U.S. patent application number 10/557455 was filed with the patent office on 2007-08-09 for novel antimicrobial peptides with heparin binding activity.
This patent application is currently assigned to DERMAGEN AB. Invention is credited to Martin Malmsten, Artur Schmidtchen.
Application Number | 20070185019 10/557455 |
Document ID | / |
Family ID | 20291321 |
Filed Date | 2007-08-09 |
United States Patent
Application |
20070185019 |
Kind Code |
A1 |
Schmidtchen; Artur ; et
al. |
August 9, 2007 |
Novel antimicrobial peptides with heparin binding activity
Abstract
The invention relates to an antimicrobial peptide with heparin
binding activity, being derived from endogenous mammalian proteins
being substantially free from antimicrobial activity selected from
the group consisting of laminin isoforms, complement factor C3,
histidin rich glycoprotein and kininogen and having from 10 to 36
amino acid residues, wherein the antimicrobial peptide consists of
at least four amino acid residues selected from the group
consisting of K, R and H. The invention also relates to
pharmaceutical compositions comprising said antimicrobial peptide
and use of the antimicrobial peptide and/or
antimicrobial/pharmaceutical composition.
Inventors: |
Schmidtchen; Artur; (Lund,
SE) ; Malmsten; Martin; (Taby, SE) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
DERMAGEN AB
c/o Milifa, Hallestad 591
Dalby
SE
SE-240 10
|
Family ID: |
20291321 |
Appl. No.: |
10/557455 |
Filed: |
May 19, 2004 |
PCT Filed: |
May 19, 2004 |
PCT NO: |
PCT/SE04/00797 |
371 Date: |
September 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60320204 |
May 19, 2003 |
|
|
|
Current U.S.
Class: |
514/2.6 ;
514/2.3; 514/2.7; 514/2.8; 514/3.4; 514/3.5; 514/4.6; 530/350 |
Current CPC
Class: |
A61K 38/57 20130101;
C07K 14/4723 20130101; Y02A 50/473 20180101; A61P 31/00 20180101;
A61P 31/10 20180101; A61P 31/04 20180101; A61P 33/14 20180101; A61K
38/1709 20130101; A61P 31/12 20180101; A61K 38/39 20130101; Y02A
50/30 20180101; C07K 14/8128 20130101 |
Class at
Publication: |
514/012 ;
530/350 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 14/195 20060101 C07K014/195 |
Foreign Application Data
Date |
Code |
Application Number |
May 19, 2003 |
SE |
0301431-3 |
Claims
1.-49. (canceled)
50. An antimicrobial peptide with heparin binding activity, the
peptide comprising a 10 to 36 amino acid residue portion of the
amino acid sequence KHNLGHGHKH ERDQGHGHQR GHGLGHGHEQ QHGLGHGHKF
KLDDDLEHQG GHVLDHGHKHKHGHGHGKH KNKGKKNGKH NGWK or a variant
thereof, wherein at least 30% of the residues of the 10 to 36 amino
acid residue portion are selected from the group consisting of K, R
and H.
51. The peptide of claim 50, wherein at least 40% of the residues
of the 10 to 36 amino acid residue portion are selected from the
group consisting of K, R and H.
52. The peptide of claim 50, wherein at least 50% of the residues
of the 10 to 36 amino acid residue portion are selected from the
group consisting of K, R and H.
53. The peptide of claim 50, wherein the 10 to 36 amino acid
residue portion comprises at least 20% H amino acid residues.
54. The peptide of claim 50, wherein the peptide consists of 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acid residues.
55. The peptide of claim 50, wherein the 10 to 36 amino acid
residue portion is selected from the group consisting of SEQ ID NO
1, 2 and 3.
56. The peptide of claim 50, wherein the 10 to 36 amino acid
residue portion is endogenous, synthetic, or semisynthetic.
57. The peptide of claim 50, wherein the peptide is linked to one
or more other antimicrobial peptide(s) or other substances.
58. The peptide of claim 50, wherein the peptide is extended by
1-100 amino acid residues at either the N- or C-terminal end, or at
both ends.
59. The peptide of claim 58, wherein the peptide is extended by
5-50 amino acid residues.
60. The peptide of claim 59, wherein the peptide is extended by 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, or 30 amino acid residues.
61. The peptide of claim 50, wherein the 10 to 36 amino acid
residue portion is modified by substitution of one to six amino
acids.
62. The peptide of claim 50, wherein the peptide(s) are modified by
amidation, esterification, acylation, acetylation, PEGylation or
alkylation.
63. A composition comprising the peptide according to claim 50, the
composition comprising a pharmaceutically acceptable buffer,
diluent, carrier, adjuvant or excipient.
64. The composition of claim 63, wherein the composition comprises
a salt.
65. The composition of claim 64, wherein the salt is selected from
the group consisting of monovalent sodium, potassium or divalent
zinc, magnesium, copper and calcium.
66. The composition of claim 65, wherein the cation in the salt is
divalent zinc.
67. The composition of claim 63, wherein the composition has a pH
from about 5.0 to about 7.0.
68. The peptide of claim 50, wherein the peptide is in the form of
a composition comprising a mixture of between 1, 2, 3 or 4
different polypeptides.
69. The composition of claim 63, additionally comprising one or
more antibiotic and/or antiseptic agent(s).
70. The composition of claim 50, in the form of granules, powders,
tablets, coated tablets, capsules, suppositories, syrups,
emulsions, gels, ointments, suspensions, creams, aerosols, droplets
or injectable forms.
71. A method of preventing, inhibiting, reducing, or destroying
microorganisms selected from the group consisting of bacteria,
viruses, parasites, fungus, and yeast, the method comprising
administering to a patient the peptide of claim 50.
72. The method of claim 71, wherein the patient has a microbial
infection.
73. The method of claim 72, wherein the microbial infection is
caused by a microorganism selected from the group consisting of
Enterococcus faecalis, Eschericia coli, Pseudomonas aeruginosa,
Proteus mirabilis, Streptococcus pneumoniae, Streptococcus pyogenes
and Staphylococcus aureus.
74. The method of claim 72, wherein the microbial infection is
caused by a microorganism selected from the group consisting of
Candida albicans of Candida parapsilosis.
75. The method of claim 72, wherein the patient is a mammal.
76. The method of claim 71, wherein the peptide is administered in
the form of a composition comprising a plaster, wound dressing,
suture, or adhesive.
77. An antimicrobial peptide with heparin binding activity, the
peptide comprising a 10 to 36 amino acid residue portion of the
amino acid sequence SVQLTEKRM DKVGKYPKEL RKCCEDGMRE NPMRFSCQRR
TRFISLGEAC KKVFLDCCNY ITELRRQHAR ASHLGLAR or a variant there of,
wherein the 10 to 36 amino acid residue portion comprises at least
four amino acids selected from the group consisting of K, R and H.
Description
FIELD OF INVENTION
[0001] The invention relates to antimicrobial peptides with heparin
binding activity, being derived from endogenous mammalian proteins
being substantially free from antimicrobial activity selected from
the group consisting of laminin isoforms, complement factor C3,
histidin rich glycoprotein and kininogen and having from 10 to 36
amino acid residues, wherein the antimicrobial peptides consists of
at least four amino acid residues selected from the group
consisting of K, R and H. The invention also relates to
pharmaceutical compositions comprising said antimicrobial peptides
and use of the antimicrobial peptides and/or
antimicrobial/pharmaceutical compositions.
BACKGROUND OF INVENTION
[0002] Several infections are successfully combated by the immune
system of a mammal such as a human being. However, in some
instances, bacteria, fungi, or viruses are not always cleared,
which may cause localised or generalised acute infections. This is
a serious concern at perinatal-, burn, or intensive care units, and
in immunocompromised individuals. In other cases, a continuous
bacterial persistence at epithelial surfaces may cause or aggravate
chronic disease. In humans, this is exemplified by chronic skin
ulcers, atopic dermatitis and other types of eczema, acne, or
genitourinary infections.
[0003] Symptomatic infections may be treated by various
medicaments. Some diseases may also be combated by for instance
vaccines. However, vaccines are not always the best treatment
option and for certain microorganisms no vaccine is available. When
no protection is available treatment of the disease is pursued.
Often the treatment is performed by the use of an antibiotic agent,
which kills the microbe. However, during the last years several
microbes have become resistant against antibiotic agents. Most
likely, resistance problems will increase in the near future.
Additionally, several individuals have developed allergy against
the antibiotic agent, thereby reducing the possibility to
effectively use certain antibiotic agents.
[0004] Epithelial surfaces of various organisms are continuously
exposed to bacteria. During recent years the innate immune system,
based on antibacterial peptides has been attributed important roles
in the initial clearance of bacteria at biological boundaries
susceptible to infection (Lehrer, R. I., and Ganz, T. (1999) Curr
Opin Immunol 11: 23-27, Boman, H. G. (2000) Immunol. Rev. 173,
5-16). Antimicrobial peptides kill bacteria by permeating their
membranes, and thus the lack of a specific molecular microbial
target minimizes resistance development.
[0005] Several antimicrobial peptides and proteins, unrelated to
the herein described peptides are known in the art.
[0006] U.S. Pat. No. 6,503,881 disclose cationic peptides being an
indolicidin analogue to be used as an antimicrobial peptide. The
cationic peptides being derived from different species, including
animals and plants.
[0007] U.S. Pat. No. 5,912,230 disclose anti-fungal and
anti-bacterial histatin-based peptides. The peptides being based on
defined portions of the amino acid sequences of naturally occurring
human histatins and methods for treatment of fungal and bacterial
infections.
[0008] U.S. Pat. No. 5,717,064 disclose methylated lysine-rich
lytic peptides. The lytic peptides being tryptic digestion
resistant and non-natural. The lytic peptides are suitable for in
vivo administration.
[0009] U.S. Pat. No. 5,646,014 disclose an antimicrobial peptide.
The peptide was isolated from an antimicrobial fraction from
silkworm hemolymph. The peptide exhibits excellent antimicrobial
activity against several bacterial strains, such as Escherichia
coli, Staphylococcus aureus and Bacillus cereus.
[0010] McCabe et al., J. Biol. Chem. Vol 277:27477-27488, 2002,
describes an 37 kDa antimicrobial and chemotactic protein,
azurocidin, containing the heparin binding consensus motifs XBBXBX
and XBBBXXBX.
[0011] WO2004016653 disclose a peptide based on the 20-44 sequence
of azurocidin. This peptide contains a loop structure linked by
disulfide bridges.
[0012] U.S. Pat. No. 6,495,516 and related patents, disclose
peptides based on the bactericidal 55 kDa protein
bactericidal/permeability increasing protein (BPI). The peptides
exerted antimicrobial effects as well as had heparin and
LPS-neutralizing capacity.
[0013] WO 01/81578 discloses numerous sequences encoding G-coupled
protein-receptor related polypeptides, which may be used for
numerous diseases.
[0014] WO 00/27415 discloses peptides being suitable for inhibition
of angiogenesis. The peptides being analogous of high molecular
weight kininogen 5. The BLASTp search shows sequences, which are
conserved or have similarities among different species such as
kininogen without any indication of the function of such conserved
regions or if they at all have any function as small peptides.
[0015] At present, over 700 different antimicrobial peptide
sequences are known
(www.bbcm.univ.trieste.it/.about.tossi/search.htm), including
cecropins, defensins magainins and cathelicidins.
[0016] Even though there is a huge amount of antimicrobial peptides
available today there is still an increased need of new improved
antimicrobial peptides. Antimicrobial peptides which can be used to
combat microbes and being resistant or tolerant against antibiotic
agents and/or other antimicrobial agents. Additionally, there is a
need for new antimicrobial peptides, which are non-allergenic when
introduced into mammals such as human beings. Bacteria have
encountered endogenously produced antimicrobial peptides during
evolution without induction of significant resistance.
SUMMARY OF THE INVENTION
[0017] According to a first aspect, the invention relates to
antimicrobial peptides with heparin binding activity, being derived
from endogenous mammalian proteins being substantially free from
antimicrobial activity selected from the group consisting of
laminin isoforms, complement factor C3, histidin rich glycoprotein
and kininogen and having from 10 to 36 amino acid residues, wherein
the antimicrobial peptides consists of at least four amino acid
residues, selected from the group consisting of K, R and H.
[0018] By providing such antimicrobial peptides, the risks for
allergenic reactions to antimicrobial peptides may be reduced due
to the fact that the peptides are derived from endogenous proteins
and/or peptides. By using short peptides the stability of the
peptide may be increased and the production costs reduced, as
compared to longer peptides and proteins, whereby the invention may
be economically advantageous. The invention originates from the
finding that peptides with heparin-binding motifs derived from
non-antimicrobial endogenous proteins exhibit antimicrobial
activities, as described by Andersson et al., Eur J Biochem, 2004,
271:1219-1226, published after the priority date of the present
application. The structural prerequisite for heparin-binding and
the presence of heparin-binding motifs in various proteins, is
generally well documented. This group of molecules includes various
laminin isoforms, fibronectin, coagulation factors, growth factors,
chemokines, histidine-rich glycoprotein, kininogen and many others
(see Andersson et al., (2004) Eur J Biochem 271; 271:1219-26 and
references therein), none of them being inherently
anti-microbial.
[0019] The antimicrobial peptides and the corresponding
antimicrobial/pharmaceutical compositions according to the
invention provide peptides and compositions, which facilitate
efficient prevention, reduction or elimination of microorganisms.
Thereby the possibility to combat microorganisms, which are
resistant or tolerant against the antibiotic agents, may be
increased. Moreover, mammals, which are allergenic against
commercially available antimicrobial agents, may be treated. By
providing antimicrobial/pharmaceutical compositions, which are
derived from endogenous proteins, the probability may be reduced or
even eliminated that a mammal will develop allergy against these
particular peptides. This makes the antimicrobial/pharmaceutical
compositions useful for several applications in which the
antimicrobial/pharmaceutical compositions contact a mammal either
as a medicament or as an additive to prevent infections.
[0020] Additionally, the use of short peptides improves
bioavailibility. Furthermore, the use of structurally distinct
heparin-binding antimicrobial peptides with specific or preferable
actions on Gram-negative and Gram-positive bacteria, or fungi,
enables specific targeting of various microorganisms, thus
minimising development of resistance and ecological problems. By
supplementing peptides that already exist in the mammal, the risk
of additional ecological pressure by novel antibiotics is further
diminished. Finally, these formulations may also enhance the effect
of endogenous antimicrobial peptides.
[0021] According to a second aspect, the invention relates to
antimicrobial/pharmaceutical compositions comprising one or more
antimicrobial peptides as defined above and an pharmaceutical
acceptable buffer, diluent, carrier, adjuvant or excipient.
[0022] According to a third aspect, the invention relates to the
use of the antimicrobial peptides and/or the
antimicrobial/pharmaceutical compositions as defined herein
after.
[0023] The inventive antimicrobial peptides increase the list of
antimicrobial agents, which aid in the choice to prevent, reduce or
eliminate microorganisms in all kind of applications including but
not limited to those that invade or infect mammals such as the
human being.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 A-C are diagrams demonstrating the antibacterial
effects of peptides on Enterococcus faecalis.
[0025] FIG. 2 A and B are petri dishes illustrating radial
diffusion assays using a set of highly active peptides.
[0026] FIG. 3A-C are diagrams and a table describing antibacterial
effects of histidine-rich peptides.
[0027] FIG. 4 A-H are electron microscopy pictures showing the
analysis of Pseudomonas aeruginosa subjected to antimicrobial
peptides.
[0028] FIG. 5 A-C are photographs showing the heparin binding
activity of peptides derived from complement C3, histidine-rich
glycoprotein and kininogen.
[0029] FIG. 6 is a photograph illustrating purification of
histidine-containing antimicrobial fragment on
nickel-sepharose.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0030] In the context of the present application and invention the
following definitions apply:
[0031] The term "nucleotide sequence" is intended to mean a
sequence of two or more nucleotides. The nucleotides may be of
genomic DNA, cDNA, RNA, semisynthetic or synthetic origin or a
mixture thereof. The term includes single and double stranded forms
of DNA or RNA.
[0032] The term "antimicrobial peptide" is intended to mean a
peptide, which comprises from about 10 to about 36 amino acid
residues, has anti-microbial and heparin binding activity and is
derived from an endogenous mammalian which inherently has no
antimicrobial effect. The "antimicrobial peptide" prevents,
inhibits, reduces or destroys a microorganism. The antimicrobial
activity can be determined by for example the method in EXAMPLE 2,
4 or 5.
[0033] The term "heparin binding affinity" is intended to mean a
peptide, which binds to a heparin either directly or indirectly.
The heparin binding activity can be determined by for example the
method in EXAMPLE 7. The invented antimicrobial peptides, which
exhibit affinity for heparin, also bind dermatan sulfate. Hence,
heparin binding antimicrobial peptides, also interact with the
endogenous glycosaminoglycan dermatan sulfate.
[0034] The term "amphipathic" is intended to mean the distribution
of hydrophilic and hydrophobic amino acid residues along opposing
faces of an .alpha.-helix structure, .beta.-strand, linear,
circular, or other secondary conformation, which result in one face
of the molecule being predominantly charged and the other face
being predominantly hydrophilic. The degree of amphipathicity of a
peptide can be assessed by plotting the sequence of amino acid
residues by various web-based algorithms, eg. those found on
http://us.expasy.org/cgi-bin/protscale.pl. The distribution of
hydrophobic residues can be visualized by helical wheel diagrams.
Secondary structure prediction algorithms, such as GORIV can be
found at www.expasy.com.
[0035] The term "cationic" is intended to mean a molecule, which
has a net positive charge within the pH range of from about 4 to
about 12.
[0036] The term "microorganism" is intended to mean any living
microorganism. Examples of microorganisms are bacteria, fungus,
virus, parasites and yeasts.
[0037] The term "antimicrobial agent" is intended to mean any
agent, which prevent, inhibit or destroy life of microbes. Examples
of antimicrobial agents can be found in The Sanford Guide to
Antimicrobial Therapy (32nd edition, Antimicrobial Therapy, Inc,
US).
[0038] In the present context, amino acid names and atom names are
used as defined by the Protein DataBank (PNB) (www.pdb.org), which
is based on the IUPAC nomenclature (IUPAC Nomenclature and
Symbolism for Amino Acids and Peptides (residue names, atom names
etc.), Eur J Biochem., 138, 9-37 (1984) together with their
corrections in Eur J Biochem., 152, 1 (1985). The term "amino acid"
is intended to indicate an amino acid from the group consisting of
alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D),
glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or
G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K),
leucine (Leu or L), methionine (Met or M), asparagine (Asn or N),
proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R),
serine (Ser or S), threonine (Thr or T), valine (Val or V),
tryptophan (Trp or W) and tyrosine (Tyr or Y), or derivatives
thereof.
DESCRIPTION
Antimicrobial Peptide
[0039] The present invention relates to antimicrobial peptides with
heparin binding activity, being derived from endogenous mammalian
proteins being substantially free from antimicrobial activity and
having from 10 to 36 amino acid residues, wherein the antimicrobial
peptides consist of at least four amino acid residues selected from
the group consisting of K, R and H. Two of the amino acid residues
may be adjecent. A distance of .about.20 .ANG. between the B amino
acid residues constitutes a prerequisite for heparin binding
irrespective of peptide conformation as reported by Margalit et
al., 1993 J Biol Chem 268, 19228-31. The use of short peptides
increase bioavailibility of shorter peptides as compared to longer
peptides or proteins, e.g., through an increased skin the
penetration capacity as well as reduces the production and
purification costs. The present antimicrobial peptides are
complements to those antimicrobial peptides, which are commercially
available today and increases the possibility to combat
microorganisms, being tolerant and/or resistant against available
antimicrobial agents. By deriving the new antimicrobial peptides
from endogenous non-antimicrobial proteins it is possible to
identify new peptides which are non-allergenic for the mammal from
which the peptide has been based.
[0040] Furthermore, increased knowledge of peptide action and
dependence of various salts and ionic environments enables design
of specific compositions, which enhance and control peptide
effects. Peptides scissored for actions on fungi will further be
advantageous in targeting specific diseases, such as yeast
infections on mucous membranes without significantly affecting
bacterial ecology at these sites. The fact that antimicrobial
peptides, act on bacterial membranes suggest that they may act
synergistically together with antibiotics. Therefore, combination
of antibiotics and peptides may have therapeutical advantages.
Finally, there is also a need of antimicrobial agents, which are
low cost and non-allergenic to be used in different kinds of
products in which it is necessary to prevent growth of
microorganisms.
[0041] Additionally, the use of structurally distinct
heparin-binding short antimicrobial peptides with specific or
preferable actions on Gram-negative and Gram-positive bacteria, or
fungi enables specific targeting of various microorganisms, thus
minimising resistance and ecological problems. By supplementing
peptides that already occur in the organism, the risk of additional
ecological pressure by novel antibiotics is further diminished. The
introduction of specific formulations that enhance peptide effects
localise and enhance exogenously supplied peptides which further
minimises the risk of side effects of peptides, such as induction
of resistance, outside the treated area. Finally, these
formulations may also enhance the effect of endogenous
antimicrobial peptides. If the antimicrobial peptides, are
developed to be used to combat microorganisms in humans, the
endogenous antimicrobial peptides are derived from human endogenous
proteins. The same applies for other animals, such as horses, cows,
pigs, or poultry. The antimicrobial peptides may be based on the
structure of a peptide and/or protein present in plasma, blood,
connective tissue and constituent cells and may be selected from
the group consisting of heparin binding proteins; laminin isoforms,
von Willebrand factor, vitronectin, protein C inhibitor,
fibronectin, coagulation factors, growth factors, chemokines,
histidin rich glycoprotein, kininogen, or complement factor C3.
[0042] The antimicrobial peptides of the invention have, a binding
affinity (Kd) to heparin of about 10 nM to about 20 .mu.M.
[0043] The peptides may have a size of 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35 or 36 amino acid residues. The length and sequence of the
peptides is dependent on the origin of the antimicrobial peptides
and which microorganism to combat, if the peptides are to be used
to prevent, inhibit, reduce or destroy the microorganism and what
kind of environment the microorganism is present in and what kind
of environment the antimicrobial peptide will encounter after
administration.
[0044] According to a first embodiment the invention relates to
antimicrobial peptides being based on kininogen proteins or
histidin rich glycoprotein, wherein at least 20% of the amino acid
residues are H. The antimicrobial peptides may comprise more than
30, 40 or even 50% H, R and/or K amino acid residues. In specific
examples 1, 2, 3, 4, 5 or 6 amino acid residues are H. For example
the antimicrobial peptide may be selected from the group consisting
of SEQ ID NO:1, 2, 3 and 4. These peptides are derived from
heparin-binding domains of the non-antimicrobial proteins kininogen
and histidine-rich glycoprotein, respectively and are rich in H
residues.
[0045] According to another embodiment the invention relates to
antimicrobial peptides being based on complement factor proteins.
For example the antimicrobial peptide may be selected from the
group consisting of SEQ ID NO: 5, 6 and 7. SEQ ID NO: 5, 6 and 7
peptides are derived from well-defined helical segments of the
complement factor C3 molecule. As has been shown by Hugli and
co-workers (Chazin et al., (1988) Biochemistry 27, 9139-48, Hugli,
Current topics in Microbiology and Immunology, 1989, 153, 181-208)
the helical regions of the C3-derived C3a molecule are defined by
segments 19-28 (represented by SEQ ID NO: 5) and 47-70 (represented
by SEQ ID NO: 6 and 7). The holoprotein C3 exerts no antimicrobial
effects. The heparin binding and antimicrobial capacity of peptide
segments derived from C3 has been disclosed recently (Andersson et
al., Eur J Biochem, 2004, 271; 271:1219-1226).
[0046] According to a third embodiment the invention relates to
antimicrobial peptides derived from the group of laminin proteins.
For example the antimicrobial peptide may be selected from the
group consisting of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 and 16.
Laminin .alpha.-chain LG-domains are composed of five (1-5)
LG-modules that have been identified as binding sites for heparin
and other cell-surface receptors (Timpl., et al., Matrix Biol,
2000, 19, 309-317). These modular proteins are synthesised during
developmental processes such as wound healing and it has been
described that proteolytic processing of LG-modules occur during
these events. A previously undisclosed antimicrobial function of
heparin-binding epitopes of LG-modules was described recently
(Andersson et al., Eur J Biochem, 2004, 271; 271:1219-1226)
[0047] Even though the peptides are derived from endogenous
proteins they may be produced as semisynthetic or even synthetic
peptides as well as in microorganisms.
[0048] The antimicrobial peptides may be extended by one or more
amino acid residues, such as 1-100 amino acid residues, 5-50 amino
acid residues or 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 amino acid
residues. Such additional amino acids may duplicate a sequence
contiguous to the sequence of the antimicrobial peptide derived
from a non-antimicrobial protein. The number to be added depends on
which microorganism to be combated in including, stability of the
peptide, toxicity, the mammal to be treated or in which product the
peptide should be in and which peptide structure the antimicrobial
peptide is based upon. The number of amino acid residues to be
added to the peptides depends also on the choice of production,
e.g., expression vector and expression hosts and the choice of
manufacturing the antimicrobial/pharmaceutical composition. The
extension may be at the N- or C-terminal part or at both parts of
the antimicrobial peptides as long as it does not disrupt the
antimicrobial effect of the peptide. The antimicrobial peptides may
also be a fusion protein, wherein the antimicrobial peptide is
fused to another peptide.
[0049] Additionally the antimicrobial peptides may be operably
linked to other known antimicrobial peptides or other substances,
such other peptides, proteins, oligosaccharides, polysaccharides,
other organic compounds, or inorganic substances. For example the
antimicrobial peptides may be coupled to a substance which protect
the antimicrobial peptides from being degraded within a mammal
prior to the antimicrobial peptides has inhibited, prevented or
destroyed the life of the microorganism.
[0050] Accordingly the antimicrobial peptides may be modified at
the C-terminal part by amidation or esterification and at the
N-terminal part by acylation, acetylation, PEGylation, alkylation
and the like.
[0051] Alternatively, peptides derived from functional
antimicrobial segments of non-antimicrobial holo-proteins may be
modified by substitution of one to six amino acids.
[0052] Examples of microorganism that are inhibited, prevented or
destroyed by the antimicrobial peptide are bacteria, both Gram
positive and Gram-negative bacteria such as Enterococcus faecalis,
Eschericia coli Pseudomonas aeruginosa, Proteus mirabilis,
Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus
aureus, viruses, parasites, fungus and yeast, such Candida albicans
and Candida parapsilosis.
[0053] The antimicrobial peptides can be obtained from a naturally
occurring source, such as from a human cell, a c-DNA, genomic
clone, chemically synthesized or obtained by recombinant DNA
techniques as expression products from cellular sources.
[0054] The antimicrobial peptides may be synthesized by standard
chemical methods, including synthesis by automated procedure. In
general, peptide analogues are synthesized based on the standard
solid-phase Fmoc protection strategy with HATU
(N-[DIMETHYLAMINO-1H-1.2.3.-TRLAZOLO[4,5-B]PYRIDIN-1-YLMETHYLELE]-N-METHY-
LMETHANAMINIUM HEXAFLUOROPHOSPHATE N-OXIDE) as the coupling agent
or other coupling agents such as HOAt-1-HYDROXY-7-AZABENZOTRIAZOLE.
The peptide is cleaved from the solid-phase resin with
trifluoroacetic acid containing appropriate scavengers, which also
deprotects side chain functional groups. Crude peptide is further
purified using preparative reversed-phase chromatography. Other
purification methods, such as partition chromatography, gel
filtration, gel electrophoresis, or ion-exchange chromatography may
be used. Other synthesis techniques, known in the art, such as the
tBoc protection strategy, or use of different coupling reagents or
the like can be employed to produce equivalent peptides.
[0055] Peptides may alternatively be synthesized by recombinant
production (see e.g., U.S. Pat. No. 5,593,866). A variety of host
systems are suitable for production of the peptide analogues,
including bacteria, such as E. coli, yeast, such as Saccharomyces
cerevisiae or pichia, insects, such as Sf9, and mammalian cells,
such as CHO or COS-7. There are many expression vectors available
to be used for each of the hosts and the invention is not limited
to any of them as long as the vector and host is able to produce
the antimicrobial peptide. Vectors and procedures for cloning and
expression in E. coli can be found in for example Sambrook et al.
(Molecular Cloning.: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1987) and Ausubel et
al. (Current Protocols in Molecular Biology, Greene Publishing Co.,
1995).
[0056] Finally, the peptides may be purified from plasma, blood,
various tissues or the like. The peptides may be endogenous, or
generated after enzymatic or chemical digestion of the purified
protein. For example, a heparin binding protein may be digested by
trypsin and the resulting antibacterial peptides further isolated
in larger scale.
[0057] A DNA sequence encoding the antimicrobial peptide is
introduced into a suitable expression vector appropriate for the
host. In preferred embodiments, the gene is cloned into a vector to
create a fusion protein. To facilitate isolation of the peptide
sequence, amino acids susceptible to chemical cleavage (e.g., CNBr)
or enzymatic cleavage (e.g., V8 protease, trypsin) are used to
bridge the peptide and fusion partner. For expression in E. coli,
the fusion partner is preferably a normal intracellular protein
that directs expression toward inclusion body formation. In such a
case, following cleavage to release the final product, there is no
requirement for renaturation of the peptide. In the present
invention, the DNA cassette, comprising fusion partner and peptide
gene, may be inserted into an expression vector. Preferably, the
expression vector is a plasmid that contains an inducible or
constitutive promoter to facilitate the efficient transcription of
the inserted DNA sequence in the host.
[0058] The expression vector can be introduced into the host by
conventional transformation techniques such as by calcium-mediated
techniques, electroporation, or other methods well known to those
skilled in the art.
[0059] The sequence encoding the antimicrobial peptide may be
derived from a natural source such as a mammalian cell, an existing
cDNA or genomic clone or synthesized. One method, which may be
used, is amplification of the antimicrobial peptide by the aid of
PCR using amplification primers which are derived from the 5' and
3' ends of the antimicrobial DNA template and typically incorporate
restriction sites chosen with regard to the cloning site of the
vector. If necessary, translational initiation and termination
codons can be engineered into the primer sequences. The sequence
encoding the antimicrobial peptide may be codon-optimized for
facilitate expression in the particular host as long as the choice
of the codons are made considering the final mammal to be treated.
Thus, for example, if the antimicrobial peptide is expressed in
bacteria, the codons are optimized for bacteria.
[0060] The expression vector should contain a promoter sequence, to
facilitate expression of the introduced antimicrobial peptide. If
necessary, regulatory sequences may also be included, such as one
or more enhancers, ribosome binding site, transcription termination
signal sequence, secretion signal sequence, origin of replication,
selectable marker, and the like. The regulatory sequences are
operably linked to each other to allow transcription and subsequent
translation. If the antimicrobial peptide is to be expressed in
bacteria, the regulatory sequences are those which are designed to
be used within bacteria and such are well-known for a person
skilled in the art. Suitable promoters, such as constitutive and
inducible promoters, are widely available and includes promoters
from T5, T7, T3, SP6 phages, and the trp, lpp, and lac operons.
[0061] If the vector containing the antimicrobial peptide is to be
expressed within bacteria examples of origin are either those which
give rise to a high copy number or those which give rise to a low
copy, for example f1-ori and col E1 ori.
[0062] Preferably, the plasmids include at least one selectable
marker that is functional in the host, which allows transformed
cells to be identified and/or selectively grown. Suitable
selectable marker genes for bacterial hosts include the ampicillin
resistance gene, chloroamphenicol resistance gene, tetracycline
resistance gene, kanamycin resistance gene and others known in the
art.
[0063] Examples of plasmids for expression in bacteria include the
pET expression vectors pET3a, pET 11a, pET 12a-c, and pET 15b
(available from Novagen, Madison, Wis.). Low copy number vectors
(e.g., pPD100) can be used for efficient over-production of
peptides deleterious to the E. coli host (Dersch et al., FEMS
Microbiol. Lett. 123:19, 1994).
[0064] Examples of suitable hosts are bacteria, yeast, insects and
mammal cells. However, often either bacteria such as E. coli is
used.
[0065] The expressed antimicrobial peptide is isolated by
conventional isolation techniques such as affinity, size exclusion,
or ionic exchange chromatography, HPLC and the like. Different
purification techniques can be found in A Biologist's Guide to
Principles and Techniques of Practical Biochemistry (eds. Wilson
and Golding, Edward Arnold, London, or in Current Protocols in
Molecular Biology (John Wiley & Sons, Inc).
Antimicrobial/Pharmaceutical Composition
[0066] Additionally the invention relates to
antimicrobial/pharmaceutical compositions comprising an
antimicrobial peptide as described above and a pharmaceutical
acceptable buffer, diluent, carrier, adjuvant or excipient.
Additional compounds may be included in the compositions. These
include, for example, chelating agents such as EDTA, EGTA or
glutathione. The antimicrobial/pharmaceutical compositions may be
prepared in a manner known in the art that is sufficiently storage
stable and suitable for administration to humans and animals. The
pharmaceutical compositions may be lyophilised e.g., through freeze
drying, spray drying or spray cooling.
[0067] "Pharmaceutically acceptable" means a non-toxic material
that does not interfere with the effectiveness of the biological
activity of the active ingredients, i.e., the antimicrobial
peptide(s). Such pharmaceutically acceptable buffers, carriers or
excipients are well-known in the art (see Remington's
Pharmaceutical Sciences, 18th edition, A. R Gennaro, Ed., Mack
Publishing Company (1990) and handbook of Pharmaceutical
Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press
(2000).
[0068] The term "buffer" is intended to mean an aqueous solution
containing an acid-base mixture with the purpose of stabilising pH.
Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS,
Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate,
glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO,
BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine,
HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO,
TAPS, TABS, TAPSO, TES, tricine.
[0069] The term "diluent" is intended to mean an aqueous or
non-aqueous solution with the purpose of diluting the peptide in
the pharmaceutical preparation. The diluent may be one or more of
saline, water, polyethylene glycol, propylene glycol, ethanol or
oils (such as safflower oil, corn oil, peanut oil, cottonseed oil
or sesame oil).
[0070] The term "adjuvant" is intended to mean any compound added
to the formulation to increase the biological effect of the
peptide. The adjuvant may be one or more of zinc, copper or silver
salts with different anions, for example, but not limited to
fluoride, chloride, bromide, iodide, tiocyanate, sulfite,
hydroxide, phosphate, carbonate, lactate, glycholate, citrate,
borate, tartrate, and acetates of different acyl composition.
[0071] The excipient may be one or more of carbohydrates, polymers,
lipids and minerals. Examples of carbohydrates include lactose,
sucrose, mannitol, and cyclodextrines, which are added to the
composition, e.g., for facilitating lyophilization. Examples of
polymers are starch, cellulose ethers, cellulose
carboxymethylcellulose, alginates, carrageenans, hyaluronic acid,
polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene
oxide, polyvinylalcohol/polyvinylacetate of different degree of
hydrolysis, and polyvinylpyrrolidone, all of different molecular
weight, which are added to the composition, e.g., for viscosity
control, for achieving bioadhesion, or for protecting the lipid
from chemical and proteolytic degradation. Examples of lipids are
fatty acids, phospholipids, mono-, di-, and triglycerides,
ceramides, sphingolipids and glycolipids, all of different acyl
chain length and saturation, egg lecithin, soy lecithin,
hydrogenated egg and soy lecithin, which are added to the
composition for reasons similar to those for polymers. Examples of
minerals are talc, magnesium oxide, zinc oxide and titanium oxide,
which are added to the composition to obtain benefits such as
reduction of liquid accumulation or advantageous pigment
properties.
[0072] The characteristics of the carrier are dependent on the
route of administration. One route of administration is topical
administration. For example, for topical administrations, a
preferred carrier is an emulsified cream comprising the active
peptide, but other common carriers such as certain
petrolatum/mineral-based and vegetable-based ointments can be used,
as well as polymer gels, liquid crystalline phases and
microemulsions.
[0073] The antimicrobial/pharmaceutical compositions may comprise
one or more peptides, such as 1, 2, 3 or 4 different peptides in
the antimicrobial/pharmaceutical compositions. By using a
combination of different peptides the antimicrobial effect may be
increased as well as decrease of the possibility that the
microorganism to combat might be resistant and/or tolerant against
the antimicrobial agent.
[0074] Histidin rich and/or kininogen based peptides, particularly
as short peptides have limited antimicrobial activity. However if
these peptides are in a composition comprising a salt and/or a pH
from about 5.0 to about 7.0, the peptides become active , i.e., an
enhanced effect is obtained by the addition of a salt and/or a
choice of a specific pH range.
[0075] The peptide as a salt may be an acid adduct with inorganic
acids, such as hydrochloric acid, sulfuric acid, nitric acid,
hydrobromic acid, phosphoric acid, perchloric acid, thiocyanic
acid, boric acid etc. or with organic acid such as formic acid,
acetic acid, haloacetic acid, propionic acid, glycolic acid, citric
acid, tartaric acid, succinic acid, gluconic acid, lactic acid,
malonic acid, fumaric acid, anthranilic acid, benzoic acid,
cinnamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid,
sulfanilic acid etc. Inorganic salts such as monovalent sodium,
potassium or divalent zinc, magnesium, copper calcium, all with a
corresponding anion, may be added to improve the biological
activity of the antimicrobial composition. An antimicrobial H-rich
peptides based on kininogen and histidine-rich glycoprotein may be
used in defined solutions, such as gel, where the pH is defined and
controlled (eg. pH 5.5-6.0) to enhance the effects of the added
antimicrobial peptides. For example a gel, ointment or bandage,
with a defined pH from about 5.0 to about 7.0, such as from about
5.5 to about 6.0 with or without an ionic environment will enhance,
control, and localise the function of the antimicrobial
peptides.
[0076] The antimicrobial/pharmaceutical compositions of the
invention may also be in the form of a liposome in which the
peptide is combined, in addition to other pharmaceutically
acceptable carriers, with amphipathic agents such as lipids, which
exist in aggregated forms as micelles, insoluble monolayers and
liquid crystals. Suitable lipids for liposomal formulation include,
without limitation, monoglycerides, diglycerides, sulfatides,
lysolecithin, phospholipids, saponin, bile acids, and the like.
Preparation of such liposomal formulations is can be found in for
example U.S. Pat. No. 4,235,871.
[0077] The antimicrobial/pharmaceutical compositions of the
invention may also be in the form of biodegradable microspheres.
Aliphatic polyesters, such as poly(lactic acid) (PLA),
poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or
poly(carprolactone) (PCL), and polyanhydrides have been widely used
as biodegradable polymers in the production of microsheres.
Preparations of such microspheres can be found in U.S. Pat. No.
5,851,451 and in EP0213303.
[0078] Alternatively, the antimicrobial peptides may be dissolved
in saline, water, polyethylene glycol, propylene glycol, ethanol or
oils (such as safflower oil, corn oil, peanut oil, cottonseed oil
or sesame oil), tragacanth gum, and/or various buffers. The
pharmaceutical composition may also include ions and a defined pH
for poteniation of action of antimicrobial peptides.
[0079] The antimicrobial/pharmaceutical compositions may be
subjected to conventional pharmaceutical operations such as
sterilisation and/or may contain conventional adjuvants such as
preservatives, stabilisers, wetting agents, emulsifiers, buffers,
fillers, etc., e.g., as disclosed elsewhere herein.
[0080] The antimicrobial/pharmaceutical compositions according to
the invention may be administered locally or systemically. Routes
of administration include topical, ocular, nasal, pulmonary,
buccal; parenteral (intravenous, subcutaneous, and intramuscular),
oral, parenteral, vaginal and rectal. Also administration from
implants is possible. Suitable antimicrobial preparation forms are,
for example granules, powders, tablets, coated tablets, (micro)
capsules, suppositories, syrups, emulsions, microemulsions, defined
as optically isotropic thermodynamically stable systems consisting
of water, oil and surfactant, liquid crystalline phases, defined as
systems characterized by long-range order but short-range disorder
(examples include lamellar, hexagonal and cubic phases, either
water- or oil continuous), or their dispersed counterparts, gels,
ointments, dispersions, suspensions, creams, aerosols, droplets or
injectable solution in ampule form and also preparations with
protracted release of active compounds, in whose preparation
excipients, diluents, adjuvants or carriers are customarily used as
described above. The pharmaceutical composition may also be
provided in bandages or plasters or the like.
[0081] The pharmaceutical compositions will be administered to a
patient in a pharmaceutically effective dose. By "pharmaceutically
effective dose" is meant a dose that is sufficient to produce the
desired effects in relation to the condition for which it is
administered. The exact dose is dependent on the, activity of the
compound, manner of administration, nature and severity of the
disorder, age and body weight of the patient different doses may be
needed. The administration of the dose can be carried out both by
single administration in the form of an individual dose unit or
else several smaller dose units and also by multiple administration
of subdivided doses at specific intervals
[0082] The pharmaceutical compositions of the invention may be
administered alone or in combination with other therapeutic agents,
such as antibiotic or antiseptic agents such as anti-bacterial
agents, anti-fungicides, anti-viral agents, and antiparasitic
agents. Examples are penicillins, cephalosporins, carbacephems,
cephamycins, carbapenems, monobactams, aminoglycosides,
glycopeptides, quinolones, tetracyclines, macrolides, and
fluoroquinolones. Antiseptic agents include iodine, silver, copper,
chlorhexidine, polyhexanide and other biguanides, chitosan, acetic
acid, and hydrogen peroxide. These agents may be incorporated as
part of the same pharmaceutical composition or may be administered
separately.
[0083] The present invention concerns both humans and other mammal
such as horses, dogs, cats, cows, pigs, camels, among others. Thus
the methods are applicable to both human therapy and veterinary
applications. The objects, suitable for such treatment may be
identified by well-established hallmarks of an infection, such as
fever, puls, culture of organisms, and the like. Infections that
may be treated with the antimicrobial peptides include those caused
by or due to microorganisms. Examples of microorganisms include
bacteria (e.g., Gram-positive, Gram-negative), fungi, (e.g., yeast
and molds), parasites (e.g., protozoans, nematodes, cestodes and
trematodes), viruses, and prions. Specific organisms in these
classes are well known (see for example, Davis et al.,
Microbiology, 3.sup.rd edition, Harper & Row, 1980). Infections
include, but are not limited to, chronic skin ulcers, infected
acute wounds and burn wounds, infected skin eczema, impetigo,
atopic dermatitis, acne, external otitis, vaginal infections,
seborrhoic dermatitis, oral infections and parodontitis, candidal
intertrigo, conjunctivitis and other eye infections, and
pneumonia.
[0084] Accordingly the antimicrobial/pharmaceutical compositions
may be used for prophylactic treatment of burn wounds, after
surgery and after skin trauma. The pharmaceutical composition may
also be included in solutions intended for storage and treatment of
external materials in contact with the human body, such as contact
lenses, orthopedic implants, and catheters.
[0085] Additionally, the antimicrobial/pharmaceutical compositions
may be used for treatment of atopic dermatitis, impetigo, chronic
skin ulcers, infected acute wound and burn wounds, acne, external
otitis, fungal infections, pneumonia, seborrhoic dermatitis,
candidal intertrigo, candidal vaginitis, oropharyngeal candidiasis,
eye infections (bacterial conjunctivitis), and nasal infections
(including MRSA carriage).
[0086] The antimicrobial/pharmaceutical compositions may also be
used to in cleansing solutions, such as lens disinfectants and
storage solutions or used to prevent bacterial infection in
association with urinary catheter use or use of central venous
catheters.
[0087] Additionally the antimicrobial compostions may be used for
prevention of infection post-surgery in plasters, adhesives,
sutures, or be incorporated in wound dressings.
[0088] The antimicrobial peptides may also be used in polymers,
textiles or the like to create antibacterial surfaces or Cosmetics,
and personal care products (soap, shampoos, tooth paste, anti-acne,
suncreams, tampons, diapers, etc) may be supplemented with the
antimicrobial/pharmaceutical compositions.
Method to Identify Antimicrobial Human Peptides and/or Proteins
[0089] The invention also relates to a method for the
identification of one or more new antimicrobial peptide, which
enables the possibility to provide mammals such as human beings
with a new set of antimicrobial peptides having low allergenicity
and being effective against the microorganism, which has invaded
the mammal. By such a method new improved antimicrobial peptides
will be available which provides a large collection of
antimicrobial agents which reduce or even eliminates the problems
of resistance and/or tolerance which are common today against the
antibiotic agents available on the market.
[0090] The method comprising the steps of; providing the endogenous
peptide and/or protein, providing heparin, mixing the endogenous
peptide and/or protein with heparin creating a peptide and/or
protein heparin complex, detecting the peptide and/or protein
heparin complex and identifying the antimicrobial human endogenous
peptide and/or protein. Additionally nickel such as nickelsepharose
may be used instead of heparin. Heparin can be presented in
solution, or connected to a matrix. In the latter case, this is
suitable for separation purposes (h.p.l.c or f.p.l.c) or Biocore
analysis. For separation purposes, Heparin-sepharose, or similar
media may be used. Since antimicrobial peptides also interact with
other glycosaminoglycans, it is possible to use these molecules,
such as dermatan or heparan sulfate, for the purification of novel
antimicrobial peptides. Heparin, heparan sulfate, and dermatan
sulfate contains interspersed and spatially defined sulfo- or
carboxyl-groups. In principal, any other polymeric compound of
similar interactive capability as these glycosaminoglycans can be
used for specific binding of antimicrobial peptides. Additionally,
H-rich peptides may be purified on Nickel-sepharose or similar
media, either alone or in combination with
heparin-chromatography.
[0091] The following examples are intended to illustrate but not to
limit the invention in any manner, shape, or form, either
explicitly or implicitly.
EXAMPLES
Microorganisms
[0092] Enterococcus faecalis 2374, Escherichia coli 37.4,
Pseudomonas aeruginosa 27.4, originally obtained from chronic
venous ulcers, and the fungus Candida albicans BM 4435 obtained
from an patient with atopic eczema, were used in the
experiments.
Example 1
Antimicrobial Peptides
[0093] The antimicrobial peptides shown in the sequence listing and
Table 1 below were synthesized by Innovagen AB, Ideon, SE-22370,
Lund, Sweden. The purity and molecular weight of these peptides was
confirmed by mass spectral analysis (MALDI.TOF Voyager).
TABLE-US-00001 TABLE 1 Origin Peptide Code C3a LRKCCEDGMR
ENPMRFSCQR RTRFIS LRK26 C3a LGEACKKVFL DCCNYITELR RQHARAS LGE27 C3a
CNYITELRRQHARASHLGLAR CNY21 Laminin-.alpha.1 SRNLSEIKLLISQARK SRN16
Laminin-.alpha.1 SRNLSEIKLL ISQARKQAAS IKVAVSADR SRN29
Laminin-.alpha.1 KDFLSIELFR GRVKV KDF15 Laminin-.alpha.1 SAVRKKLSVE
LSIRT SAV15 Laminin-.alpha.5 LGTRLRAQSR QRSRPGRWHK VSVRW LGT25
Laminin-.alpha.5 PPPPLTSASK AIQVFLLGGS RKRVL PPP25 Laminin-.alpha.5
RLRAQSRQRS RPGRWHKVSV RW RLR22 Laminin-.alpha.1 PGRWHKVSVR W PGR11
Laminin-.beta.1 RIQNLLKITNLRIKFVKL RIQ18 Fibronectin QPPRARITGY
IIKYEKPG QPP18 Von Willebrand Factor YIGLKDRKRP SELRRIASQV KYA
YIG23 Vitronectin AKKQRFRHRN RKGYR AKK15 Protein C inhibitor
SEKTLRKWLK MFKKRQLELY SEK20 Histidine-rich glycopro- GHHPHGHHPH
GHHPHGHHPH GHH20 tein Kininogen KHNLGHGHKH ERDQGHGHQR KHN20
Kininogen GGHVLDHKHGHGHGKHKNKG GGH20 Kininogen HKHGHGHGKH
KNKGKKNGKH HKH20 Synthetic sequence AKKARAAKKA RAAKKARAAK KARA
AKK24 Synthetic sequence AKKARAAKKA RAAKKARA AKK18 Synthetic
sequence AKKARAAKKA RA AKK12 Synthetic sequence ARKKAAKAAR
KKAAKAARKK AAKA ARK24 Synthetic sequence ARKKAAKAAR KKAAKA ARK16
Synthetic K->H sequence AHHAHAAHHA HAAHHAHAAH HAHA AHH24:1
Synthetic K->H sequence AHHHAAHAAH HHAAHAAHHH AAHA AHH24:2
Example 2
Antibacterial Effects of Arginine and Lysine-Rich Peptides
[0094] FIG. 1 describes bactericidal effects of arginine and
lysine-rich peptides (Sequence listing) on Enterococcus faecalis.
Bacteria were grown to mid-logarithmic phase in Todd-Hewitt (TH)
medium. Bacteria were washed and diluted in either 10 mM Tris, pH
7.4, containing 5 mM glucose Bacteria (50 .mu.l; 2.times.10.sup.6
cfu/ml) were incubated, at 37.degree. C. for 2 hours, with the
synthetic peptide at concentrations ranging from 0.03 to 60 .mu.M.
To quantify the bactericidal activity, serial dilutions of the
incubation mixture were plated on TH agar, followed by incubation
at 37.degree. C. overnight and the number of colony-forming units
was determined.
[0095] 2.times.10.sup.6 colony-forming units (CFU).times.ml.sup.-1
of E. faecalis (isolate 2374) were incubated in 50 .mu.l with
peptides at concentrations ranging from 0.03 to 60 .mu.M. (A)
Synthetic peptides derived from laminin. Effect of peptides from
the LG-domain of the .alpha.5 chain (PPP25: SEQ ID NO:13, LGT25:
SEQ ID NO:12, RLR22: SEQ ID NO:14, PGR11: SEQ ID NO:15) and al
chain (SRN16: SEQ ID NO:8, SRN29:SEQ ID NO:9, KDF15:SEQ ID NO:10,
SAV15:SEQ ID NO:11) are shown. One peptide (RIQ18: SEQ ID NO:16) is
derived from the .beta.1 chain. (B) Three peptides are derived from
the complement factor C3 (LRK26:SEQ ID NO:5, LGE27:SEQ ID NO:6 and
CNY21:SEQ ID NO:7), AKK15 from vitronectin, SEK20:SEQ ID NO:19 from
the protein C inhibitor, QPP18:SEQ ID NO:17 from fibronectin, and
YIG23:SEQ ID NO:18 from the von Willebrand factor. (C)
Antibacterial effects of heparin-binding consensus sequences
(AKKARA).sub.n (n=1-4), and (ARKKAAKA).sub.n (n=1-3). The n=1
peptides exerted no antimicrobial effects. Peptides not interacting
with heparin; GHRPLDKKREEAPSLRPA, LVTSKGDKELRTGKEKVTS, and
KNNQKSEPLIGRKKT (Andersson et al., Eur J Biochem, 2004, 271;
271:1219-1226) were not antimicrobial.
Example 3
Radial Diffusion Assay Analysis of Antimicrobial Peptides (Table
2)
[0096] Radial diffusion assays (RDA) were performed essentially as
described earlier (Andersson et al., Eur J Biochem, 2004,
271:1219-1226). Briefly, bacteria (E. coli) or fungi (C. albicans)
were grown to mid-logarithmic phase in 10 ml of full-strength (3%
w/v) trypticase soy broth (TSB) (Becton-Dickinson, Cockeysville,
Md.). The microorganisms were washed once with 10 mM Tris, pH 7.4.
4.times.10.sup.6 bacterial cfu or 1.times.10.sup.5 fungal cfu was
added to 5 ml of the underlay agarose gel, consisting of 0.03%
(w/v) TSB, 1% (w/v) low-electroendosmosistype (Low-EEO) agarose
(Sigma, St Louise Mo.) and a final concentration of 0.02% (v/v)
Tween 20 (Sigma). The underlay was poured into a O85 mm petri dish.
After agarose solidified, 4 mm-diameter wells were punched and 6
.mu.l of test sample was added to each well. Plates were incubated
at 37.degree. C. for 3 hours to allow diffusion of the peptides.
The underlay gel was then covered with 5 ml of molten overlay (6%
TSB and 1% Low-EEO agarose in dH.sub.2O). Antimicrobial activity of
a peptide is visualized as a clear zone around each well after
18-24 hours of incubation at 37.degree. C. Synthetic peptides were
tested in concentrations of 100 .mu.M to determine the
antibacterial effect relative the known peptide LL-37. To minimize
variation between experiments, a LL-37 standard (100 .mu.M) was
included on each plate. The activities of the peptides are
presented in radial diffusion units ((diameter of clear zone in
millimetres-well diameter).times.10). The results are shown in
table 2 below. TABLE-US-00002 TABLE 2 Radial diffusion Origin Code
units hCAP-18 LL-37 50 C3a LRK26 70 C3a LGE27 40 C3a CNY21 32
Laminin-.alpha.1 SRN16 77 Laminin-.alpha.1 SRN29 71
Laminin-.alpha.1 KDF15 65 Laminin-.alpha.1 SAV15 75
Laminin-.alpha.5 LGT25 85 Laminin-.alpha.5 PPP25 81
Laminin-.alpha.5 RLR22 92 Laminin-.alpha.1 PGR11 86 Laminin-.beta.1
RIQ18 93 Fibronectin QPP18 59 Von Willebrand Factor YIG23 80
Vitronectin AKK15 101 Protein C inhibitor SEK20 92 Synthetic
sequence AKK24 67 Synthetic sequence ARK24 74
Example 4
Radial Diffusion Assay of Peptides Against E. coli and C. albicans
(FIG. 2).
[0097] FIG. 2 illustrates radial diffusion assays using a set of
antimicrobial peptides. The assays were performed as above.
Antimicrobial activity of a peptide was visualized as a clear zone
around each well after 18-24 hours of incubation at 37.degree. C.
for E. faecalis bacteria panel A) and 28.degree. C. for Candida
albicans (panel B).
Example 5
Antibacterial Effects of Histidine-Rich Peptides
[0098] FIG. 3 describes bactericidal effects of histidine-rich
peptides. E. faecalis bacteria were grown to mid-logarithmic phase
in Todd-Hewitt (TH) medium. Bacteria were washed and diluted in
either 10 mM Tris, pH 7.4, containing 5 mM glucose with or without
50 .mu.M ZnCl or 10 mM MES-buffer, 5 mM glucose, pH 5.5. Bacteria
(50 .mu.l; 2.times.10.sup.6 cfu/ml) were incubated, at 37.degree.
C. for 2 hours, with the synthetic peptide at concentrations
ranging from 0.03 to 60 .mu.M (Tris-buffer with or without zinc),
or 30 and 60 .mu.M (Tris and MES-buffer). To quantify the
bactericidal activity, serial dilutions of the incubation mixture
were plated on TH agar, followed by incubation at 37.degree. C.
overnight and the number of colony-forming units was determined.
(A): effect of peptides from the heparin-binding domain of
histidine-rich glycoprotein (GHH20: SEQ ID NO:4) and kininogen
(KHN20: SEQ ID NO:3, GGH20: SEQ ID NO:2 and HKH20: SEQ ID NO:1) in
the presence or absence of 50 uM ZnCl are shown. (B): Effects of
peptides (30 and 60 uM) in 10 mM Tris, pH 7.4, containing 5 mM
glucose or 10 mM MES-buffer, 5 mM glucose, pH 5.5. The numbers
indicate % survival where 100% is control (without peptide). (C):
Effects of peptides AHH24:1 and AHH24:2 on E. faecalis in the
presence of a fixed peptide/zinc molar ratio (1:100). Peptides
without zinc exerted no antimicrobial activity.
Example 6
Analysis by Electron Microscopy of Peptide Effects
[0099] FIG. 4 shows electron microscopy analysis of Pseudomonas
aeruginosa bacteria subjected to antimicrobial peptides. (A)
Control. (B-H) Analysis of bacteria treated with peptides at
.about.50% of the required bactericidal concentration. HKH20 was
also analysed at 200%. (B) LL-37, (C) ARK24, (D) SEK20, (E) AKK24,
(F) LGT25 (G) HKH20, (H) HKH20 at 200% of bactericidal
concentration. The bar represents 1 .mu.m except for G and H (0.5
.mu.m). Electron microscopy analysis of bacteria treated with
peptides demonstrated clear differences in the morphology of
treated bacteria in comparison with the control. The cathelicidin
LL-37 caused local perturbations and breaks along P. aeruginosa
bacterial cell membranes, and occasionally, intracellular material
was found extracellularly and similar finding were obtained with
the endogenous antimicrobial peptides herein disclosed.
Example 7
Heparin Binding of Endogenous Antimicrobial Peptides (FIG. 5).
[0100] Peptides were tested for heparin binding activities.
Peptides were applied on nitrocellulose membranes (Hybond, Amersham
Biosciences). Membranes were blocked (PBS, pH 7.4, 0.25% Tween 20,
3% bovine serum albumin) for one hour and incubated with
radiolabelled heparin for one hour in the same buffer.
Histidine-rich peptides were tested for heparin-binding in the
presence or absence of 50 .mu.M ZnCl. The radioiodination of
heparin was performed as described earlier (Andersson et al., Eur J
Biochem, 2004, 271; 271:1219-1226). Unlabelled polysaccharides (2
mg/ml) were added for competition of binding. The membranes were
washed (3.times.10 min in PBS, pH 7.4, 0.25% Tween 20). A Bas 2000
radioimaging system (Fuji) was used for visualization of
radioactivity.
Unlabelled heparin (6 mg/ml) inhibited the binding of 12511 heparin
to the C3-derived peptides LRK26 and LGE27 and LL-37 (upper
part).
Example 8
Purification of Histidine-Containing Antimicrobial Fragment on
Nickel-Sepharose (FIG. 6).
[0101] Domain D5 of human kininogen, which contains peptide
epitopes KHN20, GGH20 and HKH20 was expressed in Eschericia coli
strain (BL21DE3). Protein production was induced by addition of 1
mM isopropyl-thio-.beta.-D-galactoside to exponentially growing
bacteria. After 3 h incubation bacteria were harvested by
centrifugation. The pellet was resuspended in 50 mM phosphate, 300
mM NaCl, pH 8.0 (buffer A) and bacteria were lysed by repeated
cycles of freeze-thawing. The lysate was then centrifuged at 29000
g for 30 min. The supernatant was mixed with 2 ml NiNTA-sepharose
loaded with nickel and equilibrated with buffer A. The sepharose
was loaded into a column and washed with 10 ml buffer A with 0.1%
Triton X-100, 10 ml buffer A, 5 ml buffer a with 1 M NaCl, 5 ml
buffer A, 10 ml 20% ethanol, 10 ml buffer A with 5 mM imidazol, and
buffer A with 30 mM imidazole. Protein (arrow) was eluted in 500 mM
imidazole. This domain exerts antibacterial effects against E. coli
in radial diffusion assays.
Sequence CWU 1
1
22 1 20 PRT Artificial Sequence derived from kininogen 1 His Lys
His Gly His Gly His Gly Lys His Lys Asn Lys Gly Lys Lys 1 5 10 15
Asn Gly Lys His 20 2 19 PRT Artificial Sequence derived from
kininogen 2 Gly Gly His Val Leu Asp His Lys His Gly His Gly His Gly
His Lys 1 5 10 15 Asn Lys Gly 3 20 PRT Artificial Sequence derived
from kininogen 3 Lys His Asn Leu Gly His Gly His Lys His Glu Arg
Asp Gln Gly His 1 5 10 15 Gly His Gln Arg 20 4 20 PRT Artificial
Sequence derived from histidin-rich glycoprotein 4 Gly His His Pro
His Gly His His Pro His Gly His His Pro His Gly 1 5 10 15 His His
Pro His 20 5 26 PRT Artificial Sequence derived from complement
factor C3 5 Leu Arg Lys Cys Cys Glu Asp Gly Met Arg Glu Asn Pro Met
Arg Phe 1 5 10 15 Ser Cys Gln Arg Arg Thr Arg Phe Ile Ser 20 25 6
27 PRT Artificial Sequence derived from complement factor C3 6 Leu
Gly Glu Ala Cys Lys Lys Val Phe Leu Asp Cys Cys Asn Tyr Ile 1 5 10
15 Thr Glu Leu Arg Arg Gln His Ala Arg Ala Ser 20 25 7 21 PRT
Artificial Sequence derived from complement factor C3 7 Cys Asn Tyr
Ile Thr Glu Leu Arg Arg Gln His Ala Arg Ala Ser His 1 5 10 15 Leu
Gly Leu Ala Arg 20 8 16 PRT Artificial Sequence derived from
laminin 8 Ser Arg Asn Leu Ser Glu Ile Lys Leu Leu Ile Ser Gln Ala
Arg Lys 1 5 10 15 9 29 PRT Artificial Sequence derived from laminin
9 Ser Arg Asn Leu Ser Glu Ile Lys Leu Leu Ile Ser Gln Ala Arg Lys 1
5 10 15 Gln Ala Ala Ser Ile Lys Val Ala Val Ser Ala Asp Arg 20 25
10 15 PRT Artificial Sequence derived from laminin 10 Lys Asp Phe
Leu Ser Ile Glu Leu Phe Arg Gly Arg Val Lys Val 1 5 10 15 11 15 PRT
Artificial Sequence derived from laminin 11 Ser Ala Val Arg Lys Lys
Leu Ser Val Glu Leu Ser Ile Arg Thr 1 5 10 15 12 25 PRT Artificial
Sequence derived from laminin 12 Leu Gly Thr Arg Leu Arg Ala Gln
Ser Arg Gln Arg Ser Arg Pro Gly 1 5 10 15 Arg Trp His Lys Val Ser
Val Arg Trp 20 25 13 25 PRT Artificial Sequence derived from
laminin 13 Pro Pro Pro Pro Leu Thr Ser Ala Ser Lys Ala Ile Gln Val
Phe Leu 1 5 10 15 Leu Gly Gly Ser Arg Lys Arg Val Leu 20 25 14 22
PRT Artificial Sequence derived from laminin 14 Arg Leu Arg Ala Gln
Ser Arg Gln Arg Ser Arg Pro Gly Arg Trp His 1 5 10 15 Lys Val Ser
Val Arg Trp 20 15 11 PRT Artificial Sequence derived from laminin
15 Pro Gly Arg Trp His Lys Val Ser Val Arg Trp 1 5 10 16 18 PRT
Artificial Sequence derived from laminin 16 Arg Ile Gln Asn Leu Leu
Lys Ile Thr Asn Leu Arg Ile Lys Phe Val 1 5 10 15 Lys Leu 17 18 PRT
Artificial Sequence derived from fibronectin 17 Gln Pro Pro Arg Ala
Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu Lys 1 5 10 15 Pro Gly 18 23
PRT Artificial Sequence derived from Von Willebrand Factor 18 Tyr
Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile 1 5 10
15 Ala Ser Gln Val Lys Tyr Ala 20 19 20 PRT Artificial Sequence
derived from protein C 19 Ser Glu Lys Thr Leu Arg Lys Trp Leu Lys
Met Phe Lys Lys Arg Gln 1 5 10 15 Leu Glu Leu Tyr 20 20 24 PRT
Artificial Sequence synthetic sequence 20 Ala Arg Lys Lys Ala Ala
Lys Ala Ala Arg Lys Lys Ala Ala Lys Ala 1 5 10 15 Ala Arg Lys Lys
Ala Ala Lys Ala 20 21 24 PRT Artificial Sequence synthetic sequence
21 Ala Lys Lys Ala Arg Ala Ala Lys Lys Ala Arg Ala Ala Lys Lys Ala
1 5 10 15 Arg Ala Ala Lys Lys Ala Arg Ala 20 22 15 PRT Artificial
Sequence derived from vitronectin 22 Ala Lys Lys Gln Arg Phe Arg
His Arg Asn Arg Lys Gly Tyr Arg 1 5 10 15
* * * * *
References