CA2592034A1 - Antimicrobial peptides, polypeptides comprising same, methods for preparing same, genes coding for said peptides, vectors, transformed organisms and compositions containing same - Google Patents
Antimicrobial peptides, polypeptides comprising same, methods for preparing same, genes coding for said peptides, vectors, transformed organisms and compositions containing same Download PDFInfo
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- CA2592034A1 CA2592034A1 CA002592034A CA2592034A CA2592034A1 CA 2592034 A1 CA2592034 A1 CA 2592034A1 CA 002592034 A CA002592034 A CA 002592034A CA 2592034 A CA2592034 A CA 2592034A CA 2592034 A1 CA2592034 A1 CA 2592034A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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Abstract
The invention concerns novel peptides exhibiting antimicrobial properties, polypeptides comprising said peptides, methods for preparing same, polynucleotides coding for said peptides, chimeric genes, vectors containing said polynucleotides or said chimeric genes and transformed organisms. The novel inventive peptides are peptides of formula (1): C-x(3,7)-C-x(2,4)-C-x(9,10)-C-x(3,6)-CC wherein x(3,7), x(2,4), x(9,10) and x(3,6), do not have the same values at the same time forming the peptide sequences defined by the following five formulae: (1A), (1B), (1C), (1D), and (1E): (1A): C-x(2)-x-x(1,5)-C-[RKQET]-x(2,3)-C-x(3)-[EDNL]-x(5,6)-C-x(3,6)-CC (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]-[TSNHKL]-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.
Description
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 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 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.
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 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 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.
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) 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):
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) 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):
- 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.
- 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.
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.
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.
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) 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.
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.
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) 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.
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.
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.
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 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.
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 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.
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 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.
SEQ ID NO:38 TGTTTCATAACAAATGGCACGTGTTATATGCTAACATGTGATATATCCTTGAGAATGT
TCCATGTGTGCTTTAGAAGTTTATTCTTCTGCTGT
coding for the peptide SEQ ID NO: 12.
SEQ ID NO:39 TGCCGCCTGGGCCTCCCAAAGTGCTGGGATCACAGGTGTGAGCCACCGCACCCAGCCC
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
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 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.
SEQ ID NO:38 TGTTTCATAACAAATGGCACGTGTTATATGCTAACATGTGATATATCCTTGAGAATGT
TCCATGTGTGCTTTAGAAGTTTATTCTTCTGCTGT
coding for the peptide SEQ ID NO: 12.
SEQ ID NO:39 TGCCGCCTGGGCCTCCCAAAGTGCTGGGATCACAGGTGTGAGCCACCGCACCCAGCCC
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
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
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.
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.
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 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 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 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.
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 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 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 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 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 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 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;
By "vehicle" it is understood to mean any substance that is added to the peptide(s) of the 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 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 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;
- 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 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.
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 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.
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 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 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 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 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 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.
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 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 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 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 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 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.
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.
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.
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...).
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...).
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 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:
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:
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 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
- 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 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 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.
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.
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 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:
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:
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 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
- 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 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 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.
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.
Claims (53)
1. Isolated peptide 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) 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 (1E):
(1A): C-{Y}-x(0,1)-{W}-x(1,5)-C-[RKFHQET]-x(2,3)-C-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: 11 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):
- x(n) is a peptide residue of n amino acids with n a positive whole number;
- x(n1, n2) is a peptide residue of n1 to n2 amino acids, with n1 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.
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) 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 (1E):
(1A): C-{Y}-x(0,1)-{W}-x(1,5)-C-[RKFHQET]-x(2,3)-C-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: 11 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):
- x(n) is a peptide residue of n amino acids with n a positive whole number;
- x(n1, n2) is a peptide residue of n1 to n2 amino acids, with n1 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.
2. Peptide of claim 1 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.
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.
3. Peptide as claimed in any of claims 1 and 2, 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
insofar as it is based on formula (1) and/or formula (2), wherein x(n) is a peptide residue of n amino acids with n a positive whole number; and x(n1,n2) is a peptide residue of n1 to n2 amino acids, with n1 and n2 being positive whole numbers.
insofar as it is based on formula (1) and/or formula (2), wherein x(n) is a peptide residue of n amino acids with n a positive whole number; and x(n1,n2) is a peptide residue of n1 to n2 amino acids, with n1 and n2 being positive whole numbers.
4. Peptide of claim 3, of which the amino acid sequence is SEQ ID NO: 1 CCLCHVGALQCIGYCALRLREMGTCRLAQFKCC.
5. Peptide of claim 3, of which the amino acid sequence is SEQ ID NO: 2 CRRSCRGHCNKECGLFLLEIKRCSQRQSWCC.
6. Peptide of claim 3, of which the amino acid sequence is SEQ ID NO: 3 CINWEDGSCTYQACVALEEKKKQICGMLAAHCC.
7. Peptide as claimed in any of claims 1 and 2, having the formula (4): C-x(2)-G-x(1)-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 n1 and n2 being positive whole numbers.
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 n1 and n2 being positive whole numbers.
8. Peptide of claim 7, of which the amino acid sequence is SEQ ID NO: 4 CGFGACFFLWCLAKVEQLLSKCFSVLLCC.
9. Peptide of claim 7, of which the amino acid sequence is SEQ ID NO: 5 CLYGQCGFGACLSRLFQPTDCMLCALCC.
10. Peptide of claim 7, of which the amino acid sequence is SEQ ID NO: 6 CPWGSCQSQCKAIHTDKVHICPRTDCCC.
11. Peptide of claim 7, of which the amino acid sequence is SEQ ID NO: 7 CFTGACVSRPCLPSHAGMRVCTPLPHCC.
12. Peptide of claim 7, of which the amino acid sequence is SEQ ID NO: 8 CCPGGCLWGNCVFAYHRVPALCQNLMYCC.
13. Peptide as claimed in any of claims 1 and 2, having 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 n1 to n2 amino acids, with n1 and n2 being positive whole numbers.
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 n1 and n2 being positive whole numbers.
14. Peptide of claim 13, of which the amino acid sequence is SEQ ID NO: 9 CYSLPGHYCRASCSPVINGAPFHCAAIALCC.
15. Peptide of claim 13, of which the amino acid sequence is SEQ ID NO: 10 CVDACGHVCARACSTRLEEPRLCPLGGKCC.
16. Peptide as claimed in any of claims 1 and 2, having 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
insofar as it is based on formula (1) and/or formula (2), 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 n1 to n2 amino acids, with n1 and n2 being positive whole numbers.
insofar as it is based on formula (1) and/or formula (2), 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 n1 to n2 amino acids, with n1 and n2 being positive whole numbers.
17. Peptide of claim 16, of which the amino acid sequence is SEQ ID NO: 12 CFITNGTCYMLTCDISLRMFHVCFRSLFFCC.
18. Peptide of claim 16, of which the amino acid sequence is SEQ ID NO: 13 CRLGLPKCWDHRCEPPHPAHLLICKSSFCCC.
19. Peptide of claim 16, of which the amino acid sequence is SEQ ID NO: 14 CFHLGYLFCYHFCPNFDPSHMEICSTMRFPCC.
20. Peptide as claimed in any of claims 1 and 2, of formula (7):
C-x(2)-G-x(2)-PC-x(1)-P-x(1,2)-C-x(1,2)-TP-x(1)-LK-x(1)-S-x(1)-CL-x(1,2)-L-x(2)-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 n1 and n2 being positive whole numbers.
C-x(2)-G-x(2)-PC-x(1)-P-x(1,2)-C-x(1,2)-TP-x(1)-LK-x(1)-S-x(1)-CL-x(1,2)-L-x(2)-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 n1 and n2 being positive whole numbers.
21. Peptide of claim 20, of which the amino acid sequence is SEQ ID NO: 16 CRDGVWPCWPAWCQTPKLKGSTCLSLPKCC.
22. Peptide of claim 20, of which the amino acid sequence is SEQ ID NO: 18 CRGGVSPCCPGCSQTPGLKQSSCLDLPKCC.
23. Peptide as claimed in any of claims 1 and 2, having 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(n1,n2) is a peptide residue of n1 to n2 amino acids, with n1 and n2 being positive whole numbers.
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(n1,n2) is a peptide residue of n1 to n2 amino acids, with n1 and n2 being positive whole numbers.
24. Peptide of claim 23, of which the amino acid sequence is SEQ ID NO: 19 CSNGGVNCCDYCLTGFPQTFKYCLRFHLCC.
25. Peptide of claim 23, of which the amino acid sequence is SEQ ID 15 NO: 20 CKNGLIQCCFVLCKTATYSTLILCYPRDLCC.
26. Peptide of claim 23, of which the amino acid sequence is SEQ ID NO: 21 CSKCGVNCCDYCYTGFPQTFKYCLSFCLCC.
27. Peptide of claim 23, of which the amino acid sequence is SEQ ID NO: 22 CSKGGVNCCDYCLTGFPQTFKYCLRFCLCC.
28. Peptide of claim 23, of which the amino acid sequence is SEQ ID NO: 23 CYLTLGICLKGCLFPKTSPLCSCPFICC.
29. Peptide as claimed in any of claims 1 and 2, of which the amino acid sequence is SEQ ID NO: 11 CWRLQGTCRPKCLKNEQYRILCDTIHLCC.
30. Peptide as claimed in any of claims 1 and 2, of which the amino acid sequence is SEQ ID NO: 24 CLNLSGVCRRDVCKVVEDQIGACRRRMKCC.
31. Peptide as claimed in any of claims 1 and 2, of which the amino acid sequence is SEQ ID NO: 25 CLNLFGVCRTDVCNIVEDQIGACRRRMKCC.
32. Peptide as claimed in any of claims 1 and 2, of which the amino acid sequence is SEQ ID NO: 26 CLSLSGVCRRDVCKVVEDQIGACRRRMKCC.
33. Peptide as claimed in any of claims 1 to 32, characterised in that it involves a salt and/or a derivative and/or a fragment of one of said peptides.
34. Peptide characterised in that it includes a peptide as claimed in any of claims 1 to 33.
35. Peptide of claim 34, of which the amino acid sequence is SEQ ID NO:15 CSDGILPCCPGCSETPGLKPSTCLSLLKCCC.
36. Peptide of claim 34, of which the amino acid sequence is SEQ ID NO: 17 CRDGVLPCCPGCSQTPGLKRSSCLSLPSCC.
37. Peptide of claim 34, of which the amino acid sequence is SEQ ID NO: 53 IFCRDGVWPCWPAWCQTPKLKGSTCLSLPKCCAGITGVSHHTQPK.
38. Peptide of claim 34, of which the amino acid sequence is SEQ ID NO: 54 ASCWRLQGTCRPKCLKNEQYRILCDTIHLCCVNPKYLPILTGK.
39. Peptide of claim 34, of which the amino acid sequence is SEQ ID NO: 55 GRAIPCRRSCRGHCNKECGLFLLEIKRCSQRQSWCCGQF
40. Peptide of claim 34, of which the amino acid sequence is SEQ ID NO: 56 THTHCFTGACVSRPCLPSHAGMRVCTPLPHCCQ
41. Peptide of claim 34, of which the amino acid sequence is SEQ ID NO: 57 IFCRDGVLPCCPGCSQTPGLKRSSCLSLPSCCDYR
42. Peptide of claim 34, of which the amino acid sequence is SEQ ID NO: 58 FAFCRGGVSPCCPGCSQTPGLKQSSCLDLPKCCDYRR
43. Peptide of claim 34, of which the amino acid sequence is SEQ ID NO: 59 GPAEGHCLNLSGVCRRDVCKVVEDQIGACRRKMKCCR
44. Peptide of claim 34, of which the amino acid sequence is SEQ ID NO: 60 GHCLNLFGVCRTDVCNIVEDQIGACRRRMKCCR
45. Peptide of claim 34, of which the amino acid sequence is SEQ ID NO: 61 GPAEVHCLSLSGVCRRDVCKVVEDQIGACRRRMKCCR
46. Isolated polynucleotide, characterised in that it codes for a peptide as claimed in any of claims 1 to 45.
47. Chimeric gene, characterised in that it includes bonded together operationally, at least:
- one functional promoter in a host organism;
- one polynucleotide as claimed in claim 46;
- one functional terminator element in said host organism.
- one functional promoter in a host organism;
- one polynucleotide as claimed in claim 46;
- one functional terminator element in said host organism.
48. Expression and/or cloning vector, characterised in that it includes a polynucleotide as claimed in claim 46 or a chimeric gene as claimed in claim 47.
49. Method for preparing a peptide as claimed in any of claims 1 to 45, owing to an expression vector then purified, or by chemical synthesis, or by purification using a human biological product.
50. Antimicrobial composition, characterised in that, as an active agent, it includes at least one peptide as claimed in any of claims 1 to 45.
51. Antimicrobial composition of claim 50, for preparing an anti-infectious drug.
52. Antimicrobial composition of claim 51, characterised in that it is antibacterial.
53. Antimicrobial composition of claim 51, characterised in that it is antifungal.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0413150 | 2004-12-09 | ||
| FR0413150A FR2879203A1 (en) | 2004-12-09 | 2004-12-09 | ANTIMICROBIAL PEPTIDES, POLYPEPTIDES COMPRISING SAID PEPTIDES, PROCESSES FOR PREPARING THEM, GENES ENCODING THE SAME, VECTORS, PROCESSED ORGANISMS AND COMPOSITIONS CONTAINING SAME |
| PCT/EP2005/056588 WO2006061406A2 (en) | 2004-12-09 | 2005-12-07 | Antimicrobial peptides, polypeptides comprising same, methods for preparing same, genes coding for said peptides, vectors, transformed organisms and compositions containing same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2592034A1 true CA2592034A1 (en) | 2006-06-15 |
Family
ID=34954789
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002592034A Abandoned CA2592034A1 (en) | 2004-12-09 | 2005-12-07 | Antimicrobial peptides, polypeptides comprising same, methods for preparing same, genes coding for said peptides, vectors, transformed organisms and compositions containing same |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP1819814A2 (en) |
| CA (1) | CA2592034A1 (en) |
| FR (1) | FR2879203A1 (en) |
| WO (1) | WO2006061406A2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11932594B1 (en) | 2023-10-31 | 2024-03-19 | King Faisal University | 8-((2-hydroxy-5-methylphenyl)diazenyl)naphthalene-1,3-disulfonic acid as an antioxidant compound |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001515720A (en) * | 1997-09-10 | 2001-09-25 | ザイモジェネティクス，インコーポレイティド | β-defensin |
| EP1068232B1 (en) * | 1999-02-01 | 2005-03-23 | Schering Aktiengesellschaft | Human antibiotic protein |
| DK1299541T3 (en) * | 2000-07-11 | 2008-01-21 | Ipf Pharmaceuticals Gmbh | Process for Extraction and Use of New Human Defensins as Biologically Active Proteins to Treat Infections and Other Diseases |
-
2004
- 2004-12-09 FR FR0413150A patent/FR2879203A1/en not_active Withdrawn
-
2005
- 2005-12-07 CA CA002592034A patent/CA2592034A1/en not_active Abandoned
- 2005-12-07 WO PCT/EP2005/056588 patent/WO2006061406A2/en active Application Filing
- 2005-12-07 EP EP05815800A patent/EP1819814A2/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| EP1819814A2 (en) | 2007-08-22 |
| WO2006061406A2 (en) | 2006-06-15 |
| FR2879203A1 (en) | 2006-06-16 |
| WO2006061406A3 (en) | 2006-11-30 |
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