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Publication numberUS20030186854 A1
Publication typeApplication
Application numberUS 10/296,853
PCT numberPCT/BR2001/000070
Publication dateOct 2, 2003
Filing dateMay 29, 2001
Priority dateMay 29, 2000
Also published asUS7723468, US20060276380, WO2001092290A2, WO2001092290A3
Publication number10296853, 296853, PCT/2001/70, PCT/BR/1/000070, PCT/BR/1/00070, PCT/BR/2001/000070, PCT/BR/2001/00070, PCT/BR1/000070, PCT/BR1/00070, PCT/BR1000070, PCT/BR100070, PCT/BR2001/000070, PCT/BR2001/00070, PCT/BR2001000070, PCT/BR200100070, US 2003/0186854 A1, US 2003/186854 A1, US 20030186854 A1, US 20030186854A1, US 2003186854 A1, US 2003186854A1, US-A1-20030186854, US-A1-2003186854, US2003/0186854A1, US2003/186854A1, US20030186854 A1, US20030186854A1, US2003186854 A1, US2003186854A1
InventorsSirlei Daffre, Pedro da Silva
Original AssigneeSirlei Daffre, Da Silva Pedro Ismael
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Antimicrobial peptide, process to obtain a peptide and uses therefor
US 20030186854 A1
Abstract
The invention refers to small peptides with low hemolytic activity, presenting equal antiparasitic, antifungal and antibacterial activities. More specifically, it refers to a peptide called gomesin, with 18 amino acid residues, configured as a clamp-like structure consisting of two anti-parallel beta-folded sheets joined by a beta turn, containing four invariable residues of cystein forming two disulphide bridges, optionally configurable as a cyclic chain with closed edges.
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Claims(25)
1. Peptide of the general formula:
in which:
Z1 is:
in the absence of X19, a free amino end residue or a residue with a blocked end amino group, providing resistance against protease activity;
when X19 is present, a basic, hydrophobic, polar/large or small residue, with a free amino end group available to close the edges of the molecule.
C2, C6, C11 and C15 are independently cystein, homocystein or pennicilamine;
P1 and P2 are disulphide bridges from C2 to C15 and from C6 to C11, respectively, either one or both bridges being present;
A3, A4, A5, A12, A13, A14, A16, A17 are independently basic, hydrophobic, polar/large or small residues;
A7 to A10 are residues which are able to effect a beta turn and can be independently basic, hydrophobic, polar/large or small, with at least one of them being basic and the other hydrophobic;
A18 is:
in the absence of X19: a basic, hydrophobic, polar/large or small residue, bearing a free carboxyl group end or forming an acceptable salt, or amidated with an amine of the formula NH3 or RNH2 or R2NH, in which R is independently a saturated or insaturated hydrocarbyl with one to six carbons;
when X19 is present: a basic, hydrophobic, polar/large or small residue, which free carboxyl end is involved with the closing of peptide molecule edges;
X19 may be absent or present; if present, it is a chemical link between Z1 and A18 or a chemical structure or molecule present between Z1 and A18 and linked to both, closing the amino acid chain edges of the peptide of the invention;
from about 15% to about 50% of all amino acid residues are basic;
the peptides present a positive charge of at least +1 at physiologic pH.
2. Peptide of claim 1 in which, in the absence of X19, Z1 is pyroglutamic acid.
3. Peptide of claim 1 in which, when X19 is present, Z1 is a glutamine residue.
4. Peptide of claim 1, in which A3, A4, A8, A10 and A16 are basic amino acid residues.
5. Peptide of claim 1, in which A5, A7, A12 and A14 are hydrophobic amino acid residues.
6. Peptide of claim 1, in which A13 and A17 are small amino acid residues.
7. Peptide of claim 1, in which A8 and A10 are both basic amino acid residues.
8. Peptide of claim 1 in which, in the absence of X19, A18 is a basic amino acid residue.
9. Peptide of claim 1 in which, when X19 is present, A18 is a basic amino acid residue which free carboxyl group end is involved in closing the edges of the peptide molecule.
10. Peptide of claim 4, in which the basic amino acid residue is chosen from the group consisting of arginine, lysine and histidine.
11. Peptide of claim 5, in which the hydrophobic amino acid residue is chosen from the group consisting of leucine, valine, isoleucine, methyonine, phenylalanine, tryptophan and tyrosine.
12. Peptide of claim 1, in which the A9 residue is polar, preferably chosen from the group consisting of glutamine and asparagine.
13. Peptide of claim 1, in which the small amino acid residues are chosen from the group consisting of threonine, glycine, serine and alanine.
14. Peptide of claim 1, in which:
A3, A4, A10, A16 and A18 are arginine;
A5 is leucine;
A7 and A14 are tyrosine;
A8 is lysine;
A9 is glutamine;
A12 is valine;
A13 is threonine;
A17 is glycine;
A18 is amidated arginine;
Z1 is pyroglutamic acid;
X9 is not present.
15. Peptide of claim 1, which is a cyclic chain with closed edges, in which:
A3, A4, A10, A16 and A18 are arginine;
A5 is leucine;
A7 and A14 are tyrosine;
A8 is lysine;
A9 is glutamine;
A12 is valine;
A13 is threonine;
A17 is glycine;
A18 is arginine;
Z1 is glutamic acid;
X9 is a link or chemical structure linking Z1 to A18.
16. PROCESS TO OBTAIN A PEPTIDE by means of extraction from arachnids, consisting of the stages of:
collection of hemolymph;
separation of hemocytes from plasma;
obtaining an acid extract from hemocytes;
isolation of the peptide, preferably by HPLC;
17. PROCESS TO OBTAIN PEPTIDE of claim 16, wherein said spider is an Acanthoscurria gomesiana.
18. PROCESS TO OBTAIN A PEPTIDE of the general formula
of claim 1, in which the Fmoc chemical synthesis techniques is used.
19. FORMULATION CONSISTING OF A PEPTIDE for the combat against infection in human beings, animals, and plants, especially for the combat against bacteria, fungi and parasites, consisting, among other constituents, of the peptide of the general formula:
of claim 1.
20. RECOMBINANT EXPRESSION SYSTEM for the production of a peptide of the general formula
of claim 1, in which said expression system consists of the nucleotide sequence coding the said peptide as linked to a controlling sequence regulating said expression.
21. METHOD TO PREVENT THE DEVELOPMENT OF PARASITES, FUNGI AND BACTERIA, in which said parasites, fungi and bacteria are contacted with an effective amount of a peptide of claim 1.
22. METHOD TO PREVENT THE DEVELOPMENT OF PARASITES, FUNGI AND BACTERIA of claim 21, in which the parasite is preferably a protozoary.
23. METHOD TO PREVENT THE DEVELOPMENT OF PARASITES, FUNGI AND BACTERIA of claim 21, in which the parasite is preferably a Leishmania.
24. METHOD TO INACTIVATE THE ENDOTOXIN FROM GRAM-NEGATIVE BACTERIA, in which said endotoxin is contacted by an amount of a peptide of claim 1 which is substantially effective to inactivate said endotoxin.
25. ANTIBODY which is substantially especifically reactive to the peptide of claim 1.
Description

[0001] The invention refers to small peptides with low hemolytic activity, presenting balanced activity against parasites, fungi and bacteria.

[0002] A number of peptides extracted from animals and plants have shown activity against infection. The application PCT WO9503325, published on Feb. 2, 1995, mentions peptides called protegrins, also reviewing literature on this subject, which includes references on tachyplesins, poliphemusins, defensins, β-defensins and insect defensins. The U.S. Pat. No. 5,994,306 refers to more specific protegrins.

[0003] The application PCT WO9702287, published on Jan. 23, 1997, discloses peptides called parevins and tachytegrins, which are similar to protegrins, except for cysteins on positions 6 and 15.

[0004] This invention has the object to disclose new and small peptides which are similar in some aspects to protegrins, tachytegrins and parevins, but present balanced anti-parasitic, anti-bacteria and anti-fungus activity, besides low hemolitic activity, as well as processes to obtain them and their applications. This is a peptide called gomesin, configured as a clamp-like structure consisting of two anti-parallel beta-folded sheets, joined by a beta turn, containing four invariable cysteine residues forming two disulphide bridges, with the following general formula (1):

[0005] in which:

[0006] Z1 is:

[0007] in the absence of X19, a free amino end residue or a residue with a blocked end amino group by methylation, carbamylation, acylation, acetylation, and some protecting groups like terbutyl, etc., being preferrably pyroglutamic acid, providing resistance against protease activity;

[0008] when X19 is present, a basic, hydrophobic, polar/large or small residue, with a free amino end group available to close the edges of the molecule, such as glutamine or glutamic acid.

[0009] C2, C6, C11 and C15 are independently cystein, homocystein or pennicilamine;

[0010] P1 and P2 are disulphide bridges from C2 to C15, and from C6 to C11, respectively, either one or both bridges being present;

[0011] A3, A4, A5, A12, A13, A14, A16, A17 are independently basic, hydrophobic, polar/large or small residues, provided that:

[0012] A3, A4 and A16 are preferably basic amino acid residues,

[0013] A5, A12 and A14 are preferably hydrophobic residues; and

[0014] A17 is preferably a small residue,

[0015] A7 to A10 are residues which are able to effect a beta turn and can be independently basic, hydrophobic, polar/large or small, with at least one of them being basic and the other hydrophobic, with A8 and A1o being preferably basic amino acid residues,

[0016] A18 is:

[0017] in the absence of X19: a basic, hydrophobic, polar/large or small residue, preferably a basic residue, bearing a free carboxyl group or forming an acceptable salt such as potassium, sodium, calcium, magnesium or other with an organic or inorganic ion, or amidated with an amine of the formula NH3 or RNH2 or R2NH, in which R is independently a saturated or insaturated hydrocarbyl with one to six carbons, such as methyl, ethyl, isopropyl, t-butyl, n-pentyl, cyclohexyl, 2-cyclohexenyl, 3-cyclohexenyl, 4-hexinyl and similar;

[0018] when X19 is present: a basic, hydrophobic, polar/large or small residue, preferably a basic residue, which free carboxyl group end is involved with the closing of peptide molecule edges

[0019] X19 may be absent or present; if present, it is a chemical link between Z1 and A18 or a chemical structure or molecule present between Z1 and A18 is and linked to both, closing the amino acid chain edges of the peptide of the invention;

[0020] from about 15% to about 50% of all amino acid residues should be basic;

[0021] they should present a positive net charge of at least +1 at physiological pH.

[0022] Some of the peptides of the invention can be obtained by extraction from animals, such as from the spider Acanthoscurria gomesiana, and due to this origin they are called gomesins. They can also be synthetically produced and, when containing only genetically coded amino acids, they can be produced in a recombinant way. Peptides of the invention are useful for the treatment and prevention of animal and plant infection as caused by parasites, bacteria and/or fungi. In another aspect, DNA coding peptides of the invention may be expressed in situ, in animals or plants, to fight infection. Peptides of the invention can also be used as standard for antimicrobial tests and for binding to endotoxins.

[0023] Peptides of the invention can be obtained in a recombinant way by means of peptide-coding cDNA expression in heterologous systems, as well known in the literature.

[0024] The invention also refers to useful compositions against bacteria, fungi and parasites, used in the combat against such organisms.

[0025] Peptides of the invention are generally different from others known in the art, among other reasons, for presenting the following qualities which had not been simultaneously found so far:

[0026] small structure, being therefore prone to be better distributed within tissues and less immunogenic;

[0027] they are about equally active against bacteria, fungi and parasites (while e.g. protegrins are less efficient against fungi);

[0028] they have low hemolytic activity, especially in a closed edge cyclic configuration.

[0029] Peptides of the invention contain a beta turn connecting two beta-turned sheets. As it is known by one skilled in the art, a beta fold refers tipically to a peptide segment containing residues of four amino acids reverting the amino acid chain direction. Cysteins on positions 2, 6, 11 and 15 notably provide for the existence of the beta turn by forming dissulphide bridges between them, i.e. from C2 to C15, and from C6 to C11.

[0030] As known in the literature, dissulphide bridges can be substituted by lactam bridges or any other bridge playing an equivalent role.

[0031] In an alternative embodiment, peptides of the invention can present a cyclic structure with closed edges in the peptide chain. Methods to obtain closed cyclic peptides is known in the art.

[0032] In non-cyclic peptides, as known to one skilled in the art, amino and carboxy ends may be derived. In the peptides of the invention, the amino group end may be methyled, carbamyled, acyled, acetylated or as pyroglutamic acid. Peptides of the invention may, by means of addition to the carboxyl end of the molecules, be present as inorganic salts, such as chloride, bromide, iodide, fluoride, sulphate, nitrate, phosphate, etc., or organic salts, such as acetate, formate, benzoate, etc. The acceptance of each one of the above salts depends on the desired use, which is routinely understood. The carboxyl end can also be amidated. Derivation reactions are known.

[0033] As mentioned in this description, amino acid residues of the peptides of the invention are defined under the following characteristics:

[0034] acid: the residue has a negative charge due to the loss of the H ion under physiologic pH, and the residue is attracted to an acquous solution, so to search for superficial positions in the configuration of a peptide in which it is contained, provided it is in acquous media with physiologic pH;

[0035] basic: the residue has a positive charge due to its association with the H ion under physiologic pH, and the residue is attracted to an acquous solution, so to seek superficial positions in the configuration of a peptide in which it is contained, provided that it is in acquous media with physiologic pH;

[0036] hydrophobic: the residue is not charged under physiologic pH and is repelled by an acquous solution, so to seek more internal positions in the configuration of a peptide in which it is contained, provided that the peptide is in acquous media;

[0037] polar/large: the residue is not charged under physiologic pH, but it is not sufficiently repelled by an acquous solution, so that it could seek more internal positions in the configuration of a peptide in which it is contained, provided that the peptide is in acquous media;

[0038] polar/small: neutral residue, since its side chains are not sufficiently large, even if polar groups are absent, to make them become hydrophobic. It contains four or less carbons when at least one polar group appears in the side chain and three or less carbons when this is not the case.

[0039] Concerning amino acids of naturally-occurring proteins, as used in the terms of this application, the following classification is used:

[0040] acids: aspartic acid, glutamic acid

[0041] basic:

[0042] non cyclic: arginine, lysine

[0043] cyclic: histidine

[0044] small: glycine, serine, alanine, threonine

[0045] polar/large: asparagine, glutamine

[0046] hydrophobic: tyrosine, valine, isoleucine, leucine, methyonine, phenyl alanine, tryptophan.

[0047] The functional equivalents of the peptide of the invention are also the said compounds where one or several aminoacids are enantiomers, diastereoisomers, natural aminoacids with a D-conformation, unusual aminoacids such as Ca—methyl—aminoacid, hydroxylysine, methyllysine, dimethyllysine and preferentially pyroglutamic acid at the particular position of the residue A1 at the N-terminus of the said compounds. The invention is also covering the retropeptides and the retro-inversopeptides and the synthetic aminoacids such as the ornithine, the norleucine, the cyclohexyl-alanine and omega-aminoacids.

[0048] Cystein and other amino acid residues containing sulphydryl-SH are not contemplated in the above list, since their capacitiy to form dissulphide bridges providing for a secondary structure is critical for the compounds of the invention.

[0049] Without creating limits to the scope of the invention, it is understood that the action of the peptide of the invention against microorganisms and parasites is enhanced by a larger number of hydrophobic amino acid residues.

[0050] According to some embodiments, constituents of the general formula (1) of the peptide of the invention are as follows:

[0051] a) in the absence of X19:

[0052] Z1 is pyroglutamic acid;

[0053] A3, A4, A8, A10 and A16 are basic residues, with preferably A3, A4, A10 and A16 being arginine and A8 being lysine;

[0054] A18 is a basic residue, preferably amidated arginine, optionally non-amidated;

[0055] A5, A7, A12 and A14 are hydrophobic residues, with preferably A5 being leucine, A7 and A14 being tyrosine and A12 being valine;

[0056] A9 is a polar/large residue, preferably glutamine;

[0057] A13 and A17 are small residues, preferably with A13 being threonine and A17 being glycine.

[0058] b) in the presence of X19, all components of the peptide of the invention are the same as above, except that:

[0059] Z1 is a basic amino acid, hydrophobic, polar/large or small residue with a free amino group end, e.g. glutamine.

[0060] A18 is a basic residue, with a free carboxyl end group involved in closing peptide edges.

[0061] X19 is a chemical structure present between Z1 and A18, connected to both so to close the peptide chain edges.

EXAMPLES OF PROCESSES TO OBTAIN THE PEPTIDES OF THE INVENTION

[0062] The examples given herein are intended to explain the invention, and do not add any limitation to the claims that follow at the end of this specification.

[0063] 1) Extraction of Gomesin From the Spider Acanthoscurria gomesiana.

[0064] By means of the process as described below, a gomesin corresponding specifically to the following structure within the general formula (1) is obtained, including two disulphide bridges (schematic space representation just for illustrative purposes):

[0065] in which:

[0066] Z=pyroglutamic acid

[0067] C=cystein

[0068] R=arginine

[0069] L=leucine

[0070] Y=tyrosine

[0071] K=lysine

[0072] Q=glutamine

[0073] V=valine

[0074] T=threonine

[0075] G=glycine

[0076] Ra=arginine with an amidated carboxyl group end

[0077] Hemolymph (approximately 0.4 ml/spider) of both male and female animals in different development stages was collected from pre-cooled animals by heart puncture with an apyrogenic syringe, in the presence of sodium citrate buffer (30 mmol/L, pH 4.6) containing NaCl (450 mmol/L), EDTA (10 mmol/L) and glucose (100 mmol/L). Hemocytes are removed from plasma by centrifugation at 800g for 10 minutes at 4 C. Entire hemocytes are washed once with sodium citrate buffer and lysated by concentration in a vacuum centrifuging machine.

[0078] After concentration, hemocytes are re-suspended in a 1.5 ml of 2M acetic acid supplemented with aprotinin (20 μg/ml) as protease inhibitor, being homogenized in a Dounce equipment (maximum 152μ, minimum 76μ). A second homogenization is effected by means of sonication (330s) at average intensity, kept in an ice cold water bath and the extraction is effected for 30 minutes at 4 C. under mild stiring. The supernatant obtained by means of centrifugation at 13,800g for 30 minutes at 6 C. is directly submitted to pre-purification by solid phase extraction.

[0079] Organella and cytosolic acid extracts are applied to solid phase columns connected in series, balanced in acidic water (0.05% trifluoroacetic acid). Three elutions are successively effected with 5%, 40% and 80% acetonitrile in acidic water. The 40% acetonitrile portion is concentrated by centrifugation under vacuum, reconsistuted with MiliQ water and reverse phase chromatographed in a column which is balanced with 2% acetonitrile in acidified water. Elution was performed with a linear gradient of acetonitrile from 2 to 60% in acidified water for 120 minutes, at a flow rate of 1.3 ml/min.

[0080] The active fraction against the tested bacterium Micrococcus luteus of hemocytes (AGH2) is additionally purified by filtration chromatography. Elution is made under isocratic conditions with 30% acetonitrile in acidic water at a flow rate of 0.4 ml/min.

[0081] HPLC (high pressure liquid chromatography) purification is made at room temperature. The effluent column is monitored for its absorbance at 225 nm. Fractions corresponding to absorbance peaks are collected, concentrated under vacuum and reconstituted in MilliQ water. Anti-bacterial activity of the collected fractions was monitored by a liquid growth inhibition assay according to J. Biol. Chem., 268, 14893-14897 (1993), using Micrococcus luteus as the test microorganism.

[0082] 2) Gomesin Synthesis

[0083] Peptides of the invention have been synthesized using a classic Fmoc procedure as described in J. Biol. Chem., 271, 29537-29544 (1996).

[0084] For comparison with the gomesin obtained by extraction, as mentioned before, the following peptide was synthesized:

[0085] where asterisks inidcates a C-terminal α-amide.

[0086]FIG. 1 attached shows a table regarding the activity spectrum for gomesin, both amidated (Example 1)and non-amidated (Example 2), against bacteria, fungi and yeast. The evaluation of activity against bacteria and fungi was done as described in J. Biol. Chem., 271, 29537-29544 (1996).

[0087] On this table, n. d. means “non-detected” for the tested concentration range of up to 100 μM for gomesin and 50 μM for androctonin, while n. m. means not measured.

[0088] The table shows that the peptides of the invention have anti-bacteria properties. As shown, gomesin (from Example 1 above) is effective against most tested strains, with 24 from the 27 strains being susceptible to gomesin under MIC (minimum inhibitory concentration) below 1.6 μM for half of them, under concentrations from 1.6 to 6.25 μM (42%). Anti-bacterial properties have also been verified in the peptides of the invention, e.g. by the mortality of Micrococcus luteus and Entamoeba coli D22 as shown in FIG. 2. 10 μM of the gomesin of Example 1 (solid line) or water (dotted line) have been added to an exponential stage culture of M. luteus (circles) or E. coli (triangles). Aliquotes were removed at different time intervals and the number of CFU (colony forming units) counted by plating on Luria Bertani agar plates after overnight incubation at 37 C. The kinetic of killing of gomesin, as shown after approximately one minute, is one of the advantages of the invention.

[0089] Anti-Parasitic Activity

[0090] As an example of anti-parasitic activity, the cell viability of Leishmania (leishmania) amazonensis (MRPO/BR/72/M 1841-LV-79) was evaluated, by meaking use of the MTT test [3-(4,5-dimethyltiazolyl-2)-2,5-diphenyl tetrazolio bromide] as described in J Immunol. Methods 65,55-63 (1983) and adapted for Leishmania spp, according to J. Immunol. Methods 127,11-18 (1990).

[0091] Quantities from 0.1 to 100 μM have been tested against the parasite Leishmania (leishmania) amazonensis. After incubation for one hour, the viability of the parasite was noticed as dependent on the used concentration of gomesin, as shown in FIG. 3. Synthetic gomesin of Example 1 shown as solid bars, and a fragment of hemoglobin (33-61 bovine alpha-hemoglobin) shown as hatched bars and used as a positive control, has been incubated with the parasites for one hour at 22 C. under concentrations from 0.3 to 80 μM. The viability of the parasite (%) has been measured by making use of the MTT assay as described in J. Immunol. Methods 65, 55-63 (1983) and adapted for Leishmania spp, according to J. Immunol. Methods 127, 11-18 (1990).

[0092] Antibodies

[0093] Antibodies to the peptides of the invention can be produced by making use of standard immunologic technics for the production of policlonal antibodies and, if required, by perpetuating antibody producing cells of the immunized host as a source for the production of monoclonal antibodies.

[0094] Gomesin-Containing Formulations

[0095] Peptides of the invention may be used in various compositions which are active against bacteria, fungi and parasites. They can be used individually, as a mixture with other peptides of the invention, with other microbicidal agents or both, and jointly with other ingredients known in the art, e.g. active principles such as erythromicine, tetracycline, azithromicine, cephalosporines, etc. and general constituents such as carriers, diluents, excipients, etc.

[0096] Peptides of the invention may be formulated for pharmaceutical or veterinary use.

[0097] The formulations of the invention can be presented in any form, adapted to the intended purpose as well known by an expert in the art, e.g. for topic use such as creams, oils, ointments, powders, gels, etc. or appropriately for oral, transdermal, transmucous, intramuscular, intravenous, subcutaneous, etc. administration.

1 100 1 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 1 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 2 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 2 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 3 18 PRT Arachinid BINDING (1)..(18) 3 Xaa Cys Lys Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 4 18 PRT Arachinid BINDING (1)..(18) 4 Xaa Cys His Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 5 18 PRT Arachinid BINDING (1)..(18) 5 Xaa Cys Arg Lys Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 6 18 PRT Arachinid BINDING (1)..(18) 6 Xaa Cys Arg His Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 7 18 PRT Arachinid BINDING (1)..(18) 7 Xaa Cys Arg Arg Tyr Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 8 18 PRT Arachinid BINDING (1)..(18) 8 Xaa Cys Arg Arg Val Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 9 18 PRT Arachinid BINDING (1)..(18) 9 Xaa Cys Arg Arg Ile Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 10 18 PRT Arachinid BINDING (1)..(18) 10 Xaa Cys Arg Arg Met Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 11 18 PRT Arachinid BINDING (1)..(16) 11 Xaa Cys Arg Arg Phe Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 12 18 PRT Arachinid BINDING (1)..(18) 12 Xaa Cys Arg Arg Trp Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 13 18 PRT Arachinid BINDING (1)..(18) 13 Xaa Cys Arg Arg Leu Cys Leu Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 14 18 PRT Arachinid BINDING (1)..(18) 14 Xaa Cys Arg Arg Leu Cys Val Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 15 18 PRT Arachinid BINDING (1)..(18) 15 Xaa Cys Arg Arg Leu Cys Ile Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 16 18 PRT Arachinid BINDING (1)..(18) 16 Xaa Cys Arg Arg Leu Cys Met Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 17 18 PRT Arachinid BINDING (1)..(18) 17 Xaa Cys Arg Arg Leu Cys Phe Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 18 18 PRT Arachinid BINDING (1)..(18) 18 Xaa Cys Arg Arg Leu Cys Trp Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 19 18 PRT Arachinid BINDING (1)..(18) 19 Xaa Cys Arg Arg Leu Cys Tyr Arg Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 20 18 PRT Arachinid BINDING (1)..(18) 20 Xaa Cys Arg Arg Leu Cys Tyr His Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 21 18 PRT Arachinid BINDING (1)..(18) 21 Xaa Cys Arg Arg Leu Cys Tyr Lys Asn Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 22 18 PRT Arachinid BINDING (1)..(18) 22 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Lys Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 23 18 PRT Arachinid BINDING (1)..(18) 23 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln His Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 24 18 PRT Arachinid BINDING (1)..(18) 24 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Leu Thr Tyr Cys Arg Gly Arg 1 5 10 15 25 18 PRT Arachinid BINDING (1)..(18) 25 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Tyr Thr Tyr Cys Arg Gly Arg 1 5 10 15 26 18 PRT Arachinid BINDING (1)..(18) 26 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Ile Thr Tyr Cys Arg Gly Arg 1 5 10 15 27 18 PRT Arachinid BINDING (1)..(18) 27 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Met Thr Tyr Cys Arg Gly Arg 1 5 10 15 28 18 PRT Arachinid BINDING (1)..(18) 28 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Phe Thr Tyr Cys Arg Gly Arg 1 5 10 15 29 18 PRT Arachinid BINDING (1)..(18) 29 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Trp Thr Tyr Cys Arg Gly Arg 1 5 10 15 30 18 PRT Arachinid BINDING (1)..(18) 30 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Gly Tyr Cys Arg Gly Arg 1 5 10 15 31 18 PRT Arachinid BINDING (1)..(18) 31 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Ser Tyr Cys Arg Gly Arg 1 5 10 15 32 18 PRT Arachinid BINDING (1)..(18) 32 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Ala Tyr Cys Arg Gly Arg 1 5 10 15 33 18 PRT Arachinid BINDING (1)..(18) 33 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Leu Cys Arg Gly Arg 1 5 10 15 34 18 PRT Arachinid BINDING (1)..(18) 34 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Val Cys Arg Gly Arg 1 5 10 15 35 18 PRT Arachinid BINDING (1)..(18) 35 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Ile Cys Arg Gly Arg 1 5 10 15 36 18 PRT Arachinid BINDING (1)..(18) 36 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Met Cys Arg Gly Arg 1 5 10 15 37 18 PRT Arachinid BINDING (1)..(18) 37 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Phe Cys Arg Gly Arg 1 5 10 15 38 18 PRT Arachinid BINDING (1)..(18) 38 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Trp Cys Arg Gly Arg 1 5 10 15 39 18 PRT Arachinid BINDING (1)..(18) 39 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Lys Gly Arg 1 5 10 15 40 18 PRT Arachinid BINDING (1)..(18) 40 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys His Gly Arg 1 5 10 15 41 18 PRT Arachinid BINDING (1)..(18) 41 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Thr Arg 1 5 10 15 42 18 PRT Arachinid BINDING (1)..(18) 42 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Ser Arg 1 5 10 15 43 18 PRT Arachinid BINDING (1)..(18) 43 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Ala Arg 1 5 10 15 44 18 PRT Arachinid BINDING (1)..(18) 44 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Lys 1 5 10 15 45 18 PRT Arachinid BINDING (1)..(18) 45 Xaa Cys Lys His Ile Cys Phe Lys Asn Arg Cys Trp Ser Val Cys Lys Gly Arg 1 5 10 15 46 18 PRT Arachinid BINDING (1)..(18) 46 Xaa Cys Arg His Leu Cys Tyr Lys Asn Lys Cys Val Thr Tyr Cys Arg Gly Lys 1 5 10 15 47 18 PRT Arachinid BINDING (1)..(18) 47 Xaa Cys Arg Arg Val Cys Tyr Arg Gln Arg Cys Val Gly Ile Cys His Thr Arg 1 5 10 15 48 18 PRT Arachinid BINDING (1)..(18) 48 Xaa Cys His Lys Met Cys Leu Lys Gln His Cys Tyr Ala Trp Cys Arg Gly Arg 1 5 10 15 49 18 PRT Arachinid BINDING (1)..(18) 49 Xaa Cys His Arg Leu Cys Ile His Gln Arg Cys Val Thr Tyr Cys Lys Ala Lys 1 5 10 15 50 18 PRT Arachinid BINDING (1)..(18) 50 Xaa Cys Arg Arg Tyr Cys Met Arg Gln Arg Cys Val Thr Tyr Cys Arg Gly Arg 1 5 10 15 51 18 PRT Arachinid BINDING (1)..(18) 51 Xaa Cys His Lys Phe Cys Leu Lys Asn Lys Cys Phe Ser Tyr Cys Lys Thr Arg 1 5 10 15 52 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 52 Xaa Cys Lys Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 53 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 53 Xaa Cys His Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 54 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 54 Xaa Cys Arg Lys Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 55 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 55 Xaa Cys Arg His Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 56 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 56 Xaa Cys Arg Arg Tyr Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 57 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 57 Xaa Cys Arg Arg Val Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 58 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 58 Xaa Cys Arg Arg Ile Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 59 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 59 Xaa Cys Arg Arg Met Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 60 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 60 Xaa Cys Arg Arg Phe Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 61 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 61 Xaa Cys Arg Arg Trp Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 62 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 62 Xaa Cys Arg Arg Leu Cys Leu Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 63 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 63 Xaa Cys Arg Arg Leu Cys Val Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 64 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 64 Xaa Cys Arg Arg Leu Cys Ile Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 65 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 65 Xaa Cys Arg Arg Leu Cys Met Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 66 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 66 Xaa Cys Arg Arg Leu Cys Phe Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 67 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 67 Xaa Cys Arg Arg Leu Cys Trp Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 68 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 68 Xaa Cys Arg Arg Leu Cys Tyr Arg Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 69 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 69 Xaa Cys Arg Arg Leu Cys Tyr His Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 70 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 70 Xaa Cys Arg Arg Leu Cys Tyr Lys Asn Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 71 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 71 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Lys Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 72 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 72 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln His Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 73 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 73 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Leu Thr Tyr Cys Arg Gly Xaa 1 5 10 15 74 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 74 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Tyr Thr Tyr Cys Arg Gly Xaa 1 5 10 15 75 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 75 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Ile Thr Tyr Cys Arg Gly Xaa 1 5 10 15 76 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 76 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Met Thr Tyr Cys Arg Gly Xaa 1 5 10 15 77 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 77 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Phe Thr Tyr Cys Arg Gly Xaa 1 5 10 15 78 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 78 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Trp Thr Tyr Cys Arg Gly Xaa 1 5 10 15 79 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 79 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Gly Tyr Cys Arg Gly Xaa 1 5 10 15 80 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 80 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Ser Tyr Cys Arg Gly Xaa 1 5 10 15 81 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 81 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Ala Tyr Cys Arg Gly Xaa 1 5 10 15 82 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 82 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Leu Cys Arg Gly Xaa 1 5 10 15 83 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 83 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Val Cys Arg Gly Xaa 1 5 10 15 84 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 84 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Ile Cys Arg Gly Xaa 1 5 10 15 85 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 85 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Met Cys Arg Gly Xaa 1 5 10 15 86 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 86 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Phe Cys Arg Gly Xaa 1 5 10 15 87 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 87 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Trp Cys Arg Gly Xaa 1 5 10 15 88 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 88 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Lys Gly Xaa 1 5 10 15 89 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 89 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys His Gly Xaa 1 5 10 15 90 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 90 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Thr Xaa 1 5 10 15 91 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 91 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Ser Xaa 1 5 10 15 92 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 92 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Ala Xaa 1 5 10 15 93 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 93 Xaa Cys Arg Arg Leu Cys Tyr Lys Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 94 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 94 Xaa Cys Lys His Ile Cys Phe Lys Asn Arg Cys Trp Ser Val Cys Lys Gly Xaa 1 5 10 15 95 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 95 Xaa Cys Arg His Leu Cys Tyr Lys Asn Lys Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 96 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 96 Xaa Cys Arg Arg Val Cys Tyr Arg Gln Arg Cys Val Gly Ile Cys His Thr Xaa 1 5 10 15 97 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 97 Xaa Cys His Lys Met Cys Leu Lys Gln His Cys Tyr Ala Trp Cys Arg Gly Xaa 1 5 10 15 98 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 98 Xaa Cys His Arg Leu Cys Ile His Gln Arg Cys Val Thr Tyr Cys Lys Ala Xaa 1 5 10 15 99 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 99 Xaa Cys Arg Arg Tyr Cys Met Arg Gln Arg Cys Val Thr Tyr Cys Arg Gly Xaa 1 5 10 15 100 18 PRT Arachinid DISULFID (2)..(15) C or homocysteine or penicillamine 100 Xaa Cys His Lys Phe Cys Leu Lys Asn Lys Cys Phe Ser Tyr Cys Lys Thr Xaa 1 5 10 15

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Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US7183381Oct 26, 2005Feb 27, 2007Agennix, Inc.Composition of lactoferrin related peptides and uses thereof
US7420033Feb 22, 2007Sep 2, 2008Agennix, Inc.Composition of lactoferrin related peptides and uses thereof
Classifications
U.S. Classification435/69.1, 530/317, 514/3.3, 514/3.7, 514/21.1, 514/2.3, 514/2.4, 514/2.1
International ClassificationC07K7/64, C07K7/08, A61K38/00
Cooperative ClassificationC07K7/64, A61K38/00, C07K7/08
European ClassificationC07K7/08, C07K7/64
Legal Events
DateCodeEventDescription
Sep 9, 2003ASAssignment
Owner name: UNIVERSIDADE DE SAO PAULO, BRAZIL
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:DAFFRE, SIRLEI;DA SILVA JR., PEDRO ISMAEL;REEL/FRAME:014497/0641
Effective date: 20030224