Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02592034 2007-06-08
1
ANTIMICROBIAL PEPTIDES, POLYPEPTIDES COMPRISING SAME,
METHODS FOR PREPARING SAME, GENES CODING FOR SAID
PEPTIDES, VECTORS, TRANSFORMED ORGANISMS AND
COMPOSITIONS CONTAINING SAME
Field of the invention
The field of the invention is that of genetic
engineering and anti-infective agents.
More precisely, this invention relates to new
peptides having antimicrobial properties, polypeptides
including said peptides, methods for preparing them,
polynucleotides coding for said peptides, chimeric
genes, vectors containing said polynucleotides or said
chimeric genes and transformed organisms.
The invention also relates to methods for
preparing said peptides as well as antimicrobial
compositions containing said peptides usable in human
and animal therapy, in agriculture as well as in the
agroprocessing and phytosanitary industry.
Prior art
The pathogens responsible for infectious diseases
are bacteria, fungi, viruses and parasites. The
CA 02592034 2007-06-08
2
antimicrobial agents making it possible to fight
infectious diseases resulting from these micro-
organisms are antibiotics (effective against bacteria
and fungi), antiviral agents and anti-parasitic agents.
An increase in multi-resistances can be witnessed, i.e.
a bacteria that is resistant to several families of
antibiotics at the same time.
The discovery of new antimicrobial (or anti-
infectious) agents is a necessity for fighting against
these pathogens, as concerns human health and animal
health as well as plant health.
Furthermore, although antibiotics have been used
increasingly in recent years, antibiotic therapies,
however, encounter significant problems due, in
particular, to the appearance of resistant strains of
bacteria.
The phenomenon of resistance to antibiotics,
acquired by bacteria, is a natural process governed by
chromosomal and plasmid DNA, aggravated by the
insufficiently selective use of antibiotics, which
creates a selection pressure promoting mutations which
confer resistance to the antibiotics used.
Resistance to antibiotics is a general phenomenon
observed in all bacterial species encountered by man
and is observed in various degrees with respect to all
the members of a given antibiotic family.
Thus, the increasing phenomenon of resistance by
numerous pathogenic micro-organisms to known
antibiotics and the increase in the frequency of
nosocomial diseases of bacterial and fungal origin
which involve hospitalised patients, requires the
CA 02592034 2007-06-08
3
continuous discovery of new antibiotics for fighting
against these micro-organisms.
Furthermore, the fight against pathogenic micro-
organisms, such as viruses or parasites, for example,
against which few antimicrobial agents are known in the
prior art, as well as the emergence of new pathogens,
requires continuous search for and discovery of new
anti-infectious agents.
Objectives of the invention
In particular, the invention has the objective of
proposing new antimicrobial agents, or equally referred
to as anti-infectious agents, for fighting against
infectious agents (viruses, parasites, fungi and
bacteria) and for reducing the phenomena of resistance
to antibiotics.
Thus, the invention relates to the
characterisation of new compounds having anti-
infectious properties.
The invention relates more particularly to the
characterisation of new compounds having antibacterial
properties and/or antifungal properties.
According to this invention, "antibacterial" is
understood to mean any molecule having bacteriostatic
and/or bactericidal properties. According to this
invention, "antifungal" is understood to mean any
molecule having fungistatic and/or fungicidal
properties.
In the peptide sequences presented below, the
amino acids are represented by their one-letter code,
according to the official nomenclature below.
CA 02592034 2007-06-08
4
A alanine
C cysteine
D aspartic acid
E glutamic acid
F phenylalanine
G glycine
H histidine
I isoleucine
K lysine
L leucine
M methionine
N aspargine
P praline
Q glutamine
R arginine
S serine
T threonine
V valine
4V tryptophan
Y tyrosine
Essential characteristics of the invention
Thus, the invention relates to new isolated
peptides (or proteins, the two terms will be used
equally) having the formula
(1) : C-x(3, 7) -C-x(2,4) -C-x(9, 10) -C-x(3, 6) -CC
wherein: x(3,7) is a peptide residue of 3 to 7 amino
acids; x(2,4) is a peptide residue of 2 to 4 amino
acids; x(9,10) is a peptide residue of 9 or 10 amino
acids; x(3,6) is a peptide residue of 3 to 6 amino
acids; with the condition that x(3,7), x(2,4), x(9,10)
CA 02592034 2007-06-08
and x(3,6) do not at the same time assume the values
forming the peptide sequences defined by the following
five formulas (1A), (1B), (1C), (1D), and (lE) :
(1A): C-{Y}-x(0,1)-{W}-x(1,5)-C-[RKFHQET]-x(2,3)-C-
5 x(3) - [EDFKWNL] -x(5, 6) -C-x(3, 6) -CC
wherein {Y} is an amino acid residue different from Y,
{W} is an amino acid residue different from W; x(0,1)
is a peptide bond or an amino acid residue; x(1,5) is a
peptide residue of 1 to 5 amino acids; x(2,3) is a
peptide residue of 2 or 3 amino acids; x(3) is a
peptide residue of 3 amino acids; x(5,6) is a peptide
residue of 5 or 6 amino acids; x(3,6) is a peptide
residue of 3 to 6 amino acids; [RKFHQET] is the amino
acid residue R, K, F, H, Q, E or T; and [EDFKWNL] is
the amino acid residue E, D, F, K, W, N or L; with the
condition that {Y}, {W}, x(0,1) , x(1,5) , x(2,3) , x(3) ,
x(5,6), x(3,6), [RKFHQET] and [EDFKWNL] do not at the
same time assume the values forming the following four
sequences:
SEQ ID NO : 1 I CWRLQGTCRPKCLKNEQYRILCDTIHLCC ;
SEQ ID NO : 24 CLNLSGVCRRDVCKVVEDQIGACRRRMKCC ;
SEQ ID NO : 25 CLNLFGVCRTDVCNIVEDQIGACRRRMKCC ;
SEQ ID NO : 26 CLSLSGVCRRDVCKVVEDQIGACRRRMKCC;
(1B) : C-x(3) -G-x(2) -C-x(3,4) -C-x(9, 10) -C-x(2, 5) -KCC
(1C) : C-x (4 ) -G-x-C-x ( 3 , 4 ) -C-x ( 7 , 8 ) - [ASG] - [TSNHKLR] -
C-x(5,6)-CC
(1D) : C- [LVIF] - [QE] -x(4) -C-x(3,4) -C-x(9, 10) -C-Y-
x(2) - [KR] -G-x-CC
(1E) : C-M-x(2) -G-x(2) -CWGPC-x(9) -C-x(6) -CC
with in the formulas (1B), (1C), (1D) and (1E):
CA 02592034 2007-06-08
6
- x(n) is a peptide residue of n amino acids with
n a positive whole number;
- x(nl, n2) is a peptide residue of n,, to n2 amino
acids, with nl and n2 being positive whole
numbers;
-[ASG} is the amino acid residue A, S or G;
-[TSNHKLR] is the amino acid residue T, S, N,H,
K, L or R;
-[LVIF] is the amino acid residue L, V, I or F;
-[QE] is the amino acid residue Q or E; and
-[KR] is the amino acid residue K or R,
said isolated peptide not having the sequence
CLGLPKCWNYRCEPLHLAYAFYCLLPTSCC.
A first preferred formula for the new peptides
according to formula (1) are the peptides having the
formula
(2) : C-x(3, 7) -C-x(2,4) -C-x(9, 10) -C-x(3, 6) -CC
wherein: x(3,7) is a peptide residue of 3 to 7 amino
acids including at least one amino acid chosen from A,
G, I, L or V; x(2,4) is a peptide residue of 2 to 4
amino acids different from C; x(9,10) is a peptide
residue of 9 or 10 amino acids including at least one
amino acid chosen from A, G, I, L or V; and x(3,6) is a
peptide residue of 3 to 6 amino acids; with the
condition that
- x(3,7) and x(9,10) together include at least
three amino acids chosen from A, G, I, L or V;
and
- if x(9,10)includes a C, then x(3,7) and x(3,6)
do not include a C.
CA 02592034 2007-06-08
7
A second preferred formula for the peptides
according to formulas (1) and/or (2) are the peptides
having the formula
(3): C-x(5)-G-x(1,3)-C-x(3,4)-C-x(2,3)-L-
x(5,7)-C-x(6)-CC
wherein x(n) is a peptide residue of n amino acids with
n a positive whole number; and x(nl,n2) is a peptide
residue of nl to n2 amino acids, with nl and n2 being
positive whole numbers.
The particularly preferred peptides according to
formula (3) are the peptides of the following amino
acid sequences:
(3a) SEQ ID NO :1 CCLCHVGALQCIGYCALRLREMGTCRLAQFKCC ;
(3b) SEQ ID NO :2 CRRSCRGHCNKECGLFLLEIKRCSQRQSWCC ;
(3c) SEQ ID NO :3 CINWEDGSCTYQACVALEEKKKQICGMLAAHCC.
A third preferred formula for the peptides
according to the invention is the formula
(4) : C-x(2) -G-x(l) -C-x(3,4) -C-x(9, 10) -C-x(5) -CC
wherein x(n) is a peptide residue of n amino acids with
n being a positive whole number; and x(n1,n2) is a
peptide residue of n1 to n2 amino acids, with nl and n2
being positive whole numbers.
The particularly preferred peptides according to
formula (4) are the peptides of the following amino
acid sequences:
(4a) SEQ ID NO :4 CGFGACFFLWCLAKVEQLLSKCFSVLLCC ;
(4b) SEQ ID NO :5 CLYGQCGFGACLSRLFQPTDCMLCALCC ;
(4c) SEQ ID NO :6 CPWGSCQSQCKAIHTDKVHICPRTDCCC ;
(4d) SEQ ID NO :7 CFTGACVSRPCLPSHAGMRVCTPLPHCC ;
(4e) SEQ ID NO :8 CCPGGCLWGNCVFAYHRVPALCQNLMYCC.
CA 02592034 2007-06-08
8
A fourth preferred formula for the peptides
according to the invention is the formula
(5) : C-x(4) -GH-x(1) -C-x(3) -CS-x(8, 9) -C-x(5) -CC
wherein x(n) is a peptide residue of n amino acids with
n being a positive whole number; and x(n1,n2) is a
peptide residue of nl to n2 amino acids, with nl and n2
being positive whole numbers.
The particularly preferred peptides according to
formula (5) are the peptides of the following amino
acid sequences:
(5a) SEQ ID NO :9 CYSLPGHYCRASCSPVINGAPFHCAAIALCC ;
(5b) SEQ ID NO :10 CVDACGHVCARACSTRLEEPRLCPLGGKCC.
A fifth preferred formula for the peptides
according to the invention is the formula
(6) : C- [FR] -x(1,3) -G-x(1,3) -C-x(3,4) -C-
x(9, 10) -C- (3,4) -F-x(1) -CC
wherein [FR] is the amino acid residue F or R; x(n) is
a peptide residue of n amino acids with n being a
positive whole number; and x(n1,n2) is a peptide
residue of nl to n2 amino acids, with nl and n2 being
positive whole numbers.
The particularly preferred peptides according to
formula (6) are the peptides with the following amino
acid sequences:
(6a) SEQ ID NO :12 CFITNGTCYMLTCDISLRMFHVCFRSLFFCC ;
(6b) SEQ ID NO :13 CRLGLPKCWDHRCEPPHPAHLLICKSSFCCC ;
(6c) SEQ ID NO :14 CFHLGYLFCYHFCPNFDPSHMEICSTMRFPCC.
A sixth preferred formula for the peptides
according to the invention is formula (7)
CA 02592034 2007-06-08
9
C-x(2) -G-x(2) -PC-x(1) -P-x(l, 2) -C-x(1, 2) -TP-x(1) -LK-
x(1)-S-x(1)-CL-x(1,2)-L-x(2)-CC
Wherein x x(n) is a peptide residue of n amino acids
with n being a positive whole number; and x(nl,nz) is a
peptide residue of nl to n2 amino acids, with nl and n2
being positive whole numbers.
The particularly preferred peptides according to
formula (7) are the peptides of the following amino
acid sequences:
(7a) SEQ ID NO :15 CSDGILPCCPGCSETPGLKPSTCLSLLKCCC ;
(7b) SEQ ID NO :16 CRDGVWPCWPAWCQTPKLKGSTCLSLPKCC ;
(7c) SEQ ID NO :17 CRDGVLPCCPGCSQTPGLKRSSCLSLPSCC ;
(7d) SEQ ID NO :18 CRGGVSPCCPGCSQTPGLKQSSCLDLPKCC.
A seventh preferred formula for the peptides
according to the invention is the formula
(8) : C-x(6) -C-x(3,4) -C-x(3, 5) - [QSK] -T-x(3) -C-x(5) -CC
wherein [QSK] is the amino acid residue Q, S or K; x(n)
is a peptide residue of n amino acids with n being a
positive whole number; and x(nl, nZ) is a peptide
residue of nl to n2 amino acids, with n,_ and n2 being
positive whole numbers.
The particularly preferred peptides according to
formula (8) are the peptides of the following amino
acid sequences:
(8a) SEQ ID NO :19 CSNGGVNCCDYCLTGFPQTFKYCLRFHLCC ;
(8b) SEQ ID NO :20 CKNGLIQCCFVLCKTATYSTLILCYPRDLCC ;
(8c) SEQ ID NO :21 CSKCGVNCCDYCYTGFPQTFKYCLSFCLCC ;
(8d) SEQ ID NO :22 CSKGGVNCCDYCLTGFPQTFKYCLRFCLCC ;
(8e) SEQ ID NO :23 CYLTLGICLKGCLFPKTSPLCSCPFICC.
CA 02592034 2007-06-08
Other preferred peptides according to the
invention are the peptides of the following amino acid
sequences:
SEQ ID NO :l 1 CWRL,QGTCRPKCLKNEQYRILCDTIHLCC ;
SEQ ID NO :24 CLNLSGVCRRDVCKVVEDQIGACRRRMKCC ;
SEQ ID NO :2S CLNLFGVCRTDVCNIVEDQIGACRRRIvIKCC ;
SEQ ID NO :26 CLSLSGVCRRDVCKVVEDQIGACRRRMKCC.
5 The invention also relates to the salts of the
peptides according to the invention presented
hereinabove, such as the acid addition salts, for
example, as well as their functional derivatives and
their fragments.
10 By functional derivatives of the peptides of the
invention, it is meant peptides having a post-
translational modification and/or a chemical
modification and retaining at least a portion of their
function, so that they can be used in the same way as
the peptides of the invention. The post-translational
and/or chemical modifications can be, in particular,
glycosylation, phosphorylation, oxidation, amidation,
acetylation and/or methylation. The functional
derivatives also encompass peptides having a protective
radical. According to this invention, a "protective
radical" is understood to mean any radical making it
possible to prevent the degradation of said peptides.
The derivative also encompass peptides of which
the side chain of one or more of the amino acids is
substituted with radicals which do not modify the
antimicrobial activity of the peptides of the
invention.
CA 02592034 2007-06-08
11
By fragments of the peptides of the invention, it
is understood to mean fragments of at least 10 amino
acids which have an antimicrobial activity. The
antimicrobial activity of the derivatives and fragments
of the peptides of the invention can be demonstrated by
the in vitro tests described below.
The invention also relates to any peptide (or
protein) including a peptide according to the
invention. A peptide such as this can, in particular,
include a peptide of the invention of which one and/or
both of the ends of said peptide include one or more
amino acids necessary for its expression and/or
targeting in a host organism.
Particularly preferred peptides such as this are
the peptides of the following amino acid sequences:
SEQ ID NO :53 IFCRDGV WPC WPAWCQTPKLKGSTCLSLPKCCAGITGVSHHTQPK
Including the peptide of sequence SEQ ID NO:16; and
SEQ ID NO :54 ASCWRLQGTCRPKCLKNEQYRILCDTIHLCCVNPKYLPILTGK
including the peptide of sequence SEQ ID NO:11.
SEQ IDNO :55 GRAIPCRRSCRGHCNKECGLFLLE[KRCSQRQSWCCGQF
including the peptide of sequence SEQ ID NO:2; and
SEQ ID NO :56 THTHCFTGACVSRPCLPSHAGMRVCTPLPHCCQ
including the peptide of sequence SEQ ID NO:7; and
SEQ ID NO :57 IFCRDGVLPCCPGCSQTPGLKRSSCLSLPSCCDYR
including the peptide of sequence SEQ ID NO:17; and
SEQ ID NO :58 FAFCRGGVSPCCPGCSQTPGLKQSSCLDLPKCCDYRR
including the peptide of sequence SEQ ID NO:18; and
SEQ ID NO :59 GPAEGHCLNLSGVCRRDVCKVVEDQIGACRRKMKCCR
including the peptide of sequence SEQ ID NO:24; and
CA 02592034 2007-06-08
12
SEQ ID NO :60 GHCLNLFGVCRTDVCNIVEDQIGACRRRMKCCR
including the peptide of sequence SEQ ID NO:25; and
SEQ ID NO :6I GPAEVHCLSLSGVCRRDVCKVVEDQIGACRRRMKCCR
including the peptide of sequence SEQ ID NO:26.
The invention also relates to isolated
polynucleotides coding for a peptide according to the
invention. According to this invention,
"polynucleotide" is understood to mean a DNA or RNA
type artificial or natural nucleotide sequence,
preferably of the double-stranded DNA type in
particular.
The invention also relates to isolated
polynucleotides which include modifications with
respect to one or more nucleotides resulting from
degeneracy of the genetic code and which thus code for
the same sequence of amino acids of the peptides or
polypeptides which are objects of the invention.
The invention also relates to isolated
polynucleotides coding for peptides and homologues of
the previously described polynucleotides. According to
the invention, "homologue" is understood to mean
polynucleotides having one or more modifications of
sequences in relation to those described above and
coding for a peptide the properties of which are not
significantly altered. These modifications can be
obtained according to common mutation techniques
leading, in particular, to the addition, deletion, or
substitution of one or more nucleotides in relation to
the sequences of the invention.
CA 02592034 2007-06-08
13
The invention also relates to peptides
corresponding to these polynucleotides, and homologues
of the peptides according to the invention.
The degree of homology will advantageously be at
least 80%, and preferably at least 90%, in relation to
the corresponding nucleotide sequences and/or peptide
sequences of the invention. The methods for measuring
and identifying the homologies between nucleic acid
sequences are well known to those skilled in the art.
For example, the BLAST programme can be used.
The invention also covers the complementary
nucleotide sequences of the isolated polynucleotides
defined above, as well as the corresponding RNA.
The invention also relates more particularly to
the isolated and purified polynucleotides of the
following sequences:
SEQ ID NO:27
TGTTGTCTTTGCCATGTGGGTGCTCTGCAGTGTATAGGCTACTGTGCCCTGAGACTGA
GAGAGATGGGTACCTGCAGGCTTGCACAGTTCAAGTGTTGC
coding for the peptide SEQ ID NO: 1.
SEQ ID NO:28
TGCAGGAGAAGTTGCCGAGGACACTGTAATAAAGAATGTGGATTGTTTCTCCTAGAGA
TAAAAAGATGCTCTCAGAGACAGTCATGGTGCTGT
coding for peptide SEQ ID NO: 2.
SEQ ID NO:29
TGTATTAACTGGGAAGACGGATCCTGCACTTACCAAGCCTGTGTAGCTCTAGAAGAAA
AAAAAAAGCAAATATGCGGTATGTTGGCTGCTCACTGCTGC
coding for the peptide SEQ ID NO: 3.
SEQ ID NO:30
CA 02592034 2007-06-08
14
TGTGGATTTGGAGCTTGTTTTTTTCTGTGGTGTTTGGCTAAAGTAGAGCAGCTATTGT
CTAAATGTTTTTCTGTCTTGCTATGCTGC
coding for the peptide SEQ ID NO: 4.
SEQ ID NO:31
TGTTTGTATGGCCAGTGCGGCTTTGGTGCCTGCCTGAGCCGCCTTTTTCAGCCTACTG
ATTGTATGTTGTGTGCACTCTGCTGT
coding for the peptide SEQ ID NO: 5.
SEQ ID NO:32
TGCCCATGGGGGTCCTGTCAGAGTCAGTGTAAAGCAATACACACTGACAAGGTACATA
TTTGCCCCAGAACTGACTGCTGTTGT
coding for the peptide SEQ ID NO: 6.
SEQ ID NO:33
TGCTTTACTGGTGCATGTGTGAGTAGACCTTGCCTCCCATCCCATGCTGGCATGCGTG
TGTGTACCCCGCTGCCCCACTGCTGC
coding for the peptide SEQ ID NO: 7.
SEQ ID NO:34
TGCTGTCCAGGAGGCTGCCTTTGGGGTAATTGTGTGTTTGCATACCATAGGGTACCTG
CTTTATGCCAAAACCTAATGTATTGCTGT
coding for the peptide SEQ ID NO: 8.
SEQ ID NO:35
TGTTACTCCCTTCCAGGCCACTATTGCAGAGCCTCCTGTTCACCTGTAATAAATGGTG
CGCCCTTCCATTGTGCAGCAATTGCCTTATGTTGT
coding for the peptide SEQ ID NO: 9.
SEQ ID NO:36
TGTGTGGATGCATGTGGGCACGTGTGTGCACGTGCGTGTTCAACACGTCTAGAGGAAC
CTCGGTTGTGCCCACTAGGAGGAAAGTGCTGT
coding for the peptide SEQ ID NO: 10.
SEQ ID NO:37
TGTTGGCGACTGCAAGGTACTTGCCGGCCAAAATGTCTAAAAAACGAACAATATCGTA
TTTTGTGTGATACTATACATTTGTGCTGT
coding for the peptide SEQ ID NO: 11.
CA 02592034 2007-06-08
SEQ ID NO:38
TGTTTCATAACAAATGGCACGTGTTATATGCTAACATGTGATATATCCTTGAGAATGT
TCCATGTGTGCTTTAGAAGTTTATTCTTCTGCTGT
coding for the peptide SEQ ID NO: 12.
SEQ ID NO:39
TGCCGCCTGGGCCTCCCAAAGTGCTGGGATCACAGGTGTGAGCCACCGCACCCAGCCC
5 ATTTGCTCATTTGTAAATCGAGTTTTTGTTGTTGC
coding for the peptide SEQ ID NO: 13.
SEQ ID NO:40
TGCTTTCACCTGGGTTATCTATTCTGCTATCACTTTTGCCCTAATTTTGATCCATCTC
ACATGGAAATCTGCTCCACCATGAGATTTCCATGTTGT
coding for the peptide SEQ ID NO: 14.
10 SEQ ID NO:41
TGTAGTGATGGGATTTTGCCATGTTGTCCAGGCTGTTCTGAAACTCCTGGGCTCAAGC
CATCCACTTGCCTCAGCCTCCTAAAGTGCTGTTGC
coding for the peptide SEQ ID NO: 15.
SEQ ID NO:42
TGTAGAGATGGGGTTTGGCCATGTTGGCCAGCCTGGTGTCAAACTCCTAAGCTCAAGG
GATCCACCTGCCTCAGCCTCCCAAAGTGCTGT
15 coding for the peptide SEQ ID NO: 16.
SEQ ID NO:43
TGTAGAGATGGGGTACTGCCATGTTGCCCAGGCTGCTCTCAAACTCCTGGGCTCAAGC
GATCCTCCTGCCTCAGTCTCCCAAGCTGCTGT
coding for the peptide SEQ ID NO: 17.
SEQ ID NO: 44
coding for the peptide SEQ ID NO: 18.
TGTAGAGGTGGGGTTTCACCATGTTGTCCAGGCTGCTCTCAAACTCCTGGGCTCAAAC
AGTCTTCCTGCCTTGACCTCCCGAAGTGCTGC
CA 02592034 2007-06-08
16
SEQ ID NO:45
TGCTCAAACGGCGGAGTTAATTGCTGTGATTACTGCCTTACAGGATTTCCCCAAACCT
TTAAATATTGTCTCAGATTCCACTTATGTTGT
coding for the peptide SEQ ID NO: 19.
SEQ ID NO:46
TGCAAGAATGGACTAATACAATGTTGTTTTGTCCTGTGTAAAACTGCTACTTACTCTA
CTCTGATTTTGTGTTACCCCAGAGACCTTTGCTGT
coding for the peptide SEQ ID NO:20.
SEQ ID NO:47
TGCTCAAAATGTGGAGTTAATTGCTGTGATTACTGCTATACAGGATTTCCCCAAACCT
TTAAATATTGTCTCAGCTTCTGCTTATGTTGT
coding for the peptide SEQ ID NO: 21.
SEQ ID NO:48
TGCTCAAAAGGTGGAGTTAATTGCTGTGATTACTGCCTTACAGGATTTCCTCAAACCT
TTAAATATTGTCTCAGATTCTGCTTATGTTGT
coding for the peptide SEQ ID NO: 22.
SEQ ID NO:49
TGTTATCTGACACTCGGAATCTGCTTAAAAGGCTGCCTATTCCCAAAGACTTCCCCAT
TGTGCAGCTGTCCCTTCATTTGCTGC
coding for the peptide SEQ ID NO: 23.
SEQ ID NO:50
TGTCTCAATTTGTCTGGTGTTTGCAGAAGAGATGTCTGCAAAGTAGTAGAAGATCAAA
TTGGTGCCTGCCGAAGAAGGATGAAGTGCTGT
coding for the peptide SEQ ID NO: 24.
TGTCTCAATTTGTTTGGTGTTTGCAGAACAGATGTCTGCAACATAGTAGAAGATCAAA
TTGGTGCCTGCCGAAGAAGGATGAAGTGCTGT
coding for the peptide SEQ ID NO: 25.
SEQ ID NO:52
TGTCTCAGTTTGTCTGGTGTTTGCAGAAGAGATGTCTGCAAAGTAGTAGAAGATCAAA
TTGGTGCCTGCCGAAGAAGGATGAAGTGCTGT
coding for the peptide SEQ ID NO: 26.
CA 02592034 2007-06-08
17
The invention also relates to any polynucleotide
including at least one of the polynucleotides described
above.
The invention also relates to a chimeric gene
including, bonded together operationally, at least one
constitutive or functional inducible promoter in a host
organism, one polynucleotide coding for a peptide or a
polypeptide of the invention and one functional
terminator element in this same host organism. The
various elements that a chimeric gene can contain are,
on the one hand, elements for regulating transcription,
translation and protein processing, such as a promoter,
a sequence coding for a signal peptide, or a terminator
element, and, on the other hand, a polynucleotide
coding for a protein (or peptide). The expression
"bonded together operationally" means that said
elements of the chimeric gene are bonded together in
such a way that the functioning of one of these
elements is affected by that of the other.
The invention also relates to a cloning and/or
expression vector characterized in that it contains a
polynucleotide or a chimeric gene according to the
invention, for transforming a host organism and
expressing therein a peptide or polypeptide of the
invention. The vector can be a plasmid, a phage or a
virus. Generally speaking, the principal qualities of
this vector must be a capability to maintain itself and
to self-replicate in the cells of the host organism,
and to express therein a peptide or polypeptide
according to the invention. Such cloning and/or
CA 02592034 2007-06-08
18
expression vectors are well known to those skilled in
the art and amply described in literature.
This invention also relates to a transformed host
organism containing a vector as described above. By
"host organism" it is understood to mean any uni- or
multicellular organism into which a polynucleotide or a
chimeric gene is introduced for producing a peptide or
polypeptide of the invention.
The host organism is advantageously a micro-
organism such as a yeast, a bacteria or a fungus, a
plant cell or a plant.
By "plant cell," it is understood to mean any cell
coming from a plant and capable of consisting of
undifferentiated tissues, and by "plant," any
differentiated multicellular organism capable of
photosynthesis.
By "transformed host organism," it is understood
to mean a host organism which has incorporated into its
genome the chimeric gene of the invention and, in
consequence thereof, produces a peptide or polypeptide
of the invention.
This invention also relates to a method for
preparing a peptide according to the invention, owing
to an expression vector, followed by chromatography
extraction and purification, for example.
The peptides of the invention can also be
chemically synthesized according to techniques known by
those skilled in the art. They can also be produced by
extraction from a human biological product, such as a
body fluid, for example. Extraction, for example, may
CA 02592034 2007-06-08
19
be carried out by chromatography (high-performance
liquid) or by electrophoresis.
The peptides according to the invention are
members of the R-defensin family. The defensins are
cationic peptides having an antibiotic activity in
particular. Human defensins are composed of two groups,
the a- and the R-defensins. These two groups of
defensins differ, in particular, by the spacing and
connectivity of the six cysteine residues preserved in
the mature peptide.
The peptide sequences of the R-defensins known in
the prior art generally have the basic pattern C-x(6)-
C-x(4)-C-x(9)-C-x(6)-CC described in literature
(bibliographic reference:: Selsted ME, Tang YQ, Morris
WL, McGuire PA, Novotny MJ, Smith W, Henschen AH,
Cullor JS. Purification, primary structures, and
antibacterial activities of beta-defensins, a new
family of antimicrobial peptides from bovine
neutrophils. J Biol Chem. 1993 Mar 25; 268(9):6641-8).
The peptides according to the invention also have the
preserved and characteristic pattern of six cysteine
residues. However, although related to the R-defensins
known in the prior art, the peptides of the invention
have a more complex and more variable structure, in
particular in the spacing between the cysteine
residues.
The R-defensins are expressed in various tissues
and organs. It is known in the prior art that, besides
their role in antimicrobial defence, the defensins have
the capability of modulating inflammatory responses, by
stimulating acquired immunity and by contributing to
CA 02592034 2007-06-08
tissue repair. Prior studies have demonstrated their
antimicrobial activity against a wide spectrum of
micro-organisms including bacteria, fungi, parasites
and certain viruses.
5 Furthermore, the R-defensins wide spectrum of
action as antibiotics makes it possible, in particular,
to treat infections due to multi-resistant pathogens.
Thus, the peptides according to the invention, as
members of the R-defensin family, and because of the
10 major role of the latter in the elimination of
pathogens, constitute ideal candidates capable of
serving as the basis for discovering new antimicrobial
agents.
The peptides according to the invention have been
15 tested for their antibacterial activity and, in
particular, demonstrate an activity against gram-
positive as well as gram-negative bacteria. In
addition, they have been tested for their antifungal
activity and demonstrate an activity against yeasts.
20 The invention also relates to an antimicrobial
composition including, as an active agent, at least one
peptide according to the invention, and/or one salt of
one of these peptides, and/or at least one of their
derivatives, advantageously associated in said
composition with an acceptable vehicle. The
antimicrobial composition can further include a mixture
of peptides according to the invention and/or include
at least one other active substance, such as another
antimicrobial agent, for example.
By "vehicle" it is understood to mean any
substance that is added to the peptide(s) of the
CA 02592034 2007-06-08
21
invention in order to promote the transport of the
peptide or peptides, to prevent its degradation in said
composition and to protect its antimicrobial
properties.
The vehicle is chosen on the basis of the type of
application of the composition.
This composition can be a cosmetic composition
and, in this case, the suitable vehicle is cosmetically
acceptable, and further suited for application to the
skin. In the case where a salt of one of the peptides
of the invention is used, it involves a cosmetically
acceptable salt.
The composition can also be a pharmaceutical
composition (or drug) for therapeutic use in human or
animal health and, in this case, the suitable vehicle
is pharmaceutically acceptable, suitable for
administering the peptide orally, topically,
parenterally, rectally or by inhalation. The term
"parenterally" includes applications via the
subcutaneous, intravenous and intramuscular routes. In
the case where a salt of one of the peptides of the
invention is used, this involves a pharmaceutically
acceptable salt.
Thus, the invention relates to the use of at least
one of the peptides according to the invention, and/or
their derivatives and/or their pharmacologically
acceptable salts, for preparing an anti-infectious (or
antimicrobial drug) and, in particular, an antibiotic
and/or antifungal drug.
The dosage units for these pharmaceutical
compositions may be of the order of 500 g to 500 mg of
CA 02592034 2007-06-08
22
peptide(s) according to the invention. However, the
dose for one particular subject depends on numerous
factors, e.g., such as weight, age, overall health, the
route of administration, the possible combination with
other treatments and the severity of the disease
requiring treatment.
According to another embodiment, the composition
can be an agrochemical composition and, in this case,
the vehicle is an agrochemically acceptable vehicle,
suitable for application to plants or in proximity to
plants, without damaging them.
According to another embodiment, the composition
can be a food composition, for feeding animals or
humans, and, in this case, the vehicle is an acceptable
food-related vehicle, compatible with assimilation of
the composition by ingestion. As a matter of fact,
peptides according to the invention are of interest in
the agroprocessing industry. In particular, their use
makes it possible to prevent contamination by bacteria,
yeasts and/or fungi during production, and after they
are produced, in order to preserve them.
According to yet another embodiment, the
composition can be a cleaning and/or disinfecting
composition. A composition such as this, for example,
can be used in industrial and household cleaning
products (detergents, etc.), but also in disinfecting
surgical tools, and other equipment and clinical (human
and veterinary) and hospital spaces.
The compositions according to the invention may
include conventional, non-toxic and acceptable
adjuvants, additives, and diluents well known to those
CA 02592034 2007-06-08
23
skilled in the art, such as preservatives, stabilisers,
colouring agents or antioxidants, for example.
Figures and description of an embodiment
Other characteristics and advantages of the
invention will become apparent upon reading the
following examples, given for non-limiting and
illustrative purposes, and from the appended drawings,
in which:
- figures 1A, 1B, 1C, 1D, 1E, iF and 1G show
preferred peptide sequence alignments according
to the invention;
- figure 2A shows the RT-PCR (reverse
transcription-polymerase chain reaction)
analysis of the expression of the
polynucleotides SEQ ID NO: 34, 35, 42, 43 and
48 coding, respectively, for the peptides SEQ
ID NO: 8, 9, 16, 17 and 22 in various human
tissues;
- figure 2B indicates, in the form of a table,
the compositions of the solutions used for
carrying out the RT-PCR analysis shown in
figure 2A.
- figures 3A and 3B show the study of the
antibacterial activity of a peptide according
to the invention against the bacterium bacillus
megaterium;
- figure 3C indicates, in the form of a table,
the various solutions used for carrying out the
antibacterial activity study shown in figures
3A and 3B;
CA 02592034 2007-06-08
24
- figures 4A and 4B show the study of the
antibacterial activity of the peptide SEQ ID
NO: 53 according to the invention against the
bacterium Escherichia coli 363;
- figures 5A and 5B show the study of the
antibacterial activity of the peptide SEQ ID
NO: 53 according to the invention against the
bacterium Escherichia coli D22;
- figures 6A and 6B show the study of the
antibacterial activity of the peptide SEQ ID
NO: 53 according to the invention against the
bacterium Micrococcus luteus (A270);
- figures 7A and 7B show the study of the
antifungal activity of the peptide SEQ ID NO:
54 against the yeast Saccharomyces cerevisiae
CC788-2B;
- figure 8 presents, in the form of a summary
table, the minimum inhibitory concentration of
the peptides SEQ ID NO: 53 and SEQ ID NO: 54
against various strains of bacteria;
- figure 9 presents, in the form of a summary
table, the minimum inhibitory concentration of
the peptides SEQ ID NO: 55, SEQ ID NO: 56, SEQ
ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID
NO: 60 and SEQ ID NO: 61 against four different
strains of bacteria and one strain of yeast.
Example I: Sequence homology of the preferred peptides
Figures 1A to 1F present sequence alignments of
the preferred peptides according to the invention and
show the homologies between these sequences. Formulas
CA 02592034 2007-06-08
1A to 1G show, respectively, alignments corresponding
to the preferred peptides SEQ ID NO: 1 to 23 (except
for the peptide SEQ ID NO: 11), grouped together
according to their belonging to the preferred formulas
5 (3) to (8). An alignment was also produced for the
peptide sequences SEQ ID NO: 24, 25 and 26 having
strong homology. This alignment is shown in figure 1G.
Example II: Chromosome distribution of the nucleotide
10 sequences SEQ ID NO: 27 to 52 coding, respectively, for
the peptides SEQ ID NO: 1 to 26.
It was mentioned previously that, although having
a structure close to that of the R-defensins, the
peptides according to the invention have variations in
15 the basic pattern preserved for six cysteines, as
concerns the spacing separating the cysteine residues.
The peptides according to the invention SED IQ NO:
1 to 26 also differ from the R-defensins known in the
prior art, as concerns the location of the nucleotide
20 sequences coding for these peptides in the human
genome. As matter of fact, the genes coding for the
human R-defensins known in the prior art are
substantially located on the chromosomes 6, 8 and 20,
whereas, the nucleotide sequences SEQ ID NO: 27 to 52
25 coding, respectively, for the new peptides SEQ ID NO: 1
to 26 are, in the human genome, distributed over
practically all of the 23 pairs of human chromosomes.
For each of the sequences of nucleotides SEQ ID
NO: 27 to 52, the table below indicates their human
chromosome location.
CA 02592034 2007-06-08
26
Nucleotide sequence Chromosome
SEQ ID NO: 27 3
SEQ ID NO: 28 9
SEQ ID NO: 29 10
SEQ ID NO: 30 1
SEQ ID NO: 31 2
SEQ ID NO: 32 4
SEQ ID NO: 33 12
SEQ ID NO: 34 X
SEQ ID NO: 35 2
SEQ ID NO: 36 7
SEQ ID NO: 37 8
SEQ ID NO: 38 14
SEQ ID NO: 39 19
SEQ ID NO: 40 21
SEQ ID NO: 41 4
SEQ ID NO: 42 8
SEQ ID NO: 43 10
SEQ ID NO: 44 16
SEQ ID NO: 45 3
SEQ ID NO: 46 9
SEQ ID NO: 47 12
SEQ ID NO: 48 19
SEQ ID NO: 49 20
SEQ ID NO: 50 4
SEQ ID NO: 51 8
SEQ ID NO: 52 12
Example III: Analysis of expression in various tissues
CA 02592034 2007-06-08
27
Figure 2A shows the RT-PCR (reverse transcription-
polymerase chain reaction) analysis of the expression
of the genes SEQ ID NO: 34, 35, 42, 43 and 48 coding,
respectively, for the peptides SEQ ID NO: 8, 9, 16, 17
and 22 in eight different human tissues. The tissues
chosen, i.e., the pancreas, the colon, the spleen, the
lungs and the skin, correspond to organs known in the
prior art as producers of defensins. The testicles, the
epididymis and the prostate have also been used since
recent studies have shown that there are major
defensin-producing sites in the male genital tract.
The total RNA was extracted from cells of these
tissues according to a method known in the prior art,
by means of a "Rneasy Total RNA Isolation Kit"
(Qiagen), by following the instructions thereof.
The total RNA is then treated with Dnase. This
treatment prevents the problem of contaminating the
total RNA by genomic DNA. The Dnase treatment lasts 1
hour at 37 C. The reaction medium used is described in
the table of figure 2B. A double digestion (2 x 1 hr.)
is carried out in the particular case where
oligonucleotide primers situated on a single exon are
used. The total RNA is then extracted with
phenol/chloroform and then precipitated with 0.1 volume
3M sodium acetate, pH 5.2 and 2.5 volumes 100o ethanol.
It is finally taken up in Mi11i-RO quality sterile
water.
The synthesis of complementary DNA (cDNA) carried
out next is of the "random priming" type, with
hexanucleotides hybridising according to their affinity
to the total RNA. The latter will enable the action of
CA 02592034 2007-06-08
28
the reverse transcriptase of the Moloney Murine
Leukaemia Virus (M-MLV; Gibco-BRL). The total RNA is
then denatured for 5 min. at 65 C in the presence of
40 ng hexanucleotides. It is then mixed with the
reaction medium (table, figure 2B) and incubated for
1.5 hrs. at 37 C.
A negative control was carried out in parallel for
each tissue, by omitting the addition of M-MLV reverse
transcriptase, thereby enabling anticipation of
possible contamination by genomic DNA in the PCR
reactions concerned. The RT-PCR was then carried out on
the samples thus prepared.
This reaction enables specific amplification of a
DNA fragment owing to the use of two selected
oligonucleotide primers on either side of the DNA
segment to be amplified. The polymerase chain reaction
(PCR) enables the specific amplification of a DNA
fragment owing to the use of two selected
oligonucleotide primers on either side of the DNA
segment to be amplified. From these, two primers,
synthesis reactions are catalyzed by a DNA polymerase
(Taq polymerase; Qiagen) in a reaction volume of 25 L
the composition of which is described in the table of
figure 2B. A sequential programme is established owing
to the use of a thermocycler making it possible to
carry out the following steps:
Step 1: Denaturation: 94 C/1 min
Step 2: Denaturation: 94 C/3 min
Step 3: Hybridization of the primers:
hybridization temperature (Ta) 5 C lower than the
CA 02592034 2007-06-08
29
melting temperature (Tm) of the primers used the lowest
Tm of the two primers/1 min
Step 4: Elongation: 72 C/2 min
Step 5: Repetition of steps 2 to 4, 35 times
Step 6: Final elongation: 72 C/7 min
Step 7: Waiting phase: 10 C.
Analysis of the amplification products thus
obtained is carried out via electrophoretic migration
in 1. 5 - 2% agarose gel, in the presence of 0. 01% BET
(ethidium bromide), with the pre-depositing of a
mixture of 25 L of PCR products and 5 L of a loading
buffer (50% Orange G/glycerol). Suitable molecular
weight markers are deposited in parallel. For the
purpose of verifying if the cDNA synthesis and
amplification are being carried out properly, a
specific RT-PCR for actin is carried out on the cDNA.
The results obtained after electrophoresis are
viewed under a UV lamp and shown in figure 2A as an
inverted view.
Correspondence between the detected DNA bands and
the nucleotide sequences SEQ ID NO: 34, 35, 42, 43 and
48 coding, respectively, for the peptides SEQ ID NO: 8,
9, 16, 17 and 22, were able to be established by
sequencing the PCR products contained in these bands.
Thus, after migration, the amplification products
having the expected size were extracted from the
agarose gel by using the QIAEX II kit (Qiagen). This
kit enables extraction of DNA fragments having a length
of 40-bp to 50-kb. The purified DNA is then recovered
and analysed by sequencing. The sequence reactions are
based on BigDye Terminator chemistry (Applied
CA 02592034 2007-06-08
Biosystems) and are founded on the Sanger method.
Approximately 30 ng of a PCR product fragment are
inserted into a sequencing PCR the composition of which
is indicated in the table of figure 2B. The sequences
5 are then analysed by an automatic DNA sequencer.
Sequencing of the amplification products present
in the testicles and in the epididymis, visible in
figure 2A, thus made it possible, for each of the five
transcripts, to verify the correspondences with the
10 nucleotide sequences SEQ ID NO: 34, 35, 42, 43 and 48
coding, respectively, for the peptides of sequence SEQ
ID NO: 8, 9, 16, 17 and 22.
The + sign indicates that the total RNA extract
was treated in the presence of reverse transcriptase,
15 and the - sign in the absence of reverse transcriptase
(negative control) . The 0 designator also indicates a
negative control carried out from a sample without
cDNA. The integrity of the cDNA and the success of the
RT-PCR are verified by specific amplification of R-
20 actin. The figures next to the arrows indicate the size
of each amplification product.
Figure 2A shows the presence of transcripts (in
vivo transcription of an RNAm gene) for the nucleotide
sequences SEQ ID NO: 42 and SEQ ID NO: 43 coding,
25 respectively, for the peptide sequences SEQ ID NO: 16
and SEQ ID NO: 17, by detecting the DNA fragments
having expected sizes of 105-bp (base pairs) and 85-bp,
respectively, in all of the tissues examined.
In the male genital tract, transcripts
30 corresponding to the DNA sequences SEQ ID NO: 35, 48
and 34 coding, respectively, for the peptide sequences
CA 02592034 2007-06-08
31
SEQ IN NO: 9, 22 and 8 were detected in the prostate,
by the presence of fragments of an expected length
(i.e., 156, 103 and 124 bp, respectively) . A similar
distribution is found with respect to the testicle and
the epididymis, with the exception of SEQ ID NO: 48
coding for the peptide sequence SEQ ID NO: 22,
undetected in the epididymis.
In the non-reproductive organs, the expected
fragment of 156-bp, corresponding to the DNA sequence
SEQ ID NO: 35 coding for the peptide SEQ ID NO: 9, was
detected in the colon, the spleen, the lungs and the
skin, with a relatively strong signal, while no signal
could be detected in the pancreas. The expected
fragment of 103-bp, corresponding to the DNA sequence
SEQ ID NO: 48 coding for the peptide sequence SEQ ID
NO: 22, was detected only in the lungs. The expected
fragment of 124-bp, corresponding to the DNA sequence
SEQ ID NO: 34 coding for the peptide SEQ ID NO: 8 was
detected in the spleen, the lungs and the skin, and a
very low level in the pancreas and the colon.
The existence of an expression of the peptides of
the invention, like other (3-defensins known in the
prior art, in the male genital tract, and particularly
in the epididymis, presents a major advantage in the
search for new antimicrobial peptides. One possible
additional role in the epididymal maturation of
spermatozoa, and more generally in reproductive
physiology, must be pursued more specifically.
CA 02592034 2007-06-08
32
Example IV: Measurement of the antimicrobial activity
of two peptides according to the invention, from
sequences SEQ ID NO: 53 and SEQ ID NO: 54.
1. Against the gram-positive bacterium Bacillus
megaterium MA
The anti-bacterial activity of two peptides, which
are an object of the invention, were next tested
against the bacterium Bacillus megaterium.
A first peptide of 45 amino acids corresponding to
the sequence
SEQ ID NO: 53
IFCRDGVWPCWPAWCQTPKLKGSTCLSLPKCCAGITGVSHHTQPK,
including the 30 amino acids corresponding to the
sequence SEQ ID NO: 16, was manufactured by chemical
synthesis according to a technique known in the prior
art.
A second peptide of 43 amino acids corresponding
to the sequence
SEQ ID NO: 54
ASCWRLQGTCRPKCLKNEQYRILCDTIHLCCVNPKYLPILTGK,
Including the 29 amino acids corresponding to the
sequence SEQ ID NO: 11, was also manufactured by
chemical synthesis.
The homogeneity of the peptides thus manufactured
was determined by HPLC (High-Performance Liquid
Chromatography) on a C18 Nucleosil column. The
composition of the peptides of sequence SEQ ID NO 53
and SEQ ID NO: 54 was next confirmed by analysis of the
amino acids and their molecular weight (4941.8 and
4981.9, respectively), verified by mass spectrometry.
CA 02592034 2007-06-08
33
Figures 3A and 3B present the results of the
antibacterial activity test for the peptide sequence
SEQ ID NO: 53, against the bacterium Bacillus
megaterium MA, according to the radial diffusion assay
technique. This method, described below, is a method of
choice for evaluating the antibacterial activity of a
molecule, because it proves to be very sensitive,
quantitative and reproducible. The various solutions
used for conducting this test are described in the
table of figure 3C.
A preculture of bacteria (here, Bacillus
megaterium MA) is made in aerobiosis at 37 C, for one
night, under stirring (220 rpm), in a TSB (Tryptcase
Soy Broth) medium. A volume of 75 L of the culture at
saturation is transferred into 15 mL of TSB medium
preheated to 37 C, and is incubated under stirring at
37 C for 2 hr. 30 min. Centrifuging at 900 g is carried
out at 4 C for 10 min and the bacterial pellet is taken
up with 10 mL of 10 mM cold (4 C) phosphate buffer,
which is again centrifuged and taken up with 5 mL of
10 mM cold phosphate buffer. Measurement of the optical
density at 620 nm (D0620nm) of an aliquot of 1 mL of
culture gives the approximate number of bacteria (1
unit of DO62onm) equates to approximately 2. 5x10$
bacteria/mL). The gel underlay (10 mL), maintained at
42 C and inoculated with 4x106 bacteria, is poured into
a Petri dish (80 mm in diameter) on a work area the
level of flatness of which is perfectly controlled.
Gelling occurs in approximately 2 min. The composition
of the gel underlay may be modified over the course of
experiments (variation in the salt concentration...).
CA 02592034 2007-06-08
34
We l l s are made in the agar using a gel punch (3 mm in
diameter) and a Pasteur pipette connected to a vacuum
source.
The peptide sample SEQ ID NO: 53 being tested
(5 L), taken up with 0.01% acetic acid, is deposited
in wells Nos. 4 to 9, at various concentrations. Three
deposits were made for each concentration:
- well No. 4: 800 mg/mL;
- well No. 5: 400 mg/mL;
- well No. 6: 200 mg/mL;
- well No. 7: 100 mg/mL;
- well No. 8: 50 mg/mL;
- well No. 9: 25 mg/mL.
A negative control of 0.01o acetic acid was
deposited in the four No. 1 wells, as well as two
positive controls:
- of acetic acid at 2% in well No. 2;
- a solution of a standard antibacterial peptide,
Cecropin P1 (Sigma C7927) at 25 g/mL in well
No. 3.
The Petri dishes are finally covered, turned over
and incubated for 3 hrs. at 37 C. Following this 3-hour
incubation, each gel underlay is covered with 10 mL of
gel overlay, and then, after gelling is complete, the
Petri dishes are recovered, turned over and placed in
an incubator at 37 C for 18 hrs. At the end of this
incubation, the Petri dishes are then photographed (in
the presence of a graduated scale) and then recovered
with the disinfectant solution (Figure 3A).
The presence of dark circular areas effectively
demonstrates that the peptide SEQ ID NO: 53 has an
CA 02592034 2007-06-08
antibacterial activity against the gram-positive
bacterium Bacillus megaterium.
The diameter of the dark areas is calculated at
close to 0.1 mm and, after subtracting the diameter of
5 the wells, the difference is multiplied by 10 in order
to convert the diameter of the area into units (10
units = 1 mm). A semi-logarithmic graphic
representation of the non-zero values for the areas in
which the peptide SEQ ID NO: 53 was deposited is then
10 carried out, enabling determination of the MIC (minimum
inhibitory concentration) value, equal to the value of
the intersection of the straight line of correlation
with the x-axis (Figure 3B).
The straight line of correlation of figure 3B
15 intersects the x-axis at 27.9 g/mL, which corresponds
to the minimum inhibitory concentration (MIC) of this
peptide SEQ ID NO: 53 on the growth of the bacterium
Bacillus megaterium, under experimental conditions.
The test for measuring the antibacterial activity
20 against the bacterium Bacillus megaterium was conducted
for the peptide SEQ ID NO: 54, under identical
experimental conditions. This peptide also demonstrates
an antibacterial activity against this bacterium, and
the minimum inhibitory concentration (MIC) measured is
25 12.5 g/mL.
2. Against the gram-positive bacteria Escherichia
coli 363 and Escherichia coli D22, and the gram-
positive bacterium Micrococcus luteus (A27)
30 The same tests for measuring the antibacterial
activity of the peptides SEQ ID NO: 53 and SEQ ID NO:
CA 02592034 2007-06-08
36
54 were carried out according to the radial diffusion
assay technique under the same experimental conditions,
against the following bacteria:
- Escherichia coli 363;
- Escherichia coli D22;
- Micrococcus luteus (A270).
The bacteria Escherichia coli 363 and Escherichia
coli D22 are gram-negative bacteria, and the bacterium
Micrococcus luteus (A270), like the bacterium Bacillus
megaterium, is a gram-positive bacterium.
Figures 4A and 4B, 5A and 5B, and 6A and 6B show,
respectively, the evolution of the growth of the
bacteria Escherichia coli 363, Escherichia coli D22 and
Micrococcus luteus (A270), with respect to the
concentration of peptide SEQ ID NO: 53. The
concentrations tested are the same as those used
previously, in the case of Bacillus megaterium, the
distribution in the various wells also being identical.
These results show that this peptide SEQ ID NO: 53
also has an antibacterial activity against the three
strains of bacteria Escherichia coli 363, Escherichia
coli D22 and Micrococcus luteus (A270), and, as
previously, enabled measurement of the minimum
inhibitory concentrations (MIC) in each of the cases.
Similar results were obtained for the peptide SEQ
ID NO: 54, the latter also having an antibacterial
activity against these bacterial strains.
Figure 8, in the form of a table, contains the
values for the minimum concentrations for inhibiting
growth (MIC) measured for the two peptides SEQ ID NO:
CA 02592034 2007-06-08
37
53 and SEQ ID NO: 54, against the four different
strains of bacteria:
- Bacillus megaterium;
- Escherichia coli 363;
- Escherichia coli D22; and
- Micrococcus luteus (A270).
The results obtained thus demonstrate an
antibacterial activity of the peptides SEQ ID NO: 53
and SEQ ID NO: 54, against both gram-positive as well
as against gram-negative bacteria, henceforth capable
of being used as active substances for producing an
anti-infectious drug and, more particularly an
antibiotic drug.
3. Against the yeast Saccharomyces cerevisiae CC788-2B.
An adaptation of the radial diffusion assay
technique, substantially identical to the preceding
experimental conditions, is applied to the evaluation
of the antifungal activity of the peptide SEQ ID NO:
54. A preculture of yeasts (Saccharomyces cerevisiae
CC788-2B) is made in aerobiosis at 35 C, for one day,
under stirring (220 rpm), in an SD (Sabourand Dextrose)
medium. After measurement of the absorbance at 620 nm
(DO62onm is the optical density at 620 nm) , dilution of
the preculture is carried out so as to obtain an
approximate DO620n,,, of 1 at the end of one additional
night of pre-culture. Finally, a 1/5 dilution is
carried out in order to obtain, after 3 to 4 hours of
culture, cells in exponential growth phase (DO62onm -
0.5). Centrifuging and washing operations with the
10 mM cold phosphate buffer are carried out as
CA 02592034 2007-06-08
38
described previously. A measurement of the DO62onm of a
culture aliquot of 1 mL provides information about the
approximate number of yeasts (1 DO62onm unit equates to
approximately 1x10' yeasts/mL). The gel underlay
(10 mL) inoculated with 4x106 yeasts is poured into a
Petri dish. After making the wells and depositing the
samples of the peptide SEQ ID NO: 54 being tested, 3
hours of incubation at 35 C is carried out. Each gel
underlay is next covered with 10 mL of gel overlay, and
then, the Petri dishes are recovered, turned over and
placed in an incubator at 35 C for 18 hrs.
At the end of this incubation period, as
previously, the Petri dishes are photographed and
recovered with a disinfectant solution.
Figure 7A presents the results obtained for the
peptide SEQ ID NO: 54. The sample of this peptide
(5 L), taken up in 0. 01s acetic acid, is deposited in
wells Nos. 10 to 13 at various concentrations. Two
deposits were made for each concentration:
- well No. 10: 800 g/mL
- well No. 11: 400 g/mL
- well No. 12: 200 g/mL
- well No. 13: 100 g/mL
A negative control with 0.01% acetic acid was
deposited in wells No. 14 and No. 19.
A positive control was also carried out with an
antimicrobial peptide (Magainin II), known in the prior
art, taken up in 0.01% acetic acid and deposited in
wells Nos. 15 to 18 at various concentrations. Two
deposits were made for each concentration:
- well No. 15: 800 g/mL
CA 02592034 2007-06-08
39
- well No. 16: 400 g/mL
- well No. 17: 200 g/mL
- well No. 18: 100 g/mL
The results show that the peptide SEQ ID NO: 54
does indeed have an antifungal activity against yeast
Saccharomyces cerevisiae CC788-2B, and enabled
measurement of a minimum inhibitory concentration of
107 g/mL.
Example V: Measurement of the antimicrobial activity of
seven peptides according to the invention, from
sequences SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57,
SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID
NO: 61.
The antibacterial and antifungal activity of seven
peptides, which are an object of the invention, was
also tested against the following bacteria and yeasts:
- Bacillus megaterium MA;
- Escherichia coli 363;
- Escherichia coli D22;
- Micrococcus luteus (A270);
- Saccharomyces cerevisiae CC788-2B.
The tests for measuring the antibacterial and
antifungal activity of the peptides SEQ ID NO: 55, SEQ
ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,
SEQ ID NO: 60 and SEQ ID NO: 61 were carried out
according to the radial diffusion assay technique,
under the same experimental conditions explained in
detail in Example IV.
A first peptide of 39 amino acids corresponding to
the sequence
CA 02592034 2007-06-08
SEQ ID NO :55 GRAIPCRRSCRGHCNKECGLFLLEIKRCSQRQSWCCGQF,
including the 31 amino acids corresponding to the
sequence SEQ ID NO: 2 was manufacture by chemical
synthesis (molecular weight verified by mass
5 spectrometry: 4530.9).
A second peptide of 33 amino acids corresponding
to the sequence
SEQ ID NO :56 THTHCFTGACVSRPCLPSHAGMRVCTPLPHCCQ,
incl
uding the 28 amino acids corresponding to the sequence
10 SEQ ID NO: 7, was also manufactured by chemical
synthesis (molecular weight verified by mass
spectrometry: 3551.2).
A third peptide of 35 amino acids corresponding to
the sequence
SEQ ID NO :57 IFCRDGVLPCCPGCSQTPGLKRSSCLSLPSCCDYR,
15 inc
luding the 30 amino acids corresponding to the sequence
SEQ ID NO: 17, was also manufactured by chemical
synthesis (molecular weight verified by mass
spectrometry: 3765.8).
20 A fourth peptide of 37 amino acids corresponding
to the sequence
SEQ ID NO :58 FAFCRGGVSPCCPGCSQTPGLKQSSCLDLPKCCDYRR,
including the 30 amino acids corresponding to the
sequence SEQ ID NO: 18, was also manufactured by
25 chemical synthesis (molecular weight verified by mass
spectrometry: 3983.9).
A fifth peptide of 37 amino acids corresponding to
the sequence
CA 02592034 2007-06-08
41
SEQ ID NO :59 GPAEGHCLNLSGVCRRDVCKVVEDQIGACRRKMKCCR,
including the 30 amino acids corresponding to the
sequence SEQ ID NO: 24, was also manufactured by
chemical synthesis (molecular weight verified by mass
spectrometry: 4147.7).
A sixth peptide of 33 amino acids corresponding to
the sequence
SEQ ID NO :60 GHCLNLFGVCRTDVCNIVEDQIGACRRRMKCCR,
in
cluding the 30 amino acids corresponding to the
sequence SEQ ID NO: 25, was also manufactured by
chemical synthesis (molecular weight verified by mass
spectrometry: 3769.7).
A seventh peptide of 37 amino acids corresponding
to the sequence
SEQ ID NO :61 GPAEVHCLSLSGVCRRDVCKVVEDQIGACRRRMKCCR,
including the 30 amino acids corresponding to the
sequence SEQ ID NO: 26, was also manufactured by
chemical synthesis (molecular weight verified by mass
spectrometry: 4134.6).
Figure 9, in the form of a table, contains the
values for the minimum concentrations for inhibiting
growth (MIC) measured for the seven peptides SEQ ID NO:
55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID
NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, against the
five reference micro-organisms adopted:
- Bacillus megaterium MA;
- Escherichia coli 363;
- Escherichia coli D22;
- Micrococcus luteus (A270);
- Saccharomyces cerevisiae CC788-2B.
CA 02592034 2007-06-08
42
The results obtained thus demonstrate an
antibacterial activity of the peptides SEQ ID NO: 55,
SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:
59, SEQ ID NO: 60 and SEQ ID NO: 61, against both gram-
positive bacteria as well as against gram-negative
bacteria, henceforth capable of being used as active
substances for producing an anti-infectious drug and,
more particularly an antibiotic drug. Furthermore, the
results show that the peptide SEQ ID NO: 55, SEQ ID NO:
59, SEQ ID NO: 60 and SEQ ID NO: 61 do indeed have an
antifungal activity against the yeast Saccharomyces
cerevisiae CC788-2B.
