WO1994029332A1 - Matrices en boucle a axe superenroule - Google Patents

Matrices en boucle a axe superenroule Download PDF

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WO1994029332A1
WO1994029332A1 PCT/US1994/006655 US9406655W WO9429332A1 WO 1994029332 A1 WO1994029332 A1 WO 1994029332A1 US 9406655 W US9406655 W US 9406655W WO 9429332 A1 WO9429332 A1 WO 9429332A1
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molecule
coiled
positions
peptide
group
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PCT/US1994/006655
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English (en)
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Irwin Morris Chaiken
David Gerard Myszka
Thomas James Graddis
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Smithkline Beecham Corporation
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Priority to EP94920179A priority Critical patent/EP0724593A4/fr
Priority to JP7502155A priority patent/JPH08511542A/ja
Publication of WO1994029332A1 publication Critical patent/WO1994029332A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5406—IL-4
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • 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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to polypeptides that are conformationally constricted in an intramolecular, antiparallel, coiled-coil stem loop arrangement.
  • the present invention also relates to mimics and antagonists of biological macromolecules.
  • the present invention further relates to antibodies directed to the polypeptides of the present invention as well as to DNA molecules which encode the polypeptides of the present invention.
  • Zinc Fingers or repetitive zinc-binding domains are found on many transcription factors which play a role in gene regulation.
  • proteins can be thought of as composites of conformational frameworks, or scaffolds, in which a limited number of structural elements are incorporated onto a protein surface.
  • macromolecular structures include ⁇ -sheets and ⁇ - helices. The hypervariable loops of immunoglobulins are "held” in place by ⁇ -sheet molecular scaffolds which are organized into a sandwich-like structure.
  • ⁇ -helical bundle Another common structural motif of proteins is the ⁇ -helical bundle. These bundles appear in different forms, including four- ⁇ -helical bundles, multiple bundles and parallel coil-coils. Helix bundles have been reported previously by Regan et al. (Science. 241:976-978 (1988)) and Hill et al. (Science. 249:543-546 (1990)). The four-helix bundle is a common folding motif found in the structure of functionally diverse proteins such as myohemerythrin, cytochrome c' and TMV (tobacco mosaic virus) coat protein.
  • TMV tobacco mosaic virus
  • It may be formed from four separate molecules, two helix-dimer molecules or one molecule containing a four-helix construct (see, e.g., Hecht et al., Science. 242:884-891 (1990)).
  • the arrangement of helices in naturally occurring four-helix bundles can be parallel, antiparallel or a combination thereof (see, e.g., J. Richardson, Adv. Protein Chem.. 24J67 (1981)).
  • De novo synthesized four-helix bundles tend to form loosely packed helices, rather than compact helical structures as found in nature, and thus have limited stability in solution.
  • coiled-coil a structure adopted by the proteins keratin, myosin, epidermin, fibrinogen and tropomysin.
  • coiled-coil structures are frequently found on DNA binding proteins, where this motif is referred to as a leucine zipper.
  • coiled-coil domains are found in the Jun, Fos (O'Shea et al., Science. 245:646-648 (1989)), C/EBP (Landschultz et al., Science. 240 . : 1759- 1764 (1988)) and GCN4 binding proteins (O'Shea et al., Science. 242:538-542 (1989)).
  • the coiled-coil normally occurs in a parallel orientation in fibrous proteins and DNA binding proteins and is characterized by a repeating seven-residue pattern. According to McLachlan and Stewart (J. Mol.
  • a parallel coiled-coil ⁇ -helix appears to be conformationally stable and thus may bring steric order to small molecular mimics.
  • formation of the coiled-coil is concentration dependent, and thus not a suitable framework for in vivo studies. It is thus an object of this invention to design mimics and antagonists of recognition macromolecules based upon a stable 2-helix, concentration-independent, coiled-coil motif.
  • the present invention relates to a non-naturally occurring two-helix coiled- coil stem loop template molecule which comprises a single polypeptide chain comprising a first ⁇ -helical structure in an aqueous environment, a second ⁇ -helical structure in an aqueous environment and a loop segment which connects the carboxyl terminus of said first ⁇ -helical structure to the amino terminus of said second ⁇ -helical structure wherein said first and second ⁇ -helical structures are arranged longitudinally in an antiparallel orientation such that said first and second helices form an intramolecular, monomeric, superhelical structure.
  • the ⁇ -helical structures may comprise heptad repeats, (abcdefg)n (SEQ ID NO: 10), of equal length, of the formula:
  • AAx forms a polypeptide loop segment of 4 to 15 amino acid residues. It is understood that the same position within the repeating heptad units (e.g., a ⁇ , ai, A3, may or may not be the same and are selected independently of each other.
  • the present invention relates to antibodies directed to the polypeptides of the instant invention and further to a process to elicit antibodies to small peptide molecules.
  • Such process comprises inoculation of a selected mammal with a coiled-coil stem loop polypeptide having the same sequence in the loop region as the small peptide molecule of interest.
  • this invention is a pharmaceutical composition
  • a pharmaceutical composition comprising the coiled-coil stem loop molecule as disclosed above.
  • the present invention relates to an isolated DNA molecule which encodes the coiled-coil stem loop molecules of the instant invention.
  • the present invention relates to a method to produce helical recognition mimics which comprises synthesis of the coiled-coil stem loop molecules of the instant invention.
  • recognition sequences are incorporated into the b, c, e,/and g positions of a heptad repeat.
  • the present invention relates to polypeptides produced by the method just described.
  • the present invention relates to a method to antagonize binding of a ligand to its receptor wherein the ligand or receptor has an ⁇ -helical structure involved in ligand-receptor binding, which comprises binding to said ligand or receptor a coiled-coil stem loop template molecule that mimics the conformational elements of said ⁇ -helical structure.
  • FIGURE IA Cartoon of coiled-coil stem loop template.
  • FIGURE IB Helical wheel cross section for an antiparallel coiled-coil. Letters a-g denote residue positions of an ⁇ -helical heptad repeat.
  • FIGURE lC Helical wheel cross section for a parallel coiled-coil. Letters a-g denote residue positions of an ⁇ -helical heptad repeat.
  • FIGURE 2A Coiled-coil stem loop template sequence (corresponds to SEQ ID NO: 1).
  • FIGURE 2B A papillomavirus E2 protein binding helix (SEQ ID NO:3) and amino acids 335-347 of the E2 protein of human papillomavirus-6 (SEQ ID NO: 2).
  • FIGURE 2C An IL5-like helix (SEQ ID NO:5) and amino acids 108-126 of human IL-5 (SEQ ID NO:4).
  • FIGURE 2D An IL-4 binding helix (SEQ ID NO:7) and amino acids 108- 126 of human IL-4 (SEQ ID NO:6).
  • FIGURE 3 Circular dichroism spectrum of SEQ ID NO: 1 (20 ⁇ M [micromolar]) in the presence (dashed line) and absence (solid line) of the ⁇ -helix inducing solvent, trifluoroethanol (TFE).
  • the buffer was phosphate buffered saline (PBS) and the spectra were taken at 25°C.
  • PBS phosphate buffered saline
  • TFE 1 1 (v/v).
  • FIGURE. 4 Effect of the concentration of the CCSL peptide (SEQ ID NO: 1) (also referred to as LZLS peptide) on ⁇ -helical content.
  • SEQ ID NO: 1 also referred to as LZLS peptide
  • the measured molar ellipticity [ ⁇ ] at 222 nm is reported verses the concentration of CCSL peptide from 0.2 to 500 ⁇ M in PBS.
  • the averaged value for the [ ⁇ J222 shown by the dashed line is -28,000 deg cm ⁇ dmol"l.
  • FIGURE 5A, B and C Gel filtration chromatography elution profiles for the CCSL peptide (solid line) and the (VSSLESK) 6 peptide (SEQ ID NO:9) (dashed line) loaded at a concentration of 30, 3 and 0.3 ⁇ M, respectively.
  • Superdex® ⁇ 75 column was run at 22 °C in 150 mM sodium chloride, 20 mM phosphate, pH 7.4, (PBS) at a flow rate of 0.5 mL/min and monitoring column effluent at an absorbance of 215 nm.
  • FIGURE 5D The elution profiles for the two peptides loaded at a concentration of 3 ⁇ M and run in PBS containing 50% TFE (1:1 ,v/v).
  • FIGURE 6A Selected circular dichroism spectra (CD) of the CCSL peptide taken at the indicated temperature in 150 mM sodium chloride, 20 mM phosphate, pH 7.4 (PBS).
  • FIGURE 6B Thermal melting profiles of the CCSL peptide where, [ ⁇ ] t /[ ⁇ ]5c, represents the molar ellipticity at 222 nm at the indicated temperature to the ellipticity at 5 °C.
  • the inset shows the f ⁇ ]222/f®5208 rano f° r eacn spectra verses temperature.
  • FIGURE 6C CD spectra of the CCSL peptide taken in the indicated molarity of urea in PBS.
  • FIGURE 6D Urea denaturation profile of the CCSL peptide where,
  • [ ⁇ ]M/[ ⁇ ]Q . represents the fraction of ellipticity at 222 nm at the indicated molarity of urea to the ellipticity without urea.
  • the line drawn through the data represents a nonlinear least-squares best fit assuming a two state denaturation process.
  • the inset shows the linear dependence of ⁇ G U on the concentration of urea. The free energy of unfolding in the absence of urea is estimated by extrapolating to zero.
  • FIGURE 6E Selected CD spectra of the CCSL peptide taken at the indicated pH in 150 mM sodium chloride.
  • FIGURE 6F The pH titration curves for the CCSL peptide where,
  • [ ⁇ ] p H9.3 » represents the fraction of ellipticity at 222 nm at the indicated pH to the ellipticity at pH 9.3.
  • the inset shows the [ ⁇ ]222/f®-208 rat ⁇ ° f° r eacn spectra verses pH.
  • FIGURE 7A An IL2-like helix (SEQ ID NOJ 1).
  • the dashed lines indicate hydrophobic coiled-coil interface interactions.
  • the underlined amino acids corresponds to helices A and D of human IL-2.
  • FIGURE 7B Helical wheel cross section of SEQ ID NO: 11.
  • FIGURE 8A Gel filtration chromatography elution profiles for the EL2- like helix (SEQ ID NO: 11). The elution conditions are the same as recited in FIGURE 5A, B and C.
  • FIGURE 8B Circular dichroism spectra (CD) of SEQ ID NOJ 1 taken in 150 mM sodium chloride, 20 mM phosphate, pH 7.4 (PBS).
  • FIGURE 9A An IL5Jike binding helix#2 (SEQ ID NO: 12).
  • the dashed lines indicate hydrophobic coiled-coil interface interactions.
  • the underlined amino acids corresponds to helices A and D of human IL-5.
  • FIGURE 9B Helical wheel cross section of SEQ ID NO: 12.
  • FIGURE 10A Gel filtration chromatography elution profiles for the IL5- like helix#2 (SEQ ID NO: 12). The elution conditions are the same as recited in FIGURES 5A, B and C.
  • FIGURE 10B Circular dichroism spectra (CD) of SEQ ID NO: 12 taken in
  • the present invention comprises polypeptides that are conformationally constricted and are thus useful as carriers or scaffolds to present amino acid sequences of interest in a particular three dimensional conformation.
  • the molecules of the invention assume the following conformation in an aqueous environment: an antiparallel, coiled-coil ⁇ -helical stem and an internal loop, collectively and hereinafter referred to as a coiled-coil stem loop (CCSL) or a coiled-coil recognition molecule.
  • the stem comprises an intramolecular heterodimer of two ⁇ -helical sequences in an antiparallel orientation such that said helices are capable of forming an intramolecular, and monomeric, superhelical structure, for example, like an antiparallel leucine zipper.
  • the internal loop or loop segment connects the carboxy terminus of a first ⁇ -helical structure to the amino terminus of a second ⁇ -helical structure.
  • a coiled-coil stem loop is depicted in Figure 1.
  • a coiled-coil arrangement is thought to be conformationally ordered and thus useful for bringing steric constraint to small molecular mimics.
  • the stability of such intermolecular coiled-coils is, however, dependent on peptide concentration. That is, two separate ⁇ -helices are in equilibrium with a dimeric or superhelical structure, and thus dimerization can occur only in high concentration of the individual ⁇ -helices, i.e., the monomers. At low monomeric concentration, the dimeric structure dissociates.
  • a significant advantage of the instant invention is that it eliminates this concentration dependence.
  • the molecules of the present invention comprise intramolecular antiparallel coiled-coils, which are more stable than intermolecular coiled-coils because the latter are concentration dependent.
  • Another advantage of the present invention is that the coiled-coil stem loop structure forms spontaneously in an aqueous environment to form a monomeric, intramolecular superhelical structure. Confirmation of monomeric (verses dimeric) structure can be readily determined by techniques known in the art, e.g., gel filtration chromatography, equilibrium sedimentation centrifugation, NMR, etc.
  • Recognition elements which can be mimicked in the coiled-coil stem loop include ⁇ -helical surfaces, continuous loop residues and chemical groups brought together in space by specific placement due to the coiled-coil and loop domains.
  • the coiled-coil stem loop of the present invention provides a template for molecular recognition elements.
  • the CCSL can be adapted to form specific recognition molecules (i.e., helical recognition mimics) by presenting helical recognition sequences from naturally occurring proteins.
  • helices for which mimicry can be tested with the coiled coil stem loop include, but are not limited to: papillomavirus E2 protein, a DNA-binding transcriptional regulator; interleukin 5, a cytokine which stimulates eosinophil maturation, proliferation and activation; and interleukin 4, a cytokine which activates resting B lymphocytes and is involved in immunoglobulin class switching. It is appreciated to one of skill in the art that the helical surfaces from naturally occurring proteins can be grafted onto either one or both of the coiled-coil helical sequences to form monovalent or bivalent mimics.
  • the helical recognition mimics produced by the present invention can be used to antagonize the binding site of a ligand (e.g., cell surface receptors), provided the binding site interacts with an ⁇ -helical structure which has been mimicked.
  • the present invention thus relates to a method to produce helical recognition mimics which comprises synthesis of the coiled-coil stem loop molecules of the present invention wherein the recognition sequences are preferentially (but not always) incorporated into the b, c, e,/and g positions and/or the b c', e',f and g' positions of a heptad repeat.
  • the present invention relates to polypeptides produced by said method or process.
  • the present invention relates to a method to antagonize binding of a ligand to its receptor wherein the ligand or receptor has an ⁇ -helical structure involved in ligand- receptor binding, which comprises binding to said ligand or receptor a coiled-coil stem loop template molecule that mimics the conformational elements of said ⁇ - helical structure.
  • the coiled coil stem loop can also be adapted by presenting recognition sequences into the loop segment.
  • the RGD (Arginine-Glycine- Aspartic Acid) sequence known to bind the fibrinogen receptor i.e., a ligand
  • CCSL coiled-coil stem loop
  • This molecule has the ability to bind fibrinogen receptor with an affinity at least as great as that of RGD containing linear peptides.
  • Another approach is to mimic the binding site of a receptor.
  • residues Gin 40 to Phe 43 and/or residues Val 86 to Gin 89 of the CD4 receptor protein could be placed in the loop region of the (CCSL) stem loop to test inhibition of ligand binding (e.g., HIV envelope protein) to said receptor.
  • sequence composition and length of the loop can be varied to form a diverse set of molecules which may bind to macromolecular receptors with various affinities due to differences in composition and conformation.
  • the ⁇ -helical structures of the present invention comprise a plurality of heptad repeats, of which both ⁇ -helical structures are of equal length. According to McLachlan and Stewart (J. Mol. Biol.. 98:293 (1975)), the positions within the heptad repeat are termed a, b, c, d, e, and g.
  • the ⁇ -helical structures can be represented by the formula:
  • Each heptad repeat is a right handed ⁇ -helix.
  • the geometry of each heptad in a coiled-coil conformation is a repeating unit where every seventh residue of an amino acid sequence is at a structurally equivalent position. For example, the a position always appears on the same hydrophilic interior surface of the ⁇ -helix.
  • the first and second ⁇ -helical structures interact longitudinally in an antiparallel orientation such that they are capable of forming an intramolecular superhelical structure under a range of conditions (see, e.g., the Examples section). Such structure is akin to strands of rope which form a central superhelical axis. Thus for a superhelical structure, each of the helices is slightly distorted from the central axis and the overall superhelix has a left handed twist.
  • the positions a, b, c, d, e,/and g within the heptad repeat can be categorized into three groups based upon helical geometry.
  • the profile comprises a hydrophilic exterior, a hydrophobic interior and a border of polar residues, preferably with charged side chains (i.e., R groups) to form interhelical salt bridging residues.
  • the positions of the first ⁇ -helical structure are designated a, b, c, d, e,fsmd g and the positions of the second, or antiparallel, ⁇ -helical structurere are designed with " ' ", e.g., a', b c', d e',f and g'.
  • coiled-coil stem loop molecules of the invention can be depicted as follows:
  • Positions a and d are typically hydrophobic residues, forming the hydrophobic interface of the coiled-coil and are believed to stabilize antiparallel helix dimerization in the present invention.
  • Such hydrophobic residues comprise alanine, valine, leucine, isoleucine, methionine, phenylalanine, and tryptophan as well as modifications to naturally occurring amino acids which are known in the art.
  • the hydrophobic residues are selected from the group consisting of leucine, isoleucine, methionine, valine and alanine with the proviso that at least one residue is leucine.
  • positions a and d' are hydrophobic residues independently selected from the group consisting of alanine, isoleucine, methionine and valine, then positions d and a' are leucine and similarly, when positions d and a' are hydrophobic residues independently selected from the group consisting of alanine, isoleucine, methionine and valine, then positions a and d' are leucine with the proviso that the a or d position of the first heptad repeat, which ever occurs first, and the last d' o ⁇ a' position of the last heptad repeat, whichever occurs last, is any amino acid residue.
  • d and a' are the same amino acid residue. It is still more preferable that when a or d' (or d and a 1 ) is leucine, then the other position (i.e., d or a', or a or d' , respectively) is alanine, isoleucine or valine, more prefereably alanine or valine and most preferably valine.
  • Positions e and g and e' and g' are independently selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine with the proviso that when one of two consecutive heptad positions (e.g., e., g., etc.) is so selected from the group of hydrophobic-like residues consisting of valine, leucine, isoleucine, methionine, phenylalanine and tryptophan then the other structurally equivalent heptad position (e.g., ei, g2, e'2, etc.) may not be selected from the same group of hydrophobic-like residues, and with the additional proviso that each e, and the anti
  • e and g and e' and g' are selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, serine, threonine and tyrosine. More preferably yet, e and g are charged residues 50% or greater of the time which facilitate interhelical ionic or electrostatic interactions, for example, residues which form interhelical salt bridges.
  • Such residues may comprise aspartic acid, glutamic acid, lysine, arginine, histidine and other residues which can be modified to have an overall net charge (e.g., tyrosine residues can be modified at the hydroxyl group to form carboxylic acids, amines, etc.).
  • the overall net charge of one helix i.e., all e and g residues of the first ⁇ -helical structure
  • the second ⁇ -helical structure i.e., all e' and g' residues
  • the residues at one position e.g., ei
  • a corresponding position of the coiled-coil stem loop e.g., e' ⁇ (or g ⁇ and g' ⁇ )
  • positions e, g, e' and g' comprise arginine, aspartic acid, lysine and glutamic acid, such that a positively charged residue (e.g., lysine) on one ⁇ -helix will interact with a negatively charged residue (e.g., glutamic acid) on the second (or antiparallel) ⁇ -helical structure and vice versa.
  • a positively charged residue e.g., lysine
  • glutamic acid e.g., glutamic acid
  • Positions b, c,f, b', c' and/ are less important for intramolecular dimerization of the ⁇ -helices than the other positions. They are located on the exterior surface on the coiled-coil and are exposed to the aqueous environment. Residues at these positions may be used to stabilize an ⁇ -helix, or be used as recognition surfaces to form ⁇ -helical mimics. The residues at positions b, c,f, b', c' and/ can be virtually any amino acid with the proviso that there cannot be more than one glycine or proline residue per heptad repeat.
  • b, c, f, b c' and/ are comprised of 25% or less of "strongly hydrophobic" residues (i.e., leucine, isoleucine, glycine, methionine, phenylalanine, tryptophan and valine). More preferably, b, c,f, b', c' and/ are independently selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, lysine, serine, threonine and tyrosine.
  • n is 2 - 15 or fractions (i.e., sevenths, for example 1/7, 2/7, 3/7, etc.) thereof in-between the integers 2 - 15.
  • n is 3 to 10 repeating units or fractions thereof. More preferably, n is 3 to 7 repeating units and fractions thereof (e.g., n can be 3 4/7). It is understood that the same position within the repeating heptad units (e.g., a ⁇ , ai, ai, ... may or may not be the same and is selected independently from each other.
  • the coiled-coil motif is highly tolerant of amino acid substitutions so long as the ⁇ -helix is not substantially destabilized.
  • alpha helix formers and breakers see Chou and Fasman (Biochem.. 13:21 1-222 (1974)).
  • the present invention comprises a loop segment (AAx) which connects the two ⁇ -helical structures.
  • the first and last residues (in a N-terminus to C-terminus orientation) of the non-helical region can be any amino acid.
  • one or both residues are helix-breaking or helix destabilizing residues (see, Chou and Fasman, supra), for example, glycine and proline.
  • the non-helical region or loop segment comprises from about 4 to 15 amino acid residues.
  • the non-helical region comprises from about 5 to 10 amino acid residues.
  • ⁇ -helices of this invention In addition to these general considerations for the formation of ⁇ -helices of this invention, one can further promote helix formation and stability in the molecules of this invention by including negatively charged groups at the N- terminal end and positively charged groups at the C-terminal end of the coiled-coil stem loop molecule to stabilize the dipole (see, e.g. Fairman et al., Proteins: Structure. Function and Genetics. 5.J-7 (1989)). Alternatively, one can block the charged amino and carboxyl groups of the first and last residues (i.e., NH3 + and COO") with, for example, an acetyl group and an amide group, respectively.
  • NH3 + and COO charged amino and carboxyl groups of the first and last residues
  • Another method to further stabilize the overall coiled-coil structure is to design coiled-coil stem loop molecules with residues at the amino terminus and carboxy terminus that are capable of forming covalent bonds with each other. For example, disulfide bonds or homodetic peptide cyclization.
  • the coiled-coil stem loops of this invention comprise non-naturally occurring two-helix sequences which may optionally mimic residues of naturally occurring polypeptides.
  • the CCSL template molecules may be synthesized by a variety of means, for example, by recombinant DNA technology or by chemical synthesis. Methods of well-known peptide synthesis are generally set forth by Ali et al., I Med. Chem.. 29:984 (1986) and J. Med. Chem.. 2Q:2291 (1987) and are incorporated by reference herein.
  • the peptides are prepared by the solid phase technique of Merrifield (J Am Chem Soc. 85:2149 (1964)).
  • a combination of solid phase and solution synthesis may be used, as in a convergent synthesis in which di-, tri-, terra-, or penta-peptide fragments may be prepared by solid phase synthesis and either coupled or further modified by solution synthesis.
  • the side chain functional groups e.g., -NH2, -COOH, -OH, -SH
  • the side chain functional groups are protected during the coupling reactions.
  • the ⁇ -amino group is temporarily protected as t-Butoxycarbonyl (BOC) but other acid or base labile protecting groups can be used, e.g., fluorenylmethoxycarbonyl (FMOC).
  • FMOC fluorenylmethoxycarbonyl
  • the amino side chain group of lysine is protected as benzyloxycarbonyl or p- chlorobenzyloxycarbonyl (Z or Cl-Z). Para-methylbenzyl (p-MBz) or acetomidomethyl protection is used for cysteines.
  • Benzyl ethers Hydroxy groups are protected as benzyl ethers and carboxyl groups are protected as benzyl (Bz) or cyclohexyl esters.
  • the peptides of the present invention can be synthesized either from the C- terminus or the N-terminus. Preferably it is from the C-terminus.
  • the alpha-carboxyl group Prior to coupling the alpha-carboxyl group (of a suitable protected amino acid) is activated.
  • One skilled in the art can activate the protected group in a number of ways.
  • N,N' dicyclohexylcarbodiimide DCC
  • pNp p-nitrophenyl esters
  • HOBt hydroxybenzotriazole ester
  • Osu N-hydroxy succinimidyl ester
  • Solution synthesis of peptides is accomplished using conventional methods to form amide bonds.
  • a protected Boc-amino acid which has a free carboxyl group is coupled to a protected amino acid which has a free amino group using a suitable carbodiimide coupling agent, such as N, N' dicyclohexyl carbodiimide (DCC), optionally in the presence of a catalyst such as 1 -hydroxybenzotriazole (HOBt) and dimethylamino pyridine (DMAP).
  • a suitable carbodiimide coupling agent such as N, N' dicyclohexyl carbodiimide (DCC)
  • a catalyst such as 1 -hydroxybenzotriazole (HOBt) and dimethylamino pyridine (DMAP).
  • the coupling reactions are preferably carried out at low temperature (e.g., -20°C) in such solvents as dichloromethane (DCM), dimethyl formamide (DMF), N-methyl pyrrolidone (NMP), tetrahydrofuran (THF) acetonitrile (ACN) or dioxane.
  • DCM dichloromethane
  • DMF dimethyl formamide
  • NMP N-methyl pyrrolidone
  • THF tetrahydrofuran
  • ACN acetonitrile
  • Solid phase methods the peptide is built up sequentially starting from the carboxy terminus and working towards the amino terminus of the peptide.
  • Solid phase synthesis begins by covalently attaching the C terminus of a protected amino acid to a suitable resin, such as methyl benzhydrylamine (mBHA).
  • mBHA methyl benzhydrylamine
  • the first amino acid residue is normally attached to an insoluble polymer.
  • two commonly used polymers are polystyrene (1% cross-linked with divinyl benzene) and 1 % cross-linked polyamides.
  • polymers are functionalized to contain a reactive group, e.g., -OH, -NH2 and - CH2CI to link the first amino acid of the targeted peptide (i.e., carboxy terminus).
  • a reactive group e.g., -OH, -NH2 and - CH2CI to link the first amino acid of the targeted peptide (i.e., carboxy terminus).
  • the choice of the linkage between the first amino acid and the polymer is dictated by the carboxy terminus of the peptide. For example, peptides having a carboxyl group at the C-terminus would be linked by an ester linkage and peptides with a carboxamide ending would have an amide linkage.
  • the protected amino group is hydrolyzed by mild acid treatment, and the free carboxyl of the next (protected) amino acid is coupled to this amino group. This process is carried out sequentially, without isolation of the intermediate, until the peptide of interest has been formed. The completed peptide may then be deblocked and/or cleaved from the resin in any order.
  • Preferred solvents for the coupling reactions include, but are not limited to, dichloromethane (DCM), dimethyl formamide (DMF) and N-methyl pyrrolidone (NMP).
  • DCM dichloromethane
  • DMF dimethyl formamide
  • NMP N-methyl pyrrolidone
  • the peptide is deprotected and cleaved from the resin using standard techniques (e.g., using hydrofluoric acid (HF)).
  • the preferred method for cleaving a peptide from the support resin is to treat the resin supported peptide with anhydrous HF in the presence of a suitable cation scavenger, such as anisole or dimethoxy benzene.
  • a suitable cation scavenger such as anisole or dimethoxy benzene.
  • the synthetic peptide may be cyclized using methods well known in the art. For example, there are numerous protocols for forming intramolecular bonds between cysteine residues. In performing these reactions, water, methanol, acetic acid, DMF or a suitable mixture of these solvents can be used.
  • Formation of the disulfide bond may be accomplished by several known methods. If the sulfur-containing amino acids of the linear peptide are protected differently, in such a manner as to allow formation of a mono mercaptan, cyclization may be effected by base catalyzed nucleophilic displacement of the protecting group of the sulfur-containing amino acid. Groups which are especially useful as displaceable protecting groups are thioalkyl or thioaryl groups. Exemplary of this method is the protection of one sulfur-containing amino acid by the thioethyl group, and protection of the second by a substituted benzyl group.
  • any oxidizing agent known in the art to be capable of converting a dimercaptan to a disulfide may be used.
  • exemplary of such agents are an alkali metal ferricyanide, (e.g., potassium or sodium ferricyanide), oxygen gas, diiodomethane or iodine.
  • the reaction is conducted in a suitable inert solvent, such as aqueous methanol or water, at temperatures from about 0 to 40°C, under high dilution.
  • the pH is usually maintained at about 7 to 8.
  • Cyclization may be performed upon the peptide while it is still attached to the support resin or while other functional groups are still protected, but it is preferably performed on the deprotected free peptide.
  • Acid addition salts of the peptides are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hybrobromic, sulfuric, phosphoric, acetic, maleic, succinic, or methanesulfonic.
  • the acetate salt form is especially useful. Certain of the compounds form inner salts or zwitterions which may be acceptable.
  • Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide containing the appropriate cation. Cations such as Na + , K + , Ca ⁇ + and NH4 + are examples of cations present in pharmaceutically acceptable salts.
  • the peptides can then be purified by a number of techniques. Preferred embodiments include reverse phase HPLC, counter current distribution (CCD) and crystallization. More preferably, HPLC is used. The purified products can then be analyzed for purity using HPLC, amino acid analysis and fast atom bombardment mass spectrometry (FAB-MS).
  • coding sequences for the polypeptides of the present invention can be recombinant DNA molecules which are introduced into expression vectors or phage (collectively referred to as "vectors") by operatively linking the DNA to the necessary expression control regions (e.g. regulatory regions) required for gene expression.
  • the vectors can be introduced into the appropriate host cells such as prokaryotic (e.g., bacterial), or eukaryotic (e.g., yeast or mammalian) cells by methods well known in the art (see, e.g., "Current Protocol in Molecular Biology", Ausubel et al. (eds.), Greene Publishing Assoc. and John Wiley Interscience, New York, 1989 and 1992).
  • cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice.
  • Examples of recombinant DNA vectors for cloning and host cells which they can transform include, but are not limited to, the bacteriophage ⁇ (E. coli). pBR322 (E coli). pACYC177 (E. coli . pKT230 (gram-negative bacteria), pGVl 106 (gram- negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. coli gram- negative bacteria), pHV14 (E. coli and Bacillus subtilis " ). pBD9 (Bacillus). pIJ61 (Streptomyces). pUC6 (Streptomvces). YIp5 (Saccharomvces). pAcYMl
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence.
  • the subunit antigens of the present invention can be expressed using, for example, the E. coli tac promoter or the protein A gene (spa) promoter and signal sequence. Leader sequences can be removed by the bacterial host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397.
  • regulatory sequences which allow for regulation of the expression of the protein sequences relative to the growth of the host cell.
  • Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence).
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • Modification of the sequences encoding the particular antigen of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • a vector such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., T. Maniatis et al., supra; DNA Cloning. Vols. I and II, supra; Nucleic Acid Hybridization, supra.
  • prokaryotic expression vectors are known in the art. See, e.g., U.S. Patent Nos. 4,578,355; 4,440,859; 4,436,815; 4,431 ,740; 4,431 ,739; 4,428,941 ; 4,425,437; 4,418,149; 4,41 1,994; 4,366,246; 4,342,832; see also U.K. Patent Applications GB 2,121,054; GB 2,008,123; GB 2,007,675; and European Patent Application 103,395. Yeast expression vectors are also known in the art. See, e.g., U.S. Patent Nos.
  • the proteins of the present invention are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The protein is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction. The selection of the appropriate growth conditions and recovery methods are within the skill of the art. Confirmation of ⁇ -helical formation can be accomplished by known techniques in the art, for example, by circular dichroism (CD) monitoring, fluorescence spectra monitoring, NMR, thermal denaturation and x-ray diffraction analysis.
  • CD circular dichroism
  • the recognition peptide sequences can be inserted into it and the scaffolding or conformational framework is used to limit flexibility.
  • the sequence in or adjacent to the recognition peptide could be mutated and sufficiently high-affinity species selected by an affinity capture method.
  • filamentous phage surface proteins see, e.g., Scott et al., Science. 249:386 (1990) and Devlin et al., Science. 249:404 (1990) which are hereby incorporated by reference
  • the invention can thus be used to identify amino acid residues which bind to macromolecules, including receptors.
  • compositions can antagonize the interactions of the biological macromolecules and thereby antagonize their biological activities.
  • Coiled-coil stem loops which function as antagonists can be used directly as therapeutic agents.
  • the CCSL may be an inhibitor (or modulator) of proteins, e.g., anti-inflammatory agents which neutralize cell adhesion molecules, cytokines, etc.
  • the CCSL can also be modified by selective changes (e.g., chemically or by site directed mutagenesis, i.e., insertion, deletion, substitution or rearrangements) to modulate macromolecular affinity and specificity.
  • a coiled-coil stem loop with antagonist activity also provides a chemical lead for SAR (structure activity relationships) and xenobiotic design.
  • the structure of an active coiled-coil stem loop can be predicted or determined experimentally.
  • the structure defines the organization of binding elements. These elements can be reconstituted into small organic molecules by synthetic chemistry approaches.
  • a coiled-coil stem loop which mimics the binding site of a biological macromolecule can be used as an immunogen to produce neutralizing antibodies for the activity of that molecule.
  • the coiled coil stem loop can be used to stimulate or elicit an immunogenic response (e.g., as a vaccine), by presenting an antigenic site in a configuration which stimulates production of protective antibodies.
  • a small peptide sequence i.e., less than 50 residues, preferably less than 15 residues, more preferably less than 10 residues
  • proteins encompassed by the present invention or fragments thereof comprising at least one epitope can be used to elicit or produce antibodies, both polyclonal and monoclonal.
  • a selected mammal e.g., mouse, rabbit, rat, goat, horse, primate, human, etc., preferably mouse, rat or rabbit
  • Serum from the immunized animal is collected and treated according to known procedures.
  • serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography or other known procedures.
  • Monoclonal antibodies to the proteins of the present invention, and to the fragments thereof, can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by using hybridoma technology is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., "Hybridoma Techniques” (1980); Hammerling et al., “Monoclonal Antibodies and T-cell Hybridomas" (1981); Kennett et al., "Monoclonal Antibodies” (1980); see also U.S.
  • Panels of monoclonal antibodies produced against the antigen of interest, or fragment thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc.
  • Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens which they are directed against.
  • genes encoding the monoclonals of interest may be isolated from the hybridomas by PCR techniques known in the art and cloned and expressed in the appropriate vectors.
  • the antibodies of this invention, whether polyclonal or monoclonal have additional utility in that they may be employed reagents in immunoassays, RIA,
  • ELISA ELISA, and the like.
  • “monoclonal antibody” is understood to include antibodies derived from one species (e.g., murine, rabbit, goat, rat, human, etc.) as well as antibodies derived from two (or perhaps more) species (e.g., chimeric and humanized antibodies). Chimeric antibodies, in which non-human variable regions are joined or fused to human constant regions (see, e.g. Liu et al., Proc. Natl Acad. Sci. USA. 84:3439 (1987)), may also be used in assays or therapeutically. Preferably, a therapeutic monoclonal antibody would be "humanized” as described in Jones et al., Nature.
  • compositions of the polypeptides prepared as hereinbefore described and other peptide or polypeptide derivatives may be formulated as solutions of lyophilized powders for parenteral administration.
  • Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use.
  • the liquid formulation is generally a buffered, isotonic, aqueous solution.
  • suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution.
  • Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer.
  • excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
  • excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
  • the compounds described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional proteins and art-known lyophilization and reconstitution techniques can be employed.
  • these peptides may be encapsulated, tableted or prepared in a emulsion or syrup for oral administration.
  • Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition.
  • Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
  • Liquid carriers include syrup, peanut oil, olive oil, saline and water.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the amount of solid carrier varies but, preferably, will be between about 20mg to about lg per dosage unit.
  • the pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms.
  • a liquid carrier When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension.
  • Such a liquid formulation may be administered directly or filled into a soft gelatin capsule.
  • the peptides of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
  • the physician will determine the dosage of the present therapeutic agents which will be most suitable and it will vary with the form of administration and the particular compound chosen, and furthermore, it will vary with the particular patient under patient under treatment. He will generally wish to initiate treatment with small dosages substantially less than the optimum dose of the compound and increase the dosage by small increments until the optimum effect under the circumstances is reached. It will generally be found that when the composition is administered orally, larger quantities of the active agent will be required to produce the same effect as a smaller quantity given parenterally.
  • the compounds are useful in the same manner as other serotonergic agents and the dosage level is of the same order of magnitude as is generally employed with these other therapeutic agents.
  • the therapeutic dosage will generally be from 1 to 10 milligrams per day and higher although it may be administered in several different dosage units. Tablets containing from 0.5 to 10 mg. of active agent are particularly useful.
  • the pharmaceutical composition of the invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the disease and its complications.
  • compositions containing the present compounds or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance.
  • Single or multiple administrations of the pharmaceutical compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical composition of the invention should provide a quantity of the compounds of the invention sufficient to effectively treat the patient.
  • Solid phase peptide synthesis resins were obtained from Applied Biosystems, Inc. (Foster City, CA). Protected amino acids were purchased from Bachem (Philadelphia, PA), Keystone Bio-Tec (Philadelphia, PA), and Peninsula Laboratories (Belmont, CA). Diisopropylcarbodiimide was obtained from Milligen/Biosearch (Burlington, MA). Diisopropyethylamine, hydroxybenzotriazole, l-methyl-2-pyrrolidinene, anisole, dimethyl sulfide, m- cresol, ethanedithiol, piperidine, pyridine, and buffer salts were obtained from Aldrich (Milwaukee, WI).
  • CCSL peptide SEQ ID NO: 1
  • tBoc t-butoxycarbonyl
  • MBHA p-methylbenzhyldrylamine
  • the blocking groups were removed by HF cleavage in die presence of anhydrous anisole.
  • the peptides GRGDMP (SEQ ID NO:8), (VSSLESK) ⁇ 1 (SEQ ID NO: 9), IL2-like Helix (SEQ ID NOJ 1 ) and IL5-like Helix#2 (SEQ ID NO: 12) were synthesized by similar techniques and were purified by preparative HPLC using delta-pack Cl 8 from Millipore Co. Peptide purity was verified to be greater than 90% by amino acid composition analysis (Beckman 6300
  • TFE 50% trifluoroethanol
  • Circular Dichroism spectroscopy Circular Dichroism spectroscopy. Circular Dichroism (CD) spectra were measured with a Jasco J-500C spectropolarimeter attached to a Lauda (model RMS) water bath used to control the cell temperature. The spectropolarimeter was interfaced to a Macintosh computer for data collection and manipulation. All spectra were measured at 270 to 190 nm using a 0.1 cm cell, 1-nm bandwidth and a 1-s time constant. Four scans were averaged for each spectrum and then corrected for solvent contributions. Measured rotations were converted to molar mean residue ellipticity [ ⁇ ] (degrees cm ⁇ dmol" ⁇ ) (Schmid, F.X., in Protein Structure a Practical Approach. Creighton, T.E. (ed), IRL Press, Oxford, p. 283 (1989)). All spectra were recorded with a cell temperature of 25°C unless otherwise stated. All concentrations of stock peptide solutions were determined by amino acid
  • CD spectra were recorded for 20 ⁇ M solutions of the various peptides in PBS alone and in a 50% TFE/PBS buffer, generated by diluting the PBS with TFE (1 : 1 , v/v).
  • CD spectra were measured for the peptide at concentrations of 0.2, 1, 10, 100, and 500 ⁇ M in PBS.
  • CD spectra were measured on a 20 ⁇ M concentration of the peptide in PBS in a temperature range of 5°C to 80°C. The sample cell temperature was raised at 5° C increments and incubated for 15 minutes at each temperature before taking the CD spectrum.
  • the urea denaturation studies were carried out by preparing CCSL peptide
  • SEQ ID NO: 1 stock solutions at a concentration of 20 ⁇ M in PBS alone, and with 6 M urea in PBS. Different ratios of the PBS and 6 M urea solutions were mixed to give the appropriate final urea concentration for the CD measurements. Mixed samples were incubated for 1 hour before taking the CD spectra.
  • the CCSL peptide (SEQ ID NO: 1) is highly soluble in water and PBS which is the first indication that it is assuming a folded conformation with prevents the exposure of the hydrophobic leucine and valine residues to solvent.
  • PBS the CCSL peptide (SEQ ID NO: 1 ) exhibits a circular dichroism spectrum which is typical of an ⁇ -helix with high molar ellipticity minima values at 222 (n- ⁇ *) and 208 nm ( ⁇ - ⁇ *), indicating high ⁇ -helical content (Figure 3).
  • TFE trifluoroethanol
  • TFE to increase the helical content of the CCSL peptide (SEQ ID NO: 1 ) indicates that under these conditions (i.e., pH 7.4, 25°C) the peptide is near its maximum ⁇ -helical potential.
  • CD spectra are obtained at varying concentrations of the CCSL peptide (SEQ ID NO: 1) to determine if the ⁇ -helical content of the peptide is dependent on concentration.
  • Peptides with ⁇ -helical structures that are dependent on dimerization or oligomerization show a loss of ⁇ -helical content as the peptide concentration is decreased (Zhou et al., J. Biol. Chem.. 262:2664-2670 (1992); Kaumaya et al., Biochem..22:12-23 (1990)).
  • the CCSL peptide (SEQ ID NO: 1) shows a similar mean residue ellipticity minima value at 222 nm and no significant difference in the [ ⁇ ]222/[®-208 rau0 over a 2,500 fold peptide concentration range (500 ⁇ M) ( Figure 4).
  • Figure 4 show the ⁇ -helical content of the CCSL peptide (SEQ ID NO: 1 ) is independent of concentration and, therefore, suggest that the ⁇ -helices are stabilized through the formation of an intramolecular coiled-coil.
  • the elution size corresponds to an apparent molecular weight of 6,500 daltons which is close to the calculated monomeric molecular weight of 5,789 daltons.
  • the slightly larger size determined by gel filtration is consistent with results seen for other coiled-coil peptides.
  • ⁇ - helical coiled-coils are rod-like in shape and elute more rapidly from a gel filtration column than a globular molecule of identical molecular weight (Hodges et al., I Biol. Chem.. 256:1214-1224 (1981)).
  • Both the CCSL and (VSSLESK) 6 (SEQ ID NO: 9) peptides (SEQ ID NO: 1) elute as a single peak from the gel filtration column with an elution buffer containing 50% of the helix inducing solvent TFE (Figure 5D).
  • the (VSSLESK) ⁇ (SEQ ID NO: 9) peptide elutes at the position seen for the monomeric peptide eluting in PBS ( Figure 5D). There is no indication of a dimeric species present suggesting that the TFE is stabilizing the single stranded ⁇ -helical monomer.
  • TFE the CCSL peptide (SEQ ID NO: 1) elutes at an apparent molecular weight of 11,000 daltons which is much larger than the apparent molecular weight determined in PBS (6,500 daltons).
  • the CCSL peptide (SEQ ID NO: 1) dissolved in 20 mM phosphate, pH 7.4, shows a 2 and 5% increase in ⁇ -helical content upon the addition of 150 mM and 1M sodium chloride, respectively.
  • This increased helical content with increasing ionic strength is consistent with other coiled-coil peptides and can simply be explained by the increased strength of the hydrophobic interactions in a more polar medium (Lau et al., supra); Mo et al., Biopolv ers.20:921-927 (1990)).
  • the [ ⁇ ]222/-®]208 rauo * s sensitive to whether the ⁇ -helix is single- stranded or in the form of a coiled-coil.
  • the winding of the ⁇ -helices around each other or pitch of the coiled-coil is dependent on the number of amino acids per turn of the ⁇ -helix (Phillips, G. N., Proteins: Structure. Function and Genetics. 14:425-429 (1992) and varies widely for different proteins (Seo and Cohen, Proteins: Structure. Function and Genetics. 15:223-234 (1993)).
  • Figure 6A shows the CD spectra changes recorded for the CCSL peptide (SEQ ID NO: 1) in PBS, over the temperature range of 5°C to 80°C.
  • the peptide shows a decrease in ⁇ -helical signal upon thermal denaturation reflecting the transition from a native folded state to a more disordered one.
  • the temperature melting profile shows a gentle decrease in the amount of ⁇ -helix up to 60°C and then rapid decrease above 65°C ( Figure 6B). Above 65°C the peptide precipitates out of solution which suggests that the molecule unfolds. It is thought that exposure of the hydrophobic leucine and valine residues leads to aggregation. Upon cooling to room temperature this precipitate remains.
  • Tm temperature required at which 50% of the peptide is in its unfolded form
  • Tm temperature required at which 50% of the peptide is in its unfolded form
  • This high Tm indicates that the ⁇ -helical structure of the peptide is remarkably stable.
  • increasing temperature causes a decrease in both the [ ⁇ ]222/[®]208 rat ⁇ ° anc ⁇ - n ⁇ -helical content which is consistent with the model that supercoiling and ⁇ -helical content are strongly coupled.
  • Urea is commonly used to disrupt the structure of ⁇ -helical coiled-coil peptides.
  • the urea denaturation profile shows this gradual decrease in helical content with increasing urea concentration ( Figure 6D).
  • a concentration of 2.8 M urea is required to reduce the helical content to 50%.
  • the urea denaturation curve was analyzed by assuming a two-state folding unfolding transition (Pace, C.N., Meth. Enz.. 121:266-280 (1986)), with an understanding that a more complex unfolding equilibrium may exist (Bracken et al., Biopolvmers. 22:1223-1237 (1988)).
  • the inset of Figure 6D shows the free energy associated with the unfolding of the CCSL peptide (SEQ ID NO: 1) as a function of urea concentration.
  • the value of the Gibbs free energy in the absence of denaturant ( ⁇ G U ) is estimated by linear extrapolation to zero urea.
  • the folded structure of the CCSL peptide (SEQ ID NO: 1) is stabilized, at 25°C, by a ⁇ G U of about 2.2 kcal mol ' 1 .
  • the CCSL peptide (SEQ ID NO: 1) was synthesized with Acm-blocked amino- and carboxy-terminal cysteine residues. These blocking groups were removed and a disulfide bond was formed between the cysteine residues by utilizing oxidation with iodine.
  • the disulfide form of the CCSL peptide (SEQ ID NO: 1 ) eluted from reverse-phase HPLC much faster that the Acm-blocked peptide.
  • the disulfide form of the CCSL peptide (SEQ ID NO: 1) eluted form the size exclusion column as a single peak at the same position observed for the Acm-blocked peptide, suggesting it was a folded monomer.
  • the observed mass was 16 Da higher than the expected mass of 5645 Da. This increase in the mass was probably due to oxidation of the peptide's single methionine to a sulfoxide as the Acm blocking groups were removed with iodine.
  • the CD spectrum of the disulfide form of the CCSL peptide (SEQ ID NO: 1) exhibited a small increase in ⁇ -helical content versus the reduced peptide, with no change in the [ ⁇ ]222/f®l208 rau0 -
  • the oxidized and reduced forms of the peptide showed similar thermal denaturation profiles form 4 to 60°C. However, at higher temperatures, the oxidized form showed an increased stability, which was likely the result of the formation of a disulfide bond between the otherwise open ends of the coiled-coil.
  • the binding of the CCSL peptide (SEQ ID NO: 1) and the GRGDMP (SEQ ID NO: 8) peptide to purified GPIIbllla was determined by the solid-phase receptor binding assay described by (Smith et al., J. Biol. Chem. 265:12267-12271 (1990)). In competition binding experiments with fibrinogen the GRGDMP and CCSL peptides (SEQ ID NO: 1) gave apparent dissociation constants (Rp) of 150 nM and 180 nM, respectively.
  • Rp dissociation constants
  • the high affinity of the CCSL peptide (SEQ ID NO: 1) for the GPIIbllla receptor suggests the RGD sequence present in the loop is readily available for binding.
  • the slight decrease in affinity for the CCSL peptide (SEQ ID NO: 1) verses the linear control peptide GRGDMP (SEQ ID NO: 8) may be due to the restricted conformation of the RGD sequence dictated by the CCSL peptide (SEQ ID NO: 1) stem loop structure. Binding of the CCSL peptide to the GPIIbllla receptor provides further evidence that the CCSL peptide folds into a structure which both presents and restricts the conformation of the RGD sequence.
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO: 1 : Cys Ala Ala Leu Glu Ser Glu Val Ser Ala Leu Glu Ser Glu Val Ala 1 5 10 15

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Abstract

L'invention concerne des polypeptides à configuration limitée en un agencement de boucle à axe superenroulé, antiparallèle et intramoléculaire. L'axe superenroulé comprend deux structures α-hélicoïdales disposées longitudinalement dans une orientation antiparallèle telle que les hélices puissent former une structure superhélicoïdale. Le segment de la boucle comprend un connecteur entre la terminaison carboxyle d'une première structure α-hélicoïdale et la terminaison amine d'une seconde structure α-hélicoïdale. L'invention concerne également des anticorps dirigés contre ces polypeptides et des molécules d'ADN qui codent ces derniers. L'invention concerne encore les imitateurs et les antagonistes des macromolécules biologiques.
PCT/US1994/006655 1993-06-11 1994-06-10 Matrices en boucle a axe superenroule WO1994029332A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP94920179A EP0724593A4 (fr) 1993-06-11 1994-06-10 Matrices en boucle a axe superenroule
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WO1996035783A1 (fr) * 1995-05-10 1996-11-14 Chiron Corporation Procedes de preparation de proteines oligomeres solubles
WO1999025732A2 (fr) * 1997-11-13 1999-05-27 Europäisches Laboratorium für Molekularbiologie (EMBL) Conception, par derivation de dimeres bispirales, d'antagonistes des recepteurs specifiques de cytokines en faisceau de quatre helices
WO1999051625A2 (fr) * 1998-04-02 1999-10-14 Rigel Pharmaceuticals, Inc. Peptides provoquant la formation de structures compactes
US6271198B1 (en) 1996-11-06 2001-08-07 Genentech, Inc. Constrained helical peptides and methods of making same
WO2002012337A1 (fr) * 2000-08-09 2002-02-14 European Molecular Biology Laboratory Mimetismes de peptides
US6709814B1 (en) 1998-04-02 2004-03-23 Rigel Pharmaceuticals, Inc. Peptides causing formation of compact structures
WO2006105440A2 (fr) * 2005-03-30 2006-10-05 Sirtris Pharmaceuticals, Inc. Nicotinamide riboside et analogues
WO2017037158A1 (fr) * 2015-09-01 2017-03-09 Oncoqr Ml Gmbh Lieur en superhélice
WO2017149117A1 (fr) 2016-03-04 2017-09-08 Morphosys Ag Bibliothèque de polypeptides
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JPH07502163A (ja) * 1991-08-09 1995-03-09 イー・アイ・デユポン・ドウ・ヌムール・アンド・カンパニー 植物の栄養価改良用のプログラム可能水準の必須アミノ酸類を含有する規定された構造を有する合成貯蔵蛋白質

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BIOCHEMISTRY, Volume 33, Number 9, issued 08 March 1994, MYSZKA et al., "Design and Characterization of an Intramolecular Antiparallel Coiled Coil Peptide", pages 2363-2372. *
BIOSCIENCE REPORTS, Volume 2, issued 1982, PARRY, "Coiled-Coils in Alpha-Helix-Containing Proteins: Analysis of the Residue Types Within the Heptad Repeat and the Use of These Data in the Prediction of Coiled-Coils in Other Proteins", pages 1017-1024. *
PROTEINS, Volume 7, Number 1, issued 1990, COHEN et al., "Alpha-Helical Coiled Coils and Bundles: How to Design an Alpha-Helical Protein", pages 1-15. *
See also references of EP0724593A4 *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 267, Number 9, issued 25 March 1992, KAUMAYA et al., "Design and Immunological Properties of Topographic Immunogenic Determinants of a Protein Antigen (LDH-C4) as Vaccines", pages 6338-6346. *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996035783A1 (fr) * 1995-05-10 1996-11-14 Chiron Corporation Procedes de preparation de proteines oligomeres solubles
US5837816A (en) * 1995-05-10 1998-11-17 Chiron Corporation Interleukin-2 receptor subunit ectodomain fusion protein comprising a leucine zipper domain
US6271198B1 (en) 1996-11-06 2001-08-07 Genentech, Inc. Constrained helical peptides and methods of making same
WO1999025732A2 (fr) * 1997-11-13 1999-05-27 Europäisches Laboratorium für Molekularbiologie (EMBL) Conception, par derivation de dimeres bispirales, d'antagonistes des recepteurs specifiques de cytokines en faisceau de quatre helices
WO1999025732A3 (fr) * 1997-11-13 1999-09-02 Europ Lab Molekularbiolog Conception, par derivation de dimeres bispirales, d'antagonistes des recepteurs specifiques de cytokines en faisceau de quatre helices
US6685932B1 (en) 1997-11-13 2004-02-03 Europaisches Laboratorium Molekularbiologie Coiled-coil dimer derived antagonists of 4-helix bundle cytokines, design and uses thereof
WO1999051625A2 (fr) * 1998-04-02 1999-10-14 Rigel Pharmaceuticals, Inc. Peptides provoquant la formation de structures compactes
WO1999051625A3 (fr) * 1998-04-02 2000-04-06 Rigel Pharmaceuticals Inc Peptides provoquant la formation de structures compactes
US6709814B1 (en) 1998-04-02 2004-03-23 Rigel Pharmaceuticals, Inc. Peptides causing formation of compact structures
WO2002012337A1 (fr) * 2000-08-09 2002-02-14 European Molecular Biology Laboratory Mimetismes de peptides
WO2006105440A2 (fr) * 2005-03-30 2006-10-05 Sirtris Pharmaceuticals, Inc. Nicotinamide riboside et analogues
WO2006105440A3 (fr) * 2005-03-30 2007-04-05 Sirtris Pharmaceuticals Inc Nicotinamide riboside et analogues
EP2805719A1 (fr) * 2005-03-30 2014-11-26 Glaxosmithkline LLC Nicotinamide riboside et analogues
WO2017037158A1 (fr) * 2015-09-01 2017-03-09 Oncoqr Ml Gmbh Lieur en superhélice
CN108290928A (zh) * 2015-09-01 2018-07-17 安可Qr Ml有限公司 卷曲螺旋连接体
JP2018528214A (ja) * 2015-09-01 2018-09-27 オンコクール・エムエル・ゲーエムベーハー コイルドコイルコネクタ
US11090381B2 (en) 2015-09-01 2021-08-17 Oncoqr Ml Gmbh Coiled-coil connector
CN108290928B (zh) * 2015-09-01 2022-03-29 安可Qr Ml有限公司 卷曲螺旋连接体
WO2017149117A1 (fr) 2016-03-04 2017-09-08 Morphosys Ag Bibliothèque de polypeptides
US10655125B2 (en) 2016-03-04 2020-05-19 Mor0059Us.Np Polypeptide library
US11085038B2 (en) 2016-03-04 2021-08-10 Morphosys Ag Polypeptide library
WO2019086694A1 (fr) 2017-11-06 2019-05-09 Geert Mudde Vaccin et antigène il31

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Publication number Publication date
EP0724593A1 (fr) 1996-08-07
EP0724593A4 (fr) 1997-11-26
JPH08511542A (ja) 1996-12-03

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