U.S. patent application number 14/188913 was filed with the patent office on 2015-10-22 for antibiotic peptides.
This patent application is currently assigned to AMP-THERAPEUTICS GMBH. The applicant listed for this patent is AMP-THERAPEUTICS GMBH. Invention is credited to Anna Klara Brigitte Hansen, Ralf Hoffmann, Daniel Knapp.
Application Number | 20150299256 14/188913 |
Document ID | / |
Family ID | 42124600 |
Filed Date | 2015-10-22 |
United States Patent
Application |
20150299256 |
Kind Code |
A1 |
Hoffmann; Ralf ; et
al. |
October 22, 2015 |
ANTIBIOTIC PEPTIDES
Abstract
The invention relates to a peptide or peptide derivative having
the general formula:
Sub.sub.1-X.sub.1-D.sub.2K.sub.3-P.sub.4-P.sub.5-Y.sub.6-L.sub.7-P.sub.8--
R.sub.9-P.sub.10-X.sub.2-P.sub.12-
P.sub.13-R.sub.14-X.sub.3-I.sub.16-P.sub.17/Y.sub.17-N.sub.18-N.sub.19-X.-
sub.4-Sub.sub.2, wherein X.sub.1 is a non-polar, hydrophobic group
pr a positively charged group, D.sub.2 is asparagine or glutamine,
K.sub.3, X.sub.2, and X.sub.4 are positively charged groups,
X.sub.3 is a positively charged group, proline, or a proline
derivative; L.sub.7 and I.sub.16 are non-polar, hydrophobic groups,
Y.sub.6 and Y.sub.17 are tyrosine, R.sub.9 and R.sub.14 are
arginine, N.sub.18 and N.sub.19 are asparagine or glutamine,
P.sub.4, P.sub.5, P.sub.8, P.sub.10, P.sub.12, P.sub.13, and
P.sub.17 are proline, hydroxyproline, or derivatives thereof,
wherein possibly one or two of the groups selected from D.sub.2,
P.sub.4, P.sub.5, P.sub.8, P.sub.10, P.sub.12, P.sub.13, P.sub.17,
and Y.sub.17 are replaced by an arbitrary group and/or P.sub.13 and
R.sub.14 are exchanged, Sub.sub.1 is the free or modified
N-terminus, and Sub.sub.2 is the free or modified C-terminus. The
invention further relates to the use of the peptides and peptide
derivatives in medicine, as an antibiotic, in a disinfectant or
cleaning agent, as a preservative or in a packaging material, in
pharmaceutical research, or in a screening method.
Inventors: |
Hoffmann; Ralf;
(Grosspoesna, DE) ; Knapp; Daniel; (Leipzig,
DE) ; Hansen; Anna Klara Brigitte; (Gehrden,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMP-THERAPEUTICS GMBH |
Leipzig |
|
DE |
|
|
Assignee: |
AMP-THERAPEUTICS GMBH
Leipzig
DE
|
Family ID: |
42124600 |
Appl. No.: |
14/188913 |
Filed: |
February 25, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13147095 |
Oct 7, 2011 |
8686113 |
|
|
PCT/EP10/51072 |
Jan 29, 2010 |
|
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14188913 |
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Current U.S.
Class: |
435/32 ;
435/252.3; 435/252.31; 435/252.33; 435/252.35; 435/254.2;
435/254.21; 435/325; 435/348; 435/358; 435/365; 435/69.1; 530/326;
536/23.1 |
Current CPC
Class: |
A61P 31/10 20180101;
A01N 47/44 20130101; Y02A 50/483 20180101; G01N 2500/10 20130101;
A61P 31/04 20180101; Y02A 50/473 20180101; Y02A 50/30 20180101;
C07K 14/43563 20130101; A61K 38/10 20130101; Y02A 50/475 20180101;
A61K 38/00 20130101; Y02A 50/478 20180101; A61K 47/64 20170801;
C07K 7/08 20130101; C12Q 1/18 20130101 |
International
Class: |
C07K 7/08 20060101
C07K007/08; A61K 38/10 20060101 A61K038/10; A61K 47/48 20060101
A61K047/48; A01N 47/44 20060101 A01N047/44; C12Q 1/18 20060101
C12Q001/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 29, 2009 |
DE |
10 2009 007 381.7 |
Claims
1. A peptide or peptide derivative, which contains one of the
following sequences:
Sub.sub.1-X.sub.1-D.sub.2-K.sub.3-P.sub.4-P.sub.5-Y.sub.6-L.sub.7-P.sub.8-
R.sub.9-P.sub.10-X.sub.2-P.sub.12-P.sub.13-R.sub.14-X.sub.3-I.sub.16-P.sub-
.17/Y.sub.17-N.sub.18-N.sub.19-X.sub.4-Sub.sub.2 X.sub.1 is a
residue with a nonpolar, hydrophobic side chain or with a positive
net charge or a side chain that is positively charged under
physiological conditions; D.sub.2 is an aspartic acid or glutamic
acid residue, K.sub.3 is a residue with a positive net charge or a
side chain that is positively charged under physiological
conditions, preferably lysine or arginine, X.sub.2 and X.sub.4 are
selected independently of one another from residues with a positive
net charge or a side chain that is positively charged under
physiological conditions; X.sub.3 is a residue with a positive net
charge or a side chain that is positively charged under
physiological conditions or proline or a proline derivative;
L.sub.7 is selected from residues with a nonpolar, hydrophobic side
chain, preferably leucine, isoleucine and valine, I.sub.16 is
selected from residues with a nonpolar, hydrophobic side chain,
preferably leucine, isoleucine, tert-butylgylycine and valine,
Y.sub.6 and Y.sub.17 are in each case tyrosine, R.sub.9 and
R.sub.14 are in each case arginine, N.sub.18 is asparagine or
glutamine, N.sub.19 is asparagine or glutamine or is absent,
P.sub.4, P.sub.5, P.sub.8, P.sub.10, P.sub.12, P.sub.13 and
P.sub.17 are selected independently of one another from proline and
proline derivatives or hydroxyproline and hydroxyproline
derivatives, Sub.sub.1 is the free N-terminus of the amino acid
X.sub.1 or a modified N-terminal amino group; Sub.sub.2 is the free
C-terminal carboxyl group of the C-terminal amino acid (--COOH) or
a modified C-terminal carboxyl group.
2. The peptide or peptide derivative as claimed in claim 1, wherein
P.sub.13 and R.sub.14 are exchanged, and/or one or two of the
residues selected from D.sub.2, P.sub.4, P.sub.5, P.sub.8,
P.sub.10, P.sub.12, P.sub.13, P.sub.17 and Y.sub.17 are replaced
with any residue.
3. The peptide or peptide derivative as claimed in claim 1 or 2,
wherein the N-terminus is linked to another peptide directly or via
a linker.
4. The peptide or peptide derivative as claimed in any one of
claims 1 to 3 containing at least one additional residue X.sub.5
and/or X.sub.6 in Sub.sub.2, wherein X.sub.5 is selected from the
group comprising proline, a proline derivative and a building
block, which has a positive net charge or bears a side chain that
is positively charged under physiological conditions and in that
X.sub.6 is selected from proline, a proline derivative, a polar
building block or a hydrophobic building block.
5. The peptide or peptide derivative as claimed in any one of
claims 1 to 4, characterized in that the residue X.sub.1 is
selected from arginine, lysine, .delta.-hydroxylysine,
homoarginine, 2,4-diaminobutyric acid, .beta.-homoarginine,
D-arginine, arginal, 2-amino-3-guanidinoproprionic acid,
nitroarginine, N-methylarginine, .epsilon.-N-methyllysine,
allo-hydroxylysine, 2,3-diaminopropionic acid, 2,2'-diaminopimelic
acid, ornithine, sym-dimethylarginine, asym-dimethylarginine,
2,6-diaminohexinic acid, p-aminobenzoic acid, 3-aminotyrosine,
valine, isoleucine, leucine and methionine, alanine, phenylalanine,
N-methylleucine, tert-butylglycine, cyclohexylalanine,
.beta.-alanine, 1-aminocyclohexylcarbonxylic acid,
N-methylisoleucine, norleucine, norvaline and N-methylvaline.
6. The peptide or peptide derivative as claimed in any one of
claims 1 to 5, characterized in that the residue X.sub.2 and/or the
residue X.sub.4 are selected independently of one another from
arginine, lysine, .delta.-hydroxylysine, homoarginine,
.beta.-homoarginine, D-arginine, arginal, 2,4-diaminobutyric acid,
2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine,
N-methyl-arginine, t-N-methyllysine, allo-hydroxylysine,
2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, ornithine,
sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexinic
acid, p-aminobenzoic acid and 3-aminotyrosine.
7. The peptide or peptide derivative as claimed in any one of
claims 1 to 6, characterized in that the residue X.sub.3 is
selected from arginine, lysine, .delta.-hydroxylysine,
homoarginine, .beta.-homoarginine, D-arginine, arginal,
2,4-dimaminobutyric acid, .beta.-homoarginine,
2-amino-3-guanidinopropionic acid, nitroarginine, nitrosoarginine,
N-methyl-arginine, .epsilon.-N-methyllysine, allo-hydroxylysine,
2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, ornithine,
sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexinic
acid, p-aminobenzoic acid, 3-aminotyrosine, proline,
cis-4-hydroxyproline, trans-4-hydroxyproline, cis-3-hydroxyproline,
trans-3-hydroxyproline, .beta.-cyclohexylalanine,
3,4-cis-methanoproline, 3,4-dehydroproline, homoproline and
pseudoproline.
8. The peptide or peptide derivative as claimed in any one of
claims 1 to 7, selected from the group of sequences comprising SEQ
ID NO. 5 to 9, 14 to 26, 29, 30, 32, 33, 36, 38, 40, 41, 44 to 46,
49, 50, 53 to 59, 61 to 85, 93, 94, 101, 102 and 107 to 112.
9. A peptide or peptide derivative containing an antibacterial
peptide and a cell-penetrating peptide.
10. The peptide or peptide derivative as claimed in claim 9,
wherein said antibacterial peptide is selected from the group
comprising apidaecin, drosocin, formaecin 1, pyrrhocoricin and
metalnikowin 1.
11. The peptide or peptide derivative as claimed in claim 9,
wherein said antibacterial peptide comprises a peptide or peptide
derivative as claimed in any one of claims 1 to 8.
12. The peptide or peptide derivative as claimed in any one of
claims 9 to 11, wherein said cell-penetrating peptide is selected
from the group comprising penetratin, Tat peptides, model
amphipathic peptides, transportan, SynB and
cis-.gamma.-amino-1-proline-containing peptides.
13. The peptide or peptide derivative as claimed in any one of
claims 1 to 12 selected from the group comprising SEQ ID NO. 95 to
102 and 106.
14. The peptide or peptide derivative as claimed in any one of
claims 1 to 13, characterized in that at least one of the peptide
bonds of the peptide backbone is chemically modified.
15. The peptide or peptide derivative as claimed in claim 14,
characterized in that the bond between X.sub.4-X.sub.7 is a
chemical bond that cannot be cleaved under physiological
conditions.
16. The peptide or peptide derivative as claimed in claim 14 or 15,
characterized in that the chemically modified bond is selected from
a reduced amide bond, an alkylated amide bond or a thioamide
bond.
17. The peptide or peptide derivative as claimed in any one of
claims 1 to 16, which is joined to a protein or coupled to a
polymer or bound to a carrier.
18. A multimer, in which at least two peptides or peptide
derivatives are joined together, characterized in that at least one
of the peptides or peptide derivatives is a peptide or peptide
derivative as claimed in any one of claims 1 to 17.
19. A pharmaceutical composition, characterized in that it contains
at least one peptide, peptide derivative or multimer as claimed in
any one of claims 1 to 18.
20. A method of production of a peptide or peptide derivative or
multimer as claimed in any one of claims 1 to 18, characterized in
that the peptide or peptide derivative or multimer is produced by
chemical synthesis or by means of recombinant methods.
21. The use of a peptide, peptide derivative or multimer as claimed
in any one of claims 1 to 18 for the production of a medicinal
product.
22. The use of a peptide, peptide derivative or multimer as claimed
in any one of claims 1 to 18 as disinfectant and/or cleaning agent,
as preservative and/or in a packaging material.
23. The peptide, peptide derivative and/or multimer as claimed in
any one of claims 1 to 18 for use in the treatment of microbial,
bacterial or fungal infections.
24. The use of a peptide, peptide derivative and/or multimer as
claimed in any one of claims 1 to 18 for removing contamination by
microbes, bacteria or fungi.
25. The use of a peptide, peptide derivative or multimer as claimed
in any one of claims 1 to 18 in pharmaceutical research or in a
screening process.
26. A method for identifying a substance with antibacterial or
antimycotic action, comprising the following steps: (i) carrying
out a competitive assay with: (a) a microorganism that is sensitive
to a peptide, peptide derivative or multimer as claimed in any one
of claims 1 to 18; (b) a peptide, peptide derivative or multimer as
claimed in any one of claims 1 to 18 and (c) at least one substance
to be tested by bringing (a) in contact with (b) and (c); and (ii)
selecting a test substance, which competitively displaced the
binding of the peptide, peptide derivative or multimer on the
microorganisms.
27. The method as claimed in claim 26, wherein the microorganism is
a species selected from the group of genera comprising Escherichia
coli, Enterobacter cloacae, Erwinia amylovara, Klebsiella
pneumoniae, Morganella morganii, Salmonella typhimurium, Salmonella
typhi, Shigella dysenteriae, Yersinia enterocolitica, Acinetobacter
calcoaceticus, Acinetobacter baumannii, Agrobacterium tumefaciens,
Francisella tularensis, Legionella pneumophila, Pseudomonas
syringae, Rhizobium meliloti, Pseudomonas aeruginosa, Proteus
vulgaris, Proteus mirabilis, Stenotrophomonas maltophilia and
Haemophilus influenzae.
28. A nucleic acid coding for a peptide or multimer as claimed in
any one of claims 1 to 18.
29. A host cell, comprising one or more nucleic acids as claimed in
claim 28.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 13/147,095, filed Jul. 29, 2011, which is a
National Stage Entry of PCT/EP2010/051072, filed Jan. 29, 2010,
which claims the benefit of German Patent Application No. 10 2009
007 38.7, filed Jan. 29, 2009.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
of Aug. 13, 2014 is named 01080002US2SL.txt and is 51,879 bytes in
size.
FIELD OF THE INVENTION
[0003] This invention relates to antibiotic peptides and peptide
derivatives especially for use in medicine. The invention further
relates to compositions and methods for destroying microorganisms,
such as bacteria or fungi, and methods of treating microbial
infections. The invention further comprises a method for screening
active substances.
BACKGROUND OF THE INVENTION
[0004] The occurrence of serious bacterial and fungal infections is
an increasing problem despite notable progress in antibiotic
therapy. Each year there are more than 40 million hospitalizations
in the United States of America and more than 2 million of these
patients become infected in the hospital. Antibiotic-resistant
bacteria are involved in 50-60% of these cases (Tomasz A.
Multiple-Antibiotic-Resistant Pathogenic Bacteria--A Report on the
Rockefeller-University Workshop. New England Journal of Medicine
330; 1247-51, 1994). These hospital-acquired diseases are estimated
to lead to 60 000 to 70 000 deaths in the USA and up to 10 000
deaths in Germany (Wenzel R. P, The Mortality of Hospital-Acquired
Blood-Stream Infections--Need for A New Vital Statistic.
International Journal of Epidemiology 17: 225-7, 1998).
[0005] Whereas resistant Gram-negative bacteria were the main
problem in the 1970s, in the last decade there has been an increase
in cases in which Gram-positive bacteria that are resistant to
several antibiotics play a role (Moellering R C. Emerging
resistance with gram-positive aerobic infections: Where do we go
from here? Introduction: Problems with antimicrobial resistance in
gram-positive cocci. Clinical Infectious Diseases 26: 1177-8,
1998). The current rapid development of resistant strains involves
both Gram-positive and Gram-negative pathogens (Hand W L. Current
challenges in antibiotic resistances. Adolescent Medicine 11:
427-38, 2000). Resistances developed first in species in which
single mutations were sufficient to reach clinically important
levels, e.g. Staphyloccus aureus and Pseudomonas aeruginosa; next
were bacteria in which multiple mutations were necessary, for
instance E. coli and Neisseria gonorrhoeae. This is due primarily
to the frequent use of fluoroquinolone antibiotics (Hooper D C.
Emerging mechanisms of fluoroquinolone resistance. Emerging
Infectious Diseases 7: 337-41, 2001). Another important cause of
the development of resistance in Gram-negative bacteria is the
extensive range of lactamases in Escherichia coli and Klebsiella
pneumoniae (Jones R N. Resistance patterns among nosocomial
pathogens--Trends over the past few years. Chest 119: 3978-404S,
2001). Nearly half the clinically relevant strains of Haemophilus
ducreyi, the causative agent of soft chancre, carry genes that
makes this bacterium resistant to amoxicillin, ampicillin and
several other .beta.-lactams (Prachayasittikul V, Lawung R, &
Bulow L. Episome profiles and mobilizable beta-lactamase plasmid in
Haemophilus ducreyi. Southeast Asian J Trop Med Public Health 31:
80-4, 2000). Similarly, the resistance of Salmonella enterica
serovar typhimurium to teracyclines rose from zero percent in the
year 1948 to 98% in the year 1998 (Teuber M. Spread of antibiotic
resistance with food-borne pathogens. Cellular and Molecular Life
Sciences 36: 755-63, 1999).
[0006] This explains the need for further searching for new
antibiotics. Inducible antibacterial peptides represent a field of
research in which modern biochemistry, immunology and research into
active substances come together. Peptide antibiotics, ranging in
size from 13 to more than a hundred amino acids, have been isolated
from plants, animals and microbes (Boman H G. Peptide Antibiotics
and Their Role in Innate Immunity. Annual Review of Immunology 13:
61-92, 1995). A single animal has approx. 6-10 peptide antibiotics,
with each peptide often displaying a completely different activity
spectrum (Barra D, Simmaco M, & Boman H G. Gene-encoded peptide
antibiotics and innate immunity. Do `animalcules` have defense
budgets? Febs Letters 430: 130-4, 1998). It is known that the
overwhelming number of antibacterial peptides, including the
much-studied defensins, cecropins and magainins, act by a
"lytic/ionic" mechanism. A permeabilizing effect on the bacterial
cytoplasmic membrane has been discussed as a common mechanism of
action of these "lytic" peptides (Ludtke S, He K, & Huang H.
Membrane thinning caused by magainin 2, Biochemistry 34: 16764-9,
1995; Wimley W C, Selsted M E, & White S H. Interactions
Between Human Defensins and Lipid Bilayers--Evidence for Formation
of Multimeric Pres. Protein Sciences 3: 1362-73, 1994; Shai Y.
Molecular Recognition Between Membrane-Spanning Polypeptides.
Trends in Biochemical Sciences 20: 460-4, 1995). A cationic,
amphipathic structure, which forms hydrophilic ion (proton)
channels in a lipid bilayer, is the basis of this activity. Owing
to the outflow of protons, the membrane potential that is necessary
for many fundamental life processes is disturbed and as a result
the cell is killed. Since disturbance of the membrane by these
peptides is dependent on the recognition of chiral molecules, an
amino acid exchange, which does not remove the general amphipathic
structure or basic net charge, is tolerated functionally (Wade D et
al. A11-D Amino Acid-Containing Channel-Forming Antibiotic
Peptides. Proceedings of the National Academy of Sciences of the
United States of America 87: 4761-5, 1990: Steiner H, Andreu D,
& Merrifield R B. Binding and Action of Cecropin and Cecropin
Analogs--Antibacterial Peptides from Insects. Biochimica et
Biophysica Acts 939: 260-6, 1988). At higher concentrations these
lytic peptides often have toxic action on mammalian membranes,
which limits their suitability as possible medicinal products. If
proline is inserted into the sequence of the .alpha.-helical
antimicrobial peptides, the capacity of the peptides to
permeabilize the cytoplasmic membrane of E. coli decreases as a
function of the number of proline residues. On examining this, it
is amazing that some of the most active, native antibacterial
peptides, at least with respect to some Gram-negative pathogens,
belong to the family of proline-rich peptides (Otvos L et al.
Insect peptides with improved protease-resistance protect mice
against bacterial infection. Protein Science 9: 742-9, 2000).
[0007] The side effects described above are overcome by
antimicrobial peptides (AMP), which specifically recognize a
bacterial protein or other intra- or extracellular components,
without displaying cross-reactivity with mammalian analogs. This
seems to apply to proline-rich antimicrobial peptides, including
apidaecins, drosocin and pyrrhocoricin which are originally
isolated from insects. With the enormous variation in size and
biochemical properties, it is not surprising that the
structure-activity and conformation-activity relations are the
focus of antibacterial peptide research. A complete investigation
of the natural antibacterial peptide repertoire for biological
strength is important not only for general biochemical questions,
but is also of constant interest for the pharmaceutical industry.
Despite the problems of in-vitro tests with peptide-based
antibiotics, some natural, cationic antibacterial peptides have
already reached the clinical trial phase (Boman H G. Peptide
Antibiotics and Their Role in Innate Immunity. Annual Review of
Immunology 13: 61-92, 1995). Whereas some of these peptides showed
activity as topical (local) agents in the early clinical trial
phase, others were active in systemic therapy. For example, the
cationic protein rBPI 21, which is used for parental treatment of
meningococcemia, has completed the third phase of clinical testing
(Boman H G. Peptide Antibiotics and Their Role in Innate Immunity.
Annual Review of Immunology 13: 61-92, 1995).
[0008] The family of the proline-rich peptides (e.g. apidaecin,
drosocin and pyrrhocoricin) kill bacteria not only by
permeabilization of their membrane, but bind stereospecifically to
one or more target proteins. These possible interaction partners,
up to now the heat-shock protein DnaK has been investigated
thoroughly (Kragol G et al. Identification of crucial residues for
the antibacterial activity of the proline-rich peptide,
pyrrhocoricin. European Journal of Biochemistry 269: 4226-37, 2002;
Kragol G et al. The antibacterial peptide pyrrhocoricin inhibits
the ATPase actions of DnaK and prevents chaperone-assisted protein
folding. Biochemistry 40: 3016-26, 2011), are inhibited by the
proline-rich peptides and presumably the correct protein folding is
prevented, ultimately leading to cell death. Moreover, proline-rich
peptides, in stark contrast to AMPs with defined secondary
structure such as melittin or gramicidin, seem in vitro to have
neither hemolytic nor toxic effects on eukaryotic cells. Along with
antimicrobial activity, mainly the stability in mammalian serum
(25%) has a decisive influence on the development of new
peptide-based antibiotics. For example, drosocin is broken down
within an hour, whereas pyrrhocoricin is far more stable with
respect to proteases, with half-lives of 120 minutes.
[0009] In biological experiments by Schneider and Dorn (2001)
(Schneider M & Dorn A. Differential infectivity of two
pseudomonas species and the immune response in the milkweed bug,
Oncopeltus fasciatus (Insecta: Hemiptera). Journal of Invertebrate
Pathology 78: 135-40, 2001), nymphs and pupae of the milkweed bug
Oncopeltus fasciatus from the Lygaeidae family were infected with
two different Gram-negative Pseudomonas species and their immune
response was analyzed. Whereas infection of the nymphs of O.
fasciatus with the human pathogen Pseudomonas aeruginosa resulted
in the death of all individuals after 48 h, 71% of individuals
infected with the less pathogenic Pseudomonas putide survived for
at least 96 h. If the nymphs of the milkweed bug were then infected
first with P. putide and after 24 h with P. aeruginosa, the
survival rate of the doubly infected individuals within the first
24 h rose significantly to 73%. The probable induction of synthesis
of antibacterial peptides, by which insects defend themselves,
within the scope of their innate immune system, against invading
microorganisms, was then investigated. Four peptides (Oncopeltus
antibacterial peptide 1-4) were identified with molecular weights
of 15, 8, 5 or 2 kDa and were held to be responsible for the
antibacterial action. Sequence analysis according to Edman found,
in addition to a 34 amino acid long partial sequence for peptide 1
(15 kDa), also the incomplete sequence of the proline-rich 2 kDa
peptide 4. The amino acids in positions 11 and the C-terminal
sequence starting from position 19 could not be identified
definitively. The exact molecular weight is unknown.
[0010] A selection of currently known sequences of antibiotic
peptides is presented in Table 1:
TABLE-US-00001 TABLE 1 SEQ ID Peptide Species Sequence NO. Ref.
Apidaecin 1a Apis GNNRPVYIPQPRPPHPRI 119 [1] mellifera Apidaecin 1b
Apis GNNRPVYIPQPRPPHPRL 87 [1] mellifera Drosocin Drosophila
GKPRPYSPRPTSHPRPIRV 89 [2] melanogaster Formaecin 1 Myrmecia
GRPNPVNNKPTPYPHL 120 [3] gulosa Pyrrhocoricin Pyrrhocoris
VDKGSYLPRPTPPRPIYNRN-NH.sub.2 91 [4] apterus Metalnikowin 1
Palomena VDKPDYRPRPRPPNM 121 [5] prasina Oncopeltus Oncopeltus
EVSLKGEGGSNKGFIQGSGTKTLFQDD 122 [6] antibacterial fasciatus KTKLDGT
peptide 1 Oncopeltus Oncopeltus VDKPPYLPRP(X/P)PPRRIYN(NR) 123 [6]
antibacterial fasciatus peptide 4 [1] Casteels P, Ampe C, Jacobs F,
Vaeck M, & Tempst P. Apidaecins - Antibacterial Peptides from
Honeybees. Embo Journal 8: 2387-91, 1989 [2] Bulet P et al. A Novel
Inducible Antibacterial Peptide of Drosophila Carries an
O-Glycosylated Substitution. Journal of Biological Chemistry 268:
14893-7, 1993 [3] Mackintosh J A et al. Isolation from an ant
Myrmecia gulosa of two inducible O-glycosylated proline-rich
antibacterial peptides. Journal of Biological Chemistry 273:
6139-41, 1998 [4] Cociancich S et al. Novel Inducible Antibacterial
Peptides from A Hemipteran Insect, the Sap-Sucking Bug
Pyrrhocoris-Apterus. Biochemical Journal 300: 567-75, 1994 [5]
Chernysh S, Cociancich S, Briand J P, Hetru C, & Bulet P. The
inducible antibacterial peptides of the hemipteran insect Palomena
prasina: Identification of a unique family of proline-rich peptides
and of a novel insect defensin. Journal of Insect Physiology 42:
81-9, 1996 [6] Schneider M & Dorn A. Differential infectivity
of two pseudomonas species and the immune response in the milkweed
bug, Oncopeltus fasciatus (Insecta: Hemiptera). Journal of
Invertebrate Pathology 78: 134-40, 2001
[0011] There is still a demand for new antibacterial and
antimycotic compounds, new antibacterial and antimycotic
pharmaceutical compositions, as well as methods using them, and
compounds that can be used for screening active substances, to
detect new pharmaceutical antibiotics.
[0012] The problem to be solved by the present invention is to
provide new antibiotic peptides with increased stability, to extend
the spectrum of action of the AMPs on Gram-positive bacteria and
thus make modern spectrum antibiotics available, and to introduce
the peptides into eukaryotic cells and thus combat hidden
bacteria.
DESCRIPTION OF THE INVENTION
[0013] The problem is solved by the peptides and peptide
derivatives according to the invention with the general
formula:
Sub.sub.1-X.sub.1-O.sub.2-K.sub.3-P.sub.4-P.sub.5-Y.sub.6-L.sub.7-P.sub.-
8-R.sub.9-P.sub.10-X.sub.2-P.sub.12-P.sub.13-R.sub.14-X.sub.3-I.sub.16-P.s-
ub.17/Y.sub.27-N.sub.18-N.sub.19-X.sub.4-Sub.sub.2 (Formula 1)
[0014] X.sub.1 is a residue with a nonpolar, hydrophobic side chain
or with a positive net charge or a side chain that is positively
charged under physiological conditions:
[0015] D.sub.2 is an aspartic acid or glutamic acid residue,
[0016] K.sub.3 is a residue with a positive net charge or a side
chain that is positively charged under physiological conditions,
preferably lysine or arginine,
[0017] X.sub.2 and X.sub.4 are selected independently of one
another from residues with a positive net charge or a side chain
that is positively charged under physiological conditions;
[0018] X.sub.3 is a residue with a positive net charge or a side
chain that is positively charged under physiological conditions or
proline or proline derivative;
[0019] L.sub.7 and I.sub.14 are selected independently of one
another from residues with a nonpolar, hydrophobic side chain,
preferably leucine, isoleucine and valine,
[0020] Y.sub.6 and Y.sub.17 are in each case tyrosine, R.sub.3 and
R.sub.14 are in each case arginine, N.sub.18 is asparagine or
glutamine, N.sub.19 is asparagine or glutamine or is absent,
P.sub.4, P.sub.5, P.sub.8, P.sub.10, P.sub.12, P.sub.13 and
P.sub.17 are selected independently of one another from proline and
proline derivatives or hydroxyproline and hydroxyproline
derivatives,
[0021] wherein optionally P.sub.13 and R.sub.14 are exchanged,
and/or
[0022] optionally one or two of the residues selected from D.sub.2,
P.sub.4, P.sub.5, P.sub.8, P.sub.10, P.sub.12, P.sub.13, P.sub.17,
and Y.sub.17 are replaced with any residue,
[0023] Sub.sub.1 is the free N-terminus of the amino acid X.sub.1
or a modified N-terminal amino group;
[0024] Sub.sub.2 is the free C-terminal carboxyl group of the
C-terminal amino acid (--COOH) or a modified C-terminal carboxyl
group.
[0025] In an especially preferred embodiment I.sub.16 is selected
from the group comprising leucine, isoleucine, tert-butylglycine
and valine.
[0026] In one embodiment N.sub.19 is absent when P.sub.17 is
present.
[0027] Peptides and peptide derivatives with one of the general
formulas 1 to 3 are preferred:
Sub.sub.1-X.sub.1-D.sub.2-K.sub.3-P.sub.4-P.sub.5-Y.sub.6-L.sub.7-P.sub.-
8-R.sub.9-P.sub.10-X.sub.2-P.sub.12-P.sub.13-X.sub.14-X.sub.3-I.sub.16-Y.s-
ub.17-N.sub.18-X.sub.4-Sub.sub.2 (Formula 2)
Sub.sub.2-X.sub.1-D.sub.2-K.sub.3-P.sub.4-P.sub.5-Y.sub.6-L.sub.7-P.sub.-
8-R.sub.9-P.sub.10-X.sub.2-P.sub.12-P.sub.13-R.sub.14-X.sub.3-I.sub.16-Y.s-
ub.17-N.sub.18-X.sub.4-Sub.sub.2 (Formula 3)
Sub.sub.1-X.sub.1-D.sub.2-K.sub.3-P.sub.4-P.sub.5-Y.sub.6-L.sub.7-P.sub.-
8-R.sub.9-P.sub.10-X.sub.2-P.sub.12-P.sub.13-R.sub.14-X.sub.3-I.sub.16-Y.s-
ub.17-N.sub.18-X.sub.4-Sub.sub.2 (Formula 4)
[0028] X.sub.1 is a residue with a nonpolar, hydrophobic side chain
or with a positive net charge or a side chain that is positively
charged under physiological conditions;
[0029] D.sub.2 is an aspartic acid or glutamic acid residue,
[0030] K.sub.3 is a residue with a positive net charge or a side
chain that is positively charged under physiological conditions,
preferably lysine or arginine,
[0031] X.sub.3 and X.sub.4 are selected independently of one
another from residues with a positive net charge or a side chain
that is positively charged under physiological conditions;
[0032] X.sub.3 is a residue with a positive net charge or a side
chain that is positively charged under physiological conditions or
proline or a proline derivative;
[0033] L.sub.7 and I.sub.16 are selected independently of one
another from residues with a nonpolar, hydrophobic side chain,
preferably leucine, isoleucine and valine.
[0034] In an especially preferred embodiment I.sub.16 is selected
from the group comprising leucine, isoleucine, tert-butylglycine
and valine.
[0035] Y.sub.6 and Y.sub.17 are in each case tyrosine, R.sub.9 and
R.sub.14 are in each case arginine, N.sub.18 and N.sub.19 are in
each case asparagine or glutamine, P.sub.4, P.sub.5, P.sub.8,
P.sub.10, P.sub.12, P.sub.13 and P.sub.17 are selected
independently of one another from proline and proline derivatives
or hydroxyproline and hydroxyproline derivatives.
[0036] Optionally one or two of the residues selected from D.sub.2,
P.sub.4, P.sub.5, P.sub.8, P.sub.10, P.sub.12, P.sub.13, P.sub.17
and Y.sub.17 are replaced with any amino acid residue, preferably a
neutral residue, especially preferably a neutral polar residue.
[0037] Moreover, P.sub.13 and R.sub.14 are optionally
exchanged.
[0038] Residues with a positive net charge or a side chain that is
positively charged under physiological conditions are preferably
selected from the group comprising arginine, lysine,
.delta.-hydroxylysine, homoarginine, 2,4-diaminobutyric acid,
.beta.-homoarginine, D-arginine, arginal (--COOH in arginine is
replaced with --CHO), 2-amino-3-guanidinopropionic acid,
nitroarginine (preferably N(G)-nitroarginine), nitrosoarginine
(preferably N(G)-nitrosoarginine), methylarginine (preferably
N-methyl-arginine), .epsilon.-N-methyllysine, allo-hydroxylysine,
2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, ornithine,
sym-dimethylarginine, asym-dimethylarginine, 2,6-diaminohexinic
acid, p-aminobenzoic acid and 3-aminotyrosine and less preferably
histidine, 1-methylhistidine and 3-methylhistidine. X1, X2 and X3
are preferably selected independently of one another from this
list.
[0039] The term proline derivative stands for an amino acid residue
derived from proline, which is obtained from proline preferably by
structural modification of a functional group. Preferred proline
derivatives are selected from the group comprising
.beta.-cyclohexylalanine, 3,4-cis-methanoproline,
3,4-dehydroproline, homoproline or pseudoproline. The term
hydroxyproline includes inter alia cis-4-hydroxyproline,
trans-4-hydroxyproline, cis-3-hydroxyproline and
trans-3-hydroxyproline. The term hydroxyproline derivative stands
correspondingly for an amino acid residue derived from
hydroxyproline, which is obtained from hydroxyproline preferably by
structural modification of a functional group. Preferred
hydroxyproline derivatives are selected from
hydroxy-.beta.-cyclohexylalanine and the aforementioned proline
derivatives, which are substituted with a hydroxyl group.
[0040] A neutral residue is a residue with a side chain that is
uncharged under physiological conditions.
[0041] A polar residue preferably has at least one polar group in
the side chain. These are preferably selected from the group
comprising hydroxyl, sulfhydryl, amine, amide or ester groups pr
other groups that permit the formation of hydrogen bridges.
[0042] Preferred neutral polar residues are selected from the group
comprising asparagine, cysteine, glutamine, serine, threonine,
tyrosine, citrulline, N-methylserine, homoserine, allo-threonine
and 3,5-dinitrotyrosine and .beta.-homoserine.
[0043] In a preferred embodiment P5 is selected from the group
comprising .beta.-cyclohexylalanine, 3,4-cis-methanoproline,
3,4-dehydroproline, homoproline or pseudoproline,
cis-4-hydroxyproline, cis-3-hydroxyproline, trans-3-hydroxyproline,
asparagine, cysteine, glutamine, serine, threonine, tyrosine,
citrulline, N-methylserine, homoserine, allo-threonine and
3,5-dinitrotyrosine and .beta.-homoserine.
[0044] The residues with a nonpolar, hydrophobic side chain are
uncharged residues under physiological conditions, preferably with
a hydropathy index above 0, especially preferably above 3.
Preferred nonpolar, hydrophobic side chains are selected from the
group comprising alkyl, alykylene, alkoxy, alkenoxy, alkylsulfanyl
and alkenylsufanyl residues with 1 to 10, preferably 2 to 6 carbon
atoms, or aryl residues with 5 to 12 carbon atoms. Preferred
residues with a nonpolar, hydrophobic side chain are selected from
leucine, isoleucine, valine, methionine, alanine phenylalanine,
N-methylleucine, tert-butylglycine, cyclohexylalanine,
.beta.-alanine, 1-aminocyclohexylcarboxylic acid,
N-methylisoleucine, norleucine, norvaline and N-methylvaline.
[0045] "Physiological conditions" means a pH from pH 6 to 8 and a
temperature from 30.degree. C. to 40.degree. C., preferably a
temperature of 37.degree. C., a pH of 7.4 and an osmotic pressure
of 300 mosmol/kg.
[0046] The peptides or peptide derivatives according to the
invention preferably contain at least 19 amino acid residues,
preferably up to 50 amino acid residues.
[0047] Sub.sub.1 is the free N-terminus of the amino acid X.sub.1
or a modified N-terminal amino group. Sub.sub.2 is the free
C-terminal carboxyl group of the C-terminal amino acid (--COOH) or
a modified C-terminal carboxyl group. "Modified N-terminal amino
group" and "modified C-terminal carboxyl group" mean that the amino
group or carboxyl group is altered (e.g. reduced or
substituted).
[0048] Sub.sub.1 therefore represents the free N-terminus of the
amino acid X.sub.1 or a modification of the N-terminal amino group
(which replaces the N-terminal amino group of the amino acid
X.sub.1 with Sub.sub.1) with the general formula NR.sub.1R.sub.2.
Sub.sub.1=NR.sub.1R.sub.2, wherein R.sub.1 and R.sub.2 are
independent of one another and are preferably selected from
hydrogen or from the following groups: [0049] (i) a linear,
branched, cyclic or heterocyclic alkyl group, for example methyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl or cyclohexyl; [0050]
(ii) a linear, branched, cyclic or heterocyclic alkanoyl group, for
example acetyl or methanoyl (formyl), propionyl, n-butyryl,
isobutyryl, pentanoyl, hexanoyl or cyclohexanoyl; [0051] (iii) a
reporter group, preferably a fluorescent dye (for example
fluorescein, Alexa488) or biotin; [0052] (iv) together with
COR.sub.3 (see below) a linker between N- and C-terminus to obtain
a cyclic peptide, e.g. based on guanidine, ethylene glycol
oligomers, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid,
2,2'-diaminopimelic acid, desmosinee or isodesmosinees. [0053] (v)
a linker for coupling a further peptide or peptide derivative
(Y.sub.1) by means of a specific chemical or enzymatic reaction,
e.g. based on iodo-, bromo- or chloroalkanoic acids (e.g.
iodoacetic acids) or maleimide for coupling to a thiol-containing
peptide or also another reactive group (e.g. amino group, thiol
group) for coupling a second peptide or peptide derivative (e.g. as
active ester, aldehyde or thioester) as carrier protein. [0054]
(vi) a linker as stated in (v), to which another peptide or peptide
derivative Y.sub.3 is coupled.
[0055] Examples of N-terminal modifications are acetylated,
formylated or guanylated N-termini.
[0056] Preferably another peptide or peptide derivative Y.sub.1 is
coupled via Sub.sub.1. Y.sub.1 is preferably a biopolymer (e.g.
peptide), which introduces the antimicrobial peptide according to
any one of the formulas 1 to 4 into bacteria and therefore
increases the activity of the antimicrobial peptide against this
bacterium and/or introduces it into mammalian cells and therefore
make it possible to treat bacteria that are concealed in mammalian
cells. Y.sub.1 is coupled via Sub.sub.1 to X.sub.1 of the peptide
either permanently (e.g. peptide or amidine bond for
Sub.sub.1=NH.sub.2 or thioether for Sub.sub.1=SH, iodoacetate or
maleimide) or by a compound that is cleavable under certain
conditions (for example disulfide bridges or acid-labile linkers).
Preferred sequences for Y.sub.1 are cell-penetrating peptides
(CPP), for example penetratin, Tat peptides, model amphipathetic
peptides and transportans (Langel, U. in Handbook of
Cell-Penetrating Peptides 5-28 (CRC--Taylor & Francis Group,
2006).
[0057] A linker is a designation for molecules or groups of
molecules that are employed for coupling two substances; preferred
linkers contain two reactive groups (for example iodoacetate,
maleimide, imido- or NHs-ester or hydrazide), which are joined by a
bridging molecule (e.g. polyethylene glycol) preferably with 10 to
20 carbon atoms.
[0058] Sub.sub.2 is the free C-terminal carboxyl group of the
C-terminal amino acid (--COOH) or a modified C-terminal carboxyl
group, preferably with the general formula COR.sub.3 (R.sub.3
replaces the hydroxyl group of the last amino acid),
X.sub.5-COR.sub.3 or X.sub.6-COR.sub.3 or
X.sub.3X.sub.6-COR.sub.3.
[0059] COR.sub.3 is preferably selected from the following group:
[0060] (i) carboxyl (R.sub.3 is a free hydroxyl group), an ester
(R.sub.3 is an alkoxy group), an amide (R.sub.3 is an amine) or an
imide; [0061] (ii) a linker, which together with Sub.sub.1 bridges
the N- and C-termini to a cyclic peptide; [0062] (iii) COR.sub.3,
in which R.sub.3 is either an additional amino acid residue, which
is selected from the group comprising Pro, Ile, Leu, Arg and Gln,
or in which R.sub.3 is a peptide, preferably with two to six amino
acids, of which at least one amino acid is selected from the group
comprising Pro, Ile, Leu, Arg or Gln, wherein the latter is
substituted with a member from the group with carboxyl (R.sub.3 is
a free hydroxyl group), an ester (R.sub.3 is an alcohol, such as
methanol, ethanol, propanol, iso-propanol or butanol), an amide
(R.sub.3 is an amide) or an imide (R.sub.3 is an alkylamine or
dialkylamine, such as methylamine, ethylamine, dimethylamine or
cyclohexylamine). [0063] (iv) COR.sub.3 in which R.sub.3 is an
additional, branched amino acid, to form a dimeric or oligomeric
structure, for example lysine, hydroxylysine, ornithine,
2,4-diaminobutyric acid, 2,3-diaminopropionic acid,
2,2'-diaminopimelic acid, desmosinee, isodesmosinee or a
combination of these branched amino acids. [0064] (v) a linker for
coupling another peptide or peptide derivative (Y.sub.1) by means
of a specific chemical or enzymatic reaction, e.g. based on iodo-,
bromo- or chloroalkanoic acids (e.g. iodoacetic acid) or maleimide,
for coupling to a thiol-containing peptide or also another reactive
group (e.g. amino group, thiol group) for coupling a second peptide
or peptide derivative (e.g. as active ester, aldehyde or thioester)
as carrier protein. [0065] (vi) a linker as stated in (v), onto
which another peptide or peptide derivative Y.sub.1 is coupled.
[0066] In this way, C-terminal peptide derivatives can be obtained,
such as ester (R.sub.3=alkoxy), amide (R.sub.3=amine, e.g.
--NH.sub.2) or imide (R.sub.2=alkylamine, e.g. --NHC.sub.3H.sub.7)
or a peptide, which has been extended with further amino acids,
which was selected from the group comprising Pro, Ile, Arg and Val,
or once again are modified on the C-terminus as ester, amide or
imide. Further peptide derivatives can be formed by modification of
the N-terminal or C-terminal ends of the peptides. These changes
can be for example an additional alkyl or alkanoyl group (either
with a straight chain or branched, cyclic or heterocyclic) or an
additional guanidino group or an additional macromolecule or a
reporter residue, which is coupled either permanently or via a
compound that is cleavable under certain conditions (such as
disulfide bridges or acid labile linkers).
[0067] Modification of the C-terminus preferably takes place by
thioester synthesis and subsequent substitution with primary
amines.
[0068] All natural amino acids, unnatural amino acids or amino acid
derivatives (for example imino acids), which form the peptides or
peptide derivatives according to the invention, can be either in
the L- or D-conformation. Unless specified otherwise, however, the
building blocks in the sequences are preferably in the
L-conformation.
[0069] X.sub.5 and X.sub.6 are optionally additional residues. In
the case when X.sub.5 and X.sub.6 are absent, the last arginine
(Arg) in the abovementioned sequence has a free C-terminal carboxyl
group or is joined to Sub.sub.2.
[0070] In the case when at least one residue X.sub.5 and X.sub.6 is
present, the peptide has for example one of the following general
formulas:
Y.sub.1-Sub.sub.1-X.sub.1-D-K-P-P-Y-L-P-R-P-X.sub.2-P-P-R-X.sub.3-I-Y-N--
X.sub.4-X.sub.5-X.sub.6-COR.sub.3 (Formula 5)
Y.sub.2-Sub.sub.1-X.sub.1-D-K-P-P-Y-L-P-R-P-X.sub.2-P-P-R-X.sub.3-I-Y-R--
X.sub.4-X.sub.5-COR.sub.3 (Formula 6)
Y.sub.1-Sub.sub.1-X.sub.1-D-K-P-P-Y-L-P-R-P-X.sub.2-P-P-R-X.sub.3-I-Y-N--
X.sub.4-X.sub.6-COR.sub.3 (Formula 7)
[0071] X.sub.5 is selected from proline, proline derivatives or a
neutral residue with a polar side chain ( such as asparagine,
glutamine). Preferred residues X.sub.3 are selected from the groups
comprising proline, cis-4-hydroxyproline, trans-4-hydroxyproline,
cis-3-hydroxyproline, trans-3-hydroxyproline,
.beta.-cyclohexylalanine, 3,4-cis-methanoproline,
3,4-dehydroproline, homoproline, pseudoproline as well as
asparagine, glutamine, citrulline, N-methylserine, N-methylglycine,
homoserine, dihydroxyphenylalanine, N-ethyl asparagine,
N-ethylglycine, homoserine, penicillamine,
tetrahydropyranylglycine, allo-threonine and
3,5-dinitrotyrosine.
[0072] X.sub.6 is selected from proline derivatives, a polar
residue (such as serine) or a hydrophobic residue. Preferred
residues X.sub.6 are selected from the groups comprising proline,
cis-4-hydroxyproline, trans-4-hydroxyproline, cis-3-hydroxyproline,
trans-3-hydroxyproline, .beta.-cyclohexylalanine,
3,4-cis-methanoproline, 3,4-dehydroproline, homoproline or
pseudoproline, serine, threonine, .delta.-hydroxylysine,
citrulline, homoserine or allo-threonine as well as phenylalanine,
N-methylleucine, leucine, isoleucine, valine, methionine,
tert-butylglycine, cyclohexylalanine, alanine, .beta.-alanine,
1-aminocyclohexylalanine acid, N-methylisoleucine, norleucine,
norvaline, N-methylvaline or it is a short peptide sequence
preferably with one to three residues, which are preferably
selected from proline, isoleucine or one of the residues mentioned
above.
[0073] Alternatively X.sub.6 is a branched linker, which contains
several peptide units. This is formed by the residue of an amino
acid that contains several amino groups, for example lysine,
hydroxylysine, ornithine, 2,4-diaminobutyric acid,
2,3-diaminopropionic acid, 2,2'-diaminopimelic acid, desmosinee,
isodesmosinee.
[0074] The C-terminal amino acid is for example X.sub.4 in formulas
1 to 4, X.sub.5 in formula 6 or X.sub.6 in formulas 5 and 7.
[0075] The peptides and peptide derivatives according to the
invention display, relative to the previously incompletely
determined sequence of the proline-rich antimicrobial peptide
"Oncopeltus antibacterial peptide 4", an improved antibacterial
activity, a broader spectrum of action and increased protease
resistance.
[0076] Preferred examples of the peptides and peptide derivatives
according to the invention are selected from the sequences
according to SEQ ID NO. 5 to 9, 14 to 26, 29, 30, 32, 33, 36, 38,
40, 41, 44 to 46, 49, 50, 53 to 59, 61 to 85, 93, 94, 101, 102, 107
to 112; cf. Table 2 in example 1).
[0077] The especially preferred peptide with the sequence
VDKPPYLPRPRPPRRIYNR-NH.sub.2 (SEQ ID NO. 18) is called oncocin
hereinafter. Other peptides and peptide derivatives according to
the invention are called oncocin derivatives hereinafter.
[0078] The peptides and/or multimeric peptide constructs according
to the invention, which were modified to increase the antimicrobial
or antimycotic activity and to extend the spectrum of activity to
other bacteria or fungi and to improve the stability, are
characterized by their high antibacterial and/or antimycotic
efficacy and by good metabolic stability in mammalian serum.
[0079] Suitable modification in positions 11 (X.sub.2), 15
(X.sub.3) and 19 (X.sub.4) improve the antibacterial activity of
the native Oncopeltus 4-sequence against various bacteria, as is
discussed below and is shown in the examples.
[0080] Furthermore, the residues Sub.sub.1-X.sub.1, X.sub.3 and
X.sub.4 can additionally stabilize the N- and C-terminal peptide
sequences against proteolytic degradation and thus increase the
half-life in serum.
[0081] The sequences according to the invention have a positively
charged residue X.sub.2 (position 11).
[0082] Preferred examples according to the invention are sequences
with a positively charged residue X.sub.3 (position 15), for
example the sequences selected from the sequences according to SEQ
ID NO. 18, 29, 32, 33, 46, 50, 54 to 59, 62, 65 to 74, 78, 79, 82,
107, 109, 111 and 112 or with hydroxyproline as residue X.sub.3
(position 15), for example the sequences selected from the
sequences according to SEQ ID NO. 25 and 63.
[0083] The sequences according to the invention have a positively
charged residue X.sub.4 (position 19).
[0084] Furthermore, the C-terminal carboxyl group is preferably
modified. Surprisingly, this leads to an increased half-life of the
peptides in serum.
[0085] The modification of the N- and C-termini permit coupling of
the peptides to other groups, such as for example other amino acid
sequences (possibly creating multimeric peptides or proteins) or
other biomolecules, which have the function of a carrier or label,
for example of Y.sub.1 via Sub.sub.1. In a special embodiment the
carrier molecule functions as a shuttle in order to combat
bacterial infection in mammalian cells or transport the
antibacterial peptide and peptide derivative into bacteria, into
which the antibacterial peptide cannot penetrate on its own (e.g.
Gram-positive bacterial). Examples of said cell-penetrating
peptides (CPP) are for example penetratins, Tat peptides, model
amphipathic peptides and transportans. Moreover, the site of
infection can be recognized by the coupled structure (target
molecule) and as a result the antibiotic substance is brought near
the (bacterial) cell, in order to combat it. These target molecules
are for example molecules that are known to bind to
lipopolysaccharide (LPS) molecules, which form the outside of the
Gram-negative bacteria. Known compounds for this application are
for example anchor peptides, such as the AcmA motif from
Lactobacillus or an antibody directed against lipopolysaccharide.
This last-mentioned variant is preferred, as it also has an
intrinsic antibiotic effect and can therefore be used for
increasing the activity of the peptides according to the
invention.
[0086] It is advantageous if the N-terminal amino acid, i.e.
Sub.sub.1-X.sub.1, has a residue that is positively charged in
physiological conditions, i.e. in the human body.
[0087] An example for achieving N-terminal stabilization is
acylation (Sub.sub.1=acyl--NH--), for example acetylation
(Sub.sub.1=acetyl--NH--), of the .alpha.-amino group of a
positively charged amino acid, such as ornithine or lysine
(Sub.sub.1-X.sub.1=acyl-Orn or acyl-Lys). This acylation
(preferably acetylation) leaves the positive charge in the side
chain of the amino acid intact.
[0088] Further preferred examples of the invention are sequences
with positively charged residues on X.sub.2, X.sub.3 and X.sub.4
(position 11, 15 and 19), for example the sequences that are
selected from the sequences according to SEQ ID NO. 18, 22, 58, 62,
63, 65 to 74, 78, 79, 82 and 83.
[0089] Further preferred examples of the invention are sequences
with hydroxyproline instead of proline, for example the sequences
selected from the sequences according to SEQ ID NO. 21, 22 and
24.
[0090] Especially preferred examples are peptides that bear a
positively charged amino acid positions 11, 15 and 19 (X.sub.2,
X.sub.3 and X.sub.4) (such as ornithine, arginine or lysine) and
have a modified C-terminus, in particular the peptides according to
SEQ ID NO. 18, 63, 71, 72, 74.
[0091] An especially preferred peptide contains ornithine in
position 15 (residue X.sub.3), arginine in positions 11 and 19
(residues X.sub.2 and X.sub.4) and the C-terminus as propylamide
(residue Sub.sub.2) according to SEQ ID NO. 71.
[0092] Another especially preferred peptide contains ornithine in
position 15 and 19 (residue X.sub.3 and X.sub.4 and arginine in
position 11 (residue X.sub.3) the C-terminus as amide (residue
Sub.sub.2). A preferred peptide of this kind has the sequence
according to SEQ ID NO. 72.
[0093] Another especially preferred peptide contains arginine in
position 11 (residue X.sub.2), trans-4-hydroxyproline in position
15 (residue X.sub.3) and ornithine in position 19 (residue X.sub.4)
and the C-terminus as amide (residue Sub.sub.2). A preferred
peptide of this kind has the sequence according to SEQ ID NO.
63.
[0094] Another especially preferred peptide contains ornithine in
position 15 and 19 (residue X.sub.3 and X.sub.4, arginine in
position 11 (residues X.sub.2), in position 18 glutamine instead of
asparagine and the C-terminus as amide (residue Sub.sub.2). A
preferred peptide of this kind has the sequence according to SEQ ID
NO. 74.
[0095] It can be seen from the examples that the modifications of
the C-terminus according to the invention to an amide
(Sub---NH.sub.2), surprisingly, significantly increase the
antibiotic action against E. coli and M. luteus. Preferred
sequences with an amide on the C-terminus are SEQ ID NO. 18, 22,
50, 54 to 57, 61 to 63, 65 to 70, 72 to 79 and 82.
[0096] Modifications which reduce C-terminal degration are also
preferred, for example isopropylamide on the C-terminus (Sub.sub.2)
according to SEQ ID NO. 58 and 71 and trans-4-hydroxyproline at
position(s) 8 and/or 13. The experimental results show that
apparently the amino acids at position 6 and 7 are also very
important for the antibiotic action. Thus, exchange of individual
amino acids at these positions 6 and/or 7 with alanine destroys the
efficacy in comparison with the antibiotic activity of the
oncocin.
[0097] The most preferred examples of the invention are peptides,
which offer the following advantages: [0098] (i) an increased
half-life in mammalian serum and [0099] (ii) an increased
antimicrobial activity against one or more bacterial strains,
especially human pathogens, or fungi or other microbial infections
and [0100] (iii) the peptides are not toxic to human cells,
including erythrocytes.
[0101] The action of antimicrobial peptides is very complex, as
they must pass through the cell membrane and penetrate into the
cytoplasm, in order to inhibit a special intracellular bacterial
target molecule, but without having a toxic effect on mammalian
cells and blood cells. Another important point is stability of the
peptidases or peptide derivatives against degradation by peptidases
or proteases. Therefore the ideal peptide has a high antibacterial
activity (low MIC values), no cellular toxicity, no hemolytic
activity and a half-life of several hours in blood. Compared with
the native Oncopeltus 4-sequence, the peptide derivatives according
to the invention display a more than twenty times higher
antimicrobial activity. The C-terminus is preferably modified
(amide, alkylamide, ester) and the C-terminal region after position
14 is for example altered by substitution of positions 15 and/or 19
(X.sub.3 and X.sub.4) with nonproteinogenic amino acids. At the
N-terminus, a positive charge is preferred, to achieve good
activity. The valine in position 1 (X.sub.1) of the native
Oncopeltus 4-sequence is preferable free or replaced with an
acetylated basic residue such as arginine, lysine or ornithine.
Ornithine is especially preferred, which surprisingly leads to
increased protein stability. For the same reason positions 15 and
19 of the Oncopeltus 4-sequence are preferably substituted, which
surprisingly leads to increased peptide stability. Examples are the
exchange of positions 15 and 19 (X.sub.3 and X.sub.4) for
trans-4-hydroxyproline (X.sub.3) and ornithine (X.sub.4) or both
for ornithine, which unexpectedly increases the half-life in 25%
serum by more than a factor of 10, i.e. from less than 30 min to
more than 6 h. The transfer of the C-terminus from the free acid to
a propylamide also leads to an increase in serum stability.
[0102] Preferably a cell-penetrating peptide sequence is coupled to
the peptides and peptide derivatives according to the invention.
This coupling preferably takes place via a linker, for example an
acetyl group or an alkyl group. The coupling preferably takes place
on the N-terminus of the peptide and peptide derivative according
to the invention. Preferably, for this the proline-rich peptide or
peptide derivative is derivativized at the N-terminal with
iodoacetate and the cell-penetrating peptide sequence is lengthened
at the C-terminal by a cysteine residue. The thiol group of this
cysteine then forms a thioether bridge with the acetyl group.
[0103] Cell-penetrating peptides (CPP) are relatively short
polycationic or hydrophobic peptides, attachment of which makes it
possible for prokaryotic and eukaryotic cells to pass through the
cell membrane. CPPs can have different sequences and lengths. In
most cases, however, they contain a sequence of approx. 10 to 40
amino acids and are rich in positively charged amino acids (e.g.
Arg, Lys). These short sequences are responsible for passage
through the cell membrane and are called "protein transduction
domains (PTDs)".
[0104] Preferred cell-penetrating peptides are selected from
penetratin, Tat peptides, model amphipathic peptides, transportan
(derived from galanin), SynB (derived from protegrin) and
cis-.gamma.-amino-1-proline-containing peptides.
[0105] The cell-penetrating peptide sequences used according to the
invention are preferably 8 to 20 amino acid residues long, wherein
30% to 90% of the residues have side chains that are positively
charged under physiological conditions. The remaining residues are
preferably neutral. Preferred cell-penetrating peptide sequences
are selected from:
TABLE-US-00002 SEQ ID NO. 105 RQIKIWFQNRRMKWKK-OH (a penetratin),
SEQ ID NO. 124 KLALKLALKALKAALKLA-NH.sub.2 (model amphipathic
peptide) SEQ ID NO. 125 RKKRRQRRR (a Tat peptide).
[0106] Further preferred cell-penetrating peptide sequences are
given in literature (Langel, U. in Handbook of Cell-Penetrating
Peptides 5-28 (CRC--Taylor & Francis Group, 2006), (Pujals S,
Giralt E. Proline-rich, amphipathic cell-penetrating peptides Adv
Drug Deliv Rev. 60(4-5): 473-84, 2008) and (Farrera-Sinfreu J,
Giralt E, Royo M, Albericio F. Cell-penetrating proline-rich
peptidomimetics. Methods Mol Biol. 386: 241-67, 2007), which in
this connection are incorporated as references.
[0107] In preferred examples of said peptide derivatives, the AMPs
were lengthened N-terminally before position 1 (X.sub.1) by
penetratin-cysteine (Y.sub.1) via iodoacetyl (Sub.sub.1) with
formation of a thioether bond. Preferred examples of this kind are
preferably selected from the sequences according to SEQ ID NO. 101
and 102.
[0108] Through the attachment of cell-penetrating peptide sequence,
such as penetratin, surprisingly the activity against Gram-negative
and Gram-positive bacteria is increased and the spectrum of action
is extended to other Gram-positive and Gram-negative bacteria and
additionally the antimicrobial peptides will be introduced into
mammalian calls without being cytotoxic, so that hidden bacteria,
fungi or viruses can also be reached in these cells.
[0109] Penetratin corresponds to the partial sequence R43 to K58 of
the Antennapedia homeodomain (DNA-binding region of a transcription
factor), of the fruit fly Drosophila melanogaster. The sequence of
penetratin (preferably R Q I K I W F Q N R R M K W K K--OH: SEQ ID
NO. 105) is rich in cationic amino acids and in that resembles the
sequences of many AMPs.
[0110] In this invention the cell-penetrating peptide sequence is
utilized for introducing AMPs both into bacteria and mammalian
cells. Coupled to penetratin, the AMPs are transported into
eukaryotic cells, also for treating inflections there. In addition,
toxic effects can be investigated by interaction of the AMPs with
intracellular target molecules.
[0111] The coupling of penetratin to the antimicrobial peptides
according to the invention via thioether bridge forms part of the
invention.
[0112] For this, the C-terminus of penetratin was extended by a
cysteine and was coupled to the antimicrobial peptide, labeled
N-terminally with iodoacetic acid.
[0113] As well as oncocin and its derivatives, according to the
invention other antimicrobial peptides, preferably proline-rich
peptides or peptide derivatives, for example apidaecin, drosocin,
formaecin 1, pyrrhocoricin and matalnikowin 1, can also be modified
correspondingly with a cell-penetrating peptide sequence, for
example penetratin.
[0114] Preferred apidaecin derivatives are mentioned in
PCT/EP2008/059512 (filed on Jul 21, 2008), which in this connection
is incorporated as reference.
[0115] Preferred examples of said peptide derivatives are selected
from the sequences according to SEQ ID NO. 95 to 100 and 106.
[0116] The expression "peptide", as used here, stands for a
sequence of amino acids that are linked via a peptide bond, wherein
the amino acids are preferably selected from the twenty naturally
occurring peptide-forming amino acids and in which the amino acids
can be in the L-configuration or D- configuration, or in the case
of isoleucine and threonine also in the D-allo-configuration (only
inversion of one of the two chiral centers).
[0117] The expression peptide derivative (or peptidomimetric) used
in the description of the invention comprises not only peptides
that are modified with Y.sub.1, Sub.sub.1 and Sub.sub.2 on the N-
or C-terminus, as described above. In addition it comprises
peptides that have been altered by substitutions and/or
modifications of one or more amino acid residues by chemical
groups, wherein said chemical groups are different from the natural
protein-forming amino acid residues, for example nonproteinogenic
.alpha.-amino acids, .beta.-amino acids or peptides with an altered
backbone. The term "altered backbone" means that at least one
peptide bond has been modified chemically, i.e. has been replaced
with a bond that is not cleavable under physiological conditions,
and cannot be cut by endoproteases.
[0118] Preferably the noncleavable bond is a modified peptide bond,
for example a reduced peptide bond, an alkylated amide bond or a
thioamide bond. A reduced amide bond is a peptide bond in which the
carbonyl group (C.dbd.O) has been reduced to a hydroxyl group
(HCOH) or a methylene group (CH.sub.2). An alkylated amide bond is
a peptide bond alkylated either on the nitrogen (N-alpha) or carbon
atom (C-alpha). The alkyl residue preferably has 1 to 3 carbon
atoms. An example is N-methylation.
[0119] Moreover, the term "altered backbone" comprises other groups
that are suitable for forming a covalent bond both with the COOH
group of the preceding amino acid residue and the NH.sub.2 group of
the next amino acid residue, and which therefore do not necessarily
maintain the peptide backbone structure, for example sugar amino
acid-dipeptide isostere, azapeptides, 6-homopolymers,
gamma-peptides, Y-lactam-analogs, oligo(phenylene ethylene)s,
vinylog sulfone peptides, poly-N-substituted glycines or
oligocarbamates.
[0120] Modifications of the backbone are in positions 14 to 19,
R-X.sub.3-I.sub.16-Y.sub.17-N.sub.18-X.sub.4. Therefore preferably
at least one of the bonds between X.sub.3-I.sub.16 (e.g. Arg--Ile),
N.sub.18-X.sub.4 (e.g. Asn-Arg), X.sub.4-NH.sub.2 (e.g.
Arg-NH.sub.2), X.sub.6-X.sub.7 (e.g. Arg-Leu or Arg-Ile) is
modified. These bonds are preferably selected from the group of
reduced amide bonds, alkylated amide bonds or thioamide bonds.
[0121] The peptides and peptide derivatives according to the
invention can be linear, i.e. a sequence in which the first and the
last amino acid of the sequence possess a free NH.sub.2 and COOH
group or have been modified with Sub.sub.1 and Sub2. Alternatively
the peptides are cyclic, i.e. the first and the last amino acid are
linked via a peptide bond or a linker.
[0122] The methods of producing the abovementioned novel
antibiotically active compounds also form part of the
invention.
[0123] The peptides or peptide derivatives of the present invention
can be produced either synthetically or, where applicable,
recombinantly by conventional methods. Special examples of carrying
out the invention are disclosed in detail below, in the
experimental section. Preferably the peptides or peptide
derivatives of the present invention are produced conventionally by
the known synthesis techniques, as described for example by
Merrifield (Merrifield R B. Solid-phase peptide synthesis. 1.
Synthesis of a Tetrapeptide. Journal of the American Chemical
Society 85: 2149-6, 1963).
[0124] Alternatively, the peptides described in the present
invention are produced by recombinant techniques, wherein a DNA
fragment that contains a nucleic acid sequence that codes for one
of the peptides described above is cloned, and is expressed e.g. in
a microorganism or a host cell. The coding nucleic acid sequences
can be produced synthetically (Stemmer W P C, Crameri A, Ha K D,
Brennan T M, & Heyneker H L. Single-Step Assembly of a Gene and
Entire Plasmid from Large Numbers of Oligodeoxyribonucleotides.
Gene 164: 49-53, 1995) or can be obtained by side-specific
mutagenesis of an existing nucleic acid sequences (e.g. sequence
that codes for the wild-type Oncopeltus 4). The coding sequence
thus produced can be amplified by RNA (or DNA) with correspondingly
produced primers in a polymerase chain reaction (PCR) by known
techniques. After purification, for example by agarose gel
electrophoresis, the PCR product is ligated into a vector and
finally the host cell is transformed with the corresponding
recombinant plasmid. Recombinant techniques are known for various
host cells, for example E. coli, Bacillus, Lactobacillus,
Streptomyces, mammalian cells (e.g. CHO (Chinese hamster ovary) or
COS-1 cells), yeast cells (e.g. Saccharomyces, Schizophyllum),
insect cells or viral expression systems (e.g., Baculovirus
System). Further suitable host cells and methods for
transformation, cultivation, amplification, screening, product
production and purification can be selected from the literature by
any person skilled in the art (Gething M J & Sambrook J.
Cell-Surface Expression of Influenza Hemagglutinin from a Cloned
Dna Copy of the Rna Gene. Nature 293: 620-5, 1981). After
conventional recombinant production, the peptides of the present
invention can be isolated from the host cells, either by classical
cell lysis techniques or from the cell medium by conventional
methods, e.g. liquid chromatography, in particular affinity
chromatography. The antimicrobial peptide can be expressed as
individual peptide or as oligomer. The oligomers can contain
several peptide sequences, which are linked via the N- or
C-terminus, or even contain an N- or C-terminal tag, which allows
easier purification of the recombinant peptides or protein
constructs. Conventional techniques of molecular biology and
site-directed mutagenesis can be used, to modify the sequence
further and thus obtain the desired non-native peptide sequences.
All these recombinant techniques are known by a person skilled in
the art and have already been applied for many antimicrobial
peptides including apidaecin (Maeno M, Taguchi S, & Momose H.
Production of Antibacterial Peptide Apidaecin Using the Secretory
Expression System of Streptomyces. Bioscience Biotechnology and
Biochemistry 57:1206-7, 1993), perinerin (Zhou Q F, Luo X G, Ye L,
& Xi T. High-level production of a novel antimicrobial peptide
perinerin in Escherichia coli by fusion expression. Current
Microbiology 54: 366-70, 2007) and defensin (Si L G, Liu X C, Lu Y
Y, Wang G Y, & Li W M. Soluble expression of active human
beta-defensin-3 in Escherichia coli and its effects on the growth
of host cells. Chinese Medical Journal 120: 708-13, 2007).
[0125] It is also possible for amino acids that do not occur
naturally to be introduced into the peptides by gene technology.
This was described in detail by Noren et al. and Ellman et al.
(Noren C J, Anthonycahill S J, Griffith M C, & Schultz P G. A
General Method for Site-Specific Incorporation of Unnatural
Amino-Acids Into Proteins. Science 244: 182-8, 1989; Ellman J.
Mendel D, Anthonycahill S, Noren C J, & Schultz P G.
Biosynthetic Method for Introducing Unnatural Amino-Acids
Site-Specifically Into Proteins. Methods in Enzymology 202: 301-36,
1991).
[0126] Next, the peptides can be isolated from the host cell
culture or the in-vitro translation system. This can be achieved
with the usual techniques for protein purification and isolation
that are known from the prior art. Such techniques can for example
comprise immuno-adsorption or affinity chromatography. It is also
possible to provide the peptides, during synthesis, with a tag
(e.g. histidine taq), which allows rapid binding and purification.
The tag can later be split off enzymatically, to obtain the active
peptide sequence.
[0127] If the peptide itself cannot be encoded or expressed, but is
very similar to an encodable or expressible peptide, the method can
first be applied to the similar peptide, which is subsequently
converted in one or more steps chemically or enzymatically to the
desired peptide or peptidomimetic. Some more comprehensive accounts
of these methods of producing the peptides described here are
described in the literature (Anderson W F. Human gene therapy.
Nature 392: 25-30, 1998; Pharmaceutical Biotechnology (eds.
Crommelin D J A & Sindelar R D) pp. 8-20, 53-70, 123-152,
167-180) Harwood Academic Publishers, 1997; Protein Synthesis:
Methods and Protocols (ed. Martin R) 1-144, Humana Press, 1998;
Amino Acid and Peptide Synthesis (ed. Jones J) 1-89, Oxford
University Press, 1997; Solid-Phase Peptide Synthesis (e. Fields G
B) 1-780, Academic Press, 1997.
[0128] The peptides and peptide derivatives according to the
invention can be used individually, in combination, as multimers or
as breanched multimers. Reasonable combinations of the peptides
according to the invention comprise concatamers, in which the
peptides according to the invention are linked together
sequentially or via spacers, e.g. in the form of a peptide dimer or
a peptide trimer etc. (miltimer), with the individual peptides
strung together. This multimer can be made up from peptides or
peptide derivatives with identical sequences or different sequences
according to any of the formulas 1 to 4.
[0129] Individual peptides or peptide derivatives can be coupled to
a biocompatible protein, for example human serum albumin, humanized
antibodies, lipsomes, micelles, synthetic polymers, nanoparticles
and phages. Alternatively, multimers in which the peptides or
peptide derivatives according to the invention are combined
individually, can be produced in the form of dendrimers or
clusters, with three or more peptides bound to one center.
[0130] In one embodiment, several peptides or peptide derivatives
according to any of the formulas 1 to 4 described above can be
produced as multimeric constructs or arrangement. Thus, for
example, optionally amino acids (e.g. Gly-Ser-) or other spacers
based on amino acids or other chemical compouns can be attached to
the N- or C-terminus, in order to link two or more peptides
together or couple them to a carrier. This arrangement can assume
the form of one or more of the synthetic peptides described above
couples to a carrier protein. Alternatively, an arrangement
contains several peptides, each expressed as multiple antigenic
peptide, optionally coupled to a carrier protein. In another
variant the selected peptides are linked sequentially and are
expressed as recombinant protein or polypeptide. In one embodiment,
several peptides are linked sequentially, with or without amino
acids as spacers in between, to obtain a larger recombinant
protein. Alternatively the recombinant protein can be fuesed to a
carrier protein.
[0131] In another embodiment the multimeric constructs contain at
least two of the peptides defined above (which can be the same or
different peptides of any of the formulas 1 to 4), wherein one
peptide is coupled via any amino acid to the other peptides. Any
number of further peptides can be attached to any further amino
acids of these peptides. In another embodiment of a multimeric
arrangement, which contains at least tow peptides, the second or
the further peptides is/are coupled to a branched structure of the
other peptides of the basic structure. Alternatively, each further
peptide is linked covalently via the group Sub.sub.1 or Sub.sub.2
to another peptide of the arrangement.
[0132] In another embodiment of a multimeric construct or of an
arrangement with at least two peptides, of least one or more
peptides are bound to a carrier. In another embodiment one or more
of the stated peptides are a synthetic peptide, which is fused to a
carrier protein. Furthermore, there is the alternative of combining
several of the peptides described above with or without flanking
sequences sequentially to a linear polypeptide. The peptides or the
polypeptide are either coupled to the same carrier or different
peptides can be coupled individually as peptides to one or
different immunologically inert carrier proteins.
[0133] Suitable carriers improve the stability, the administration
or the production, or alter or improve the activity spectrum of the
peptides. Examples of carriers are human albumin, polyethylene
glycol or other biopolymers or other naturally or non-naturally
occurring polymers. In one embodiment, the main component of the
carrier is preferably a protein or other molecule that increases
the stability of the peptide. A person skilled in the art can
easily select a suitable coupling unit of carrier and peptide.
[0134] In another embodiment the peptides are arranged in the form
of a multiple antigenic peptide (MAP), which can for example be
constructed by the "MAP" concept described by Tam et al. (Tam J P,
Mora A L, & Rao C. Lipidation as a novel approach to mucosal
immunization. Modulation of the Immune Response to Vaccine Antigens
92: 109-16, 1998). This system uses a central unit of lysine
residues, onto which several copies of the same peptide according
to the invention are synthesized (see e.g. Posnett D N, Mcgrath H,
& Tam j P. A Novel Method for Producing Anti-peptide
Antibodies--Production of Site-Specific Antibodies to the T-Cell
Antigen Receptor Beta-Chain, Journal of Biological Chemistry 263:
1719-25, 1988). Each MAP contains several copies of one or more of
the peptides according to the invention. One embodiment of a MAP
contains at least three, but preferably four or more peptides. A
person skilled in the art can easily produce any number of
multimeric compounds according to the peptides identified in the
above formulas. All such multimeric arrangements and constructs
form part of the present invention.
[0135] Further combinations in the form of multimers can be
produced on the surface of particles, wherein the peptides or
peptidomimetics are presented on their surface. The particle can
then function as carrier of a peptide or peptidomimetic and can act
simultaneously as detectable marker. Multimers can for example be
obtained by N-terminal biotinylation of the N-terminal end of the
peptide or peptidomimetic chains and subsequent complexation with
streptavidin. As streptavidin can bind four biotin molecules or
conjugates with high affinity, very stable tetrameric peptide
complexes can be obtained with this method. Multimers can be
produced from identical or different peptides or peptidomimetics
according to the invention. Preferably the multimers according to
the invention contain two or more peptides or peptidomimetics, in
which each component contributes to the biocidal activity (target
recognition, antimicrobial activity, purification).
[0136] Another object of the present invention is the use of the
peptides or peptide derivatives according to the invention in
medicine or pharmacy, e.g. for therapy with an antibiotic or in a
composition with antimicrobial (in particular bactericidal)
activity.
[0137] The invention also comprises a peptide, peptide derivative
and/or multimer according to the invention for use in the treatment
of microbial, bacterial or fungal infections.
[0138] The present invention further relates to pharmaceutical
compositions that contain one or more peptides or peptide
derivatives according to the invention or multimeric constructs
independently of the presence of other active pharmaceutical
ingredients.
[0139] The use of the peptides according to the invention as a
pharmaceutical and/or for the production of an active substance
that can be used as an antibiotic is also part of the present
invention.
[0140] the peptides can also be used individually in pharmaceutical
products. Alternatively, one or more peptides as described above
can be fused or conjugated to another compound, in order to
increase the pharmacokinetics or bioavailability, without inducing
an immune response. Any number of individual peptides or multimeric
constructs can be mixed together to produce a single
composition.
[0141] A pharmaceutical composition according to the invention
contains a therapeutically effective amount of one or more peptides
or peptide derivatives of the present invention. Once composed, the
pharmaceutical composition according to the invention can be
administered to the subject directly, to treat microbial (in
particular bacterial) infections. For this, a therapeutically
effective amount of a composition according to the invention is
administered to the subject to be treated.
[0142] The compositions according to the invention are intended for
treating infections of a mammal, including humans, infected with
bacteria or fungi.
[0143] At least one or alternatively also several peptides or
multimeric constructs according to the invention can be mixed with
a pharmacologically acceptable vehicle or other components to form
a composition with antimicrobial (in particular antibacterial or
fungicidal) action. For the use of such a composition, the selected
peptide is preferably produced synthetically or also recombinantly,
as described above.
[0144] The direct administration of this composition takes place
either topically (on the surface of the skin) or by some other
route of administration, for example oral, parental, subcutaneous,
sublingual, intralesional, intraperitoneal, intravenous,
intramuscular, pulmonary or interstitial into the tissue.
[0145] The pharmaceutical composition can contain further suitable
and pharmaceutically acceptable vehicles, fillers or solvents and
can have the form of a capsule, tablet, pastille, coated tablet,
pill, drops, suppository, powder, spray, vaccine, ointment, paste,
cream, inhalant, patch, aerosol or the like. Pharmaceutically
acceptable excipients can include solvents, diluents or other
liquid binders such as dispersion or suspension aids, surfactants,
isotonically active substances, thickeners or emulsifiers,
preservatives, encapsulating agent, solid binders or glidants,
depending on what is most suitable for the particular dosage and at
the same time is compatible with the peptide, peptidomimetic
(peptide derivative), peptide conjugate or peptidomimetic
conjugate.
[0146] The pharmaceutical composition therefore preferably contains
a pharmaceutically acceptable vehicle. The term "pharmaceutically
acceptable vehicle" also comprises a vehicle for administration of
the therapeutic composition, for example antibodies or
polypeptides, genes or other therapeutic agents. The term refers to
any pharmaceutical vehicle which does not itself trigger the
production of antibodies that could be dangerous for the individual
to whom the recipe has been administered, and does not possess any
unreasonable toxicity. Suitable "pharmaceutically acceptable
vehicles" can be large, slowly degradable macromolecules, for
example proteins, polysaccharides, polylactonic acids, polyglycolic
acids, polymeric amino acids, amino acid copolymers and inactivated
viral constituents. Said vehicles are familiar to a person skilled
in the art.
[0147] Salts of the peptides or functionally equivalent compounds
can be produced by known methods, which typically means that the
peptides, peptidomimetics, peptide conjugates or peptidomimetic
conjugates are mixed with a pharmaceutically acceptable acid to
form an acid salt or with a pharmaceutically acceptable base to
form a basic salt. Whether an acid or a base is pharmaceutically
acceptable can easily be established by a person skilled in the
art, knowing the application and the recipe. For instance, not all
acids and bases that are acceptable for ex vivo applications can
also be transferred to therapeutic recipes. Depending on the
particular application, pharmaceutically acceptable acids can be of
both an organic and inorganic nature, e.g. formic acid, acetic
acid, propionic acid, lactic acid, glycolic acid, oxalic acid,
pyrovic acid, succinic acid, maleic acid, malonic acid, cinnamic
acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric
acid, perchloric acid, phosphoric acid and thiocyanic acid, which
form ammonium salts with the free amino groups of peptides and
functionally equivalent compounds. Pharmaceutically acceptable
bases that form carboxylates with free carboxylic acid groups of
the peptides and functionally equivalent compounds, comprise
ethylamine, methylamine, dimethylamine, triethylamine,
isopropylamine, diisopropylamine and other mono-, di- and
trialkylamines as well as arylamines. Moreover, pharmaceutically
acceptable solvents are included.
[0148] Pharmaceutically acceptable salts can be used, for example
salts of mineral acids, such as hydrochlorides, hydrobromides,
phosphates, sulfates and the like; but also salts of organic acids,
such as acetates, propionates, malonates, benzoates and the like. A
detailed discussion of pharmaceutically acceptable ingredients is
given in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J.,
1991).
[0149] Pharmaceutically acceptable vehicles in the therapeutic
compositions can contain liquids, for example water, salt water,
glycerol and ethanol. Other excipients can also be added, such as
moistening agents or emulsifiers; pH buffering substances and
similar compounds can be present in said agents. Typically, the
therapeutic compositions are prepared either in liquid form or as
suspension for injection, and solid forms for dissolving or
suspending in carrier liquids before injection are also possible.
Liposomes are also included in the definition of a
"pharmaceutically acceptable vehicle".
[0150] For therapeutic treatment, peptides, peptide derivatives,
peptide conjugates or peptide derivative conjugates, as described
above, can be produced and can be administered to a subject who
requires this. The peptide, peptide derivative, peptide conjugate
or peptide derivative conjugate can be administered to a
subject/patient in any suitable form, preferably as a
pharmaceutical composition, which is adapted to the dosage form and
is present at a dose appropriate to the desired treatment.
[0151] The pharmaceutical compositions of the present invention can
contain other active compounds, for example conventional
antibiotics (e.g. vancomycin, streptomycin, tetracycline,
penicillin) or other antimicrobially active compounds, such as
fungicides, e.g. itraconazole or miconazole. Other compounds that
alleviate symptoms accompanying the infection, such as fever
(salicylic acid) or rash, can also be added.
[0152] Alongside the therapeutic use for the treatment of
infections, or also in biological warfare, it is further possible
to use the peptides or peptide derivatives according to the
invention in disinfectants and/or cleaning agents (e.g. a
bactericidal composition), which can be used for disinfecting
and/or cleaning surfaces and/or objects. Another field of
application is packaging, where peptides can be bound to packaging
material or incorporated therein, or as preservatives for other
materials that can readily be degraded by microorganisms. The
peptides or peptide derivatives according to the invention are
suitable in particular for the packaging of foodstuffs, as they
have no toxic action on contact or if ingested.
[0153] Another object of the present invention is a method for
treating mammals that are infected with microbes (in particular
bacteria or fungi), including the administration of an effective,
therapeutically active amount of the pharmaceutically active
composition according to the invention.
[0154] In connection with the present invention, bacterial or
fungal infections can be selected inter alia from the group
comprising infections of the urogenital tract, infections of the
blood ("blood-stream infections"), sepsis, respiratory tract
infections, peritonitis, wound infections, infections of the
digestive tract and meningitis.
[0155] The term "therapeutically effective amount" used here
designates the amount of a therapeutic agent, i.e. a peptide,
peptidomimetic, peptide conjugate or peptidomimetic conjugate
according to the invention, which is able to reduce or completely
prevent the multiplication and colony formation of the bacteria or
can achieve a measurable therapeutic or prophylactic success. The
effect can for example be determined for biopsies in culture, by
testing the bacterial activity or with some other suitable method
of assessment of the extent and degree of a bacterial infection.
The precise effective amount for a subject depends on their weight
and state of health, the type and the extent of the disease and the
therapeutic agent or the combination of several therapeutic agents
that were selected for the treatment. In particular, the
compositions according to the invention can be used for reducing or
preventing bacterial infections and/or biological or physical
concomitant effects (e.g. fever). Methods of establishing the
initial dose by a physician are known from the prior art. The doses
established must be safe and successful.
[0156] The amount of a protein, peptide or nucleic acid according
to the invention that is necessary for an antibacterially effective
dose can be established taking into account the pathogen that
causes the infection, the severity of the infection, and the
patient's age, weight, sex, general physical condition etc. The
necessary amount of the active component, to be antibacterially and
antimycotically effective without notable side effects, depends on
the pharmaceutical recipe used and the optional presence of other
ingredients, such as antibiotics, antimycotics etc. For the areas
of application according to the invention, an effective dose can be
between 0.01 .mu.g/kg and 50 mg/kg, preferably between 0.5 .mu./kg
and 10 mg/kg of the peptide, peptidomimetic, peptide conjugate or
peptidomimetic conjugate in the individual being treated.
[0157] Initial doses of the peptides, peptidomimetics, multimers,
peptide conjugates or peptidomimetic conjugates according to the
invention can optionally be monitored by repeated administration.
The frequency of the dosages depends on the factors identified
above and is preferably between one and six doses per day over a
treatment period from about three days to a maximum of one
week.
[0158] In another, alternative composition, the peptides,
peptidomimetics, peptide conjugates or peptidomimetic conjugates or
mixtures according to the invention are administered by controlled
or continuous release from a matrix, which has been introduced into
the subject's body.
[0159] In one embodiment, a compound according to the invention is
administered through the skin. This method of administration is
noninvasive and patient-friendly, and at the same time it
apparently leads to an increased bioavailability of the compound
compared with oral administration, especially if the compound is
not stable in the environment of the digestive system or if it is
too large to be absorbed efficiently from the intestine. Absorption
through the skin is for example possible in the nose, the cheek,
under the tongue, on the gums or in the vagina. Corresponding
dosage forms can be obtained by known techniques; they can be
processed into nasal drops, nasal sprays, implants, films, patches,
gels, ointments or tablets. Preferably, for absorption through the
skin, the pharmaceutical vehicle will contain one or more
components that adhere to the skin and thereby prolong the contact
time of the dosage form with the absorbing surface, so as to
increase the intake by absorption.
[0160] In another embodiment the compounds are administered in a
defined amount by the pulmonary route, e.g. by means of an inhaler,
atomizer, aerosol spray or a dry-powder inhaler. Suitable
formulations can be prepared by known methods and techniques.
Transdermal or rectal delivery, as well as application in the eye,
may be appropriate in some cases.
[0161] It may be advantageous for the substances according to the
invention to be administered more effectively by advanced drug
delivery or targeting methods. Thus, if the digestive tract is to
be avoided, the dosage form can contain any substance or mixture
that increases the bioavailability. This can for example be
achieved by reducing degradation, e.g. by means of an enzyme
inhibitor or an antioxidant. It is better if the bioavailability of
the compound is achieved by an increase in the permeability of the
barrier to absorption, generally the mucosa. Substances that
facilitate penetration can act in various ways; some increase the
fluidity of the mucosa, whereas others expand the interstices
between the mucosal cells.
[0162] Yet others reduce the viscosity of the mucus on the mucosa.
The preferred absorption accelerators include amphiphilic
substances such as cholic acid derivatives, phospholipids, ethanol,
fatty acids, oleic acid, fatty acid derivatives, EDTA, carbomers,
polycarbophil and chitosan.
[0163] Indications for which the peptides, peptide derivatives,
conjugates or multimers according to the invention can be used are
bacterial infections both with Gram-positive and with Gram-negative
bacteria, for example Escherichia coli, Enterobacter cloacae,
Erwinia amylovora, Klebsiella pneumoniae, Morganella morganii,
Salmonella typhimurim, Salmonella typhi, Shigella dysenteriae,
Yersinia enterocolitica, Acinetobacter calcoaceticus, Acinetobacter
baumannii, Agrobacterium tumefaciens, Francisella tularensis,
Legionella pneumophila, Pseudomonas syringae, Rhizobium meliloti,
Haemophilus influenzae, Stenotrophomonas maltophilia, Pseudomonas
aeruginosa, Proteus vulgaris or Proteus mirabilis.
[0164] The invention also relates to the use of a peptide, peptide
derivative or multimer according to the invention in biochemical,
biotechnological, medical or pharmaceutical research or in
screening, in particular for identifying substances that have a
potential antibacterial or antimycotic action.
[0165] Therefore the invention also relates to a method of
identifying compounds with antibacterial or antimycotic action,
which comprises the following: [0166] (i) carrying out a
competitive assay with: [0167] (a) a microorganism that is
sensitive to a peptide, peptide derivative or multimer according to
the invention, [0168] (b) a peptide, peptide derivative or multimer
according to the invention, [0169] (c) at least one compound that
is to be tested, by bringing (a) into contact with (b) and (c); and
[0170] (ii) selecting a test compound that displaces the peptide,
peptide derivative or multimer competitively from the
microorganism.
[0171] This screening process identifies test compounds that can
compete with the peptide or the multimeric construct according to
the invention for binding to the unknown receptor of the pathogen.
In this way, small molecules that bind specifically to the same
site as the peptide can be identified effectively in
high-throughput screening. The test compounds presumably have the
same mechanism of action as the original peptide sequence and
therefore are also active against multiresistant microbes, which
are eradicated by the peptides or peptide derivatives according to
the invention.
[0172] The screening process is carried out by known methods, but
uses at least one peptide, peptide derivative or multimer according
to the invention. Preferably, for this the peptide, peptide
derivative or multimer according to the invention is provided with
a fluorescent, radioactive or other detectable marker. The binding
behavior of the labeled peptide, peptide derivative or multimer to
the microorganism is compared in the presence and in the absence of
the test substance(s).
[0173] Preferably, the test compounds that compete for binding with
the peptide according to the invention or a multimeric construct
are then identified and are tested for their antibacterial or
antimycotic action.
[0174] In one embodiment, the fluorescence is measured in a
competitive assay after formation of a dimer (BIFC; bimolecular
fluorescence complementation). The method permits direct
visualization of intracellular protein interactions, which was
demonstrated for the example of the SH3 domain from c-Abl tyrosine
kinase with natural and artificial target molecules in E. coli
(Morell M, Espargaro A, Aviles F X, & Ventura S. Detection of
transient protein-protein interactions by biomolecular fluorescence
complementation: The Al-SH3 case. Proteomics 7: 1023-36, 2007).
This test system is sufficiently sensitive even to be able to
detect the interactions between proteins with low expression level
in E. coli. It is based on adduct formation of two fragments of the
yellow fluorescent protein (YFP), after the SH3 domain has bound to
its partner. As soon as these two proteins have bound to one
another, the two fragments of YFP form a complex, whose structure
is very similar to the native protein. This can be seen from the
observed fluorescence of the YFP complex, as the individual
fragments do not fluoresce. A similar construct can also be
designed for searching for compounds that compete for the binding
site with the peptides and peptide derivatives described in the
present invention. High-throughput screening can easily be
transferred by a person skilled in the art to a microtiter plate in
the 386-well format.
[0175] In another embodiment the peptides are used in a suitable
competitive assay, to determined the capacity of the test compounds
to displace the peptides competitively from the unknown receptor of
the pathogens. Where desired, microorganisms (e.g. bacterium, virus
or fungus), which are known to bind to the selected peptide(s),
e.g. strains of E. coli or K. pneumoniae, can (depending on the
assay chosen) be immobilized directly or indirectly on a suitable
surface, e.g. in an ELISA format. Corresponding surfaces for
immobilization are well known. For example, an inert particle
("bead") can be used. The ligand can also, however, be bound to a
96-well microtiter plate. Then selected amounts of the test
compounds and of the peptides according to the invention are
brought in contact with the immobilized microorganisms and the
compounds that compete with the peptides for binding to the
microorganisms are selected. If these test compounds, which compete
with the peptides for receptor binding on bacteria or fungi, are
identified, they can be investigated further for their
antibacterial or antimycotic action. Suitable methods for this are
described below in the examples.
[0176] In another aspect the invention provides an isolated nucleic
acid molecule, whose sequence codes for a peptide or multimer
according to the invention. The nucleic acid coding for the
antibacterial or antimycotic peptide or multimeric construct
according to the invention is linked operatively with a regulatory
sequence, which controls its expression in the host cell. Another
object of the invention is a host cell that has been transfected or
transformed with the nucleic acid molecule described above.
[0177] The invention will be explained below with the following
examples, without the invention being limited thereto:
EXAMPLES
Example 1
Peptide Synthesis
[0178] All chemicals for peptide synthesis were, unless stated
otherwise, obtained from Fluka Chemie GmbH (Buchs, Switzerland) at
highest possible purity.
[0179] All peptides and peptide derivatives were synthesized by
conventional solid-phase peptide synthesis using the Fmoc/.sup.tBu
strategy (Fields G B & Noble R L. Solid-Phase Peptide Synthesis
Utilizing 9-Fluorenylmethoxycarbonyl Amino-Acids. International
Journal of Peptide and Protein Research 35: 151-214, 1990) on a
Syro 2000 multiple peptide synthesis robot (MultiSynTechn GmbH,
Witten, Germany). All standard-Fmoc amino acids were obtained from
MultiSynTechn GmbH (Witten, Germany) or Orpegen Pharma GmbH
(Heidelberg, Germany). 2-Amino-3-guanidinopropionic acid ((Agp:
Iris Biotech Biotech GmbH, Marktredwitz, Germany),
.beta.-homoarginine (.beta.Har, Fluka Chemie GmbH, Buchs,
Switzerland), homoarginine (Har), N-methylarginine (N--Me--Arg) and
nitroarginine (Arg(NO.sub.2), Bachem A G, Bubendorg, Switzerland)
were used as special arginine homologs. trans-4-Hydroxyproline
(t-4-Hyp) and 2,3-diaminopropionic acid (Dap) were obtained from
Novabiochem (Merck Biosciences GmbH, Darmstadt, Germany).
[0180] The peptides were synthesized either as acid on Wang resin
(1.23 mmol/g) or as acid amide on Rink-amide
4-methylbenzylhydrylamine (MBHA) resin (0.67 mmol/g), from the
company MultiSynTech GmbH (Witten, Germany). For later
functionalization with primary amines, peptides were derivativized
as peptide thioesters and for this the first amino acid was coupled
to 4-sulfamino-butyryl-aminomethyl resin (SAB AM, 1.1 mmol/g;
Novabiochem, Merck Biosciences GmgH, Darmstadt, Germany).
[0181] On Want resin, the first C-terminal amino acid (5
equivalents(eq)) was coupled with 5 eq 2,6-dichlorobenzoyl chloride
(Merck KGaA, Darmstadt, Germany) and 8.25 eq pyridine in
dichloromethane (DCM; Biosolve B V, Valkenswaard, The Netherlands)
(Sieber P. An Improved Method for Anchoring of
9-Fluorenylmethoxycarbonyl-Amino Acids to 4-Alkoxybenzyl Alcohol
Resins. Terahedron Letters 28: 6147-50, 1987). The
functionalization of the SAB AM resin with the C-terminal amino
acid (5 eq) was carried out at -20.degree. C. by activation with 5
eq (benzotriazol-1-yloxy)-tripyrrolidinophosphonium
hexafluorophosphate (PyBOP, Novabiochem, Merck Biosciences GmbH,
Darmstadt, Germany) and 10 eq N-ethyldiisopropylamine (DIPEA) in
DCM (Backes B J & Ellman J A. An Alkanesulfonamide Safety-Catch
Linker for Solid-Phase Synthesis. The Journal of Organic Chemistry
64: 2322-30, 1999).
[0182] The first amino acid was coupled to Rink-amide resin by
activation of in each case 8 eq amino acid dissolved in 0.5 mol/L
of 1-hydroxybenzotriazole (HOBt) with 8 eq
N,N'-diisopropylcarbodiimide (DIC) in dimethylformamide (DMF;
Biosolve B V, Valkenswaard, The Netherlands), as described in the
automatic synthesis on previously loaded Wang and SAB AM resin.
[0183] The side-chain protective groups used were triphenylmethyl
(trityl) for Cys, Asn, His and Gln, tert-butyl ether (.sup.tBu) for
Tyr, Ser and Thr, tert-butyl ester (O.sup.tBu) for Asp and Glu ,
.omega.-N-2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf)
for Arg, .omega.-N-2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc)
for .beta.Har and Har, tert-butyloxy-carbonyl (Boc) for Lys, Orn,
and Agp or .omega.-N-4-methoxy-2,3,6-trimethylphenyl-sulfonyl (Mtr)
for N--Me--Arg. The temporary Fmoc protective group was cleaved
with 40% piperidine (Biosolve BV, Valkenswaard, The Netherlands) in
DMF (v/v) for 5 min and again with fresh 20% piperidine in DMF
(v/v) for 10 min.
[0184] The N-termini of the peptides or peptide derivatives were
acetylated, formylated or iodoacetylated by dissolving 8 eq acetic
acid, formic acid or iodoacetic acid in 0.5 mol/L HOBT/DMF and
activating them with 8 eq DTC in DMF. The N-terminus of the
peptides or peptide derivatives was quantitated according to
Gausepohl et al. (Gausepohl, H., Pieles, H., & Frank, R. W. in
Peptides: chemistry, structure and biology (eds. Smith, J. A. &
Rivier, J. E.) 523 (ESCOM, Leiden, 1992)) each with 10 eq
2-(.sup.3H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate (HBTU, MultiSynTech GmbH, Witten, Germany) and
DIPEA in DMF. The N-termini were modified with the fluorescent dye
5,6-carboxyfluorescein (5 eq) with 5 eq HBTU and 10 eq DIPEA in
DMF.
[0185] The completeness of N-terminal modifications was verified by
the Kaiser test. For this, a little resin was incubated with 0.28
mol/L ninhydrin (Riedel de Haen, Seelze, Germany) in ethanol (Carl
Roth GmbH+Co. K G, Karlsruhe, Germany), 0.2 mmol/L potassium
cyanide in pyridine and 76% phenol in ethanol in the ratio (1:1:2)
at 95.degree. C. If a blue coloration appeared, which indicates
free primary amino groups, the coupling was repeated.
[0186] On completion of synthesis of the peptides or peptide
derivatives, the resins were carefully washed with DMF and DCM and
dried under vacuum. The resin-bound peptides were cleaved with a
mixture of water, m-cresol, thioanisole and ethanedithiol
(5:5:5:2.5) in 87.5% trifluoroacetic acid (TFA) at room temperature
for 4 h and at the same time the side chains were deprotected. The
peptides and peptide derivatives were precipitated with cold
diethyl ether and centrifuged at 3000*g. The pellet was washed
twice with cold ether, dried and dissolved in 0.1% aqueous TFA (UV
spectroscopy). The samples were stored at -20.degree. C.
[0187] Prior to cleavage of the peptides on the SAB AM resin, after
cleavage of the Fmoc group the N-terminus was protected with 20 eq
di-tert-butyldicarbonate (Boc.sub.2O, Fluka Chemie GmbH, Buchs,
Switzerland) and 10 eq DIPEA in DMF. 100 eq of iodoacetonitrile and
20 eq DIPEA in DMF were added to the washed resin in order to
activate the sulfamyl linker by alkylation (Teruya K, Murphy A C,
Burlin T, Appella E, & Mazur S J. Fmoc-based chemical synthesis
and selective binding to supercoiled DNA of the p53 c-terminal
segment and its phosphorylated and acetylated derivatives. Journal
of Peptide Science 10: 479-93, 2004). For cleavage of the peptide,
50 eq propylamine in DMF (10% v/v) was added to the resin. After
the DMF had been removed, the raw peptide was dissolved in a
solution of water, m-cresol, thioanisole and ethanedithiol
(5:5:5:2.5) in 87.5% TFA and all side-protective groups and the
N-terminal Boc protective group were split off. The peptides were
precipitated with cold diethyl ether and centrifuged at
3000.times.g. The precipitate was washed twice with cold ether,
dried and dissolved in 0.1% aqueous TFA (UV spectroscopy). The
samples were stored at -20.degree. C.
[0188] The cleaved peptides and peptide derivatives were purified
by RP-HPLC on an Akta HPLC system (Amersham Bioscience GmbH,
Freiburg, Germany) with a Jupiter C18 5 .mu.m 300 .ANG.,
250.times.10 mm or Jupiter C18 15 .mu.m, 300 .ANG., 250.times.21 mm
column (Phenomenex Inc., Torrance, USA).
[0189] The solvent used in each case was 0.1% aqueous TFA (eluent
A) and 60% aqueous acetonitrile (Biosolve B V, Valkenswaard, The
Netherlands) with 0.1% TFA (eluent B). A typical linear gradient
started at 5% eluent B and elution was carried out with an increase
of 1% B per minute with a flow rate of 10 mL/min (250.times.21 mm
column) or 5 mL/min (250.times.10 mm column). Detection was
performed at 220, 230 and 240 .mu.m. The purified peptides were
analyzed with the same HPLC system with a Jupiter C18 5 .mu.m, 300
.ANG., 150.times.4.6 mm column (Phenomenex Inc., Torrance, USA). It
was eluted at a flow rate of 1 mL/min with a linear gradient of
5-95% B in 30 min and detected at 220 nm. In addition, the purity
was determined by matrix-assisted laser desorption/ionization with
time-of-flight mass spectrometry (MALDI-TOF-MS; 4700 Proteomic
Analyzer, Applied Biosystems GmbH, Darmstadt, Germany). For this,
0.5 .mu.L of peptide solution was co-crystallized with 0.5 .mu.L of
.alpha.-cyanchydroxycinnamic acid (Bruker Daltonik GmbH; Bremen,
Germany) as matrix (5.3 mg/mL in 50% acetonitrile in 0.1% aqueous
TFA).
[0190] Thioether linkage of the penetratin derivatives was
performed by incubation of the purified penetratin-Cys monomer with
4 eq of purified iodoacetylated AMP in degassed phosphate-buffered
saline (PBS, pH 7.4) at 4.degree. C. under nitrogen. The reaction
was monitored by RP-HPLC and the penetratin construct was purified
after complete reaction of the penetratin-Cys monomer. The
(penetratin-Cys), dimer was obtained in parallel.
[0191] Starting from the incompletely determined sequence of the
proline-rich antimicrobial peptide "Oncopeltus antibacterial
peptide 4" first derivatives of peptide 4 (Table 2) were firstly
synthesized with the C-terminal amino acid N.sub.18N.sub.19R.sub.20
and the carboxyl function was not altered. Among the modifications
at position 11, the cationic amino acids Lys and Arg showed
surprisingly good properties. The derivatives displayed
antibacterial activity against E. coli and M. luteus in the lower
micromolar range. For further derivatization of the C-terminus, the
sequence derivativized with Arg 11 was selected and derivatives
with different arrangement of Asn and Arg were synthesized. The
derivative shortened on the C-terminus by Asn (SEQ ID NO. 18)
showed amazingly high activity. As C-terminal acid amide, moreover,
it attained a surprisingly high serum stability. The peptide with
the sequence VDKPPYLPRPRPPRRIYNR-NH.sub.2 (SEQ ID NO. 18) is called
oncocin hereinafter and was further improved mainly with respect to
serum stability but also antimicrobial activity.
TABLE-US-00003 TABLE 2 SEQ ID Synthesis E. coli M. luteus NO.
number Sequence BL21 A1 10240 123* O. VDKPPYLPRP(X/P)PPRRIYN(NR)
fasciatus 2* A25 A4 VDKPPYLPRP P PPRRIYN NR-OH 128 64 4* A25 A5
VDKPPYLPRP T PPRRIYN NR-OH 128 64 8 A28 B2 VDKPPYLPRP H PPRRIYN
NR-OH 64 32 5 A29 B1 VDKPPYLPRP K PPRRIYN NR-OH 16 16 14 A25 A6
VDKPPYLPRP R PPRRIYN NR-OH 8 16 16 A29 A6 VDKPPYLPRP R PPRRIYN
RN-OH 8 16 18 A33 B3 VDKPPYLPRP R PPRRIYN R-NH.sub.2 4 8
*Comparative example.
TABLE-US-00004 TABLE 3 Review of the synthesized peptide sequences
SEQ ID Synthesis NO. number Sequence 1* A21 B2
VDKPPYLPRPPPPRRIYN-NH.sub.2 2* A25 A4 VDKPPYLPRPPPPRRIYNNR-OH 3*
A33 B1 VDKPPYLPRP-4tHyp-PPRRIYNR-OH 4* A25 A5
VDKPPYLPRPTPPRRIYNNR-OH 5 A29 B1 VDKPPYLPRPKPPRRIYNNR-OH 6 A28 B1
VDKPPYLPRPKPPRRIYNRN-OH 7 A35 A3 VDKPPYLPRPKPPRRIYNR-NH.sub.2 8 A28
B2 VDKPPYLPRPHPPRRIYNNR-OH 9 A28 B3 VDKPPYLPRPHPPRRIYNRN-OH 10* A31
B2 VDKPPYLPRPYPPRRIYNR-OH 11* A31 B3 VDKPPYLPRPYPPRRIYNR-OH 12* A31
B4 VDKPPYLPRPQPPRRIYNR-OH 13* A31 B5 VDKPPYLPRPFPPRRIYNR-OH 14 A25
A6 VDKPPYLPRPRPPRRIYNNR-OH 15 A29 A2 VDKPPYLPRPRPPRRIYNNR-NH.sub.2
16 A29 A6 VDKPPYLPRPRPPRRIYNRN-OH 17 A35 A2
VDKPPYLPRPRPPRRIYNRN-NH.sub.2 18 A33 B3
VDKPPYLPRPRPPRRIYNR-NH.sub.2 19 A35 A4 VDKPPYLPRPRPPRRIYNR-OH 20
A31 A4 VDKPPYLPRPRPPRPIYNR-OH 21 A31 B1
VDKPPYL-4tHyp-RPRPPRRIYNR-OH 22 A34 A2
VDKPPYL-4tHyp-RPRPPRRIYNR-NH.sub.2 23 A33 A6
VDK-4tHyp-PYLPRPRPPRRIYNR-OH 24 A34 A3
VDKPPYLPRPRP-4tHyp-RRIYNR-NH.sub.2 25 A53 E6
VDKPPYLPRPRPPR-4tHyp-IYNR-NH.sub.2 26 A53 F3
VDKPPYLPRPRPPRRIYNON-NH.sub.2 27* A35 A6
VDKPPYLPRPRPPRRIYN-NH.sub.2 28* A51 A6 VDKPPYLPRPRPPRRIYN-OH 29 A56
A1 ADKPPYLPRPRPPRRIYNR-NH.sub.2 30 A56 A2
VAKPPYLPRPRPPRRIYNR-NH.sub.2 31* A56 A3
VDAPPYLPRPRPPRRIYNR-NH.sub.2 32 A56 A4 VDKAPYLPRPRPRRIYNR-NH.sub.2
33 A56 A5 VDKAPAYLPRPRPPRRIYNR-NH.sub.2 34* A56 A6
VDKPPALPRPRPPRRIYNR-NH.sub.2 35* A56 B1
VDKPPYAPRPRPPRRIYNR-NH.sub.2 36 A56 B2 VDKPPYLARPRPPRRIYNR-NH.sub.2
37* A56 B3 VDKPPYLPAPRPPRRIYNR-NH.sub.2 38 A56 B4
VDKPPYLPRARPPRRIYNR-NH.sub.2 39* A56 B5
VDKPPYLPRPAPPRRIYNR-NH.sub.2 40 A56 B6 VDKPPYLPRPRAPRRIYNR-NH.sub.2
41 A56 C1 VDKPPYLPRPRPARRIYNR-NH.sub.2 42* A57 D5
VDKPPYLPRPRPPARIYNR-NH.sub.2 43* A57 D6
VDKPPYLPRPRPPRAIYNR-NH.sub.2 44 A56 C4 VDKPPYLPRPRPPRRAYNR-NH.sub.2
45 A56 C5 VDKPPYLPRPRPPRRIANR-NH.sub.2 46 A56 C6
VDKPPYLPRPRPPRRIYAR-NH.sub.2 47* A56 D1
VDKPPYLPRPRPPRRIYNA-NH.sub.2 48* A35 B1 VDKPPYLPRPRPPRPIRV-OH 49
A70 A1 VDKPPYLPRPRPPRRIYPQPRPPHPRL-NH.sub.2 50 A51 B1
VDKPPYLPRPRPPRRIYNO-NH.sub.2 51* A51 B2
VDKPPYLPRPRPPRRIYNN-NH.sub.2 52 A51 B3
VDKPPYLPRPRPPRRIYNDap(Ac)-NH.sub.2 53 A56 E1
VDKPPYLPRPRPPRRIYNH-NH.sub.2 54 A59 A2
VDKPPYLPRPRPPRRIYNAgp-NH.sub.2 55 A59 A3
VDKPPYLPRPRPPRRIYNArg-(NO.sub.2)-NH.sub.2 56 A59 A4
VDKPPYLPRPRPPRRIYN(N-Me-Arg)-NH.sub.2 57 A59 A5
VDKPPYLPRPRPPRRIYNHar-NH.sub.2 58 A66 D2 propyl.
VDKPPYLPRPRPPRRIYNR-NHC.sub.3H.sub.7 59 A45 A6
VDKPPYLPRPRPRPRIYNR-NH.sub.2 60* A45 A5 VDKPPYLPRPRPPRPIYNR-NH2 61
A45 A3 VDKPPYLPRPRPPROIYNR-NH.sub.2 62 A51 B4
VDKPPYLPRPRPPR-betHArg-IYNR-NH.sub.2 63 A53 F1
VDKPPYLPRPRPPR-4tHyp-IYNO-NH.sub.2 64 A66 C5
VDKPPYLPRPRPPRHIYNH-NH.sub.2 65 A70 B4
VDKPPYLPRPRPPRAgpIYNHar-NH.sub.2 66 A70 C1
VDKPPYLPRPRPPRAgpIYNAgp-NH.sub.2 67 A70 B5
VDKPPYLPRPRPPRHarIYNHar-NH.sub.2 68 A70 C2
VDKPPYLPRPRPPRHarIYNAgp-NH.sub.2 69 A70 C3
VDKPPYLPRPRPPROIYNAgp-NH.sub.2 70 A70 B6
VDKPPYLPRPRPPROIYNHar-NH.sub.2 71 A66 D3 propyl.
VDKPPYLPRPRPPROIYNR-NHC.sub.3H.sub.7 72 A53 F2
VDKPPYLPRPRPPROIYNO-NH.sub.2 73 A66 C6 VDKPPYLPRPRPPROLYNO-NH.sub.2
74 A66 D1 VDKPPYLPRPRPPROIYQO-NH.sub.2 75 A56 D2
VEKPPYLPRPRPPRRIYNR-NH.sub.2 76 A56 D3 VDRPPYLPRPRPPRRIYNR-NH.sub.2
77 A56 D4 VDKPPYIPRPRPPRRIYNR-NH.sub.2 78 A56 D5
VDKPPYLPRPRPPRRLYNR-NH.sub.2 79 A56 D6 VDKPPYLPRPRPPRRIYQR-NH.sub.2
80 A32 D3 ac. Ac-VDKPPYLPRPRPPRRIYNR-OH 81 A32 D3 fo.
For-VDKPPYLPRPRPPRRIYNR-OH 82 A45 A2 ODKPPYLPRPRPPRRIYNR-NH.sub.2
83 A45 A2 ac. Ac-ODKPPYLPRPRPPRRIYNR-NH.sub.2 84 A45 A4 guan.
Guan-VDKPPYLPRPRPPRRIYNR-NH.sub.2 85 A62 B3 guan.
Guan-VDKPPYLPRPRPPROIYNO-NH.sub.2 86* A35 A5
DKPPYLPRPRPPRRIYNR-NH.sub.2 87* A54 E1 GNNRPVYIPQPRPPHPRL-OH 88*
A60 E4 CF CF-GNNRPVYIPQPRPPHPRL-OH 89* A34 B3
GKPRPYSPRPTSHPRPIRV-OH 90* A60 E2 CF CF-GKPRPYSPRPTSHPRPIRV-OH 91*
A35 A1 VDKGSYLPRPTPPRPIYNRN-NH.sub.2 92* A60 E6 CF
CF-VDKGSYLPRPTPPRPIYNRN-NH.sub.2 93 A33 B3
VDKPPYLPRPRPPRRIYNR-NH.sub.2 94 A60 F2 CF
CF-VDKPPYLPRPRPPRRIYNR-NH.sub.2 95 A60 E4 F5
RQIKIWFQNRRMKWKKC-(CH.sub.2CO-GNNRPVYIPQPRPPHPRL-OH)-OH 96 A60 E4
F6 CF CF-RQIKIWFQNRRMKWKKC-(CH.sub.2CO-GNNRPVYIPQPRPPHPRL-OH)-OH 97
A60 E2 F5 RQIKIWFQNRRMKWKKC-(CH.sub.2CO-GKPRPYSPRPTSHPRPIRV-OH)-OH
98 A60 E2 F6 CF
CF-RQIKIWFQNRRMKWKKC-(CH.sub.2CO-GKPRPYSPRPTSHPRPIRV-OH)-OH 99 A60
E6 F5
RQIKIWFQNRRMKWKKC-(CH.sub.2CO-VDKGSYLPRPTPPRPIYNRN-NH.sub.2)-OH 100
A60 E6 F6 CF
CF-RQIKIWFQNRRMKWKKC-(CH.sub.2CO-VDKGSYLPRPTPPRPIYNRN-NH.sub.2)-OH
101 A60 F2 F5
RQIKIWFQNRRMKWKKC-(CH.sub.2CO-VDKPPYLPRPRPPRRIYNR-NH.sub.2)-OH 102
A60 F2 F6 CF
CF-RQIKIWFQNRRMKWKKC-(CH.sub.2CO-VDKPPYLPRPRPPRRIYNR-NH.sub.2)-OH
103* A60 F5 (RQIKIWFQNRRMKWKKC-OH).sub.2 104* A60 F5 CF
(CF-RQIKIWFQNRRMKWKKC-OH).sub.2 105* A60 G1 RQIKIWFQNRRMKWKK-OH
106* RQIKIWFQNRRMKWKKC(CH.sub.2CO-ffSGDRSGYSSRGS-OH)-OH 107 A74 C6
VDKPPYLPRPRP-4tHyp-ROIYNO-NH.sub.2 108 A74 D1
VDKPPYLPRPRP-4tHyp-R-4tHyp-IYNO-NH.sub.2 109 A76 B3
VDKPPYLPRPRPPRO-Tle-YNO-NH.sub.2 110 A76 B5
VDKPPYLPRPRP-4tHyp-R-4tHyp-Tle-YNO-NH.sub.2 111 T1 D9
VDKPPYLPRPRPPRrIYNR-NH.sub.2 112 T1 DI1
VDKPPYLPRPRPPRrIYNr-NH.sub.2 113* A82 A4
ONYIORPPRPRPLYPPKDV-NH.sub.2 114* A82 A5
ONYI-4tHyp-RPPRPRPLYPPKDV-NH.sub.2 115* T1 C7
vdkppylprprpprriynr-NH.sub.2 116* T1 C9
vdkppylprprpproiyno-NH.sub.2 117* T1 C11
rnyirrpprprplyppkdv-NH.sub.2 118* T1 D1
onyiorpprprplyppkdv-NH.sub.2
[0192] The single-letter code was used for the amino acid residues,
with O in the amino acid chain standing for ornithine;
[0193] Propyl stands for a propylamide on the C-terminus
(Sub.sub.2=Or.sub.3=NHC.sub.3H.sub.7); Ac=acetyl group, for=formyl
group, guan=guanidino group and CF=5,6-carboxyfluorescein Examples
of modified N-termini (modified alpha-amino group of the N-terminal
amino acid, Sub.sub.1=acetyl--NH, formyl--NH, guanidine or
5,6-carboxyfluorescein),
[0194] .beta.Har: .beta.-homoarginine, the beta-amino acid homologs
to arginine,
[0195] Agp: 2-amino-3-guanidinopropionic acid, Har: homoarginine
and Arg(NO.sub.2): nitroarginine are homologs of arginine,
[0196] N--Me--Arg: N-methyl-arginine--an arginine methylated on the
peptide bond, [0197] 4tHyp: trans-4-hydroxyproline,
[0198] Tle: tert-butyl glycine
[0199] Dap(Ac): 2,3-diaminopropionic acid with acetylated amino
function in the side chain,
[0200] (CH.sub.2CO): acetyl linker to SH group of cysteine,
[0201] f: .alpha.-aminocarproinic acid,
[0202] lower-case letters stand for the corresponding D-amino
acids,
[0203] comparative examples are marked with *.
Example 2
Stability
Serum Stability in 25% Mouse Serum
[0204] The serum stability studies were performed as double
determination according to Hoffmann et al. (Hoffman R, Vasko M,
& Otvos L. Serum stability of phosphopeptides. Analytica
Chimica Acta 352: 319-25, 1997) in mouse serum and 25% aqueous
mouse serum (PAA Laboratories GmbH; Pasching, Austria). For this,
the peptides and peptidomimetics were dissolved in water, mouse
serum was added and the peptide concentration was adjusted to 75
.mu.g/mL. While shaking continuously, the mixture was incubated at
37.degree. C. After 0, 30, 60, 120, 240 and 360 minutes, in each
case in aliquot was taken and mixed with 15% aqueous
trichloroacetic acid (Carl Roth GmbH & Co. KG; Karlsruhe,
Germany). After incubation on ice for a further 10 minutes, the
precipitated serum proteins were centrifuged off (5 min, 13400 rpm,
MiniSpin, Eppendorf AG, Hamburg, Germany). The supernatant was
removed, neutralized with 1 mol/L aqueous sodium hydroxide solution
(Fluka Chemie GmbH, Buchs, Switzerland) and stored at -20.degree.
C. until analysis.
[0205] The supernatants were analyzed by RP-HPLC with a linear
acetonitrile gradient (Biosolve BV, Valkenswaard, The Netherlands)
in the presence of 0.1% trifluoroacetic acid (TFA, UV-grade, Fluka
Chemie GmbH, Buchs, Switzerland) as ion-pairing reagent. The
fractions were co-crystallized with .alpha.-cyanohydroxycinnamic
acid (Bruker Daltonik GmbH; Bremen, Germany) as matrix (5.3 mg/mL
in 50% acetonitrile in 0.1% aqueous TFA) and were analyzed with a
tandem mass spectrometer (MALDI-TOF/TOF-MS, 4700 Proteomics
Analyzer; Applied Biosystems GmbH, Weiterstadt, Germany) in the
positive-ion reflector mode. The proportion of intact peptides and
their degradation products or metabolites could thus be identified
and quantified at the individual time points. The control used was
25% aqueous mouse serum, which was analyzed in parallel for the
same time intervals.
[0206] Table 4 shows the half-lives of oncocin (SEQ ID NO. 18) and
selected oncocin derivatives in 25% mouse serum. The listed
Oncopeltus 4 derivatives with SEQ ID NO. 14 and 19 have the lowest
stability, with a half-life of less than 30 min. Here, the arginine
at position 19 (residue X.sub.4) is cleaved first. The stability of
the degradation product VDKPPYLPRPRPPRRIYN-OH (SEQ ID NO. 28) then
increases to 120 min, but this fragment still only has very low
antimicrobial activity (64 .mu.g/mL E. coli). On amidation of the
C-terminus (Sub.sub.2) in oncocin, besides the activity against E.
coli and M. luteus (4 and 8 .mu.g/mL respectively), the stability
also increases to 60 min, a surprisingly high value for peptide
derivatives. Through amidation with propylamide (SEQ ID NO. 58),
the half-life of oncocin was even lengthened to 120 min.
TABLE-US-00005 TABLE 4 Serum stability: half-lives of oncocin and
selected oncocin derivatives in 25% mouse serum E. SEQ coli M. ID
Synthesis BL21 Luteus Half- NO. No. Sequence A1 10240 life 14 A25
A6 VDKPPYLPRPPRRIYNNR-OH 8 16 <30 min 19 A35 A4
VDKPPYLPRPRPPRRIYNR-OH 32 64 <30 min 17 A35 A2
VDKPPYLPRPRPPRRIYNRN-NH.sub.2 16 128 45 min 18 A33 B3
VDKPPYLPRPRPPRRIYNR-NH.sub.2 4 8 60 min 24 A34 A3
VDKPPYLPRPRP-4tHyp-RRIYNR-NH.sub.2 8 8 60 min 61 A45 A3
VDKPPYLPRPRPPROIYNR-NH.sub.2 8 16 90 min 28* A51 A6
VDKPPYLPRPRPPRRIYN-OH 64 128 120 min 50 A51 B1
VDKPPYLPRPRPPRRIYO-NH.sub.2 8 16 120 min 51* A51 B2
VDKPPYLPRPRPPRRIYNN-NH.sub.2 16 64 120 min 58 A66 D2
VDKPPYLPRPRPPRRIYNR-NHC.sub.3H.sub.7 4 8 >120 min propyl. 60*
A45 A5 VDKPPYLPRPRPPRPIYNR-NH.sub.2 8 64 120 min 62 A51 B4
VDKPPYLPRPRPPR-betaHArg-IYNR-NH.sub.2 4 16 150 min 63 A53 F1
VDKPPYLPRPRPPR-4tHyp-IYNO-NH.sub.2 4 8 >360 min 71 A66 D3
VDKPPYLPRPRPPROIYNR-NHC.sub.3H.sub.7 4 8 >360 min propyl. 72 A53
F2 VDKPPYLPRPRPPROIYNO-NH.sub.2 8 16 >360 min *comparative
examples.
[0207] The substitution of position 19 (residue X.sub.4) for the
nonproteinogenic amino acid ornithine (SEQ ID NO. 61) increased the
stability to 120 min half-life. The substitution of Arg15 for
ornithine increased the stability of this oncocin derivative (SEQ
ID NO. 50) to 90 min half-life. The derivatives with proline (SEQ
ID NO. 60) or .beta.-homoarginine (SEQ ID NO. 62) at position 15
(residue X.sub.3) were not degraded to half until after 120 or 150
min. The substitution of the proline at position 13 in
4-trans-hydroxyproline (SEQ ID NO. 24) did not have any negative
effect on the stability of oncocin.
[0208] By combining these modifications at position 15 and 19
(residues X.sub.3 and X.sub.4) it was possible to synthesize very
stable peptide derivatives, for which half-lives of over 360 min
were determined. The activity of the preferred examples SEQ ID NO.
63 and 72 with Orn19 and Hyp15 or Orn15 with MIC values of 4 or 8
mg/mL against E. coli was comparable to oncocin. The combination of
Orn15 with propylamidated C-terminus (SEQ ID NO. 71) represents
another very preferred example of the invention.
[0209] Serum Stability in 100% Mouse Serum
TABLE-US-00006 TABLE 5 Serum stability: half-lives of oncocin and
selected oncocin derivatives in 100% mouse serum E. SEQ coli M. ID
Synthesis BL21 luteus NO. No. Sequence A1 10240 Half-life 18 A33 B3
VDKPPYLPRPRPPRRIYNR-NH.sub.2 4 8 30 min 24 A34 A3
VDKPPYLPRPRP-4tHyp-RRIYNR-NH.sub.2 8 8 35 min 61 A45 A3
VDKPPYLPRPRPPROIYNR-NH.sub.2 8 16 65 min 58 A66 D2
VDKPPYLPRPRPPRRIYNR-NHC.sub.3H.sub.7 4 8 60 min propyl. 67 A70 B5
VDKPPYLPRPRPPR-Har-IYN-Har-NH.sub.2 4 4 55 min 63 A53 F1
VDKPPYLPRPRPPR-4tHyp-IYNO-NH.sub.2 4 8 >480 min 60% 71 A66 D3
VDKPPYLPRPRPPROIYNR-NHC.sub.3H.sub.7 4 8 150 min propyl. 72 A53 F2
VDKPPYLPRPRPPROIYNO-NH.sub.2 8 16 175 min 25 A53 E6
VDKPPYLPRPRPPR-4tHyp-IYNR-NH.sub.2 16 8 240 min 107 A74 C6
VDKPPYLPRPRP-4tHyp-ROIYNO-NH.sub.2 4 4 n.d. 108 A74 D1
VDKPPYLPYPYP-4tHyp-R-4tHyp-IYNO-NH.sub.2 2 32 150 min 109 A76 B3
VDKPPYLPRPRPPRO-Tle-YNO-NH.sub.2 4 4 330 min 110 A76 B5
VDKPPYLPRPRP-4tHyp-R-4tHyp-Tle-YNO-NH.sub.2 2 16 >480 min
70%
[0210] By exchanging the arginine residues at position 15 and 19
with ornithine or trans-4-hydroxyproline (SEQ ID NO. 63 and 72),
the half-life in 25% aqueous mouse serum could be increased to more
than 6 hours (see above). After incubation in 100% mouse serum, on
exchanging R.sub.15O and R.sub.19O a half-life of 175 minutes was
determined for these sequences. By exchanging R.sub.15Hyp and
R.sub.19O (SEQ ID NO. 63), 60% of the amount of peptide used was
still detected after 480 min. Another derivative of oncocin with
P.sub.13Hyp, R.sub.13Hyp, R.sub.19O and I.sub.16Tle (SEQ ID NO.
110) still contained even 60% or 70% of the original amount of
peptide after 480 min. The activity of both derivatives against E.
coli was very good, at 4 and 2 .mu.g/mL.
[0211] Stability Against Bacterial Proteases
[0212] Preparation of Bacterial Lysate
[0213] For the bacterial lysate, 500 mL of nutrient broth (Carl
Roth GmbH+Co. K G, Karlsruhe) was inoculated with E. coli BL21 A1
and incubated overnight at 37.degree. C. 2.times.250 mL bacterial
suspension were centrifuged for 25 min at 5000 rpm and 4.degree. C.
in a Beckman Avanti.TM. J-20-XP centrifuge with JLA-10.500 rotor
(Beckman Coulter, Fullerton, U.S.A.). The pellets were each
suspended in 30 mL PBS (pH 7.4) and centrifuged again in a Beckman
Allegra.TM. 2IR centrifuge (Beckman Coulter, Fullerton, U.S.A.).
The pellets were each suspended in 10 mL PBS and were disrupted
with ultrasound (Vibra-cell.TM. microtip, Fisher Bioblock
Scientific, Illkirch, France) 2.times.5 min (750 W; amplitude 40%;
2 s on/3 s off) on ice. The bacterial lysates were combined and 1
mL aliquots were centrifuged at 15400 rpm for 20 min at 4.degree.
C. (Beckman Allegra.TM.). The supernatants were removed and stored
at -20.degree. C. The protein content of the bacterial lysate was
determined by protein determination according to Bradford.
[0214] Protein Determination According to Bradford (M. M. Bradford;
(1976): Analytische Biochemie, 72, 248-254)
[0215] A 2 mg/mL BSA solution in PBS was prepared as the stock
solution for the standard series. This was diluted with PBS, to
give 6 standard solutions from 10 to 100 .mu.g/mL BSA. A dilution
series in PBS was prepared from the sample to be analyzed. In each
case 50 .mu.L of sample or standard solution was pipetted into a
polystyrene microplate. For the Bradford reagent, 0.01% Coomassie
Brilliant Blue G250 was dissolved in 5% ethanol, 8.5% o-phosphoric
acid was added and it was topped up with doubly-distilled water.
The mixture was then incubated for 1 h at 60.degree. C. and left to
stand for a further 12 h at RT, and then filtered. 200 .mu.L of the
Bradford reagent was added to each well and incubated in the dark
for 15 min. The absorption was measured at 595 nm against a blank
value (50 .mu.L PBS+200 .mu.L Bradford reagent).
[0216] Determination of the Stability in Bacterial Lysate
[0217] A solution of 0.15 .mu.g/mL peptide in bacterial lysate with
protein content of 1.5 mg/mL or 0.5 mg/mL was incubated at
37.degree. C. After 0, 30, 60, 120, 240 or 360 min, 200 .mu.L was
taken from each and the proteins were precipitated with 50 .mu.L of
15% trichlorocacetic acid. Then it was incubated for 10 min at
4.degree. C. and then centrifuged for 5 min at 13000 rpm in a
Minispin desktop centrifuge. 210 .mu.L was taken from the
supernatant and neutralized with 1 mol/L sodium hydroxide solution.
The intact peptide and the degradation products were analyzed by
HPLC. 60 .mu.L of 3% aqueous acetonitrile with 0.1% trifluoroacetic
acid (TFA) was added to the solution and 250 .mu.L of the mixture
was injected. After chromatographic separation, the constituents
were identified by MALDI-TOF-MS.
TABLE-US-00007 TABLE 6 Stability agaiast bactatial proteases: half-
lives of oncocin and selected oncocin derivatives after incubation
in E. coli lysate with 0.5 mg/mL protein concentration E. SEQ coli
M. ID Synthesis BL21 luteus Half- NO. No. Sequence A1 10240 life 18
A33 B3 VDKPPYLPRPRPPRRIYNR-NH.sub.2 4 8 60 min 25 A53 E6
VDKPPYLPRPRPPR-4tHyp-IYNR-NH.sub.2 16 8 n.d. 72 A53 F2
VDKPPYLPRPRPPROIYNO-NH.sub.2 8 16 125 min 73 A66 C6
VDKPPYLPRPRPPROLYNO-NH.sub.2 6 16 90 min 107 A74 C6
VDKPPYLPRPRP-4tHyp-ROIYNO-NH.sub.2 4 4 215 min 108 A74 D1
VDKPPYLPRPRP-4tHyp-R-4tHyp-IYNO-NH.sub.2 2 32 >240 min 60% 109
A76 B3 VDKPPYLPRPRPPRO-Tle-YNO-NH.sub.2 4 4 >240 min 90% 110 A76
B5 VDKPPYLPRPRP-4tHyp-R-4tHyp-Tle-YNO-NH.sub.2 2 16 >240 min
90%
[0218] In order to investigate the stability against bacterial
proteases, a lysate was prepared from an overnight culture of E.
coli BL21 A1 and the peptides were incubated in it. Using Bradford
protein determination, the protein concentration was adjusted to
0.5 to 1.5 mg/mL.
[0219] For the native oncocin (SEQ ID NO. 18), a half-life of 60
minutes was determined in the lysate with a total protein
concentration of 0.5 mg/mL. By exchanging R.sub.15O and R.sub.19O
(SEQ ID NO. 72) the half-life was doubled to 115 min. In the next
step, the proline at position 13 of a cleavage site of the proline
endopeptidase was exchanged for trans-4-hydroxyproline (SEQ ID NO.
107), with further increase in stability to 215 min. By combining
P.sub.13Hyp, R.sub.15Hyp and R.sub.19O, a half-life of more than
240 min was achieved.
[0220] In another derivative with R.sub.15O and R.sub.19O, the
isoleucine at position 16 was exchanged for tert-butylglycine (SEQ
ID NO. 109), increasing the stability to more than 240 min. The
derivatives with the combination P.sub.13Hyp, R.sub.15Ryp,
R.sub.19O and I.sub.16Tle (SEQ ID NO. 110) also attained half-lives
of more than 240 min in lysate with protein concentration of 0.5
mg/mL. Surprisingly, the oncocin derivative had positive effects on
the antibacterial activity against E. coli and reduced the MIC
value for SEQ ID NO. 108 and 110 to 2 .mu.g/mL.
Example 3
Antibacterial Tests
Inhibition Zone Test (Agar Diffusion Assay)
[0221] The purified peptides and peptide derivatives were diluted
in water to a final concentration of 500 .mu.g/mL. The test
microbes were plated out from a culture in the logarithmic growth
phase at a concentration of approx. 3.times.10.sup.5 cells/mL in 1%
tryptic soy broth and 1.2% agarose (Fluka Chemie GmbH, Buchs,
Switzerland). With spacing of 3 cm, in each case 10 .mu.L of
aqueous peptide solution (500 .mu.g/mL) or 10 .mu.L of water and
antibiotic solution as controls were added dropwise. After
incubation for 20 h at 37.degree. C., the inhibition zone diameter
(IZO) was determined. All tests were performed in aerobic
conditions.
[0222] Using the alanine scan of the oncocin sequence, various
residues were identified, whose exchange for alanine led to a
marked decrease in antimicrobial activity against E. coli BL21 A1
(FIG. 1) and M. luteus 10240 (FIG. 2).
[0223] In FIG. 1 and FIG. 2, the sequence of oncocin
VDKPPYLPRPRPPRRIYNR (SEQ ID NO. 18) is plotted on the X-axis. Each
amino acid represents the corresponding peptide with the alanine
exchanged at this position. For example, the column Val1 stands for
the peptide ADKPPYLPRPRPPRRIYNR-NH.sub.2. (SEQ ID NO. 29), to which
the value of the IZD is assigned on the Y-axis. The larger the
diameter of the inhibition zone, the higher the activity of the
peptide.
[0224] In the agar diffusion assays against E. Coli BL21 A1 (FIG.
1) it was found that both the alanine exchange at positions Lys3,
Tyr6-Arg9 and Arg11 reduced the activity markedly compared with
oncocin (IZD 1.7 cm). These derivatives (SEQ ID NO. 31, 34 to 37
and 39) only partially inhibit the growth of E. coli on the agar
plate, sometimes with grown-over inhibition zones with 1.0 to 1.2
cm diameter. The peptide Ala15 had the smallest inhibition zone
without growth (1.3 cm) SEQ ID NO. 43). All other positions can be
altered without reducing the activity, wherein however an increase
in activity, expansion of the spectrum, stabilization and a better
in-vivo distribution can be achieved. Positive effects were
achieved here mainly at the N-terminal Va11 and at the C-terminal
Arg19, when protease-stable stable amino acids are incorporated and
the serum stability is thus increased, without losing the
antimicrobial activity.
[0225] The agar diffusion assays against the Gram-positive
bacterium M. luteus (FIG. 2) showed that as a result of alanine
exchange, there are both positive and negative effects on
antimicrobial activity. Positions important for activity are
distributed over the whole sequence; here, the peptides inhibit
growth completely with minimally smaller inhibition zones relative
to onconin (1.8 cm). Mainly, the exchange of Lys or Arg and
therefore the loss of a positive charge in the peptide reduces the
antimicrobial activity. The largest increase in activity, at 0.7
cm, was achieved by exchanging Ile16 and Asn18 (SEQ ID NO. 44, 46).
Changes at these positions are found in examples with in each case
Orn15 and Orn19 and a substitution in position 16 for Leu (SEQ ID
NO. 73) or position 18 for Gln (SEQ ID NO. 74). SEQ ID NO. 74
attains, by these three substitutions, an activity that corresponds
to that of oncocin, wherein the Gln18 can compensate the small
negative effect of the ornithine substitution.
[0226] Growth Inhibition Assay
[0227] The minimum inhibitory concentrations (MIC) of the
antimicrobial peptides and peptide derivatives were determined in
microdilution assays. These used a continuous peptide dilution in
sterile flat-bottomed 96-well microtiter plates (polystyrene,
Greiner Bio-One GmbH, Firckenhausen, Germany) and a total volume of
100 .mu.L per well. The aqueous peptide or peptidomimetic solutions
were diluted with 1% TSB water, to obtain a final concentration of
256 .mu.g/mL. 50 .mu.L of the peptide or peptidomimetic solution
was pipetted into the first well of each series and stirred. From
this solution, 50 .mu.L was transferred to the second well, stirred
and again 50 .mu.L was transferred to the next well, and so on. A
double dilution series is obtained, beginning at 256 .mu.g/mL in
the first well to 125 ng/mL in the twelfth well. The bacteria, e.g.
E. coli BL21 Al, were cultured overnight at 37.degree. C. in
nutrient broth (N B, Carl Roth GmbH+Co. K G, Karleruhe, Germany).
50 .mu.L or a 5.times.10.sup.6 bacteria/mL suspension in 1% TSB was
added to each well of the microtiter plate and thus a final
concentration of the peptides or peptidomimetics from 128 .mu.g/mL
(well 1) to 62.5 ng/mL (well 12) was established in each series.
The plates were incubated at 37.degree. C. for 20 h and then the
absorption was measured at 595 nm with a TECAN microtiter plate
spectrophotometer (Tecan Trading A G, Mannedorf, Switzerland). The
MIC values of all peptides and peptidomimetics were determined in
triplicate. Sterile water was used as negative control. The MIC
value represents the lowest peptide concentration at which no
bacterial growth is observed after an incubation time of 20 h at
37.degree. C.
[0228] The MIC values of the peptides and peptide derivatives
against Escherichia coli BL21 A1 and Micrococcus luteus ATCC 10240
are given in Table 7.
TABLE-US-00008 TABLE 7 Antimicrobial activity against E. coli BL21
A1 and M. luteus 10240. Minimum inhibitory concentration (MIC)
determined in 1% TSB. E. SEQ coli M. ID Synthesis BL21 luteus NO.
number Sequence A1 10240 1* A21 B2 VDKPPYLPRPPPPRRIYN-NH.sub.2 128
n.d. 2* A25 A4 VDKPPYLPRPPPPRRIYNNR-OH 128 64 3* A33 B1
VDKPPYLPRP-4tHyp-PPRRIYNR-OH 128 32 4* A25 A5
VDKPPYLPRPTPPRRIYNNR-OH 128 64 5 A29 B1 VDKPPYLPRPKPPRRIYNNR-OH 16
16 6 A28 B1 VDKPPYLPRPKPPRRIYNRN-OH 32 16 7 A35 A3
VDKPPYLPRPKPPRRIYNR-NH.sub.2 8 16 8 A28 B2 VDKPPYLPRPHPPRRIYNNR-OH
64 32 9 A28 B3 VDKPPYLPRPHPPRRIYNRN-OH 64 16 10* A31 B2
VDKPPYLPRPYPPRRIYNR-OH 128 16 11* A31 B3 VDKPPYLPRPNPPRRIYNR-OH 64
64 12* A31 B4 VDKPPYLPRPQPPRRIYNR-OH 64 64 13* A31 B5
VDKPPYLPRPFPPRRIYNR-OH 64 32 14 A25 A6 VDKPPYLPRPRPPRRIYNNR-OH 8 16
15 A29 A2 VDKPPYLPRPRPPRRIYNNR-NH.sub.2 8 8 16 A29 A6
VDKPPYLPRPRPPRRIYNRN-OH 8 16 17 A35 A2
VDKPPYLPRPRPPRRIYNRN-NH.sub.2 16 128 18 A28 A4/A33 B3
VDKPPYLPRPRPPRRIYNR-NH.sub.2 4 8 19 A35 A4 VDKPPYLPRPRPPRRIYNR-OH
32 64 20 A31 A4 VDKPPYLPRPRPPRPIYNR-OH 8 16 21 A31 B1
VDKPPYL-4tHyp-RPRPPRRIYNR-OH 16 16 22 A34 A2
VDKPPYL-4tHyp-RPRPPRRIYNR-NH.sub.2 8 4 23 A33 A6
VDK-4tHyp-PYLPRPRPPRRIYNR-OH 8 16 24 A34 A3
VDKPPYLPRPRP-4tHyp-RRIYNR-NH.sub.2 8 8 25 A53 E6
VDKPPYLPRPRPPR-4tHyp-IYNR-NH.sub.2 16 8 26 A53 F3
VDKPPYLPRPRPPRRIYNON-NH.sub.2 8 8 27* A35 A6
VDKPPYLPRPRPPRRIYN-NH.sub.2 32 64 28* A51 A6 VDKPPYLPRPRPPRRIYN-OH
64 128 29 A56 A1 ADKPPYLPRPRPPRRIYNR-NH.sub.2 8 4 30 A56 A2
VAKPPYLPRPRPPRRIYNR-NH.sub.2 16 4 31* A56 A3
VDAPPYLPRPRPPRRIYNR-NH.sub.2 64 32 32 A56 A4
VDKAPYLPRPRPPRRIYNR-NH.sub.2 8 8 33 A56 A5
VDKPAYLPRPRPPRRIYNR-NH.sub.2 8 8 34* A56 A6
VDKPPALPRPRPPRRIYNR-NH.sub.2 128 16 35 A56 B1
VDKPPYAPRPRPPRRIYNR-NH.sub.2 128 16 36 A56 B2
VDKPPYLARPRPPRRIYNR-NH.sub.2 32 8 37* A56 B3
VDKPPYLPAPRPPRRIYNR-NH.sub.2 32 32 38 A56 B4
VDKPPYLPRARPPRRIYNR-NH.sub.2 32 8 39* A56 B5
VDKPPYLPRPAPPRRIYNR-NH.sub.2 64 16 40 A56 B6
VDKPPYLPRPRAPRRIYNR-NH.sub.2 16 8 41 A56 C1
VDKPPYLPRPRPARRIYNR-NH.sub.2 16 8 42* A57 D5
VDKPPYLPRPRPPARIYNR-NH.sub.2 16 32 43* A57 D6
VDKPPYLPRPRPPRAIYNR-NH.sub.2 32 16 44 A56 C4
VDKPPYLPRPRPPRRAYNR-NH.sub.2 16 16 45 A56 C5
VDKPPYLPRPRPPRRIANR-NH.sub.2 8 16 46 A56 C6
VDKPPYLPRPRPPRRIYAR-NH.sub.2 8 8 47* A56 D1
VDKPPYLPRPRPPRRIYNA-NH.sub.2 32 16 48* A35 B1 VDKPPYLPRPRPPRPIRV-OH
128 8 49 A70 A1 VDKPPYLPRPRPPRRIYPQPRPPHPRL-NH.sub.2 4 4 50 A51 B1
VDKPPYLPRPRPPRRIYNO-NH.sub.2 8 16 51* A51 B2
VDKPPYLPRPRPPRRIYNN-NH.sub.2 16 64 52* A51 B3
VDKPPYLPRPRPPRRIYNDap(Ac)-NH.sub.2 32 64 53 A56 E1
VDKPPYLPRPRPPRRIYNH-NH.sub.2 16 6 54 A59 A2
VDKPPYLPRPRPPRRIYNAgp-NH.sub.2 8 8 55 A59 A3
VDKPPYLPRPRPPRRIYNArg-(NO.sub.2)-NH.sub.2 8 16 59 A59 A4
VDKPPYLPRPRPPRRIYN(N-Me-Arg)-NH.sub.2 8 4 57 A59 A5
VDKPPYLPRPRPPRRIYNHar-NH.sub.2 8 8 58 A66 D2 propyl.
VDKPPYLPRPRPPRRIYNR-NHC.sub.3H.sub.7 4 8 59 A45 A6
VDKPPYLPRPRPRPRIYNR-NH.sub.2 8 16 60* A45 A5
VDKPPYLPRPRPPRPIYNR-NH.sub.2 8 64 61 A45 A3
VDKPPYLPRPRPPROIYNR-NH.sub.2 8 16 62 A51 B4
VDKPPYLPRPRPPRbetaHArgIYNR-NH.sub.2 4 16 63 A53 F1
VDKPPYLPRPRPPR-4tHyp-IYNO-NH.sub.2 4 8 64 A66 C5
VDKPPYLPRPRPPRHIYNH-NH.sub.2 8 32 65 A70 B4
VDKPPYLPRPRPPRAgpIYNHar-NH.sub.2 4 2 66 A70 C1
VDKPPYLPRPRPPRAgpIYNAgp-NH.sub.2 8 4 67 A70 B5
VDKPPYLPRPRPPRHarIYNHar-NH.sub.2 4 4 68 A70 C2
VDKPPYLPRPRPPRHarIYNAgp-NH.sub.2 4 4 69 A70 C3
VDKPPYLPRPRPPROIYNAgp-NH.sub.2 4 4 70 A70 B6
VDKPPYLPRPRPPROIYNHar-NH.sub.2 4 4 71 A66 D3 propyl.
VDKPPYLPRPRPPROIYNR-NHC.sub.3H.sub.7 4 8 72 A53 F2
VDKPPYLPRPRPPROIYNO-NH.sub.2 8 8 73 A66 C6
VDKPPYLPRPRPPROLYNO-NH.sub.2 8 16 74 A66 D1
VDKPPYLPRPRPPROIYQO-NH.sub.2 4 8 75 A56 D2
VEKPPYLPRPRPPRRIYNR-NH.sub.2 16 8 76 A56 D3
VDRPPYLPRPRPPRRIYNR-NH.sub.2 16 8 77 A56 D4
VDKPPYIPRPRPPRRIYNR-NH.sub.2 32 9 78 A56 D5
VDKPPYLPRPRPPRRLYNR-NH.sub.2 8 16 79 A56 D6
VDKPPYLPRPRPPRRIYQR-NH.sub.2 8 8 80 A32 D3 ac.
Ac-VDKPPYLPRPRPPRRIYNR-OH >256 16 81 A32 D3 fo.
For-VDKPPYLPRPRPPRRIYNR-OH 128 32 82 A45 A2
ODKPPYLPRPRPPRRIYNR-NH.sub.2 8 8 83 A45 A2 ac.
Ac-ODKPPYLPRPRPPRRIYNR-NH.sub.2 16 16 84 A45 A4 guan.
Guan-VDKPPYLPRPRPPRRIYNR-NH.sub.2 32 n.d. 85 A62 B3 guan.
Guan-VDKPPYLPRPRPPROIYNO-NH.sub.2 64 n.d. 86* A35 A5
DKPPYLPRPRPPRRIYNR-NH.sub.2 32 8 87* A54 E1 GNNRPVYIPQPRPPHPRL-OH 2
64 89* A34 B3 GKPRPYSPRPTSHPRPIRV-OH 4 0.5 91* A35 A1
VDKGSYLPRPTPPRPIYNRN-NH.sub.2 16 128 95 A60 E4 F5
RQIKIWFQNRRMKWKKC(CH.sub.2CO-GNNRPVY 8 8 IPQPRPPHPRL-OH)-OH 97 A60
E2 F5 RQIKIWFQNRRMKWKKC(CH.sub.2CO-GKPRPYS 16 1 PRPTSHPRPIRV-OH)-OH
99 A60 E6 F5 RQIKIWFQNRRMKWKKC(CH.sub.2CO-VDKGSYL 32 4
PRPTPPRPIYNRN-NH.sub.2)-OH 101 A60 F2 F5
RQIKIWFQNRRMKWKKC(CH.sub.2CO-VDKPPYL 32 4
PRPRPPRRIYNR)-NH.sub.2)-OH 103* A60 F5 (RQIKIWFQNRRMKWKKC-OH).sub.2
>128 >128 107 A74 C6 VDKPPYLPRPRP-4tHyp-ROIYNO-NH.sub.2 4 4
108 A74 D1 VDKPPYLPRPRP-4tHyp-R-4tHyp- 2 32 IYNO-NH.sub.2 109 A76
B3 VDKPPYLPRPRPPRO-Tle-YNO-NH.sub.2 4 4 110 A76 B5
VDKPPYLPRPRP-4tHyp-R-4tHyp-Tle- 2 16 YNO-NH.sub.2 111 T1 D9
VDKPPYLPRPRPPRrIYNR-NH.sub.2 4 4 112 T1 D11
VDKPPYLPRPRPPRrIYNr-NH.sub.2 4 n.d. 113 A82 A4
ONYIORPPRPRPLYPPKDV-NH.sub.2 >256 64 114 A82 A5
ONYI-4tHyp-RPPRPRPLYPPKDV-NH.sub.2 >256 128 115 T1 C7
vdkppylprprpprriynr-NH.sub.2 64 16 116 T1 C9
vdkppylprprpproiyno-NH.sub.2 64 32 117 T1 C11
rnyirrpprprplyppkdv-NH.sub.2 64 32 118 T1 D1
onyiorpprprplyppkdv-NH.sub.2 256 32
[0229] Propy stands for a propylamide on the C-terminus
(Sub.sub.2=OR.sub.3=NHC.sub.3H.sub.7); Ac=acetyl group, for=formyl
group, guan=guanidino group and CF=5,6-carboxyfluorescein are
examples of modified N-termini (modified alpha-amino group of the
N-terminal amino acid, Sub.sub.1=acetyl-NH, formyl-NH, guanidino or
5,6-carboxyfluorescein),
[0230] .beta.Har: .beta.-homoarginine, the beta-amino acid homolog
to arginine,
[0231] Agp: 2-amino-3-guanidinopropionic acid, Har: homoarginine
and Arg(NO.sub.2): nitroarginine are homologs of arginine,
[0232] N--Me--Arg: N-methyl-arginine--an arginine methylated at the
peptide bond,
[0233] 4tHyp: trans-4-hydroxyproline,
[0234] Tle: tert-butylglycine
[0235] Dap(Ac): 2,3-diaminopropionic acid with acetylated amino
function in the side chain,
[0236] (CH.sub.2CO): acetyl linker on SH group of cysteine,
[0237] f: .alpha.-aminocapronic acid,
[0238] lower-case letters stand for the corresponding D-amino
acids.
[0239] SEQ ID NO. 2 corresponds to the native Oncopeltus 4
sequence. SEQ ID NO. 1 is an N-terminally shortened derivative of
the native Oncopeltus 4 sequence. The sequences with SEQ ID NO. 1,
2, 3, 4, 10 to 13, 17, 28, 31, 34, 35, 39 and 48 are comparative
examples, these and others are marked with * in Table 7. The
sequences with SEQ ID NO. 8 to 9, 80 81 and 85 are less preferred
examples of the invention. The other sequences shown in Table 7 are
peptides or peptide derivatives preferred according to the
invention. The examples most preferred are SEQ ID NO. 18, 22, 24,
26, 29, 58, 62, 63, 65 to 71, 74, 79, 82 and 107 to 112.
[0240] By exchanging Proll (residue X.sub.2) in the initial
sequence (SEQ ID NO. 2) for the cationic amino acids Lys and Arg
(SEQ ID NO. 8 and 14), surprisingly, higher activity against E.
coli BL21 AI and M. luteus 10240 was achieved. Exchange for His
(SEQ ID NO. 5) and a Thr frequently occurring at this position in
proline-rich AMP (SEQ ID NO. 4) did not have a positive effect. The
derivative with Arg at position 11 had, at 8 .mu.g/mL (E. coli),
the lowest MIC so far and was accordingly altered C-terminally.
Amidation of the C-terminus only altered the activity against M.
luteus negatively by one dilution step. Peptide derivatives without
Arg and thus lacking a positive charge at the last or penultimate
position (SEQ ID NO. 27, 28, 47, 51) lost activity by a factor of 8
to 16, mainly against M. luteus and are not included among the
preferred examples.
[0241] SEQ ID NO. 16 with Arg at position 19 (residue X.sub.4) and
Asn at 20 was as active as SEQ ID NO. 14. Unexpectedly, amidation
of the C-terminus (Sub.sub.2) does not have a negative influence on
the activity (SEQ ID NO. 15). Amidation of the C-terminus
(Sub.sub.2) even leads to increased activity (cf. SEQ ID NO. 18
with SEQ ID NO. 19). C-terminal amidation has in addition a
significantly positive effect on stability. Thus, amidated peptide
derivatives have an up to 30 min longer half-life than the
corresponding peptide with free acid function (example 2). The
peptide derivative SEQ ID NO. 18 had, at 4 or 8 .mu.g/mL against E.
coli or M. luteus respectively, the lowest MIC values, at a
half-life of 60 min in 25% aqueous mouse serum and was designated
as oncocin (Table 4).
[0242] In the serum stability test, oncocin was cleaved
C-terminally at position 15 (residue X.sub.3) and 19 (residue
X.sub.4) and the peptides VDKPPYLPRPRPPR-OH (corresponds to SEQ ID
NO. 126) and VDPPYLPRPRPPRRIYN-OH (corresponds to SEQ ID NO. 28)
were identified as the main degradation products. The derivative
shortened by Arg19, with a MIC of 64 .mu.g/mL, still only had very
low antimicrobial activity against E. coli. Derivatives with
arginine or other cationic amino acids at position 19 (residue
X.sub.4) showed, surprisingly, increased stability. However, a
derivative with His at position 19 (residue X.sub.4; SEQ ID NO. 53)
with a MIC of 16 .mu.g/mL against E. coli had 4 times lower
activity than oncocin. Preferred examples are substitutions with
Agp, Arg (NO.sub.2), N--Me--Arg and Har (SEQ ID NO. 54 to 57) at
position 19 (residue X.sub.4) substituted peptide derivatives whose
MIC values correspond to the values of oncocin and therefore
unexpectedly do not have a negative influence on the activity of
the peptides. The preferred, least expensive example was the
substitution of Arg19 for ornithine in SEQ ID NO. 50. This
derivative is far more stable than oncocin (60 min) at almost the
same activity. Another preferred example for stabilization of the
C-terminus was amidation of the carboxyl function (Sub.sub.2) as
propylamide (SEQ ID NO. 58). The antimicrobial activity is
maintained and the half-life is also more than 120 min.
[0243] In the derivatives stabilized at position 19 (residue
X.sub.4), in further examples position 15 (residue X.sub.3) was
substituted with arginine derivatives or other cationic amino
acids. Preferred examples are SEQ ID NO. 65 to 70, with various
combinations of Agp, Arg(NO.sub.2), N--Me--Arg, Har and Orn. The
most cost-effective preferred example is SEQ ID NO. 72, with
ornithine at position 15 and 19 (residue X.sub.3 and X.sub.4) and a
half-life of more than 360 min, with activity comparable to oncocin
(MIC 8 .mu.g/mL E. coli). SEQ ID NO. 74 had, additionally to Orn15
and Orn16, a substitution of glutamine at position 18 for
asparagine, which resulted in slightly higher activity (MIC 4
.mu.g/mL E. coli). The combination of Orn at position 15 (residue
X.sub.3) and amidation of the C-terminus (Sub.sub.2) with
propylamine in SEQ ID NO. 71 is another much preferred example with
surprisingly high activity (4 .mu.g/mL E. coli) and very high serum
stability (>360 min). A noncationic substitution at position 15
(residue X.sub.3) with hydroxyproline was carried out in the
preferred SEQ ID NO. 63 in combination with Orn at position 19
(residue X.sub.4). This derivative also has high activity (4
.mu.g/mL E. coli, 8 .mu.g/mL M. luteus) and excellent stability
(>360 min). Substitution of the proline in position 4, 8 or 13
(SEQ ID NO. 21 to 24) of oncocin with hydroxyproline has no effect
on the MIC values, and does not reduce the proteases resistance of
the second labile cleavage site at position 15 (residue X.sub.3).
Although this exchange with hydroxyproline has neither an effect on
the MIC values nor an effect on serum stability, unexpectedly this
exchange reduces cellular toxicity and hemolysis.
[0244] A preferred example for modification of the N-terminus in
oncocin is SEQ ID NO. 82 with substitution of position 1 (residue
X.sub.1) in Orn, with the same activity against oncocin.
Acetylation, methanoylation (formylation) or guanidation of the
N-terminal amino function (Sub.sub.1; SEQ ID NO. 80, 81, 83, 84,
85) reduced the activity to e.g. 128 .mu.g/ mL or 32. (E. coli or
M. luteus; SEQ ID NO. 81).
[0245] Penetratin was coupled via a thioether bridge to the amino
function of the N-termini of the antimicrobial peptides. This
modification was able to extend the activity spectrum of the
peptides with up to now little activity against M. luteus, to
include this bacterium. The MIC value for pyrrhocoricin (SEQ ID NO.
91) was reduced to the greatest extent from 128 .mu.g/mL to 4
.mu.g/mL for penetratin-pyrrhocoricin (SEQ ID NO. 98), which is
equivalent to a 32-fold increase in activity. Penetratin-apidaecin
(8 .mu.g/mL, SEQ ID NO. 94) was also 8 times more active than the
unmodified apidaecin 1b (64 .mu.g/mL SEQ ID NO. 87). For oncocin
(SEQ ID NO. 18) with a MIC value of 8 .mu.g/mL, still a 2-fold
increase in activity to 4 .mu.g/mL was observed for
penetratin-oncocin (SEQ ID NO. 100). The high activity of drosocin
(0.5 .mu.g/mL, SEQ ID NO. 89) was maintained in the
pentetratin-drosocin construct (1 .mu.g/mL, SEQ ID NO. 96).
[0246] The exchange of proline at position 13 and 15 for
trans-4-hydroxyproline and Arg19 for ornithine (SEQ ID NO. 108)
leads surprisingly, at 2 .mu.g/mL, to an increase in activity
against E. coli compared with the native oncocin sequence (4
.mu.g/mL, SEQ ID NO. 18) while the stability remains high. If the
isoleucine at position 16 is replaced with tert-butylglycine SEQ ID
NO. 110), the MIC value of 2 .mu.g/mL against E. coli is
maintained. It is mainly the increased stability that is of
interest here.
[0247] The activity of the peptide with SEQ ID NO. 107 shows the
same antibiotic action against M. Luteus as oncocin (SEQ ID NO.
18).l The sequences SEQ ID NO. 113 to 118 are comparative examples.
The derivatives synthesized with D-amino acids (SEQ ID NO. 115 and
116) show no activity against E. coli and only slight activity
against M. luteus (64 or 128 .mu.g/mL).
[0248] Both the all D-peptides with D-amino acids in the native
order (SEQ ID NO. 115 and 116) and the retro-inverse synthesized
peptides (SEQ ID NO. 117 and 118) show only slight activity against
E. coli (64-256 .mu.g/mL). However, all peptides synthesized with
D-amino acids (SEQ ID NO. 115 to 118) still display, with MIC
values between 16-32 .mu.g/mL, relatively good activity against M.
luteus, which is located in the region of the L-peptides with five
positive net charges. The net charge-dependent activity and the MIC
values of the D-peptides against M. luteus might indicate a target
protein-nonspecific mechanism of action in this Gram-positive
bacterium.
TABLE-US-00009 TABLE 8 Antimicrobial activity against pathogenic
Escherichia coli DSM 10233, Klebsiella pneumoniae DSM 681 and
Pseudomonas aeruginosa DSM 3227. Minimum inhibitory concentration
(MIC) determined in 1% TSB. SEQ ID E. coli K. pneumoniae P.
aeruginosa NO. Sequence DSM 10233 DSM 681 DSM 3227 18
VDKPPYLPRPRPPRRIYNR-NH.sub.2 16 4 >32 72
VDKPPYLPRPRPPROIYNO-NH.sub.2 32 4 >32 63
VDKPPYLPRPRPPR-4tHyp-IYNO-NH.sub.2 1 2 16-32 107
VDKPPYLPRPRP-4tHyp-ROIYNO-NH.sub.2 16 4 >32 108
VDKPPYLPRPRP-4tHyp-R-4tHyp-IYNO-NH.sub.2 1 2 16-32 111
VDKPPYLPRPRPPRrIYNR-NH.sub.2 4 4 n.d. 117
rnyirrpprprplyppkdv-NH.sub.2 >32 >32 n.d.
[0249] The peptides shown in Table 8 were investigated for their
MIC against pathogenic bacteria such as E. coli DSM 10233, K.
pneumonias DSM 681 and P. seruginosa DSM 3227. All derivatives with
4-trans-hydroxyproline at position 15 (SEQ ID NO. 63, 108, 113 and
114) have, with MIC values between 1-4 .mu.g/mL, surprisingly up to
16 times higher activity against E. coli DSM 10233 than onconin
(SEQ ID NO. 18).
[0250] Table 10 gives the MIC values of some peptides and peptide
derivatives against multiresistant bacterial strains. The tests
were in this case carried out in Mueller-Hinton medium (1/4
concentrated), which is equivalent to 1% TSB. Some of the resistant
bacterial strains tested are shown in Table 9 and the peptides
tested are shown in Table 11.
TABLE-US-00010 TABLE 9 Multiresistant Gram-negative bacteria tested
E. coli D31 (J. Wilson) .beta.-lactamase overproducer (ESBL+) -
resistant to .beta.- lactamase inhibitors E. coli ATCC BAA-457
trimethoprim-sulfomethoxazole- resistant E. coli 045-849 SENTRY
ciprofloxacin- and trimethoprim-sulfomethoxazole- resistant K.
pneumoniae ATCC 27799 gentamicin-, cephalothin- and naladixic
acid-resistant K. pneumoniae ATCC 700603 produces .beta.-lactamase
SHV-18 (ESBL+) - resistant to .beta.- lactamase inhibitors K.
pneumoniae 012-3132 fluoroquinolone-resistant S. typhimurium ATCC
700408 multi-resistant (e.g. ampicillin, chloramphenicol) S.
typhimurium S5 (J. Weiser) multi-resistant (e.g. cefotaxime,
tobramycin)
TABLE-US-00011 TABLE 10 Antimicrobial activity against
multiresistant bacteria. MIC in 1/4 Muller-Hinton medium. SEQ ID E.
coli D31 E. coli SEQ E. coli 045-849 K. pneumoniae K. pneumoniae K.
pneumoniae S. typhimurium NO. J. Wilson 102ATCC # BAA-4 57 SENTRY
ATCC 27799 K6 ATCC 700603 012-3131 ATCC 700408 87* 1 1 0.5 4 16 16
0.13 89* 4 2 2 8 8 4 0.5 18 2 4 4 16 8 4 0.25 24 1 4 2 8 8 2 0.13
15 16 32 4 64 32 16 0.5 22 2 4 4 16 8 4 0.5 14 32 64 4 128 64 32
n.d 20 4 32 8 64 128 64 n.d 23 8 32 8 64 32 16 n.d 21 32 64 16 128
64 32 n.d 16 32 32 4 128 64 32 n.d 5 64 128 16 128 128 64 n.d SEQ
ID S. typhimurium S. typhimurium P. mirabilis P. vulgaris P.
aeruginosa P. aeruginosa S. saprophyticus NO. s5 (J. Weiser) ATCC
14028 ATCC 7002 ATCC 6896 39324 10 J. Wilson 15305 87* 0.5 0.5 >
> 64 32 > 89* 1 8 > > 32 16 >64 18 4 2 128 128 4 8
16 24 2 2 128 128 4 2 16 15 8 4 > > n.d 16 n.d 22 8 2 128 128
n.d 16 n.d 14 n.d n.d n.d n.d n.d n.d n.d 20 n.d n.d n.d n.d n.d
n.d n.d 23 n.d n.d n.d n.d n.d n.d n.d 21 n.d n.d n.d n.d n.d n.d
n.d 16 n.d n.d n.d n.d n.d n.d n.d 5 n.d n.d n.d n.d n.d n.d n.d
n.d: not determined *apidaecin 1b (SEQ ID NO. 87) and drosocin (SEQ
ID NO. 89) for comparison.
TABLE-US-00012 TABLE 11 Sequences tested SEQ ID NO. Sequence 87
GNNRPVYIPQPRPPHPRL-OH 89 GKPRPYSPRPTSHPRPIRV-OH 18
VDKPPYLPRPRPPRRIYNR-NH.sub.2 24 VDKPPYLPRPRP-4tHyp-RRIYNR-NH.sub.2
15 VDKPPYLPRPRPPRRIYNNR-NH.sub.2 22
VDKPPYL-4tHyp-RPRPPRRIYNR-NH.sub.2 14 VDKPPYLPRPRPPRRIYNNR-OH 20
VDKPPYLPRPRPPRPIYNR-OH 23 VDK-4tHyp-PYLPRPRPPRRIYNR-OH 21
VDKPPYL-4tHyp-RPRPPRRIYNR-OH 16 VDKPPYLPRPRPPRRIYNRN-OH 5
VDKPPYLPRPKPPRRIYNNR-OH
[0251] The preferred example SEQ ID NO. 18, 22 and 24 showed,
surprisingly, an at least as high or higher activity against the
three different Gram-negative multiresistant bacterial species E.
coli, Klabsiella pneumoniae and Salmonella typhimurium. Oncocin
(SEQ ID NO. 18) had MIC values of 2 .mu.g/mL against
.beta.-lactamase-overproducing E. coli D31, 4 .mu.g/mL against
fluoroquinoline-resistant K. pneumoniae 012-3132 and 0.25 .mu.g/mL
against multiresistant S. typhimurium ATCC 700408 bacteria. With
one exception (SEQ ID NO. 15), all the amidated peptides tested
showed good MIC values against the E. coli and K. pneumoniae
strains. Deletion of Asn19 from SEQ ID NO. 2, which leads to
oncocin (SEQ ID NO. 18), surprisingly increased the activity of the
peptides further. Amidation of the C-terminus not only increases
the activity against E. coli and K. pneumoniae, but in addition
increases the stability of the Oncopeltus 4 or oncocin
derivatives.
[0252] With Pseudomonas seruginosa, Proteus mirabilis and Proteus
vulgaris, further Gram-negative bacteria were tested and the
activity of the preferred examples SEQ ID NO. 18 and 24 was
determined. Oncocin (SEQ ID NO. 18) was active both against P.
seruginosa 39324 (MIC 4 .mu.g/mL) and against P. mirabilis and P.
vulgaris (128 .mu.g/mL). Oncocin was also active, at 16 .mu.g/mL,
against Gram-positive Staphylococcus saprophyticus 15305 bacteria.
Therefore the peptides according to the invention display a
surprisingly broad spectrum of action.
TABLE-US-00013 TABLE 11 Antimicrobial activity against pathogenic
Escherichia coli DSM 10233, Klebsiella pneumoniae DSM 681 and
Pseudomonas aeruginosa DSM 3227. Minimum inhibitory concentration
(MIC) determined in 1% TSB. SEQ ID K. pneumoniae P. aeruginosa NO.
Sequence E. coli DSM 681 DSM 3227 18 VDKPPYLPRPRPPRRIYNR-NH.sub.2
16 4 >32 72 VDKPPYLPRPRPPROIYNO-NH.sub.2 32 4 >32 63
VDKPPYLPRPRPPR-4tHyp-IYNO-NH.sub.2 1 2 16-32 107
VDKPPYLPRPRP-4tHyp-ROIYNO-NH.sub.2 16 4 >32 108
VDKPPYLPRPRP-4tHyp-R-4tHyp-IYNO-NH.sub.2 1 2 16-32 111
VDKPPYLPRPRPPRrIYNR-NH.sub.2 4 4 n.d. 117
rnyirrpprprplyppkdv-NH.sub.2 >32 >32 n.d.
[0253] Derivatives of oncocin show very good activity against
pathogenic bacteria such as E. coli DSM 10233, K. pneumoniae DSM
681 and P. aeruginosa DSM 3227. Substitution with
4-trans-hydroxyproline at position 15 (SEQ ID NO. 63 and 108) leads
to surprisingly high MIC values against E. coli DSM 10233 between
1-4 .mu.g/mL, these are 16 times higher than for oncocin (SEQ ID
NO. 18). These derivatives also display high activity against K.
pneumoniae DSM 681 and P. aeruginosa DSM 3227, illustrating the
broad spectrum of action of the peptides according to the
invention.
Example 4
Fluorescence Microscopy
[0254] HeLa and SH-SY5Y cells were plated out in 96-well microtiter
plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and incubated
overnight. On the next day, 5,6-carboxyfluorescein-labeled peptides
or penetratin constructs were dissolved in fresh medium (40
.mu.mol/L) and the cells were incubated therein for 2 h. The cells
were washed twice with PBS and were investigated in PBS by
fluorescence microscopy.
[0255] Parameters for fluorescence microscopy [0256] Microscope:
Leica DMI6000B (Leica Mikrosystems GmbH, Wetzlar, Germany) [0257]
Light source: Leica EL6000 with metal-halogen lamp [0258]
Objective: N PLAN L 20.times.0.40 corr [0259] Software: Leica
Application Suite 2.1.8.; Adobe Photoshop CS
[0260] The fluorescence micrographs in FIG. 3 show that after
incubation with 40 .mu.mol/L 5,6-carboxyfluorescein-labeled oncocin
(SEQ ID NO. 94), fluorescence is not detectable in HeLa or in
SH-SY5Y cells (FIG. 3B and F). Moreover, no fluorescence and
therefore no internalization of these antimicrobial proline-rich
peptides could be detected for 5,6-carboxyfluorescein-labeled
apidaecin 1b, pyrrhocoricin and drosocin (SEQ ID NO. 88, 90 and
92). In contrast, after incubation of both cell lines with
5,6-carboxyfluorescein-labeled penetratin-oncocin (SEQ ID NO. 102),
strong fluorescence in the cell interior was observed in the
micrographs (FIG. 3D and E), providing evidence of internalization
of the whole construct.
[0261] The penetratin constructs with apidaecin 1b, pyrrhocoricin
and drosocin (SEQ ID NO. 96, 98 and 100) also internalize in both
cell lines and show definite fluorescence.
Example 5
Confocal Laser Scanning Microscopy
Bacteria
[0262] A bacterial suspension that had been cultured overnight was
diluted to 150.times.10.sup.6 cells/mL and the
5,6-carboxyfluorescein-labeled peptides and penetratin constructs
were added (final concentration of 30 .mu.mol/L). In order to
quench the fluorescence of the molecules outside of the bacteria.
60 eq of 5,6-carboxytetramethylrhodamine (TAMRA; Merck, Darmstadt,
Germany) was added. The fluorescence resulting from internalization
of the labeled peptides in the bacteria was investigated
immediately with a TCS SP5 confocal laser scanning microscope from
the company Leica Microsystems GmbH (Wetzlar, Germany).
[0263] After incubation for 20 min with
5,6-carboxyfluorescein-labeled oncocin, the peptide had accumulated
in the bacterial membrane (FIG. 4B). The quenching effect through
fluorescence resonance energy transfer (FRET) between
5,6-carboxyfluorescein and TAMRA, which remains outside the cell,
is therefore lost and a fluorescence signal can be detected. After
a further 30 min, the peptide had accumulated inside the cell (FIG.
4D). The 5,6-carboxyfluorescein-labeled apidaecin 1B, pyrrhocoricin
and drosocin sequences (SEQ ID NO. 96, 98 and 100) also internalize
in E. coli and produce a definite fluorescence. In contrast, the
labeled penetratin constructs reach the interior of the cell more
slowly and cause much weaker fluorescence after comparable
incubation times (FIG. 4F). This observation correlates with the
minimum inhibitory concentrations determined. Thus, the penetratin
construct of oncocin (SEQ ID NO. 100) had, at 32 .mu.g/mL, an 8
times higher MIC value than oncocin (4 .mu.g/mL; SEQ ID NO. 18) and
thus considerably lower activity against E. coli (Table 7). Entry
of the penetratin homodimer could not be detected by fluorescence
microscopy even after 90 min (FIG. 4H). This shows that in the
penetratin construct, the respective proline-rich peptide sequence
transports penetratin as cargo into the bacterial cell.
[0264] In contrast, the 5,6-carboxyfluorescein-labeled penetratin
homodimer enters Gram-positive M. luteus cells and produces a
definite fluorescence signal there after incubation for 1 h (FIG.
5J). The labeled pyrrhocoricin cannot be detected in M. luteus
after the same incubation time (FIG. 5B). With penetratin as
transporter in the labeled penetratin-pyrrhocoricin, the derivative
accumulates in M. luteus and produces a strong fluorescence signal
(FIG. D). The MIC value drops from 128 .mu.g/mL for pyrrhocoricin
to 4 .mu.g/mL for penetratin-pyrrhocoricin, which corresponds to a
32-fold increase in activity (Table 7). In parallel, and 8-fold
increase in activity for apidaecin 1b (64 .mu.g/mL) was determined
in the penetratin-apidaecin derivative (8 .mu.g/mL).
[0265] HeLa and SH-SY5Y Cells
[0266] The cells were cultured on glass-bottomed culture dishes
from the company MatTek Corporation (Ashland, Mass., USA) and were
incubated with 5,6-carboxyfluorescein-labeled penetratin constructs
(10 .mu.mol/L for SH-SY5Y or 7 .mu.mol/L for HeLa) for 2 h or 24 h.
The medium was removed, washed twice with PBS and fresh medium was
added. For staining the cell nucleus, the dye Hoechst 33342 (Fluka
Chemie GmbH, Buchs, Switzerland) was added and it was incubated for
a further 15 min. The fluorescence was analyzed immediately with
the TCS SP5 confocal laser scanning microscope. All images were
recorded in a sequential scan mode and the batch of images was
analyzed with the Leica Application Suite Advanced Fluorescence
1.7.1 software (Leica Microsystems) and Adobe Photoshop CS (Adobe
Systems GmbH, Munich, Germany).
[0267] The results show that the antimicrobial peptides, which had
been extended N-terminally by 5,6-carboxyfluorescein-penetratin,
penetrate into the cells within 2 hours. Conversely, the
antimicrobial peptides only labeled with 5,6-carboxyfluorescein,
without the penetratin sequence, could not be detected in the
cells, i.e. these peptides cannot penetrate through the external
cell membrane into the tested cell lines. Transport into the
interior of the cell only takes place via the penetratin sequence.
Staining of the cell nuclei with the dye Hoechst 33342 and of the
mitochondria with the dye MitoTracker Red CMXRos ruled out
localization of the peptides in these compartments. After quite a
long incubation time (24 h) the fluorescence employed by the
5,6-carboxyfluorescein was concentrated near the cell nucleus. By
staining the Golgi body, partial co-localization could be
detected.
Example 6
Cytotoxicity
[0268] MTT Assays with HeLa and SH-SY5Y Cells
[0269] The cytotoxicity of the peptides, peptidomimetics and
penetratin constructs was determined with the "Cell Proliferation
Kit I" from the company Roche Diagnostics GmbH (Mannheim, Germany).
The method is based on reduction of the yellow
methylthiazolyldiphenyl tetrazolium bromide (MTT) by cellular
oxidoreductases of metabolically active cells (Vistica D T et al.
Tetrazolium-Based Assays for Cellular Viability--A Critical
Examination of Selected Parameters Affecting Formazan Production.
Cancer Research 51: 2515-20, 1991; Sister T F, Sawyer B, &
Strauli U. Studies on Succinate-Tetrazolium Reductase Systems. 3.
Points of Coupling of 4 Different Tetrazolium Salts. Biochimics et
Biophysica Acta 11: 383-6, 1963; Berridge M V & Tan A S.
Characterization of the Cellular Reduction of
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(Mtt)--Subcellular Localization, Substrate Dependence, and
Involvement of Mitochondrial Electron Transport in Mtt Reduction.
Archives of Biochemistry and Biophysics 303: 474-82, 1993). The
formation of the water-insoluble purple-colored formazan product is
proportional to the number of viable cells and can be detected
photometrically after cell lysis.
[0270] Cell culture was carried out in cell culture bottles (25
cm.sup.2) with a filter cap or in 96-well microtiter plates
(Grainer Bio-One GmbH, Frickenhausen, Germany) at 37.degree. C.
under 5% CO.sub.2 and 95% air humidity. All media and additives
were obtained from PAA Laboratories (Pasching, Austria). In each
case 1% of nonessential amino acids and 1% of
penicillin/streptomycin were added to MEM/glutamine medium with 5%
fetal calf serum (HeLa) or DMEM/HAM's F-12 medium with 15% fetal
calf serum (SH-SY5Y).
[0271] HeLa or SH-SY5Y cells were plated out in sterile 96-well
microtiter plates with a concentration of 2.times.10.sup.4
cells/well and were incubated overnight. The cells were washed once
with sterile PBS and the peptides, dissolved in 100 .mu.L fresh
medium, were added. 12% PBS or 12% DMSO in medium served as
negative or positive control, respectively. After incubation (24
h), 10 .mu.L of MTT reagent was added to give a final concentration
of 0.5 mg and it was incubated at 37.degree. C. for a further four
hours. With a 10% sodium dodecylsulfate solution in 0.01 mol/L
hydrochloric acid, the cells and the crystalline formazan were
dissolved and the absorption was determined after 16 h at 590 and
650 nm with the Paradigm.TM. Microplate Reader (Beckman Coulter
GmbH, Wals, Austria).
[0272] The surprising results show that none of the antimicrobial
proline-rich peptides tested at 600 .mu.g/mL has toxic effects on
SH-SY5Y or HeLa cells (FIG. 6). The experiments were carried out
three times independently as triple determination and the
proportion of metabolically active cells was normalized to the
negative control 12% PBS in medium. The results are confirmed by
the fact that the fluorescence-labeled derivatives of these
peptides could not be detected in the interior of these cells after
an incubation time of one hour (see example 5). Interaction with
extracellular target molecules or receptors in the external cell
membrane could thus also be ruled out for SH-SY5Y and HeLa
cells.
[0273] The cytotoxicity tests of the penetratin constructs were
carried out in three independent experiments of a dilution series
50-400 .mu.g/mL as triple determination. The penetratin monomer
(SEQ ID NO. 105) and a penetratin-tau sequence, which served as
control, showed no toxic action against HeLa cells up to the
highest concentration of 400 .mu.g/mL (FIG. 6). With the
penetration constructs with antimicrobial peptides, negligibly
small toxic effects were observed between 100 and 400 .mu.g/mL. The
penetratin homodimer (SEQ ID NO. 103) was almost 100% toxic at 400
.mu.g/mL, whereas penetratin-drosocin and penetratin-oncocin (SEQ
ID NO. 97 and 101) displayed slight toxicities. In the
investigations with SH-SY5Y cells, at 400 .mu.g/mL the penetratin
monomer and the penetratin-tau construct reduced the proportion of
growing cells to 70%. All penetratin-AMP constructs showed,
surprisingly, no toxic action in the preferred concentrations.
[0274] Hemolysis Test
[0275] Another possibility for investigating the cytotoxicity of
the peptides and peptide derivatives is the hemolysis test. The
hemolytic activity is investigated on human erythrocytes (Ryge T S
& Hansen P R. Potent antibacterial lysine-peptoid hybrids
identified from a positional scanning combinatorial library.
Bioorganic & Medicinal Chemistry 14: 4444-51, 2006), which the
Leipzig University Hospital (Germany) made available as human
erythrocyte concentrate in sodium chloride-adenine-glucose-mannitol
buffer (stored at 4.degree. C.). The erythrocytes were centrifuged
off at 1000 g and washed three times with ten times the volume of
cold phosphate-buffered saline (PBS, pH 7.4). The erythrocytes were
diluted to a final concentration of 1% in PBS. One hundred
microliters of erythrocyte suspension was pipetted into each
V-shaped well of a 96-well polypropylene microtiter plate (Greiner
Bio-One GmbH). Then 100 .mu.L of the peptides dissolved in PBS was
added to each position, to obtain a dilution series from 600
.mu.g/mL to 4.7 .mu.g/mL in seven dilution steps. The microtiter
plate was incubated at 37.degree. C. for 1 h and then centrifuged
at 1000*g. 100 .mu.L was taken from the supernatant, transferred to
a 96-well flat-bottomed polystyrene microtiter plate (Greiner
Bio-One GmbH) and the absorption was determined at 405 nm in a
Sunrise microtiter plate reader (Tecan Trading A G, Mannedorf,
Switzerland), to evaluate the release of the home group. PBS or
0.1% Triton X-100.RTM. ((p-tert-octylphenoxy) polyethoxyethanol;
Fluka Chemie GmbH, Buchs, Switzerland) and malittin
(SIGMA-Aldrich-Laborchemikalien, Taufkirchen, Germany) were used as
negative or positive controls. All hemolysis tests were carried out
twice independently as double determination and the degree of
hemolysis was determined from the following equation (Park Y et al.
A Leu-Lys-rich antimicrobial peptide: activity and mechanism.
Biochimica et Biophysica Acta-Proteins and Proteomics 1645: 172-82,
2003):
(E.sub.peptide-E.sub.pos)/(E.sub.Eciton-E.sub.PBS).times.100%
E=extinction at 405 nm
[0276] None of the antimicrobial proline-rich peptides analyted
showed hemolytic activity up to a concentration of 600 .mu.g/mL
(Table 12). This means, even at the 100-fold higher concentrations
than the MIC values determined, no lysis of human erythrocytes was
observed. The hemolysis rates of all the peptides were, relative to
Triton X-100.degree., only about 1%, which is within the error
limits of this test. The nonionic surfactant Triton X-100.RTM. was
used as positive control, as it completely disrupts red blood cells
in this test setup within one hour. Melittin, the honeybee venom,
has an .alpha.-helical peptide, a strongly lytic action on
biological membranes and disrupts both prokaryotic and eukaryotic
cell membranes even at concentrations of 5 .mu.g/mL. This cell test
shows that the peptides and peptide derivatives can be used in high
concentrations in the blood, without having side effects on human
erythrocytes.
TABLE-US-00014 TABLE 12 Degree of hemolysis of selected
antimicrobial peptides Degree of hemolysis (%) at SEQ ID NO. Name
600 .mu.g/mL 18 Onocin 1.5 87 Apidaecin 1b 1.2 89 Drosocin 1.1
Melittin 96.3 Triton X- 100 100 .RTM.
BRIEF DESCRIPTION OF THE DRAWINGS
[0277] FIG. 1 shows the antibacterial activity of the oncocin
analogs (Ala-Scan) against E. coli BL21 AI in agar diffusion
assays. Cross-hatched bars indicate partial inhibition.
[0278] FIG. 2 shows the antibacterial activity of oncocin analogs
(alanine scan) against Micrococcus luteus ATCC 10240 in agar
diffusion assays.
[0279] FIGS. 3A-3H show fluorescence micrographs of HeLa and
SH-SY5Y cells after incubation with 5, 6-carboxyfluorescein-labeled
oncocin (CF-oncocin SEQ ID NO. 94) and penetratin-oncocin
(CF-penetratin-oncocin SEQ ID NO. 102). Top row: phase contrast;
bottom row: fluorescence (517 nm emission). A, B: SH-SY5Y incubated
with CF-oncocin; C, D: SH-SY5Y with CF-penetratin-oncocin; E, F:
HeLa incubated with CF-oncocin; G, H: HeLa incubated with
CF-penetratin-oncocin. Bars correspond to 20 .mu.m.
[0280] FIGS. 4A-BH show confocal laser scanning micrographs of E.
coli BL21AI. Peptide concentration 30 .mu.mol/L; TAMRA
concentration 180 .mu.mol/L. Top row: phase contrast; bottom row:
fluorescence. A, B: 20 min incubation with CF-oncocin; C, D: 50 min
incubation with CF-oncocin; E, F: 50 min incubation with
CF-penetratin-oncocin; G, H: 90 min incubation with CF-penetratin
homodimer. Bars correspond to 5 .mu.m.
[0281] FIGS. 5A-5J show confocal laser scanning micrographs of M.
luteus 10240. Peptide concentration 30 .mu.ol/L; TAMRA
concentration 180 .mu.mol/L. Top row: phase contrast; bottom row:
fluorescence. A, G: CF-pyrrhocoricin; C, D: CF
penetratin-pyrrhocoricin; E, F: CF-drosocin; G, H:
CF--penetratin-drosocin; I, J: CF-penetratin homodimer. Bars
correspond to 5 .mu.m.
[0282] FIG. 6 shows the results of the cytotoxicity test for the
antimicrobial peptides against SH-SY5Y (cross-hatched column) and
HeLa cells (black column) determined with the "Cell Proliferation
Kit I", Test after 24 h incubation with 600 .mu.g/mL oncocin,
oncocin R150 R190, drosocin and apidaecin 1b (SEQ ID NO. 18, 72, 89
and 87) in medium. Positive controls 12% DMSO and 100 .mu.g/mL
melittin. Normalized to negative control 12% PBS.
[0283] FIG. 7 shows the results of the cytotoxicity test for the
penetratin constructs against HeLa cells determined with the "Cell
Proliferation Kit I". Test after 24 h incubation with 50-400
.mu.g/mL penetratin-drosocin (SEQ ID NO. 96), penetratin-apidaecin
1b (SEQ ID NO. 94), penetratin-pyrrhocoricin (SEQ ID NO. 98),
penetratin-oncocin (SEQ ID NO. 100), penetratin homodimer (SEQ ID
NO. 102), penetratin (SEQ ID NO. 105) in medium. Negative control
12% PBS and positive control 12% DMSO.
[0284] FIG. 8 shows the results of the cytotoxicity test for the
penetratin constructs against SH-SY5Y cells determined with the
"Cell Proliferation Kit I". Test after 24 h incubation with 50-400
.mu.g/mL penetratin-drosocin (SEQ ID NO. 96), penetratin-apidaecin
1b (SEQ ID NO. 94), penetratin-pyrrhocoricin (SEQ ID NO. 98),
penetratin-oncocin (SEQ ID NO. 100), penetratin homodimer (SEQ ID
NO. 102), penetratin (SEQ ID NO. 105), penetratin-tau (SEQ ID NO.
106) in medium. 12% PBS used as negative control and 12% DMSO as
positive control.
[0285] FIG. 9 shows the results of the hemolysis test for the
peptides oncocin, drosocin and apidaecin 1b (SEQ ID NO. 18, 89 and
87). Peptide dilution series 4.7-600 .mu.g/mL. Positive controls:
melittin and Triton X-100.RTM., negative control PBS.
[0286] FIG. 10: shows the results of the cytotoxicity test for the
antimicrobial peptides against HeLa cells determined with the "Cell
Proliferation Kit I". Test after 24 h incubation with 600 .mu.g/mL
oncocin and oncocin derivatives (SEQ ID NO. 18, 63, 72, 107 to
110), and the comparative examples apidaecin 1b and drosocin (SEQ
ID NO. 87 and 89) in medium. Positive controls 12% DMSO and 100
.mu.g/mL melittin. Normalized to negative control 12% PBS. The
diagram shows the mean value of two independent tests with
triplicates.
Sequence CWU 1
1
126118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Pro Pro
Pro Arg Arg Ile 1 5 10 15 Tyr Xaa 220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Pro Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Asn Arg 20 319PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Val Asp Lys Pro Pro Tyr Leu
Pro Arg Pro Xaa Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Arg
420PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Thr Pro
Pro Arg Arg Ile 1 5 10 15 Tyr Asn Asn Arg 20 520PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Lys Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Asn Arg 20 620PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 6Val Asp Lys Pro Pro Tyr Leu
Pro Arg Pro Lys Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Arg Asn 20
719PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Lys Pro
Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 820PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro His Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Asn Arg 20 920PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 9Val Asp Lys Pro Pro Tyr Leu
Pro Arg Pro His Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Arg Asn 20
1019PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Tyr Pro
Pro Arg Arg Ile 1 5 10 15 Tyr Asn Arg 1119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Asn Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Arg 1219PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 12Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Gln Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Arg 1319PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Phe Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Arg 1420PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 14Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Asn Arg 20
1520PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro
Pro Arg Arg Ile 1 5 10 15 Tyr Asn Asn Xaa 20 1620PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Arg Asn 20 1720PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 17Val Asp Lys Pro Pro Tyr Leu
Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Arg Xaa 20
1819PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro
Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 1919PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Arg 2019PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 20Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Pro Ile 1 5 10 15 Tyr Asn Arg 2119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 21Val
Asp Lys Pro Pro Tyr Leu Xaa Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Arg 2219PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 22Val Asp Lys Pro Pro Tyr Leu Xaa Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 2319PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Val
Asp Lys Xaa Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Arg 2419PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 24Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Xaa Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 2519PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Xaa Ile 1 5 10
15 Tyr Asn Xaa 2620PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 26Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa Xaa 20
2718PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro
Pro Arg Arg Ile 1 5 10 15 Tyr Xaa 2818PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 28Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn 2919PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 29Ala Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 3019PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 30Val
Ala Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 3119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 31Val Asp Ala Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 3219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Val
Asp Lys Ala Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 3319PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 33Val Asp Lys Pro Ala Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 3419PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Val
Asp Lys Pro Pro Ala Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 3519PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 35Val Asp Lys Pro Pro Tyr Ala Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 3619PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 36Val
Asp Lys Pro Pro Tyr Leu Ala Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 3719PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 37Val Asp Lys Pro Pro Tyr Leu Pro Ala
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 3819PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Val
Asp Lys Pro Pro Tyr Leu Pro Arg Ala Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 3919PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 39Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Ala Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 4019PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Ala Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 4119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 41Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Ala Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 4219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 42Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Ala Arg Ile 1 5 10
15 Tyr Asn Xaa 4319PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 43Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Ala Ile 1 5 10 15 Tyr Asn Xaa 4419PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 44Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ala 1 5 10
15 Tyr Asn Xaa 4519PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 45Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Ala Asn Xaa 4619PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 46Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Ala Xaa 4719PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 47Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 4818PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 48Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Pro Ile 1 5 10
15 Arg Val 4927PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 49Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Pro Gln Pro Arg Pro Pro
His Pro Arg Xaa 20 25 5019PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 50Val Asp Lys Pro Pro Tyr Leu
Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa
5119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro
Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 5219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 5319PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 53Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 5419PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 5519PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 55Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 5619PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 56Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 5719PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 57Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 5819PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 58Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 5919PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 59Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Arg Pro Arg Ile 1 5 10 15 Tyr Asn Xaa 6019PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 60Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Pro Ile 1 5 10
15 Tyr Asn Xaa 6119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 61Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Xaa Ile 1 5 10 15 Tyr Asn Xaa 6219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 62Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Xaa Ile 1 5 10
15 Tyr Asn Xaa 6319PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 63Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Xaa Ile 1 5 10 15 Tyr Asn Xaa 6419PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 64Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg His Ile 1 5 10
15 Tyr Asn Xaa 6519PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 65Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Xaa Ile 1 5 10 15 Tyr Asn Xaa 6619PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 66Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Xaa Ile 1 5 10
15 Tyr Asn Xaa 6719PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 67Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Xaa Ile 1 5 10 15 Tyr Asn Xaa 6819PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 68Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Xaa Ile 1 5 10
15 Tyr Asn Xaa 6919PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 69Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Xaa Ile 1 5 10 15 Tyr Asn Xaa 7019PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 70Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Xaa Ile 1 5 10
15 Tyr Asn Xaa 7119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 71Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Xaa Ile 1 5 10 15 Tyr Asn Xaa 7219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 72Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Xaa Ile 1 5 10
15 Tyr Asn Xaa 7319PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 73Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Xaa Leu 1 5 10 15 Tyr Asn Xaa 7419PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 74Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Xaa Ile 1 5 10
15 Tyr Gln Xaa 7519PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 75Val Glu Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 7619PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 76Val
Asp Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 7719PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 77Val Asp Lys Pro Pro Tyr Ile Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 7819PRTArtificial
SequenceDescription of Artificial Sequence
Synthetic peptide 78Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro
Pro Arg Arg Leu 1 5 10 15 Tyr Asn Xaa 7919PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 79Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Gln Xaa 8019PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 80Xaa Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Arg 8119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 81Xaa
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Arg 8219PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 82Xaa Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 8319PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 83Xaa
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 8419PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 84Xaa Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 8519PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 85Xaa
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Xaa Ile 1 5 10
15 Tyr Asn Xaa 8618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 86Asp Lys Pro Pro Tyr Leu Pro Arg Pro
Arg Pro Pro Arg Arg Ile Tyr 1 5 10 15 Asn Xaa 8718PRTApis mellifera
87Gly Asn Asn Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro His Pro 1
5 10 15 Arg Leu 8818PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 88Xaa Asn Asn Arg Pro Val Tyr Ile Pro
Gln Pro Arg Pro Pro His Pro 1 5 10 15 Arg Leu 8919PRTDrosophila
melanogaster 89Gly Lys Pro Arg Pro Tyr Ser Pro Arg Pro Thr Ser His
Pro Arg Pro 1 5 10 15 Ile Arg Val 9019PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 90Xaa
Lys Pro Arg Pro Tyr Ser Pro Arg Pro Thr Ser His Pro Arg Pro 1 5 10
15 Ile Arg Val 9120PRTPyrrhocoris
apterusMOD_RES(20)..(20)Asparagine amide 91Val Asp Lys Gly Ser Tyr
Leu Pro Arg Pro Thr Pro Pro Arg Pro Ile 1 5 10 15 Tyr Asn Arg Xaa
20 9220PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 92Xaa Asp Lys Gly Ser Tyr Leu Pro Arg Pro Thr Pro
Pro Arg Pro Ile 1 5 10 15 Tyr Asn Arg Xaa 20 9319PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 93Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 9419PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 94Xaa Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 9518PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 95Xaa
Asn Asn Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro Pro His Pro 1 5 10
15 Arg Leu 9618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 96Xaa Asn Asn Arg Pro Val Tyr Ile Pro
Gln Pro Arg Pro Pro His Pro 1 5 10 15 Arg Leu 9719PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 97Xaa
Lys Pro Arg Pro Tyr Ser Pro Arg Pro Thr Ser His Pro Arg Pro 1 5 10
15 Ile Arg Val 9819PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 98Xaa Lys Pro Arg Pro Tyr Ser Pro Arg
Pro Thr Ser His Pro Arg Pro 1 5 10 15 Ile Arg Val 9920PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 99Xaa
Asp Lys Gly Ser Tyr Leu Pro Arg Pro Thr Pro Pro Arg Pro Ile 1 5 10
15 Tyr Asn Arg Xaa 20 10020PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 100Xaa Asp Lys Gly Ser Tyr
Leu Pro Arg Pro Thr Pro Pro Arg Pro Ile 1 5 10 15 Tyr Asn Arg Xaa
20 10119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 101Xaa Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg
Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa 10219PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 102Xaa
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Arg Ile 1 5 10
15 Tyr Asn Xaa 10317PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 103Arg Gln Ile Lys Ile Trp Phe Gln Asn
Arg Arg Met Lys Trp Lys Lys 1 5 10 15 Xaa 10417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 104Xaa
Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10
15 Xaa 10516PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 105Arg Gln Ile Lys Ile Trp Phe Gln Asn
Arg Arg Met Lys Trp Lys Lys 1 5 10 15 10614PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 106Xaa
Xaa Ser Gly Asp Arg Ser Gly Tyr Ser Ser Arg Gly Ser 1 5 10
10719PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 107Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg
Pro Xaa Arg Xaa Ile 1 5 10 15 Tyr Asn Xaa 10819PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 108Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Xaa Arg Xaa Ile 1 5 10
15 Tyr Asn Xaa 10919PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 109Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Xaa Xaa 1 5 10 15 Tyr Asn Xaa
11019PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 110Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg
Pro Xaa Arg Xaa Xaa 1 5 10 15 Tyr Asn Xaa 11119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 111Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg Xaa Ile 1 5 10
15 Tyr Asn Xaa 11219PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 112Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Xaa Ile 1 5 10 15 Tyr Asn Xaa
11319PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 113Xaa Asn Tyr Ile Xaa Arg Pro Pro Arg Pro Arg
Pro Leu Tyr Pro Pro 1 5 10 15 Lys Asp Xaa 11419PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 114Xaa
Asn Tyr Ile Xaa Arg Pro Pro Arg Pro Arg Pro Leu Tyr Pro Pro 1 5 10
15 Lys Asp Xaa 11519PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 115Val Asp Lys Pro Pro Tyr Leu Pro Arg
Pro Arg Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Xaa
11619PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 116Val Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg
Pro Pro Arg Xaa Ile 1 5 10 15 Tyr Asn Xaa 11719PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 117Arg
Asn Tyr Ile Arg Arg Pro Pro Arg Pro Arg Pro Leu Tyr Pro Pro 1 5 10
15 Lys Asp Xaa 11819PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 118Xaa Asn Tyr Ile Xaa Arg Pro Pro Arg
Pro Arg Pro Leu Tyr Pro Pro 1 5 10 15 Lys Asp Xaa 11918PRTApis
mellifera 119Gly Asn Asn Arg Pro Val Tyr Ile Pro Gln Pro Arg Pro
Pro His Pro 1 5 10 15 Arg Ile 12016PRTMyrmecia gulosa 120Gly Arg
Pro Asn Pro Val Asn Asn Lys Pro Thr Pro Tyr Pro His Leu 1 5 10 15
12115PRTPalomena prasina 121Val Asp Lys Pro Asp Tyr Arg Pro Arg Pro
Arg Pro Pro Asn Met 1 5 10 15 12234PRTOncopeltus fasciatus 122Glu
Val Ser Leu Lys Gly Glu Gly Gly Ser Asn Lys Gly Phe Ile Gln 1 5 10
15 Gly Ser Gly Thr Lys Thr Leu Phe Gln Asp Asp Lys Thr Lys Leu Asp
20 25 30 Gly Thr 12320PRTOncopeltus fasciatusMOD_RES(11)..(11)Pro
or any other naturally occurring amino acid 123Val Asp Lys Pro Pro
Tyr Leu Pro Arg Pro Xaa Pro Pro Arg Arg Ile 1 5 10 15 Tyr Asn Asn
Arg 20 12418PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 124Lys Leu Ala Leu Lys Leu Ala Leu Lys
Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Xaa 1259PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 125Arg
Lys Lys Arg Arg Gln Arg Arg Arg 1 5 12614PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 126Val
Asp Lys Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Arg 1 5 10
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