EP0661988A1 - Composition and treatment with biologically active peptides and chelating agents - Google Patents

Composition and treatment with biologically active peptides and chelating agents

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Publication number
EP0661988A1
EP0661988A1 EP93900822A EP93900822A EP0661988A1 EP 0661988 A1 EP0661988 A1 EP 0661988A1 EP 93900822 A EP93900822 A EP 93900822A EP 93900822 A EP93900822 A EP 93900822A EP 0661988 A1 EP0661988 A1 EP 0661988A1
Authority
EP
European Patent Office
Prior art keywords
peptide
ala
amino acids
basic
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93900822A
Other languages
German (de)
French (fr)
Other versions
EP0661988A4 (en
Inventor
Barry Berkowitz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Magainin Pharmaceuticals Inc
Original Assignee
Magainin Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Magainin Pharmaceuticals Inc filed Critical Magainin Pharmaceuticals Inc
Publication of EP0661988A4 publication Critical patent/EP0661988A4/en
Publication of EP0661988A1 publication Critical patent/EP0661988A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1751—Bactericidal/permeability-increasing protein [BPI]

Definitions

  • This invention relates to biologically active peptides and proteins, and more particularly to compositions and uses
  • composition comprising at least one biologically active amphiphilic peptide or biologically active protein, and a chelating agent.
  • the peptide or protein is preferably an ion channel-forming peptide or protein.
  • a process of inhibiting growth of a target cell in a host which comprises administering to a host at least one biologically active amphiphilic peptide or biologically active protein and a chelating agent.
  • the peptide or protein is
  • the biologically active amphiphilic peptide or biologically active protein and the chelating agent are administered in amounts effective to inhibit growth of a target cell in a host.
  • magnesium ions decrease the activity of certain biologically active amphiphilic peptides as antibacterial agents. It is believed that such decrease in activity may be due to competition between the ions, which are positively charged, and the peptides for negative charges on the bacteria. Applicant also has found that when a chelating agent, which binds ions such as calcium and/or magnesium ions, is added to the biologically active amphiphilic peptide, the activity of the biologically active peptide is enhanced, or potentiated, whereas when known calcium channel-blocking agents, such as verapamil and diltiazem, are added to such peptides, no improvement in the biological activity of the peptides was obtained.
  • a chelating agent which binds ions such as calcium and/or magnesium ions
  • potentiate means either that the biologically active amphiphilic peptide or protein is effective in increasing the biological activity of the chelating agents against a target cell so thereby the chelating agent may be employed in an amount lower than which would be required for preventing, destroying or inhibiting growth of a target cell and/or that the peptide or protein may be employed in an amount lower than which would be required for preventing, destroying, or inhibiting growth of a target cell.
  • Chelating agents which may be employed in accordance with the present invention are agents which are capable of chelating calcium. Such chelating agents may also increase the
  • Such chelating agents include ethylene dinitrilo tetraacetic acid (EDTA), and ethylene glycol bis- ⁇ -aminoethyl ether N,N,N',N'-tetraacetic acid (EGTA).
  • EDTA ethylene dinitrilo tetraacetic acid
  • EGTA ethylene glycol bis- ⁇ -aminoethyl ether N,N,N',N'-tetraacetic acid
  • the biologically active amphiphilic peptides employed in the present invention are generally water soluble to a concentration of at least 20 mg/ml at neutral pH in water.
  • the structure of such peptide provides for flexibility of the peptide molecule. When the peptide is placed in water, it does not assume an amphiphilic structure. When the peptide encounters an oily surface or membrane, the peptide chain folds upon itself into a rod-like structure, sometimes referred to as an ⁇ -helical structure.
  • such peptides have at least 10 amino acids, and preferably at least 20 amino acids. In most cases, such peptides do not have in excess of 50 amino acids.
  • biologically active peptides or proteins employed in the present invention are ion channel-forming
  • An ion channel-forming peptide or protein or ionophore is a peptide or protein which increases the
  • an ion channel-forming peptide or protein is a peptide or protein which has ion
  • amphiphilic peptide or protein is a peptide which
  • the administration of the biologically active amphiphilic peptides or proteins and chelating agent to a target cell may be by direct administration to the target cell or systemic or topical administration to a host which includes the target cell, in order to prevent, destroy, or inhibit the growth of a target cell.
  • compositions of the present invention may be any compositions of the present invention.
  • compositions may be used as antimicrobial agents, or in particular as anti-bacterial agents, or as
  • antimicrobial means that the compositions of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes such as, for example, bacteria.
  • compositions employed in the present invention produce effects adverse to the normal biological functions of the target non-host cell, including death or destruction and prevention of the growth or proliferation of the non-host target cell when contacted with the compositions.
  • compositions of the present invention have a broad range of potent antibiotic activity against a plurality of
  • microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative microorganisms including Gram-positive and Gram-negative
  • compositions of the present invention allow a method for treating or controlling microbial infection caused by organisms which are sensitive to the peptides herein described.
  • Such treatment may comprise administering to a host organism or tissue susceptible to or affiliated with a microbial infection an antimicrobial amount of at least one of the peptides and a chelating agent.
  • compositions may also be used as preservatives or sterilants of materials susceptible to microbial contamination.
  • compositions may be administered in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution.
  • a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution.
  • compositions may be used topically or systemically and may be in any suitable form such as a liquid, solid,
  • compositions may also be used in combination with adjuvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful
  • microorganisms including bacteria.
  • compositions of the present invention may be any compositions of the present invention.
  • a host in particular an animal, in an effective antibiotic and/or anti-microbial amount.
  • composition in accordance with the invention will contain an effective anti-microbial amount of one or more of the hereinabove described peptides which have such activity.
  • compositions of the present invention may be used in the treatment of external burns and to treat and/or prevent skin and burn infections.
  • the compositions may be used to treat skin and burn infections caused by organisms such as, but not limited to, P. aeruginosa, S. aureus, and other Streptococcus species.
  • compositions are also useful in the prevention or treatment of eye infections.
  • infections may be caused by bacteria such as, but not limited to, P. aeruginosa, S. aureus, and N. gonorrhoeae.
  • compositions may also be administered to plants in an effective antimicrobial amount to prevent or treat microbial contamination thereof.
  • the peptide or protein is employed to provide peptide dosages of from 1 mg to 500 mg per kilogram of host weight, when administered systemically.
  • the peptide or protein is used in a concentration of from .05% to 10%.
  • the chelating agent such as those hereinabove described, or derivatives or analogues thereof, when used topically, is
  • the chelating agent is generally employed in a concentration of about .001% to about 25%.
  • the chelating agent is generally employed in an amount of from 1 mg/kg to about 2 grams/kg of host weight per day.
  • the peptide used in conjunction with a chelating agent such as those hereinabove described, or derivatives or analogues thereof is a basic
  • polypeptide having at least ten amino acids wherein the polypeptide includes both hydrophobic and hydrophilic amino acids.
  • the polypeptide may have at least sixteen amino acids wherein the polypeptide includes at least eight hydrophobic amino acids and at least eight
  • hydrophobic amino acids are in groups of two adjacent amino acids, and each group of two hydrophobic amino acids is spaced from another group of two hydrophobic amino acids by at least one amino acid other than a hydrophobic amino acid (preferably at least two amino acids) and generally by no greater than four amino acids, and the amino acids between pairs of hydrophobic amino acids may or may not be hydrophilic.
  • the hydrophilic amino acids are generally also in groups of two adjacent amino acids in which at least one of the two amino acids is a basic hydrophilic amino acid, with such groups of two hydrophilic amino acids being spaced from each other by at least one amino acid other than a hydrophilic amino acid (preferably at least two amino acids) and generally no greater than four amino acids, and the amino acids between pairs of hydrophilic amino acids may or may not be hydrophobic.
  • the polypeptide comprises a chain of at least four groups of amino acids, with each group consisting of four amino acids. Two of the four amino acids in each group are hydrophobic amino acids, and two of the four amino acids in each group are hydrophilic, with at least one of the hydrophilic amino acids in each group being a basic hydrophilic amino acid and the other being a basic or neutral hydrophilic amino acid.
  • the hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, lie, Leu, Met, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
  • the neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gln, Ser, and Thr.
  • hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
  • Each of the groups of four amino acids may be of the sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each hydrophobic amino acids and may be the same or different, one of C or D is a basic hydrophilic amino acid, and the other of C or D is a basic or neutral hydrophilic amino acid and may be the same or different.
  • the polypeptide chain may comprise 5 or 6 groups of this sequence. In each group, each of A, B, C and D may be the same in some or all of the groups or may be different in some or all of the groups.
  • the polypeptide chain preferably has at least 20 amino acids, and no greater than 50 amino acids. It is to be
  • polypeptide does not have to consist entirely of the groups described above.
  • the polypeptide may have amino acids extending from either or both ends of the noted groups forming the polypeptide chain and/or there may be amino acids between one or more of the at least four groups and still remain within the scope of the invention.
  • the groups of amino acids may be repeating groups of amino acids, or the amino acids in the various groups may vary provided that in each group of the at least four groups of amino acids there are two hydrophobic and two hydrophilic amino acids as hereinabove noted.
  • the biologically active polypeptide comprises a chain including at least four groups of amino acids, each containing four amino acids. Two of the four amino acids in each. group are hydrophobic, at least one amino acid is basic hydrophilic, and the remaining one is basic or neutral hydrophilic, with the polypeptide chain preferably having at least 20 amino acids but no greater than 50 amino acids.
  • each of the at least four groups of amino acids which are in the peptide chain is of the sequence A-B-C-D, B-C-D-A, C-D-A-B or D-A-B-C wherein A and B are hydrophobic amino acids, one of C or D is basic hydrophilic amino acid, and the other of C or D is basic or neutral hydrophilic amino acid.
  • the resulting polypeptide chain may have one of the following sequences:
  • X 2 is A-, D-A- or C-D-A-
  • Y 2 is -B, -B-C or B-C-D
  • X 3 is B-, A-B-, D-A-B-
  • Y 3 is -C, -C-D, -C-D-A
  • X 4 is C-, B-C-, A-B-C-
  • Y 4 is -D, -D-A, -D-A-B
  • n is at least 4.
  • the peptide chain may include amino acids between the hereinabove noted groups of four amino acids provided that the spacing between such groups and the charge on the amino acids does not change the characteristics of the peptide chain which provide amphiphilicity and a positive charge and do not adversely affect the folding characteristics of the chain to that which is significantly different from one in which the hereinabove noted group of four amino acids are not spaced from each other.
  • amino acids between the hereinabove noted groups of four amino acids provided that the spacing between such groups and the charge on the amino acids does not change the characteristics of the peptide chain which provide amphiphilicity and a positive charge and do not adversely affect the folding characteristics of the chain to that which is significantly different from one in which the hereinabove noted group of four amino acids are not spaced from each other.
  • the peptide may have amino acids extending from either end of the chain.
  • the chains may have a Ser-Lys sequence before the "Ala” end, and/or an Ala-Phe sequence after the "Lys" end.
  • Other amino acid sequences may also be attached to the "Ala” and/or the "Lys" end.
  • the chain may have, for example, a C-D sequence before the first A-B-C-D group.
  • other amino acid sequences may be attached to the "A" and/or the "D" end of one of these polypeptide chains.
  • amino acids in the chain which space one or more groups of the hereinabove noted four amino acids from each other.
  • the peptides may be produced by known techniques and
  • the peptides m y be Synthesized on an automatic synthesizer. Journal of the American Chemical Society, Vol. 85 Pages 2149-54(1963). It is also possible to produce such peptides by genetic engineering techniques.
  • the peptide employed in conjunction with a chelating agent such as those hereinabove described, or derivatives or analogues thereof may be a magainin peptide.
  • a magainin peptide is either a magainin such as Magainin I, II or III or an analogue or derivative thereof.
  • the magainin peptides may include the following basic peptide structure X 12 - - R 11 -R 11 -R 12 -R 13 -R 11 -R 14 -R 12 -R 11 -R 14 - R 12 -R 11 -R 11 -R 14a -(R 15)n -R 14a -R 14 - - wherein R 11 is a hydrophobic amino acid, R 12 is a basic hydrophilic amino acid; R 13 is a hydrophobic, neutral
  • R 14 and R 14a are hydrophobic or basic hydrophilic amino acids
  • R 15 is glutamic acid or aspartic acid, or a hydrophobic or basic hydrophilic amino acid
  • n is 0 or 1.
  • R 13 is a hydrophobic or neutral hydrophilic amino acid
  • R 14a is a hydrophobic or neutral hydrophilic amino acid
  • R 15 is glutamic acid or aspartic acid.
  • a magainin peptide may include the following structure:
  • R 11 , R 12 , R 14 , and R 14a are as previously defined.
  • a magainin peptide may also have the following structure:
  • X 12 is as previously defined and Z 12 is: (i) R 16 where R 16 is a basic hydrophilic amino acid or asparagine or glutamine; or
  • R 16 -R 17 where R 17 is a neutral hydrophilic amino acid, a hydrophobic amino acid, or a basic hydrophilic amino acid.
  • R 17 is a neutral hydrophilic amino acid.
  • a magainin peptide may also have the following structure:
  • the magainin peptides may also include the following basic peptide structure X 13 :
  • R 11 , R 12 , R 13 , R 14 , and R 14a are amino acids as hereinabove described.
  • the magainin peptide may also include the following
  • the magainin peptides generally include at least fourteen amino acids and may include up to forty amino acids.
  • a magainin peptide preferably has 22 or 23 amino acids. Accordingly, the hereinabove described basic peptide structures of a magainin peptide may include additional amino acids at the amino end or at the carboxyl end, or at both ends.
  • Magainin peptides are described in Proc. Natl. Acad Sci. Vol. 84 pp. 5449-53 (Aug. 1987).
  • magainin peptides refers to the basic magainin structure as well as derivatives and analogs thereof, including but not limited to the representative derivatives or analogs.
  • a chelating agent employed in conjunction with a chelating agent may be a PGLa peptide or an XPF peptide.
  • a PGLa peptide is either PGLa or an analogue or derivative thereof.
  • the PGLa peptides preferably include the following basic peptide structure X 14 :
  • R 11 , R 12 , R 14 , and R 17 are as previously defined.
  • the PGLa peptides generally include at least seventeen amino acids and may include as many as forty amino acids. Accordingly, the hereinabove described basic peptide structure for a PGLa peptide may include additional amino acids at the amino end or at the carboxyl end or at both the amino and carboxyl end.
  • a PGLa peptide may have the following structure:
  • Y 14 is (i) R 11 ;
  • R 11 is as previously defined.
  • a PGLa like peptide may also have the following structure:
  • R 11 is as previously defined.
  • a PGLa peptide may also have the following structure:
  • An XPF peptide is either XPF or an analogue, or derivative thereof.
  • the XPF peptides preferably include the following basic peptide structure X 16 :
  • R 11 , R 12 , R 14 , R 15 and R 17 are as previously defined and R 18 is glutamine or asparagine.
  • the XPF peptides generally include at least nineteen amino acids and may include up to forty amino acids. Accordingly, the hereinabove described basic peptide structure of XPF may include additional amino acids at the amino end, or at the carboxyl end or at both the amino and carboxyl ends.
  • an XPF peptide may include the following structure:
  • An XPF peptide may include the following structure:
  • An XPF peptide may also have the following structure:
  • X 16 , Y 16 , and Z 16 are as previously defined: a is 0 or
  • XPF or PGLa peptides which are characterized by the following as listed in the accompanying sequence listing:
  • the peptide employed in conjunction with a chelating agent such as those hereinabove described may be a CPF peptide or appropriate analogue or derviative thereof.
  • CPF peptides A basic CPF peptide structure as well as analogues and derivatives thereof are herein sometimes referred to collectively as CPF peptides.
  • the CPF peptide is preferably one which includes the
  • R 21 is a hydrophobic amino acid
  • R 22 is a hydrophobic amino acid or a basic hydrophilic amino acid
  • R 23 is a basic hydrophilic amino acid
  • R 24 is a hydrophobic or neutral hydrophilic amino acid
  • R 25 is a basic or neutral hydrophilic amino acid.
  • the hydrophobic amino acids may be Ala, Cys, Phe, Gly, lie, Leu, Met, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
  • the neutral hydrophilic amino acids may be Asn, Gln, Ser, and Thr.
  • the basic hydrophilic amino acids may be Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and
  • the CPF peptide may include only the hereinabove noted amino acids or may include additional amino acids at the amino end or carboxyl end or both the amino and carboxyl end. In general, the peptide does not include more than 40 amino acids.
  • the CPF peptides including the above basic peptide structure may have from 1 to 4 additional amino acids at the amino end.
  • Y 20 -X 20 - wherein X 20 is the hereinabove described basic peptide structure and Y 20 is
  • the carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13
  • the basic structure may have from 1 to 6 additional amino acids at the carboxyl end, which may be represented as follows:
  • X 20 is the hereinabove defined basic peptide structure and Z 20 is
  • R 21 and R 24 are as previously defined, and R 26 is proline or a hydrophobic amino acid.
  • Preferred peptides may be represented by the following structural formula:
  • X 20 , Y 20 and Z 20 are as Previously defined and a is 0 or 1 and b is 0 or 1.
  • CPF peptides which may be employed in the present invention are represented by the following:
  • CPF peptide includes the basic peptide structure as well as analogues or derivatives thereof.
  • the peptide may include one of the following basic structures X 31 through X 37 wherein:
  • X 31 is -[R 31 -R 32 -R 32 -R 33 -R 31 -R 32 -R 32 ]- n ;
  • X 32 is -[R 32 -R 32 -R 33 -R 31 -R 32 -R 32 -R 31 ]- n ;
  • X 33 is - [R 32 -R 33 -R 31 -R 32 -R 32 -R 31 -R 32 ]- n ;
  • X 34 is -[ R 33 -R 31 -R 32 -R 32 -R 31 -R 32 -R 32 ]- n ;
  • X 35 is -[ R 31 -R 32 -R 32 -R 31 -R 32 -R 32 -R 33 ]- n ;
  • X 36 is -[ R 32 -R 32 -R 31 -R 32 -R 32 -R 33 -R 31 ]- n ;
  • X 37 is -[R 32 -R 31 -R 32 -R 32 -R 33 -R 31 -R 32 ]- n ;
  • R 31 is a basic hydrophilic amino acid
  • R 32 is a
  • R 33 is a neutral hydrophilic or
  • n is from 2 to 5.
  • the basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
  • the hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, lie. Leu, Met, Val, Trp and Tyr,norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
  • the neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gln, Ser and Thr.
  • the peptide when the peptide includes the structure X 31 , the peptide may include the following
  • Y 31 -X 31 wherein X 31 is as hereinabove described, and Y 31 is:
  • the peptide when the peptide includes the structure X 31 , the peptide may include the following structure:
  • X 31 -Z 31 wherein X 31 is as hereinabove described, and Z 31 is: (i) R 31 ;
  • the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide may Include, the following structure:
  • the peptide when the peptide includes the structure X 33 , the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 33 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 34 , the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 34 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 35 , the peptide may include the following structure :
  • Y 35 -X 35 wherein X 35 is as hereinabove described, and Y 35 is:
  • the peptide when the peptide includes the structure X 35 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 36 , the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 36 , the peptide may include the following structure:
  • the peptide may include the following structure:
  • the peptide when the peptide includes the structure X 37 , the peptide may includes the structure
  • Y 37 -X 37 wherein X 37 is as hereinabove described, and Y 37 is:
  • the peptide when the peptide includes the structure X 37 , the peptide may include the following structure :
  • the peptide may include the following structure:
  • n 3 + preferably the peptide has one of the following structures:
  • the biologically active amphiphilic peptide includes the following basic structure X 40 :
  • the peptide may include the following structure:
  • Y 40 -X 40 wherein X 40 is as hereinabove described, and Y 40 is:
  • the peptide may include the following structure:
  • X 40 -Z 40 wherein X 40 is as hereinabove described and Z 40 is: (i ) R 31 ;
  • the peptide has the following structural formula as indicated in the sequence listing contained herein:
  • the peptide has the following structural formula as indicated in the sequence listing contained herein:
  • the peptide has one of the following structural formulae as indicated in the sequence listing contained herein:
  • n is from 2 to 5.
  • n is 3, and the peptide has the following structural formula:
  • the peptide is selected from the group consisting of the following structural formulae as given in the accompanying sequence listing:
  • the peptide includes the following basic structure X 50 :
  • R 41 is a hydrophobic amino acid
  • R 42 is a basic hydrophilic or neutral hydrophilic amino acid
  • the peptide includes the basic structure
  • Y 50 -X 50 wherein X 50 is as hereinabove described and Y 50 is:
  • R 41 is leucine.
  • R 42 is lysine.
  • Representative, examples of such peptides include those having the following structures:
  • the peptide includes the following basic structure X 52 :
  • R 41 is a hydrophobic amino acid
  • R 42 is a basic hydrophilic or neutral hydrophilic amino acid
  • R 41 is leucine. In another embodiment,
  • R 42 is lysine
  • the peptide includes the basic structure
  • Y 52 -X 52 wherein X 52 is as hereinabove described, and Y 52 is (i) R 42 ;
  • the peptide may have the following structure:
  • the peptide includes the basic structure X 52 -Z 52 , wherein X 52 is as hereinabove described, and
  • the peptide may have the following structure:
  • the peptide may include the
  • a is 0 or 1
  • b is 0 or 1.
  • the above peptides may be acetylated with a CH 3 CO-group at the N-terminal.
  • each of the amino acid residues may be a D-amino acid residue or glycine.
  • the peptide employed in conjunction with a chelating agent such as those hereinabove described, or derivatives or analogues thereof is a cecropin.
  • the cecropins and analogues and derivatives thereof are described in Ann. Rev. Microbiol 1987, Vol. 41 pages 103-26, in particular p. 108 and Christensen at al PNAS Vol. 85 p. 5072-76, which are hereby incorporated by reference.
  • cecropin includes the basic structure as well as analogues and derivatives.
  • a chelating agent such as those hereinabove described, or derivatives or analogues thereof is a sarcotoxin.
  • the sarcotoxins and analogues and derivatives thereof are
  • sarcotoxin includes the basic materials as well as analogues and derivatives.
  • Ion channel-forming proteins or peptides which may be employed include defensins, also known as human neutrophil antimicrobial peptides (HNP), major basic protein (MBP) of eosinophils, bactericidal permeability-increasing protein (BPI), a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein, lactoferrin, B- 2 -binding protein, eosinophil cationic protein (ECP), and eosinophil-derived
  • defensins also known as human neutrophil antimicrobial peptides (HNP), major basic protein (MBP) of eosinophils, bactericidal permeability-increasing protein (BPI), a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein, lactoferrin, B- 2 -binding protein, eos
  • Defensins are described in Selsted, et al., J. Clin. Invest., Vol. 76, pgs. 1436-1439 (1985).
  • MBP proteins are described in Wasmoen, et al., J. Biol. Chem., Vol. 263, pgs.
  • BPI proteins are described in Ooi, et al., J. Biol. Chem., Vol. 262, pgs. 14891-14894 (1981). Perform is described in Henkart, et al. J. Exp. Med., 160:75 (1984), and in Podack, et al. J. Exp. Med., 160:695 (1984). Lactoferrin,
  • B 12 -binding protein eosinophil cationic protein
  • eosinphil-derived neurotoxin are described in Elsbach, et al., Inflammation: Basic Principles and Clinical Correlates, Gallin, et al., eds.; pgs. 445-471 (1988). The above articles are hereby incorporated by reference.
  • ion channel-forming proteins includes the basic structures of the ion channel-forming proteins as well as
  • the method comprises administering to a host (human or animal) that is being treated with a biologically active peptide or protein an ion selected from the group consisting of calcium ions and magnesium ions.
  • the ion is administered in an amount effective to
  • Such a method is particularly applicable to a method of selective treatment.
  • the peptide or protein may be biologically effective in one or more body parts or organs of a host, but not in others.
  • the ion would be administered to those body parts or organs in which onedid not desire the peptide or protein to- have biological effect.
  • the ion may be administered in an amount up to about 2 grams/kg of host weight.
  • Peptide (1) is (SEQ ID NO: 27)
  • Peptide (2) is (SEQ ID NO: 97) amide-terminated
  • Peptide (3) is (LysIle Ala Gly LysIle Ala) 3 ,
  • each amino acid residue of Peptide (3) is a D-amino acid residue or glycine, were prepared at a concentration of 512 ⁇ g/ml in sterile deionized distilled water and stored at -70°C.
  • the stock peptide solutions are diluted in serial dilutions (1:2) down the wells of a microtiter plate so that the final concentrations of peptides in the wells are 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, 128, and 256 ⁇ g/ml. 1-5 x 10 5 CFUs/ml of P.
  • aeruginosa ATCC 27853 were added to the wells in full strength or half-strength Mueller Hinton broth (MHB), or in half-strength MHB broth to which 0.002 M Ca 2+ , or 0.002 M Mg 2+ , or 0.001 M Ca 2+ and 0.001M Mg 2+ has been added.
  • the organisms are from a mid-log culture.
  • the inoculum is standardized spectrophotometrically at
  • concentration is defined as the lowest concentration of peptide which produces a clear well in the microtiter plate, The MIC values are given in Table I below.
  • Example 2 The procedure of Example 1 was repeated, except that the assays were carried out in full strength Mueller Hinton Broth (MHB) or cation-adjusted Mueller Hinton Broth (CAMHB). Peptides (1) and (2) were tested for MIC against P. aeruginosa 27853 in each of these broths without further additives, and in each of these broths to which was added 0.05mM, 0.5mM, or 5mM of the calcium channel blockers verapamil or diltiazem. The results are given in Table II below.
  • Peptides (1), (2), (4), (5), and (6) were tested for activity against P. aeruginosa strain 27853 in full or
  • Peptide (4) is amide-terminated Magainin II (SEQ ID NO: 7).
  • Peptide (5) has the following structural formula:
  • Peptide (6) is SEQ ID NO:21, amide-terminated.
  • the results of this assay are given in Table III below:
  • the checkerboard assay was carried out in a 96-well
  • microtiter plate having 12 rows and 8 columns of wells. 100 ⁇ l of plain broth is added to every row of wells. 100 ⁇ l of the desired peptide at 512 ⁇ g/ml is added to the top well, and serially diluted through row 11. 50 ⁇ l of the EDTA in varying
  • Peptides (1) and (2) were tested alone or in combination with EDTA for activity against P. aeruginosa strain 27853.
  • the MIC of Peptide (1) was 64 ⁇ g/ml, and the MIC of EDTA was 4mM.
  • FIC Fractional Inhibitory Index
  • FIC MIC of peptide in combination MIC of EDTA in combination _
  • An FIC value of 0.5 or less is indicative of synergy, a value of greater than 0.5 but less than 2 is indicative of indifference, and a value greater than 2 is indicative of antagonism.
  • Peptide (1) and EDTA were found to be inhibitory, and the FIC values of each combination are given herewith.
  • the assay was also performed to determine the MIC values of Peptide (2), EDTA, and inhibitory combinations thereof.
  • the MIC of Peptide (2) against P. aeruginosa was 4 ⁇ g/ml, and the MIC of EDTA was 8mM.
  • the following combinations of Peptide (2) and EDTA were also found to be inhibitory, and FIC values are given therefor:
  • aeruginosa strain 27853 as employed alone or in combination with 3mM, or 4mM or 5mM EGTA.
  • the procedure followed was that of Example 1, and all testing was done using P. aeruginosa grown in full strength MHB broth. The results of this assay are given in Table IV below.
  • Example 5 The procedure of Example 5 was repeated, except the MIC values of Peptides (1) and (2) were determined for Peptides (1) and (2) alone and in combination with 0.0625mM, 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, or 3mM EGTA. The results of this assay are given in Table V below.
  • ADDRESSEE Carella, Byrne, Bain, Gilfillan,
  • NAME/KEY Magainin II peptide.
  • NAME/KEY magainin peptide
  • NAME/KEY magainin peptide
  • NAME/KEY magainin peptide

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Abstract

Combinations of biologically active amphiphilic peptides or proteins and chelating agents are useful as antimicrobial agents.

Description

COMPOSITION AND TREATMENT WITH
BIOLOGICALLY ACTIVE PEPTIDES AND CHELATING AGENTS
This invention relates to biologically active peptides and proteins, and more particularly to compositions and uses
involving biologically active peptides and chelating agents.
In accordance with an aspect of the present invention, there is provided a composition comprising at least one biologically active amphiphilic peptide or biologically active protein, and a chelating agent. The peptide or protein is preferably an ion channel-forming peptide or protein.
In accordance with another aspect of the present invention, there is provided a process of inhibiting growth of a target cell in a host which comprises administering to a host at least one biologically active amphiphilic peptide or biologically active protein and a chelating agent. The peptide or protein is
preferably an ion channel-forming peptide or protein. The biologically active amphiphilic peptide or biologically active protein and the chelating agent are administered in amounts effective to inhibit growth of a target cell in a host.
Although the scope of the present invention is not intended to be limited to any theoretical reasoning, Applicant believes that certain ions such as, for example, calcium ions and
magnesium ions, decrease the activity of certain biologically active amphiphilic peptides as antibacterial agents. It is believed that such decrease in activity may be due to competition between the ions, which are positively charged, and the peptides for negative charges on the bacteria. Applicant also has found that when a chelating agent, which binds ions such as calcium and/or magnesium ions, is added to the biologically active amphiphilic peptide, the activity of the biologically active peptide is enhanced, or potentiated, whereas when known calcium channel-blocking agents, such as verapamil and diltiazem, are added to such peptides, no improvement in the biological activity of the peptides was obtained.
The term "potentiate", as used herein, means either that the biologically active amphiphilic peptide or protein is effective in increasing the biological activity of the chelating agents against a target cell so thereby the chelating agent may be employed in an amount lower than which would be required for preventing, destroying or inhibiting growth of a target cell and/or that the peptide or protein may be employed in an amount lower than which would be required for preventing, destroying, or inhibiting growth of a target cell.
Chelating agents which may be employed in accordance with the present invention are agents which are capable of chelating calcium. Such chelating agents may also increase the
permeability of the peptide or protein through the membrane of a target cell, such as a bacterium, as described in Marvin, et al., "Enhanced Penetration of Externally Added Macromolecules Through the Outer Membrane of Gram-Negative Bacteria," found in Actor, et al. (eds.), Antibiotic Inhibition of Bacterial Cell Surface
Assembly and Eunction, (1988), pgs. 431-435. Such chelating agents include ethylene dinitrilo tetraacetic acid (EDTA), and ethylene glycol bis-β-aminoethyl ether N,N,N',N'-tetraacetic acid (EGTA).
The biologically active amphiphilic peptides employed in the present invention are generally water soluble to a concentration of at least 20 mg/ml at neutral pH in water. In addition, the structure of such peptide provides for flexibility of the peptide molecule. When the peptide is placed in water, it does not assume an amphiphilic structure. When the peptide encounters an oily surface or membrane, the peptide chain folds upon itself into a rod-like structure, sometimes referred to as an α-helical structure.
In general, such peptides have at least 10 amino acids, and preferably at least 20 amino acids. In most cases, such peptides do not have in excess of 50 amino acids.
In general, the biologically active peptides or proteins employed in the present invention are ion channel-forming
peptides or proteins. An ion channel-forming peptide or protein or ionophore is a peptide or protein which increases the
permeability for ions across a natural or synthetic lipid
membrane. B. Christensen et al . PNAS Vol. 85 P. 5072-76 (July, 1988) describes methodology which indicates whether or not a peptide or protein has ion channel-forming properties and is therefore an ionophore. As used herein an ion channel-forming peptide or protein is a peptide or protein which has ion
channel-forming properties as determined by the method of
Christensen et al.
An amphiphilic peptide or protein is a peptide which
includes both hydrophobic and hydrophilic peptide regions.
The administration of the biologically active amphiphilic peptides or proteins and chelating agent to a target cell may be by direct administration to the target cell or systemic or topical administration to a host which includes the target cell, in order to prevent, destroy, or inhibit the growth of a target cell.
The compositions of the present invention may be
administered to a host; for example a human or non-human animal, in an amount effective to inhibit growth of a target cell. Thus, for example, the compositions may be used as antimicrobial agents, or in particular as anti-bacterial agents, or as
antibiotics.
The term "antimicrobial" as used herein means that the compositions of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes such as, for example, bacteria.
The term "antibiotic" as used herein means that the
compositions employed in the present invention produce effects adverse to the normal biological functions of the target non-host cell, including death or destruction and prevention of the growth or proliferation of the non-host target cell when contacted with the compositions.
The compositions of the present invention have a broad range of potent antibiotic activity against a plurality of
microorganisms including Gram-positive and Gram-negative
bacteria. The compositions of the present invention allow a method for treating or controlling microbial infection caused by organisms which are sensitive to the peptides herein described. Such treatment may comprise administering to a host organism or tissue susceptible to or affiliated with a microbial infection an antimicrobial amount of at least one of the peptides and a chelating agent.
Because of the antibiotic and antimicrobial properties of the compositions, they may also be used as preservatives or sterilants of materials susceptible to microbial contamination.
The compositions may be administered in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution. Such
pharmaceutical compositions may be used topically or systemically and may be in any suitable form such as a liquid, solid,
semi-solid, injectable solution, tablet, ointment, lotion, paste, capsule, or the like. The compositions may also be used in combination with adjuvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful
microorganisms including bacteria.
The compositions of the present invention may be
administered to a host; in particular an animal, in an effective antibiotic and/or anti-microbial amount.
Depending on the use, a composition in accordance with the invention will contain an effective anti-microbial amount of one or more of the hereinabove described peptides which have such activity.
The compositions of the present invention may be used in the treatment of external burns and to treat and/or prevent skin and burn infections. In particular, the compositions may be used to treat skin and burn infections caused by organisms such as, but not limited to, P. aeruginosa, S. aureus, and other Streptococcus species.
The compositions are also useful in the prevention or treatment of eye infections. Such infections may be caused by bacteria such as, but not limited to, P. aeruginosa, S. aureus, and N. gonorrhoeae.
The compositions may also be administered to plants in an effective antimicrobial amount to prevent or treat microbial contamination thereof.
In general, the peptide or protein is employed to provide peptide dosages of from 1 mg to 500 mg per kilogram of host weight, when administered systemically. When administered topically, the peptide or protein is used in a concentration of from .05% to 10%.
The chelating agent, such as those hereinabove described, or derivatives or analogues thereof, when used topically, is
generally employed in a concentration of about .001% to about 25%. When used systemically, the chelating agent is generally employed in an amount of from 1 mg/kg to about 2 grams/kg of host weight per day. In accordance with a preferred embodiment, the peptide used in conjunction with a chelating agent such as those hereinabove described, or derivatives or analogues thereof is a basic
(positively charged) polypeptide having at least ten amino acids wherein the polypeptide includes both hydrophobic and hydrophilic amino acids. In one embodiment, the polypeptide may have at least sixteen amino acids wherein the polypeptide includes at least eight hydrophobic amino acids and at least eight
hydroplilic amino acids. Still more particularly, the
hydrophobic amino acids are in groups of two adjacent amino acids, and each group of two hydrophobic amino acids is spaced from another group of two hydrophobic amino acids by at least one amino acid other than a hydrophobic amino acid (preferably at least two amino acids) and generally by no greater than four amino acids, and the amino acids between pairs of hydrophobic amino acids may or may not be hydrophilic.
The hydrophilic amino acids are generally also in groups of two adjacent amino acids in which at least one of the two amino acids is a basic hydrophilic amino acid, with such groups of two hydrophilic amino acids being spaced from each other by at least one amino acid other than a hydrophilic amino acid (preferably at least two amino acids) and generally no greater than four amino acids, and the amino acids between pairs of hydrophilic amino acids may or may not be hydrophobic.
In accordance with a particularly preferred embodiment, the polypeptide comprises a chain of at least four groups of amino acids, with each group consisting of four amino acids. Two of the four amino acids in each group are hydrophobic amino acids, and two of the four amino acids in each group are hydrophilic, with at least one of the hydrophilic amino acids in each group being a basic hydrophilic amino acid and the other being a basic or neutral hydrophilic amino acid.
The hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, lie, Leu, Met, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha). The neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gln, Ser, and Thr. The basic
hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
Each of the groups of four amino acids may be of the sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each hydrophobic amino acids and may be the same or different, one of C or D is a basic hydrophilic amino acid, and the other of C or D is a basic or neutral hydrophilic amino acid and may be the same or different. In a preferred embodiment, the polypeptide chain may comprise 5 or 6 groups of this sequence. In each group, each of A, B, C and D may be the same in some or all of the groups or may be different in some or all of the groups.
The polypeptide chain preferably has at least 20 amino acids, and no greater than 50 amino acids. It is to be
understood, however, that the polypeptide does not have to consist entirely of the groups described above. The polypeptide may have amino acids extending from either or both ends of the noted groups forming the polypeptide chain and/or there may be amino acids between one or more of the at least four groups and still remain within the scope of the invention.
The groups of amino acids may be repeating groups of amino acids, or the amino acids in the various groups may vary provided that in each group of the at least four groups of amino acids there are two hydrophobic and two hydrophilic amino acids as hereinabove noted.
Thus, in a preferred embodiment, the biologically active polypeptide comprises a chain including at least four groups of amino acids, each containing four amino acids. Two of the four amino acids in each. group are hydrophobic, at least one amino acid is basic hydrophilic, and the remaining one is basic or neutral hydrophilic, with the polypeptide chain preferably having at least 20 amino acids but no greater than 50 amino acids.
In one embodiment, each of the at least four groups of amino acids which are in the peptide chain is of the sequence A-B-C-D, B-C-D-A, C-D-A-B or D-A-B-C wherein A and B are hydrophobic amino acids, one of C or D is basic hydrophilic amino acid, and the other of C or D is basic or neutral hydrophilic amino acid. The resulting polypeptide chain, therefore, may have one of the following sequences:
(X1)a(A-B-C-D)n(Y1)b
(X2)a(B-C-D-A)n(Y2)b
(X3)a(C-D-A-B)n(Y3)b
(X4)a(D-A-B-C)n(Y4)b wherein X, is D; C-D- or B-C-D-, Y1 is -A or
-A-B or -A-B-C
X2 is A-, D-A- or C-D-A-
Y2 is -B, -B-C or B-C-D
X3 is B-, A-B-, D-A-B-
Y3 is -C, -C-D, -C-D-A
X4 is C-, B-C-, A-B-C-
Y4 is -D, -D-A, -D-A-B
a is 0 or 1; b is 0 or 1
and n is at least 4.
It is to be understood that the peptide chain may include amino acids between the hereinabove noted groups of four amino acids provided that the spacing between such groups and the charge on the amino acids does not change the characteristics of the peptide chain which provide amphiphilicity and a positive charge and do not adversely affect the folding characteristics of the chain to that which is significantly different from one in which the hereinabove noted group of four amino acids are not spaced from each other. As representative examples of peptides in accordance with the present invention, there may be mentioned.
I Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser- Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys (SEQ ID
NO:1)
II Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser- Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe- Ser-Lys-(SEQ ID NO: 2)
III Phe-Ser-Lys-Ala-Phe-Ser- Lys-Ala-Phe-Ser-Lys-Ala- Phe-Ser-Lys-Ala-(SEQ ID NO: 3)
IV Ser-Lys-Ala-Phe-Ser-Lys-Ala- Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala- Phe-Ser-Lys-Ala-Phe-(SEQ ID NO: 4)
V Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser- Lys-Ala-Phe-Ser-(SEQ ID NO: 5)
The peptide may have amino acids extending from either end of the chain. For example, the chains may have a Ser-Lys sequence before the "Ala" end, and/or an Ala-Phe sequence after the "Lys" end. Other amino acid sequences may also be attached to the "Ala" and/or the "Lys" end.
Similarly, in any polypeptide chain having at least four groups of amino acids of the sequence as described above, the chain may have, for example, a C-D sequence before the first A-B-C-D group. Also other amino acid sequences may be attached to the "A" and/or the "D" end of one of these polypeptide chains. Also there may be amino acids in the chain which space one or more groups of the hereinabove noted four amino acids from each other.
The peptides may be produced by known techniques and
obtained in substantially pure form. For example, the peptides m y be Synthesized on an automatic synthesizer. Journal of the American Chemical Society, Vol. 85 Pages 2149-54(1963). It is also possible to produce such peptides by genetic engineering techniques.
In accordance with another preferred embodiment, the peptide employed in conjunction with a chelating agent such as those hereinabove described, or derivatives or analogues thereof may be a magainin peptide.
A magainin peptide is either a magainin such as Magainin I, II or III or an analogue or derivative thereof. The magainin peptides may include the following basic peptide structure X12 - - R11-R11-R12-R13-R11-R14-R12-R11-R14- R12-R11-R11-R11-R14a-(R15)n-R14a-R14 - - wherein R11 is a hydrophobic amino acid, R12 is a basic hydrophilic amino acid; R13 is a hydrophobic, neutral
hydrophilic, or basic hydrophilic amino acid; R14 and R14a are hydrophobic or basic hydrophilic amino acids, R15 is glutamic acid or aspartic acid, or a hydrophobic or basic hydrophilic amino acid, and n is 0 or 1. In a preferred embodiment, R13 is a hydrophobic or neutral hydrophilic amino acid, R14a is a
hydrophobic amino acid, and R15 is glutamic acid or aspartic acid.
Thus, for example, a magainin peptide may include the following structure:
Y12 - X12,
where X12 is the hereinabove described basic peptide
structure and Y12 is
(i) R12
(ii) R14a - R12;
(iii) R11-R14a-R12; or
(iv) R14-R11-R14a-R12
where R11 , R12, R14, and R14a are as previously defined.
A magainin peptide may also have the following structure:
-X12-Z12- wherein X12 is as previously defined and Z12 is: (i) R16 where R16 is a basic hydrophilic amino acid or asparagine or glutamine; or
(ii) R16-R17 where R17 is a neutral hydrophilic amino acid, a hydrophobic amino acid, or a basic hydrophilic amino acid. Preferably, R17 is a neutral hydrophilic amino acid.
A magainin peptide may also have the following structure:
(Y12)a - X12 - (Z12)b
where X12, Y12, and Z12 are as previously defined, and a is
0 or 1 and b is 0 or 1.
The magainin peptides may also include the following basic peptide structure X13:
R14-R11-R14a-R12-R11-R11-R12-R13-R11-R14- R12-R11-R11-R12-,
wherein R11, R12, R13, R14, and R14aare amino acids as hereinabove described.
The magainin peptide may also include the following
structure X13-Z13; wherein X13 is the hereinabove described basic peptide structure and Z13 is
(R11)n-R11)n-(R11)n-(R14a)n-(R15)n-(R14a)n-
(R14)n-(R16)Rn1-6(R17)n-, wherein R11, R14, R14a, R15, R16, and R17 are amino acids as hereinabove described, and n is 0 or 1, and each n may be the same or different.
The magainin peptides generally include at least fourteen amino acids and may include up to forty amino acids. A magainin peptide preferably has 22 or 23 amino acids. Accordingly, the hereinabove described basic peptide structures of a magainin peptide may include additional amino acids at the amino end or at the carboxyl end, or at both ends.
As representative examples of such magainin peptides, there may be mentioned the following peptides, the structures of which are given in the accompanying sequence listing as well as
appropriate analogues and derivatives thereof:
(a) (NH2) (SEQ ID NO:6) (OH) or (NH2)
(Magainin I) (b) (NH2) (SEQ ID NO: 7) (OH) or (NH2)
(Magainin II)
(c) (NH2) (SEQ ID NO:8) (OH) or (NH2)
(Magainin III)
The following are examples of peptide derivatives or analogs of the basic structure:
(d) (NH2) (SEQ ID NO:9) (OH) or (NH2)
(e) (NH2) (SEQ ID NO: 10) (OH) or (NH2)
(f) (NH2) (SEQ ID NO: 11) (OH) or (NH2)
Magainin peptides are described in Proc. Natl. Acad Sci. Vol. 84 pp. 5449-53 (Aug. 1987). The term "magainin peptides" as used herein refers to the basic magainin structure as well as derivatives and analogs thereof, including but not limited to the representative derivatives or analogs.
In accordance with a further embodiment, the peptide
employed in conjunction with a chelating agent may be a PGLa peptide or an XPF peptide.
A PGLa peptide is either PGLa or an analogue or derivative thereof. The PGLa peptides preferably include the following basic peptide structure X14:
- R11-R17-R12-R11-R14-R11-R11- R11-R14-R12-R11-R11-R12-R11- R11-R11-R12- where R11, R12, R14, and R17 are as previously defined.
The PGLa peptides generally include at least seventeen amino acids and may include as many as forty amino acids. Accordingly, the hereinabove described basic peptide structure for a PGLa peptide may include additional amino acids at the amino end or at the carboxyl end or at both the amino and carboxyl end.
Thus, for example, a PGLa peptide may have the following structure:
-Y14-X14- where X14 is as previously defined and
Y14 is (i) R11;
(ii) R14- R11
where R11 is as previously defined.
For example, a PGLa like peptide may also have the following structure:
-X14-Z14- where X14 is as previously defined; and Z14 is :
(i) R11; or
(ii) R11-R11
where R11 is as previously defined.
A PGLa peptide may also have the following structure:
(Y14 )a-X14-(Z14)b
where X 14; Y14 and Z14 are as previously defined, a is 0 or
1 and b is 0 or 1.
An XPF peptide is either XPF or an analogue, or derivative thereof. The XPF peptides preferably include the following basic peptide structure X16 :
--R11-R17-R12-R11-R14-R18-R17- R11-R14-R12-R11-R11-R12- R11-R11- R11-R12-R15-R11--,
wherein R11, R12, R14, R15 and R17 are as previously defined and R18 is glutamine or asparagine.
The XPF peptides generally include at least nineteen amino acids and may include up to forty amino acids. Accordingly, the hereinabove described basic peptide structure of XPF may include additional amino acids at the amino end, or at the carboxyl end or at both the amino and carboxyl ends.
Thus, for example, an XPF peptide may include the following structure:
-Y16-X16- where X16 is as previously defined and Y16 is
(i) R11 or
(ii) R14-R11 where R11 and R14 are is as previously defined.
An XPF peptide may include the following structure:
-X16-Z16- where X16 is as previously defined and Z16 is
(i) R11; or
(ii) R11-R18; or
(iii) R11-R18-Proline; or
(iv) R11-R18-Proline-R12
An XPF peptide may also have the following structure:
(Y16)a-X16(Z16)b
where X16, Y16 , and Z16 are as previously defined: a is 0 or
1 and b is 0 or 1.
Preferred are XPF or PGLa peptides, which are characterized by the following as listed in the accompanying sequence listing:
PGLa : (SEQ ID NO: 12) (NH2)
XPF : (SEQ ID NO: 13)
A review of XPF and PGLa can be found in Hoffman et al, EMBO J. 2:711-714, 1983; Andreu et al, J. Biochem. 149:531-535, 1985; Gibson et al J. Biol. Chem. 261:5341-5349, 1986; and Giovannini et al, Biochem J. 243 : 113-120, 1987.
In accordance with yet another embodiment, the peptide employed in conjunction with a chelating agent such as those hereinabove described, may be a CPF peptide or appropriate analogue or derviative thereof.
A basic CPF peptide structure as well as analogues and derivatives thereof are herein sometimes referred to collectively as CPF peptides.
The CPF peptide is preferably one which includes the
following peptide structure X20:
-R21-R21-R22-R22-R21-R21-R23-R21- -R21-R21-R23-R21-R21-R24-R25-R21- wherein R21 is a hydrophobic amino acid; R22 is a hydrophobic amino acid or a basic hydrophilic amino acid;
R23 is a basic hydrophilic amino acid; and
R24 is a hydrophobic or neutral hydrophilic amino acid; and
R25 is a basic or neutral hydrophilic amino acid.
The hereinabove basic structure is hereinafter symbolically indicated as X20.
The hydrophobic amino acids may be Ala, Cys, Phe, Gly, lie, Leu, Met, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
The neutral hydrophilic amino acids may be Asn, Gln, Ser, and Thr.
The basic hydrophilic amino acids may be Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and
-aminophenylalanine.
The CPF peptide may include only the hereinabove noted amino acids or may include additional amino acids at the amino end or carboxyl end or both the amino and carboxyl end. In general, the peptide does not include more than 40 amino acids.
The CPF peptides including the above basic peptide structure may have from 1 to 4 additional amino acids at the amino end.
Accordingly, such preferred peptides may be represented by the structural formula:
Y20-X20- wherein X20 is the hereinabove described basic peptide structure and Y20 is
(i) R25-, or
(ii) R22-R25; or
(iii) R21-R22-R25; or
(iv) R22-R21-R22-R25; preferably
Glycine -R21-R22-R25- wherein R21, R22, and R25 are as Previously defined.
The carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13
additional amino acids. In a preferred embodiment, the basic structure may have from 1 to 6 additional amino acids at the carboxyl end, which may be represented as follows:
-X20-Z20 wherein
X20 is the hereinabove defined basic peptide structure and Z20 is
(i) R21-,
(ii) R21-R21-;
(iii) R21-R21-R24;
(iv) R21-R21-R24-R24;
(v) R21-R21-R24-R24-R26;
(vi) R21-R21-R24-R24-R26-Gln; or
(vii) R21-R21-R24-R24-R26-Gln-Gln,
wherein R21 and R24 are as previously defined, and R26 is proline or a hydrophobic amino acid.
Preferred peptides may be represented by the following structural formula:
(Y20)a-X20-(Z20)b
wherein X20, Y20 and Z20 are as Previously defined and a is 0 or 1 and b is 0 or 1.
Representative examples of CPF peptides which are useful in the present invention have been described in the literature and comprise the following sequences the structures of which are given in the accompanying sequence listing:
(SEQ ID NO: 14)
(SEQ ID NO:15)
(SEQ ID NO: 16)
(SEQ ID NO: 17)
(SEQ ID NO: 18)
(SEQ ID NO: 19)
(SEQ ID NO:20.)
(SEQ ID NO:21)
(SEQ ID NO:22) ( SEQ ID NO : 23 )
( SEQ ID NO : 24 )
( SEQ ID NO : 25 )
( SEQ ID NO : 26 )
A review of the CPF peptides can be found in Richter, K., Egger, R., and Kreil (1986) J. Biol. Chem. 261, 3676-3680;
Wakabayashi, T. Kato, H., and Tachibaba, S. (1985) Nucleic Acids Research 13, 1817-1828; Gibson, B.W., Poulter, L., Williams, D.H., and Maggio, J.E. (1986) J. Biol. Chem. 261, 5341-5349.
CPF peptides which may be employed in the present invention are represented by the following:
Glyl2Ser3LeuGly4AlaLeuLysAla5LeuLysIleGly678LeuGlyGly9(10)
GlnGln
Where :
1 = Phe, Leu
2 = Gly, Ala
3 = Phe, Leu
4 = Lys, Leu
5 = Ala, Gly, Thr
6 = Ala, Thr
7 = His, Asn
8 = Ala, Met, Phe, Leu
9 = Ala, Ser, Thr
10 = Pro, Leu
The numbered amino acids may be employed as described in any combination to provide either a basic CPF peptide structure or an analogue or derivative. The term CPF peptide includes the basic peptide structure as well as analogues or derivatives thereof.
In accordance with yet another embodiment, the peptide may include one of the following basic structures X31 through X37 wherein:
X31 is -[R31-R32-R32-R33-R31-R32-R32]-n;
X32 is -[R32-R32-R33-R31-R32-R32-R31]-n; X33 is - [R32-R33-R31-R32-R32-R31-R32]-n;
X34 is -[ R33-R31-R32-R32-R31-R32-R32]-n;
X35 is -[ R31-R32-R32-R31-R32-R32-R33]-n;
X36 is -[ R32 -R32-R31-R32-R32-R33-R31]-n; and
X37 is -[R32-R31-R32-R32-R33-R31-R32]-n;
wherein R31 is a basic hydrophilic amino acid, R32 is a
hydrophobic amino acid, R33 is a neutral hydrophilic or
hydrophobic amino acid, and n is from 2 to 5.
The basic hydrophilic amino acids may be selected from the class consisting of Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
The hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, lie. Leu, Met, Val, Trp and Tyr,norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
The neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gln, Ser and Thr.
In accordance with one embodiment, when the peptide includes the structure X31, the peptide may include the following
structure:
Y31-X31, wherein X31 is as hereinabove described, and Y31 is:
(i) R32;
(ii) R32-R32;
(iii) R31-R32-R32;
(iv) R33-R31-R32-R32;
(v) R32-R33-R31-R32-R32; or
(vi) R32-R32-R33-R31-R32-R32, wherein R31, R32, and R33 are as hereinabove described
In accordance with another embodiment, when the peptide includes the structure X31, the peptide may include the following structure:
X31-Z31, wherein X31 is as hereinabove described, and Z31 is: (i) R31;
R31;
(ii) R31-R32;
(iii) R31-R32-R32;
(iv) R31-R32-R32-R33;
(v) R31-R32-R32-R33-R31; or
(vi) R31-R32-R32-R33-R31-R32.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y31)a -X31-(Z31)b, wherein Y31 and Z31 are as previously defined, a is 0 or 1, and b is 0 or 1.
When the peptide includes the structure X32, the peptide may include the following structure:
Y32 - X32, wherein X32 is as hereinabove described, and Y32 is:
(i) R31;
(ii) R32-R31;
(iii) R32-R32-R31;
(iv) R31-R32-R32-R31;
(v) R33-R31-R32-R32-R31; or
(vi) R32-R33-R31-R32-R32-R31.
In another embodiment, when the peptide includes the
structure X32, the peptide may include the following structure:
X32 - Z32, wherein X32 is as hereinabove described, and Z32 is:
(i) R32;
(ii) R32-R32;
(iii) R32-R32-R33;
(iv) R32-R32-R33-R31;
(v) R32-R32-R33-R31-R32; or
(vi) R32-R32-R33-R31-R32-R32.
In accordance with yet another embodiment, the peptide may Include, the following structure:
(Y32)a - X32 - (Z32)b, wherein Y32 and Z32 are as Previously defined, a is 0 or 1, and b is 0 or 1. In accordance with another embodiment, when the peptide includes the structure X33, the peptide may include the following structure:
Y33 - X33 wherein X33 is as hereinabove described, and Y33 is:
(i) R32;
(ii) R31-R32;
(iii) R32-R31-R32;
(iv) R32-R32-R31-R32;
(v) R31-R32-R32-R31-R32; or
(vi) R33-R31-R32-R32-R31-R32, wherein R31, R32, and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X33, the peptide may include the following structure:
X33 - Z33 wherein X33 is as hereinabove described, and Z33 is:
(i) R32;
(ii) R32-R33;
(iii) R32-R33-R31;
(iv) R32-R33-R31-R32;
(v) R32-R33-R31-R32-R32; or
(vi) R32-R33-R31-R32-R32-R31.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y33)a - X33 - (Z33)b, wherein Y33 and Z33 are as Previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with yet another embodiment, when the peptide includes the structure X34, the peptide may include the following structure:
Y34 - X34, wherein X34 is as hereinabove described, and Y34 is:
(i) R32. (ii) R32-R32 ;
( iii ) R31-R32-R32 ;
(iv) R32-R31-R32-R32 ;
(v) R32-R32-R31-R32-R32; or
(vi) R31-R32-R32-R31-R32-R32, wherein R31, R32 and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X34, the peptide may include the following structure:
X34-Z34, wherein X34 is as hereinabove described, and Z34 is:
(i) R33;
(ii) R33-R31;
(iii) R33-R31-R32;
(iv) R33-R31-R32-R32;
(v) R33-R31-R32-R32-R31; or
(vi) R33-R31-R32-R32-R32-R32.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y34)a- X34- (Z34)b, wherein X34 and Z34 are as previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with a further embodiment, when the peptide includes the structure X35, the peptide may include the following structure :
Y35-X35, wherein X35 is as hereinabove described, and Y35 is:
(i) R33;
(ii) R32-R33;
(iii) R32-R32-R33;
(iv) R31-R32-R32-R33;
(v) R32-R31-R32-R32-R33; or
(vi) R32-R32-R31-R32-R32-R33, wherein R31, R32, and R33 are as hereinabove described. In accordance with another embodiment, when the peptide includes the structure X35, the peptide may include the following structure:
X35 - Z35 wherein X35 is as hereinabove described, and Z35 is:
(i) R31;
(ii) R31-R32;
(iii) R31-R32-R32;
(iv) R31-R32-R32-R31;
(v) R31-R32-R32-R31-R32; or
(vi) R31-R32-R32-R31-R32-R32.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y35)a- X35 (Z35)b , wherein X35 and Z35 are as previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with a further embodiment, when the peptide includes the structure X36, the peptide may include the following structure:
Y35 - X36 wherein X36 is as hereinabove described, and Y36 is:
(i) R31;
(ii) R33-R31;
(iii) R32-R33-R31;
(iv) R32-R32 -R33-R31;
(v) R31-R32-R32-R33-R31; or
(vi) R32-R31-R32-R32-R33-R31, wherein R31, R32, and R33 are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X36, the peptide may include the following structure:
X36-Z36, wherein X36 is as hereinabove described, and Z36 is:
(i) R32;
(ii) R32-R32; (iii ) R32-R32-R31 ;
( iv) R32-R32-R31-R32 ;
(v) R32-R32-R31-R32-R32; or
(vi) R32-R32-R31-R32-R32-R33.
In accordance with yet another embodiment, the peptide may include the following structure:
( Y36)a- X36 (Z36)b, wherein Y36 and Z36 are as Previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with one embodiment, when the peptide includes the structure X37, the peptide may includes the structure
Y37-X37, wherein X37 is as hereinabove described, and Y37 is:
(i) R32;
(ii) R31-R32;
(iii) R33-R31-R32;
(iv) R32-R33-R31-R32;
(v) R32-R32-R33-R31-R32; or
(vi) R31-R32-R32-R33-R31-R32, wherein R31, R32, and R33 are as hereinabove described.
In accordance with a further embodiment, when the peptide includes the structure X37, the peptide may include the following structure :
X37 - Z37 wherein X37 is as hereinabove described, and Z37 is:
(i) R32;
(ii) R32-R31;
(iii) R32-R31-R32;
(iv) R32-R31-R32-R32;
(v) R32-R31-R32-R32-R33; or
(vi) R32-R31-R32-R32-R33-R31.
In accordance with yet another embodiment, the peptide may include the following structure:
(Y37) - X37 (Z37)b, wherein Y37 and Z37 are as previously defined, a is 0 or 1, and b is 0 or 1. In a preferred embodiment, n is 3, and most preferably the peptide has one of the following structures:
(Lys Ile Ala Gly Lys Ile Ala)3-NH2 (SEQ ID NO: 27)
(Lys Ile Ala Lys Ile Ala Gly)3-NH2 (SEQ ID NO:28)
(Lys Ile Ala Gly Lys Ile Gly)3-NH2 (SEQ ID NO:29)
(Lys Leu Ala Gly Lys Leu Ala)3-NH2 (SEQ ID NO: 30)
(Lys Phe Arg Gly Lys Phe Ala)3-NH2 (SEQ ID NO: 31)
(Lys Ala Leu Ser Lys Ala Leu)3-NH2 (SEQ ID NO: 32)
(Lys Leu Leu Lys Ala Leu Gly)3-NH2 (SEQ ID NO: 33)
(Lys Ala Ile Gly Lys Ala Ile)3-NH2 (SEQ ID NO:34)
(Gly Ile Ala Lys Ile Ala Lys)3-NH2 (SEQ ID NO: 35)
(Lys Ile Ala Lys Ile Phe Gly)3-NH2 (SEQ ID NO: 36)
(Gly Ile Ala Arg Ile Ala Lys)3-NH2 (SEQ ID NO:37)
(Lys Phe Ala Arg Ile Ala Gly)3-NH2 (SEQ ID NO:38)
(Gly Phe Ala Lys Ile Ala Lys)3-NH2 (SEQ ID NO: 39)
(Lys Ile Ala Gly Orn Ile Ala)3-NH2 (SEQ ID NO: 40)
(Lys Ile Ala Arg Ile Ala Gly)3-NH2 (SEQ ID NO: 41)
(Orn Ile Ala Gly Lys Ile Ala)3-NH2 (SEQ ID NO:42)
(Gly Ile Ala Arg Ile Phe Lys)3-NH2 (SEQ ID NO: 43)
(Lys Nle Ala Gly Lys Nle Ala)3-NH2 (SEQ ID NO: 44)
(Lys Nle Ala Gly Lys Ile Ala)3-NH2 (SEQ ID NO:45)
(Lys Ile Ala Gly Lys Nle Ala)3-NH2 (SEQ ID NO:46)
(Lys Nva Ala Gly Lys Nva Ala)3-NH2 (SEQ ID NO: 47)
(Lys Nva Ala Gly Lys Ile Ala)3-NH2 (SEQ ID NO: 48)
(Lys Leu Leu Ser Lys Leu Gly)3-NH2 (SEQ ID NO: 49)
(Lys Leu Leu Ser Lys Phe Gly)3-NH2 (SEQ ID NO:50)
(Lys Ile Ala Gly Lys Nva Ala)3-NH2 (SEQ ID NO:51)
(His Ile Ala Gly His Ile Ala)3-NH2 (SEQ ID NO:52)
(Ala Gly Lys Ile Ala Lys Ile)3-NH2 (SEQ ID NO:53)
(Ile Ala Lys Ile Ala Gly Lys)3-NH2 (SEQ ID NO:54)
(Lys Ile Ala Gly Arg Ile Ala)3-NH2 (SEQ ID NO: 55)
(Arg Ile Ala Gly Arg Ile Ala)3-NH2 (SEQ ID NO: 56)
(Lys Val Ala Gly Lys Ile Ala)3-NH2 (SEQ ID NO:57)
(Lys Ile Ala Gly Lys Val Ala)3-NH2 (SEQ ID NO: 58) (Ala Lys Ile Ala Gly Lys Ile)3-NH2 (SEQ ID NO:59) (Orn Ile Ala Gly Orn Ile Ala)3-NH2 (SEQ ID NO: 60)
(Lys Phe Ala Gly Lys Ile Ala)3-NH2 (SEQ ID NO: 61)
(Lys Ile Ala Gly Lys Phe Ala)3-NH2 (SEQ ID NO: 62)
(Lys Cha Ala Gly Lys Ile Ala)3-NH2 (SEQ ID NO: 63)
(Lys Nle Ala Lys Ile Ala Gly)3-NH2 (SEQ ID NO: 64)
(Arg Ile Ala Gly Lys Ile Ala)3-NH2 (SEQ ID NO:65)
(Har Ile Ala Gly Har Ile Ala)3-NH2 (SEQ ID NO: 66)
(Xaa Ile Ala Gly Lys Ile Ala)3-NH2 (SEQ ID NO: 67)
(Lys Ile Ala Gly Xaa Ile Ala)3-NH2 (SEQ ID NO: 68)
In (SEQ ID NO:67) and (SEQ ID NO:68), Xaa is
p-aminophenylalanine.
In accordance with another embodiment, the biologically active amphiphilic peptide includes the following basic structure X40 :
R31-R32-R32-R33-R34-R32-R32-R31-R32-R32-R32-R34-R32-R32, wherein R31, R32, and R33 are as hereinabove described, and R34 is a basic hydrophilic or hydrophobic amino acid.
In accordance with one embodiment, the peptide may include the following structure:
Y40-X40, wherein X40 is as hereinabove described, and Y40 is:
(i) R32;
(ii) R32-R32;
(iii) R34-R32-R32;
(iv) R33-R34-R32-R32;
(v) R32-R33-R34-R32-R32;
(V) R32-R32-R33-R34-R32-R32, or
(vii) R31-R32-R32-R33-R34-R32-R32, wherein R31, R32, R33 and
34 are as hereinabove described.
In accordance with another embodiment, the peptide may include the following structure:
X40-Z40, wherein X40 is as hereinabove described and Z40 is: (i ) R31 ;
( ii ) R31-R32 ;
(iii ) R31-R32-R32 ;
( iv) R31-R32-R32-R33;
(v) R31-R32-R32-R33-R34;
(vi) R32-R32-R32-R33-R34-R32, or
(vii) R31-R32-R32-R33-R34-R32-R32. wherein R31, R32, R33, and R34 are as hereinabove described.
In accordance with yet another embodiment the peptide may include the following structure:
( Y40)a-X40-(Z40)b, wherein Y40 and Z40 are as Previously defined, a is 0 or 1, and b is 0 or 1. In a preferred
embodiment, the peptide has the following structural formula as indicated in the sequence listing contained herein:
(SEQ ID NO:69)-NH2.
In another preferred embodiment, the peptide has the following structural formula as indicated in the sequence listing contained herein:
(SEQ ID NO:70)-NH2.
In accordance with a further embodiment, the peptide has one of the following structural formulae as indicated in the sequence listing contained herein:
(SEQ ID NO:71)-NH2
(SEQ ID NO:72)-NH2
(SEQ ID NO:73)-NH2
(SEQ ID NO:74)-NH2
(SEQ ID NO:75)-NH2
(SEQ ID NO:76)-NH2
(SEQ ID NO:77)-NH2
(SEQ ID NO:78)-NH2
(SEQ ID NO:79)-NH2
(SEQ ID NO:80)-NH2
(SEQ ID NO:81)-NH2
(SEQ ID NO:82)-NH2 ( SEQ ID NO : 83 ) -NH2
( SEQ ID NO : 84) -NH2
( SEQ ID NO : 85 ) -NH2
In accordance with another embodiment the peptide has the following structural formula:
-(LysIle Ala Lys LysIle Ala)n-, wherein n is from 2 to 5. Preferably, n is 3, and the peptide has the following structural formula:
(LysIle Ala Lys Lys Ile Ala)3-NH2.
(SEQ ID NO: 86)
In accordance with another embodiment, the peptide is selected from the group consisting of the following structural formulae as given in the accompanying sequence listing:
(SEQ ID NO:87)-NH2
(SEQ ID NO:88)-NH2
(SEQ ID NO:89)-NH2
(SEQ ID NO:90)-NH2.
In accordance with another embodiment, the peptide includes the following basic structure X50 :
R41-R42-R42-R41-R42-R42-R41-R41-R42-R41-R41,
wherein R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
In one embodiment, the peptide includes the basic structure
Y50-X50, wherein X50 is as hereinabove described and Y50 is:
(i) R41;
(ii) R42-R41; or
(iii) R42-R42-R41, wherein R41 and R42 are as hereinabove describe.
In one embodiment, R41 is leucine. In another embodiment, R42 is lysine. Representative, examples of such peptides include those having the following structures:
(SEQ ID NO: 91)
(SEQ ID NO: 92)
(SEQ ID NO: 93) ( SEQ ID NO : 94) .
In yet another embodiment, the peptide includes the following basic structure X52 :
R42-R41-R42-R42-R41-R41-R42-R42-R41-R42-R42,
wherein R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
In one embodiment, R41 is leucine. In another embodiment,
R42 is lysine.
In one embodiment, the peptide includes the basic structure
Y52-X52, wherein X52 is as hereinabove described, and Y52 is (i) R42;
(ii) R41-R42; or
(iii) R41-R41-R42;
(iv) R42-R41-R41-R42; or
(v) R42-R42-R41-R41-R42.
In one embodiment, the peptide may have the following structure:
Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu
5 10
Leu Lys Lys Leu Arg Arg
15
(SEQ ID NO: 95).
In another embodiment, the peptide includes the basic structure X52-Z52, wherein X52 is as hereinabove described, and
Z52 is :
(i) R41;
(ii) R41-R41; or
(iii) R41-R41-R42;
(iv) R41-R41-R42-R42; or
(v) R41-R41-R42-R42-R41.
In one embodiment, the peptide may have the following structure:
Lys Leu Lys Lys Leu Leu Lys Lys Leu
5
Lys Lys Leu Leu Lys Lys Leu
10 15 ( SEQ ID NO: 96 ) .
In another embodiment, the peptide may include the
structure :
(Y52)a-X52-(Z52)b, wherein X52, Y52, and Z52 are as
hereinabove described, and a is 0 or 1, and b is 0 or 1.
The above peptides may be acetylated with a CH3CO-group at the N-terminal.
In accordance with another embodiment, each of the amino acid residues may be a D-amino acid residue or glycine.
In still another embodiment, the peptide employed in conjunction with a chelating agent such as those hereinabove described, or derivatives or analogues thereof, is a cecropin. The cecropins and analogues and derivatives thereof are described in Ann. Rev. Microbiol 1987, Vol. 41 pages 103-26, in particular p. 108 and Christensen at al PNAS Vol. 85 p. 5072-76, which are hereby incorporated by reference.
The term cecropin includes the basic structure as well as analogues and derivatives.
In yet another embodiment, the peptide employed in
conjunction with a chelating agent such as those hereinabove described, or derivatives or analogues thereof is a sarcotoxin. The sarcotoxins and analogues and derivatives thereof are
described in Molecular Entomology, pages 369-78, in particular p. 375 Alan R. Liss Inc. (1987), which is hereby incorporated by reference.
The term sarcotoxin includes the basic materials as well as analogues and derivatives.
Ion channel-forming proteins or peptides which may be employed include defensins, also known as human neutrophil antimicrobial peptides (HNP), major basic protein (MBP) of eosinophils, bactericidal permeability-increasing protein (BPI), a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein, lactoferrin, B-2-binding protein, eosinophil cationic protein (ECP), and eosinophil-derived
neurotoxin. Defensins are described in Selsted, et al., J. Clin. Invest., Vol. 76, pgs. 1436-1439 (1985). MBP proteins are described in Wasmoen, et al., J. Biol. Chem., Vol. 263, pgs.
12559-12563 (1988). BPI proteins are described in Ooi, et al., J. Biol. Chem., Vol. 262, pgs. 14891-14894 (1981). Perform is described in Henkart, et al. J. Exp. Med., 160:75 (1984), and in Podack, et al. J. Exp. Med., 160:695 (1984). Lactoferrin,
B12-binding protein, eosinophil cationic protein, and
eosinphil-derived neurotoxin are described in Elsbach, et al., Inflammation: Basic Principles and Clinical Correlates, Gallin, et al., eds.; pgs. 445-471 (1988). The above articles are hereby incorporated by reference.
The term ion channel-forming proteins includes the basic structures of the ion channel-forming proteins as well as
analogues or derivatives.
In accordance with another aspect of the present invention, there is provided a method of antagonizing the biological
activity of a biologically active amphiphilic peptide or protein, such as those hereinabove described, in a host. The method comprises administering to a host (human or animal) that is being treated with a biologically active peptide or protein an ion selected from the group consisting of calcium ions and magnesium ions. The ion is administered in an amount effective to
antagonize the biological activity of the peptide or protein.
Such a method is particularly applicable to a method of selective treatment. For example, one may wish for the peptide or protein to be biologically effective in one or more body parts or organs of a host, but not in others. In such cases, the ion would be administered to those body parts or organs in which onedid not desire the peptide or protein to- have biological effect. As an illustrative example, if one desired the peptide or protein to be effective in the stomach of the host but not in the bloodstream, one would administer the ion to the bloodstream in an amount effective in antagonizing the biological activity of the peptide or protein in the bloodstream. The ion may be administered in an amount up to about 2 grams/kg of host weight.
The invention will now be described with respect to the following examples; however, the scope of the present invention is not intended to be limited thereby.
Example 1
The procedure for the following antibacterial assay is based upon the guidelines of the National Committee for Clinical
Laboratory Standards, Document M7-T2, Volume 8, No. 8, 1988.
Stock solutions of the following Peptides (1) through (3), wherein:
Peptide (1) is (SEQ ID NO: 27)
Peptide (2) is (SEQ ID NO: 97) amide-terminated; and
Peptide (3) is (LysIle Ala Gly LysIle Ala)3,
wherein each amino acid residue of Peptide (3) is a D-amino acid residue or glycine, were prepared at a concentration of 512 μg/ml in sterile deionized distilled water and stored at -70°C.
The stock peptide solutions are diluted in serial dilutions (1:2) down the wells of a microtiter plate so that the final concentrations of peptides in the wells are 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, 128, and 256 μg/ml. 1-5 x 105 CFUs/ml of P.
aeruginosa ATCC 27853 were added to the wells in full strength or half-strength Mueller Hinton broth (MHB), or in half-strength MHB broth to which 0.002 M Ca2+, or 0.002 M Mg2+, or 0.001 M Ca2+ and 0.001M Mg 2+ has been added. The organisms are from a mid-log culture. The inoculum is standardized spectrophotometrically at
600nm and is verified by colony counts. The plates are incubated for 16-20 hours at 37°C, and the minimal inhibitory concentration
(MIC) for each peptide is determined. Minimal inhibitory
concentration is defined as the lowest concentration of peptide which produces a clear well in the microtiter plate, The MIC values are given in Table I below.
The above results indicate that calcium and magnesium ions decrease the activity of Peptides (1) through (3) against P.
aeruginosa.
Example 2
The procedure of Example 1 was repeated, except that the assays were carried out in full strength Mueller Hinton Broth (MHB) or cation-adjusted Mueller Hinton Broth (CAMHB). Peptides (1) and (2) were tested for MIC against P. aeruginosa 27853 in each of these broths without further additives, and in each of these broths to which was added 0.05mM, 0.5mM, or 5mM of the calcium channel blockers verapamil or diltiazem. The results are given in Table II below.
The above results indicate that the known calcium channel blockers verapamil and diltiazem have little or no effect upon the activity of Peptides (1) or (2) against P. aeruginosa.
Example 3
Peptides (1), (2), (4), (5), and (6) were tested for activity against P. aeruginosa strain 27853 in full or
half-strength Mueller Hinton Broth according to the procedure of Example 1, and in Mueller Hinton Broth to which 0.034mM, or 0.34mM, or 3.4mM of ethylene dinitrilo tetraacetic acid (EDTA) or ethylene glycol bis (B-aminoethyl ether) NNN'N,-tetraacetic acid is added. Peptide (4) is amide-terminated Magainin II (SEQ ID NO: 7). Peptide (5) has the following structural formula:
Gly Ile Gly D-Lys D-Phe Leu His Ser
5
Ala D-Lys D-Lys D-Phe Gly D-Lys
10
Ala D-Phe Val Gly Glu Ile Met Asn Ser-NH2
15 20
Peptide (6) is SEQ ID NO:21, amide-terminated. The results of this assay are given in Table III below:
TABLE III MIC (μg/ml)
The above results indicate that, with the exception of Peptides (2) and (5), EGTA at a concentration of 3.4mM enhances the activity of the peptides against P. aeruginosa.
Example 4
The procedure for this assay, which is a checkerboard assay, is performed as described in Antibiotics and Laboratory Medicine, 2nd ed., Victor Lorian, M.D. Editor, pgs. 540-546 (1986).
The checkerboard assay was carried out in a 96-well
microtiter plate having 12 rows and 8 columns of wells. 100μl of plain broth is added to every row of wells. 100μl of the desired peptide at 512 μg/ml is added to the top well, and serially diluted through row 11. 50μl of the EDTA in varying
concentrations (obtained through serial dilutions), are added to each appropriate column of wells. 50 μl of P. aeruginosa strain 27853 is then added to each well. The plate is then incubated at 35°-37°C for 18 to 24 hours, and the wells are then examined for the presence of visible growth; i.e., turbidity.
Peptides (1) and (2) were tested alone or in combination with EDTA for activity against P. aeruginosa strain 27853. In the checkerboard assay in which Peptide (1) and EDTA were tested for activity against P. aeruginosa, the MIC of Peptide (1) was 64μg/ml, and the MIC of EDTA was 4mM.
Combinations of varying amounts of Peptide (1) and EDTA which resulted in inhibition of the growth of P. aeruginosa, and the FIC values of each combination, were then determined.
FIC, or Fractional Inhibitory Index, is determined as follows:
FIC = MIC of peptide in combination MIC of EDTA in combination _______________________________________________________________
MIC of peptide alone + MIC of EDTA alone
An FIC value of 0.5 or less is indicative of synergy, a value of greater than 0.5 but less than 2 is indicative of indifference, and a value greater than 2 is indicative of antagonism. The following combinations of Peptide (1) and EDTA were found to be inhibitory, and the FIC values of each combination are given herewith.
Combination FIC
16μg/ml Peptide (1) plus 0.125mM EDTA 0.28
4μg/ml Peptide (1) plus 0.25mM EDTA 0.125
2μg/ml Peptide (1) plus 0.5mM EDTA 0.156
1μg/ml Peptide (1) plus ImM EDTA 0.27
lμg/ml Peptide (1) plus 2mM EDTA 0.52
The assay was also performed to determine the MIC values of Peptide (2), EDTA, and inhibitory combinations thereof. The MIC of Peptide (2) against P. aeruginosa was 4μg/ml, and the MIC of EDTA was 8mM. The following combinations of Peptide (2) and EDTA were also found to be inhibitory, and FIC values are given therefor:
Combination FIC
2μg/ml Peptide (2) plus 0.25mM EDTA 0.53
2μg/ml Peptide (2) plus 0.5mM EDTA 0.56
2μg/ml Peptide (2) plus ImM EDTA 0.63
1μg/ml Peptide (2) plus 2mM EDTA 0.50
0.5μg/ml Peptide (2) plus 4mM EDTA 0.63
The above results suggest that there is a synergistic interaction between Peptide (1) and EDTA when employed in combination to inhibit growth of P. aeruginosa.
Example 5
Peptides (1) and (2) were tested for MIC against P.
aeruginosa strain 27853, as employed alone or in combination with 3mM, or 4mM or 5mM EGTA. The procedure followed was that of Example 1, and all testing was done using P. aeruginosa grown in full strength MHB broth. The results of this assay are given in Table IV below.
Example 6
The procedure of Example 5 was repeated, except the MIC values of Peptides (1) and (2) were determined for Peptides (1) and (2) alone and in combination with 0.0625mM, 0.125mM, 0.25mM, 0.5mM, 1mM, 2mM, or 3mM EGTA. The results of this assay are given in Table V below.
It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Berkowitz, Barry
(ii) TITLE OF INVENTION: Composition and Treatment with Biologically Active Peptides and Chelating Agents.
(iii) NUMBER OF SEQUENCES: 97
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Carella, Byrne, Bain, Gilfillan,
Cecchi & Stewart
(B) STREET: 6 Becker Farm Road
(C) CITY: Roseland
(D) STATE: New Jersey
(E) COUNTRY: USA
(F) ZIP: 07068
(V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch diskette
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: DW4.V2
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 07/803,629
(B) FILING DATE: 9-DEC-1991
(C) CLASSIFICATION:
(Vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Olstein, Elliot M.
(B) REGISTRATION NUMBER: 24,025
(C) REFERENCE/DOCKET NUMBER: 421250-146
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700
(B) TELEFAX: 201-994-1744
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide- or carboxy-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Ala Phe Ser Lys Ala Phe Ser Lys
5
Ala Phe Ser Lys Ala Phe Ser Lys Ala
10 15
Phe Ser Lys
20
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide- or carboxy-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ala Phe Ser Lys Ala Phe Ser Lys Ala Phe
5 10
Ser Lys Ala Phe Ser Lys Ala Phe Ser Lys
15 20
Ala Phe Ser Lys
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide- or carboxy-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Phe Ser Lys Ala Phe Ser Lys Ala Phe Ser
5 10
Lys Ala Phe Ser Lys Ala
15
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide- or carboxy-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Ser Lys Ala Phe Ser Lys Ala Phe Ser Lys
5 10
Ala Phe Ser Lys Ala Phe Ser Lys Ala Phe
15 20
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide- or carboxy-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Lys Ala Phe Ser Lys Ala Phe Ser Lys Ala
5 10
Phe Ser Lys Ala Phe Ser
15
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Magainin I peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proceedings of the National Academy
of Sciences
(D) VOLUME: 84
(F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777 (I) FILING DATE: 04-MAR-1987
(J) PUBLICATION DATE: 07-MAR-1989
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6
Gly Ile Gly Lys Phe Leu His Ser Ala Gly
5 10
Lys Phe Gly Lys Ala Phe Val Gly Glu Ile
15 20
Met Lys Ser
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Magainin II peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(X) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proceedings of the National Academy
of Sciences
(D) VOLUME: 84
(F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777
(I) FILING DATE: 04-MAR-1987
(J) PUBLICATION DATE: 07-MAR-1989 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7
Gly Ile Gly Lys Phe Leu His Ser Ala Lys
5 10
Lys Phe Gly Lys Ala Phe Val Gly Glu Ile
15 20
Met Asn Ser
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Magainin III peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proceedings of the National Academy
of Sciences
(D) VOLUME: 84
(F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777
(I) FILING DATE: 04-MAR-1987
(J) PUBLICATION DATE: 07-MAR-1989 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8
Gly Ile Gly Lys Phe Leu His Ser Ala Lys
5 10
Lys Phe Gly Lys Ala Phe Val Gly Glu Ile
15 20
Met Asn
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: magainin peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(X) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proceedings of the National Academy
of Sciences
(D) VOLUME: 84
(F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777
(I) FILING DATE: 04-MAR-1987
(J) PUBLICATION DATE: 07-MAR-1989 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9
Ile Gly Lys Phe Leu His Ser Ala Lys Lys
5 10
Phe Gly Lys Ala Phe Val Gly GluIle Met
15 20
Asn Ser
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: magainin peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proceedings of the National Academy
of Sciences
(D) VOLUME: 84
(F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777
(I) FILING DATE: 04-MAR-1987
(J) PUBLICATION DATE: 07-MAR-1989 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10
Gly Lys Phe Leu His Ser Ala Lys Lys Phe
5 10
Gly Lys Ala Phe Val Gly Glu Ile Met Asn
15 20
Ser
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: magainin peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(X) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proceedings of the National Academy
of Sciences
(D) VOLUME: 84
(F) PAGES: 5449-5453
(G) DATE: AUG - 1987
(H) DOCUMENT NUMBER: US 4810777
(I) FILING DATE: 04-MAR-1987
(J) PUBLICATION DATE: 07-MAR-1989 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 Lys Phe Leu His Ser Ala Lys Lys Phe Gly
5 10
Lys Ala Phe Val Gly Glu Ile Met Asn Ser
15 20
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: PGLa peptide.
(D) OTHER INFORMATION: amide-terminated
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Hoffman, et al.
(C) JOURNAL: EMBO J.
(D) VOLUME: 2
(F) PAGES: 711-714
(G) DATE: 1983
(A) AUTHOR: Andreu, et al.
(C) JOURNAL: Journal of Biochemistry
(D) VOLUME: 149
(F) PAGES: 531-535
(G) DATE: 1985
(A) AUTHOR: Gibson, et al.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349 (G) DATE : 1986
(A) AUTHOR: Giovannini, et al.
(C) JOURNAL : Biochem J.
(D) VOLUME: 243
(F) PAGES: 113-120
(G) DATE: 1987
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 Gly Met Ala Ser Lys Ala Gly Ala Ile Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: XPF peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Hoffman, et al.1
(C) JOURNAL: EMBO J.
(D) VOLUME: 2
(F) PAGES: 711-714
(G) DATE: 1983
(A) AUTHOR: Andreu, et al.
(C) JOURNAL: Journal of Biochemistry (D) VOLUME: 149
(F) PAGES: 531-535
(G) DATE: 1985
(A) AUTHOR: Gibson, et al.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(A) AUTHOR: Giovannini, et al.
(C) JOURNAL: Biochem J.
(D) VOLUME: 243
(F) PAGES: 113-120
(G) DATE: 1987
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13
Gly Trp Ala Ser Lys Ile Gly Gln Thr Leu
5 10
Gly Lys Ile Ala Lys Val Gly Leu Lys Glu
15 20
Leu Ile Gln Pro Lys
25
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated (x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL : J. Biol. Chem.
(D) VOLUME : 261
(F) PAGES : 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL : Nucleic Acids Research
(D) VOLUME : 13
(F) PAGES : 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME : 261
(F) PAGES : 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING : DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14 Gly Phe Gly Ser Phe Leu Gly Leu Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Ala
15 20
Leu Gly Gly Ala Pro Gln Gln
25 (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S .
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261 (F) PAGES: 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15
Gly Leu Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Gly Leu Lys Ile Gly Ala His Leu
15 20
Leu Gly Gly Ala Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680 (G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES : 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16 Gly Leu Ala Ser Leu Leu Gly Lys Ala Leu
5 10
Lys Ala Gly Leu Lys Ile Gly Thr His Phe
15 20
Leu Gly Gly Ala Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17
Gly Leu Ala Ser Leu Leu Gly Lys Ala Leu
5 10
Lys Ala Thr Leu Lys Ile Gly Thr His Phe
15 20
Leu Gly Gly Ala Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(X) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13 (F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18
Gly Phe Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Met
15 20
Leu Gly Gly Thr Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated (x) PUBLICATION INFORMATION: (A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19 Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Ala
15 20
Leu Gly Gly Ala Pro Gln Gln
25 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(X) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261 (F) PAGES: 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20
Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Ala
15 20
Leu Gly Gly Ser Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680 (G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES : 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES : 5341-5349
(G) DATE: 1986
(H) DOCUMENT NUMBER: W090/04407
(I) FILING DATE: 16-OCT-1989
(J) PUBLICATION DATE: 03-MAY-1990
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 Gly Phe Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Leu
15 20
Leu Gly Gly Thr Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDDESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(ix) EEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22
Gly Phe Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Ala
15 20
Leu Gly Gly Ala Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(X) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13 (F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23
Gly Phe Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Met
15 20
Leu Gly Gly Ala Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(X) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K. Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME : 261
(F) PAGES : 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL : Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES : 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES : 5341-5349
(G) DATE: 1986
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24 Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Ala
15 20
Leu Gly Gly Ser Leu Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(X) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K.
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13
(F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25
Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Gly Leu Lys Ile Gly Thr Asn Phe
15 20
Leu Gly Gly Ala Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CPF peptide.
(D) OTHER INFORMATION: amide- or carboxy- terminated
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Richter, K
Egger, R.
Kreil
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 3676-3680
(G) DATE: 1986
(A) AUTHOR: Wakabayashi, T.
Kato, H.
Tachibaba, S.
(C) JOURNAL: Nucleic Acids Research
(D) VOLUME: 13 (F) PAGES: 1817-1828
(G) DATE: 1985
(A) AUTHOR: Gibson, B.W.
Poulter, L.
Williams, D.H.
Maggio, J.E.
(C) JOURNAL: J. Biol. Chem.
(D) VOLUME: 261
(F) PAGES: 5341-5349
(G) DATE: 1986
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Gly Leu Ala Ser Leu Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys Ile Gly Ala Asn Ala
15 20
Leu Gly Gly Ser Pro Gln Gln
25
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10 Gly Lys Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Lys Ile Ala Lys Ile Ala Gly Lys Ile Ala
5 10
Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Lys Ile Ala Gly Lys Ile Gly Lys Ile Ala
5 10
Gly Lys Ile Gly Lys Ile Ala Gly Lys Ile
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: Lys Leu Ala Gly Lys Leu Ala Lys Leu Ala
5 10
Gly Lys Leu Ala Lys Leu Ala Gly Lys Leu
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE :
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Lys Phe Ala Gly Lys Phe Ala Lys Phe Ala
5 10
Gly Lys Phe Ala Lys Phe Ala Gly Lys Phe
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Lys Ala Leu Ser Lys Ala Leu Lys Ala Leu
5 10
Ser Lys Ala Leu Lys Ala Leu Ser Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Lys Leu Leu Lys Ala Leu Gly Lys Leu Leu
5 10
Lys Ala Leu Gly Lys Leu Leu Lys Ala Leu
15 20
Gly
(2) INFORMATION EOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Lys Ala Ile Gly Lys Ala Ile Lys Ala Ile
5 10
Gly Lys Ala Ile Lys Ala Ile Gly Lys Ala
15 20
Ile
(2) INFORMATION FOR SEQ ID NO: 35 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Gly Ile Ala Lys Ile Ala Lys Gly Ile Ala
5 10
Lys Ile Ala Lys Gly Ile Ala Lys Ile Ala
15 20
Lys
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
Lys Ile Ala Lys Ile Phe Gly Lys Ile Ala
5 10 Lys Ile Phe Gly Lys Ile Ala Lys Ile Phe
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Gly Ile Ala Arg Ile Ala Lys Gly Ile Ala
5 10
Arg Ile Ala Lys Gly Ile Ala Arg Ile Ala
15 20
Lys
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Lys Phe Ala Arg Ile Ala Gly Lys Phe Ala
5 10
Arg Ile Ala Gly Lys Phe Ala Arg Ile Ala
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: Gly Phe Ala Lys Ile Ala Lys Gly Phe Ala
5 10
Lys Ile Ala Lys Gly Phe Ala Lys Ile Ala
15 20
Lys
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE :
(D) OTHER INFORMATION: amide-terminated, Xaa is ornithine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
Lys Ile Ala Gly Xaa Ile Ala Lys Ile Ala
5 10
Gly XaaIle Ala Lys Ile Ala Gly Xaa Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Lys Ile Ala Arg Ile Ala Gly Lys Ile Ala
5 10
Arg Ile Ala Gly Lys Ile Ala Arg Ile Ala
5 20
Gly
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE:, amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is ornithine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
Xaa Ile Ala Gly Lys Ile Ala Xaa Ile Ala
5 10
Gly Lys Ile Ala Xaa Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Gly Ile Ala Arg Ile Phe Lys Gly Ile Ala
5 10
Arg Ile Phe Lys Gly Ile Ala Arg Ile Phe
15 20
Lys
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is norleucine ( (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
Lys Xaa Ala Gly Lys Xaa Ala Lys Xaa Ala
5 10
Gly Lys Xaa Ala Lys Xaa Ala Gly Lys Xaa
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is norleucine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Lys Xaa Ala Gly Lys Ile Ala Lys Xaa Ala
5 10 Gly Lys Ile Ala Lys Xaa Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is norleucine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Lys Ile Ala Gly Lys Xaa Ala Lys Ile Ala
5 10
Gly Lys Xaa Ala Lys Ile Ala Gly Lys Xaa
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE: (D) OTHER INFORMATION: amide-terminated, Xaa is norvaline
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Lys Xaa Ala Gly Lys Xaa Ala Lys Xaa Ala
5 10
Gly Lys Xaa Ala Lys Xaa Ala Gly Lys Xaa
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is norvaline
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
Lys Xaa Ala Gly Lys Ile Ala Lys Xaa Ala
5 10
Gly Lys Ile Ala Lys Xaa Ala Gly Lys Xaa
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: Lys Leu Leu Ser Lys Leu Gly Lys Leu Leu
5 10
Ser Lys Leu Gly Lys Leu Leu Ser Lys Leu
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Lys Leu Leu Ser Lys Phe Gly Lys Leu Leu
5 10
Ser Lys Phe Gly Lys Leu Leu Ser Lys Phe
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 51
(i) SEQUENCE CHARACTERISTICS (A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is norvaline
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
Lys Ile Ala Gly Lys Xaa Ala LysIle Ala
5 10
Gly Lys Xaa Ala Lys Ile Ala Gly Lys Xaa
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
His Ile Ala Gly His Ile Ala His Ile Ala
5 10 Gly His Ile Ala His Ile Ala Gly His Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: Ala Gly Lys Ile Ala Lys Ile Ala Gly Lys
5 10
Ile Ala Lys Ile Ala Gly Lys Ile Ala Lys
15 20
Ile
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:Ile Ala Lys Ile Ala Gly Lys Ile Ala Lys
5 10
Ile Ala Gly Lys Ile Ala Lys Ile Ala Gly
15 20
Lys
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55: Lys Ile Ala Gly Arg Ile Ala Lys Ile Ala
5 10
Gly Arg Ile Ala Lys Ile Ala Gly Arg Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE: (D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: Arg Ile Ala Gly Arg Ile Ala Arg Ile Ala
5 10
Gly Arg Ile Ala Arg Ile Ala Gly Arg Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Lys Val Ala Gly Lys Ile Ala Lys Val Ala
5 10
Gly Lys Ile Ala Lys Val Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: Lys Ile Ala Gly Lys Val Ala Lys Ile Ala
5 10
Gly Lys Val Ala Lys Ile Ala Gly Lys Val
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
Ala Lys Ile Ala Gly Lys Ile Ala Lys Ile
5 10
Ala Gly Lys Ile Ala Lys Ile Ala Gly Lys
15 20
Ile
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is ornithine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Xaa Ile Ala Gly Xaa Ile Ala Xaa Ile Ala
5 10
Gly Xaa Ile Ala Xaa Ile Ala Gly Xaa Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
Lys Phe Ala Gly Lys Ile Ala Lys Phe Ala
5 10
Gly Lys Ile Ala Lys Phe Ala Gly Lys Ile
15 20.
Ala (2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
Lys Ile Ala Gly Lys Phe Ala Lys Ile Ala
5 10
Gly Lys Phe Ala Lys Ile Ala Gly Lys Phe
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is cyclohexylalanine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
Lys Xaa Ala Gly Lys Ile Ala Lys Xaa Ala 5 10
Gly Lys Ile Ala Lys Xaa Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is norleucine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
Lys Xaa Ala Lys Ile Ala Gly Lys Xaa Ala
5 10
Lys Ile Ala Gly Lys Xaa Ala Lys Ile Ala
15 20
Gly
(2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
Arg Ile Ala Gly Lys Ile Ala Arg Ile Ala
5 10
Gly Lys Ile Ala Arg Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is homoarginine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
Xaa Ile Ala Gly Xaa Ile Ala Xaa Ile Ala
5 10
Gly Xaa Ile Ala Xaa Ile Ala Gly Xaa Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE: amide-terminated, Xaa is p-aminophenylalanine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
Xaa Ile Ala Gly Lys Ile Ala Xaa Ile Ala
5 10
Gly Lys Ile Ala Xaa Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE: amide-terminated, Xaa is p-aminophenylalanine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
Lys Ile Ala Gly Xaa Ile Ala Lys Ile Ala
5 10
Gly Xaa Ile Ala Lys Ile Ala Gly Xaa Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS (A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
Lys Leu Ala Ser Lys Ala Gly Lys Ile Ala Gly
5 10
Lys Ile Ala Lys Val Ala Leu Lys Ala Leu
15 20
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is ornithine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala Gly
5 10
Xaa Ile Ala Lys Ile Ala Gly Lys Ile Ala
15 20 (2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Arg Ile Ala Lys Ile Ala Gly Lys Ile
15. 20
Ala
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is norleucine (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Xaa Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is norvaline
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Xaa Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide ( ix) FEATURE :
(D) OTHER INFORMATION: amide-terminated, Xaa is ornithine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
Lys Phe Ala Gly Lys Phe Ala Lys Phe Ala Gly
5 10
Xaa Phe Ala Lys Phe Ala Gly Lys Phe Ala
15 20
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is ornithine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
Lys Ile Ala Gly Lys Phe Ala Lys Ile Ala
5 10
Gly Xaa Phe Ala Lys Ile Ala Gly Lys Phe
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE. CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa at residue 6, 13, and 20 is norleucine, Xaa at residue 12 is ornithine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
Lys Ile Ala Gly Lys Xaa Ala Lys Ile Ala
5 10
Gly Xaa Xaa Ala Lys Ile Ala Gly Lys Xaa
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
Lys Met Ala Ser Lys Ala Gly Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala
15 20 Leu
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78
Lys Ile Ala Ser Lys Ala Gly Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala Leu
15 20
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is norleucine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: Lys Ile Ala Ser Lys Ala Gly Lys Xaa Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is norleucine
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
Lys Leu Ala Ser Lys Ala Gly Lys Xaa Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (ix) FEATURE :
(D) OTHER INFORMATION: amide-terminated, Xaa is norleucine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
Lys Xaa Ala Ser Lys Ala Gly Lys Xaa Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala Leu
15 20
(2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated, Xaa is p-aminophenylalanine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Xaa Ile Ala Lys Ile Ala Gly Lys Ile
15
Ala
(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
Lys Ile Ala Gly Ala Ile Ala Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Ala Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala (2) INFORMATION FOR SEQ ID NO: 85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:
Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Ile Ala Gly Ala Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
Lys Ile Ala Lys Lys Ile Ala Lys Ile Ala
5 10 Lys Lys Ile Ala Lys Ile Ala Lys Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 87:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:
Ala Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 88:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88: Lys Ile Ala Gly Lys Ile Ala Ala Ile Ala
5 10
Gly Lys Ile Ala Lys Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide-terminated
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
Lys Ile Ala Gly Lys Ile Ala Lys Ile Ala
5 10
Gly Lys Ile Ala Ala Ile Ala Gly Lys Ile
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE: (D) OTHER INFORMATION: amide-terminated
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
Gly Met Ala Ser Lys Ala Gly Lys Ile Ala
5 10
Gly Lys Ile Ala Lys Val Ala Leu Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at N-terminus.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
Leu Lys Lys Leu Lys Lys Leu Leu Lys Leu
5 10
Leu
(2) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at N-terminus.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
Leu Leu Lys Lys Leu Lys Lys Leu Leu Lys
5 10
Leu Leu
(2). INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at N-terminus. (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:
Lys Leu Leu Lys Lys Leu Lys Lys Leu Leu
5 10
Lys Leu Leu
(2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at N-terminus.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu
5 10
Leu Lys Leu Leu
(2) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at N-terminus. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu
5 10
Leu Lys Lys Leu Arg Arg
15
(2) INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: May be a C-terminal amide, and/or may be acetylated at N-terminus.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
Lys Leu Lys Lys Leu Leu Lys Lys Leu Lys
5 10
Lys Leu Leu Lys Leu Leu
15
(2) INFORMATION FOR SEQ ID NO: 97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: amide- or carboxy-terminated (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: Gly Ile Gly Lys Phe Leu Lys Lys Ala Lys
5 10
Lys Phe Gly Lys Ala Phe Val Lys Ile Met
15 20
Lys Lys

Claims

WHAT IS CLAIMED IS:
1. A process of inhibiting growth of a target cell in a host comprising:
administering to a host at least one biologically active amphiphilic peptide or biologically active protein; and
a chelating agent, said biologically active amphiphilic peptide or protein and said chelating agent being administered in a combined amount effective to inhibit growth of a target cell in a host.
2. The process of Claim 1 wherein the peptide is a basic polypeptide having at least ten amino acids, wherein said basic polypeptide includes hydrophobic amino acids and hydrophilic amino acids.
3. The process of Claim 2 wherein the peptide is a basic polypeptide having at least sixteen amino acids, wherein said basic polypeptide includes at least eight hydrophobic amino acids and at least eight hydrophilic amino acids.
4. The process of Claim 3 wherein said polypeptide
comprises a chain of at least four groups of amino acids, each of said at least four groups consisting of four amino acids, wherein two of the four amino acids in each group are hydrophobic amino acids, and two of the four amino acids in each group are
hydrophilic amino acids, with at least one of the hydrophilic amino acids in each group being a basic hydrophilic amino acid and the other hydrophilic amino acid being a basic or neutral hydrophilie amino acid.
5. The process of Claim 4 wherein each of said groups of four amino acids is of the sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each hydrophobic amino acids and may be the same or different, one of C or D is a basic hydrophilic amino acid, and the other of C or D is a basic or neutral hydrophilic amino acid and may be the game or different.
6. The process of Claim 1 wherein the peptide is a
magainin peptide.
7. The process of Claim 1 wherein the peptide is a cecropin.
8. The process of Claim 1 wherein the peptide is a sarcotoxin.
9. The process of Claim 1 wherein the peptide is a XPF peptide.
10. The process of Claim 1 wherein the peptide is a PGLa peptide.
11. The process of Claim 1 wherein the peptide is a CPF peptide.
12. The process of Claim 1 wherein the peptide includes one of the following basic structures X31 through X37, wherein:
X31 is -[R31-R32-R32-R33-R31-R32-R32]n-;
X32 is -[R32-R32-R33-R31-R32-R32-R31]n-;
X33 is -[R32-R33-R31-R32-R32-R31-R32]n-;
X34 is -[R33-R31-R32-R32-R31-R32-R32]n-;
X35 is -[R31-R32-R32-R31-R32-R32-R33]n-;
X36 is -[R32-R32-R31-R32-R32-R33-R31]n-; and
X37 is -[R32-R31-R32-R32-R33-R31-R32]n, wherein R31 is a basic hydrophilic amino acid, R32 is a hydrophobic amino acid,
R33 is a neutral hydrophilic, hydrophobic, or basic hydrophilic amino acid, and n is from 2 to 5.
13. The process of Claim 1 wherein the peptide includes the following basic structure X40:
R31-R32-R32-R33-R34-R32-R32-R31-R32-R32-R32-R34-R32-R32, wherein R31 is a basic hydrophilic amino acid, R32 is a hydrophobic amino acid, and R33 is a neutral hydrophilic, hydrophobic or basic hydrophilic amino acid, and R34 is a basic hydrophilic or
hydrophobic amino acid.
14. The process of Claim 1 wherein the peptide includes the following structural formula:
-(LysIle Ala Lys Lys Ile Ala)n-,
wherein n is from 2 to 5.
15. The process of Claim 14 wherein the peptide has the following structural formula:
(Lys Ile Ala Lys Lys Ile Ala)3.
16. The process of Claim 1 wherein the peptide is selected from the group consisting of:
(SEQ ID NO:87)-NH2;
(SEQ ID NO:88)-NH2;
(SEQ ID NO:89)-NH2; and
(SEQ ID NO:90)-NH2.
17. The process of Claim 1 wherein the peptide includes the following basic structure X50:
R41-R42-R42-R41-R42-R42-R41-R41-R42-R41-R41,
wherein R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
18. The process of Claim 17 wherein the peptide includes the basic structure Y50-X50, wherein X50 is the basic peptide structure of Claim 17, and Y50 is:
(i) R41;
(ii) R42-R41; or
(iii) R42-R42-R41.
19. The process of Claim 1 wherein the peptide includes the following basic structure X52 :
R42-R41-R42-R42-R41-R41-R42-R42-R41-R42-R42,
wherein R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
20. The process of Claim 19 wherein the peptide includes the basic structure Y52-X52, wherein X52 is the basic peptide structure of Claim 19, and Y52 is:
(i) R42;
(ii) R41-R42;
(iii) R41-R41-R42;
(iv) R42-R41-R41-R42; or
(v) R42-R42-R41-R41-R42.
21. The process of Claim 19 wherein the peptide includes the basic structure X52-Z52, wiιerein X52 is tlle basic peptide structure of Claim 19, and Z52 is:
(i) R41;
(ii) R41-R41;
(iii) R41-R41-R42;
(iv) R41-R41-R42-R42; or
(v) R41-R41-R42-R42-R41
22. A composition comprising:
(a) at least one biologically active amphiphilic peptide or biologically active protein; and
(b) a chelating agent.
23. The composition of Claim 22 wherein said components (a) and (b) are present in a combined amount effective to inhibit growth of a target cell in a host.
24. The composition of Claim 22 wherein the peptide is a basic polypeptide having at least ten amino acids, wherein said basic polypeptide includes hydrophobic amino acids and
hydrophilic amino acids.
25. The composition of Claim 24 wherein the peptide is a basic polypeptide having at least sixteen amino acids, wherein said basic polypeptide includes at least eight hydrophobic amino acids and at least eight hydrophilic amino acids.
26. The composition of Claim 25 wherein said polypeptide comprises a chain of at least four groups of amino acids, each of said at least four groups consisting of four amino acids, wherein two of the four amino acids in each group are hydrophobic amino acids, and two of the four amino acids in each group are
hydrophilic amino acids, with at least one of the hydrophilic amino acids in each group being a basic hydrophilic amino acid and the other hydrophilic amino acid being a basic or neutral hydrophilic amino acid.
27. The composition of Claim 26 wherein each of said groups of four amino acids is of the sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each hydrophobic amino acids and may be the same or different, one of C or D is a basic hydrophilic amino acid, and the other of C or D is a basic or neutral hydrophilic amino acid and may be the same or different.
28. The composition of Claim 22 wherein the peptide is a magainin peptide.
29. The composition of Claim 22 wherein the peptide is a cecropin.
30. The composition of Claim 22 wherein the peptide is a sarcotoxin.
31. The composition of Claim 22 wherein the peptide is a XPF peptide.
32. The composition of Claim 22 wherein the peptide is a PGLa peptide.
33. The composition of Claim 22 wherein the peptide is a CPF peptide.
34. The composition of Claim 22 wherein the peptide includes one of the following basic structures X31 through X37, wherein:
X 31 is -[R31-R32-R32-R33-R31-R32-R32]n- X32 is -[R32-R32-R33-R31-R32-R32-R31]n-;
X33 is -[R32-R33-R31-R32-R32-R31-R32]n-;
X34 iS -[R33-R31-R32-R32-R31-R32-R32]n-;
X35 is -[R31-R 32-R32-R31-R32-R32-R33]n-;
X36 is -[R32-R32-R31-R32-R32-R33-R31]n-; and
X 37 is -[R32-R31-R32-R32-R33-R31-R32]n-; wherein R 31 is a basic hydrophilic amino acid, R32 is a hydrophobic amino acid, R33 is a neutral hydrophilic, hydrophobic, or basic hydrophilic amino acid, and n is from 2 to 5.
35. The composition of Claim 22 wherein the peptide includes the following basic structure X40:
R31-R32-R32-R33-R34-R32-R32-R31-R32-R32-R32-R34-R32-R32, wherein R31 is a basic hydrophilic amino acid, R32 is a
hydrophobic amino acid, and R33 is a neutral hydrophilic, hydrophobic, or basic hydrophilic amino acid, and R34 is a basic hydrophilic or hydrophobic amino acid.
36. The composition of Claim 22 wherein the peptide includes the following basic structure X50:
R41-R42-R42-R41-R42-R42-R41-R41-R42-R41-R41,
wherein R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
37. The composition of Claim 36 wherein the peptide includes the basic structure Y50-X50, wherein X50 is the basic peptide structure of Claim 36, and Y 50 is:
(i) R41;
(ii) R42-R41;
(iii) R42-R42-R41.
38. The composition of Claim 22 wherein the peptide includes the following basic structure X52:
R42-R41-R42-R42-R41-R41-R42-R42-R41-R42-R42,
wherein R41 is a hydrophobic amino acid, and R42 is a basic hydrophilic or neutral hydrophilic amino acid.
39. The composition of Claim 38 wherein the peptide includes the basic structure Y52-X52,wherein X52 is the basic peptide structure of Claim 38, and Y52 is:
(i) R42;
(ii) R41-R42;
( iii ) R41-R41-R42 ;
(iv) R42-R41-R41-R42; or
(v) R42-R42-R41-R41-R42-
40. The composition of Claim 38 wherein the peptide includes the basic structure X52-Z52, wherein X52 is the basic peptide structure of Claim 38, and Z52 is:
(i) R41;
(ii) R41-R41;
(iii) R41-R41-R42;
(iv) R41-R41-R42-R42; or
(v) R41-R41-R42-R42-R41.
41. The process of Claim 1 wherein said chelating agent is selected from the class consisting of ethylene
dinitrilo-tetraacetic acid and ethylene glycol bis-(β-aminoethyl ether) NNN'N'-tetraacetic acid.
42. The composition of Claim 22 wherein said chelating agent is selected from the class consisting of ethylene
dinitrilo-tetraacetic acid and ethylene glycol bis-(β-aminoethyl ether) NNN'N'-tetraacetic acid.
43. The composition of Claim 22 wherein the peptide includes the following structural formula:
-(Lys Ile Ala Lys Lys Ile Ala)n-,
wherein n is from 2 to 5.
44. The composition of Claim 43 wherein the peptide has the following structural formula:
(LysIle Ala Lys LysIle Ala)3.
45. The composition of Claim 26 wherein the peptide is selected from the group consisting of:
(SEQ ID NO:87)-NH2;
(SEQ ID NO:88)-NH2;
(SEQ ID NO:89)-NH2; and
(SEQ ID NO:90)-NH2.
46. A method of antagonizing the biological activity of a biologically active amphiphilic peptide or biologically active protein in a host, comprising:
administering to a host being treated with said
bioloigcally-active peptide or protein, said peptide or protein being an ion channel-forming peptide or protein, an ion selected from the group consisting of calcium ions and magnesium ions in an amount effective to antagonize the biological activity of said peptide or protein.
EP93900822A 1991-12-09 1992-12-03 Composition and treatment with biologically active peptides and chelating agents Withdrawn EP0661988A1 (en)

Applications Claiming Priority (3)

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US80362991A 1991-12-09 1991-12-09
US803629 1991-12-09
PCT/US1992/010427 WO1993011783A1 (en) 1991-12-09 1992-12-03 Composition and treatment with biologically active peptides and chelating agents

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EP0661988A1 true EP0661988A1 (en) 1995-07-12

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US6057291A (en) 1995-06-02 2000-05-02 University Of British Columbia Antimicrobial cationic peptides
JP3860838B2 (en) 1995-08-23 2006-12-20 ユニバーシティー オブ ブリティッシュ コロンビア Antimicrobial cationic peptide and screening method thereof
US5688489A (en) * 1995-09-15 1997-11-18 Resolution Pharmaceuticals, Inc. Non-receptor mediated imaging agents
US6010876A (en) * 1996-11-06 2000-01-04 The Regents Of The University Of California Clavanins
US6040293A (en) * 1996-11-06 2000-03-21 The Regents Of The University Of California Clavanins
WO2005074968A2 (en) * 2004-02-10 2005-08-18 Universiteit Maastricht Medical use of basic peptides
GB0415985D0 (en) * 2004-07-19 2004-08-18 Taylor Russell Methods of manufacture and applications of a chelating compounds or substances that when used as a treatment for burns will assist in the growth
WO2009094634A1 (en) 2008-01-24 2009-07-30 Esperance Pharmaceuticals Lytic domain fusion constructs and methods of making and using same
PT105332A (en) * 2010-10-12 2012-04-12 Cev Biotecnologia Das Plantas S A AGENTS FOR USE WITH ANTIMICROBIALS
AU2011315525B2 (en) * 2010-10-12 2016-05-12 Consumo Em Verde - Biotecnologia Das Plantas, S.A. Antimicrobial protein
AU2013337926B2 (en) 2012-10-30 2017-12-21 Esperance Pharmaceuticals, Inc. Antibody/drug conjugates and methods of use

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EP0661988A4 (en) 1995-02-10

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