encountered amino acids, which are not
encoded by the genetic code, include, for example, .beta.-Alanine
(.beta.-Ala), or other omega-amino acids, such as 3-aminopropionic,
2,3-diaminopropionic (2,3-diaP), 4-aminobutyric and so forth,
I-aminisobutyric acid (Aib), sarcosine (Sar) or N-methyl glycine
(MeGly), ornithine (Orn), citrulline (Cit), t-butylalanine (t-BuA),
t-butylglycine (t-BuG), N-methylisoleucine (N-MeIle), phenylglycine
(Phg), and cyclohexylalanine (Cha), norleucine (Nle),
2-naphthylalanine (2-Nal); 1,2,3,4-tetrahydroisoquinoline-3-carbox-
ylic acid (Tic); .beta.-2-thienylalanine (Thi); methionine
sulfoxide (MSO); and homoarginine (Har). These also fall
conveniently into particular categories.
[0088] Based on the above definitions,
[0089] Sar or MeGly, .beta.-Ala, and Aib are small;
[0090] t-BuA, t-BuG, N-MeIle, Nle, Mvl, Cha, Phg, Nal, Thi and Tic
are hydrophobic;
[0091] Orn, 2,3-diaP and Har are basic;
[0092] Cit, Acetyl Lys, and MSO are polar/large.
[0093] The various omega-amino acids are classified according to
size as small (.beta.-Ala and 3-aminopropionic) or as large and
hydrophobic (all others).
[0094] Other amino acid substitutions of those encoded in the gene
can also be included in peptide compounds within the scope of the
invention and can be classified within this general scheme
according to their structure.
[0095] In all of the peptides of the invention, one or more amide
linkages (--CO--NH--) may optionally be replaced with another
linkage which is an isostere such as --CH.sub.2NH--, --CH.sub.2S--,
--CH.sub.2CH.sub.2, --CH.dbd.CH-- (cis and trans), --COCH.sub.2--,
--CH(OH)CH.sub.2-- and --CH.sub.2SO--. This replacement can be made
by methods known in the art. The following references describe
preparation of peptide analogs which include these
alternative-linking moieties: Spatola, A. F., Vega Data (March
1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general
review); Spatola, A. F., in Chemistry and Biochemistry of Amino
Acids Peptides and Proteins, B. Weinstein, eds., Marcel Dekker, New
York, p. 267 (1983) (general review); Morley, J. S., Trends Pharm
Sci (1980) pp. 463-468 (general review); Hudson, D., et al., Int J
Pept Prot Res (1979) 14:177-185 (--CH.sub.2NH--,
--CH.sub.2CH.sub.2--); Spatola, A. F., et al., Life Sci (1986)
38:1243-1249 (--CH.sub.2--S); Hann, M. M., J Chem Soc Perkin Trans
I (1982) 307-314 (--CH--CH--, cis and trans); Almquist, R. G., et
al., J Med Chem (1980) 23:1392-1398 (--COCH.sub.2--);
Jennings-White, C., et al., Tetrahedron Lett (1982) 23:2533
(--COCH.sub.2--); Szelke, M., et al., European Application EP 45665
(1982) CA:97:39405 (1982) (--CH(OH)CH.sub.2--); Holladay, M. W., et
al., Tetrahedron Lett (1983) 24:4401-4404 (--C(OH)CH.sub.2--); and
Hruby, V. J., Life Sci (1982) UB:189-199 (--CH2--S--).
[0096] In addition to analogs which contain isosteres in place of
peptide linkages, the peptides or proteins of the invention include
peptide mimetics in general, such as those described by Olson, G.
L. et al. J Med Chem (1993) 36:3039-3049 and retro-inverso type
peptides as described by Chorev, M. et al. Science (1979)
204:1210-1212; and Pallai, P. V. et al., Int J Pept Protein Res
(1983) 21:84-92.
[0097] One class of preferred embodiments of the compounds
invention includes the "unmodified" forms where positions 8 and 13
are independently cysteine, homocysteine or penicillamine residues,
especially in the disulfide bonded form.
[0098] In addition, or alternatively, each of A.sub.7 and A.sub.14
is a hydrophobic acid, preferably Ile, Val, Leu, NLe, Trp, Tyr or
Phe or is a small amino acid, Ala, Gly, Ser or Thr.
[0099] In another set of preferred embodiments, all of
A.sub.1-A.sub.3 are not present or at least one, and preferably two
of A.sub.1-A.sub.3 is a hydrophobic amino acid, preferably Ile,
Val, Leu, NLe, Trp, Tyr or Phe.
[0100] In another set of preferred embodiments, C.sub.4 and/or
C.sub.17 is not present or, if present, is a cysteine, homocysteine
or penicillamine or a hydrophobic amino acid, preferably Ile, Val,
Leu, NLe, Trp, Tyr or Phe, or a small amino acid, preferably S, A,
G or T.
[0101] In another set of preferred embodiments, C.sub.5 and/or
C.sub.16 is a cysteine, homocysteine or penicillamine or a
hydrophobic amino acid, preferably Ile, Val, Leu, NLe, Trp, Tyr or
Phe, or a small amino acid, preferably S, A, G or T.
[0102] In another set of preferred embodiments, A.sub.9-A.sub.12
contain at least one hydrophobic amino acid residue, preferably
Phe, Tyr or Trp.
[0103] Other preferred embodiments include those wherein each of
A.sub.1 and A.sub.9 is independently selected from the group
consisting of R, K and Har; more preferably, both A.sub.1 and
A.sub.9 are R; however, each of A.sub.1 may be absent.
[0104] In another class of preferred embodiments, each of A.sub.2
and A.sub.3 is independently selected from the group consisting of
G, A, S and T; more preferably, A.sub.2 and A.sub.3 are G; however,
A.sub.2 and/or A.sub.3 may be absent.
[0105] In another set of preferred embodiments, one of A.sub.9 and
A.sub.12 is R, K, Har, Orn or H preferably R and the other is I, V,
L, NLe, W, Y or F, preferably R, F or W, or is S, G, A or T.
[0106] In another set of preferred embodiments, each of A.sub.10
and A.sub.11 is independently proline or a small, basic or
hydrophobic amino acid, preferably R, G, W or P.
[0107] A.sub.18 is preferably absent, but when present, is
preferably R, K or Har, most preferably R.
[0108] Also preferably when all four amino acids A.sub.1-A.sub.3
and C.sub.4 are present, A.sub.1 is basic, C.sub.4 is C or basic,
and A.sub.2 and A.sub.3 are small amino acids, or at least one of
A.sub.1-A.sub.3 and C.sub.4 is hydrophobic. Preferred embodiments
of A.sub.1-A.sub.3 include R-G-G, K-G-S, K-S-G, and the like.
[0109] As described above, the compounds of Formula (1) are either
in cyclic or noncyclic (linearalized) form or may be modified
wherein 1 or 2 of the cysteines, homocysteine or penicillamine at
C.sub.8 and C.sub.13 are replaced by a small, hydrophobic, or a
basic amino acid residue. Such modification is preferred when
compounds containing only one disulfide bond are prepared. If the
linearalized forms of the compound of Formula (1) are prepared, or
if linearalized forms of those modified peptides which contain at
least two cysteines are prepared, it is preferred that the
sulfhydryl groups be stabilized by addition of a suitable reagent.
Preferred embodiments for the hydrophobic amino acid to replace
cysteine, homocysteine or penicillamine residues at C.sub.8 and/or
C.sub.13 are I, V, L and Nle, preferably I, V or L. Preferred small
amino acids to replace the cysteine, homocysteine or penicillamine
residues include G, A, S and T, more preferably G. Preferred basic
amino acids are R and K.
[0110] Where the compounds of the invention have two disulfide
bridges, particularly preferred are the pairs of bridges:
[0111] a) C.sub.5-C.sub.16 and C.sub.8-C.sub.13;
[0112] b) C.sub.5-C.sub.17 and C.sub.8-C.sub.13;
[0113] c) C.sub.5-C.sub.8 and C.sub.13-C.sub.16;
[0114] d) C.sub.4-C.sub.16 and C.sub.8-C.sub.13; and
[0115] e) C.sub.4-C.sub.17 and C.sub.8-C.sub.13.
[0116] Especially preferred are the bridges:
[0117] C.sub.5-C.sub.16 and C.sub.8-C.sub.13; and
[0118] C.sub.4-C.sub.17 and C.sub.8-C.sub.13.
[0119] Where the compound has only one disulfide bridge,
particularly preferred are:
[0120] C.sub.4-C.sub.17 or C.sub.5-C.sub.16.
[0121] Particularly preferred compounds of the invention, including
the N-terminal acylated and C-terminal amidated forms thereof are
the parevins, wherein C.sub.5 and C.sub.16 are both cysteine,
homocysteine or penicillamine and the tachytegrins wherein both
C.sub.4 and C.sub.17 are cysteine, homocysteine or penicillamine.
Also preferred are the disulfide forms of these compounds,
cis-parevins wherein the two disulfide bridges are C.sub.5-C.sub.8
and C.sub.13-C.sub.16; the trans-parevins wherein the disulfide
bridges are C.sub.5-C.sub.16 and C.sub.8-C.sub.13; and the
trans-tachytegrins wherein the disulfide bridges are
C.sub.4-C.sub.17 and C.sub.8-C.sub.13. Particularly preferred are
the following parevins and tachytegrins:
[0122] Parevin 1 trans ______ and cis . . . 1
[0123] Parevin 2 trans ______ and cis . . . 2
[0124] Parevin 3 trans ______ and cis . . . 3
[0125] Parevin 4 trans ______ and cis . . . 4
[0126] Parevin 5 trans ______ and cis . . . 5
[0127] Tachytegrin-1 trans ______ 6
[0128] Tachytegrin-2 trans ______ 7
[0129] Tachytegrin-3 trans ______ 8
[0130] Tachytegrin-4 trans ______ 9
[0131] Tachytegrin-5 trans ______ 10
[0132] Particularly preferred are cis- and trans-parevin-1 and
trans-tachytegrin-1.
[0133] Typical compounds of the invention include the linear and
cyclic (where possible) forms of the following compounds:
5 RGGRCLYCRRRFCVVCGR (SEQ ID NO:11); RGGCRLYCRRRFCVVGCR (SEQ ID
NO:12); RGGRCLYCRRRFCIVCG (SEQ ID NO:13); RGGCRLYCRRRFCIVGC (SEQ ID
NO:14); RGGGCLYCRRRFCVVCGR (SEQ ID NO:15); RGGCGLYCRRRFCVVGCR (SEQ
ID NO:16); RGGRCLYCRGWICFVCGR (SEQ ID NO:17); RGGCRLYCRGWICFVGCR
(SEQ ID NO:18); RGGRCLYCRPRFCVVCGR (SEQ ID NO:19);
RGGCRLYCRPRFCVVGCR (SEQ ID NO:20); RGGRCVYCRRRFCVVCG (SEQ ID
NO:21); RGGCRVYCRRRFCVIGC (SEQ ID NO:22); KGGRCLYCRRRFCVVCG (SEQ ID
NO:23); KGGCRIYCRRRFCVIGC (SEQ ID NO:24); RGGXCLYCRRRFCVVC (SEQ ID
NO:25); RGGCXLYCRRRFCVIC (SEQ ID NO:26); RGGXCLYCXRRFCVVCGR (SEQ ID
NO:27); RGGCXLYCXRRFCVIGCR (SEQ ID NO:28); RGGRCVYCRXRFCVVCGR (SEQ
ID NO:29); RGGCRVYCRXRFCVVGCR (SEQ ID NO:30); RGGRCLYCRKKWCVVCGR
(SEQ ID NO:31); RGGCRLYCRKKWCVVGCR (SEQ ID NO:32);
RGGRCLYCRXRYCVVCGR (SEQ ID NO:33); RGGCRLYCRXRYCVVACR (SEQ ID
NO:34); RGSGCLYCRRKWCVVCGR (SEQ ID NO:35); RGSCGLYCRRKWCVVGCR (SEQ
ID NO:36); RATRCIFCRRRFCVVCGR (SEQ ID NO:37); RATCRIFCRRRFCVIGCR
(SEQ ID NO:38); RGGKCVYCRXRFCVVCGR (SEQ ID NO:39);
RGGCKVYCRXRFCVIGCR (SEQ ID NO:40); RATRCIFCrRRFCVVCGr (SEQ ID
NO:41); RATCRIFCrRRFCVVGCr (SEQ ID NO:42); RGGKCVYCRxRFCVVCGR (SEQ
ID NO:43); RGGCKVYCRxRFCVVGCR (SEQ ID NO:44); rggrclycrrrfcvvcgr
(SEQ ID NO:45); rggcrlycrrrfcvvgcr (SEQ ID NO:46);
rggrclycrrrfcivcg (SEQ ID NO:47); rggcrlycrrrfcivgc (SEQ ID NO:48);
rgggclycrrrfcvvcgr (SEQ ID NO:49); rggcglycrrrfcvvgcr (SEQ ID
NO:50); rggrclycrgwicfvcgr (SEQ ID NO:51); rggcrlycrgwicfvgcr (SEQ
ID NO:52); RGGCLRYCRPRFCVRVCR (SEQ ID NO:53); RGGCRLYCRRRFCVVGCR
(SEQ ID NO:54); RGVCLRYCRGRFCVRLCR (SEQ ID NO:55);
RGRVCLRYCRGRFCVRLCFR (SEQ ID NO:56); RWRVCLRYCRGRFCVRLCLR (SEQ ID
NO:57); RGWRVCLKYCRGRFCVKLCLR (SEQ ID NO:58); RGGRVCLRYCRGKFCVRLCLR
(SEQ ID NO:59); RGGRCLYARRRFAVVCGR (SEQ ID NO:60); RGGRCLYARRRFSIVC
(SEQ ID NO:61); RGGGCLYSRRRFAVVCGR (SEQ ID NO:62); RGGRCLYARRRFGVVC
(SEQ ID NO:63); KGGRCLYVRRRFIVVC (SEQ ID NO:64); RGGXCLYARRRFVGCV
(SEQ ID NO:65); RGGXCLYAXRRFSVVCR (SEQ ID NO:66);
RGGCXLYAXRRFSVVGCR (SEQ ID NO:67); RGGRCVYVRXRFLVCVGR (SEQ ID
NO:68); RGGRCLYSRKKWAVSCGR (SEQ ID NO:69); RGGRCLYSRXRYSVICGR (SEQ
ID NO:70); RGSGCIYCRRKWGVVGCR (SEQ ID NO:71); RATRCIFSRRRFSVVCGR
(SEQ ID NO:72); RGGKCVYGRXRFSVVCGR (SEQ ID NO:73);
RATRCIFGrRRFGVVCGr (SEQ ID NO:74); RGGKCVYLRxRFLVVCGR (SEQ ID
NO:75); RGGRCVFLRPRIGVVCGR (SEQ ID NO:76); RGGCLRYAVPRFAVRVCR (SEQ
ID NO:77); RGGCLRYTKPKFTVRVCR (SEQ ID NO:78); RGGCLRYAVGRFAVRVCR
(SEQ ID NO:79); RGGCLRYARZRFAVRVCR (SEQ ID NO:80);
RGFCLRYTVPRFTVRFCVR (SEQ ID NO:81); RGFCLRYKVGRFKVRFCVR (SEQ ID
NO:82); RGFCLRYZVGRFZVRFCVR (SEQ ID NO:83); RGGCLRYARZRFAVRVCR (SEQ
ID NO:84); RGGCLRYAVGRFAVRVCR (SEQ ID NO:85); RGGRCLYCRRRFCVVGCR
(SEQ ID NO:86); RGGCRLYCRRRFCVVCGR (SEQ ID NO:87);
RGGRCLYCRRRFCVCVGR (SEQ ID NO:88); RGGCRLYCRRRFCVCVGR (SEQ ID
NO:89); RGGRLCYCRRRFCVVCGR (SEQ ID NO:90); RGGRLCYCRRRFCVVGCR (SEQ
ID NO:91); RGGCRLYCRRRFCVVGC (SEQ ID NO:92); RGGRCLYCRRRFCVVGC (SEQ
ID NO:93); RGGCRLYCRRRFCVVCG (SEQ ID NO:94); RGGRCLYCRRRFCVCVG (SEQ
ID NO:95); RGGCRLYCRRRFCVCVG (SEQ ID NO:96); RGGRLCYCRRRFCVVCG (SEQ
ID NO:97); RGGRLCYCRRRFCVVGC (SEQ ID NO:98); RGGGCLYCRRRFCVVGCR
(SEQ ID NO:99); RGGGCLYCRRRFCVCVGR (SEQ ID NO:100);
RGGCGLYCRRRFCVCVGR (SEQ ID NO:101); RGGGLCYCRRRFCVVCGR (SEQ ID
NO:102); RGGGLCYCRRRFCVVGCR (SEQ ID NO:103);
[0134] and N-terminal acylated and C-terminal amidated or
esterified forms thereof, where uppercase letters represent L-amino
acids, lower case letters represent D-amino acids, X is Har, x is
D-Har and Z is MeGly.
[0135] Particularly preferred are the cyclic forms (where possible)
and C-terminal amidated forms of the above compounds, especially
the cyclic forms (where possible) and C-terminal amidated forms
of:
6 RGGCLRYCRPRFCVRVCR (SEQ ID NO:53); RGGCRLYCRRRFCXTVGCR (SEQ ID
NO:54); RGVCLRYCRGRFCVRLCR (SEQ ID NO:55); RGRVCLRYCRGRFCVRLCFR
(SEQ ID NO:56); RWRVCLRYCRGRFCVRLCLR (SEQ ID NO:57);
RGWRVCLKYCRGRFCVKLCLR (SEQ ID NO:58); RGGRVCLRYCRGKFCVRLCLR (SEQ ID
NO:59); RGGCLRYAVPRFAVRVCR (SEQ ID NO:77); RGGCLRYTKPKFTVRVCR (SEQ
ID NO:78); RGGCLRYAVGRFAVRVCR (SEQ ID NO:79); RGGCLRYARZRFAVRVCR
(SEQ ID NO:80); RGFCLRYTVPRFTVRFCVR (SEQ ID NO:81);
RGFCLRYKVGRFKVRFCVR (SEQ ID NO:82); RGFCLRYZVGRFZVRFCVR (SEQ ID
NO:83); RGGCLRYARZRFAVRVCR (SEQ ID NO:84); and RGGCLRYAVGRFAVRVCR
(SEQ ID NO:85),
[0136] where upper and lower case letters and X and Z are as
previously defined.
[0137] Preparation of the Invention Compounds
[0138] The invention compounds are essentially peptide backbones
which may be modified at the N- or C-terminus and also may contain
one or two cystine disulfide linkages. The peptides may first be
synthesized in noncyclized form. These peptides may then be
converted to the cyclic peptides if desired by standard methods of
cystine bond formation. As applied to the compounds herein, "cyclic
forms" refers to those forms which contain cyclic portions by
virtue of the formation of disulfide linkages between cysteine
residues in the peptide. If the straight-chain forms are preferred,
it is preferable to stabilize the sulfhydryl groups for any
peptides of the invention which contain two or more cysteine
residues.
[0139] Standard methods for synthesis of peptides can be used. Most
commonly used currently are solid phase synthesis techniques;
indeed, automated equipment for systematically constructing peptide
chains can be purchased. Solution phase synthesis can also be used
but is considerably less convenient. When synthesized using these
standard techniques, amino acids not encoded by the gene and
D-enantiomers can be employed in the synthesis. Thus, one very
practical way to obtain the compounds of the invention is to employ
these standard chemical synthesis techniques.
[0140] In addition to providing the peptide backbone, the N- and/or
C-terminus can be derivatized, again using conventional chemical
techniques. The compounds of the invention may optionally contain
an acyl group, preferably an acetyl group at the amino terminus.
Methods for acetylating or, more generally, acylating, the free
amino group at the N-terminus are generally known in the art; in
addition, the N-terminal amino acid may be supplied in the
synthesis in acylated form.
[0141] At the carboxy terminus, the carboxyl group may, of course,
be present in the form of a salt; in the case of pharmaceutical
compositions this will be a pharmaceutically acceptable salt.
Suitable salts include those formed with inorganic ions such as
NH.sub.4.sup.+, Na.sup.+, K.sup.+, Mg.sup.++, Ca.sup.++, and the
like as well as salts formed with organic cations such as those of
caffeine and other highly substituted amines. However, when the
compound of formula 1 contains a multiplicity of basic residues,
salt formation may be difficult or impossible. The carboxy terminus
may also be esterified using alcohols of the formula ROH wherein R
is hydrocarbyl (1-6C) as defined above. Similarly, the carboxy
terminus may be amidated so as to have the formula --CONH.sub.2,
--CONHR, or --CONR.sub.2, wherein each R is independently
hydrocarbyl (1-6C) as herein defined. Techniques for esterification
and amidation as well as neutralizing in the presence of base to
form salts are all standard organic chemical techniques.
[0142] If the peptides of the invention are prepared under
physiological conditions, the side-chain amino groups of the basic
amino acids will be in the form of the relevant acid addition
salts.
[0143] Formation of disulfide linkages, if desired, is conducted in
the presence of mild oxidizing agents. Chemical oxidizing agents
may be used, or the compounds may simply be exposed to the oxygen
of the air to effect these linkages. Various methods are known in
the art. Processes useful for disulfide bond formation have been
described by Tam, J. P. et al., Synthesis (1979) 955-957; Stewart,
J. M. et al., "Solid Phase Peptide Synthesis" 2d Ed. Pierce
Chemical Company Rockford, Ill. (1984); Ahmed A. K. et al., J Biol
Chem (1975) 250:8477-8482 and Pennington M. W. et al., Peptides
1990, E. Giralt et al., ESCOM Leiden, The Netherlands (1991)
164-166. An additional alternative is described by Kamber, B. et
al., Helv Chim Acta (1980) 63:899-915. A method conducted on solid
supports is described by Albericio Int J Pept Protein Res (1985)
26:92-97.
[0144] A particularly preferred method is solution oxidation using
molecular oxygen. This method has been used by the inventors herein
to refold the compounds of the invention.
[0145] If the peptide backbone is comprised entirely of
gene-encoded amino acids, or if some portion of it is so composed,
the peptide or the relevant portion may also be synthesized using
recombinant DNA techniques. The DNA encoding the peptides of the
invention may itself be synthesized using commercially available
equipment; codon choice can be integrated into the synthesis
depending on the nature of the host.
[0146] Synthesized and recombinantly produced forms of the
compounds may require subsequent derivatization to modify the N-
and/or C-terminus and, depending on the isolation procedure, to
effect the formation of cystine bonds as described hereinabove.
Depending on the host organism used for recombinant production,
some or all of these conversions may already have been
effected.
[0147] For recombinant production, the DNA encoding the peptides of
the invention is included in an expression system which places
these coding sequences under control of a suitable promoter and
other control sequences compatible with an intended host cell.
Types of host cells available span almost the entire range of the
plant and animal kingdoms. Thus, the compounds of the invention
could be produced in bacteria or yeast (to the extent that they can
be produced in a nontoxic or refractile form or utilize resistant
strains) as well as in animal cells, insect cells and plant cells.
Indeed, modified plant cells can be used to regenerate plants
containing the relevant expression systems so that the resulting
transgenic plant is capable of self protection vis--vis these
infective agents.
[0148] The compounds of the invention can be produced in a form
that will result in their secretion from the host cell by fusing to
the DNA encoding the peptide, a DNA encoding a suitable signal
peptide, or may be produced intracellularly. They may also be
produced as fusion proteins with additional amino acid sequence
which may or may not need to be subsequently removed prior to the
use of these compounds as antimicrobials or antivirals.
[0149] Thus, the compounds of the invention can be produced in a
variety of modalities including chemical synthesis, recombinant
production, isolation from natural sources, or some combination of
these techniques.
[0150] Any members of the invention class which coincidentally
occur naturally must be supplied in purified and isolated form. By
"purified and isolated" is meant free from the environment in which
the peptide normally occurs (in the case of such naturally
occurring peptides) and in a form where it can be used practically.
Thus, "purified and isolated" form means that the peptide is
substantially pure, i.e., more than 90% pure, preferably more than
95% pure and more preferably more than 99% pure or is in a
completely different context such as that of a pharmaceutical
preparation.
[0151] Antibodies
[0152] Antibodies to the peptides of the invention may also be
produced using standard immunological techniques for production of
polyclonal antisera and, if desired, immortalizing the
antibody-producing cells of the immunized host for sources of
monoclonal antibody production. Techniques for producing antibodies
to any substance of interest are well known. It may be necessary to
enhance the immunogenicity of the substance, particularly as here,
where the material is only a short peptide, by coupling the hapten
to a carrier. Suitable carriers for this purpose include substances
which do not themselves produce an immune response in the mammal to
be administered the hapten-carrier conjugate. Common carriers used
include keyhole limpet hemocyanin (KLH), diphtheria toxoid, serum
albumin, and the viral coat protein of rotavirus, VPG. Coupling of
the hapten to the carrier is effected by standard techniques such
as contacting the carrier with the peptide in the presence of a
dehydrating agent such as dicyclohexylcarbodiimide or through the
use of linkers such as those available through Pierce Chemical
Company, Chicago, Ill.
[0153] The peptides of the invention in immunogenic form are then
injected into a suitable mammalian host and antibody titers in the
serum are monitored. It should be noted, however, that some forms
of the peptides require modification before they are able to raise
antibodies, due to their resistance to antigen processing. For
example, peptides containing two cystine bridges may be
nonimmunogenic when administered without coupling to a larger
carrier and may be poor immunogens even in the presence of potent
adjuvants and when coupled in certain formats such as using
glutaraldehyde or to KLH. Any lack of immunogenicity may therefore
result from resistance to processing to a linear form that can fit
in the antigen-presenting pocket of the presenting cell.
Immunogenicity of these forms of the peptides can be enhanced by
cleaving the disulfide bonds.
[0154] Polyclonal antisera may be harvested when titers are
sufficiently high. Alternatively, antibody-producing cells of the
host such as spleen cells or peripheral blood lymphocytes may be
harvested and immortalized. The immortalized cells are then cloned
as individual colonies and screened for the production of the
desired monoclonal antibodies.
[0155] Recombinant techniques are also available for the production
of antibodies, and thus, the antibodies of the invention include
those that can be made by genetic engineering techniques. For
example, single-chain forms, such as F.sub.v forms, chimeric
antibodies, and antibodies modified to mimic those of a particular
species, such as humans, can be produced using standard methods.
Thus, the antibodies of the invention can be prepared by isolating
or modifying the genes encoding the desired antibodies and
producing these through expression in recombinant host cells, such
as CHO cells.
[0156] The antibodies of the invention are, of course, useful in
immunoassays for determining the amount or presence of the
peptides. Such assays are essential in quality controlled
production of compositions containing the peptides of the
invention. In addition, the antibodies can be used to assess the
efficacy of recombinant production of the peptides, as well as
screening expression libraries for the presence of peptide encoding
genes.
[0157] Compositions Containing the Invention Peptides and Methods
of Use
[0158] The peptides of the invention are effective in inactivating
a wide range of microbial and viral targets, including
gram-positive and gram-negative bacteria, yeast, protozoa and
certain strains of virus. Accordingly, they can be used in
disinfectant compositions and as preservatives for materials such
as foodstuffs, cosmetics, medicaments, or other materials
containing nutrients for organisms. For use in such contexts, the
peptides are supplied either as a single peptide, in admixture with
several other peptides of the invention, or in admixture with
additional antimicrobial agents or both. In general, as these are
preservatives in this context, they are usually present in
relatively low amounts, of less than 5%, by weight of the total
composition, more preferably less than 1%, still more preferably
less than 0.1%.
[0159] The peptides of the invention are also useful as standards
in antimicrobial assays and in assays for determination of
capability of test compounds to bind to endotoxins such as
lipopolysaccharides.
[0160] For use as antimicrobials or antivirals for treatment of
animal subjects, the peptides of the invention can be formulated as
pharmaceutical or veterinary compositions. Depending on the subject
to be treated, the mode of administration, and the type of
treatment desired--e.g., prevention, prophylaxis, therapy; the
invention peptides are formulated in ways consonant with these
parameters. A summary of such techniques is found in Remington's
Pharmaceutical Sciences, latest edition, Mack Publishing Co.,
Easton, Pa.
[0161] The peptides of the invention can also be used as active
ingredients in pharmaceutical compositions useful in treatment of
sexually transmitted diseases, including those caused by Chlamydia
trachomatis, Treponema pallidum, Neisseria gonorrhoeae, Trichomonas
vaginalis, Herpes simplex type 2 and HIV. Topical formulations are
preferred and include creams, salves, oils, powders, gels and the
like. Suitable topical excipient are well known in the art and can
be adapted for particular uses by those of ordinary skill.
[0162] In general, for use in treatment or prophylaxis of STDs, the
peptides of the invention may be used alone or in combination with
other antibiotics such as erythromycin, tetracycline, macrolides,
for example azithromycin and the cephalosporins. Depending on the
mode of administration, the peptides will be formulated into
suitable compositions to permit facile delivery to the affected
areas. The tachytegrins may be used in forms containing one or two
disulfide bridges or may be in linear form. In addition, use of the
enantiomeric forms containing all D-amino acids may confer
advantages such as resistance to those proteases, such as trypsin
and chymotrypsin, to which the peptides containing L-amino acids
are less resistant.
[0163] The peptides of the invention can be administered singly or
as mixtures of several peptides or in combination with other
pharmaceutically active components. The formulations may be
prepared in a manner suitable for systemic administration or
topical or local administration. Systemic formulations include
those designed for injection (e.g., intramuscular, intravenous or
subcutaneous injection) or may be prepared for transdermal,
transmucosal, or oral administration. The formulation will
generally include a diluent as well as, in some cases, adjuvants,
buffers, preservatives and the like. The tachytegrins can be
administered also in liposomal compositions or as
microemulsions.
[0164] If administration is to be oral, the peptides of the
invention must be protected from degradation in the stomach using a
suitable enteric coating. This may be avoided to some extent by
utilizing amino acids in the D-configuration, thus providing
resistance to protease. However, the peptide is still susceptible
to hydrolysis due to the acidic conditions of the stomach; thus,
some degree of enteric coating may still be required.
[0165] The peptides of the invention also retain their activity
against microbes in the context of borate solutions that are
commonly used in eye care products. Also, it is important that the
peptides retain their activity under physiological conditions
including relatively high saline and in the presence of serum. In
addition, the peptides are dramatically less cytotoxic with respect
to the cells of higher organisms as compared with their toxicity to
microbes. These properties, make them particularly suitable for in
vivo and therapeutic use.
[0166] By appropriately choosing the member or members of the
peptide class of the invention, it is possible to adapt the
antimicrobial activity to maximize its effectiveness with respect
to a particular target microbe. As used herein, "microbe" will be
used to include not only yeast, bacteria, and other unicellular
organisms, but also viruses. The particular peptide used can also
be chosen to be advantageous in a particular context, such as low
salt or physiological salt, the presence or human serum, or
conditions that mimic the conditions found in blood and tissue
fluids.
[0167] The peptides of the invention may also be applied to plants
or to their environment to prevent virus- and microbe-induced
diseases in these plants. Suitable compositions for this use will
typically contain a diluent as well as a spreading agent or other
ancillary agreements beneficial to the plant or to the
environment.
[0168] Thus, the peptides of the invention may be used in any
context wherein an antimicrobial and/or antiviral action is
required. This use may be an entirely in vitro use, or the peptides
may be administered to organisms.
[0169] In addition, the antimicrobial or antiviral activity may be
generated in situ by administering an expression system suitable
for the production of the peptides of the invention. Such
expression systems can be supplied to plant and animal subjects
using known techniques. For example, in animals, pox-based
expression vectors can be used to generate the peptides in situ.
Similarly, plant cells can be transformed with expression vectors
and then regenerated into whole plants which are capable of their
own production of the peptides.
[0170] The peptides of the invention are also capable of
inactivating endotoxins derived from gram-negative bacteria--i.e.,
lipopolysaccharides (LPS) and may be used under any circumstances
where inactivation of LPS is desired. One such situation is in the
treatment or amelioration of gram-negative sepsis.
[0171] Conditions Relevant to Antimicrobial/Antiviral Activity
[0172] It has been stated above that as used herein "antimicrobial"
activity refers to inhibition with respect both to traditional
microorganisms and to viruses, although occasionally,
"antimicrobial" and "antiviral" are both specifically
indicated.
[0173] Media for testing antimicrobial activity are designed to
mimic certain specific conditions. The standard buffer medium,
medium A, uses an underlay agar with the following composition: 0.3
mg/ml of trypticase soy broth powder, 1% w/v agarose and 10 mM
sodium phosphate buffer (final pH 7.4). This will be designated
either "medium A" or "standard in vitro conditions" herein.
[0174] All of the remaining media contain these same components.
However, in addition:
[0175] A second medium contains 100 mM NaCl in order to mimic the
salt levels in blood and tissue fluids. This will be designated
"medium B" or "salt medium" herein.
[0176] A third medium is supplemented with 2.5% normal human serum;
however, it is of low ionic strength and thus does not mimic body
fluids. This medium will be designated "medium C" or
"serum-containing medium" herein.
[0177] A fourth medium contains 80% RPMI-1640, a standard tissue
culture medium which contains the principal ions and amino acids
found in blood and tissue fluids. In addition, it contains 2.5%
normal human serum. This will be designated "medium D" or
"physiological medium" herein.
[0178] Particularly preferred is the amidated form of this
peptide.
[0179] Summary
[0180] The peptides of the invention therefore represent a
peculiarly useful class of compounds because of the following
properties:
[0181] 1) They have an antimicrobial effect with respect to a broad
spectrum of target microbial systems, including viruses, including
retroviruses, bacteria, fungi, yeast and protozoa.
[0182] 2) Their antimicrobial activity is effective under
physiological conditions--i.e., physiological saline and in the
presence of serum.
[0183] 3) They are much less toxic to the cells of higher organisms
than to microbes.
[0184] 4) They can be prepared in nonimmunogenic form thus
extending the number of species to which they can be
administered.
[0185] 5) They can be prepared in forms which are resistant to
certain proteases suggesting they are antimicrobial even in
lysosomes.
[0186] 6) They can be prepared in forms that resist degradation
when autoclaved, thus simplifying their preparation as components
of pharmaceuticals.
[0187] 7) They can be modified in amino acid sequence so as to
optimize the specificity with respect to target.
[0188] 8) They can be modified structurally so as to accommodate
the conditions under which antimicrobial activity is to be
exhibited.
[0189] The following examples are intended to illustrate but not to
limit the invention.
EXAMPLE 1
Synthesis of the Invention Compounds
[0190] The peptides of the invention are synthesized using
conventional Fmoc chemistry on solid-phase supports. The crude
synthetic peptides are refolded, purified and characterized as
follows.
[0191] The crude synthetic peptide is reduced by adding an amount
of dithiothreitol (DTT) equal in weight to that of the synthetic
peptide, which has been dissolved at 10 mg/ml in a solution
containing 6M guanidine HCl, 0.5M Tris buffer and 2 mmol EDTA, pH
8.05 and incubated for 2 hours at 52.degree. C. under nitrogen. The
mixture is passed through a 0.45 um, filter, acidified with 1/20
v/v glacial acetic acid and subjected to conventional RP HPLC
purification with a C18 column.
[0192] The HPLC-purified, reduced peptides are partially
concentrated by vacuum centrifugation in a Speed Vac and allowed to
fold for 24 hours at room temperature and air. The folding is
accomplished in 0.1M Tris, pH 7.7 at 0.1 mg peptide/ml to minimize
formation of interchain cystine disulfides. The folded compounds
are concentrated and acidified with 5% acetic acid. The purity of
the final products is verified by AU-PAGE, analytical HPLC and
FAB-mass spec.
[0193] Using this procedure, the compounds trans-parevin-1 (or the
"hairpin" isoform), cis-parevin-1 (or "cloverleaf" isoform) and
trans-tachytegrin-1 were prepared. These compounds are of the
formulas
[0194] Trans-parevin-1: 11
[0195] Cis-parevin-1: 12
[0196] Trans-tachytegrin-1: 13
EXAMPLE 2
Antimicrobial Activity
[0197] The radial diffusion assay in agarose gels is conducted
using radiodiffusion and gel overlay techniques as described by
Lehrer, R. I. et al. J Immunol Meth (1991) 137:167-173. Briefly,
the underlay agars used for all organisms had a final pH of 7.4 and
contained 10 mM sodium phosphate buffer, 1% w/v agarose and 0.30
ug/ml trypticase soy broth powder (BBL Cockeysville, Md.). In some
cases, the underlay was supplemented with 100 mM NaCl. The units of
activity in the radial diffusion assay were measured as described;
10 units correspond to a 1 mm diameter clear zone around the sample
well. FIGS. 1-6 show the results against five test organisms in
units described as above. A synthetic protegrin (PG-1) containing
two cystines (sPG-1) or PG-1 in linear form were used as
controls.
[0198] FIG. 1 shows the results for trans-parevin and tachytegrin
with respect to E. coli both with and without the addition of 100
mM NaCl. Both of these peptides were slightly more effective than
sPG-1 although slightly less effective than linear PG-1 in the
absence of salt. However, in the presence of 100 mM NaCl, all four
peptides were comparably effective.
[0199] FIG. 2 shows the results of the same determination with
respect to L. monocytogenes. With respect to this organism, all
four peptides were roughly similarly effective in the absence of
salt; the presence of 100 mM NaCl, however, greatly reduced the
effectiveness of linear PG-1. The remaining three peptides remained
effective under these conditions.
[0200] FIG. 3 shows the results of the same experiment using C.
albicans as the target organism. All four peptides were comparably
effective in the absence of salt; again, the effectiveness of
linear PG-1 was greatly reduced in the presence of 100 mM NaCl,
while the remaining three peptides maintained their effectiveness
under these conditions.
[0201] FIGS. 4-6 show the results of similar experiments using, as
test peptides, the two isomers of parevin, trans-parevin (hairpin)
and cis-parevin (cloverleaf). sPG-1 was used as a control. As shown
in FIG. 4, the two parevins were comparably effective in the
absence of salt and both were more effective than sPG-1. In the
presence of 100 mM NaCl, all three peptides maintained their
effectiveness and were comparable.
[0202] FIG. 5 shows results of the same experiment conducted with
B. subtilis as target organism. Again, both forms of parevin were
comparably effective and both were slightly more effective than
sPG-1; in the presence of 100 mM NaCl, all three peptides remained
effective antimicrobials and had about the same activity.
[0203] The results obtained with respect to S. typhimurium are also
similar, as shown in FIG. 6. Again, the two parevins were more
effective than sPG-1 in the absence of salt and all three peptides
had comparable effectiveness when 100 mM NaCl was added.
EXAMPLE 3
Ability to Bind Endotoxin
[0204] The compounds of the invention are tested for their ability
to bind the lipid polysaccharide (LPS) of the gram-negative
bacterium E. coli strain 0.55B5, using the Limulus amebocyte lysate
(LAL) test for endotoxins conducted in the presence and absence of
the test compounds. The test is conducted using the procedure
described in Sigma Technical Bulletin No. 210 as revised in
December 1992 and published by Sigma Chemical Company, St. Louis,
Mo.
[0205] The LAL test is based on the ability of LPS to effect
gelation in the commercial reagent E-Toxate which is prepared from
the lysate of circulating amebocytes of the Horseshoe Crab Limulus
polyphemus. As described in the technical bulletin, when exposed to
minute quantities of LPS, the lysate increases in opacity as well
as viscosity and may gel depending on the concentration of
endotoxin. The technical bulletin goes on to speculate that the
mechanism appears analogous to the clotting of mammalian blood and
involves the steps of activation of a trypsin-like preclotting
enzymes by the LPS in the presence of calcium ion, followed by
enzymic modifications of a "coagulogen" by proteolysis to produce a
clottable protein. These steps are believed tied to the
biologically active or "pyrogenic" portion of the molecule. It has
been shown previously that detoxified LPS (or endotoxin) gives a
negative LAL test.
[0206] The test compounds are used at various concentrations from
0.25 ug-10 ug in a final volume of 0.2 ml and the test mixtures
contained LPS at a final concentration of 0.05 endotoxin unit/ml
and E-Toxate.TM. at the same concentration. The test compounds are
incubated together with the LPS for 15 minutes before the
E-Toxate.TM. is added to a final volume after E-Toxate.TM. addition
of 0.2 ml. The tubes are then incubated for 30 minutes at
37.degree. C. and examined for the formation of a gel.
[0207] In a follow-up experiment, the concentration of LPS is
varied from 0.05-0.25 endotoxin units (E.U.).
EXAMPLE 4
Antimicrobial Activity Under Conditions Suitable for Treatment of
the Eye
[0208] Contact lens solutions are typically formulated with borate
buffered physiological saline and may or may not contain EDTA in
addition. The compounds of the invention are tested generally in
the assay described in Example 2 wherein all underlay gels contain
25 mM borate buffer, pH 7.4, 1% (v/v) trypticase soy broth (0.3
ug/ml TSB powder) and 1% agarose. Additions include either 100 mM
NaCl, 1 mM EDTA or a combination thereof. Other test compounds used
as controls are the defensin NP-1 and lysozyme, and dose response
curves are determined.
EXAMPLE 5
Preparation of Enantio Trans-parevin
[0209] Using standard solid phase techniques, a peptide having the
amino acid sequence of trans-parevin, but wherein every amino acid
is in the D form is prepared. This form is tested against E. coli,
L. monocytogenes, C. albicans and other microbes in the absence and
presence of protease and otherwise as described for the
radiodiffusion assay in agarose gels set forth in Example 2.
EXAMPLE 6
Activity Against STD Pathogens
[0210] The compounds of the invention are tested for antimicrobial
activity against various STD pathogens. These include HIV-1,
Chlamydia trachomatis, Treponema pallidum, Neisseria gonorrhoeae,
Trichomonas vaginalis, Herpes simplex type 2, Herpes simplex type
1, Hemophilus ducreyi, and Human papilloma virus. The results are
provided in a form wherein "active" means that the peptide is
effective at less than 10 10 ug/ml; moderately active indicates
that it is active at 10-25 ug/ml; and slightly active means
activity at 25-50 ug/ml. If no effect is obtained at 50-200 ug/ml
the compound is considered inactive.
[0211] The compounds of the invention are tested for their
antimicrobial activity against Chlamydia using the "gold standard"
chlamydial culture system for clinical specimens described by
Clarke, L. M. in Clinical Microbiology Procedures Handbook II
(1992), Isenberg, H. T. Ed. Am. Soc. Microbiol. Washington, D.C.;
pp. 8.0.1 to 8.24.3.9.
[0212] In the assays, C. trachomatis serovar L2 (L2/434Bu)
described by Kuo, C. C. et al. in Nongynococcal Urethritis and
Related Infections (1977), Taylor-Robinson, D. et al. Ed. Am. Soc.
Microbiol. Washington, D.C., pp. 322-326 is used. The seed is
prepared from a sonicated culture in L929 mouse fibroblast cells,
and partially purified by centrifugation. Since host protein is
still present in the seed aliquots, each seed batch is titered at
the time of preparation with serial ten-fold dilutions to
2.times.10.sup.-9. The seed containing 9.2.times.10.sup.6 IFU/ml is
thawed quickly at 37.degree. C. and diluted to 10.sup.-2 with
sucrose/phosphate salts/glycine to produce IFU of about 200 after
room temperature preincubation and to dilute background eukaryotic
protein.
[0213] In the initial assays, the peptides to be tested are
prepared as stock solutions in 0.01% glacial acetic acid. 100 ul of
the diluted chlamydial seed are aliquoted into 1.5 ml eppendorf
tubes and 200 ul of the antibiotic peptide was added per tube.
Aliquots of the peptide stock (and controls) are incubated with the
seed at room temperature for one hour, two hours and four hours.
About 10 minutes before the end of each incubation period,
maintenance media are aspirated from the McCoy vials in preparation
for standard inoculation and culture. Culture is then performed in
the presence and absence of the peptides; in some cases, the
peptides are added to final concentration in the culture media in
addition to the preculture incubation. The test is evaluated
microscopically.
[0214] In another series of experiments, various concentrations of
tachytegrin (1 ug, 12.5 ug, 25 ug and 50 ug) are used in the
two-hour preincubation.
[0215] The effect of the presence of serum is also tested. The
Chlamydia seed is preincubated for two hours with and without 10%
FBS and also with or without test compound at 25 ug.
[0216] The experiments are repeated but adding 25 ug of compound
after the start of the chlamydial culture, i.e., after
centrifugation and final medium mix and one hour into the beginning
of the 48-hour culture period. Finally, the compound (at 25 ug) is
added to the chlamydial seed and the mix then immediately
cultured.
[0217] The effect of serum is particularly important since for a
topical agent to be effective in combatting Chlamydia infection, it
must act in the presence of serum.
[0218] In addition, there are several mouse-based models for
Chlamydia infection which can be used to assess the efficacy of the
tachytegrins. These include those described by Patton, D. L. et al.
in Chlamydial Infections (1990) Bowie, W. R. et al. Eds. Cambridge
Universe providing a source of complement. Ten ul of a suspension
of T. pallidum containing about 5.times.10.sup.7/ul organisms is
added to each tube and the mixtures with the appropriate peptides
are incubated at 34.degree. C. under 95% N.sub.2 and 5% CO.sub.2.
At time zero, just prior to incubation, 4 hours and 16 hours, 25
randomly selected organisms are examined for the presence or
absence of motility. The 50% immobilizing end point (IE.sub.50) is
calculated to indicate the concentration needed to immobilize 50%
of the spirochetes. Tachyplesin IE.sub.50s are 5.231 ug and 2.539
ug for 0 and 4 hours, in contrast to HNP and NP preparations which
show little immobilizing ability.
[0219] For Herpes Simplex Virus, using viral stocks prepared in
VERO cells, grown in minimal essential medium (MEM) with 2% fetal
calf serum, the effect of various peptides on HSV 1 MacIntyre
strain, a pool of ten clinical HSV 1 isolates, HSV-2G, and a pool
of ten clinical HSV 2 isolates, all sensitive to 3 uM acyclovir are
tested. Two fibroblast cell lines, human W138 and equine CCL57, are
used as targets and tests are done by direct viral neutralization
and delayed peptide addition.
[0220] In the direct neutralization format, the virus is
preincubated with the peptides for 90 min before it is added to the
tissue culture monolayers. In the delayed peptide addition format,
the virus is added and allowed 50 min to adsorb to the target
cells, then the monolayers are washed and peptides are added for 90
min. Finally, the monolayer is washed to remove the peptide and the
cells are fed with peptide-free MEM and cultured until the
untreated infected monolayers exhibit 4+ cytopathic effect (CPE)
(about 60 hours).
[0221] For Trichomonas vaginallis, strain C1 (ATCC 30001) is grown
as described by Gorrell, T. E. et al., Carlsberg Res Comm (1984)
49:259-268. In experiments performed in RPMI +1% heat-activated
fetal calf serum, within a few minutes after exposure to 50 ug/ml
PG-1, T. vaginallis (heretofore vigorously motile) becomes
stationary. Soon thereafter, the organisms become permeable to
trypan blue, and, over the ensuing 15-30 minutes, lyse. As
expected, such organisms fail to grow when introduced into their
customary growth medium (Diamond's medium). Organisms exposed to 25
ug/ml of PG-3 retain their motility.
EXAMPLE 7
Antiretroviral Activity
[0222] The invention compounds are tested for antiviral activity
against strains of HIV using the method described in Miles, S. A.
et al., Blood (1991) 78:3200-3208. Briefly, the mononuclear cell
fraction is recovered from normal donor leukopacs from the American
Red Cross using a Ficoll-hypaque density gradient. The mononuclear
cells are resuspended at 1.times.10.sup.6 cells per ml in RPMI 1640
medium with 20% fetal bovine serum, 1% penn/strep with fungizone
and 0.5% PHA and incubated 24 hours at 37.degree. C. in 5%
CO.sub.2. The cells are centrifuged, washed and then expanded for
24 hours in growth medium.
[0223] Non-laboratory adapted, cloned HIV.sub.JR-CSF and
HIV.sub.JR-FL are electroporated into the human peripheral blood
mononuclear cells prepared as described above. Titers are
determined and in general, multiplicities of infection (MOI) of
about 4,000 infectuous units per cell are used (which corresponds
to 25-40 picograms per ml HIV p24 antigen in the supernatant).
[0224] In the assay, the HIV stocks prepared as above are diluted
to the correct MOI and the PBM are added to 24 well plates at a
concentration of 2.times.10.sup.6 per ml. One ul total volume is
added to each well. The peptide to be tested is added in growth
medium to achieve the final desired concentration. Then the
appropriate number of MOI are added. To assay viral growth, 200 ul
of supernatant is removed on days 3 and 7 and the concentration of
p24 antigen is determined using a commercial assay (Coulter
Immunology, Hialeah, Fla.). Controls include duplicate wells
containing cells alone, cells plus peptide at 5 ug/ml cells with
virus but not peptide and cells with virus in the presence of AZT
at 10.sup.-5 M-10.sup.-8 M.
[0225] The time of addition of peptide can be varied. Cells
pretreated for 2 hours prior to addition of virus, at the time of
addition of virus, or 2 hours after infection show antiviral
activity for the peptide.
EXAMPLE 8
Preparation and Activity of Tachytegrins
[0226] Several illustrative tachytegrins were synthesized as
described in Example 1 and tested for activity against
Staphylococcus aureus (MRSA), Pseudomonas (Psa), VREF, Candida and
E.coli as described in Example 2. The results shown in Table 1 as
minimal inhibitory concentration (MIC) in ug/ml were obtained with
the C-terminal amidated forms except for the last two which were
tested as the free acids, as indicated by *. In all peptides in
Table 1, X is MeGly.
7TABLE 1 SEQUENCE MRSA Psa VREF Candida E. Coli RGGCLRYAVPRFAVRVCR
SEQ ID NO:77 >128 0.05 RGGCLRYTKPKFTVRVCR SEQ ID NO:78
RGGCLRYAVGRFAVRVCR SEQ ID NO:79 RGGCLRYARXRFAVRVCR SEQ ID NO:80
>32 5.7 RGFCLRYTVPRFTVRFCVR SEQ ID NO:81 1.88 0.57 0.99
RGFCLRYKVGRFKVRFCVR SEQ ID NO:82 >64 2.7 RGFCLRYXVGRFXVRFCVR SEQ
ID NO:83 RGGCLRYCRPRFCVRVCR SEQ ID NO:53 9.8 0.18 9.68 0.2
RGGCRLYCRRRFCVVGCR SEQ ID NO:54 53.3 3.3 4 4 RGVCLRYCRGRFCVRLCR SEQ
ID NO:55 8 2 RGRVCLRYCRGRFCVRLCFR SEQ ID NO:56 6.7 1
RWRVCLRYCRGRFCVRLCLR SEQ ID NO:57 4 4 RGWRVCLKYCRGRFCVKLCLR SEQ ID
NO:58 RGGRVCLRYCRGKFCVRLCLR SEQ ID NO:59 8 0.75 RGGCLRYAVGRFAVRVCR*
SEQ ID NO:77 >32 5.3 RGFCLRYXVGRFXVRFCVR* SEQ ID NO:83 >32
12
[0227]
Sequence CWU 0
0
* * * * *