BIOLOGICALLY ACTIVE PEPTIDES HAVING N-TERMNΔL SUBSTITUTIONS
This invention relates to biologically active peptides. More particularly, this invention relates to biologically active peptides having N-terminal (or amino-terminal) substitutions.
In accordance with an aspect of the present invention, there is provided an N-terminal substituted peptide or protein having the formula: W
I I
T - N - X, wherein X is a biologically active peptide or protein. The peptide or protein is preferably an ion channel-forming peptide or protein. T is a lipophilic moiety, and W is T or hydrogen.
The term "lipophilic," as used herein, means that the lipophilic moiety enhances the interaction of the peptide or protein with a lipid membrane, such as, for example, a cell membrane.
Lipophilic moieties which may be employed, include, but are not limited to, any moiety which may be placed on the N-terminal of the peptide through a condensation reaction with nitrogen. The lipophilic moiety T may be, for example, a carboxylic acid, a phosphoric acid, preferably an alkylphosphoric acid, a phosphonic acid,
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preferably an alkylphosphonic acid, a sulfonic acid, preferably an alkylsulfonic acid, or an alkyl group . Preferably, T is : 0
I I
R - C -, wherein R is a hydrocarbon having at least two and no more than 16 carbon atoms.
In one embodiment, R is an alkyl group. The alkyl group may be a straight chain or branched chain alkyl group; or a cycloalkyl group. For example, R may be CH„(CH2) -, wherein n is from 1 to 14. Preferably, n is from 4 to 9, and most preferably n is 6, whereby T is an octanoyl group.
In another embodiment, R is an aromatic (including phenyl and naphthyl), or an alkyl aromatic group. For example, R may be 0 -(CH-) -, wherein z is from 0 to 6.
Preferably, z is 1 or 2.
In one embodiment, W is hydrogen.
Applicant has found, that when biologically active peptides have substitutions at the N-terminal such as those hereinabove described, such peptides have increased biological activity against target cells, viruses, and virally-infected cells, as compared with unsubstituted peptides or peptides substituted at the N-terminal with an acetyl group. Applicant also has found that the N-terminal substitutions hereinabove described significantly increase the biological activity of "short"
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peptides, i.e. peptides having no more than 14 amino acid residues.
As hereinabove stated, the biologically active peptides or proteins of the present invention are preferably ion channel-forming peptides. 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, pgs. 5072-5076 (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 ion channel-forming protein is a peptide or protein which has ion channel-forming properties as determined by the method of Christensen, et al.
An a phiphilic peptide or protein is a peptide or protein which includes both hydrophobic and hydrophilic peptide or protein regions.
The ion channel-forming 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. Such peptides are capable of forming an alpha-helical structure. When the peptide is placed in water, it does not assume an amphiphilic structure. When the peptide
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encounters an oily surface or membrane, the peptide chain folds upon itself into a rodlike structure.
In general, such peptides have at least 7 amino acids, and in many cases have at least 20 amino acids. In most cases, such peptides do not have in excess of 40 amino acids.
The peptides and/or analogues or derivatives thereof 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, virus, or virally-infected cell. Thus, for example, the peptides and/or analogues or derivatives thereof may be used as antimicrobial agents, anti-viral agents, anti-bacterial agents, anti-tumor agents, anti-parasitic agents, spermicides, as well as exhibiting other bioactive functions.
The term "antimicrobial" as used herein means that the polypeptides or proteins of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes such as bacteria, fungi, viruses, or the like.
The term "anti-bacterial" as used herein means that the peptides or proteins employed in the present invention produce effects adverse to the normal biological fimctions of bacteria, including death or destruction and prevention of the growth or proliferation of the bacteria when contacted with the peptides or proteins.
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The term "antibiotic" as used herein means that the peptides or proteins employed in the present invention produce effects adverse to the normal biological functions of the non-host cell, tissue or organism, including death or destruction and prevention of the growth or proliferation of the non-host cell, tissue, or organism when contacted with the peptides or proteins.
The term "spermicidal" as used herein means that the peptides or proteins employed in the present invention, inhibit, prevent, or destroy the motility of sperm.
The term "anti-fungal" as used herein means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of fungi.
The term "antiviral" as used herein means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of viruses, or of virally-infected cells.
The term "anti-tumor" as used herein means that the peptides or proteins inhibits the growth of or destroys tumors, including cancerous tumors.
The term "anti-parasitic" as used herein means that the peptides or proteins employed in the present invention inhibit, prevent, or destroy the growth or proliferation of parasites.
The peptides or proteins of the present invention have a broad range of potent antibiotic activity against
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a plurality of microorganisms including gram-positive and gram-negative bacteria, fungi, protozoa, and the like, as well as parasites. The peptides or proteins of the present invention allow a method for treating or controlling microbial infection caused by organisms which are sensitive to the peptides or proteins. 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 or proteins .
Because of the antibiotic, antimicrobial, antiviral, and antibacterial properties of the peptides or proteins, they may also be used as preservatives or sterilants or disinfectants of materials susceptible to microbial or viral contamination.
The peptides or proteins and/or derivatives or analogues thereof 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, inject ble solution, tablet, ointment, lotion, paste, capsule, or the like. The peptide or protein compositions may also be sed in combination with adjuvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused
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by harmful microorganisms including protozoa, viruses, and the like, as well as by parasites.
The peptides or proteins of the present invention may be administered to a host; in particular a human or non-human animal, in an effective antibiotic and/or anti-tumor and/or anti-fungal and/or anti-viral and/or anti-microbial and/or antibacterial and/or anti-parasitic and/or spermicidal amount.
Depending on the use, a composition in accordance with the invention will contain an effective anti-microbial amount and/or an effective spermicidal amount and/or an effective anti-fungal amount and/or an effective anti-viral amount and/or an effective anti-tumor amount and/or an effective anti-parasitic and/or an effective antibiotic amount of one or more of the peptides or proteins of the present invention which have such activity. The peptides or proteins may be administered by direct application of the peptides or proteins to the target cell or virus or virally-infected cell, or indirectly applied through systemic administration.
The peptides or proteins of the present invention may also be employed in promoting or stimulating healing of a wound in a host.
The term "wound healing" as used herein includes various aspects of the wound healing process.
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These aspects include, but are not limited to, increased contraction of the wound, increased deposition of connective tissue, as evidenced by, for example, increased deposition of collagen in the wound, and increased tensile strength of the wound, i.e., the peptides or proteins increase wound breaking strength. The peptides or proteins of the present invention may also be employed so as to reverse the inhibition of wound healing caused by conditions which depress or compromise the immune system.
The peptides or proteins 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 peptides or proteins may be used to treat skin and burn infections caused by organisms such as, but not limited to, P. aeruginosa and S. aureus.
The peptides or proteins 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. by fungi such as but not limited to C. albicans and A. fumigatus, by parasites such as but not limited to A. castellan!. or by viruses.
The peptides or proteins may also be effective in killing cysts, spores, or trophozoites of infection - causing organisms. Such organisms include, but are not limited to Acanthamoeba which forms trophozoites or
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cysts, C. albicans. which forms spores, and A. fumigatus, which forms spores as well.
The peptides or proteins may also be administered to plants in an effective antimicrobial or antiviral or antiparasitic amount to prevent or treat microbial or viral or parasitic contamination thereof.
The peptides or proteins, when used in topical compositions, are generally present in an amount of at least 0.1%, by weight. In most cases, it is not necessary to employ the peptide in an amount greater than 2.0%, by weight.
In employing such compositions systemically (intramuscular, intravenous, intraperitoneal), the active peptide or protein is present in an amount to achieve a serum level of the peptide of at least about 5 ug/ml. In general, the serum level of peptide or protein need not exceed 500 ug/ml. A preferred serum level is about 100 ug/ml. Such serum levels may be achieved by incorporating the peptide or protein in a composition to be administered systemically at a dose of from 1 to about 10 mg/kg. In general, the peptide(s) or protein(s) need not be administered at a dose exceeding 100 mg/kg.
The peptides or proteins may be produced by known techniques and obtained in substantially pure form. For example, the peptides may be synthesized on an automatic peptide synthesizer. Journal of the American Chemical Society. Vol. 85, pgs. 2149-54 (1963). It is also
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possible to produce such peptides or proteins by genetic engineering techniques. The codons encoding specific amino acids are known to those skilled in the art, and therefore DNA encoding the peptides may be constructed by appropriate techniques, and one may clone such DNA into an appropriate expression vehicle (e.g., a plasraid) which is transfected into an appropriate organism for expression of the peptide or protein.
Upon production or synthesis of the peptide or protein, the N-terminal (NH- or amino terminal) of the peptide is reacted such that the lipophilic moiety is attached to the N-terminal of the peptide. For example, the reaction may be a condensation reaction with an amine. When the lipophilic moiety T is 0
I I
R - C -, the N-terminal is reacted with a carboxylic acid of the formula R-C OH, wherein R is a hydrocarbon having at least 2 carbon atoms. The reaction may be carried out in the presence of a coupling agent, such as, for example, DCC, or DIC, and HOBT, or in the presence of an acid chloride. Such a reaction results in the formation of an N-terminal substituted peptide or protein having the structural formula hereinabove described.
In one embodiment, X is a peptide which is a basic (positively charged) polypeptide having at least sixteen amino acids wherein the polypeptide includes at least
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eight hydrophobic amino acids and at least eight hydrophilic 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
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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, Pro, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha). The neutral hydrophilic amino acids may be selected from the class consisting of Asn, Gin, Ser, Thr and homoserine (Hse). 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 one 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
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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 the biologically active polypeptide may comprise 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 a 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
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(X4)a(D-A-B-C)n(Y4)b wherein X. is D; C-D- or B-C-D-, Y is -A or -A-B or
-A-B-C
X2 is A-, D-A- or C-D-A-
Y is -B, -B-C or B-C-D
X3is B-, A-B-, D-A-B-
Y3 is -C, -C-D, -C-D-A
X^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 groups of four amino acids are not spaced from each other.
As representative examples of such peptides, 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 N0: 1)
II Ala-Phe-Ser-Lys-Ala-Phe-Ser-Lys-Ala-Phe-Ser-
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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.
In accordance with another embodiment, X is a magainin peptide.
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A magainin peptide is either a magainin such as magainin I, II or III or an analogue or derivative thereof. The magainin peptides preferably include the following basic peptide structure X.-
"' Ri Ri R12"R13"Ri R14'R12"Rll" R14"R12"Ri Ri Ri R14a"(R15}n"R14a"R14 "" wherein R.. is a hydrophobic amino acid, R-„ is a basic hydrophilic amino acid; R.. is a hydrophobic, neutral hydrophilic, or basic hydrophilic amino acid; R-, and R., are hydrophobic or basic hydrophilic amino acids; R - is glutamic acid or aspartic acid, or a hydrophobic or a basic hydrophilic amino acid, and n is 0 or 1. In a preferred embodiment, R-_ is a hydrophobic or neutral hydrophilic amino acid, R-., is a hydrophobic amino acid, and R-_ is glutamic acid or aspartic acid.
Thus, for example, a magainin peptide may include the following structure:
~Y12~X12~ where X.« is the hereinabove described basic peptide structure and ϊ-- is
where R-., R.,, R., and R,/ are as previously defined.
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A magainin peptide may also have the following structure:
"X12_Z12" wherein X-_ is as previously defined and Z-„ is:
(i) R., where R.- is a basic hydrophilic amino acid or asparagine or glutamine.
(ii) R---R-- where R-_ is a neutral hydrophilic amino acid, a hydrophobic amino acid, or a basic hydrophilic amino acid. Preferably, R-_ is a neutral hydrophilic amino acid.
A magainin peptide may also have the following structure:
where X-2, Y-,. and Z.„ 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 X-_:
"R14'RirR14a"R12'Ri RH"R12'R13" Rll"R14"R12"Rll"Rll"R12"' whβrein Rll'R12,R13' R14' and
R,, are amino acids as hereinabove described. 14a
The magainin peptide may also include the following structure X..-Z.,; wherein X13 is the hereinabove described basic peptide structure and Z., is
<Rll <^l (^l < n-<R15 < n-<R1 - ( R16)n"(R17)n whβrθin Rll* R14' R14a' R15 ' R16« ∞* R17
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are 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 peptides having the following primary sequences as given in the accompanying sequence listing as well as appropriate analogues and derivatives thereof:
(a) (SEQ ID NO:6) (OH) or (NH2) (Magainin I)
(b) (SEQ ID NO:7) (OH) or (NH2 (Magainin II)
(c) (SEQ ID NO:8) (OH) or (NH2 (Magainin III)
The following are examples of peptide derivatives or analogs of the basic structure:
(d) (SEQ ID NO:9) (OH) or (NH2)
(e) (SEQ ID NO: 10) (OH) or (NH2
(f) (SEQ ID NO:11) (OH) or ( H2)
Magainin peptides are described in Proc. Natl. Acad Sci. Vol. 84 pp. 5449-53 (Aug. 87). The term "magainin
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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, X 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., :
- R n-R 17-R12-Rι Ri4"Ri4'Rιr
Ri Ri4"Ri2"Rn"RirRi2'Rir
where R-...f R-i?' RiΛ' and Ri7 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 X., is as previously defined and
Y14 is
(i) Rn;
(ϋ) R14'R11
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where R- - and R. , are as previous ly defined. For example, a PGLa peptide may also have the following structure:
"X14"Z14" where X. , is as previous ly defined; and Z- , is :
( i) Rn ; or
( ii) Rn-Rn where R.. is as previously defined.
A PGLa peptide may also have the following structure:
(Y ~X14~^Z14^ 1 14ja l b
where X,#; Y.,/ and Z., 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 X.,:
'"Ri R17"R12"RH'R14"R18'R17" Rι Ri4"Ri2'RιrRn"Ri2" RI RI RιrRi2-(Ri5 Rn"' wherein Rll' 12* 14' R15 and 17 arβ βS Previ°usly defined and R-- is glutamine or asparagine or a basic hydrophilic, or hydrophobic amino acid and, n is 0 or 1.
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
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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 X-, is as previously defined and Y., is
(i) Rn or
(ii) R14-Rn where R- - and R., are as previously defined.
An XPF peptide may include the following structure:
'16
An XPF peptide may also have the following structure:
where X1fi» Y1ft and Z., 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 primary amino acid sequences as given in the accompanying sequence listing:
PGLa : (SEQ ID NO:12) H2)
XPF : (SEQ ID NO:13)
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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, X is a CPF peptide or appropriate analogue or derviative thereof.
CPF peptides as well as analogues and derivatives thereof are herein sometimes referred to collectively as CPF peptides.
The CPF peptide may be one which includes the following basic peptide structure X-,-,:
"R2 R2 R22'R22"R2l'R2rR23"R2r
"R2l"R2 R23"R2 R2 R24'R25"R2 wherein R-.. is a hydrophobic amino acid;
R_„ is a hydrophobic amino acid or a basic hydrophilic amino acid;
R_- is a basic hydrophilic amino acid;
R_, is a hydrophobic or neutral hydrophilic amino acid; and
R__ is a basic or neutral hydrophilic amino acid.
The hereinabove basic structure is hereinafter symbolically indicated as X_0.
The hydrophobic amino acids are Ala, Cys, Phe, Gly, lie, Leu, Met, Val, Trp, Tyr, norleucine (Nle), norvaline (Nva), and cyclohexylalanine (Cha).
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The neutral hydrophilic amino acids are Asn, Gin, Ser, Thr, and homoserine (Hse).
The basic hydrophilic amino acids are Lys, Arg, His, Orn, homoarginine (Har), 2,4-diaminobutyric acid (Dbu), and p-aminophenylalanine.
The CPF peptide may include only the hereinabove noted amino acids or may include additional amino acids at the amino and/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 structure preferably have from 1 to 4 additional amdLno acids at the amino end.
Accordingly, such preferred peptides may be represented by the structural formula:
Y20 " X20 " wherein X__ is the hereinabove described basic peptide structure and Y... is
(i) R25-, or
(ii) R22'R25_; or
(iii) R2l"R22"R25' or
(iv) R22_R2l"R22"R25; PreferablY Glycine - R21-R22-R25. wherein R21> 22 ant^ R ?ς are as Prβvi°usly 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.
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In a preferred embodiment, the basic structure may have from 1 to 7 additional amino acids at the carboxyl end, which may be represented as follows:
" 20 " Z20 wherein
X is the hereinabove defined basic peptide structure and Z20 is
(i) R21-, or
(ii) R 2i"R2l"; or
(iii) R2l"R2l"R24; or
(v) R 2 R 2rR 24-R24-R26; °r (vi) R21-R21-R24-R24-R26-Gln; or
(vii) R21-R21-R24-R24-R26-Gln-Gln, wherein R and R_4 are as previously defined, and R_, is proline or a hydrophobic amino acid.
Preferred peptides may be represented by the following structural formula
^20^ " X20 " ^20^
wherein X-,n» Y«n and Z__ are as previously defined and a is 0 or 1 and b is 0 or 1.
Representative examples of CPF peptides which may be employed, some of which have been described in the literature, include the following sequences as given in the accompanying sequence listing: (SEQ ID NO:14) (SEQ ID NO:15)
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(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.
In accordance with yet another embodiment, X is a peptide which includes one of the following basic structures X,- through X__ wherein:
X32 1S '[R32'R32"R33~R3 R32"R32"R31
X33 is "[R32'R33"R3 R32"R32'R3l"R32
X34 is "lR33'R3l"R32"R32"R3 R32"R32
X35 iS "[R3 R32'R32"R3l"R32'R32"R33 and
X36 1S "[R32"R32"R3 R32'R32'R33"R31
X37 iS "[R32"R3 R32'R32"R33"R3l'R32
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wherein R,- is a basic hydrophilic amino acid, R,-. is a hydrophobic amino acid, R_, is a neutral hydrophilic, basic 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-aminopheny1a1anine.
The hydrophobic amino acids may be selected from the class consisting of Ala, Cys, Phe, Gly, lie, Leu, Met, Pro, 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, Gin, Ser, Thr, and homoserine (Hse).
In accordance with one embodiment, when the peptide includes the structure X-.,» the peptide may include the following structure:
Y,..-X,-., wherein X,- is as hereinabove described, and Y_. is:
(i) R32;
(ii) R32-R32;
(iii) R31-R32-R32;
(iv) R33-R 31-R32-R32;
(v) R 32-R 33-R31-R32-R32; or
(vi) R 32- 32-R 33-R 31-R32-R 32. "herein 3ι» R32' ^ R_. are as hereinabove described
-26-
SUBSTITUTESHEET
In accordance with another embodiment, when the peptide includes the structure X-,,> the peptide may include the following structure:
X- . -Z- . , wherein X,- is as hereinabove described, and Z_- is:
(i) R31;
(ii) R31-R32;
(iii) R31-R32-R32;
(iv) R31-R32-R 32-R 33;
(v) R 31-R 32-R32-R33-R31; or
(vi) R31-R32-R32-R 33-R 3 R 32-
In accordance with yet another embodiment, the peptide may include the following structure:
(T3lYX3 (Z3lV whθrein Y31 ^ Z31 a" as previously defined, a is 0 or 1, and b is 0 or 1.
When the peptide includes the structure X-.->. the peptide may include the following structure:
Y_2 - X«2» wherein X__ is as hereinabove described, and Y32 is:
(i) R31;
(ii) R32-R31;
(in) R32-R32-R31; (iv) R31-R32-R32-R31;
(V) R33-R3 R32-R32_R31; °r (Vi) R 32-R33-R3 R32'R32"R31-
■27-
SUBST ΠJTE SHEET
In another embodiment, when the peptide includes the structure -j - the peptide may include the following
*32
(iv) R32-R32-R 33-R31;
(v) R32-R32"R33-R31-R32; °r
(vi) R 32-R 32-R 33-R 3 R 32-R 32-
In accordance with yet another embodiment, the peptide may include the following structure:
(Y32}a " X32 " (Z32V whβrein Y 32 and Z32 a" βS previously defined, a is 0 or 1, and b is 0 or 1.
In accordance with another embodiment, when the peptide includes the structure X__, the peptide may include the following structure:
Y,, - X_3 wherein X__ is as hereinabove described,
(iv) R32-R32-R31-R32;
(v) R3 R 32-R 32-R 31-R 32; or
(vi) R 33-R 3 32-R 32-R 3 R32. wherein R31 > R32» and R_- are as hereinabove described.
-28-
SUBSTΠTUTE SHEET
In accordance with another embodiment, when the peptide includes the structure X-,-,. the peptide may include the following structure:
X-_ - Z__ wherein X__ is as hereinabove described, and Z33 is:
(i) R32; (ii) R32-R33; (iii) R32-R33-R31;
( iv) R 32-R 33-R 31-R 32;
(v 32-R 33-R 31- 32-R 32; °r
(vi) R 32-R 33-R 31-R 32-R 32-R 31-
In accordance with yet another embodiment, the peptide may include the following structure:
"Wa ' X33 " i Z > whθrein Y 33 "* Z33 arβ βS 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:
Y,, - X~4, wherein X„ , is as hereinabove described, and Y-. is: 34
(i) R32;
(11) R32-R32; (iii) R31-R32- 32J
(v) R32-R32-R3 32-R325 °r
-29-
SUBSTITUTE SHEET
(V± R 3 R32-R32-R3 R32"R32' wherein R31' R32 and R are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X-, > the peptide may include the following structure:
X_,-Z_,, wherein X„, is as hereinabove described, 34 34 34 and Z„, is: 34
(i) R33; (ii) R33-R31; (iii) R33-R31-R32;
(lv) R33'R3l"R32"R32;
(v) R33-R31-R 32-R32- 31; or (vi) 33- 31-R 32-R 32-R 31-R 32-
In accordance with yet another embodiment, the peptide may Include the following structure:
(Y34)a" X34" (Z345b' whβrein X 34 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 X,_, the peptide may include the following structure:
Y_,-X__, wherein X__ is as hereinabove described, and Y__ is:
(i) R33;
(ii) R32-R33;
(iii) R32-R32-R33;
(v) R 32-R 31-R 32"R 32-R 33; or
-30-
SUBSTITUTESHEET
(V1) R32-R32'R3 R32"R32_R33' whβrein R31' R32' « R_, are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure X,,, the peptide may include the following structure:
X__ - Z._ wherein X__ is as hereinabove described, and Z,_ is:
(i) R31; ii) R31-R32;
(iii) R31-R32-R32;
(iv) R31-R32-R32-R31;
(v) R31-R32-R32-R31-R32; °r
(vi) R31-R32-R32-R31-R32-R32-
In accordance with yet another embodiment, the peptide may include the following structure:
(Y«) - X35 (Z35 t,> wherein X3_ and Z-. 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 X,,, the peptide may
Jo include the following structure:
Ygfi - X-6 wherein X,, is as hereinabove described, and Y--g is:
(i) R31;
(ii) R33-R31;
(ill) R32-R33-R31;
(v) R 31-R 32-R 32-R 33-R 31; or
-31-
SUBSΠTUTE SHEET
(vi) R32 "R31 "R32"R32"R33"R31' herein R 31> R32» and R__ are as hereinabove described.
In accordance with another embodiment, when the peptide includes the structure XQ6> the peptide may include the following structure: ~, -Z-, _ wherein X. , is as hereinabove described, 36 3o JO and Z_- is:
Jo
(i) R32; (ϋ) R32-R32; (iii) R32-R32-R31J (iv) R32-R32-R31-R32; (v) R32-R32-R 31- 32-R32; °r
(vi) R32-R32-R31-R32-R32-R33- In accordance with yet another embodiment, the peptide may include the following structure:
(Y3βY X36 (Z36}b' Whβrein Y36 ^ Z36 a" 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 X,7, the peptide may includes the structure Y__-Xg_, wherein Xg7 is as hereinabove described, and Y__ is:
(i) R32;
(ii) R3i~R32'
(v) R 32-R32-R33-R 31-R32; or
-32-
SUBSTITUTESHEET
(vi) R 31 "R32"R32"R33"R3l"R32' hereln R 31» R 32' and R_, are as hereinabove described.
In accordance with a further embodiment, when the peptide includes the structure X-,7, the peptide may include the following structure:
X,_ - Z__ wherein X__ is as hereinabove described, and Z,_ is:
(i) R32;
(ii) R32-R31;
(iii) R32-R31-R32;
(v) R 32-R 31-R32-R32- 33; or (vi) R32-R 31-R32-R32-R33-R 31-
In accordance with yet another embodiment, the peptide may Include the following structure:
(Y37)fi- 37
whβrβin γ 37 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 is of one of the following structures as given in the accompanying sequence listing:
(Lys He Ala Gly Lys He Ala)3 (SEQ ID NO:27).
(Lys He Ala Lys He Ala Gly)3 (SEQ ID NO:28).
(Lys He Ala Gly Lys He Gly)g (SEQ ID NO:29).
(Lys Leu Ala Gly Lys Leu Ala)3 (SEQ ID N0:30).
(Lys Phe Ala Gly Lys Phe Ala)3 (SEQ ID NO:31).
(Lys Ala Leu Ser Lys Ala Leu)3 (SEQ ID NO:32).
(Lys Leu Leu Lys Ala Leu Gly)3 (SEQ ID NO:33).
-33-
SUBSTITUTESHEET
SUBSTITUTE SHEET
(Lys Phe Ala Gly Lys He Ala)3 (SEQ ID NO:61).
(Lys He Ala Gly Lys Phe Ala)3 (SEQ ID NO:62).
(Lys Cha Ala Gly Lys He Ala)3 (SEQ ID NO:63).
(Lys Nle Ala Lys He Ala Gly).. (SEQ ID NO:64).
(Arg He Ala Gly Lys He Ala)3 (SEQ ID NO:65).
(Har He Ala Gly Har He Ala)3 (SEQ ID NO:66).
(Xaa He Ala Gly Lys He Ala)3 (SEQ ID NO:67).
(Lys He Ala Gly Xaa He Ala)3 (SEQ ID NO:68). In (SEQ ID NO:67) and (SEQ ID NO:68), Xaa is p-aminophenylalanine.
In accordance with another embodiment, X is a peptide which Includes the following basic structure X,_:
R3 R32"R32"R33"R34"R32"R32"R3 R32"R32'R32"R34"R32"R32« wherein R31, R,-, and R_3 are as hereinabove described, and R«, is a basic hydrophilic or hydrophobic amino acid.
In accordance with one embodiment, the peptide may include the following structure:
Y,0-X,_, wherein X,_ is as hereinabove described, and Y,_ is: 40
(i) R32;
(11) R32-R32;
(iii) R34-R 32- 32;
(v) R32"R33"R34~R32"R32; (vi) R 32- 32-R33-R3 -R32-R32' °r
-35-
SUBSΠT ΠΈ SHEET
(vii) R31-R32-R32-R33-R34-R32-R32,wherein R31, R32>
33 an 34 are as hereinabove described.
In accordance with another embodiment, X is a peptide which includes the following structure:
X «"Z n, wherein X,Λ is as hereinabove described and 40 40 40
Z40 is:
(i) R31; (ii) R31-R32;
(iii) R31-R32-R32;
(v) R31-R32-R32-R33-R34;
(vi) R 31-R32-R32'R33'R3 "R32; or
(vii) R31-R32-R32-R33-R34"R32*R32* Whβrθln R31« R32«
R,_, and R,. are as hereinabove described. 33 34
In accordance with yet another embodiment the peptide may Include the following structure:
(Y40 a"X40'(Z40)b» wherβin Y 0 ^ 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 given in the accompanying sequence listing:
(SEQ ID NO: 69)
In another preferred embodiment, the peptide has the following structural formula as given in the accompanying sequence listing:
(SEQ ID NO: 70)
■36-
SUBSTITUTESHEET
In accordance with a further embodiment, the peptide has one of the one of the following structural formulae as given in the accompanying sequence listing:
In accordance with another embodiment, X is a peptide which Includes one of the following structural formulae:
(1) - (Lys He Ala Lys Lys He Ala) n'
(11) - (Lys Phe Ala Lys Lys Phe Ala) -, and (iii) - (Lys Phe Ala Lys Lys He Ala) -, wherein n is from 2 to 5. Preferably, n is 3, and the peptide has one of the following structural formulae:
-37-
SUBSΠT ΠΈ SHEET
(Lys He Ala Lys Lys He Ala)
(SEQ ID NO: 86)
(Lys Phe Ala Lys Lys Phe Ala)
(SEQ ID NO: 87)
(Lys Phe Ala Lys Lys He Ala) _
(SEQ ID NO: 88)
In accordance with another embodiment, the X is a peptide which is selected from the group consisting of the following structural formulae as given in the accompanying sequence listing:
(SEQ ID NO: 89)
(SEQ ID NO: 90)
(SEQ ID NO: 91)
(SEQ ID NO: 92)
In accordance with yet another embodiment, X is a cecropin or sarcotoxin.
The term cecroplns Includes the basic structure as well as analogues and derivatives thereof. The cecropins and analogues and derivatives thereof are described in Ann. Rev. Microbiol. 1987, Vol. 41, pages 103-26, in particular page 108, and In Christensen, et al., PNAS Vol. 85, pgs. 5072-76, which are hereby incorporated by reference.
The term sarcotoxins includes the basic materials as well as analogues and derivatives thereof. The sarcotoxins and analogues and derivatives thereof are described in Molecular Entomology, pages 369-78, in
-38-
SUBSTITUTESHEET
particular page 375, Alan R. Liss, Inc. (1987), which is hereby incorporated by reference.
In accordance with another embodiment, X is melittin or an analogue or derivative thereof.
Melittin is an amphipathic peptide consisting of 26 amino acid residues, and is isolated from honeybee (Apis ellifera) venom. Habermann, et al., Hoppe-Seyler's Zeitschrift Phvsiol. Chem.. Vol. 348, pgs. 37-50 (1987). Melittin has the following structural formula as represented by the three-letter amino acid code: Gly He Gly Ala Val Leu Lys Val Leu
5 Thr Thr Gly Leu Pro Ala Leu He Ser 10 15
Trp He Lys Arg Lys Arg Gin Gin
20 25
(SEQ ID NO: 93)
In another embodiment, X is a amphiphilic peptide which includes the following basic structure X__:
R4l'R42'R42"R4 R42"R42"R4 R4 R42"R4 R4r
R41 is a hydrophobic amino acid, and R,_ is a basic hydrophilic or neutral hydrophilic amino acid.
In one embodiment, the peptide includes the basic structure Y__-X_0 wherein X__ is as hereinabove described and Y__ is: i) R41;
(ii) R42"R41; or ■39-
SUBSTITUTESHEET
(iii) R4 -R4--R4-, wherein R4- and R4- are as hereinabove described.
In one embodiment, R. , is leucine. In another
41 embodiment, R4_ is lysine. Representative examples of peptides in accordance with this aspect of the present invention include those having the following structures:
(SEQ ID NO: 94)
(SEQ ID NO: 95)
(SEQ ID NO: 96)
(SEQ ID NO: 97)
In accordance with another embodiment, X is an amphiphilic peptide which includes the following basic structure X '.
R42"R4 R42"R42"R4 R4rR42"R42~R4l'R42"R42' whθreln R4- is a hydrophobic amino acid and R4_ is a basic hydrophilic or neutral hydrophilic amino acid.
In one embodiment R, -, is leucine. In another
41 embodiment, R, ~ is lysine.
In one embodiment, the peptide includes the basic structure Y__-X_2, wherein X_2 is as hereinabove described, and Y_2 is:
-40-
SUBSTITUTESHEET
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.: 98)
In another embodiment, the peptide includes the basic structure X__ - Z_2, wherein X__ is as hereinabove described, and Z__ is:
In one embodiment, the peptide may have the following structure:
Lys Leu Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu Leu Lys Lys Leu 5 10
15
(SEQ ID NO: 99)
In another embodiment, the peptide may include the structure:
-41-
SUBSTITUTESHEET
Whβrein X52 « Y52 and Z52 are as hereinabove described, and a is 0 or 1, and b is 0 or
1.
In another embodiment X is a biologically active amphiphilic peptide which includes the following basic structure X_, : 54
R4 R42"R42 "R4 R41 "R42"R42"R4 R42"R42"R4 R4 R42 "R42"
R43' » wherein R, , and R, „ are as hereinabove described, and R.. 41 42 43 is a neutral hydrophilic amino acid.
In one embodiment, the peptide may have the following structure:
(SEQ ID NO: 100)
In another embodiment, the peptide may have the following structure:
(SEQ ID NO: 101)
In another embodiment, X is a biologically active amphiphilic peptide which includes the following basic structure X..:
R4 R42"R4 R4l"R42"R42"R4 R4rR42'R42"R44' whβrβin R41 and R.„ are as hereinabove described, and R, , is a 42 44 neutral hydrophilic amino acid or proline.
In one embodiment, the peptide may include the structure:
X- . -Z- , , wherein X_fi is as hereinabove described, and Z_, is:
■42-
SUBSTITUTESHEET
( ii) -R 42-R42; (iii) -R 42-R42-R41 ;
(v) -R 42-R 42-R 41- 41-R 42;
(vi) -R42"R42"R4 R4 R42-R 2; °r (vii) R42-R42-R41-R41-R42-R42-R41.
In a preferred embodiment, the peptide may have one of the following structures:
(SEQ ID NO: 102); or
(SEQ ID NO: 103).
In another embodiment, X is a biologically active amphiphilic peptide which includes the following basic structure X__:
R4 R4ΓR42'R42"R4 R42_R42"R41"R41'R42"R42"R41"R43' wherein R 1 , R „, and R,, are as hereinabove described.
In one embodiment, the peptide Includes the structure X_g-Z_a, wherein X__ is as hereinabove described, and Z__ is:
( i -R41;
(ii) -R41-R45; (ill) -R41-R45-R45;
(iv) -R41-R45-R45-R43;
(v) -R41-R45-R45-R43-R41;
(vi) -R41-R45-R45-R43-R41-R43
(vii) -R41-R45-R45-R43-R 1-R43-R43;
(viii) -R41-R45-R45-R43-R41-R43-R43-R45; or
-43-
SUBSTITUTESHEET
(ix) -R41-R45-R45-R43-R41-R43-R43-R45-R43, wherein
R., and R, _ are as hereinabove described, and R. _ is 41 43 45 proline.
In one embodiment, the peptide has the following structure:
(SEQ ID NO: 104).
In another embodiment, X is a biologically active amphiphilic peptide which includes the following basic structure X,.:
R4 R4 R43"R42'R4 R4 R4 R4 R4I'R4 R42"R4 R4 R42"
R 2"R4l"R4rR42"R42"R4 ' whβrein R41« R42 > and R43 are as hereinabove described. In one embodiment, the peptide may have the following structure:
(SEQ ID NO: 105).
In accordance with another embodiment, X is a peptide which Includes the following basic structure X, : oZ
"R4 R42"R42"R4 R42"R42'R41"' wherein R. , and R,„ are as hereinabove described. 41 42
In one embodiment the peptide includes the following structure Y,„ - Xfi2, where X.. is as hereinabove described, and Y,_ is:
(i) 41;
(ii) R42-R42;
(iii) R42-R42-R 41; or
(iv) R41-R 2-R42-R4r
-44-
SUBSTITUTESHEET
Representative examples of such peptides include the following, the sequences of which are given in the accompanying sequence listing:
In one embodiment, the peptide includes the structure Xfi2-Z,_, wherein X,. is as hereinabove descibed, and Z,_ is:
(ii) R41-R42;
(iii) R41-R42-R42; or
(iv)
R4l"R42'R42-R41' whβre R41 and R42 arβ as hereinabove described.
A representative example includes the following peptide having the structural formula given below and listed in the accompanying sequence listing: (SEQ ID NO: 112)
In another embodiment, the peptide has the structure
(Y62V 62 (Z62)b, wherein Xg2, Yg2 and Zg2 are as hereinabove described, a is 0 or 1, and b is 0 or 1.
Representative examples of such peptides include the following, the structures of which are given in the accompanying sequence listing:
-45-
SUBSΠTUTE SHEET
( SEQ ID NO : 113)
( SEQ ID NO : 114)
( SEQ ID NO : 115 )
(SEQ ID NO: 116) In another embodiment, X is a peptide having the following structural formula:
(SEQ ID NO: 117) In accordance with yet another embodiment, X is an ion channel-forming peptide or protein.
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), and a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein. 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 (1987). Perforin is described in Henkart, et al., J. Exp. Med.. 160: 75 (1984), and in Podack, et al., J. Exp. Med.. 160:695 (1984). The above articles are hereby incorporated by reference.
-46-
SUBSTITUTESHEET
The term ion channel-forming proteins includes the basic structures of the ion channel-forming proteins as well as analogues and derivatives.
In accordance with yet another embodiment, each of the amino acid residues of the peptides or proteins may be a D-amino acid or glycine. Although the scope of this particular embodiment is not to be limited to any theoretical reasoning, it is believed that the above-mentioned peptides or proteins, when consisting entirely of D-amino acid or glycine residues, may have increased resistance to proteolytic enzymes while retaining their activity. Such peptides thus may be administered orally. Also, in accordance with another embodiment, all of the amino acid residues may be D-amino acid or glycine residues, or L-amino acid or glycine residues.
It is also to be understood that the peptides or proteins may be administered in combination with one another.
In accordance with another embodiment, the N-terminal substituted peptides or proteins of the present invention may be employed in combination with an ion having phamacological properties for the purposes hereinabove described.
An ion having pharmacological properties is one which when introduced into a target cell or virus or virally-infected cell inhibits and/or prevents and/or
-47-
SUBSTITUTE SHEET
destroys the growth of the target cell, virus, or virally-infected cell.
Such an ion having pharmacological properties is one which in the absence of an ion channel forming peptide is unable to cross a natural or synthetic lipid membrane; in particular a cell or virus membrane, in sufficient amounts to affect a cell or virus adversely.
The peptide or protein and ion having pharmacological properties may be administered as a single composition or in separate compositions, and the single or separate compositions may include additional materials, actives and/or inactives, in addition to the peptide or protein and ion having pharmacological properties. As representative examples of ions having pharmacological properties which may be employed, there may be mentioned fluoride, peroxide, bicarbonate, silver, zinc, mercury, arsenic, copper, platinum, antimony, gold, thallium, nickel, selenium, bismuth, and cadmium ions.
The peptide or protein and the ion having pharmacological properties, whether administered or prepared in a single composition or in separate compositions, are employed in amounts effective to inhibit and/or prevent and/or destroy the growth of the target cell, virus, or virally-infected cell. In effect, the ion potentiates the action of the peptide, i.e., the amount of ion is effective to reduce the maximum effective concentration of the peptide or protein for
-48-
SUBSTITUTESHEET
inhibiting growth of a target cell, virus, or virally-infected cell.
The ion having pharmacological properties, when used topically, is generally employed in a concentration of from 0.05% to 2.0%. When used systemically, the ion is generally employed in an amount of from 1 to 10 mg. per kg. of host weight. Peptide or protein dosages may be within the ranges hereinabove described.
It is also to be understood that the peptide or protein and ion having pharmacological properties, may be delivered or administered in different forms; for example, the ion may be administered orally, while the peptide may be administered by IV or IP.
As representative examples of administering the peptide or protein and ion for topical or local administration, the peptide could be administered in an amount of up to about 1% weight to weight and the ion delivered in an amount of about 50mM (about 0.1%). Alternatively, the ion, in the form of a salt such as sodium fluoride, could be administered orally in conjunction with systemic administration of the peptide or protein. For example, the peptide or protein may be administered IV or IP to achieve a serum dose of 100 micrograms per milliliter (10 milligrams per kilogram) in conjunction with an oral dose of ion, in particular, sodium fluoride, of 10 meq per kilogram.
■49-
SUBSTITUTESHEET
In accordance with another embodiment, the peptides or proteins of the present invention may be administered to a host in combination with an antibiotic selected from the class consisting of bacitracins, gramacidin, polymyxin, vancomycin, teichoplanin, aminoglycosides, hydrophobic antibiotics, penicillins, raonobactams, or derivatives or analogues thereof.
The bacitracins, gramacidin, polymyxin, vancomycin, teichoplanin, and derivatives and analogues thereof, are a group of polypeptide antibiotics. A preferred bacitracin is bacitracin A.
Aminoglycoside antibiotics include tobramycin, kanamycin, amikacin, the gentamiclns (e.g., gentamicin C-, gentamicin C„, gentamicin C- ), netilmicin, and derivatives and analogues thereof. The preferred aminoglycosides are tobramycin and the gentamiclns. The aminoglycosides, and the bacitracins hereinabove described, tend to be hydrophilic and water-soluble.
Penicillins which may be employed include, but are not limited to benzyl penicillin, ampicillin, methicillin (dimethoxyphenyl penicillin), tlcaricillin, penicillin V (phenoxymethyl penicillin), oxacillin, cloxacillin, dicloxacillin, flucloxacillln, amoxicillin, and amidinocillin. Preferred penicillins which may be employed are benzyl penicillin and ampicillin. A preferred monobactam which may be employed is aztreonam.
-50-
SUBSTITUTESHEET
As representative examples of hydrophobic antibiotics which may be used in the present invention, there may be mentioned macrolides such as erythromycin, rσxythromycin, clarithromycin, etc. ; 9-N-alkyl derivatives of erythromycin; midecamycin acetate; azithromycin; flurithromycin; rifabutin; rokitamycin; a 6-0-methyl erythromycin A known as TE-031 (Taisho); rifapentine; benzypiperazinyl rifamycins such as CGP-7040, CGP-5909, CGP-279353 (Ciba-Geigy); an erythromycin A derivative with a cyclic carbamate fused to the C-j. C^ position of a macrolide ring known as A-62514 (Abbott); AC-7230 (Toyo Jozo); benzoxazinorifamycin; difficidin; dirithromycin; a 3-N-piperdinomethylzaino methyl rifamycin SV known as FCE-22250 (Faπnitalia); M-119-a (Kirln Brewery); a 6-0-methyl-l-4"-0-carbamoyl erythromycin known as A-63075 (Abbott); 3-formylrifamycin SV-hydrazones with diazabicycloalkyl side chains such as CGP-27557 and CGP-2986 (Ciba-Geigy); and 16-membered macrolides having a 3-0-alpha-L-cladinosyl moiety, such as 3-0-alpha-L-cladinosyldeepoxy rosaramicin; tylosins and acyl demycinosyl tylosins.
In addition to the macrolides hereinabove described, rifamycin, carbenicillin, and nafcillin may be employed as well.
Other antibiotics which may be used (whether or not hydrophobic) are antibiotics which are 50-S ribosome
-51-
SUBSTITUTESHEET
inhibitors such as lincomycin; clindamycin; and chloramphenicol; etc.; antibiotics which have a large lipid like lactone ring, such as mystatin; pimaricin, etc.
The peptide or protein and antibiotic may be adminstered by direct administration to a target cell or by 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. Target cells whose growth may be prevented, inhibited, or destroyed by the administration of the peptides and antibiotic include Gram-positive and Gram-negative bacteria as well as fungal cells.
The antibiotic, such as those hereinabove described, or derivatives or analogues thereof, when used topically, is generally employed in a concetration of about 0.1% to about 10%. When used systemically, the antibiotic or derivative or analogue thereof is generally employed in an amount of from 1.25 mg. to about 45 mg. per kg. of host weight per day. Peptide or protein dosages may be those as hereinabove described.
As representative examples of administering the peptide or protein and antibiotic for topical or local administration, the peptide or protein could be administered in an amount of from about 0.1% to about 10% weight to weight, and the antibiotic is delivered in an amount of from about 0.1% to about 10% weight to weight.
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In accordance with another embodiment, the peptides or proteins of the present invention may be administered in combination with an antiparasitic agent or an antifungal agent.
Antiparasitic agents which may be employed include, but are not limited to, anti-protozoan agents. Examples of specific anti-parasitic agents which may be employed include, but are not limited to, pentamidine isethionate, and propamidine isethionate (Brolene).
Anti-fungal agents which may be employed include, but are not limited to, ketoconazole. It is also to be understood that certain anti-parasitic agents, may also have anti-fungal activity, and that certain anti-fungal agents may have anti-parasitic activity.
In accordance with another embodiment, the peptides or proteins of the present invention may be administered in combination with an antibiotic which inhibits DNA gyrase, which is an enzyme involved in the formation of bonds between individual coiling strands of replicating bacterial DNA. Thus, DNA gyrase is necessary for the normal replication of bacterial DNA, and, therefore, antibiotics which inhibit DNA gyrase inhibit the normal replication of bacterial DNA.
Examples of antibiotics which inhibit DNA gyrase include nalidixic acid, oxolinic acid, cinoxacin, and quinolone antibiotics which include ciprofloxacin, norfloxacin, ofloxacin, enoxacin, pefloxacin,
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lomefloxacin, fleroxacin, tosulfloxacin, temafloxacin, and rufloxacin.
The present invention will be further described with respect to the following example; however, the scope of the invention is not to be limited thereby.
EXAMPLE
Table I, which follows, indicates the Minimal Inhibitory Concentration (MIC) in μg/ml of Peptides (SEQ ID NO: 27), (SEQ ID NO: 110), (SEQ ID NO: 113), and (SEQ ID NO: 118), against S.aureus strain ATCC 25923, P^ aeruginosa strain ATCC 27853, and E.coli ATCC strain 25922. The peptides are unsubstituted at the N-terminal, substituted with an acetyl group at the N-terminal as indicated by Ac-, or substituted with an octanoyl group at the N-terminal as indicated by Oct-.
The procedure for the 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 peptides (SEQ ID N0:27), (SEQ ID NO: 110), (SEQ ID NO: 113), and (SEQ ID NO: 118), with and without the appropriate substitutions, are prepared at a concentration of 512 μg/ml in sterile deionized distilled water and stored at -70°C. Each peptide is a C-terminal amide.
The stock peptide solution is diluted in serial dilutions (1:2) down the wells of a microtiter plate so
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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 either S. aureus ATCC 25923, E. coli ATCC 25922, or P. aeruginosa ATCC 27853 were added to the wells in full strength Mueller Hinton broth (BBL 11443) from a mid-log culture. The inoculum is standardized spectrophotometrically at 600 nm 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 minimal inhibitory concentration of each of (SEQ ID NO: 27), (SEQ ID NO: 110), (SEQ ID NO: 113), and (SEQ ID NO: 118) with and without the appropriate substitutions is given in Table I below.
■55-
SUBSTITUTESHEET
Table I
Minimal Inhibitory Concentration
( μg/ml)
Peptide S. aureus P. aeruginosa E. coli
(SEQ ID NO: 27)-NH2 8,16 64,128 8
Ac- (SEQ ID NO: 27)-NH2 32 128 8
Oct-(SEQ ID NO: 27)-NH2 2 4 2,4
(SEQ ID NO: 110)-NH2 >256 32,64 64,128
Ac-(SEQ ID NO: 110) - H2 256 8,16 32,64
Oct-(SEQ ID NO: 110)-NH2 8 4 16
(SEQ ID NO: 113)-NH2 16,32 8,16 32
Ac- (SEQ ID NO: 113)-NH2 32 64 64
Oct-(SEQ ID NO: 113)-NH2 8 4 16,32
(SEQ ID NO: 118)-NH2 >256 256 256
Ac-(SEQ ID NO: 118)-NH2 256 256 256
Oct-(SEQ ID NO: 118)-NH2 8 8 32
The above results indicate that when a biologically active peptide is substituted with a lipophilic moiety of the present invention, the peptid has increased biological activity against a variety of microorganisms.
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.
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SUBSΠTUTE SHEET
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Karl, U. Prasad
(ii) TITLE OF INVENTION: Biologically Active Peptides Having N-Terminal Substitutions
(iii) NUMBER OF SEQUENCES: 118
(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:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Olsteln, Elliot M.
(B) REGISTRATION NUMBER: 24,025
(C) REFERENCE/DOCKET NUMBER: 421250
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700
(B) TELEFAX: 201-994-1744
(2) INFORMATION FOR SEQ ID N0:1: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(x) PUBLICATION INFORMATION:
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SUBSTITUTESHEET
(H) DOCUMENT NUMBER: W089/11290
(I) FILING DATE: 19-MAY-1989
(J) PUBLICATION DATE: 30-NOV-1989
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ala Phe Ser Lys Ala Phe Ser Lys Ala Phe 5 10
Ser Lys Ala Phe Ser Lys Ala Phe Ser Lys 15 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
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: W089/11290
(I) FILING DATE: 19-MAY-1989
(J) PUBLICATION DATE: 30-N0V-1989
(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
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: W089/11290
(I) FILING DATE: 19-MAY-1989
(J) PUBLICATION DATE: 30-NOV-1989
(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: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
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SUBSTITUTESHEET
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: W089/11290
(I) FILING DATE: 19-MAY-1989
(J) PUBLICATION DATE: 30-NOV-1989
(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
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: W089/11290
(I) FILING DATE: 19-MAY-1989
(J) PUBLICATION DATE: 30-NOV-1989
(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.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proc. Nat. Acad. Sci.
(D) VOLUME: 84
(F) PAGES: 5449-5453
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SUBSTITUTESHEET
(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 He Gly Lys Phe Leu His Ser Ala Gly
5 10
Lys Phe Gly Lys Ala Phe Val Gly Glu He 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.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proc. Nat. Acad. Sci.
(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 He Gly Lys Phe Leu His Ser Ala Lys
5 10
Lys Phe Gly Lys Ala Phe Val Gly Glu He 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.
(x) PUBLICATION INFORMATION:
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SUBSTITUTESHEET
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proc. Nat. Acad. Sci.
(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 He Gly Lys Phe Leu His Ser Ala Lys
5 10
Lys Phe Gly Lys Ala Phe Val Gly Glu He 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.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proc. Nat. Acad. Sci.
(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: He Gly Lys Phe Leu His Ser Ala Lys Lys
5 10
Phe Gly Lys Ala Phe Val Gly Glu He 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
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(ix) FEATURE:
(A) NAME/KEY: magainin peptide.
(x) PUBLICATION INFORMATION:
(A) AUTHOR: Zasloff, Michael
(C) JOURNAL: Proc. Nat. Acad. Sci.
(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 He 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
(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 He 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
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SUBSTITUTESHEET
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Gly Met Ala Ser Lys Ala Gly Ala He Ala
5 10
Gly Lys He 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.l
(C) JOURNAL: EMBO J.
(D) VOLUME: 2
(F) PAGES: 711-714
(G) DATE: 1983
(A) AUTHOR: Andreu, et al . (C) JOURNAL: Journal of Biochemistry
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SUBSTITUTE SHEET
(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 He Gly Gin Thr Leu
5 10
Gly Lys lie Ala Lys Val Gly Leu Lys Glu
15 20 Leu He Gin 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.
(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. -64-
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(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 He Gly Ala Asn Ala
15 20 Leu Gly Gly Ala Pro Gin Gin 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
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SUBSTITUTESHEET
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Gly Leu Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Gly Leu Lys He Gly Ala His Leu 15 20
Leu Gly Gly Ala Pro Gin Gin 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.
(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: 16-0CT-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 He Gly Thr His Phe
15 20 Leu Gly Gly Ala Pro Gin Gin 25
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SUBSTITUTE SHEET
(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.
(x) PUBLICATION INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Gly Leu Ala Ser Leu Leu Gly Lys Ala Leu
5 10
Lys Ala Thr Leu Lys He Gly Thr His Phe 15 20
Leu Gly Gly Ala Pro Gin Gin 25
(2) INFORMATION FOR SEQ ID NO:18: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
-67-
SUBSTITUTESHEET
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Gly Phe Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys He Gly Ala Asn Met 15 20
Leu Gly Gly Thr Pro Gin Gin 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.
(x) PUBLICATION INFORMATION: (A) AUTHOR: Richter, K.
-68-
SUBSTITUTESHEET
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys He Gly Ala Asn Ala 15 20
Leu Gly Gly Ala Pro Gin Gin 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.
(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.
-69-
SUBSΠTUTE SHEET
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys He Gly Ala Asn Ala 15 20
Leu Gly Gly Ser Pro Gin Gin 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
SUBSTITUTESHEET
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Gly Phe Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys He Gly Ala Asn Leu 15 20
Leu Gly Gly Thr Pro Gin Gin 25
(2) INFORMATION FOR SEQ ID NO:22: (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.
(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
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SUBSΠTUTE SHEET
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Gly Phe Ala Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys He Gly Ala Asn Ala 15 20
Leu Gly Gly Ala Pro Gin Gin 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.
(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 He Gly Ala Asn Met
15 20
Leu Gly Gly Ala Pro Gin Gin 25
(2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS:
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SUBSTITUTE SHEET
(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.
(x)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Ala Leu Lys He Gly Ala Asn Ala 15 20
Leu Gly Gly Ser Leu Gin Gin 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.
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SUBSΠTUTE SHEET
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Gly Phe Gly Ser Phe Leu Gly Lys Ala Leu
5 10
Lys Ala Gly Leu Lys He Gly Thr Asn Phe
15 20 Leu Gly Gly Ala Pro Gin Gin 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.
(x) PUBLICATION INFORMATION: (A) AUTHOR: Richter, K Egger, R. Kreil Chem.
hi, -74-
SUBSTITUTESHEET
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 He Gly Ala Asn Ala 15 20
Leu Gly Gly Ser Pro Gin Gin 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Lys He Ala Gly Lys He Ala Lys He Ala
5 10
Gly Lys He Ala Lys He Ala Gly Lys He
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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Lys He Ala Lys He Ala Gly Lys He Ala
5 10
Lys He Ala Gly Lys He Ala Lys He Ala
15 20
Gly
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SUBSΠTUTE SHEET
(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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Lys He Ala Gly Lys He Gly Lys He Ala
5 10
Gly Lys He Gly Lys He Ala Gly Lys He
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
(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
(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
-76-
SUBSTITUTESHEET
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(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
(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 FOR 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Lys Ala He Gly Lys Ala He Lys Ala He
5 10
Gly Lys Ala He Lys Ala He Gly Lys Ala
15 20
He
(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
-77-
SUBSTITUTE SHEET
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: Gly He Ala Lys He Ala Lys Gly He Ala
5 10
Lys He Ala Lys Gly He Ala Lys He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Lys He Ala Lys He Phe Gly Lys He Ala
5 10
Lys He Phe Gly Lys He Ala Lys He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Gly He Ala Arg He Ala Lys Gly He Ala
5 10
Arg He Ala Lys Gly He Ala Arg He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Lys Phe Ala Arg He Ala Gly Lys Phe Ala
5 10
Arg He Ala Gly Lys Phe Ala Arg He Ala
15 20
Gly
-78-
SUBSTITUTESHEET
(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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Gly Phe Ala Lys He Ala Lys Gly Phe Ala
5 10
Lys He Ala Lys Gly Phe Ala Lys He 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: Xaa is ornithine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: Lys He Ala Gly Xaa He Ala Lys He Ala
5 10
Gly Xaa He Ala Lys He Ala Gly Xaa He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Lys He Ala Arg He Ala Gly Lys He Ala
5 10
Arg He Ala Gly Lys He Ala Arg He Ala
15 20
Gly
(2) INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
-79-
SUBSΠTUTE SHEET
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: Xaa is ornithine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: Xaa He Ala Gly Lys He Ala Xaa He Ala
5 10
Gly Lys He Ala Xaa He Ala Gly Lys He
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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Gly He Ala Arg He Phe Lys Gly He Ala
5 10
Arg He Phe Lys Gly He Ala Arg He 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: 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
-80-
SUBSTITUTESHEET
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
( ii) MOLECULE TYPE : peptide
(ix) FEATURE:
(D) OTHER INFORMATION: Xaa is norleucine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: Lys Xaa Ala Gly Lys He Ala Lys Xaa Ala
5 10
Gly Lys He Ala Lys Xaa Ala Gly Lys He 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: Xaa is norleucine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: Lys He Ala Gly Lys Xaa Ala Lys He Ala
5 10
Gly Lys Xaa Ala Lys He 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: 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:
-81-
SUBSTITUTESHEET
( 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: Xaa is norvaline.
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48: Lys Xaa Ala Gly Lys He Ala Lys Xaa Ala
5 10
Gly Lys He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 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
(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
-82-
SUBSTITUTESHEET
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: Xaa is norvaline.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: Lys He Ala Gly Lys Xaa Ala Lys He Ala
5 10
Gly Lys Xaa Ala Lys He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: His He Ala Gly His He Ala His He Ala
5 10
Gly His He Ala His He Ala Gly His He 15 20
Ala
(2) INFORMATION FOR SEQ ID NO:53: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: Ala Gly Lys He Ala Lys He Ala Gly Lys
5 10
He Ala Lys He Ala Gly Lys He Ala Lys 15 20
He
(2) INFORMATION FOR SEQ ID NO:54: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
-83-
SUBSTITUTESHEET
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
He Ala Lys He Ala Gly Lys He Ala Lys
5 10
He Ala Gly Lys He Ala Lys He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO-55: Lys He Ala Gly Arg He Ala Lys He Ala
5 10
Gly Arg He Ala Lys He Ala Gly Arg He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: Arg He Ala Gly Arg He Ala Arg He Ala
5 10
Gly Arg He Ala Arg He Ala Gly Arg He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: Lys Val Ala Gly Lys He Ala Lys Val Ala 5 10
-84-
SUBSTITUTESHEET
Gly Lys He Ala Lys Val Ala Gly Lys He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: Lys He Ala Gly Lys Val Ala Lys He Ala
5 10
Gly Lys Val Ala Lys He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: Ala Lys He Ala Gly Lys He Ala Lys He
5 10
Ala Gly Lys He Ala Lys He Ala Gly Lys 15 20
He
(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: Xaa is ornithine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
Xaa He Ala Gly Xaa He Ala Xaa He Ala
5 10
Gly Xaa He Ala Xaa He Ala Gly Xaa He
15 20
Ala
-85-
SUBSTITUTESHEET
(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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
Lys Phe Ala Gly Lys He Ala Lys Phe Ala
5 10
Gly Lys He Ala Lys Phe Ala Gly Lys He
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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
Lys He Ala Gly Lys Phe Ala Lys He Ala
5 10
Gly Lys Phe Ala Lys He 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: Xaa is cyclohexylalanine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
Lys Xaa Ala Gly Lys He Ala Lys Xaa Ala
5 10
Gly Lys He Ala Lys Xaa Ala Gly Lys He
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:64: (i) SEQUENCE CHARACTERISTICS:
-86-
SUBSTITUTESHEET
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: Xaa is norleucine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
Lys Xaa Ala Lys He Ala Gly Lys Xaa Ala
5 10
Lys He Ala Gly Lys Xaa Ala Lys He Ala
15 20
Gly
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 21 AMINO ACIDS
(B) TYPE: amino acids
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: Arg He Ala Gly Lys He Ala Arg He Ala
5 10
Gly Lys He Ala Arg He Ala Gly Lys He
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: Xaa is homoarginine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: Xaa He Ala Gly Xaa He Ala Xaa He Ala
5 10
Gly Xaa He Ala Xaa He Ala Gly Xaa He
15 20
Ala
(2)INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS::
-87-
SUBSTITUTESHEET
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE: Xaa is p-aminophenylalanine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: Xaa He Ala Gly Lys He Ala Xaa He Ala
5 10
Gly Lys He Ala Xaa He Ala Gly Lys He
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: Xaa is p-aminophenylalanine
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: Lys He Ala Gly Xaa He Ala Lys He Ala
5 10
Gly Xaa He Ala Lys He Ala Gly Xaa He
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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: Lys Leu Ala Ser Lys Ala Gly Lys He Ala Gly
5 10
Lys He 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:
-88-
SUBSΠTUTE SHEET
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: Xaa is ornithine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
Lys He Ala Gly Lys He Ala Lys He Ala Gly
5 10
Xaa He Ala Lys He Ala Gly Lys He 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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: Lys He Ala Gly Lys He Ala Lys He Ala
5 10
Gly Arg He Ala Lys He Ala Gly Lys He 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: Xaa is norleucine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
Lys He Ala Gly Lys He Ala Lys He Ala
5 10
Gly Xaa He Ala Lys He Ala Gly Lys He 15 20
Ala
(2) INFORMATION FOR SEQ ID NO:73: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
-89-
SUBSΠTUTE SHEET
( ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: Xaa is norvaline.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: Lys He Ala Gly Lys He Ala Lys He Ala
5 10
Gly Xaa He Ala Lys He Ala Gly Lys He 15 20
Ala
(2) INFORMATION FOR SEQ ID NO:74: (1) 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: Xaa is ornlthine.
(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: (1) 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: Xaa is ornlthine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: Lys He Ala Gly Lys Phe Ala Lys He Ala
5 10
Gly Xaa Phe Ala Lys He Ala Gly Lys Phe 15 20
Ala
(2) INFORMATION FOR SEQ ID NO:76: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
-90-
SUBSΠTUTE SHEET
(D) TOPOLOGY: linear
( ii) MOLECULE TYPE : peptide
(ix) FEATURE:
(D) OTHER INFORMATION: Xaa at residues 6, 13, and 20 is norleucine; Xaa at residue
12 is ornlthine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: Lys He Ala Gly Lys Xaa Ala Lys He Ala
5 10
Gly Xaa Xaa Ala Lys He Ala Gly Lys Xaa 15 20
Ala
(2) INFORMATION FOR SEQ ID NO:77: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: Lys Met Ala Ser Lys Ala Gly Lys He Ala
5 10
Gly Lys He Ala Lys Val Ala Leu Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO:78: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78 Lys He Ala Ser Lys Ala Gly Lys He Ala
5 10
Gly Lys He Ala Lys Val Ala Leu Lys Ala Leu 15 20
(2) INFORMATION FOR SEQ ID NO:79: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
-91-
SUBSTITUTESHEET
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: Xaa is norleucine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: Lys He Ala Ser Lys Ala Gly Lys Xaa Ala
5 10
Gly Lys He Ala Lys Val Ala Leu Lys Ala Leu 15 20
(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: Xaa is norleucine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: Lys Leu Ala Ser Lys Ala Gly Lys Xaa Ala
5 10
Gly Lys He Ala Lys Val Ala Leu Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO:81: (1) 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: Xaa is norleucine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: Lys Xaa Ala Ser Lys Ala Gly Lys Xaa Ala
5 10
Gly Lys He 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
-92-
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(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: Xaa is p-aminophenylalanine.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: Lys He Ala Gly Lys He Ala Lys He Ala
5 10
Gly Xaa He Ala Lys He Ala Gly Lys He
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:83:
(1) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: Lys He Ala Gly Ala He Ala Lys He Ala
5 10
Gly Lys He Ala Lys He Ala Gly Lys He
15 20
Ala
(2) INFORMATION FOR SEQ ID NO:84:
(1) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: Lys He Ala Gly Lys He Ala Lys He Ala
5 10
Gly Ala He Ala Lys He Ala Gly Lys He
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
-93-
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(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: Lys He Ala Gly Lys He Ala Lys He Ala
5 10
Gly Lys He Ala Lys He Ala Gly Ala He
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 86: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:
Lys He Ala Lys Lys He Ala Lys He Ala
5 10
Lys Lys He Ala Lys He Ala Lys Lys He 15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 87: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:
Lys Phe Ala Lys Lys Phe Ala Lys Phe Ala
5 10
Lys Lys Phe Ala Lys Phe Ala Lys Lys Phe 15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 88: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
-94-
SUBSTITUTESHEET
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88: Lys Phe Ala Lys Lys He Ala Lys Phe Ala
5 10
Lys Lys He Ala Lys Phe Ala Lys Lys He Ala 15 20
(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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
Ala He Ala Gly Lys He Ala Lys He Ala
5 10
Gly Lys He Ala Lys He Ala Gly Lys He
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
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: Lys He Ala Gly Lys He Ala Ala He Ala
5 10
Gly Lys He Ala Lys He Ala Gly Lys He
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 91: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: Lys He Ala Gly Lys He Ala Lys He Ala
5 10
Gly Lys He Ala Ala He Ala Gly Lys He
15 20
Ala
(2) INFORMATION FOR SEQ ID NO: 92: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
-95-
SUBSTITUTESHEET
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
Gly Met Ala Ser Lys Ala Gly Lys He Ala
5 10
Gly Lys He Ala Lys Val Ala Leu Lys Ala
15 20
Leu
(2) INFORMATION FOR SEQ ID NO: 93: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE
(A) ORGANISM: Apis me11ifera
(vii) FEATURE
(A) NAME/KEY: melittin peptide
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Habermann, E.
Jentsch, J.
(B) TITLE: Sequenzanalyse des Melittins aus den tryptischen and peptischen
Spaltstucken
(C) JOURNAL: Hoppe-Seyler's Zeitschrift
Physio1. Chem
(D) VOLUME: 348
(F) PAGES: 37-50
(G) DATE: 1987
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: Gly He Gly Ala Val Leu Lys Val Leu
5 Thr Thr Gly Leu Pro Ala Leu He Ser Trp 10 15
He Lys Arg Lys Arg Gin Gin 20 25
(2) INFORMATION FOR SEQ ID NO: 94: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
-96-
SUBSTITUTESHEET
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 94: Leu Lys Lys Leu Lys Lys Leu Leu Lys Leu
5 10
Leu
(2) INFORMATION FOR SEQ ID NO: 95: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 95: Leu Leu Lys Lys Leu Lys Lys Leu Leu Lys
5 10
Leu Leu
(2) INFORMATION FOR SEQ ID NO: 96: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 96:
Lys Leu Leu Lys Lys Leu Lys Lys Leu Leu
5 10
Lys Leu Leu
(2) INFORMATION FOR SEQ ID NO: 97:
(1) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 97:
Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu
5 10
Leu Lys Leu Leu
(2) INFORMATION FOR SEQ ID NO: 98:
(1) SEQUENCE CHARACTERISTICS: : (A) LENGTH: 16 amino acids -97-
SUBSΠTUTE SHEET
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 98: Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu Leu Lys Lys Leu
5 10
Arg Arg 15
(2) INFORMATION FOR SEQ ID NO: 99: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 99:
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: 100: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 100:
Leu Lys Lys Leu Leu Lys Lys Leu Lys Lys
5 10
Leu Leu Lys Lys Asn
15
(2) INFORMATION FOR SEQ ID NO: 101: (i) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
-98-
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(D) OTHER INFORMATION: Xaa is homoserine. (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 101:
Leu Lys Lys Leu Leu Lys Lys Leu Lys Lys
5 10
Leu Leu Lys Lys Xaa
15
(2) INFORMATION FOR SEQ ID NO: 102: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 102:
Leu Lys Leu Leu Lys Lys Leu Leu Lys Lys
5 10
Asn Lys Lys Leu Leu Lys Lys Leu
15
(2) INFORMATION FOR SEQ ID NO: 103: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 103:
Leu Lys Leu Leu Lys Lys Leu Leu Lys Lys
5 10
Pro Lys Lys Leu Leu Lys Lys Leu
15
(2) INFORMATION FOR SEQ ID NO: 104: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
-99-
SUBSTITUTESHEET
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 104:
Leu Leu Lys Lys Leu Lys Lys Leu Leu Lys
5 10
Lys Leu Gin Gly Pro Pro Gin Gly Gin Ser
15 20
Pro Gin
(2) INFORMATION FOR SEQ ID NO: 105: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 105:
Leu Ala Ser Lys Ala Gly Ala He Ala Gly 5 10
Lys He Ala Lys Lys Leu Leu Lys Lys Leu 15 20
(2) INFORMATION FOR SEQ ID NO: 106: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 106: Leu Lys Lys Leu Lys Lys Leu 5
(2) INFORMATION FOR SEQ ID NO: 107: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 107: Leu Leu Lys Lys Leu Lys Lys Leu 5
(2) INFORMATION FOR SEQ ID NO: 108: (i) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
-100-
SUBSTITUTESHEET
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 108: Lys Leu Leu Lys Lys Leu Lys Lys Leu 5
(2) INFORMATION FOR SEQ ID NO: 109: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 109: Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu 5 10
(2) INFORMATION FOR SEQ ID NO: 110: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 110: Leu Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu 5 10
(2) INFORMATION FOR SEQ ID NO: 111: (1) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 111: Ala Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu 5 10
(2) INFORMATION FOR SEQ ID NO: 112: (1) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
-101-
SUBSTITUTESHEET
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 112: Leu Lys Lys Leu Lys Lys Leu Leu Lys Lys Leu 5 10
(2) INFORMATION FOR SEQ ID NO: 113: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 113: Leu Leu Lys Lys Leu Lys Lys Leu Leu Lys Leu Leu 5 10
(2) INFORMATION FOR SEQ ID NO: 114: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:'
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 114: Lys Leu Leu Lys Lys Leu Lys Lys Leu Leu Lys Lys
5 10
Leu
(2) INFORMATION FOR SEQ ID NO: 115: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 115: Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu Leu
5 10
Lys Lys Leu
(2) INFORMATION FOR SEQ ID NO: 116: (i) SEQUENCE .CHARACTERISTICS: :
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
-102-
SUBSTITUTESHEET
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 116: Leu Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu Leu
5 10
Lys Lys Leu 15
(2) INFORMATION FOR SEQ ID NO: 117: (i) SEQUENCE CHARACTERISTICS: :
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 117: Leu Lys Lys Leu Leu Lys Lys Leu Lys Lys Leu
5 10
Leu Lys Arg
(2) INFORMATION FOR SEQ ID NO: 118: (i) SEQUENCE CHARACTERISTICS::
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION:SEQ ID NO: 118: Lys He Ala Lys Lys He Ala Lys He Ala
5 10
Lys Lys He Ala
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