WO1997001578A1 - Procede d'identification des peptides qui affectent les interactions proteine-proteine et des peptides modulant l'activite complementaire - Google Patents

Procede d'identification des peptides qui affectent les interactions proteine-proteine et des peptides modulant l'activite complementaire Download PDF

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WO1997001578A1
WO1997001578A1 PCT/US1996/010958 US9610958W WO9701578A1 WO 1997001578 A1 WO1997001578 A1 WO 1997001578A1 US 9610958 W US9610958 W US 9610958W WO 9701578 A1 WO9701578 A1 WO 9701578A1
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PCT/US1996/010958
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Ronald T. Ogata
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Medical Biology Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to modulating protein activity, and specifically relates to a general method of identifying regions of contact in protein-protein binding complexes, peptides or peptide analogs that modulate these protein-protein interactions, and peptides or peptide analogs that modulate complement activity.
  • a fundamental component of essentially all biological processes is the specific interaction of proteins with other molecules, in particular other proteins.
  • Specific protein-protein interactions are essential for cellular maintenance, regulation, reproduction and death. Protein-protein interactions are required for basic processes such as intercellular adhesion and communication, signal transduction, and gene replication, expression, and regulation.
  • Protein-protein binding interactions occur at specific regions of contact on the protein surfaces and identification of the amino acid residues in these contact regions is important for controlling the binding interaction. For example, knowledge of the residues known to be involved in a contact site ma ⁇ be used to synthesize a relatively short peptide corresponding to the protein segment that includes these residues. This "interface" peptide may be used directly to compete with and thus inhibit that specific protein-protein binding interaction. Interactions that have been successfully inhibited in this manner include those between the herpes virus protein Vmw65 and the Oct-1 protein (Haigh, A. et al., 1990, Nature 344:257-259), the subunits of ribonucleotide reductases from viral and mammalian sources (Dutia.
  • An interface peptide may also be used indirectly to elicit an antibody which binds specifically to the intact protein. Such an antibodv ma ⁇ be used to inhibit the protein-protein binding reaction.
  • An interface peptide ma ⁇ be used as a structural model for design of a modified peptide. a peptide analog (Bianchi. E. et al., 1995, J. Mol. Biol. 247:154-60), or peptidomimetic inhibitor (McDonnell, J.M. et al., 1996, Nature Structural Biol. 3(5):419-426; Nakanishi, H.
  • an interface peptide may inhibit or substitute for the normal receptor-ligand interaction, and hence attenuate or mimic the normal cellular response depending on th ⁇ signalling mechanism ot the ligand-receptor binding.
  • Currently used methods for identifying residues at the protein-protein interface typically include direct anal ⁇ sis of the three-dimensional structure of the protein-protein complex (e.g., X-ra ⁇ crystallography or NMR spectroscop ⁇ ), or systematically testing the binding activity of protein fragments or natural or engineered variants of these proteins.
  • synthetic peptides were used to block protein-protein interactions
  • crystallographic data and genetically engineered modified proteins were used to identif ⁇ interface residues in all cases except the herpes virus ⁇ bonucleotide reductase; in that case, a peptide designed to elicit an antibod ⁇ response was fortuitously found to inhibit subunit association as well (Dutia, B.M.
  • the complement s ⁇ stem part of the mammalian humoral immune system, lyses microorganisms and infected cells by forming holes in the their plasma membranes.
  • the complement s ⁇ stem consists of more than twenty plasma and membrane-bound proteins which interact to trigger and modulate complement activity. While normal complement function is essential for health, regulation of the effects of comolement activation is also important. For example, uncontrolled complement activation can lead to adverse s ⁇ mptoms such as continuous inflammatory reactions in a variety of diseases (Vogt, W., 1985, Trends Pharm. Sci. 6: 114-119).
  • complement is the primary mediator of the hyperacute rejection of xenogeneic transplants which limits the clinical and research usefulness of xenotransplantation (R ⁇ an, U.S., 1994, Xeno 2: 19 22; Platt, J.L. et al., 1990, Immunol. Today 11: 450456).
  • xenotransplantation as a viable surgical procedure will certainly require drugs that limit complement activation.
  • therapeutic treatments of the r ⁇ pertusion injury associated with myocardial infarction will likely include modulation of the effects of complement activation (Hoffle, J.W. et al., 1994, Annu. Rev. Pharmacol. Toxicol. 34:1740).
  • complement modulators Despite a clear need for complement modulators, most of the currently available methods for inhibiting or depleting complement are not suitable for clinical applications because of their toxicity, undesirable side-effects or lack of efficacy in plasma (Vogt, W., 1985, Trends Pharm. Sci. 6:114-119).
  • One complement inhibitor being clinically tested is soluble complement receptor type 1 (sCRI), which can suppress h ⁇ peracute rejection in xenotransplantation (Weisman, H.F. et al., 1990. Science 249.146 151 ; Pruitt, S.K. et al., 1994, Transplantation 57:363-370).
  • sCRI soluble complement receptor type 1
  • sCR1 inhibits the complex C3 and C5 activating enzvmes (convertases) b ⁇ facilitating their dissociation.
  • sCRI is a large (240 kDa) protein and ther ⁇ is a need for small peptide inhibitors which are more easii ⁇ synthesized, chemically modified, or mimicked b ⁇ small organic peptidomimetic molecules (Nakanishi, H. et al.. 1993, Gene 137:51- 56).
  • the complement proteins C3, C4, and C5 are excellent targets for functional intervention by complement inhibitors because they pla ⁇ central roles in activation and regulation of this s ⁇ stem. Although the three proteins have distinct functions, their ammo acid sequences ar ⁇ closely related, and the ⁇ are encoded b ⁇ genes that doubtless evolved from a common ancestor (Campbell, R.D. et al.. 1988, Ann. Rev. Immunol. 6:161-195). Because of their close structural relationship, the C3. C4 and C5 proteins form a protein family. All three proteins interact with a number of other proteins.
  • C3 binds to or transiently interacts with more than a half-dozen soluble proteins including another molecule of itself during complement activation and attenuation, and to a similar number of cell-surface-bound proteins, which mediate immune-clearance, inflammatory, and complement regulatory activities.
  • researchers have identified the locations of sites within C3, C4 and C5 that interact with other complement proteins, including those in C3 that are recognized b ⁇ inactivating proteases and their cofactors, cell bound receptors and catal ⁇ tic subunits in the complex complement convertases (Alsenz, J. et al., 1992, Dev. Comp. Immunol. 16:63-76). Insertion/Deletion Sequ ⁇ nces in Protein Families
  • indels are r ⁇ vealed when ammo acid sequences are aligned to maximize their sequence identity. Alignments of relatively short sequ ⁇ nces can be carried out manually. Computer programs such as PILEUP (Genetics Computer Group, Madison, Wl) and CLUSTAL W (Thompson, J.D. et al., 1994, Nuc. Acids Res. 22: 4673) are useful for aligning multiple long sequences. In aligning related sequences of different lengths, gaps are introduced into one or more family members to optimize the alignment of the total sequenc ⁇ s. These gaps are indels.
  • PILEUP Genetics Computer Group, Madison, Wl
  • CLUSTAL W Thimpson, J.D. et al., 1994, Nuc. Acids Res. 22: 4673
  • Indels have be ⁇ n shown to occur in portions of th ⁇ ammo acid sequ ⁇ nc ⁇ of a prot ⁇ in that appear on the surface of the protein in its native folded state.
  • indels are short (1 to 5 residues) and occur at the protein surface as reverse turns or coils within loops rather than within secondary structural elements ( ⁇ -helices and ⁇ strands), because these properti ⁇ s minimize perturbations of th ⁇ core protein structure (Pascarella, S. & Argos, P., 1992. J. Mol. Biol. 224. 461471; Sibanda. B.L & Thornton. J.M.. 1993, J. Mol. Biol. 229: 428-447).
  • the mpthod relies on identification of mdels generally comprising selecting ammo acid sequences for at least two proteins that contain similar ammo acid sequences in at least a portion of the sequ ⁇ c ⁇ s and wherein on ⁇ s ⁇ qu ⁇ nc ⁇ is that of a targ ⁇ t prot ⁇ in.
  • Th ⁇ similar s ⁇ quences include identical and/or conserv ⁇ d ammo acid r ⁇ sidu ⁇ s, and th ⁇ similar s ⁇ qu ⁇ nc ⁇ s ar ⁇ aligned b ⁇ matching th ⁇ identical and/or conserv ⁇ d ammo acid r ⁇ sidu ⁇ s
  • sites are identified that contain insertions and/or deletions of ammo acid residu ⁇ s in o ⁇ prot ⁇ in relative to another protein, wherein the insertions and/or deletions define an del.
  • An del associat ⁇ d p ⁇ ptide sequence can eith ⁇ r span or flank an mdel.
  • Indel associated peptides include peptides of about 4 to about 20 am o acid residues in length that are located within 30 ammo acid residues, or preferabl ⁇ within 20, 15, 10, 9, 8, 7, 6 or 5 or less ammo acid residues of an indel id ⁇ ntifi ⁇ d in th ⁇ ammo acid s ⁇ qu ⁇ c ⁇ of a target protein.
  • a method for identifying molecul ⁇ s that affect biological activity of a target protein includes the steps of obtaining information regarding th ⁇ location of an indel in an ammo acid sequ ⁇ nc ⁇ of a targ ⁇ t prot ⁇ in, obtaining a p ⁇ ptid ⁇ fragment of the target protein, the peptide fragment having a sequ ⁇ c ⁇ that is locat ⁇ d in th ⁇ ammo acid sequenc ⁇ of the target protein within 30 ammo acids or less of the del, or obtaining a peptidomimetic or peptid ⁇ analog of th ⁇ p ⁇ ptid ⁇ fragment, and screening the peptide fragment, the peptidomimetic, or the peptid ⁇ analog for its affect on biological or biochemical activity of the target protein.
  • the scre ⁇ tng st ⁇ p includes analyzing for modulation of protein activity, inhibition of protein activity, activation or potentiation of protein activity, competition for binding to a prot ⁇ in, binding to a prot ⁇ in or ligand, substitution for a substrat ⁇ of th ⁇ targ ⁇ t protein, substitution for a ligand of the target protein, or making an anti peptid ⁇ antibody capabl ⁇ of modulating a biological activity of th ⁇ target protein.
  • the p ⁇ ptid ⁇ fragment, the peptidomimetic, or the p ⁇ ptid ⁇ analog directiy affects the target protein whereas in another embodim ⁇ nt the peptid ⁇ fragment, th ⁇ peptidomimetic, or the peptid ⁇ analog indirectly affects the target protein
  • Another pref ⁇ rr ⁇ d ⁇ mbodim ⁇ nt furth ⁇ r comprises the step of synthetically constructing a peptide, peptid ⁇ analog, or peptidomimetic that aff ⁇ cts th ⁇ biological or biochemical activity of th ⁇ target protein.
  • One pref ⁇ rr ⁇ d ⁇ mbodime ⁇ t is a method for making a pharmaceutical composition, including the step of obtaining a moiecul ⁇ id ⁇ ntifi ⁇ d as having biological or biochemical activity in accordance with th ⁇ steps of this method and combining the mol ⁇ cul ⁇ with a pharmaceutically acceptable carrier.
  • This method ma ⁇ further include the step of packaging the molecule in unit-dosage form.
  • the target protem is a protem of the mammalian complement system.
  • th ⁇ targ ⁇ t prot ⁇ in which is a orot ⁇ in of th ⁇ mammalian compi ⁇ m ⁇ nt system is C2, C3.
  • th ⁇ p ⁇ ptide fragment has a sequ ⁇ nce of about 4 to about 20 ammo acid residu ⁇ s in i ⁇ ngth.
  • the p ⁇ ptid ⁇ fragment has a s ⁇ qu ⁇ c ⁇ of about 10 to about 20 am o acid r ⁇ sidu ⁇ s in l ⁇ ngth.
  • the peptide fragment has a sequence of about 4 to about 15 ammo acid residu ⁇ s in length.
  • the peptid ⁇ fragment has a sequenc ⁇ of about 5 to about 18 ammo acid r ⁇ sidu ⁇ s in i ⁇ ngth.
  • the peptide fragment is located within about 20 ammo acid residues of an indel.
  • the peptide fragment is located within about 15 ammo acid residu ⁇ s of an mdel.
  • th ⁇ p ⁇ ptide fragment is located within about 12 ammo acid residu ⁇ s of an indel.
  • th ⁇ p ⁇ ptid ⁇ fragment is iocated within about 10 ammo acid residues of an indel.
  • the peptid ⁇ fragm ⁇ nt is iocat ⁇ d within about 9 ammo acid residues of an mdel.
  • the peptide fragment is Iocated within about 8 ammo acid residues of an indel.
  • the peptide fragment is Iocated within about 7 ammo acid residues of an indel.
  • th ⁇ p ⁇ ptid ⁇ fragm ⁇ nt is iocat ⁇ d within about 6 ammo acid residues of an mdel.
  • the peptide fragment is Iocated within about 5 or less ammo acid residues ot an i ⁇ d ⁇ l or has a s ⁇ que ⁇ ce that spans the mdel or has a sequenc ⁇ Iocated within the indel.
  • a m ⁇ thod of identifying interface peptides for a target protein including the steps of identifying the location of an mdel in the ammo acid sequence of a target prot ⁇ in, selecting an ammo acid sequenc ⁇ from the target protein sequenc ⁇ overlapping or iocated within about 30 ammo acid residues or less of an ammo- or carbox ⁇ l-t ⁇ rminus of an indel, obtaining a molecule that is a peptide having the sel ⁇ ct ⁇ d ammo acid s ⁇ qu ⁇ ce, a peptid ⁇ analog of the sel ⁇ cted ammo acid sequence, or a peptidomimetic of the selected am o acid sequenc ⁇ , and evaluating the peptid ⁇ , p ⁇ ptide analog or peptidomimetic in an assay to measure a change in activity of the target protein, wh ⁇ r ⁇ in th ⁇ change in activity is
  • th ⁇ p ⁇ ptid ⁇ having the selected ammo acid sequence is about 4 to about 20 ammo acid residues m length.
  • the peptide having the selected ammo acid sequence is about 10 to about 20 amino acid residues in length.
  • the p ⁇ ptid ⁇ having the sel ⁇ ct ⁇ d ammo acid sequenc ⁇ is about 4 to about 15 ammo acid residues in length, in another embodiment, the peptid ⁇ having the selected ammo acid sequence is about 5 to about 18 ammo acid residues in length.
  • the selected ammo acid seque ⁇ c ⁇ has an ammo- or carboxyl-terminal residue within about 20 ammo acid residues of one terminus of the indel.
  • the peptide is Iocated within about 15 ammo acid residues of an ammo- or carboxyl-termmus of an ind ⁇ i.
  • the peptide is iocated withm about 10 ammo acid residu ⁇ s of an ammo- or carboxyl-termmus of an mdel.
  • the peptid ⁇ is locat ⁇ d within about 9 amino acid r ⁇ sidu ⁇ s of an am o- or carboxyl-termmus of an mdel.
  • the peptide is Iocated within about 8 ammo acid residu ⁇ s of an ammo- or carboxyl- termmus of an indel.
  • the peptide is Iocated within about 7 ammo acid residues of an am o or carboxyl terminus of an indel In another embodiment, the peptide is Iocated within about 6 ammo acid residues of an ammo or carboxyl terminus of an indel In on ⁇ ⁇ mbodim ⁇ nt, th ⁇ p ⁇ ptid ⁇ is locat ⁇ d within about 5 ammo acid r ⁇ sidu ⁇ s or l ⁇ ss of an ammo or carboxyl terminus of an mdel.
  • the method furth ⁇ r includes the step of making antibodies to the peptide, peptide analog or peptidomimetic, wherein the antibodies are capable of modulating an activity of the targ protein.
  • the change in activit ⁇ of the target protein is a decr ⁇ ase in activit ⁇ , an increas ⁇ in activit ⁇ , utilization of a substrate different than the substrate normally utilized by the target protein, or binding to a ligand differently than the ligand binding activit ⁇ ordinarily demonstrated b ⁇ the target protein
  • the target protein is a protein of the mammalian compiem ⁇ nt syst ⁇ m and most pr ⁇ f ⁇ rably is C2, C3, C4, C5 or Factor B.
  • a peptide for modulating activit ⁇ of the complement s ⁇ stem of a mammal comprising a sequ ⁇ nce of about 4 to about 25 ammo acid residues that occurs in an ammo acid sequence of a mammalian comolem ⁇ nt protein, the peptide having an ammo acid sequence in which an ammo- or carboxyl-terminal residue is Iocated within about 15 ammo acid r ⁇ sidues of an indel of the mammalian complement protein.
  • the p ⁇ ptid ⁇ moduiat ⁇ s activity of th ⁇ mammalian complement system by directly or indirectly inhibiting an activity of a protein of the mammalian compi ⁇ ment s ⁇ stem.
  • the peptide modulates activit ⁇ of the mammalian complem ⁇ nt system by directl ⁇ or indirectly ⁇ nha ⁇ cmg an activity of a protein of the mammalian complem ⁇ nt syst ⁇ m.
  • the indel occurs within the ammo acid sequence of a C2, C3, C4, C5 or Factor B protein
  • the peptide further includes another molecule attached to the peptid ⁇
  • Anoth ⁇ r embodim ⁇ t is an antibody that specifically recognizes such a peptide with another molecule attached to the peptide.
  • Another preferred embodim ⁇ nt is a p ⁇ ptid ⁇ analog or peptidomimetic molecule of a peptide according to this asp ⁇ ct of th ⁇ invention.
  • Anoth ⁇ r ⁇ mbodiment is an antibod ⁇ that specifically recognizes such a peptide analog or peptidomim ⁇ tic molecule.
  • Another preferred embodiment is a pharmaceutical composition including a peptide, peptide analog or peptidomim ⁇ tic molecule according to this aspect of the invention.
  • a preferred embodiment is a pharmaceutical composition including an antibody that specifically recogmz ⁇ s a peptide, peptide analog or peptidomim ⁇ tic mol ⁇ cul ⁇ according to this asp ⁇ ct of the invention.
  • the peptide is Iocated within about 12 ammo acid residu ⁇ s of an md ⁇ l of th ⁇ mammalian compi ⁇ ment prot ⁇ i ⁇ .
  • the peptide is Iocated within about 10 ammo acid residu ⁇ s of an indel of the mammalian complement protein.
  • a p ⁇ ptid ⁇ having a s ⁇ quence of any one of: SEQ ID NO:53, SEQ ID N0.54, SEO ID N0.55, SEQ ID N0:56, SEQ ID NO:57, SEQ ID N0.58, SEQ ID N0:72, SEQ ID N0:73, SEQ ID N0:74 or SEQ ID N0.75 BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows a prototype indel. revealed by alignment of portions of the ammo acid s ⁇ quences of the human ("hum") and mouse C mus”) C3, C4, and C5 proteins, using the alignment program CLUSTAL W.
  • the aligned ammo acid sequences correspond to sequenc ⁇ s around mdel number 7 in FIG. 2A-2E and includ ⁇ humC3 (SEQ ID NO:1), musC3 (SEQ ID NO 2), humC4 (SEQ ID NO 3), musC4 (SEQ ID N0:4), humC5 (SEQ ID N0:5) and musC5 (SEQ ID N0:6).
  • FIG. 2A 2E shows th ⁇ alignment of the complet ⁇ ammo acid s ⁇ qu ⁇ nc ⁇ s of th ⁇ compl ⁇ m ⁇ nt prot ⁇ ins human C3 (hC3; SEQ ID NO:7), mous ⁇ C3 (mC3.
  • FIG. 3A-30 shows the alignment of ammo acid sequences of the DNA pol ⁇ merases from herpes simplex virus
  • HSV HSV t ⁇ pe 1 (HSV-1; SEQ ID N0:13), HSV type 2 (HSV 2; SEQ ID N0:14), HSV type 6 (HSV-6; SEQ ID N0:15), baculovirus (Baculo; SEQ ID 1.0:16), ictalund herpes virus 1 (CCV; SEQ ID N0:17), Chonstoneura bie ⁇ nis entomopoxvirus (ENTPOX; SEQ ID N0:18) and hepatitis B virus (HepB; SEQ ID N0:19).
  • FIG. 4A4C shows th ⁇ alignment ot am o acid s ⁇ qu ⁇ nces of the human 03 (HuC3; SEQ ID N0:7), mouse C3 (MuC3; SEQ ID N0:8), rat C3 (RATC3; SEQ ID N0.20) and guinea pig C3 (GUIPIG; SEQ ID N0:21) proteins.
  • Asterisks ( * ) mark identical residues and p ⁇ riods (.) mark conserved r ⁇ sidues.
  • FIG. 5A-5C shows th ⁇ alignment of ammo acid sequ ⁇ nc ⁇ s of retroviral polyprot ⁇ ins of the immunodeficiency viruses of humans (HIV-1, SEQ ID N0:22; and HIV-2, SEQ ID N0:23), Simian (Simia ⁇ lV; SEQ ID N0:24), chimp
  • FIG. 6A-6B shows the alignment of part of the ammo acid sequ ⁇ c ⁇ s of retroviral pol ⁇ proteins of th ⁇ immunodeficiency viruses of humans (HIV-1 , SEQ ID N0:22; and HIV-2, SEQ ID N0.23), Molone ⁇ murine leukemia virus (MOLONEY; SEQ ID N0:28) and Friend murine leukemia virus (F-MULV; SEQ ID N0:29).
  • Asterisks ( * ) mark identical residues and periods (.) mark conserved residues.
  • FIG. 7A-7B shows the alignment of ammo acid s ⁇ quences of ribo ⁇ ucl ⁇ otid ⁇ r ⁇ ductases from HSV-1 (SEQ ID NO: 1).
  • HSV-2 (SEQ ID N0:31), Epstein-Barr virus (EBV; SEQ ID NQ:32), human (SEQ ID ..0:33), vaccinia virus (Vaccinia; SEQ ID N0:34), mouse (Mus; SEQ ID N0:35), ⁇ east (Yeast; SEQ ID N0:36), Escherichia coli (Coli; SEQ ID NO:31), HSV-2 (SEQ ID N0:31), Epstein-Barr virus (EBV; SEQ ID NQ:32), human (SEQ ID ..0:33), vaccinia virus (Vaccinia; SEQ ID N0:34), mouse (Mus; SEQ ID N0:35), ⁇ east (Yeast; SEQ ID N0:36), Escherichia coli (Coli; SEQ ID
  • FIG. 8A-8B shows the alignment of ammo acid sequ ⁇ nc ⁇ s of herpes virus Vmw65 (VP16) proteins for HSV-1
  • HSV TYPE1/F HSV TYPE1/F; SEQ ID N0:39
  • HSV 2 HSV TYPE2JHG52; SEQ ID N0:40
  • bovine virus BvHV Type1/P8 2; SEQ ID N0:41
  • Varicella-Zoster virus Var-ZosV/Dumas; SEQ ID N0:42
  • EqHV Type4 SEQ ID N0.43
  • FIG. 9 graphically shows inhibition of compl ⁇ m ⁇ nt activit ⁇ (CH50) b ⁇ th ⁇ C3 peptides 11-4 ( • ), II 5 (C .
  • FIG. 10 shows the peptid ⁇ concentrations giving 50% inhibition of compi ⁇ ment function as measured b ⁇ hemolysis for the individual peptid ⁇ s listed under th ⁇ bars on th ⁇ X-axis.
  • FIG. 11 A-1 I B shows th ⁇ a gnm ⁇ nt of ammo acid s ⁇ qu ⁇ nc ⁇ s of human C2 (humC2; SEQ ID NQ:45), mouse C2 (musC2; SEQ ID N0:46), human factor B (humBf; SEQ ID N0:47), mouse factor B (musBf; SEQ ID N0.48) and zebrafish factor B (ZebBf; SEQ ID N0:49).
  • FIG. 12 graphically shows the inhibition of compl ⁇ m ⁇ nt hemolytic activity b ⁇ th ⁇ C2 peptide E1 for concentrations of peptide indicated on the X-axis used without serum ( » ) or with serum ( ⁇ , 0 and ⁇ ).
  • a general method for identifying portion of proteins that are involved in specific protein-protein interactions or contacts with other molecules. Identifying tnese interactive portions of proteins constitutes a critical step in designing small molecule inhibitors of protein-protein interactions. This method involves the analysis of only the primary structures of relat ⁇ d proteins and has been demonstrated to be effective in predicting the ammo acid sequenc ⁇ s of inhibitory peptides for the complement s ⁇ stem.
  • the method describ ⁇ d her ⁇ for identifying regions of specific protein- protein contact relies on analysis of l ⁇ ngth polymorphisms and on determination of the locations of indels in protein families. This general method provid ⁇ s a means for identifying interactive sit ⁇ s on prot ⁇ ins with a high probability of success. For purposes of this application, certain terms are defined as follows.
  • Identity means that all of the ammo acid residues at a single position are identical wh ⁇ n two or more protein sequ ⁇ nc ⁇ s ar ⁇ aligned. Two or more protein sequences ma ⁇ have onl ⁇ limited regions of identity and gaps may b ⁇ introduced into one or mor ⁇ s ⁇ qu ⁇ nces to aid in aiignm ⁇ nt of identical residues.
  • Consed ammo acids means that non-identical ammo acid r ⁇ sidues share sufficient chemical similarity that they can be considered to form a functional group. That is, when ammo acid residues for two or more protein sequ ⁇ nces are compared, a single position of alignment ma ⁇ contain non-identical ammo acids but th ⁇ residues may be conserved for that position and functionally similar because of their chemical and/or structural similarity.
  • the following groups of am o acids form groups of conserved am o acids: Phe (F) and T ⁇ r (Y); lie (I), Leu (L), Val (V) and Met (M), Ala (A), Ser (S) and Thr (T); Asn (N), His (H), Arg (R), L ⁇ s (K) and Gin (Q); and Asn (N), Asp (D), Glu (E) and Gin (Q).
  • conserved ammo acids for an ⁇ giv ⁇ n position m ⁇ ans that ail of the ammo acid residu ⁇ s at a single position are m ⁇ mbers of a single group of conserv ⁇ d am o acids although an ⁇ combination of ammo acids within that conserved group is permissible.
  • the definition of cons ⁇ rv ⁇ d residues used here follows closely that defined by the PAM250 matrix (Da ⁇ hoff, M.O., et al. 1978. In Atlas of Protein Sequence and Structure. Vol. 5, suppl. 3 (Da ⁇ hoff, M.O., ed) p. 345, NBRF, Washington, DC).
  • “Alignment” of ammo acid se ⁇ uences allows for insertion of spaces in one or more of the sequences of ammo acid residues to maximize the number of positions having identical and/or conserved residues in the compared protein sequ ⁇ nc ⁇ s using methods well known in th ⁇ art.
  • the identical or conserved residues do not have to be pres ⁇ nt in all of th ⁇ s ⁇ qu ⁇ ces ior an ⁇ particular location (i.e., an identical or conserved residue ma ⁇ be identified even if it is identical or conserved for that locus oni ⁇ for two of the compared sequ ⁇ nces).
  • An "interface peptid ⁇ " is a p ⁇ ptide that includes an ammo acid sequence that occurs in a protein at or near a specific protein-protem contact region
  • An interface oeptid ⁇ can specifically inhibit a protein-protein interaction by acting as a competitive inhibitor of the native protein or can serve as a model for making a modified peptide, a peptidomimetic or anti-peptid ⁇ antibodies.
  • An “indel” is a r ⁇ gion of ammo acid s ⁇ quence that includes all “deleted” residu ⁇ s as shown in at least one am o acid sequence when aligned with another ammo acid sequ ⁇ c ⁇ (s), wh ⁇ r ⁇ th ⁇ s ⁇ que ⁇ ces share overall similar sequenc ⁇ s of ammo acid r ⁇ sidues. That is, the compared sequences contain some identical or conserved ammo acid residues occurring at the same or approximately th ⁇ sam ⁇ positions in th ⁇ compar ⁇ d prot ⁇ in sequences.
  • An mdel spans the farthest extent of d ⁇ l ⁇ t ⁇ d (or ins ⁇ rt ⁇ d) ammo acid r ⁇ sidu ⁇ s as discuss ⁇ d in detail below and shown in FIG. 1. in cases where insertion/deletions are less than ten residu ⁇ s apart (e.g., see mdel 13 in FIG. 2A-2E), the entire segm ⁇ nt containing th ⁇ multiple insertions/deletions is regarded as a single ind ⁇ l Indels can also occur at th ⁇ ammo- and carboxyl-terminal ends of a protein.
  • a "div ⁇ rg ⁇ nt r ⁇ gion" is a portion of an ammo acid s ⁇ qu ⁇ nc ⁇ which shows dissimilarities in the ammo acid residues of at least two members of a protein family whose ammo acid sequ ⁇ c ⁇ s are compared.
  • the divergent region is bordered b ⁇ segments of ammo acid sequences having high sequ ⁇ nc ⁇ cons ⁇ rvation among th ⁇ famii ⁇ members.
  • An mdel is th ⁇ focal point of a div ⁇ rg ⁇ nt region. That is, a divergent region is a segment of the ammo acid sequ ⁇ nc ⁇ including th ⁇ indel that is relatively polymorphic among the compared sequences and is bounded b ⁇ highly conserved regions.
  • locat ⁇ d within means that at least one residue of the peptid ⁇ is co ⁇ tam ⁇ d within the length of the sequence from at least one end of the mdel. For example, if a peptide is located within 30 ammo acids ot an indel, at least one residue of the peptide is Iocated within the sequence defined by residu ⁇ s 1 to 30 away from th ⁇ indel.
  • an md ⁇ l is shown as the region that includes all "deleted" r ⁇ sidu ⁇ s. shown by spaces occupi ⁇ d by ast ⁇ nsks ( * ), with th ⁇ md ⁇ l b ⁇ mg th ⁇ focal point of a divergent region among the protem famil ⁇ members which is bordered b ⁇ segments having high sequence conservation among the famiiy members.
  • the spaces are introduced into the ammo acid sequenc ⁇ s to maximize the alignment of the flanking am o acid residu ⁇ s of th ⁇ related proteins.
  • the indel shown spans the farthest extent of deleted (or inserted) am o acid residues, shown by the arrow svmbols (" ⁇ " and " > "), with "N” and "C” under the arrow symbols identifying tne ammo and carboxyl termini of the mdel. reso ⁇ ctiv ⁇ l ⁇ .
  • the position marked with an arrow ( l ) is defined as being six residues away from the ammo terminal end of the mdel.
  • This figure compares small portions of the human and mous ⁇ ammo acid s ⁇ qu ⁇ c ⁇ s of th ⁇ complement proteins C3, C4 and C5. Because of the multiple interactive properties of thes ⁇ proteins and their close sequence similarity, this protein famil ⁇ is used to demonstrate the utility of the disclosed method for identifying regions of proteins that are involved in interactions with other proteins.
  • FIG. 2A-2E shows the alignment of th ⁇ compl ⁇ t ⁇ ammo acid s ⁇ quences of human and mouse C3, C4, and C5 proteins, which reveals the positions of indels in this protein family.
  • the residues shown in lower case letters in C3 and C4 have previously been identified by other methods as interactive sites in these proteins. Some of these sites are listed in Table 1 with summaries of th ⁇ functional f ⁇ atur ⁇ s, thus showing that th ⁇ reported interactive sites are generally mdel-proximal as defined herein.
  • CCD Complement receptor 1
  • CR2 Complement receptor 2
  • CR3 Complem ⁇ nt r ⁇ c ⁇ ptor 3
  • MCP M ⁇ mbran ⁇ cofactor prot ⁇ in
  • indels primarily as loops on protein surfaces ied me to hypothesize that indels are in general involved in the interactions of proteins with oth ⁇ r molecules.
  • indels provide focal points for identifying am o acid residues within a protein sequence that are iik ⁇ iy to be involved in interactions with other molecules.
  • the disclosed method for identifying likely sites of protein-protem interactions is bas ⁇ d on alignments of the ammo acid sequ ⁇ nc ⁇ s of r ⁇ iated proteins and identification of indels in the protein family
  • Protein sequ ⁇ c ⁇ s in databanks such as GENBANK and SWISS-PROT are readii ⁇ available to those skilled in the art for use with this method which provid ⁇ s a rapid and ⁇ ff ⁇ ctive approach to defining sites of protein protein interactions compar ⁇ d to curr ⁇ ntl ⁇ available procedures.
  • This method has been used to identify linear ammo acid sequences of peptid ⁇ s that significantly aff ⁇ ct (inhibit or enhance) complem ⁇ t activit ⁇ .
  • the method for identifying protein protein and protein substrate interactive sites includes the steps of aligning the ammo acid sequ ⁇ nc ⁇ of the target protein of interest with the sequ ⁇ nc ⁇ s of on ⁇ or more closely r ⁇ iated proteins, and identifying indels in the protein family using this alignment. Because indels usually mark protein segments at the surface of a naturally folded protein, they aiso mark regions of potential protein interactive sites. Although only a subset of 'nde' nr n-imai r ⁇ in ⁇ . may actually be involved in a particular protein prot ⁇ in interaction, focusing on indels greatl ⁇ reduces the task of finding the desired contact s ⁇ te(s) compar ⁇ d to currentl ⁇ used methods.
  • s ⁇ nthetic peptides containing md ⁇ l-associat ⁇ d s ⁇ quences ma ⁇ be co ⁇ struct ⁇ d and tested for modulating a particular protein prot ⁇ in interaction b ⁇ measuring the activity of th ⁇ prot ⁇ in of interest, in the pres ⁇ nc ⁇ and absence of peptide, in an assay in which that interaction is obligatory
  • the peptides may be used to elicit anti-
  • SUBSTTTUTE SHEET (RULE 26) peptide antibodies which are similarly t ⁇ sted
  • the peptide ma ⁇ also be use ⁇ as a structural mod ⁇ l for construction of modified peptides or peptidomimetics which are tested in the same manner.
  • Peptide-based tests for involvement of a particular indel-proximal region may fail to reveal interface peptide activity even if the region is involved in a protein-protem interaction becaus ⁇ the assay ma ⁇ require the peptide to be in a three-dimensional conformation like that of the corresponding segm ⁇ nt within th ⁇ nativ ⁇ protein.
  • Additional methods for mdel testing include engineering proteins with s ⁇ qu ⁇ c ⁇ modifications at the mdel. No matter which assa ⁇ is chosen, focusing on indels greatl ⁇ simplifies the search for interactive sites
  • the location of an mdel is defined as follows. From the point of view of an ammo acid s ⁇ qu ⁇ nc ⁇ containing a deletion relative to another prot ⁇ in (defined as the insertion- containing member) with which it shares a similar ammo acid sequence, the indel site begins at th ⁇ first residue that is missing in the deletion containing member(s) relative to the insertion containing m ⁇ mb ⁇ r(s) and ends at the last residue that is missing in the deletion containing member(s) relative to the insertion containing m ⁇ mber(s) (illustrated in FIG.
  • the mdel will be a single ammo acid r ⁇ sidu ⁇ in l ⁇ ngth.
  • the indel will have ammo and carboxyl termini (as indicated with the "N" and "C” in FIG. 1).
  • the length of the mdel is irrelevant to its identification or its predictive utility for identifying mdel-associat ⁇ d peptides.
  • Peptides associated with such indels ma ⁇ be proximal to eith ⁇ r th ⁇ ammo t ⁇ rmmus or th ⁇ carboxyl terminus or may span th ⁇ indel.
  • Indel- associated peptides that flank an mdel do not necessarily begin or end immediately adjacent to the ammo- or carbox ⁇ l-terminus of the mdel but can have an ammo or carboxyl terminus withm about 30 ammo acid residues of the indel.
  • Indels can also occur at th ⁇ ammo and carboxyl termini of a protein as indicated b ⁇ length polymorphisms at the termini of related proteins (e.g., the carbox ⁇ l termini of the protein s ⁇ quences shown in FIG. 7A 7B).
  • the process of identifying indels involves selection of sequ ⁇ nc ⁇ s to b ⁇ aligned, for ⁇ xample, from databanks such as GENBANK. Selection of sequ ⁇ nc ⁇ s may be based on functional similarities of proteins, gen ⁇ tic relatedness, random scanning of database sequ ⁇ nc ⁇ s for related sequ ⁇ nces or other well known molecular biology methods or combinations of methods
  • the seiect ⁇ d s ⁇ qu ⁇ nc ⁇ s are aligned usually by using computer programs (e.g., PILEUP or CLUSTAL W programs) but may also b ⁇ aligned manually
  • this process adequat ⁇ l ⁇ identifies the positions of most indels, it will be understood by thos ⁇ skilled in the art that the location of some indels may be somewhat uns ⁇ stematic depending on th ⁇ method ( ⁇ g., algorithm) us ⁇ d to align th ⁇ ammo acid s ⁇ qu ⁇ nc ⁇ s and on the particular protein family members s ⁇ lected This variability
  • SUBSTTTUTE SHEET (RULE 26) In selecting individual protein family members for comparison, sequenc ⁇ similarity and the functional properties of the proteins should be considered.
  • the proteins must have sufficiently similar s ⁇ quences to provide regions of unequivocal sequ ⁇ nc ⁇ alignment but must contain the length poi ⁇ morphisms caused bv insertions and/or deletions.
  • the sequence similarities among the proteins is poor.
  • the number and precise locations of indels is ambiguous.
  • the alignment of the very similar C3 proteins from diff ⁇ r ⁇ nt sp ⁇ ci ⁇ s in FIG. 4A-4C shows only a single mdel greater than one residue in length. Therefore, the alignment of C3 with C4 and C5 in FIG. 2A-2E is more informative of potential locations of interactive sites in C3 and functionally important sites that are unique for each member of this family.
  • protems for alignment can alter the appearance of indels as shown in FIGS. 5 and 6, in which diff ⁇ r ⁇ nt subs ⁇ ts of retroviral polyprotems are aligned.
  • FIGS. 5 and 6 show that when all eight sequences are included in the analysis, the numb ⁇ r and locations of indels are somewhat ambiguous. In contrast, when only four of these sequences are compared in FIG. 6A-6B the indels become more clearly defined.
  • indels are us ⁇ d as guid ⁇ s in designing s ⁇ nthetic interlace p ⁇ ptides that modulate the interaction betw ⁇ en two proteins
  • guid ⁇ s indels are us ⁇ d as guid ⁇ s in designing s ⁇ nthetic interlace p ⁇ ptides that modulate the interaction betw ⁇ en two proteins
  • a practitioner skilled in the art ma ⁇ use personal discretion in selecting interface pept ⁇ d ⁇ (s). the followmg two guidelines are useful.
  • First utilize sequences contained within the region of sequence divergence surrounding each mdel as describ ⁇ d abov ⁇ and illustrated in FIG. 1. That is, use only mdel and flanking divergent sequences bounded by highly conserv ⁇ d sequences.
  • I picture th ⁇ divergent region as being at or near the protein surface, and henc ⁇ that its seque ⁇ c ⁇ can vary without gr ⁇ atly p ⁇ rturb g th ⁇ protein conformation and is available for interactions with other protems.
  • the conserved residues are inferred to be invariant because the ⁇ ar ⁇ closer to the prot ⁇ in cor ⁇ , taking part in intramolecular interactions that maintain the overall protein conformation and are physically l ⁇ ss available for mtermolecular interactions.
  • select peptides that correspond in sequence to segments withm the divergent region that are intermediate in hydropathy (i.e., amphipathic peptides).
  • H ⁇ dropath ⁇ can b ⁇ d ⁇ t ⁇ rm ⁇ d by any of a nu o ⁇ r of well known methods. As shown in Tables 3 and 4 (discussed below), most effective peptide inhibitors have intermediate hydropathies.
  • Indel associated peptid ⁇ s include peptides of about 4 to about 20 ammo acid residues in length that are Iocated within 30 am o acid residues, or preferably within 20, 15, 10, 9, 8, 7, 6 or 5 or less residu ⁇ s of an d ⁇ i identified in the ammo acid sequence of a target protein.
  • the seiect ⁇ d p ⁇ ptide includes about 15 am o acid r ⁇ sidu ⁇ s and is Iocated within about 6 residu ⁇ s of an mdel.
  • an interface peptide is identified using the method and guidelines, it ma ⁇ be modified with respect to length and sequenc ⁇ for maximum effect using techniques well known to those skilled in the art. Modification b ⁇ inclusion of conserv ⁇ d r ⁇ sidu ⁇ s, presumably from th ⁇ cor ⁇ of the folded protein, may be desirable to stabilize the conformation of the peptide in a form similar to that of the native protein. Peptid ⁇ s may also b ⁇ ac ⁇ tyiated at their ammo terminus and/or amidated at their carboxyl t ⁇ rm us.
  • Standard solid-phase p ⁇ ptide synth ⁇ tic techniques allow for essentially unlimited quantities of the s ⁇ nthesized p ⁇ ptid ⁇ of interest to be chemically produced (e.g., see Erickson & Merrifield. The Proteins, 3rd Ed.. Vol. 2. Chapt. 3. Academic Press, New York, 1976; and Merrifield & Barany, The P ⁇ ptid ⁇ s: Analysis, Synthesis. Biology, Vol. 1 , Chapt. 1, Gross & Me ⁇ nhof ⁇ r, ⁇ ds., Acad ⁇ mic Pr ⁇ ss, N ⁇ w York, 1980).
  • Peptide analogs and peptidomimetics are molecules that are modeled on a known peptide and synthesized generally to force a desired conformation on a peptide or peptide-like molecule b ⁇ introducing conformational restraints (McDonnell, J.M. et al., 1996, Nature Structural Biol. 3(51:419-426; Nakanishi, H. ⁇ t al., 1993, Gene 137:51-56; Liuzzi, M. ⁇ t al., 1994, Nature 372:695-698). That is, a succ ⁇ ssful peptide analog or peptidomimetic includes appropriate functional groups positioned on a relatively rigid molecular fram ⁇ work.
  • Conformational restraints may be introduced in a variet ⁇ of wa ⁇ s, all well known in the art, including using ammo acid residues that display strong conformational te ⁇ d ⁇ ncies, covalently cyclizing a peptide backbone (e.g., by introducing a disulfide bond between two cysteine residues in a peptide sequ ⁇ nc ⁇ ), and introducing bulk ⁇ chemical groups as side chains or as terminal modifications to a peptid ⁇ (e.g., introducing o ⁇ or more (C e H 5 )CH 2 groups at the am o-terminus of a peptide).
  • Peptid ⁇ analogs and peptidomimetics ma ⁇ be designed in a variety of ways yielding molecules that mimic the peptid ⁇ of mt ⁇ r ⁇ st.
  • On ⁇ type of analog consists of the ammo acid seouenc ⁇ of the identified peptide of mterest with cystem ⁇ r ⁇ sidues add ⁇ d at th ⁇ am o- and carbox ⁇ l termini of the peptide.
  • the resulting peptide is then oxidized to join covalently the terminal c ⁇ stein ⁇ sulphydryl sid ⁇ chains, producing intrachai ⁇ disulfide bridge and forming a cyclic peptide.
  • the corresponding linear version of the peptide is easily produced by reducing the disulfide bond with a reducing agent such as dithiothreitol (DTT) or by reductive alkylation with vinylpyridine (McDonnell, J.M. et al., 1996, Nature Structural Biol. 3(51:419426).
  • a reducing agent such as dithiothreitol (DTT) or by reductive alkylation with vinylpyridine (McDonnell, J.M. et al., 1996, Nature Structural Biol. 3(51:419426).
  • Peptide analogs ma ⁇ contain some or all D-amino acid residues substituted for the L-ammo acid residues in the peptide sequence of interest.
  • D-amino acid containing peptides are oft ⁇ n preferr ⁇ d for in vivo use becaus ⁇ of their greater serum stability compared to peptid ⁇ s containing L-ammo acids.
  • One type of D-amino acid containing peptide analog is a retro-e ⁇ antiom ⁇ c peptide consisting of onl ⁇ D-amino acid residues occurring in the reverse order of the sequence of L-amino acids in the peptide of interest.
  • Such a retro-D-ammo acid peptide is an isomer of the corresponding L-ammo acid peptide, having reversed peptide bond orientation but theoretically identical side-chain topology as in the corresponding L amino acid peptide.
  • th ⁇ p ⁇ ptid ⁇ includes a ⁇ -X tn that causes the peptide chain to reverse direction, for example, b ⁇ limiting the distance betw ⁇ n th ⁇ C ⁇ of th ⁇ first r ⁇ sidu ⁇ and C ⁇ of th ⁇ fourth r ⁇ sidue to about 4 to 7 A.
  • Such p ⁇ ptid ⁇ s ar ⁇ readily produced by a macrocyclization reaction involving th ⁇ use of an azetidinone as an activated ⁇ st ⁇ r during peptide s ⁇ nthesis (Nakanishi, H. et al., 1993, Gene 137.51-56).
  • peptides containing a 6 turn can also be s ⁇ thesiz ⁇ d to produce -5-tur ⁇ peptidomimetics.
  • a peptide analog may be produced by using a scrambled sequ ⁇ nc ⁇ of th ⁇ p ⁇ ptid ⁇ of interest and introducing one or more D ammo acid residues into the peptid ⁇ .
  • Synthesis of peptid ⁇ analogs and peptidomimetics is w ⁇ ll known in th ⁇ art and generally involves: (1) molecular modeling of a known peptide sequence which can be performed using any cf a variety of molecular modeling computational chemistr ⁇ methods ( ⁇ .g., those available as DiscoverTM and HomologyTM from Biosym Technologies Inc. or the PROCHECK program of Laskowski, R.A. et al., 1993, J. Appl. Crystallogr. 26:283-291), and (2) chemical s ⁇ nthesis using well known p ⁇ ptide s ⁇ nthesis or organic s ⁇ nthesis techniqu ⁇ s.
  • peptide analogs and peptidomimetics ma ⁇ b ⁇ produce ⁇ using standard protein synthetic chemistry on an automated system (e.g., Applied Bios ⁇ stems 430A peptide s ⁇ nthesizer) but including one or more D-ammo acid residues or bv modifying the svnthesis to include a macrocyclization reaction as discussed above.
  • the synthesized peptides are then readily purified by anv of a variety of techniques such as reverse phase high pressure liquid chromatography (RP HPLC). Screening ot existing ch ⁇ mical libraries or natural products for molecules that mimic the peptide of interest is an alternative method of identifying peptidomimetics without synth ⁇ sizmg the molecules de novo.
  • candidate indel-proximal interface peptid ⁇ s is t ⁇ xact but can readily be determined b ⁇ a practitioner skilled in th ⁇ art using th ⁇ t ⁇ chniqu ⁇ s de. - ib ⁇ d herein.
  • the amount of experimentation ne ⁇ d ⁇ d to confirm th ⁇ in vitro or in vivo a ivity of pot ⁇ tial interface peptid ⁇ s is significantly reduced becaus ⁇ of th ⁇ relatively high probability of predicting mterfac ⁇ peptide sequences, in practice, th ⁇ activity of an individual peptid ⁇ , peptide analog or peptidomim ⁇ tic ma ⁇ d ⁇ p ⁇ nd on its three-dimensional conformation, its solubility in physiological conditions and its ability to be chemically or biologically synthesized and purified.
  • interf ac ⁇ peptides that have be ⁇ us ⁇ d to inhibit (1) the HIV protease (Zhang, Z.-Y. et al., 1991, J. Biol. Chem. 266:15591 15594; Babe, L.M. et al., 1992, Prot. Sci. 1:1244-1253; Schramm, H.J. et al., 1993, Biochem. Biophys. Res. Commun. 194:595-600), (2) HIV reverse transcriptase (Divita, G. et al., 1994, J. Biol. Chem.
  • inhibitory peptid ⁇ s ⁇ quences had been select ⁇ d on th ⁇ basis of X ray crystallographic structural information, b ⁇ fortuitous observation of inhibitory activity and anaiog ⁇ to related proteins, and b ⁇ individual testing of modified proteins.
  • the present invention includes a useful method for identifying peptides that can inhibit the interactions of proteins with other molecules including substrates and other proteins, such as in enz ⁇ matic protein protem mteractions. This can be especially important in mo ⁇ ulati ⁇ g activation of proteins involved in complex biological Drocesses such as complement activation.
  • inhibitory peptid ⁇ s may be useful for preventing and treating human pathological conditions associated with activation ot the complement system including tissue rejection associated with xenotransplantation ot organs, limb and gut ischemia, iscnemia-reperfusion following myocardial infarction, stroke, aneurysm, hemorrhagic shock, and crush or thermal injury, anaph ⁇ iaxis, and an ⁇ of a variet ⁇ of chronic inflammation conditions.
  • Autoimmune disorders associated with increased complement activit ⁇ including s ⁇ stemic lupus ⁇ r ⁇ thmatosis rheumatoid arthritis and multiple sclerosis may also be regulated by inhibitory peptid ⁇ s or antibodies g ⁇ n ⁇ rat ⁇ d using p ⁇ ptid ⁇ s ⁇ esigned b ⁇ th ⁇ mdel method.
  • inhibitory peptid ⁇ s or antibodies g ⁇ n ⁇ rat ⁇ d using p ⁇ ptid ⁇ s ⁇ esigned b ⁇ th ⁇ mdel method may also be regulated by inhibitory peptid ⁇ s or antibodies g ⁇ n ⁇ rat ⁇ d using p ⁇ ptid ⁇ s ⁇ esigned b ⁇ th ⁇ mdel method.
  • a pharmaceutical composition containing a biologically active peptide, peptide analog or peptidomimetic compound identified by identifying an del-associat ⁇ d p ⁇ ptide sequ ⁇ nce an effective amount of the peptid ⁇ , p ⁇ ptid ⁇ analog, peptidomimetic or mixtures ther ⁇ of, is admixed with a physiologically acc ⁇ ptable carrier suitable for administration to mammals including humans.
  • the peptides, peptide analogs or peptidomimetics may be covalently attached to each other, to other peptides or protein carriers or to other carriers such as b ⁇ incorporation into lipid vesicles.
  • the peptid ⁇ , peptide analog or peptidomimetics are mixed with an adjuvant or adsorbent as is w ⁇ ll known in vaccine art.
  • the peptides, peptide analogs and/or peptidomimetics may be delivered to the mammal to be tr ⁇ at ⁇ d in any of a va ⁇ ty of known m ⁇ thods including but not limit ⁇ d to s ⁇ stemic delivery via i.v., i.m., i.d. or s.c, or i.p. injection of a solution, suspension or lipid encapsulated form.
  • p ⁇ ptid ⁇ s that potentiate or enhanc ⁇ protein-protem interactions. Because peptides near to or overlapping with an del sequ ⁇ nc ⁇ r ⁇ pr ⁇ se ⁇ t sections of protein that have a higher potential for b ⁇ ing involved in prot ⁇ in prot ⁇ in interactions, such p ⁇ ptid ⁇ s ma ⁇ also be used to potentiate an ⁇ reaction dependent on protein-protem interaction.
  • peptide mediated activity is that an indel-proximal p ⁇ ptide select ⁇ d for a target protein binds to a receptor of that target protein and acts either as an agonist or an antagonist of the normal cellular respons ⁇ associated with protein receptor binding.
  • a growth factor p ⁇ ptid ⁇ identified b ⁇ its mdel proximity may instigate or potentiate cell division when the peptide binds to the cellular receptor for th ⁇ growth factor prot ⁇ in.
  • Th ⁇ d ⁇ l method is used to select and design peptid ⁇ s, peptide analogs and peptidomem ⁇ tics that are then tested for their ability to potentiate or enhanc ⁇ cellular respons ⁇ s.
  • Such t ⁇ sting involves th ⁇ us ⁇ of well- known in vivo or in vitro assays available for a wide variety of known cellular respons ⁇ s.
  • a va ⁇ ty of animal mod ⁇ ls ar ⁇ available to t ⁇ st th ⁇ ⁇ fficac ⁇ of p ⁇ ptid ⁇ s id ⁇ ntifi ⁇ d b ⁇ th ⁇ md ⁇ l m ⁇ thod for their rel ⁇ vanc ⁇ to th ⁇ tr ⁇ atm ⁇ t of human m ⁇ dical conditions.
  • These include in vivo animal mod ⁇ ls include the
  • SUBSTTTUTE SHEET (RULE 26) following nonlimitmg models for: acute myocardial infarction (Weisman. H.F. et al., 1990, Science 249: 146); rejection of xenograft transoiants (Leventhal, J. et al., 1993, Transplantation 55: 857); ischemia related to stroke (Chang, L. et al.. 1992, J. Cerebr. Blood Flow Metab. 12. 1030); cardiooulmo ⁇ ary bypass (Nilsson, L. et al., 1990. Artif. Organs 14: 46); pancreatitis (Steer, M . 1992, Yale J. Biol. Med.
  • Inhibitory peptides, peptidomimetics, antibodies to peptides or combinations thereof ma ⁇ be administered b ⁇ a variet ⁇ of m ⁇ thods well known in th ⁇ art including intravenous injection, oral and mtranasal routes, intraperitoneal, intradermal, intramuscular and subcutaneous injection.
  • a variety of delivery systems may be employed including injection of pharmacologically acceptabl ⁇ solutions or suspensions, encapsulation in liposomes or by controlled release methods w ⁇ ll known in the art.
  • In vitro treatment of donor organs with prior to transplantation into a recipient is contemplated.
  • compositions containing peptides, peptidomimetics, antibodies to peptides or combinations ther ⁇ of in th ⁇ range of about 10 ⁇ g/kg to 1 g/kg ma ⁇ include other active ingredients including antibiotics, immunosuppressive drugs and growth factors.
  • Animals made transgenic for inhibitory peptides where the peptid ⁇ is ⁇ xpr ⁇ ssed in the animal's organs ma ⁇ also serve as a sourc ⁇ of organs for xenotransplantation to avoid h ⁇ p ⁇ racut ⁇ rejection (Morgan, B.P., 1995, Immunol. Today 16: 257).
  • the disclosed method is a general m ⁇ thod for identifying pot ⁇ ntiai interactive sites in a protein and for using this information to guide the design of peptid ⁇ s, peptide analogs, or peptidomimetics that inhibit, enhance, or mimic the activity of a target protein.
  • the method includes the following steps. First, the known ammo acid sequenc ⁇ s of two or more proteins that share similar ammo acid sequences are compared as described earlier. One of the ammo acid sequences is that of the target protein for which modulating/mimicking peptid ⁇ s ar ⁇ sought.
  • Th ⁇ similarity b ⁇ tw ⁇ the protem sequenc ⁇ s bas ⁇ d on s ⁇ qu ⁇ nc ⁇ s of identical and/or cons ⁇ rv ⁇ d am o acid r ⁇ sidu ⁇ s between proteins for comparison should be in the range of about 0.25% to about 70% for identity, about 1 % to about 20% for conserv ⁇ d ammo acid residues and about 1 % to about 80% for a combination of identical and co ⁇ s ⁇ rv ⁇ d ammo acid residues.
  • the preferred ranges are about 3% to about 25% sequence identity and most preferably about 5% to about 15% sequence identity between the compared ammo acid sequenc ⁇ s.
  • Pr ⁇ f ⁇ r ably the compared sequ ⁇ nc ⁇ s hav ⁇ about 4% to about 15% cons ⁇ rv ⁇ d ammo acid r ⁇ sidues and most preferably about 8% to about 12% conserv ⁇ d ammo acid residues.
  • th ⁇ s ⁇ numb ⁇ rs represent the average of identical and/or conserv ⁇ d ammo acid r ⁇ sidu ⁇ s ov ⁇ r the entire ammo acid sequ ⁇ nc ⁇ s of th ⁇ compar ⁇ d prot ⁇ ins and that limited portions of the sequences ma ⁇ hav ⁇ greater or lesser percentag ⁇ s of identical or cons ⁇ rv ⁇ d ammo acid r ⁇ sidu ⁇ s.
  • a peptid ⁇ sequenc ⁇ that corresponds to a s ⁇ gm ⁇ nt of th ⁇ protein sequ ⁇ ce and includes ammo acid r ⁇ sidues that span or occur withm about one to about ten residues of an am o terminus or a carboxyl terminus of an md ⁇ l ar ⁇ s ⁇ iect ⁇ d.
  • P ⁇ ptid ⁇ s are select ⁇ d using th ⁇ two guid ⁇ lin ⁇ s d ⁇ scribed earlier, and are s ⁇ nthesiz ⁇ d using an ⁇ of a variet ⁇ of molecular genetic and chemical methods. Using well-known assays, the effects of the peptid ⁇ s on the activity of the target protein or proteins that interact with the target protein are evaluated.
  • the total p ⁇ rc ⁇ ntag ⁇ of identical or cons ⁇ rv ⁇ d amino acid sequence between two or more proteins may be relatively low
  • alignment of ev ⁇ n limited portions of th ⁇ proteins ma ⁇ identif ⁇ i ⁇ deis useful for identifying peptides or other molecules that can affect the activities of the proteins.
  • Indels are highly variable in size, ranging from one to about 50 residues in length. The length of the indel is not relevant; the identification of an indel, no matter how short or long, is a key step leading to selection of indel-proximal p ⁇ ptid ⁇ s.
  • the sel ⁇ cted peptide sequ ⁇ nce ma ⁇ range in size from about 4 to 25 am o acid residu ⁇ s in l ⁇ ngth including peptides of about 4 to 15 or 4 to 20 ammo acid residu ⁇ s in l ⁇ ngth.
  • a p ⁇ ptid ⁇ of an ⁇ I ⁇ ngth whose ammo and carbox ⁇ termini are both within about 15, 12 or 10 residues of an indel is included within the scope of this invention if the peptid ⁇ sequenc ⁇ spans a larg ⁇ ind ⁇ l.
  • sequence "A” and “B” are shown at an indel (indicated b ⁇ asterisks) and a peptide from sequence "B” that spans the indel is underlined in sequence "B".
  • Sequence "A” AAA_ AAAAAAAA* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
  • the peptid ⁇ corresponding to the underlined segment in sequenc ⁇ "B" is 30 residues long and within 6 residues of each end of th ⁇ ind ⁇ l.
  • a longer peptide is included in the invention because of its proximity to the indel by spanning all of or a portion of the mdel.
  • any p ⁇ ptide that has proximity to the indel, including thos ⁇ completely Iocated within the s ⁇ qu ⁇ c ⁇ that corresponds to the indel are within the scope of this invention.
  • a p ⁇ ptid ⁇ iocated within the indel would be a seri ⁇ s of residues from "B" sequence that corresponds to the sequenc ⁇ indicat ⁇ d by th ⁇ series of asterisks in the "A" sequence.
  • indel-proximal peptide ma ⁇ be based on peptide characteristics including length, soiubilit ⁇ and ease of s ⁇ nthesis or purification, all of which are routine determinations for those skilled in the art of peptide s ⁇ nthesis.
  • the method of delivery of the peptide (e.g., in an aqueous solution or emulsion) ma ⁇ determine the particular indel-proximal peptid ⁇ s ⁇ que ⁇ ce s ⁇ lected.
  • an overlapping s ⁇ t of p ⁇ ptides proximal to that indel is s ⁇ l ⁇ ct ⁇ d and ⁇ ach is t ⁇ st ⁇ d for its ability to alt ⁇ r th ⁇ activit ⁇ of the target protein in a variet ⁇ of assays.
  • an effectiv ⁇ md ⁇ l-proximal p ⁇ ptid ⁇ may span th ⁇ i ⁇ d ⁇ l sit ⁇ or flank the indel site.
  • Peptides that flank an mdel site ma ⁇ occur proximal to eith ⁇ r th ⁇ ammo or carbox ⁇ l t ⁇ rminus of the indel and ma ⁇ have at least one residue within about one. five.
  • FIG. 2A-2E An alignment of the C3/C4/C5 famil ⁇ of complement proteins using th ⁇ CLUSTAL W program revealed over 30 indels as shown in FIG. 2A-2E. Indels were ⁇ som ⁇ what arbitrarily defined her ⁇ as loci where one member of the family had an insertion or deletion equal to or greater than two residues in length. However, it will be understood that other definitions are also predictive of inhibitory peptides based on this general procedure of identification, including where one memb ⁇ r of th ⁇ family has an insertion or deletion equal to or greater than one ammo acid residue in length. In FIG.
  • th ⁇ ind ⁇ ls are identified b ⁇ a solid lin ⁇ under the last im ⁇ of protem sequ ⁇ nce for each section of six sequ ⁇ nces and labelled with consecutiv ⁇ numbers.
  • Previously report ⁇ d interactive sites in C3, C4, and C5 shown as lower case lett ⁇ rs for the ammo acid residues in FIG.
  • Th ⁇ s ⁇ include the thioester sit ⁇ and the proteolytic site that occurs eight residu ⁇ s upstream of the thioester in the C3 ⁇ -chain that generates the C3d fragm ⁇ nt from C3dg, which may not b ⁇ physiologically important (Law, S.K.A., 1988, J. Cell Sci. Suppl. 9:67-97; D ⁇ Bruijn, M.H.L. & Fey, G.H., 1985, Proc. Natl. Acad. Sci. USA 82:708-712).
  • Another isot ⁇ pe-specific site in C4, which ma ⁇ be involved in intramolecular interactions (Law, S.K.A.
  • ⁇ nz ⁇ m ⁇ s and antisera can be purified using well known techniqu ⁇ s.
  • Most compl ⁇ me ⁇ t reagents are commercially available (e.g., from Advanced Research Technologies, San Diego, CA).
  • Other commercial sources include: human Cis (Enzyme res ⁇ arch Labs. Inc.. South B ⁇ nd, IN), human factor B (Caibiochem, La Jolla, CA), cobra venom factors (Diamedix. Miami. FL or Quid ⁇ l. San Di ⁇ go, CA), human factor D, (Quidel, San
  • SUBSTTTUTE SHEET (RULE 26) Di ⁇ go, CA), and goat antisera against murine C3 (Organon Tek ⁇ ika-Capel, West Chest ⁇ r, PA) and numan C3 and C5 (Quidel, San Diego, CA).
  • S ⁇ nthetic peptid ⁇ s ma ⁇ b ⁇ obtained from Chiron Mimotopes (San Di ⁇ go, CA).
  • the peptides were s ⁇ nthesiz ⁇ d using well-known methods (Geysen, H.M. et al., 1984, Proc. Natl. Acad. Sci. USA 81:3998; Geysen, H.M. et al., 1983, J. Immunol. Meth. 102: 259).
  • th ⁇ individual peotides e.g., I1 1
  • Table 3 The names of th ⁇ individual peotides (e.g., I1 1 ) used in Table 3 correspond to the nam ⁇ s over the underlined peptide sequences in FIG. 2A-2E. Some of the peptides were ⁇ ac ⁇ t ⁇ iat ⁇ d at th ⁇ ammo t ⁇ rminus (indicated b ⁇ "Ac-" in Table
  • Ther ⁇ for ⁇ , p ⁇ ptid ⁇ s s ⁇ l ⁇ ct ⁇ d on th ⁇ basis of th ⁇ g ⁇ n ⁇ rai md ⁇ i method could be produced b ⁇ a variet ⁇ of procedures well known to thos ⁇ skilled in th ⁇ art and th ⁇ invention should not b ⁇ consid ⁇ r ⁇ d limited to an ⁇ one particular method of making the peptid ⁇ s so identified.
  • Peptid ⁇ s identified in Example 2 w ⁇ r ⁇ t ⁇ st ⁇ d for inhibition of complement activity using the hemolytic assay for complement activity.
  • the peptides were add ⁇ d to th ⁇ hemolytic assa ⁇ and their ability to inhibit lysis of erythrocytes or red blood c ⁇ lls (RBC) by compl ⁇ m ⁇ nt activity was m ⁇ asur ⁇ d.
  • sh ⁇ p RBC coat ⁇ d with rabbit anti-sh ⁇ p RBC antibodies were ⁇ ⁇ xpos ⁇ d to dilute human s ⁇ rum.
  • Complement is activated on erythrocyte surfaces b ⁇ the rabbit antibod ⁇ resulting in lysis of the red blood cells. Th ⁇ amount of complement in the serum is determined b ⁇ the ext ⁇ nt of ⁇ r ⁇ throc ⁇ t ⁇ lysis. If p ⁇ ptid ⁇ s inhibit any step in the lytic pathway, this inhibition reduc ⁇ s th ⁇ appar ⁇ nt compl ⁇ m ⁇ nt activity of th ⁇ s ⁇ rum resulting in l ⁇ ss erythrocyte lysis. Erythrocyte lysis or hemolytic assays were performed using the "EZ Complement" kit (Diam ⁇ dix, Miami, FL).
  • the level of hemolysis was measur ⁇ d as th ⁇ absorbance of th ⁇ solution at 415 nm after centrifugation of the sample to remove intact erythroc ⁇ tes. Th ⁇ l ⁇ v ⁇ l of hemolysis was expressed as th ⁇ fraction
  • FIG. 9 shows the hemolvsis inhibition r ⁇ sults for C3 p ⁇ ptid ⁇ s 114, II 5, III 8C and III 11. Th ⁇ lytic activity of the peptide alone was negligible for these peptid ⁇ s. Th ⁇ data for the peptides (II-4, II 5, III-8C and 111-11) shown in FIG. 9 show that increasing concentrations of peptide resulted in decreasing complement activity.
  • FIG. 9 The results in FIG. 9 were selected to show the range of respons ⁇ s found among the most strongly inhibiting peptid ⁇ s. Results for all the peptides are listed in Table 3, which presents the approximate peptide concentration at which 50% inhibition of erythrocyte lysis (IH 5g ) was s ⁇ n. Th ⁇ s ⁇ r ⁇ sults ar ⁇ shown graphically in FIG. 10.
  • the hemolytic results suggest that specific peptid ⁇ -prot ⁇ m interactions interfere with the specific protein- protem interactions that are necessary for complement function. Inhibition of hemolysis was dose-dependent, and the concentration of peptid ⁇ necessary for 50% inhibition va ⁇ d substantially with each peptide (from about 25 ⁇ M to greater than about 600 ⁇ M).
  • Tabl ⁇ 3 includes data for a single human C4 peptid ⁇ (p ⁇ ptide C4 B1) and two human C5 peptid ⁇ s (p ⁇ ptides C5-D5 and C5 D2, respectively), that were ⁇ also tested for inhibition of hemolytic activity. A total of seven C5 peptides have been examm ⁇ d to date. Peptide C4 B1. the only C4 peptide chosen for study, shows moderate inhibitory activity. Thus, in a single attempt, th ⁇ indel-proximal method of peptid ⁇ identification successfully Iocated an inhibitory peptid ⁇ in a second member of the C3/C4/C5 family.
  • Peptide C5 D5 shows the greatest inhibitory activity of the C5 peptid ⁇ s test ⁇ d to dat ⁇ although its activity is relatively w ⁇ ak.
  • p ⁇ ptide C5 D2 shows strong potentiating activity in the hemolytic assay, possibly due to inhibition of C5 interaction with a natural inhibitor protein in human serum.
  • Bas ⁇ d on th ⁇ r ⁇ sults obtain ⁇ d with peptides C5 D5 and C5 02, the disclosed method can be used to identify peptides that have potentiating as well as attenuating effects in a complex biochemical s ⁇ stem.
  • Th ⁇ inhibitor ⁇ p ⁇ ptid ⁇ s did not show any pattern of favore ⁇ orientation with regard to ind ⁇ ls. That is, inhibitor ⁇ p ⁇ ptides spann ⁇ d and flanked both the N- and C- termini of indels and came from indels where there were deletions and insertions in the C3 sequenc ⁇ reiativ ⁇ to the C4 and/or C5 sequences. Moreover, there was no preferred amino acid composition. The p ⁇ ptide length chosen arbitrarily in thes ⁇ ⁇ xampl ⁇ s was 13 to 15 am o acid r ⁇ sidu ⁇ s.
  • the single-l ⁇ tt ⁇ r amino acid cod ⁇ is used with Acet ⁇ lat ⁇ d (Ac-) amino t ⁇ rmi ⁇ i and amidat ⁇ d (-NH 2 ) carboxy termini.
  • Peptide concentration giving 50% inhibition of hemoi ⁇ tic activit ⁇ (Cry in about 0.15% human serum, which gives about 30% hemoi ⁇ sis of input targ ⁇ t EA in th ⁇ absence of peptide.
  • C2 and factor B are structurail ⁇ similar proteins (about 100,000 m.w.) that pla ⁇ analogous roles in the classical and alternative oathwa ⁇ s, respectively, of compi ⁇ ment activation and whose genes arose from a common ancestor (Campbell, R.D. et al., 1988, Ann. Rev. Immunol. 6:161-195).
  • members of the C2/factor B complement protein famil ⁇ were aligned as shown in FIG. 11A-
  • the mdel proximal method of peptide identification has be ⁇ n successfully used to guide the d ⁇ sign of p ⁇ ptid ⁇ inhibitors for a ⁇ oth ⁇ r protein family in the compi ⁇ ment system b ⁇ efficiently identifying prot ⁇ in interactive regions and inhibitory p ⁇ ptides which are presumably interface peptid ⁇ s.
  • Antibodies that recog z ⁇ th ⁇ inhibitory p ⁇ ptid ⁇ s id ⁇ ntifi ⁇ d b ⁇ the ind ⁇ l method ma ⁇ also be useful reagents for inhibiting protein acttvit ⁇ . Such antibodies would be ⁇ xpected to bind to the i ⁇ del-proximai portion of th ⁇ nativ ⁇ prot ⁇ in, thus blocking activit ⁇ associated with that portion of the protein. Polyclonal and monoclonal antibodies can be generated against the purified peptides designed b ⁇ using the indel method.
  • Monoclonal antibodies us ⁇ ful in th ⁇ present inv ⁇ ntion can b ⁇ produc ⁇ d and isolated b ⁇ processes which are well known in the art, such as thos ⁇ discuss ⁇ o b ⁇ Milstein and Kohler [Nature 256:495-497, 1975).
  • Monoclonal antibodies ma ⁇ be more us ⁇ ful b ⁇ caus ⁇ of th ⁇ specificity associat ⁇ d with such antibodies and the ability to produce antibodies with th ⁇ sam ⁇ specificity from a clonal cell line.
  • Peptides design ⁇ d b ⁇ using the indel method that have inhibitor ⁇ activity in an in vitro or in vivo assa ⁇ are s ⁇ nth ⁇ siz ⁇ d a ⁇ o monoclonal antibodies are then generated using the purified peptid ⁇ s b ⁇ standard proc ⁇ dur ⁇ s.
  • mice sucn as Balb/c female mice or other mouse strains or even other suitable animals are immunized with an amount of a peptide of ter ⁇ st, such as thos ⁇ id ⁇ ntifi ⁇ d in Exampl ⁇ s 3 and 4, to initiate an immune respons ⁇ .
  • a peptide of ter ⁇ st such as thos ⁇ id ⁇ ntifi ⁇ d in Exampl ⁇ s 3 and 4
  • Th ⁇ animals are immunized with the peptid ⁇ s mix ⁇ d with a suitable adjuvant or with p ⁇ ptides conjugated to a carrier molecule
  • the peptide dosage and immunization schedul ⁇ for producing us ⁇ ful quantiti ⁇ s of suitable spl ⁇ noc ⁇ tes can be readily determmed by one skilled in the art depending on the animal strain used.
  • the siz ⁇ and spacing of dos ⁇ s of p ⁇ ptid ⁇ are generally microgram quantiti ⁇ s with a minimum dosage for initiating an immune respons ⁇ typically in th ⁇ rang ⁇ of 0.1 100 ⁇ g/ammal.
  • an initial immunization with approximately 50 ⁇ g of peptid ⁇ may be followed b ⁇ a seri ⁇ s of five injections for h ⁇ pe ⁇ mmumzation.
  • An adjuvant with approximately 50 ⁇ g of peptid ⁇ may be followed b ⁇ a seri ⁇ s of five injections for h ⁇ pe ⁇ mmumzation.
  • the ammai is monitored for production of anti- peptide specific serum antibodies using well Known techniqu ⁇ s (e.g., ELISA or radioimmunoassa ⁇ ) normally about one to two weeks after immunization.
  • Known techniqu ⁇ s e.g., ELISA or radioimmunoassa ⁇
  • a second dose of the same antigen is given to elicit a p ⁇ ptid ⁇ -sp ⁇ cific secondary immune response which is also detected by standard immunoassays.
  • th ⁇ animal is kiil ⁇ d and its spl ⁇ en cells are isolat ⁇ d and fus ⁇ d with m ⁇ eloma cells (e.g., th ⁇ murine cell line Sp2/0-Ag14) to produce h ⁇ bridoma cell lines capable of reproduction in vitro to produce anti-peptide antibodies.
  • m ⁇ eloma cells e.g., th ⁇ murine cell line Sp2/0-Ag14
  • Th ⁇ m ⁇ eloma cell line s ⁇ l ⁇ ct ⁇ d should b ⁇ compatibl ⁇ with th ⁇ spleen c ⁇ lls, and optimally should be a cell line of the same speci ⁇ s as th ⁇ spl ⁇ n c ⁇ lls.
  • th ⁇ murine cell line Sp2/0-Ag14 has been found to be effective for use with mouse spleen cells, other myeloma c ⁇ ll lin ⁇ s can alternatively b ⁇ used. See, for example, Nature. 276: 269-270 (1978) and U.S. Pat ⁇ nt No. 5,472,868, for fusion partner c ⁇ lls and m ⁇ thods of using such c ⁇ lls to produce hybridomas.
  • Spleen cells are fused with an appropriate m ⁇ eloma cell lin ⁇ using polyethylene glycol.
  • a selective medium e.g., containing h ⁇ poxanthin ⁇ , aminopterin and th ⁇ midine
  • the supernatant from each of a plurality of hyb ⁇ doma-containing tissue culture wells is ⁇ valuated for the presence of antibody specific to th ⁇ p ⁇ ptid ⁇ of int ⁇ r ⁇ st using an ⁇ of a numb ⁇ r of w ⁇ li known immu ⁇ oassa ⁇ s.
  • H ⁇ bridomas that produc ⁇ antibodies that specifically recognize the peptide used as an immunogen are cloned (e.g., b ⁇ limiting dilution) for subsequent production of antibodies in vitro or in vivo.
  • Anti-p ⁇ ptide antibodies are produced eith ⁇ r in vitro b ⁇ tissue culture of th ⁇ sel ⁇ cted cell lines or in vivo b ⁇ gen ⁇ rating ascites fluid in mice i ⁇ ject ⁇ d with th ⁇ h ⁇ bridoma c ⁇ ll line.
  • the monoclonal antibod ⁇ ma ⁇ then b ⁇ isolat ⁇ d in accordance with techniques known in the art.
  • Purified Anti-peptid ⁇ antibodies are identifi ⁇ d as thos ⁇ capable of modulating a biological s ⁇ stem, such as the complement s ⁇ stem. using the appropriate in vitro or in vivo assa ⁇ for the targ ⁇ t prot ⁇ in or prot ⁇ in s ⁇ st ⁇ m.
  • th ⁇ monoclonal antibodies ar ⁇ t ⁇ sted for their ability to enhanc ⁇ or inhibit hemolysis in the assa ⁇ as described in Example 3.
  • the anti peptide antibod ⁇ is added into the hemolysis assa ⁇ alon ⁇ or in combination with th ⁇ corresponding p ⁇ ptide against which th ⁇ antibod ⁇ was rais ⁇ d.
  • the ⁇ are test ⁇ d for their ability to mediat ⁇ a therapeutic eff ⁇ ct as in the in vivo assay d ⁇ scribed in Example 6.
  • the anti peptide antibod ⁇ is injected befor ⁇ , simultaneous or after injection of the corresponding peptid ⁇ or inst ⁇ ad of th ⁇ p ⁇ ptid ⁇ during th ⁇ antibodv challenge and modulation of the inflammation response is monitored relative to the appropriate control reactions.
  • the Arthus model of complement mediated inflammation is caused by interaction of tissue antigen with circulating antibod ⁇ .
  • This in vivo model of compiem ⁇ nt activation is characterized b ⁇ formation ot immune complexes resulting in complem ⁇ nt activation, inflammatory c ⁇ ll recruitment, ⁇ d ⁇ ma and tissu ⁇ damag ⁇ (Bailey, P. & Sturm, A., 1983, Biochem. Pharm. 32. 475).
  • a passive Arthus reaction is established in an animal (e.g., a guinea pig) by first injecting an antigen i.v. and then challenging with an a ⁇ tigen-sp ⁇ cific antibody.
  • P ⁇ ptid ⁇ inhibition of the complement- mediated respons ⁇ is measured when peptid ⁇ is injected i.d. before or simultaneous with antibody challenge and then biops ⁇ tissue taken from the antibod ⁇ challenge sit ⁇ is assay ⁇ d for inflammation.
  • mice Male guinea pigs (about 300 g) are anesth ⁇ tiz ⁇ d by injection of sodium pentobarbital (40 mg/kg, i.p.) and then injected i.v. with ovalbumin (20 mg/kg) and I25 l labeled bovine s ⁇ rum albumin ( ,25 l BSA, 1 ⁇ Ci).
  • Antibody challenge immediately follows by injecting animals m the dorsal region with pol ⁇ cional anti ovalbumin antibody (10 mg, i.d.), with or without an mdel identified peptide in the ⁇ M range indicated as effective complement inhibition by the h ⁇ moi ⁇ sis assa ⁇ (s ⁇ Exampi ⁇ 3).
  • the animals are huma ⁇ ei ⁇ killed and skin tissue from the antibod ⁇ challenge site is ⁇ xcis ⁇ d b ⁇ biopsy punch (about 2 5 mm).
  • Inflammation is measured b ⁇ leakage of 125 l- BSA into the skin tissue det ⁇ rmm ⁇ d by standard radiochemicai counting procedures for ,25 l; percent inhibition b ⁇ peptide is measured b ⁇ comparing leakag ⁇ of 125 l BSA into th ⁇ skin tissu ⁇ in the pres ⁇ nc ⁇ of peptide with that seen in the absence of peptide
  • cobra venom factor 200 U/kg, i.p.
  • th ⁇ venom factor results in suppression of the complement respons ⁇ in animals that receive antigen and antibod ⁇ without complement inhibitory peptide.
  • Peptides identified as b ⁇ ing compi ⁇ ment inhibitory in th ⁇ h ⁇ moi ⁇ sis assa ⁇ show compl ⁇ m ⁇ nt inhibition in this in vivo mod ⁇ l in a dos ⁇ d ⁇ p ⁇ nd ⁇ nt manner with maximal inhibition comparable to the animal that rec ⁇ ived cobra venom factor befor ⁇ initiation of the Arthus response.
  • a series of 250 300 g male guinea pigs are anesthetiz ⁇ d (with sodium pentobarbital, at 40 mg/kg, i.p.) and th ⁇ n injected i.v.
  • One of the ammais is the cobra v ⁇ nom factor control which r ⁇ ceived cobra v ⁇ nom factor (200 U/kg, i.p.) at about 24 hr b ⁇ for ⁇ injection of the ovalbumin and 125 l BSA.
  • the oth ⁇ r animals are individually i v. injected with 0.1 ⁇ g, 0.5 ⁇ g, 1 ⁇ g, 5 ⁇ g and 10 ⁇ g of peptid ⁇ ill 8C (SEQ ID N0:53), or no peptide.
  • Inflammation is m ⁇ asur ⁇ d by l ⁇ akag ⁇ of ,25 l BSA into th ⁇ skin tissue determined b ⁇ standard radiochemicai counting procedures for ,25 l to det ⁇ rmme the p ⁇ rc ⁇ ntag ⁇ of inhibition of inflammation m ⁇ diated by the peptide III 8C dosage.
  • peptidomimetic moiecui ⁇ s can exhibit increased potency as inhibitory molecules for protein-protein interactions
  • peptidomimetics of an inhibitory peptid ⁇ identified using th ⁇ ind ⁇ l m ⁇ thod and the hemolytic assay are selected and screened for increased inhibitory activit ⁇ .
  • Peptidomimetics of p ⁇ ptid ⁇ 11-1 are selected from a combinatorial peptide library in which the internal five ammo acid residu ⁇ s (VPVTV) are randomized (Bianchi, E. et al., 1995, J. Mol. Biol. 247: 154-160) producing a library of peptid ⁇ s that ar ⁇ analogs of p ⁇ ptid ⁇ 11-1.
  • VTV ammo acid residu ⁇ s
  • Th ⁇ library of p ⁇ ptid ⁇ s can b ⁇ produced either using molecular genetic techniques (e.g., s ⁇ nthesis of the partially randomized DNA sequenc ⁇ s coding for th ⁇ peptide 11-1 analogs followed by expr ⁇ ssion of the peptides m cells and purification of the peptid ⁇ s) or b ⁇ chemically synthesizing the partially randomized p ⁇ ptid ⁇ s.
  • Ch ⁇ mical s ⁇ nth ⁇ sis allows additional ⁇ xpa ⁇ sion of the repertoire by allowing inclusion of non-coded am o acids or organic isoste ⁇ c replacement groups (e.g., a 4(5)-acy! ⁇ midazoie ring for replacement of an amide bond).
  • peptid ⁇ analogs and peptidomimetics are tested in vitro for their inhibitory activity relative to peptide 11-1 in a hemolytic assa ⁇ essentially as described in Examples 3 and 4 and in an in vivo assay essentiall ⁇ as describ ⁇ d in Exampi ⁇ 6. Som ⁇ of th ⁇ partially randomiz ⁇ d peptidomimetics and analogs of peptide 11-1 show increas ⁇ d inhibition of compi ⁇ ment function in the hemolysis assa ⁇ , some show increased inhibition the animal model and some show increased inhibitor ⁇ activit ⁇ in both assa ⁇ s.
  • Peptid ⁇ 1-8 (SEQ ID 1.0:63) is us ⁇ d to s ⁇ th ⁇ siz ⁇ a r ⁇ tro-D-ami ⁇ o acid p ⁇ ptid ⁇ that is an isomer of the corresponding L-ammo acid 1-8 p ⁇ ptide. That is, a peptide is s ⁇ thesiz ⁇ d using onl ⁇ D-ammo acids and having a s ⁇ qu ⁇ nce LKTSIGNKPPEKLD (SEQ ID N0:78) which is the reverse of the s ⁇ qu ⁇ nc ⁇ of p ⁇ ptid ⁇ 1-8.
  • Th ⁇ 1-8 p ⁇ ptid ⁇ and th ⁇ retro-D-peptide of 1-8 are compar ⁇ d in th ⁇ h ⁇ mol ⁇ tic assa ⁇ as d ⁇ scrib ⁇ d in Exampi ⁇ 3.
  • Th ⁇ 1-8 shows little or no inhibition of hemolysis, requiring a peptide concentration of about 300 ⁇ M to produce 50% inhibition of the hemol ⁇ tic activit ⁇ in 0.15% human serum.
  • the retro-D-p ⁇ ptid ⁇ of 1-8 havmg reversed p ⁇ ptide bond orientation of th ⁇ corresponding L-amino acid peptide shows increased inhibition because it produces 50% inhibition at a concentration of about 150 ⁇ M in the same t ⁇ pe of r ⁇ action.
  • Another peptide analog is produced as a covalently cyclized version of peptid ⁇ I-5 having th ⁇ im ⁇ ar s ⁇ quence CRLLKAGRQVREPGQC (SEQ ID NO:79) (i.e., addition of cystem ⁇ r ⁇ sidu ⁇ s at the ammo- and carbox ⁇ l-termmi of peptide I-5).
  • the linear peptide having SEQ ID 1.0:79 is synthesized using conventional solid-phase protein synthesis and purified b ⁇ reverse phase HPLC using standard methods. The purified peptide is then oxidized to covendingl ⁇ cyclize th ⁇ terminal cysteine sulphydryl side chains to form an intracham disulfide bridge (such as described b ⁇ McDonnell, J.M.
  • the linear version of the peptide having SEQ ID N0:79 also shows limited inhibition of hemolysis, requiring a peptid ⁇ concentration of about 350-400 ⁇ M to produce 50% inhibition of the hemol ⁇ tic activit ⁇ in 0.15% human serum.
  • the cyclized version of the peptide having SEQ ID N0:79 produces no inhibition of hemoiysis and enhanc ⁇ s h ⁇ mol ⁇ tic activity about 2-fold at a concentration of about 100 ⁇ M in the same type of r ⁇ action.
  • the physical form of the peptide having SEQ ID NO:79 substantially affects its ability to modulate protein activity in this assa ⁇ .
  • EXAMPLE 8 Indel-associated peptid ⁇ substitution for a taroet protein's receptor
  • a series of peptid ⁇ s are ge ⁇ rat ⁇ d for the sequence of compiem ⁇ nt C3 prot ⁇ m n ⁇ ar ind ⁇ l 13 as shown in FIG. 2A-2E, such as p ⁇ ptid ⁇ s il-5 (SEQ ID N0:56) and 1-8 (SEQ ID N0:63), and cyclized peptidomimetics of these peptides in which the am o- and carboxyl-termini D-c ⁇ st ⁇ m ⁇ s joined b ⁇ a disulfide bridge are sy ⁇ thesiz ⁇ d using techniqu ⁇ s described earlier.
  • Peptide 1-1 (SEQ ID NO:65), overlapping mdel 16 in FIG.
  • Th ⁇ s ⁇ binding assa ⁇ s show that purifi ⁇ d C3 binds to compl ⁇ m ⁇ nt r ⁇ ceptors CR1, CR2 and CR3, neither the linear peptid ⁇ 1-11 or its cyclized peptidomimetic bind to an ⁇ of the receptors, and the linear forms of peptides 11-5 and 1-8 bind to all three C3 receptors but nev ⁇ r mor ⁇ than about 40-50% of th ⁇ efficiency of binding as with purified C3 protein.
  • Th ⁇ c ⁇ ciized peptidomimetic of p ⁇ ptid ⁇ il-5 binds to ail thr ⁇ receptors but l ⁇ ss ⁇ fficiently than its linear form whereas th ⁇ cyclized peptidomimetic of peptid ⁇ 1-8 binds slightly mor ⁇ efficiently to all three receptors than the linear peptide.
  • Thes ⁇ results show that peptides identified by th ⁇ mdel-association method and peptidomimetics modeled on such peptid ⁇ s can substitut ⁇ for a ligand or substrate of th ⁇ targ ⁇ t protein.
  • EXAMPLE 9 Pharmaceutical composition including an indel-proximal identified peptide or peptide analog or peptidomimetic ther ⁇ of
  • P ⁇ ptid ⁇ s, p ⁇ ptid ⁇ analogs or peptidomimetic molecul ⁇ s according to th ⁇ present invention are id ⁇ ntified, synthesized and tested for biological activity in an in vitro and/or in vivo assa ⁇ as describ ⁇ d in the previous examples or using other well known methods and assays.
  • peptid ⁇ For delivery of a peptid ⁇ , p ⁇ ptid ⁇ analog or peptidomimetic molecule (hereafter generally referred to as "peptid ⁇ "), the peptide is dissolved in a buff ⁇ r ⁇ d salt solution (e.g., phosphat ⁇ buff ⁇ r ⁇ d saline) at a dose of 1 ⁇ g/ml to 100 ⁇ g/ml depending on the activity of the peptid ⁇ as determin ⁇ d in an in vivo or in vitro assa ⁇ .
  • a buff ⁇ r ⁇ d salt solution e.g., phosphat ⁇ buff ⁇ r ⁇ d saline
  • the p ⁇ ptid ⁇ solution is th ⁇ n injected in 0.5-5 ml aliquots into the mammal including a human to be treated via an ⁇ of a variet ⁇ well known routes (i.v., i.m., s.c, ⁇ tc.) depending on the t ⁇ pe of treatment.
  • a variet ⁇ well known routes i.v., i.m., s.c, ⁇ tc.
  • a peptide that inhibits compiem ⁇ nt activity e.g., p ⁇ ptide 111-11
  • a patient experiencing iscnemia-reperfusion following myocardial infarction may be treated with a complement-inhibiting p ⁇ ptid ⁇ delivered b ⁇ catheterization to deliver the peptide selectively to the affected area.
  • the peptide is preferably coupled to ovalbumin grade V at about a 10:1 molar ratio using N-succ ⁇ m ⁇ dyl 3-(2-p ⁇ r ⁇ dyld ⁇ th ⁇ o) propionate (SPDP).
  • SPDP N-succ ⁇ m ⁇ dyl 3-(2-p ⁇ r ⁇ dyld ⁇ th ⁇ o) propionate
  • SPDP (40 mM in 99% ethanol is added dropwis ⁇ to ovalbumin (dissolv ⁇ d in 0.2M NaH 2 P0 4 , pH 8.5) to form a SPDP-ovalbumin conjugate that is purified by column chromatography and coupl ⁇ d to p ⁇ ptide (1 mg/ml in 10% acetic acid) b ⁇ allowing the peptide and SPDP-ovalbumin to incubate for about 12 hr at RT.
  • the peptid ⁇ -SPDP-ovalbumm conjugate (100 ⁇ g/0.5 ml of phosphate buffer ⁇ d saline, with or without an adjuvant) is injected i.m. in a seri ⁇ s of three injections about one week apart to induce an anti peptide immun ⁇ response in the mammal receiving the peptide.
  • MOLECULE TYPE pepcide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • Vi ORIGINAL SOURCE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peDtide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • Trp Asp Ile Pro Glu Leu Val Asn Met Gly Gin Trp Lys Ile Arg Ala
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • Pro Gly Asn Ser Asp Pro Asn Met lie Pro Asp Gly Asp Phe Asn Ser
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
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Abstract

L'invention se rapporte à un procédé d'identification des peptides, des analogues peptidiques et des peptidomimétiques qui présentent une forte probabilité d'inhibition, d'amélioration ou d'imitation de l'activité d'une protéine cible. Ce procédé consiste à comparer les séquences d'acides aminés de protéines apparentées en vue d'identifier les séquences associées par insertion/suppression (Insertion/Deletion, 'Indel'). On décrit par ailleurs les séquences aminées des peptides qui modulent l'activité du système complémentaire.
PCT/US1996/010958 1995-06-29 1996-06-27 Procede d'identification des peptides qui affectent les interactions proteine-proteine et des peptides modulant l'activite complementaire WO1997001578A1 (fr)

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Application Number Priority Date Filing Date Title
AU63977/96A AU6397796A (en) 1995-06-29 1996-06-27 Method for identifying peptides that affect protein-protein interactions and complement-modulating peptides

Applications Claiming Priority (4)

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US67495P 1995-06-29 1995-06-29
US60/000,674 1995-06-29
US66361796A 1996-06-14 1996-06-14
US08/663,617 1996-06-14

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WO1997001578A1 true WO1997001578A1 (fr) 1997-01-16

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WO2003045991A2 (fr) * 2001-11-23 2003-06-05 Syn.X Pharma, Inc. Marqueurs de biopolymeres precurseurs du facteur c3 du complement permettant de predire la maladie d'alzheimer
WO2003070949A1 (fr) * 2002-02-22 2003-08-28 Adprotech Limited Vecteurs d'immunisation pour chats
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US11918624B2 (en) * 2020-06-10 2024-03-05 Kelsius Laboratories LLC Therapeutic composition for use in the treatment of COVID-19 and other cytokine storm associated disorders

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Cited By (15)

* Cited by examiner, † Cited by third party
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US8032347B2 (en) 2001-11-01 2011-10-04 The University Of British Columbia Methods and apparatus for protein sequence analysis
WO2003038724A2 (fr) * 2001-11-01 2003-05-08 The University Of British Columbia Appareil d'analyse d'une sequence proteique, procedes, supports lisibles par ordinateur, programmes informatiques, signaux et structures de donnees
WO2003037926A1 (fr) * 2001-11-01 2003-05-08 The University Of British Columbia Diagnostic et traitement de maladies infectieuses par l'intermediaire de proteines differenciees d'insertion/suppression
WO2003038724A3 (fr) * 2001-11-01 2004-02-19 Univ British Columbia Appareil d'analyse d'une sequence proteique, procedes, supports lisibles par ordinateur, programmes informatiques, signaux et structures de donnees
US8105789B2 (en) 2001-11-01 2012-01-31 The University Of British Columbia Diagnosis and treatment of infectious diseases through indel-differentiated proteins
US7572589B2 (en) 2001-11-01 2009-08-11 The University Of British Columbia Diagnosis and treatment of infectious diseases through indel-differentiated proteins
WO2003045991A2 (fr) * 2001-11-23 2003-06-05 Syn.X Pharma, Inc. Marqueurs de biopolymeres precurseurs du facteur c3 du complement permettant de predire la maladie d'alzheimer
WO2003045991A3 (fr) * 2001-11-23 2003-09-04 Syn X Pharma Inc Marqueurs de biopolymeres precurseurs du facteur c3 du complement permettant de predire la maladie d'alzheimer
WO2003070949A1 (fr) * 2002-02-22 2003-08-28 Adprotech Limited Vecteurs d'immunisation pour chats
WO2009056631A3 (fr) * 2007-11-02 2009-08-20 Novartis Ag Molécules et méthodes pour moduler un constituant de complément
WO2009056631A2 (fr) * 2007-11-02 2009-05-07 Novartis Ag Molécules et méthodes pour moduler un constituant de complément
US20160333082A1 (en) * 2014-01-08 2016-11-17 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services ANTIBODY TARGETING CELL SURFACE DEPOSITED COMPLEMENT PROTEIN C3d AND USE THEREOF
US10035848B2 (en) * 2014-01-08 2018-07-31 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Antibody targeting cell surface deposited complement protein C3d and use thereof
US11384139B2 (en) 2014-01-08 2022-07-12 The United States of Americans represented by the Secretary, Department of Health and Human Services Antibody targeting cell surface deposited complement protein C3d and use thereof
US11918624B2 (en) * 2020-06-10 2024-03-05 Kelsius Laboratories LLC Therapeutic composition for use in the treatment of COVID-19 and other cytokine storm associated disorders

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