WO2007068240A2 - Peptides destines a interagir avec des structures bispiralees alpha-helicoidales et/ou des sequences bispiralees, agents derives de ceux-ci, et leur utilisation - Google Patents

Peptides destines a interagir avec des structures bispiralees alpha-helicoidales et/ou des sequences bispiralees, agents derives de ceux-ci, et leur utilisation Download PDF

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WO2007068240A2
WO2007068240A2 PCT/DE2006/002295 DE2006002295W WO2007068240A2 WO 2007068240 A2 WO2007068240 A2 WO 2007068240A2 DE 2006002295 W DE2006002295 W DE 2006002295W WO 2007068240 A2 WO2007068240 A2 WO 2007068240A2
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sequence
peptide
coiled
coil
peptides
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PCT/DE2006/002295
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WO2007068240A3 (fr
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Carsten Mahrenholz
Michael Portwich
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Charite-Universitätsmedizin Berlin
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Peptides for interaction with alpha-helical coiled-coil structures and / or coiled-coil sequences means derived therefrom and their use
  • the invention relates to peptides for the attachment and influencing of coiled-coil structures and the association with coiled-coil sequences, in particular from virus fusion proteins and cellular signal transducers, agents derived therefrom and their use, preferably for the detection, labeling and inhibition of Coiled-coil structures and sequences in a biological or biotechnological system.
  • Fields of application of the invention are molecular biological research, diagnostic medicine and the pharmaceutical industry.
  • the ⁇ -helical coiled-coil is a structure that is formed in many processes of association in nature [for review, see Arndt et al., 2005, Burkhard et al., 2001, Lupas, 1996] was first predicted in 1953 by Francis Crick with the evaluation of the X-ray diffraction pattern of ⁇ -keratin: Crick described two right-handed ⁇ -helices, which through a "knobs into holes packing" of amino acid residues a left-handed superhelix form. The first amino acid sequence of a coiled-coil was published in 1972 by tropomyosin, a short regulator protein in muscle contraction.
  • the first high-resolution structure (1, 8 A) of a coiled-coil was published in 1991 by O'Shea and co-workers for the two ⁇ -helices GCN4 leucine zipper. Since then, detailed structures for three- and five-strand coiled-coil have also been published. Coincidentally, all structures show amphipathic ⁇ -helices with a light supercoil, which are associated parallel or antiparallel along hydrophobic contact surfaces.
  • the amphipathic character of the helices is due to the heptadic amino acid motif (-a-b-c-d-e-f-g-) n in the primary structure, which repeats itself every two helix turns.
  • the hydrophobic residues at positions a and d extend their side chains to the adjacent helix and each of these side chains (the "button”) juts into a space surrounded by four side chains of the opposite helix (the "buttonhole”), leaving Crick's button -in a buttonhole pack was confirmed (see Figure 1).
  • amino acid residues at positions e and g shield the hydrophobic contact surfaces and stabilize the structure by forming inter- and intra-brelonic salt bridges.
  • the supercoil that is, the slight twisting of each helix, results in a turn length of 3.5 amino acid residues rather than the average of 3.6 Amino acid residues in globular ⁇ helices. This first allows the side chains to be brought over several heptads into an equivalent position with respect to the superhelical axis.
  • the leucine zipper All living cells respond to changes in their environment with a rapid change in their transcriptional programming, which is characterized by activation or repression of their gene expression. DNA-binding proteins play an important role here. In eukaryotes, this includes the family of bZip (basic region leucine z / pper) transcription factors that bind to promoter elements. Common to them is a bZip domain that modulates the association with DNA and has a length of 60-80 amino acid residues. The bZip domain consists of two functional regions, an N-terminal basic region that binds to the DNA, and a C-terminal dimerization domain, the so-called leucine zipper ("leucine zipper”) domain.
  • leucine zipper leucine zipper
  • bZip transcription factors can homo- or heterodimerize in parallel along the leucine zipper domain to form a short ⁇ -helical Coiied coil, the so-called leucine zipper.
  • X-ray crystal structures of bZip domains associated with DNA show that the two ⁇ -helices of the leucine zipper are N-terminally extended. This positions basic amino acid residues opposite each other and forms a bimolecular domain that binds sequence-specifically in the major groove of the DNA.
  • Three well-characterized bZip transcription factors are, for example, GCN4 in yeast and c-Jun and c-Fos in cells of higher organisms.
  • GCN4 which can form a homodimer, stimulates the transcription of more than 35 enzymes for amino acid biosynthesis enzymes when the cell is deficient in amino acids. Recent studies show that GCN4 can induce transcription of at least one-tenth of the genes of the yeast genome.
  • the transcription factors c-Jun and c-Fos show different homospecific associations.
  • c-Jun in contrast to c-Fos, is able to form homodimers. If both proteins are present in the same number, but outweighs the Heteroassociation to form the activator protein 1 (AP-1), which is involved in a variety of cellular processes such as differentiation, and in particular oncogenesis and apoptosis.
  • AP-1 activator protein 1
  • Figure 2 shows a helix-wheel representation of the GCN4 leucine zipper. While the leucine side chains in position d protrude directly into the opposite helix, the residues in position a are slightly inclined to the side. This can explain why predominantly ß-branched side chains are found at this position, which stabilize the hydrophobic contact surfaces by closer contact with the opposite helix.
  • the majority of the bZip transcription factors have an asparagine residue at position a of the third heptade of their leucine zipper domain. The importance of this asparagine for the dimerization of the GCN4 leucine zipper has been demonstrated by several studies.
  • Interhelical salt bridges are formed between oppositely charged residues of the position g (i) of one helix with the position e (i ' + 5) of the opposite helix (see helix-wheel representation of the GCN4-leucine zipper Figure 1).
  • Intrahelical salt bridges can occur between positions i and i + 3 and between i and i + 4.
  • the GCN4 leucine zipper shows two interhelical salt bridges between oppositely charged residues of positions K15 (g ') of one helix and E20 (e) (one-letter abbreviation for amino acids, position in heptad and numbering according to helix-wheel representation) opposite helix, as well as between E22 (g ' ) and K27 (e).
  • An intrahelical salt bridge is formed within a helix between K8 (g) and E11 (c).
  • the amino acid residue E22 (g) is in the crystal structure both in close proximity to R25 (c) and to K27 (e ' ), so that it is assumed that an inter- and intra-skeletal salt bridge [O ' Shea et al. 1991].
  • electrostatic interactions is dependent on the immediate environment of the side chains involved.
  • electrostatic interactions between surfaces that are largely in contact with an aqueous solution are often weak, while such interactions of buried residues may contribute to the stability of a protein.
  • the charged side chains of the coiled-coil can therefore contribute to the stability of the structure, since they are not completely surrounded by water due to their close proximity to the hydrophobic contact surfaces.
  • GCN4Jun and GCN4Fos Two synthetic peptide constructs (GCN4Jun and GCN4Fos) were synthesized with an N-terminal cysteine label. All three possible combinations (GCN4Jun-GCN4Jun, GCN4Fos-GCN4Fos and GCN4Jun-GCN4Fos) were linked and purified by disulfide bonds. Both homodimerically linked leucine zippers (GCN4Jun-GCN4Jun and GCN4Fos-GCN4Fos) showed lower pH stability than the GCN4Jun-GCN4Fos heterodimer [O'Shea et al. 1992]. In a similar experiment, the contribution of two salt bridges in the N-terminal portion of the leucine zipper to the formation of the heterodimer has been particularly demonstrated [John et al. 1994].
  • the parallel orientation of the ⁇ -helices in the GCN4 leucine zipper was first shown in 1989 by O 'Shea et al. They synthesized the GCN4-leucine zipper domain firstly N-terminally and secondly C-terminally labeled with cysteine. After mixing the two peptides in oxidizing medium, and the concomitant dimerization of the domains via a disulfide bond, the chromatographic separation showed a preference for the formation of homodimers between the same species, ie for the formation of parallel ⁇ -helices.
  • a similar experiment with the leucine zipper domains of c-Jun and c-Fos also demonstrated the parallel orientation of a heterodimer [Genz et al. 1989].
  • alpha-helical coiled-coil Viral fusion proteins. Many of the more closely characterized viruses invade their target cells through the formation of a naturally omnipresent protein structure, the so-called alpha-helical coiled-coil.
  • the structural formation of these alpha-helical coiled-coils is similar to the leucine zipper along characteristic sequence sections of the viral envelope proteins, which have a repetitive motif of seven characteristic amino acid residues (abcdefg) n over several heptads [Chan et al., 1998]. Upon contact of the corresponding amino acid residues, these sequence segments form helical secondary structures, which in turn spiral around each other to form a trimeric superhelix [Bullough et al., 1994, Chen et al., 1999]. Only through this coiled-coil interaction is the binding of the virus to the host cell and the fusion of the viral membrane with the Target membrane allows. This is the prerequisite for the release of the viral genome into the cytoplasm of the host cell and thus the basis of
  • envelope proteins gp41 of HIV / SIV, hemagglutinin of influenza, GP2 of the Ebola virus, Env-TM protein of the MMVL (Moloney murine leukemia virus) and the F (fusion) protein of the paramyxovirus SV5 and of the RSV For example, the envelope proteins gp41 of HIV / SIV, hemagglutinin of influenza, GP2 of the Ebola virus, Env-TM protein of the MMVL (Moloney murine leukemia virus) and the F (fusion) protein of the paramyxovirus SV5 and of the RSV
  • the specificity of heterodimerization / oligomerization makes corresponding coiled-coil domains interesting for another approach to drug delivery [Moll et al. 2001].
  • a therapeutic component for. A radionuclide chelate complex [Goldenberg 2003]
  • a targeting moiety e.g. As an antibody
  • the route guidance component is brought into the organism for the specific recognition and marking of the therapy site.
  • the active ingredient component is supplied and localized by the formation of the coiled-coil in the immediate vicinity of the therapy site.
  • a disadvantage of the prior art is that the common peptides for the abrogation and / or attachment of / to coiled-coil structures and the association with coiled-coil sequences have amino acid sequences with more than 15 amino acid residues. Since, in general, the immunogenicity of peptides increases with the number of amino acid residues and the yield and purity of the reaction products of a peptide synthesis decreases with increasing sequence length, these peptides have the disadvantage that they can cause undesirable immunological reactions in their pharmacological use and also costly in the Production are.
  • the object of the invention is therefore novel substances for the abrogation and / or addition of coiled-coil structures and the association with coiled-coil sequences, agents derived therefrom and their use, preferably for the Identification, labeling and influencing of coiled-coil structures and sequences in a biological or biotechnological system.
  • the core of the invention is the finding that short-chain synthetic peptides with a maximum length of 15 amino acid residues can associate specifically, in particular also heterospecifically, with alpha-helical coiled-coil structures and / or sequences.
  • short peptides with a specific sequence influence natural alpha-helical coiled-coil structures (eg the homodimeric GCN4 leucine zipper or the trimerization region of an at least partially extracellular protein, eg of viruses) or with a natural one and / or coiled-coil sequence deduced therefrom (eg with a cysteine-labeled GCN4 leucine zipper domain), eg heterospecific, especially if the (coiled-coil) target structure has more than 60 amino acid residues or the (coiled-coil) ) Target sequence comprises more than 30 contiguous residues of a typical alpha-helical coiled-coil sequence motif.
  • natural alpha-helical coiled-coil structures eg the homodimeric GCN4 leucine zipper or the trimerization region of an at least partially extracellular protein, eg of viruses
  • a natural one and / or coiled-coil sequence deduced therefrom eg with a cysteine-lab
  • Natural coiled-coil sequences are, in particular, the sequence sequences of the known polypeptide chains or the amino acid sequences of the open reading frames of the sequenced genomes, in particular of organisms or viruses, which comprise one or more coiled-coil motifs with the heptadic coiled-coil sequence sequence (abcdefg) n , where n> 1 (the coiled-coil sequence may be formed in particular also with a not divisible by 7 number of amino acid residues, which is divisible by (7n + x), where x is a number from 1 to 7 is) or n ⁇ 2, in particular n> 3, particularly suitable n ⁇ 4, and this can also be determined, for example, using known mathematical algorithms in a simple manner. For example.
  • the coiled-coil sequence of hemagglutinin from influenza includes three coiled-coil motifs (A, B, and C) consisting of several coiled-coil sequence sequences useful for forming the trimeric structure and its biological Function contribute.
  • the coiled-coil sequences derived therefrom and largely corresponding to one another are, in particular, natural coiled-coil sequences which, for example, are caused by a mutation, in particular substitution, at one or more, preferably a few, sequence positions.
  • Heterospecific association means in particular the property that the peptides according to the invention essentially prefer an association with the target structure or sequence, which preferably takes place via an association constant of> 10 3 M -1 , to a homospecific association also to be understood when the peptide of the invention associated with a target sequence comprising more than 15 amino acid residues, wherein the target sequence or structure optionally also includes the sequence of the peptide of the invention as a sequence section.
  • Terminus of the peptide are or form the N-terminus and the residues e, f, g are closer to the C-terminus of the peptide or form the C-terminus), of which a and / or d hydrophobic and e and / or g are charged and n is a number from 1 to 3, or parts thereof.
  • n is to be understood such that the formula (I) describes in principle all possible peptides of length 2 to 15 amino acid residues, which are described as contiguous
  • Sequence of the formula (abcdefg) i (abcdefg) 2 (abcdefg) 3 are derived, so for example, the 15mer (bcdefg) i (abcdefg) 2 (ab) 3 , the 14mer gi (abcdefg) 2 (abcdef) 3 , the 13mer (bcdefg) i (abcdefg) 2, the 12 mer (abcdefg) 1 (abcde) 2, etc., the 8mer (fg) i (abcdef) 2 the 7mer (cdefg) i (ab) 2 etc, which includes 3mer (gave) or 2mer (de) ,
  • sequences are particularly suitable in which at least two contiguous amino acid residues ga and / or de occur, whereby the specific association properties are particularly promoted.
  • the peptides of the invention are preferably formed with a free N-terminus and / or a free C-terminus.
  • the free N-terminus is particularly easy to prepare in the synthesis of the peptide and also facilitates the preparation of one of the means described below, since the free N-terminus about, preferably covalent, allows connection of a (cross) linker, so for example one Succinimidyl ester which, in particular covalently, is associated with a dye, a solid or another of the markers described below.
  • a free C-terminus is used, for example, when a peptide of the invention is to be linked via electrostatic interactions with a label carrying an opposite charge.
  • a chemical modification of the N- or C-terminus may be desirable, for example, an acetylation of the N-terminus or an amide at the C-terminus.
  • the C-terminal amide can be prepared in the simplest manner during the acid cleavage of the peptide from the solid phase on which it was synthesized, acetylation of the N-terminus, for example by addition of acetic anhydride prior to peptide cleavage, in particular the structural formation of the support of the invention in association with the target sequence.
  • the peptides according to the invention have an extremely high potential, since they have low synthesis effort and in high yield compared to longer known peptides which associate with a natural coiled-coil sequence can be produced.
  • a further advantage of the synthetic origin is that these peptides can be provided in a simple manner, in particular also with modified, for example phosphorylated or glycosylated amino acid residues, and / or non-natural, for example D-amino acid residues, preferably the analogues of the proteinogenic amino acid residues, in their sequence can, for example, which improves their Assotiationseigenschaften, increases their protease stability and / or a biological signal effect of the peptides of the invention is inducible.
  • Proteinogenic amino acid residues are to be understood as meaning, in particular, the residues of the gene-encoded amino acid residues, the residues of valine, leucine, glycine, isoleucine, methionine, phenylalanine, alanine, tryptophan, and proline (which, due to its helix-interrupting properties, may be suitable only in exceptional cases ) are substantially hydrophobic and the residues of asparagine, glutamic acid, glutamine, histidine, lysine, arginine, aspartic acid, cysteine, serine, threonine and tyrosine are substantially hydrophilic.
  • hydrophilic residues aspartic acid, glutamic acid, lysine and arginine are charged, with aspartic acid and glutamic acid being negatively charged or acidic, and lysine and arginine being positively charged and basic, respectively. Hisitidine is not charged under physiological conditions.
  • proteinogenic amino acid residues which are modified, for example the residues of phosphorylated or glycosylated amino acids, are suitable, in particular if these occur in nature, for example phosphoserine, threonine and / or tyrosine, preferably at the positions b, c and / or f.
  • the peptides according to the invention also have outstanding therapeutic properties since they fulfill the essential requirements which are imposed on a therapeutic peptide: its short sequence has less immunogenicity than longer peptides, in particular with 20 amino acid residues and more so that it comes in contact with components of an immune system, even only an at least reduced immune response in its therapeutic administration, especially if in addition to the targeting residues a, d, e and / or g residues b, c and / or f are chosen so that they induce only a minimal immune response, so for example. Due to their sequence or structure can not interact with MHC complexes or the sequence of these residues of a occurring in the target organism natural sequence is at least similar.
  • the invention Peptides for various needs can be used in which natural or derived coiled-coil sequences to be neutralized or inhibited in their function, since their defined important positions a, d, e and / or g, in principle with all possible amino acid residues, the have the properties of the invention are occupied, any combination of which allows a unique specificity with respect to the association with the target sequence.
  • the peptides according to the invention are also able to associate specifically with transmembrane proteins, preferably with those natural or derived sequences thereof which are spread over several contiguous heptads (abcdefg).
  • n have the coiled-coil-like residues at positions a, d, e and / or g and hydrophobic residues at positions Jb, c and / or f.
  • a therapeutic administration of the peptides according to the invention is then preferably carried out together with a suitable carrier substance, in particular with albumin, lipids or vesicles.
  • hydrophobic residues further increase membrane permeability, which makes the peptide according to the invention particularly suitable for association with target sequences in the interior of a cell, for example with transcription factors or metabolic factors, in particular with bZip proteins, e.g. in Eurokaryotes, or with bacterial M or CA protein.
  • extracellular domains of transmembrane proteins not directly associated with the membrane are target structures of the peptide of the invention, e.g. Coiled-coil sequences of viral fusion proteins or bacterial cell wall proteins
  • the residues b, c, and / or f of the peptides of the invention are chosen so that a Membran reconnectkeit at least not promoted, so that, for example., At least partially, with (negatively) charged residues are occupied.
  • Particularly suitable for association with natural or derived coiled-coil sequences are peptides when they are formed from a minimum of 7 and a maximum of 14 amino acid residues, since by the choice of suitable amino acid residues and a length between one and two coiled-coil heptads one especially specific association is achievable.
  • a length between 7 and a maximum of 13 amino acid residues is suitable, preferably also a length of at most 12 amino acids, more preferably a length of up to 10 or 11 amino acid residues.
  • positions a and / or d of the peptide according to the invention are preferably formed with hydrophobic branched amino acid residues (for example of leucine, isoleucine and / or valine) which allow a particularly stable association with the target sequence, the position (s) in particular as leucine and the position (s) are preferably formed as residues with ⁇ -branched side chains (isoleucine or valine).
  • hydrophobic branched amino acid residues for example of leucine, isoleucine and / or valine
  • a salt bridge to an oppositely charged residue of the target sequence but also a charged residue, in particular of aspartic acid, glutamic acid, lysine or arginine in at least one position a or d be suitable, since this allows a particularly specific binding ,
  • an arginine residue it can contribute to the stability and specificity of the association by forming a salt bridge and a hydrogen bond in two different ways.
  • a hydrophilic, especially charged, residue at at least one position a or d can at least reduce the homo-oligomerization of the peptide according to the invention, whereby a particularly high concentration of free peptide is available for association with the natural or derived coiled-coil sequence ,
  • the positions e and / or g are occupied by at least one negatively or positively charged residue.
  • Each charged radical at a position e and / or g which is preferably formed as an aspartic acid, glutamic acid, lysine and / or arginine radical, can thereby be converted into a particularly stable and specific one by the formation of a salt bridge to an oppositely charged radical of the target sequence Associate with the natural or derived coiled-coil sequence and at the same time stabilize by its methylene group, the hydrophobic interactions of the adjacent positions, in particular of a or d, with the hydrophobic residues of the target structure.
  • the positions e and / or g are preferably selected such that they are specifically associated with oppositely charged residues of the target sequence, in particular at the g and / or e positions of the natural or derived coiled-coil Sequence, can interact.
  • An arginine residue at at least one of the positions e and / or d furthermore has the advantage that it can also interact with a hydrophilic residue of the target structure at the same time, for example with a position a of the coiled-coil target sequence.
  • the positions e and g are chosen in particular so that a homo-oligomerization of the peptide according to the invention is at least hindered, ie in the case of a possible structural formation opposite charged radicals.
  • the radicals b, c and / or f are essentially occupied by hydrophilic radicals, whereby an improved solubility in aqueous media can be achieved and by electrostatic, in particular intra- and / or interhelical interactions, the association with the target sequence is supported.
  • the invention further relates to peptides selected from the group of peptides according to Tables 3-5.
  • Length analysis of amino acid residues 638 to 673 of the HIV protein gp41 has completely surprisingly revealed that certain amino acid sequence of less than 36 amino acid residues (see Table 3), especially less than 16 amino acid residues, are as or better suited for association with the trimerizing one Sequence of amino acid residues 638 to 673 of HIV protein gp41, such as the 36 amino acid residue starting peptide.
  • the invention therefore also relates to pharmaceutical compositions comprising at least one peptide selected from the group of peptides according to Table 3, and optionally a pharmaceutically acceptable carrier and / or diluent and / or excipient.
  • the use of at least one peptide selected from the group of peptides according to Table 3 is particularly suitable for the manufacture of a medicament for the treatment, prevention or alleviation of AIDS or its comorbidities in man or other primates, wherein the peptide derived from the Group of peptides is selected according to Table 3, is able to at least reduce the formation of the homotrimeric structure of gp41 by association with gp41, in particular to inhibit.
  • the invention relates to a method for the treatment, prevention or alleviation of AIDS or its comorbidities in humans or other primates comprising administering to a human a therapeutically suitable amount of at least one peptide selected from the group of peptides according to Table 3 or primates in need of treatment or alleviation of AIDS or its comorbidities or the prevention of AIDS and its comorbidities, in particular to a patient infected with HIV or suffering from AIDS or concomitant disease.
  • the administration of the at least one peptide or the pharmaceutical composition or the medicament is preferably carried out orally, intravenously or rectally.
  • a mapping of ESAT-6 from Mycobacterium tuberculosis and a length analysis of amino acid residues 7 to 46 of this protein has unexpectedly shown that certain amino acid sequence of length 41 amino acid residues and less (see Table 4) for the abrogation and inhibition of the complex consisting of Esat-6 and Cfp-10 from Mycobacterium tuberculosis.
  • the invention therefore also relates to pharmaceutical compositions comprising at least one peptide selected from the group of peptides according to Tables 4, and optionally a pharmaceutically acceptable carrier and / or diluent and / or excipient.
  • At least one peptide selected from the group of peptides according to Table 4 is particularly suitable for the preparation of a medicament for the treatment, prevention or alleviation of tuberculosis in humans or other primates, wherein the at least one peptide is capable of by at least reducing, in particular inhibiting, the formation of the heterotetrameric structure of the Esat-6 / Cfp-10 complex by association with Esat-6 and / or Cfp-10.
  • the invention relates to a method for the treatment, prevention or alleviation of tuberculosis in humans or other primates comprising the administration of a therapeutically suitable amount of at least one peptide according to Table 4 to a human or primate receiving a treatment or palliation of tuberculosis or Prevention of tuberculosis needed, in particular to a patient infected with Mycobacterium tuberculosis or suffering from tuberculosis.
  • the administration of the at least one peptide or the pharmaceutical composition or the medicament is preferably carried out orally, intravenously or rectally.
  • DELERRIRELEARIK peptide has surprisingly been found to inhibit fusion of the influenza virus with its target cells. Subsequent optimization of this sequence by substitution analyzes has also produced sequences that are particularly suitable (see Table 5).
  • the invention therefore also relates to pharmaceutical compositions comprising at least one peptide selected from the group of peptides according to Table 5, and optionally a pharmaceutically acceptable carrier and / or diluent and / or excipient.
  • At least one peptide selected from the group of peptides according to Table 5 or DELERRIRELEARIK is particularly suitable for the manufacture of a medicament for the treatment, prevention or alleviation of influenza in humans or other primates, wherein the at least one peptide is preferably in is able, by association with hemagglutinin, to at least reduce, in particular inhibit, the formation of the trimeric structure of hemagglutinin.
  • the invention relates to a method for the treatment, prevention or alleviation of influenza in humans or other primates, comprising the administration to a human or at least one of a therapeutically suitable amount of at least one peptide selected from the group of peptides according to Table 5 or DELERRIRELEARIK A primate in need of treatment or alleviation of influenza or prevention of influenza, in particular to a patient infected with influenza virus or having influenza.
  • the administration of the at least one peptide or the pharmaceutical composition or the medicament is preferably carried out orally, intravenously or rectally.
  • Various methods preferably on a solid phase with protective group chemistry, are suitable for the preparation of the peptides according to the invention. Particularly good yields can be achieved if it is produced by one of the common synthesis processes, in particular according to Merrifield. Solid-phase-based preparation processes also have the advantage that excess reactive building blocks can be removed during the synthesis by a simple exchange of solutions or washing steps, and the protective group chemistry allows a stepwise construction of the peptide backbone.
  • the peptide according to the invention is preferably purified by customary processes, in particular by preparative HPLC, and characterized analytically, preferably by mass spectrometry and / or analytical HPLC.
  • peptides are also suitable according to the invention whose sequence (abcdefg) n is completely or predominantly or largely, in particular almost, identical to the portion of the amino acid sequence of a protein from the proteome of the organism or the virus, that is, for example, from the proteome sequences of a vertebrate, in particular human, a parasite, a bacterium, a fungus or a virus, in particular those which cause or derive from a human disease.
  • a peptide according to the invention associates with a homospecific oligomerizing target sequence
  • its sequence is preferably identical to or derived from a sequence segment of this target sequence.
  • it is desired to influence a heterospecific (intermolecular or intramolecular) oligomerizing sequence segment of the target protein it is advantageous to use a peptide according to the invention whose sequence is identical to or derived from a (coiled-coil) sequence segment of the natural association partner of the target protein.
  • Peptides which are derived from a natural coiled-coil sequence according to the invention are in particular also to be understood as meaning peptides which either completely or partially from, for example, corresponding, D-amino acids or other non-natural amino acid residues are formed.
  • proteome sequences are to be understood as meaning, in particular, the natural or derived (alpha-helical) coiled-coil sequences described above, preferably when they are present in their natural environment, e.g. in a cell, a homo-oligomeric coiled-coil structure, for example, a homodimer, trimer, tetramer or pentamer can form, such as a leucine zipper or a trimeric coiled-coil structure of a virus coat protein.
  • the peptides according to the invention are advantageously formed in such a way that they already have residues at their sequence positions which allow them to enter into specific and defined interaction with the target sequence.
  • the sequence is from one of the coiled sequences of viral hemagglutinin from influenza, gp41 from HIV or SIV, TM from Visna, GP2 from the Ebola virus, Env-TM protein from MMVL, F protein from SV5 or RSV, and / or GP derived from the Marburg virus
  • the peptides of the invention allow specific association with the coiled-coil sequence from which they are derived, and thus result in a reduction in the biological function of the trimeric viral fusion protein when in contact therewith to be brought.
  • viruses which have at least one coiled-coil sequence in their envelope or fusion proteins and which are equally vulnerable with the aid of the peptides according to the invention are: ARV, BIV, BLV, BPIV-3, BPIV-3, BRSV, BRSV, CAEV, CDV, EIAV, FeLV, FIV, GALV, HIV-1, HPIV-1, HPIV-1, HPIV-2, HPIV-3, HPIV-4a, HPIV-4b, HRSV, HTLV-1, HTLV- 2, Measles, MoMLV, MPMV, Mumps, NDV, PDV, PPRV, PRV 1 PTLV, PVM, Rous, RPV, Sendai, TRTV, and their related strains.
  • the peptides are particularly suitable according to the invention, the sequence of a homo- and / or heterospecifically oligomerizing coiled-coil sequence the desired Target structure of the cascade or from their natural or mutated association partner derived.
  • the peptide according to the invention is particularly suitable for the targeted influencing of erroneous signal cascades, which are found, for example, in many cancer cells.
  • extracellular proteins, particularly those which transmit signals are susceptible to interference by the peptides of the invention.
  • the peptides according to the invention which are derived from the coiled-coil sequences of transmembrane proteins, in particular from cell wall proteins of bacteria.
  • the peptide according to the invention can also be used in the treatment of bacterial infections if, by association with a bacterial protein with which it comes into contact, it interferes with the metabolism of the bacterium or alters its surface condition.
  • agents are suitable which are derived from the peptide according to the invention, wherein the peptide according to the invention preferably via hydrophobic and / or electrostatic interaction or in particular a covalent Bond, associated with a marker.
  • the marker has chemically, physically and / or biologically active properties which permit detection, labeling and / or treatment of natural or coiled-coil sequences derived therefrom.
  • the marker as a carrier material, linker molecule, dye, contrast agent, chemotherapeutic agent, radionuclide, toxin, antibiotic lipid, carbohydrate, biotin, amino acid, nucleotide, oligonucleotide, peptide, protein, microparticles, vesicles, cell organelle, virus, minimal virus and / or whole cell is formed, which makes the marked mite! is adaptable to the most diverse needs.
  • biotin or a preferably hetero-oligomerizing, in particular heterodimerizing, peptide is used as a marker
  • the amount of defective or misdirected alpha-helical coiled-coil sequences, in particular of viruses or cancer cells can be determined by investigation and / or therapy essential drug added in a second / subsequent step when the drug is bound to streptavidin or a domain derived therefrom, or to a protein or heterooligomerizing, especially heterodimerizing, peptide specifically associated with the peptide marker.
  • the marker is formed, for example, as one of the common antibiotics or a structure derived therefrom, for example penicillin, and the sequence of the peptide according to the invention is consistent with or derived from a coiled-coil sequence of a bacterial cell wall or transmembrane protein, eg from M protein of Streptococcus, the substance according to the invention makes possible a particularly efficient therapy of bacterial infections.
  • a substance according to the invention which is composed of a cell death-inducing substance, for example a toxin or messenger substance (for example a hormone), optionally a linker, and the peptide according to the invention whose sequence has a coiled-coil sequence of a viral Fusion protein, in particular one of the aforementioned virus proteins, matches or is derived therefrom.
  • a cell death-inducing substance for example a toxin or messenger substance (for example a hormone), optionally a linker
  • the peptide according to the invention whose sequence has a coiled-coil sequence of a viral Fusion protein, in particular one of the aforementioned virus proteins, matches or is derived therefrom.
  • the agent according to the invention is particularly suitable for the detection, labeling and treatment of natural or derived Coield coil sequences.
  • a fluorescent dye can be coupled, for example, during the synthesis, preferably N-terminal via a peptide bond, or after the synthesis of the peptide of the invention, for example.
  • the fluorescent dye with a Linker, for example, with a maleimide group or a Succinimidylester connected.
  • shorter-chain molecules are also present between the label and the peptide according to the invention, for example amino acid residues, e.g. of aminopropionic acid or aminohexanoic acid as a spacer, whereby an advantageous spatial separation between the marker and the peptide according to the invention is achieved.
  • amino acid residues e.g. of aminopropionic acid or aminohexanoic acid as a spacer
  • the peptides are covalently or via hydrophobic and / or electrostatic Wechse für with a lipidic vesicle, eg a bilayer, a matrix, eg PEG or derived structures and / or a microparticle, such as a magnetosome, which also a particular good absorption, for example.
  • a lipidic vesicle eg a bilayer
  • a matrix eg PEG or derived structures
  • / or a microparticle such as a magnetosome
  • the agent is then also administered in admixture with one or more other components which may affect the target structure or the cells or viruses which comprise the target structure, for example with pharmacologically active molecules or molecular complexes such as oligonucleotides, proteins / peptides or an antibiotic, which are embedded in the vesicle or the matrix, and / or also with components which ensure a particularly good uptake via the intestine, for example a bile acid or bile acid derivatives.
  • intake via the respiratory tract is also suitable, for example when the peptides or compositions according to the invention are supplied as an aerosol with the aid of an inhaler.
  • the marker is formed as a peptide or protein, for example as an antibody, in particular a humanized and / or bispecific antibody or as a humanized and / or bispecific antibody fragment which, for example, is linked to the peptide according to the invention via a (cross) linker, see above
  • a linker for example as an antibody, in particular a humanized and / or bispecific antibody or as a humanized and / or bispecific antibody fragment which, for example, is linked to the peptide according to the invention via a (cross) linker.
  • proteins fulfill a variety of desired functions, such as the association and / or induction of immune-specific cells, for example.
  • macrophages or B cells if Marker as, for example, bispecific directed against these cells, antibody or as an antigen, in particular peptidic epitope is formed.
  • humanized and / or bispecific antibodies or humanized and / or bispecific antibody fragments are used, as these cause a deleterious immune response in therapy, e.g. of humans, at least
  • a further aspect of the invention relates to a method for identifying alpha-helical coiled-coil sequences, wherein a, preferably planar, carrier surface comprising at least one peptide immobilized on the carrier surface according to one of claims 1-28 or at least one immobilized agent according to one of claims 29-36, with a test solution in Contact containing a natural or derived alpha-helical coiled-coil sequence.
  • the natural or derived alpha-helical coiled-coil sequence in the test solution which is formed, for example, as part of a natural protein, a structure derived therefrom, eg mutated in the sequence, or as a synthetic peptide, is preferably with a label is provided or is detected by adding a labeled substance, for example.
  • An antibody or an agent according to the invention is preferably provided, for example, as part of a natural protein, a structure derived therefrom, eg mutated in the sequence, or as a synthetic peptide.
  • peptides of the invention can be identified particularly easily, especially if they are part of a peptide library.
  • coiled-coil sequences of target proteins or of their association partners are scanned or screened with the aid of preferably overlapping peptides of the inventive length.
  • the identification of a coiled-coil sequence to be scanned advantageously takes place by derivation from the structure published in the databases or evaluation of the sequence of the target protein or its association partner published in the databases with the aid of known coiled-coil prediction programs, which, for example, also can be used over the Internet.
  • the selection of the peptide sequences to be synthesized takes place depending on the degree of their sequence overlap, that is to say, for example, whether they overlap with one, two, or more amino acid residues with the two closest (offset) peptide sequences in the native sequence.
  • Particularly suitable for accurate analysis are peptide overlaps in which the staggered sequences closest in the native sequence are matched except for one amino acid residue.
  • overlapping sequences for example in vitro or in a similar manner, are to be tested for their suitability, it is advantageous to use support materials to which they are bound, in particular covalently, for example as a peptide library. The testing is then carried out, for example, by incubation or contact with a solution which contains the target structure, its association partner and / or parts thereof.
  • the test also uses labeled synthetic peptides (coiled-coil domains) whose sequence matches or is derived from a sequence segment of the target structure or its association partner, in particular with a length of more than 15 amino acid residues, preferably more than 30 amino acid residues.
  • labeled synthetic peptides coil-coil domains
  • peptides are used which have been cleaved off from the solid phase after their synthesis and, if appropriate, labeling and which are freely mobile in solution.
  • mixtures of different peptides in solution or the use of different peptide concentrations allow particularly rapid identification of a suitable for the desired purposes of the invention peptide.
  • the determination of the association or, for example, biological function is carried out by comparing the signals or measured values obtained, in particular also with a negative control which has been tested in parallel and which, for example, consists of a peptide sequence not according to the invention.
  • an optimization of the peptides or agents according to the invention for example by amino acid substitutions, preferably at the positions a, d, e and / or g, length analyzes and / or insertion of non-natural residues in the identified sequence is suitable, wherein the screening for suitable peptides according to the invention in an analogous manner in vitro, in situ, in vivo or similar thereto.
  • length analysis is meant the use of at least two surface bound peptides or agents obtained by synthesizing N- and / or C-terminal truncated sequences of a peptide sequence of the invention.
  • Particularly suitable for the method according to the invention is if the identification and / or optimization takes place on or with the same solid phase at which the peptide or agent to be tested has been synthesized. As a result, it is also no longer necessary, for example, to split off the peptides or agents from the solid phase and, if appropriate, to immobilize them on another support, which makes possible a particularly simple handling and implementation of the method.
  • a target protein is selected, eg hemagglutinin from H6N3 (Seq ID No. 1), and possible sequence sections of the protein with at least one coiled Coii motif detected, eg Seq ID no. 2 in H3N6, which includes, for example, the suitable coiled-coil motifs according to claim 12.
  • the determined coiled-coil motif (s) of the target protein is synthesized as a library with peptides of the inventive length overlapping in its sequence on one or more solid phases (see, for example, the peptide screening of SEQ ID NO: 2) the embodiments).
  • a synthetically accessible sequence portion of the coiled-coil motif is synthesized on a solid phase, e.g. Seq. ID No 3 from H3N6, and, unless already done during the synthesis, provided with a fluorescent dye, e.g. Tetramethylrhodamin, and cleaved from the solid phase and optionally purified.
  • the dye-labeled sequence is subsequently dissolved, the solution brought to the solid-phase-bound peptides, and possibly visualized dye signals on the surface and optionally recorded.
  • the thus-determined peptide sequences of the invention e.g. the peptides AAIDQINGKLNRVI, KLNRVIEKTNEKFH, EKTNEKFHQIEKEF, FSEVEGRIQDLEKY,
  • DTKIDLWSYNAELL, SYNAELLVALENQH are used as soluble peptides on the solid phase, e.g. a synthetic resin synthesized, possibly provided with a marker / coupled, and can be used for the desired purpose.
  • Another typical use concerns optimization of detected sequences, e.g. the above-mentioned sequences, by amino acid substitutions, which is preferably carried out in such a way that a peptide library containing variants of the sequences determined is tested in the same way, wherein preferably each variant contains one or more substitutions at the positions a, d, e and / or g, especially non-natural amino acid residues.
  • a peptide library containing variants of the sequences determined is tested in the same way, wherein preferably each variant contains one or more substitutions at the positions a, d, e and / or g, especially non-natural amino acid residues.
  • Another typical use is the substitution of one or, preferably, several amino acid residues in a sequence of the invention directly selected from the target protein or its associating partner, eg the sequence IEKTNEKFHQIEKE from H3N6 and the testing of the variants for their association properties, eg in contact with the coiled-coil sequence Seq ID No 3 from the homooligomerizing target protein H3N6.
  • the identified variants with the desired properties in particular those which show a higher association than the starting sequence, for example the peptides of the sequence IEKLKEKFHQLKKK, IEKLEEKFHQLKKK, LKLNENFHQLKKK, are subsequently synthesized and used as soluble and possibly labeled peptides.
  • peptides are also typically selected from a restricted or inappropriate starting sequence which, for example, is substantially homo-oligomerized and / or does not exhibit the desired association properties, e.g. the sequence DELERRIRELEARIK, preferably by substitutions at positions a, d, e, and / or g, more preferably at the positions adjacent in a homo-oligomeric structure / button-in-buttonhole packing, and in association with the target sequence or structure, eg the GCN4 leucine zipper.
  • the identified sequences e.g. Peptides according to claim 14, whose C-terminal end consists of five amino acid residues which are not the LEARIK starting sequence, e.g. the sequences according to claim 15, are subsequently synthesized in substance, optionally provided with a marker, and used.
  • variants of a determined, predicted or non-associating starting sequence eg DELERRIRELEARIK
  • peptides preferably as peptide library, in particular systematically shortened amino acid sequences of the starting sequence of the N- and / or C-terminus of the starting sequence and tested for association, eg the length analysis the sequence DELERRIRELEARIK on association with the GCN4 leucine zipper (see embodiments).
  • Associated peptides with, in comparison to the starting sequence, truncated peptide sequences, for example, the peptides according to claim 16 or 17 are selected, prepared as freely soluble peptides and, optionally provided with a marker used.
  • Another aspect of the invention relates to compositions for the production of drugs for the detection, labeling, treatment and / or prevention of a disease or infection, which is associated with gene products comprising a coiled-coil sequence, this according to the invention at least one substance according to one of claims 1-28 or at least one agent according to any one of claims 29-36, especially in a therapeutically suitable dose.
  • Disease or infection are to be understood as meaning, in particular, viral or bacterial infections, for example of humans, whose pathogens are susceptible to attack by the peptide according to the invention or the agents derived therefrom.
  • diseases associated with gene products having a coiled-coil sequence eg, (mutated) proteins of signaling cascades in aberrant (body) cells, are recognizable, markable, and / or treatable.
  • peptide and / or agent according to the invention are used for the preparation of drugs for the detection, labeling and influencing of coiled-coil structures and sequences in a biological system, in particular for the treatment of coiled-coil-mediated diseases.
  • therapeutically suitable amounts of the peptide and / or agent according to the invention are used for the preparation of drugs for the detection, labeling and influencing of coiled-coil structures and sequences in a biological system.
  • Sequences used by the peptide or the agent is brought into contact with a biological or biotechnological system, for example, in or with a test solution, which may be at least partially from body fluid.
  • peptides or agents derived therefrom can be brought into a biological or biotechnological system, for example by addition, in particular injection, pipetting or aerosol, of solutions or emulsions containing a peptide / agent , preferably freely mobile in solution, or by administration as a solid, preferably in the form of tablets or suppositories.
  • Suitable solid-phase-bound peptide libraries are, in particular, those arrangements which contain peptides / agents which have been synthesized on the same solid phase at or on which the use according to the invention takes place.
  • peptides which are linked to a coupling group e.g. the thiol group of cysteine or an acidic function are provided, and in a subsequent step in the synthesis and cleavage of the solid phase on the, in particular functionalized, carrier and there, for example.
  • a coupling group e.g. the thiol group of cysteine or an acidic function
  • inventive peptide (s) and / or agent are immobilized on the, preferably planar, solid phase carrier which is, for example, at least partially, predominantly or completely immobilized Glass, plastic and / or as a membrane, for example with a surface which is formed essentially of cellulose, nitrocellulose or PVDF.
  • biologically active components e.g. Cells or their constituents, cell culture supernatants, tissues, etc., immobilized on a functionalized solid phase, and subsequently contacted for specific properties, e.g., by contact with a solution of a suitable agent of the invention. a viral infection, to be studied.
  • detection reagents e.g. Coloring reagents, proteins or peptides suitable, preferably labeled, in particular monoclonal and / or polyclonal antibodies, labeled streptavidin and / or labeled peptides with a coiled-coil sequence are suitable, which are preferably formed as one of the agents according to the invention.
  • the reading is preferably carried out by means of a sensitive apparatus, in particular with a suitable apparatus which can measure and / or evaluate optical, radiographic and / or scintigraphic signals, e.g. an ELISA reader or a scanner, an X-ray machine or a scintillator.
  • a suitable apparatus which can measure and / or evaluate optical, radiographic and / or scintigraphic signals, e.g. an ELISA reader or a scanner, an X-ray machine or a scintillator.
  • test kits having a support surface according to one of claims 51 to 56 with at least one The binding peptide according to any one of claims 1 to 28 or at least one agent bound thereto according to any one of claims 29 to 36 and a detection reagent according to any one of claims 59 to 61 and in particular also include a user manual.
  • the invention now makes possible, for the first time, a selective, efficient and gentle diagnosis and therapy of natural (protein) target structures with a coiled-coil sequence, in particular from viral fusion proteins, cellular signal transducers (including transcription factors), transmembrane, cell wall or metabolic proteins, extracellular proteins, or derived therefrom Structures or diseases that are triggered by them.
  • the invention allows a better effectiveness of screening for early detection of erroneous or misdirected coiled-coil target sequences, which are also easily applicable in routine diagnostics.
  • the invention also allows for the first time a rapid and simple identification of peptides of the invention and agents with a wide variety of desired properties, which are immobilized, for example, on a biochip and used for the detection of pathogens or misfired cells.
  • a virus-host interaction can be inhibited by the peptide according to the invention without inducing a harmful immune response of the target organism.
  • the short peptides serve as lead structures for transformation into peptoid and organo-chemical structures. These inhibitors are derived, for example, from simple peptide array experiments.
  • this approach allows the development of new ways to combat viral-mediated and other diseases.
  • the knowledge gained here is due to the uniform structural properties of the alpha helical coiled-coil in all living systems and viruses, e.g. the typical button-in buttonhole pack, universal, e.g. also on bacterial systems and coiled-coil-mediated metabolic pathways, adaptable.
  • short peptides with a specific sequence influence natural alpha-helical coiled-coil structures (eg the homodimeric GCN4 leucine zipper) or with a natural and / or derived coiled-coil sequence (eg with a cysteine-labeled GCN4 leucine zipper domain), especially if the (coiled-coil) target structure has more than 60 amino acid residues or the (coiled-coil) target sequence more than 30 contiguous residues of a typical coiled-coil alpha-helical sequence motif includes.
  • natural alpha-helical coiled-coil structures eg the homodimeric GCN4 leucine zipper
  • a natural and / or derived coiled-coil sequence eg with a cysteine-labeled GCN4 leucine zipper domain
  • Natural coiled-coil sequences are to be understood as meaning, in particular, the sequence sequences of the known polypeptide chains or the amino acid sequences of the open reading frames of the sequenced genomes, in particular of organisms or viruses, which have a typical coiled-coil sequence of amino acid residues over several contiguous heptads for example, also using known mathematical algorithms can be determined easily.
  • the coiled-coil sequences derived therefrom and largely corresponding to one another are, in particular, natural coiled-coil sequences which are caused, for example, by a mutation, in particular substitution, at one or more, in particular a few, or all sequence positions.
  • a significant advantage of the invention is the combinatorial access to short, non-natural coiled-coil sequences with biotechnological and / or biological function. As has been shown, even coiled-coil domains or sequences can be optimized at the amino acid level for the desired properties. For example. allows the invention to appropriately alter the stability and specificity of a given or engineered peptide sequence at the amino acid level.
  • the invention also offers for the first time a possibility for the investigation of such systems. This is particularly because it also allows the combinatorial study and identification of peptides of the invention containing nonproteinogenic building blocks.
  • the invention opens up a possibility that appears extremely interesting: specifically, the controlled influence on the most varied alpha-helical coiled-coil systems in nature.
  • Embodiments of the invention Synthesis of the GCN4 leucine zipper domain and a dye-labeled coiled-coil sequence from H3N6.
  • the amino acid sequence C ⁇ ßRMKQLEDKVEELLSKNYHLENEVARLKKLVG one-letter code for amino acids, sequence from N-terminus to C-terminus
  • cysteine for labeling
  • spacer consisting of two molecules of 3-aminopropionic acid (designated ⁇ )
  • ⁇ the native leucine zipper Domain of GCN4 from saccharomyces cerevisiae
  • the coiled-coil sequence of H3N6 was selected according to the structure containing the trimerizing regions in the center of hemagglutinin, and checked with different prediction programs and set and composed of the residues according to Seq ID. 3 was coupled to the N-terminal of the activated dye tetramethylrhodamine via a peptide bond. The sequence with the coiled-coil sequence from gp41 sat down accordingly from the residues according to Seq ID no.
  • the synthesis was carried out according to a standard Fmoc-Protoll with PyBOP activation of the amino acid building blocks using a peptide synthesizer (Abimed (Langenfeld, Germany)) to each 50 .mu.mol Tenta Gel S Ram polystyrene resin with a capacity of 0.25 mmol / g (Rapp Polymere (Tübingen, Germany)).
  • N-terminally protected amino acid building blocks with the following side protection groups were used: OfBu for aspartic acid and glutamic acid, tBU for serine, threonine and tyrosine, Boc for histidine and lysine, Trt for asparagine and glutamine, and Pbf for arginine.
  • the solution for cleavage of the side groups and for peptide cleavage from the resin was composed of TFA (trifluoroacetic acid) / phenol / triisopropylsilane / water in a volume ratio of 9.4 / 0.1 / 0.3 / 0.2.
  • the cleaved peptide was then precipitated with fe / t-butyl methyl ether, washed with diethyl ether and centrifuged (centrifuge from Beckmann (Munich, Germany)).
  • the products were identified by means of MALDI-TOF mass spectrometry (Mass spectrometer from Applied Biosystems (Foster City, USA)) and the purity of analytical HPLC (equipment from Waters (Milford, USA)) was tested at the same solvent gradient. Purified peptides were lyophilized (Lyophilizer from Steris (Mentor, USA)).
  • Dye labeling of the peptide was accomplished by Michael addition by reaction of the thiol group of the N-terminal cysteine residue with the maleimide group of the dye derivative tetramethylrhodamine-6-maleimide (Molecular Probes (Eugene, USA)) in sodium phosphate buffer (10 mM, pH 7.4 ). The peptides were used in 1.5-fold excess with respect to the dye. To reduce the disulfide bonds formed, a 1.5 fold excess of tris (2-carboxyethyl) phosphine hydrochloride was also added. After the reaction, residual maleimide groups were inactivated by addition of a 10-fold excess of 2-mercaptoethanol. The residual thiol groups of the mercaptoethanol were finally oxidized by adding a ten-fold excess of DMSO. The chromatographic purification and characterization of the products by analytical HPLC and MALDI-TOF was carried out as described above.
  • DELERRIRELEARIK synthesizes i. peptides were prepared with the following sequences shortened by the N- and / or C-terminus:
  • a so-called retro-sequence was synthesized, i. H. the peptide contained the starting sequence (see above) in reverse order (from the C-terminus to the N-terminus). Furthermore, a so-called diced sequences were synthesized. This sequence contained all the amino acid building blocks of the corresponding native domain in a random order. The signal value obtained for this diced sequence after incubation with dye-labeled GCN4-leucine zipper domain was subtracted as a control value from the remaining signal values obtained.
  • a library derived from the DELERRIRELEARIK sequence was synthesized containing all possible sequence combinations with F (one-letter code) or L at position D1 (numbering from N to C terminus), F or I at position R5, F at position E9, P or Y at position L10, F, P or Y at position or W at position 114 (library no.8), the respective individual substitutions previously being determined by means of a complete substitution analysis of the sequence DELERRIRELEARIK (library (9)), ie with a library comprising all possible single-point exchanges with the 20 genetically encoded amino acid residues.
  • the library (6) was ligated with the protein complex of Esat-6 and Cfp-10, libraries (3) and (4) were labeled with the dye-labeled sequence Seq ID no. 3 incubated.
  • the library (3) was subsequently regenerated and also incubated with the dye-labeled sequence ⁇ DELERRIRELEARIK (10 ⁇ M in blocking buffer). Negative controls were carried out in the same way by incubation with the dissolved dye (see above).
  • the signal patterns obtained were recorded by fluorescence measurement at 600 nm for 1 second using a Lumi imager (libraries (1) and (2), see Figure 3 and Table 1) or recording of the association data and densitometric determination of the signal values for the variants as described in the literature [Töpert et al. 2003] (libraries (3) and (4), see Figures 4 and 5 and Table 2).
  • Virus fusion experiments were performed as described in Schreiber et al., (Biophysical Journal 81 (2001) 1360-72) using peptide concentrations of 1 ⁇ M or 10 ⁇ M
  • Table 1 Exemplary Results for Libraries (1) and (2). Signal values (in BLU values x 10 '3 after subtraction of the control signal) obtained after incubation of the double-exchange peptide library at the DELERRIRELEARIK positions with dye-labeled GCN4-leucine zipper domain (Table 1-19).
  • the left column of the table indicates the amino acid residues inserted into the sequence in place of the original glutamate residue E.
  • the first row shows the amino acid residues used in place of the isoleucine residue I of the starting sequence.
  • the bottom line of the table shows exemplary association signals of the length analysis, wherein, inter alia, two peptides with the sequence LERRIRELEARIK or RRIRELEARIK showed a significantly increased association compared to the starting sequence.
  • the value at the bottom right of the table reflects the association signal of the diced sequence.
  • Table 2 Exemplary 2,205 IEKTNENFHQLKKK 48 1, 077 IEKLNENNHQLKKK 440 Results from library 2,179 IEW-EENFHQI-KKK 528 1, 076 1EKTKENKHQIKKK 300 (4). Signals and sequences 2,096 IEKLEEKFHQIKKK 484 1, 026 IEKTEENFHQLKKK 168 of 660 aufrubring ⁇
  • Figure 1 Button-in-buttonhole packing of the GCN4 leucine zipper (see O ' Shea et al., 1991). This representation is obtained when a piece of paper is wrapped around each of the two helices and the ⁇ -C atoms of the amino acid residues are identified by circles. The color of the circles indicates the helix affiliation, the inscription indicates the number of the heptad and the position of the remainders. The two papers are placed one on top of the other so that they can reflect the packing of the dimer interface. The rest at the position 2a protrudes z.
  • Figure 2 Helix-wheel representation of the GCN4 leucine zipper.
  • the line of sight is from the N-terminus, capital letters and numbers indicate the amino acid residues and their positions in the primary structure (numbering from the N-terminus to the C-terminus) of the GCN4-leucine zipper domain, lowercase the positions in the heptad.
  • the nearest amino acid residues to the viewer are surrounded by a circle.
  • Two crossed arrows represent the interactions of the hydrophobic contact surfaces, dashed arrows mark the inter- and intrahelical salt bridges.
  • Figure 3 Solid-phase bound variants of a de novo constructed coiled-coil domain after incubation of a fluorescently labeled natural Coiled-coil structure, the dimeric GCN4 leucine zipper.
  • the library included variants of a short de novo constructed coiled-coil sequence that differed from the starting sequence in two amino acid positions.
  • Optical signals showed the association of variants with the dissolved labeled GCN4 domain and thus the inhibition of natural dimerization.
  • Figure 4 Screening of Seq ID no. 2 from hemagglutinin with a library of solid phase-linked peptides of overlapping sequences (peptides # 1-54) after incubation with the dye-labeled peptide of the sequence
  • FIG. 5 Peptide library (3600 peptides) with solid-phase-bound variants of the sequence IEKTNEKFHQIEKE, containing all possible substitutions at the positions a, d, e and / or g with the following amino acid residues:
  • Figure 6 Length analysis of the peptide sequence 638 to 673 of the HIV protein gp41 after incubation with the dye-labeled sequence Seq ID No.6 (detail)
  • Figure 7 Binding signals of selected sequences from library # ⁇ after incubation with dye-labeled sequence of Seq ID No.3.
  • Figure 8 Binding signals of library (3) after incubation with dye-labeled sequence ⁇ DELERRIRELEARIK. The following sequences showed binding: QDLEKYVEDTKIDL *
  • Figure 9 Example result of virus fusion tests (according to Schreiber et al., 2001). While the DELERRIRELEARIK sequence clearly inhibited the fusion, the controls did not show any inhibition of viral activity.
  • Seq ID No.1 Hemagglutinin - Influenza A virus (H3N6):
  • Seq ID No.2 Structure ABC (40-105) according to Bullough et al., Nature vol 3711994): STQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDLWSYNAELLVALENQH
  • Seq ID No.3 QAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYV
  • Seq ID No.4 (gp41 from HIV-1):
  • PA Hughson FM, Skehel JJ, Wiley DC. Structure of influenza haemagglutinin atthe pH of membrane fusion. Nature 371 37-43 (1994).
  • Coiled coils a highly versatile protein folding motif.

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  • Biomedical Technology (AREA)
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  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne de nouveaux peptides qui servent à supprimer des structures bispiralées et/ou réalisent une réaction d'addition avec celles-ci, et s'associent à des séquences bispiralées, des agents dérivés de ces peptides, et leur utilisation, de préférence pour reconnaître, marquer et agir sur des structures bispiralées et des séquences bispiralées dans un système biologique ou biotechnologique, en particulier des peptides de formule générale (abcdefg)n, dans laquelle a-g sont des radicaux acides aminés dont a et/ou d sont hydrophobes, et e et/ou g sont chargés, et n est un nombre de 1 à 3, ou des parties de ces peptides comprenant au minimum 2 et au maximum 15 radicaux acides aminés. L'invention a également pour objet des compositions pharmaceutiques contenant ces peptides et l'utilisation de tels peptides pour préparer des médicaments destinés à traiter une infection par le VIH, le virus de la grippe ou Mycobacterium tuberculosis.
PCT/DE2006/002295 2005-12-18 2006-12-18 Peptides destines a interagir avec des structures bispiralees alpha-helicoidales et/ou des sequences bispiralees, agents derives de ceux-ci, et leur utilisation WO2007068240A2 (fr)

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DE112006003448T DE112006003448A5 (de) 2005-12-18 2006-12-18 Peptide für die Wechselwirkung mit alpha-helikalen Coiled-Coil-Strukturen und/oder Coiled-Coil-Sequenzen, davon abgeleitete Mittel und ihre Verwendung

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DE102005060920A DE102005060920A1 (de) 2005-12-18 2005-12-18 Peptide für die Wechselwirkung mit alpha-helikalen Coiled-Coil-Strukturen und/oder Coiled-Coil-Sequenzen, davon abgeleitete Mittel und ihre Verwendung
DE102005060920.1 2005-12-18

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CN102305855A (zh) * 2011-05-19 2012-01-04 中山大学 体外检测结核分枝杆菌感染的试剂和方法
CN104781668A (zh) * 2012-08-29 2015-07-15 亚利桑那州评议委员会,亚利桑那州法人团体,代理和代表亚利桑那州立大学 免疫特征分析:通向早期诊断和健康监测的途径
WO2018084247A1 (fr) * 2016-11-04 2018-05-11 国立研究開発法人理化学研究所 Inducteur d'immunité
US11371990B2 (en) 2016-11-11 2022-06-28 Cowper Sciences Inc. Methods for identifying candidate biomarkers
US11747334B2 (en) 2016-06-20 2023-09-05 Cowper Sciences Inc. Methods for differential diagnosis of autoimmune diseases
US11774446B2 (en) 2016-06-20 2023-10-03 Cowper Sciences Inc. Methods for diagnosis and treatment of autoimmune diseases
US11971410B2 (en) 2017-09-15 2024-04-30 Arizona Board Of Regents On Behalf Of Arizona State University Methods of classifying response to immunotherapy for cancer
US11976274B2 (en) 2019-10-02 2024-05-07 Arizona Board Of Regents On Behalf Of Arizona State University Methods and compositions for identifying neoantigens for use in treating and preventing cancer

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Publication number Priority date Publication date Assignee Title
JP2010531362A (ja) * 2007-06-25 2010-09-24 ジ アドミニストレーターズ オブ ザ トゥレーン エデュケーショナル ファンド インフルエンザを阻害する組成物および方法
JP2013241432A (ja) * 2007-06-25 2013-12-05 Administrators Of The Tulane Educational Fund インフルエンザを阻害する組成物および方法
CN102305855A (zh) * 2011-05-19 2012-01-04 中山大学 体外检测结核分枝杆菌感染的试剂和方法
CN104781668A (zh) * 2012-08-29 2015-07-15 亚利桑那州评议委员会,亚利桑那州法人团体,代理和代表亚利桑那州立大学 免疫特征分析:通向早期诊断和健康监测的途径
US11747334B2 (en) 2016-06-20 2023-09-05 Cowper Sciences Inc. Methods for differential diagnosis of autoimmune diseases
US11774446B2 (en) 2016-06-20 2023-10-03 Cowper Sciences Inc. Methods for diagnosis and treatment of autoimmune diseases
WO2018084247A1 (fr) * 2016-11-04 2018-05-11 国立研究開発法人理化学研究所 Inducteur d'immunité
US11371990B2 (en) 2016-11-11 2022-06-28 Cowper Sciences Inc. Methods for identifying candidate biomarkers
US11971410B2 (en) 2017-09-15 2024-04-30 Arizona Board Of Regents On Behalf Of Arizona State University Methods of classifying response to immunotherapy for cancer
US11976274B2 (en) 2019-10-02 2024-05-07 Arizona Board Of Regents On Behalf Of Arizona State University Methods and compositions for identifying neoantigens for use in treating and preventing cancer

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DE102005060920A1 (de) 2007-06-21
DE112006003448A8 (de) 2010-09-09
DE112006003448A5 (de) 2008-09-25

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