WO2007068240A2

WO2007068240A2 - Peptides interacting with alpha-helical coiled-coil structures and/or coiled-coil sequences, substances derived therefrom, and use thereof

Info

Publication number: WO2007068240A2
Application number: PCT/DE2006/002295
Authority: WO
Inventors: Carsten Mahrenholz; Michael Portwich
Original assignee: Charite-Universitätsmedizin Berlin
Priority date: 2005-12-18
Filing date: 2006-12-18
Publication date: 2007-06-21
Also published as: DE112006003448A8; WO2007068240A3; DE112006003448A5; DE102005060920A1

Abstract

The invention relates to novel peptides for neutralizing and/or attaching to coiled-coil structures and associating with coiled-coil sequences, substances that are derived therefrom, and the use thereof, preferably for detecting, marking, and influencing coiled-coil structures and sequences in a biological or biotechnological system, particularly peptides of general formula (abcdefg)<SUB>n</SUB>, wherein a to g represent amino acid radicals, among which a and/or d are hydrophobic while e and/or g are loaded, and n represents a number between 1 and 3, or fractions thereof that are composed of a minimum of 2 and a maximum of 15 amino acid radicals. The invention further relates to pharmaceutical compositions containing novel peptides and the use of such peptides for producing medicaments used for the treatment of HIV, influenza virus, or Mycobacterium tuberculosis infections.

Description

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 α-helical coiled-coil, literally translated as "spiral spiral," 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 α-keratin: Crick described two right-handed α-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. If the sequence is subdivided into individual amino acid heptads of the sample (-a-b-c-d-e-f-g-), a recurring motif of predominantly hydrophobic residues at positions a and d over the entire polypeptide chain of the protein results. Subsequent studies showed that at positions a and d of the coiled coil, on average, more than 80 percent of the amino acid residues are hydrophobic and often bear branched side chains (leucine, isoleucine and valine). The remaining positions are predominantly occupied by hydrophilic residues, wherein in the positions e and g often charged residues, in particular of glutamic acid or lysine, can be found.

This information led to the de novo design and chemical synthesis of shorter coiled-coil motifs as a model system for protein design research. For example, it has been shown that a 35 amino acid residue long peptide can form a more stable structure than the 284 amino acid residue long tropomyosin [Lau et al. 1984]. Generally, the number of characteristic heptads of the coiled-coil motif varies considerably.

A function of the coiled-coil in protein folding and protein interaction has been demonstrated, in addition to transcription factors, also for such diverse proteins as intermediate filaments, SNARE complexes, spindle pole bodies, tRNA synthetases or transmembrane proteins. Evaluations using bioinformatics methods predict that nine percent of all yeast proteins interact through a coiled-coil and it is estimated that between five and ten percent of all putative open reading frames of the sequenced genomes have a coiled-coil motif.

Structure of the α-helical coiled-coil. In 1981, a three-stranded coiled-coil was visible for the first time in the crystal structure of hemagglutinin in the influenza virus, followed in the same year by the two-stranded coiled-coil at the center of the catabolite gene activator protein (CAP) dimer.

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 α-helices GCN4 leucine zipper. Since then, detailed structures for three- and five-strand coiled-coil have also been published. Coincidentally, all structures show amphipathic α-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).

The 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 α helices. This first allows the side chains to be brought over several heptads into an equivalent position with respect to the superhelical axis.

As model systems for α-helical coiled-coil structures and sequences, the so-called leucine zipper and viral fusion proteins will be explained in more detail below.

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. Depending on their specificity, bZip transcription factors can homo- or heterodimerize in parallel along the leucine zipper domain to form a short α-helical Coiied coil, the so-called leucine zipper. X-ray crystal structures of bZip domains associated with DNA show that the two α-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. Thus, 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.

In 1988, Landschulz and co-workers compared the primary structures of a number of DNA-binding proteins and discovered a homologous region with a repeating pattern of leucine at every seventh position over four to five heptads, concluding that this is a dimerization domain, with dimerization via amphipathic α helices in such a way that in each case two leucine side chains of opposite helices such as the teeth of a zipper fit together, hence the name leucine zipper or leucine zipper. The crystal structure of the homodimeric GCN4 leucine zipper, which consists of two 33 amino acid long α helices, complemented the molecular interactions in the manner predicted by Crick [O ^' Shea et al. 1991], where the hydrophobic side chains of the amino acid residues in the positions a and d form the hydrophobic contact surfaces.

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. Thus, it has been shown that the substitution by the hydrophobic branched residues of valine or leucine stabilizes the leucine zipper in thermal experiments, but a mixture of homodimers and trimers is formed. Alanine exchange leads to the same homo-oligomerization, but has a slightly destabilizing effect on the structure. Interestingly, a dimer / trimer mixture is also observed when asparagine is replaced by the physicochemically similar glutamine. The substitution by lysine at this position, however, leads to the formation of homodimers, but show a lower stability. The positions e and g adjacent to the hydrophobic contact surfaces which have charged amino acid residues contribute to the stability of the leucine zipper in two ways. On the one hand, their side chains have methylene groups, which can protect the hydrophobic contact surfaces from water, on the other hand, they can form both inter- and intramolecular salt bridges. 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].

Generally, in a protein, the contribution of electrostatic interactions is dependent on the immediate environment of the side chains involved. Thus, 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. Conversely, can be reduced by the formation of the salt bridge, the contact of the hydrophobic contact surfaces with solvent.

The destabilization by opposing, equally charged ion pairs in positions g and e 'and g' and e, respectively, has been demonstrated by studies in which the amino acid residues of positions e and g of the GCN4-leucine zipper domain are replaced by the corresponding amino acid residues of c -Jun or c-Fos were exchanged. While c-Fos has five negatively charged residues at these positions, c-Jun shows three positively charged residues there. The Crystal structure of the dimerized bZip domains of c-Jun and c-Fos, bound to DNA, shows four salt bridges between opposite residues in the leucine zipper heterodimer.

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 α-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 α-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].

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 the

Virus replication.

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

(Respiratory syncytial virus).

It has been shown that the coiled-coil sequences of these envelope proteins are highly conserved and even slight mutations in these domains lead to unfavorable phenotypes. Furthermore, it is known that long-chain peptides derived from the native sequences interfere with the trimeric association, but are associated with an immune response of the target organism [for review, see, p. McGaughey et al., 2004]. Thus, they are only partially suitable for inhibiting the virus-host interaction.

Medical applications of the α-helical coiled-coil. Due to their structural simplicity, stability and specificity, leucine zippers and other coiled-coil structures have come to the fore for potential applications. As shown by the example of the inactivation of the onco-suppressor p53, the addition of a chimera coiled-coil can disrupt the structure and function of a self-associating target protein in the cell [Contegno et al. 2002]. Also in the medical field, coiled-coil systems are considered as potential pharmacon suppliers, for example for the controlled release of active substances. Thus, they have been shown to produce a reversibly contracting polypeptide-only hydrogel [Petka et al. 1998]. If they are incorporated into a water-soluble polymer network, a targeted unfolding of the coiled-coil and the concomitant contraction leads to a reduction in volume of the hydrogel of up to 90 percent [Tang et al. 2001; Wang et al. 1999; Wang ef a /. 2001].

In particular, the specificity of heterodimerization / oligomerization makes corresponding coiled-coil domains interesting for another approach to drug delivery [Moll et al. 2001]. In each case, a therapeutic component, for. A radionuclide chelate complex [Goldenberg 2003], and a targeting moiety, e.g. As an antibody, with a provided heterodimerizing domain. First, the route guidance component is brought into the organism for the specific recognition and marking of the therapy site. Subsequently, the active ingredient component is supplied and localized by the formation of the coiled-coil in the immediate vicinity of the therapy site. Such two-step approaches may enhance the therapeutic effect of pharmacological treatment and reduce toxicity to other, unaffected tissues [Goodwin & Meares 2001; Knox et al. 2000]. Heterodimerizing leucine zipper domains have advantages over existing systems. The biotin / streptavidin binding system, for example, has, in addition to a possible immune response to streptavidin, the disadvantage that the protein is trapped by endogenous biotin. In addition to combinatorial approaches, the in vivo heterodimerization of proteins fused to coiled-coil motifs has been shown several times [Behncken et al. 2000; Ghosh et al. 2000; Katz et al. 1998; Scott et al. 1996].

Since, in addition to infections, various human diseases are associated with a change in α-helical coil coil structures, e.g. in diabetes mellitus [Hua ef al. 2000; Sato et al. 2003], it is desirable to provide substances that make these diseases easily visible and / or treatable.

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.

This object is achieved by peptides according to claim 1 and claim 18 and their use, compositions according to claim 29 and 36 and method for their use according to claims 38 and 45. The further claims are preferred variants of the invention.

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.

By completely surprisingly obtained results it has been found that 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 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. 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.

From the recurring sequence of the coiled-coil motif and the identically formed structures of the coiled-coil, it follows that the peptides of the general formula according to the invention of claim 1 and of the length of at least 2 and at most 15 amino acid residues are capable of all possible ones specifically associate natural or derived coiled-coil sequences. These peptides have the general formula (I)

(abcdefg) n (I),

in which a-g are amino acid residues (where the residues a, b, c are closer to the N-

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.

The number 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) ,

If the peptide according to the invention is formed with a sequence of less than 5 amino acid residues, then 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. However, 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. While 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. Among the 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. But in particular also 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.

In particular, 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. At the same time, 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.

If residues b, c and / or d are essentially occupied by hydrophobic residues, then 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. The 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.

Where 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 Membrangängigkeit 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. For a variety of requirements, 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.

The choice of the amino acid residues is preferably carried out such that at least one position a _n or d _{n is} a hydrophobic residue, preferably of leucine, isoleucine or valine, wherein the formula (I), from which the peptide according to the invention is derived, from the three possible heptadentals (1) = (a (hydrophobic) bcd (h _y drophob) efg), (2) = (a (hydrophobic) bcd (hydrophii) efg) and (3) = (a (hydro _P hii) bcd (hydrophobic ) ef9) ' ⁿ 27 different combinations (1) (1) (1), (1) (1) (2) (3) (3) (3) may be composed, that is, for example, as a Fomel the contiguous sequence ( 1) (3) (2):

(a (hydrophobic) bcd (hydrophobic) © fg) i (a (hydrophilic) bcd (hydrophobic) ^β fg) 2 (a (hydrophobic) bcd (hydrophilic) efg) 3

Thus, from (1) (3) (1) or (1) (3) (2), for example, a peptide having the sequence (bcd (hydrophobic) g) i (a (hydrophii) bcd (hydrophobic) efg) 2 ( a (hydrophobic) bc) ₃ or a part thereof selected, which allows a particularly specific association due to a possible electrostatic interaction, which it can enter with its hydrophilic a position to at least one hydrophilic residue of the target sequence. The same applies according to the invention for the position d.

The 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 β-branched side chains (isoleucine or valine).

It is particularly suitable, for example, if at least one position a or d is occupied by a hydrophilic asparagine (N), glutamine (Q), serine (S) or threonine residue (T), that is, for example, the peptide from the sequence

(bcd (hydrophobic) g) i (Nbcd ( _h ydrophobic) efg) 2 (a (hydrophobic) bc) 3 θder (bcd (hydrophobic) g) i (a (hydrophobic) bcNfg) 2 (a (hydrophobic) bc) 3 or formed as a part thereof.

For certain functions, for example for the formation of 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 ,

If, in particular, an arginine residue is chosen, it can contribute to the stability and specificity of the association by forming a salt bridge and a hydrogen bond in two different ways.

Advantageously, 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 ,

In addition to the positions a and d, which preferably contain as many hydrophobic positions as possible and as few hydrophilic positions as necessary, the positions e and / or g are occupied by at least one negatively or positively charged residue. The general formula (I) from which the peptide according to the invention is derived is preferably selected from the three possible heptadearcies (1) = (abcde ( _ge iad) fg (geiaden)),

(2) = (abcde (loaded) fg (not charged)) and (3) = (abcdΘ _(n ot loaded) fg (charged)) composed in the 27 different combinations. 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.

In a particularly favorable embodiment of the peptide according to the invention, 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. In addition, 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.

The use of 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.

In addition, 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.

The use of the 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.

The use of 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.

In addition, 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.

For the association with natural or derived alpha-helical coiled-coil sequences, in particular from eukaryotes or higher systems or organisms which comprise them, eg animals or plants, or from prokaryotes or archaebacteria or viruses, 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. If it is desired that 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. If 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.

The 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.

By deducing such sequences from the natural coiled-coil repertoire, 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. For example, if 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. As a result, the virus is vulnerable both as a particle (virion), or during its amplification in the host cell. Further examples of 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.

If, for example, mutated proteins from signal cascades (so-called signal transducers) at the beginning receptors and at the end of transcription factors are affected, 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. As a result, 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. Likewise, extracellular proteins, particularly those which transmit signals, are susceptible to interference by the peptides of the invention.

The same applies to 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. As a result, 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.

For the detection, labeling and treatment of natural or coiled-coil sequences derived therefrom, according to another aspect of the invention, 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. According to the invention, 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.

It is particularly suitable if 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. If, for example, 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. If 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. The same applies to 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.

If the marker is formed or comprises a solid phase carrier, cross-linker, fluorescent dye, cysteine, antibody and / or enzyme or structure derived therefrom, then 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. When the peptide is N- or C-terminal labeled with a cysteine residue, and the fluorescent dye with a Linker, for example, with a maleimide group or a Succinimidylester connected. Preferably, 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.

In a further development of the invention, the peptides are covalently or via hydrophobic and / or electrostatic Wechsewirkung 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. In an oral intake, and / or a particularly good / long persistence of the compositions of the invention in the organism is made possible. Preferably, 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. For various applications, however, 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.

If 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 The substance allows a particularly effective detection, labeling and / or treatment of foreign or misdirected target sequences of the body, since proteins fulfill a variety of desired functions, such as the association and / or induction of immune-specific cells, for example. Of 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.

As the desired immune response-inducing markers with antibody function, preferably 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.

By this method peptides of the invention can be identified particularly easily, especially if they are part of a peptide library. To identify peptides or agents according to the invention and / or possible target sequences, in a further development of the invention, 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. If 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. Advantageously, 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. For in situ or in vivo or similar tests, in particular 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. In particular, 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.

At the same time or subsequently, 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.

By 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, in particular for in vitro and / or in situ tests, 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.

In a typical use for the identification of peptides or agents according to the invention, 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. For a screening of the sequence, 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,

EKYVEDTKIDLWSY, KYVEDTKIDLWSYN, YVEDTKIDLWSYNA,

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. In this way, it is also possible to identify particularly simply optimized peptide sequences which are subsequently synthesized as peptide according to the invention or means for their use and, if appropriate, provided with a marker.

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. As a result, it is also possible to directly select for peptides / agents with the property according to the invention without prior experiments and to use the identified sequences. -

Similarly, 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.

Another typical use is the determination of the optimal sequence length of the peptide or agent of the invention. For this purpose, variants of a determined, predicted or non-associating starting sequence, eg DELERRIRELEARIK, are synthesized as 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. However, other 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.

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, preferably therapeutically suitable amounts of the peptide and / or agent according to the invention are used.

According to a further aspect, at least one peptide according to one of claims 1 to 28 or at least one agent according to one of claims 29 to 36 for the detection, labeling and treatment of or action on alpha-helical coiled-coil structures or coiled-coil structures. 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. Under the biological system are essentially organisms, cell tissues, cells, cell components, DNA, RNA, cDNA, mRNA, cRNA, proteins and / or peptides and structures derived therefrom to understand the biotechnological system are all possible research arrangements to understand their Aid properties of natural coiled-coil structures or coiled-coil sequences or structures or sequences derived therefrom, in particular cell culture test systems and / or ELISAs or dye-based ELISA Similar systems in microtiter plates, screening of (micro) arrays, preferably on biochips, or biosensors.

In principle, all possible methods are suitable for contacting, with the aid of which 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.

It is particularly useful for certain, e.g., diagnostic, requirements when using at least two peptides or agents that are part of a peptide library or that make up the peptide library, particularly when the peptide library is a solid-phase bound Library is formed, whereby a particularly simple implementation of the inventive use for several peptides to be tested / means is achievable, especially if the peptide / agent in different concentrations on the solid phase or in solution and / or the test solution to different concentrations of containing analyte.

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. For the preparation of many, at least partially similar test platforms, especially biochips, it is convenient to use 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. Via a chemical reaction, e.g. as a microarray, immobilized

It is particularly suitable for the use according to the invention if at least one, preferably more than two, 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.

Also possible are corresponding arrangements of biologically active components to be tested, 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.

For detection and labeling according to the invention, a wide variety of 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.

For a particularly simple implementation of the invention, in particular the detection and labeling of natural or coiled coil sequences derived therefrom, according to a further aspect of the invention, preferably standardized, test kits are suitable, 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. In addition, 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. For example, a virus-host interaction can be inhibited by the peptide according to the invention without inducing a harmful immune response of the target organism. In addition, 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.

In particular, 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.

Due to the completely unforeseen results it was found that 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 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.

From the recurring sequence of the alpha-helical coiled-coil motif and the identically formed structures of the coiled-coil shows that the peptides of the general formula I according to claim 1 and the length of at least 2 and a maximum of 15 amino acid residues are capable to specifically associate with all possible natural or derived coiled-coil sequences.

In addition, 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.

Since the short-chain peptides or agents according to the invention have significance for a very wide variety of possible applications, for example as pharmaceuticals or biomarkers, 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.

In general, 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.

Further details and advantageous features of the invention will become apparent from the following embodiments.

Embodiments of the invention Synthesis of the GCN4 leucine zipper domain and a dye-labeled coiled-coil sequence from H3N6. The amino acid sequence CβßRMKQLEDKVEELLSKNYHLENEVARLKKLVG (one-letter code for amino acids, sequence from N-terminus to C-terminus) is composed of cysteine for labeling, a spacer consisting of two molecules of 3-aminopropionic acid (designated β) and the native leucine zipper Domain of GCN4 from saccharomyces cerevisiae (amino acid residues 249-279) together. 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 purification of the peptides was carried out using a reverse phase HPLC system (Waters (Milford, USA)) with fraction collector (Pharmacia (Freiburg, Germany)) under a solvent gradient of 95% A / 5% B to 40% A / 60% B (A = H ₂ O / 0.05% TFA (v / v); B = acetonitrile / 0.05% TFA (v / v)). 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)).

Labeling of the GCN4 Leuin Zipper Domain. 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.

To carry out negative controls, tetramethylrhodamine-6-maleimide was treated with mercaptoethanol, remaining thiol groups were oxidized with an excess of DMSO and the reacted dye was correspondingly purified and characterized.

Preparation of the peptides to be tested. The stepwise synthesis of solid-phase-bound peptides was performed by semiautomatic SPOT synthesis on Whatman 50 cellulose membranes (Whatman (Maidstone, UK) as described in the literature [Kramer & Schneider-Mergener 1998].) The LISA software was used to generate the peptide sequences (Jerini (Berlin, Germany)) and an automatic pipettor (Abimed (Langenfeld, Germany)) for the synthesis: (1) peptides of the formula (bcdefg) i (abcdefg) 2 (ab) 3 derived from the sequence ββDELERRIRELEARIK (cf. Variants with all possible simultaneous double exchanges of the 20 proteasegenic amino acid residues (with the exception of cysteine and in a library (s) of proline) were synthesized at the following positions: Double exchanges were inserted at the following sequence positions (written in bold): (a) DELERRIRELEARIK

(b) DELERRIRELEARIK (C) DELERRIRELEARIK

(d) DELERRIRELEARIK

(e) DELERRIRELEARIK

In addition, were

(2) A solid-phase bound length analysis of the sequence was performed

DELERRIRELEARIK synthesizes, i. peptides were prepared with the following sequences shortened by the N- and / or C-terminus:

DELERRIRELEARI

ELERRIRELEARIK

DELERRIRELEAR

ELERRIRELEARI

LERRIRELEARIK

DELERRIRELEA

ELERRIRELEAR

LERRIRELEARI

ERRIRELEARIK

DELERRIRELE

ELERRIRELEA

LERRIRELEAR

ERRIRELEARI

RRIRELEARIK

Similarly, length analyzes of the sequence having amino acid residues 638 to 673 of HIV protein gp41 (library (5)) and Seq ID No. 7 (library (6)) were also synthesized.

As a control, 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.

(3) The sequence Seq ID no. 2 was screened by overlapping peptides: STQAAIDQINGKLN (peptide no.1) TQAAIDQINGKLNR (peptide no.2)

QAAIDQINGKLNRV (...) AAIDQINGKLNRVI AIDQINGKLNRVIE IDQINGKLNRVIEK DQINGKLNRVIEKT QINGKLNRVIEKTN INGKLNRVIEKTNE NGKLNRVIEKTNEK GKLNRVIEKTNEKF KLNRVIEKTNEKFH LNRVIEKTNEKFHQ etc. SYNAELLVALENQH (peptide no. 54)

(4) variants of the sequence IEKTNEKFHQIEKE, including all possible substitutions at the positions a, d, e and / or g with the following amino acid residues (total 3600 peptides):

IEKTNEKFHQIEKE (source sequence) abcdefgabcdefg (positions)

E LE EE LK K (inserted residues)

K K NK

L L

N N

Analogously, 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.

Conducting association studies. The cellulose-bound peptide libraries were incubated for two hours with blocking buffer (10% v / v) blocking reagent (CRB (Norwich, UK)) and 1% (w / v) sucrose in TBS (aqueous solution 137 mM NaCl, 2, 7 mM KCl and 50 mM tris (hydroxymethyl) aminomethane, pH 8.0)). Subsequently, the dye-labeled domain, 10 μM in blocking buffer, became the library added: The libraries (1) and (2) were respectively labeled with the dye-labeled sequence of the GCN4 leucine zipper, the library (5) was labeled with the dye-labeled sequence Seq ID no. 6, 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 ββDELERRIRELEARIK (10 μM in blocking buffer). Negative controls were carried out in the same way by incubation with the dissolved dye (see above).

After incubation overnight, 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).

(1) While the peptide of the starting sequence showed only minor interaction with the dissolved leucine zipper domain of GCN4, surprisingly, variants showed a strong association. For example, the DELERRIRELRARTK and DELERRIRELRARSK and DELERRIRELNARRK and DELERRIRELQARRK variants, respectively, showed clearly visible signals compared to the output sequence (Figure 3 and Table 1).

(2) It was also found, quite unexpectedly, that two peptides with a shortened synthesized starting sequence (LERRIRELEARIK or RRIRELEARIK) showed increased association signals compared to the starting sequence with the GCN4-leucine zipper domain (Table 2).

(3) Quite surprisingly, high-affinity associating peptides (peptides # 40-54) were identified in the C-terminal region of the screened sequence (Seq ID No. 2), as well as four other regions containing at least one distinctly associative peptide (Figure 4).

(4) It was found, quite unexpectedly, that variants of the starting sequence IEKTNEKFHQIEKE, which had only a slight association with SEQ ID NO. 3, significantly enhanced with Seq ID. 3 associated (Figure 5 and Table 2). (5) Surprisingly, highly affinitive peptides were identified in the C-terminal region of the screened sequence (Seq ID No. 6) (Figure 6, Table 3).

(6) Quite surprisingly, certain peptides (Table 4) showed association with the constituents of the esat-6 / cfp-10 complex.

(8) Quite unexpectedly, certain peptides (Table 5) showed association with the dye-labeled Seq. ID No 3

Virus fusion experiments were performed as described in Schreiber et al., (Biophysical Journal 81 (2001) 1360-72) using peptide concentrations of 1 μM or 10 μM

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 aufeinanderfolgen¬

2,042 IEKTEEKFHQLKKK 128 1, 009 IEKTNEKKHQIKKK 20 the spots / dots from the upper ranks of the Biblio1,793 IEKTEENFHQIKKK 164 0,948 IEKTNEKNHQLKKK 40 thek (Figure 5), ranked 1, 729 IEKLKENFHQLKKE 647 0,932 IEKLKEKNHQLKKK 640 by signal intensity and 1, 641 IEKTKEKLHQLKKK 256 0,917 IEKTNEKFHQLEKK 6 order of arrangement. 1,640 IEKLNEKLHQLKKK 376 0,853 IEKLNEKNHQIKKK 396

1, 545 IEKTKENNHQIKKK 316 0,846 IEKLKEKKHQLKKK 624

Dot 1, 520 IEKTKENKHQLKKK 304 0.835 IEKLKENKHQIKKE 659 BLU no.

1, 479 IEKLNEKKHQIKKK 380 0,813 IEKLNENLHQIKKK 412

6,000 IEKLKEKFHQLKKK 608

1, 397 IEKLKENFHQLEKK 646 0,802 IEKTKEKFHQLEKK 246

5,475 IEKLKEKFHQIKKK 604

1, 379 IEKLNEEFHQIKKK 444 0,796 IEKTNENLHQLKKK 56

4,784 IEKLEEKFHQLKKK 488

1, 376 IEKTKENNHQLKKK 320 0,779 IEKTKENFHQIEKK 282

4,318 IEKLNENFHQLKKK 408

1, 368 lEKTKEKLHQIKKK 252 0,770 IEKTKEEFHQLKKK 328

4,239 IEKTKENFHQIKKK 284

1, 279 IEKLKEKFHQLEKK 606 0,764 IEKLKEKLHQLKKK 616

3,974 IEKTKEKFHQLKKK 248

1, 255 IEKLNEKKHQLKKK 384 0,762 IEKTNENKHQIKKK 60

3,934 1EKLNEKFHQIKKK 364

1, 238 IEKTKEEFHQIKKK 324 0,749 IEKTNEKKHQLKKK 24

3,894 IEKTKENFHQLKKK 288

1, 226 IEKLNEEFHQLKKK 448 0,740 IEKLNKFHQLKKE 367

3,728 IEKLKENFHQIKKK 644

1, 221 IEKLKENLHQLKKK 656 0,722 IEKLNENNHQIKKK 436

3,625 IEKLNEKFHQLKKK 368

1, 215 IEKLNENLHQLKKK 416 0,707 IEKLEEKFHQLKKE 487

3,577 IEKLNENFHQIKKK 404

1, 213 IEKTKEKKHQLKKK 264 0.683 IEKLNENKHQIKKK 420

3,308 IEKTKEKFHQIKKK 244

1, 208 IEKTKEKKHQIKKK 260 0,680 IEKTNENKHQLKKK 64

3,246 IEKLEENFHQIKKK 524

1, 192 IEKLKENKHQIKKK 660 0,678 IEKTEENNHQIKKK 196

2,897 IEKTNEKFHQLKKK 8

1, 185 IEKLNEKLHQIKKK 372 0,659 1EKLEEKFHQIEKK 482

2,886 IEKNENFHQIKKK 44

1, 185 IEKLNENFHQLKKE 407 0,637 IEKTNEKFHQ1EKE

2,820 IEKLKEKFHQLKKE 607

1, 182 IEKLKENLHQIKKK 652 0,635 IEKLKENKHQIEKK 658

2,742 IEKLKENFHQLKKK 648

1, 181 IEKLNENKHQLKKK 424 0.618

2,645 IEKTEEKFHQIKKK 124 IEKTNEKLHQLKKK 16

1, 077 1EKTNENEHQ1EKK 66 0.606

2,444 IEKTNEKFHQIKKK 4 IEKTKEKNHQIEKK 274 , 601 IEKLNENMHQIEKK 434 0.191 IEKLEEKLHQIKKK 492 0 IEKTNEKEHQIEKK 26, 588 IEKTNENNHQLKKK 80 0.183 IEKLKEKNHQIEKK 634 0 IEKTNEKEHQIKKE 27, 578 IEKLKEKNHQIKKK 636 0.176 IEKLEEEFHQLKKK 568 0 IEKTNEKEHQIKKK 28, 574 IEKLKEKFHQIKKE 603 0.171 IEKTKENNHQIEKE 313 0 IEKTNEKEHQLEKE 29, 562 IEKLKEKNHQLKKE 639 0.168 IEKTKENLHQLEKK 294 0 IEKTNEKEHQLEKK 30 , 560 IEKTKEKKHQIKKE 259 0.162 IEKLKENFHQIKKE 643 0 IEKTNEKNHQIEKE 33, 558 IEKTNEKNHQIKKK 36 0.150 IEKTEENKHQLKKK 184 0 IEKTNEKNHQIEKK 34, 557 IEKTKENLHQLKKK 296 0.150 IEKTNENNHQIKKK 76 0 IEKTNEKNHQIKKE 35, 554 IEKTEEKLHQLKKK 136 0.150 IEKTEEKKHQLKKK 144 0 IEKTNEKNHQLEKE 37, 550 IEKLNEKFHQIEKK 362 0.150 IEKTEENKHQIKKK 180 0 IEKTNEKNHQLEKK 38 , 530 IEKLNEKNHQLKKK 400 0.150 276 0 IEKTKEKNHQIKKK IEKTNEKNHQLKKE 39, 529 IEKTKENNHQLKKE 319 0.150 366 0 IEKLNEKFHQLEKK IEKTNENFHQIEKE 41, 510 IEKTKEKKHQIEKK 258 0.145 609 0 IEKLKEKLHQIEKE IEKTNENFHQIEKK 42, 503 IEKTKENKHQIKKE 299 0.141 649 0 IEKLKENLHQIEKE IEKTNENFHQIKKE 43 499 247 0.126 IEKTKEKFHQLKKE IEKLKEN FHQIEKK 642 0 IEKTNENFHQLEKK 46, 495 IEKTEENNHQLKKE 199 0.123 IEKTNEEKHQIKKE 99 0 IEKTNENLHQIEKE 49, 480 IEKTKENLHQIKKK 292 0.118 IEKLKEKLHQLEKK 614 0 IEKTNENLHQIKKE 51, 479 IEKTEEEFHQIKKK 204 0.109 lEKTNEKKHQLKKE 2 233 0 IEKTNENLHQLEKE 53, 479 IEKTNENLHQIKKK 52 0.109 IEKLEEKFHQLEKK 486 0 IEKTNENLHQLEKK 54, 472 IEKTKEKKHQLEKK 262 0.109 IEKTEENLHQLKKK 176 0 IEKTNENKHQIEKE 57, 468 IEKTEEKNHQIKKK 156 0.109 lEKLEENKHQLKKK 5 54444 0 IEKTNENKHQIEKK 58, 467 IEKTKEKFHQIEKK 242 0.104 IEKTNEKEHQLKKE 31 0 IEKTNENKHQIKKE 59, 444 IEKTNEKLHQIKKK 12 0.082 lEKTNENFHQLKKE 4477 0 IEKTNENKHQLEKE 61, 436 IEKTKEKNHQLKKK 280 0.082 IEKTKEKLHQLKKE 255 0 IEKTNENKHQLEKK 62, 434 IEKLNEEKHQIKKK 460 0.082 303 0 IEKTKENKHQLKKE IEKTNENKHQLKKE 63, 429 IEKLNENFHQIKKE 403 0.075 32 0 IEKTNEKEHQLKKK IEKTNENEHQIKKE 67, 410 IEKTKEKFHQIKKE 243 0.056 309 0 IEKTKENEHQLEKE IEKTNENEHQIKKK 68, 386 IEKLKEKFHQIEKK 602 0.053 55 0 IEKTNENLHQLKKE IEKTNENEHQLEKE 69, 385 IEKLKENLHQLEKK 654 0.044 149 0 IEKTEEKEHQLEKE IEKTNENEHQLEKK 70 , 384 IEKTEEKFHQLKKE 127 0.044 IEKTKEKLHQIKKE 251 0 IEKTNENEHQLKKE 71, 366 IEKLKEKKHQIKKK 620 0.043 IEKLNEKNHQIEKK 394 0 IEKTNENEHQLKKK 72, 361 IEKLKEKEHQLEKE 629 0.041 IEKTNEKKHQLEKK 22 0 IEKTNENNHQIEKE 73, 342 IEKTEEKNHQLKKK 160 0.041 IEKLNEKFHQIKKE 363 0 IEKTNENNHQIEKK 74, 338 IEKTNEKKHQIKKE 19 0.041 IEKLNEKKHQIKKE 379 0 IEKTNENNHQIKKE 75 , 332 IEKTEENNHQLEKK 198 0.041 IEKLEEKLHQLKKK 496 0 IEKTNENNHQLEKE 77, 332 IEKLEEENHQLKKE 599 0.041 IEKLEENLHQLKKK 536 0 IEKTNENNHQLEKK 78, 316 IEKLKEKLHQIKKK 612 0.041 IEKLEEEKHQLEKK 582 0 IEKTNENNHQLKKE 79, 297 IEKTKEKKHQLKKE 263 0.015 IEKLNEKLHQIEKK 370 0 IEKTNEEFHQIEKE 81, 294 IEKTKENKHQLEKK 302 0.014 IEKTEENEHQLEKK 190 0 IEKTNEEFHQIKKE 83 , 291 IEKTKEELHQIEKE 329 0.013 65 0 IEKTNENEHQIEKE IEKTNEEFHQLEKE 85 288 289 0.012 IEKTKENLHQIEKE IEKLKENLHQLEKE 653 0 IEKTNEEFHQLEKK 86, 285 IEKTEEKFHQIKKE 123 0.005 50 0 IEKTNENLHQIEKK IEKTNEEFHQLKKE 87, 276 IEKTKEKEHQLEKE 269 0.005 330 0 IEKTKEELHQIEKK IEKTNEEFHQLKKK 88 263 82 0 IEKTNEEFHQIEKK IEKTNEKFHQIKK E 3 0 lEKTNEELHQIEKE 89, 260 IEKTNEEFHQIKKK 84 0 IEKTNEKFHQLEKE 5 0 IEKTNEELHQIEKK 90, 258 IEKTEEKKHQIKKK 140 0 IEKTNEKFHQLKKE 7 0 IEKTNEELHQIKKE 91, 258 IEKTEENLHQIKKK 172 0 IEKTNEKLHQIEKE 9 0 IEKTNEELHQIKKK 92, 250 IEKLKEKKHQIKKE 619 0 IEKTNEKLHQIEKK 10 0 IEKTNEELHQLEKE 93, 246 IEKLEENFHQIKKE 523 0 IEKTNEKLHQIKKE 11 0 IEKTNEELHQLEKK 94, 236 IEKLNENFHQLEKK 406 0 IEKTNEKLHQLEKE 13 0 IEKTNEELHQLKKE 95, 235 IEKTKEKNHQLKKE 279 0 IEKTNEKLHQLEKK 14 0 IEKTNEELHQLKKK 96, 221 IEKTNENFHQLEKE 45 0 IEKTNEKLHQLKKE 15 0 IEKTNEEKHQIEKE 97, 219 IEKLEEKKHQLKKK 504 0 IEKTNEKKHQIEKE 17 0 IEKTNEEKHQIEKK 98, 214 IEKTNEKFHQIEKK 2 0 IEKTNEKKHQIEKK 18 0 IEKTNEEKHQIKKK 100, 202 IEKTEEKLHQIKKK 132 0 IEKTNEKKHQLEKE 21 0 IEKTNEEKHQLEKE 101, 191 IEKTEEENHQIKKK 236 0 IEKENEKEHQIEKE 25 0 IEKTNEEKHQLEKK 102 IEKTNEEKHQLKKE 103 0 IEKTEENLHQLEKE 173 O IEKTEEENHQLEKK 238 IEKTNEEKHQLKKK 104 0 IEKTEENLHQLEKK 174 O IEKTEEENHQLKKE 239 IEKTNEEEHQIEKE 105 0 IEKTEENLHQLKKE 175 O IEKTEEENHQLKKK 240 IEKTNEEEHQIEKK 106 0 IEKTEENKHQIEKE 177 O 1EKTKEKFHQIEKE 241 IEKTNEEEHQIKKE 107 0 IEKTEENKHQIEKK 178 O IEKTKEKFHQLEKE 245 IEKTNEEEHQIKKK 108 0 IEKTEENKHQIKKE 179 O IEKTKEKLHQIEKE 249 IEKTNEEEHQLEKE 109 0 IEKTEENKHQLEKE 181 O IEKTKEKLHQIEKK 250 IEKTNEEEHQLEKK 110 0 IEKTEENKHQLEKK 182 O IEKTKEKLHQLEKE 253 IEKTNEEEHQLKKE 111 0 IEKTEENKHQLKKE 183 O IEKTKEKLHQLEKK 254 IEKTNEEEHQLKKK 112 0 IEKTEENEHQIEKE 185 O IEKTKEKKHQIEKE 257 IEKTNEENHQIEKE 113 0 IEKTEENEHQIEKK 186 O IEKTKEKKHQLEKE 261 IEKTNEENHQIEKK 114 0 IEKTEENEHQIKKE 187 O IEKTKEKEHQIEKE 265 IEKTNEENHQIKKE 1 15 0 IEKTEENEHQIKKK 188 O IEKTKEKEHQ1EKK 266 IEKTNEENHQIKKK 116 0 IEKTEENEHQLEKE 189 O IEKTKEKEHQIKKE 267 IEKTNEENHQLEKE 1 17 0 IEKTEENEHQLKKE 191 O IEKTKEKEHQIKKK 268 IEKTNEENHQLEKK 118 0 IEKTEENEHQLKKK 192 O IEKTKEKEHQLEKK 270 IEKTNEENHQLKKE 1 19 0 IEKTEENNHQIEKE 193 O IEKTKEKEHQLKKE 271 IEKTNEENHQLKKK 120 0 1EKTEENNHQIEKK 194 O IEKTKEKEHQLKKK 272 IEKTEEKFHQIEKE 121 0 IEKTEENNHQIKKE 195 O IEKTKEKNHQIEKE 273 IEKTEEKFHQIEKK 122 0 1EKTEENNHQLEKE 197 O IEKTKEKNHQIKKE 275 IEKTEEKFHQLEKE 125 0 IEKTEENNHQLKKK 200 O IEKTKEKNHQLEKE 277 IEKTEEKFHQLEKK 126 0 IEKTEEEFHQIEKE 201 O IEKTKEKNHQLEKK 278 IEKTEEKLHQIEKE 129 0 IEKTEEEFHQIEKK 202 O 1EKTKENFHQIEKE 281 IEKTEEKLHQIEKK 130 0 IEKTEEEFHQIKKE 203 O IEKTKENFHQIKKE 283 IEKTEEKLHQIKKE 131 0 IEKTEEEFHQLEKE 205 O IEKTKENFHQLEKE 285 IEKTEEKLHQLEKE 133 0 IEKTEEEFHQLEKK 206 O IEKTKENFHQLEKK 286 IEKTEEKLHQLEKK 134 0 IEKTEEEFHQLKKE 207 O IEKTKENFHQLKKE 287 IEKTEEKLHQLKKE 135 0 1EKTEEEFHQLKKK 208 O IEKTKENLHQIEKK 290 IEKTEEKKHQIEKE 137 0 IEKTEEELHQIEKE 209 O IEKTKENLHQIKKE 291 IEKTEEKKHQIEKK 138 0 IEKTEEELHQIEKK 210 HEALTHCARE 293 IEKTEEKKHQIKKE 139 0 IEKTEEELHQIKKE 211 O IEKTKENLHQLKKE 295 IEKTEEKKHQLEKE 141 0 IEKTEEELHQIKKK 212 HEALTH CARE HEALTH 297 IEKTEEKKHQLEKK 142 0 IEKTEEELHQLEKE 213 O IEK TKENKHQIEKK 298 IEKTEEKKHQLKKE 143 0 IEKTEEELHQLEKK 214 O IEKTKENKHQLEKE 301 IEKTEEKEHQIEKE 145 0 IEKTEEELHQLKKE 215 O IEKTKENEHQIEKE 305 IEKTEEKEHQIEKK 146 0 IEKTEEELHQLKKK 216 O IEKTKENEHQIEKK 306 IEKTEEKEHQIKKE 147 0 IEKTEEEKHQIEKE 217 O IEKTKENEHQIKKE 307 IEKTEEKEHQIKKK 148 0 IEKTEEEKHQIEKK 218 O IEKTKENEHQIKKK 308 IEKTEEKEHQLEKK 150 0 1EKTEEEKHQIKKE 219 O IEKTKENEHQLEKK 310 IEKTEEKEHQLKKE 151 0 IEKTEEEKHQIKKK 220 O IEKTKENEHQLKKE 311 IEKTEEKEHQLKKK 152 0 IEKTEEEKHQLEKE 221 O IEKTKENEHQLKKK 312 IEKTEEKNHQIEKE 153 0 lEKTEEEKHQLEKK 222 O IEKTKENNHQIEKK 314 IEKTEEKNHQIEKK 154 0 IEKTEEEKHQLKKE 223 O IEKTKENNHQIKKE 315 IEKTEEKNHQIKKE 155 0 IEKTEEEKHQLKKK 224 O IEKTKENNHQLEKE 317 IEKTEEKNHQLEKE 157 0 IEKTEEEEHQIEKE 225 O IEKTKENNHQLEKK 318 IEKTEEKNHQLEKK 158 0 IEKTEEEEHQIEKK 226 O IEKTKEEFHQIEKE 321 IEKTEEKNHQLKKE 159 0 IEKTEEEEHQIKKE 227 O IEKTKEEFHQIEKK 322 IEKTEENFHQIEKE 161 0 IEKTEEEEHQIKKK 228 O IEKTKEEFHQIKKE 323 IEKTEENFHQIEKK 162 0 IEKTEEEEHQLEKE 229 O IEKTKEEFHQLEKE 325 IEKTEENF HQIKKE 163 0 IEKTEEEEHQLEKK 230 O IEKTKEEFHQLEKK 326 IEKTEENFHQLEKE 165 0 IEKTEEEEHQLKKE 231 O IEKTKEEFHQLKKE 327 IEKTEENFHQLEKK 166 0 IEKTEEEEHQLKKK 232 O IEKTKEELHQIKKE 331 IEKTEENFHQLKKE 167 0 IEKTEEENHQIEKE 233 O IEKTKEELHQIKKK 332 IEKTEENLHQIEKE 169 0 1EKTEEENHQ1EKK 234 O IEKTKEELHQLEKE 333 IEKTEENLHQ1EKK 170 0 IEKTEEENHQIKKE 235 O IEKTKEELHQLEKK 334 IEKTEENLHQIKKE 171 0 IEKTEEENHQLEKE 237 O IEKTKEELHQLKKE 335 IEKTKEELHQLKKK 336 0 lEKLNENLHQLEKE 413 O IEKLNEENHQLEKK 478 IEKTKEEKHQIEKE 337 0 IEKLNENLHQLEKK 414 O IEKLNEENHQLKKE 479 IEKTKEEKHQIEKK 338 0 IEKLNENLHQLKKE 415 O IEKLNEENHQLKKK 480 IEKTKEEKHQIKKE 339 0 IEKLNENKHQIEKE 417 O IEKLEEKFHQIEKE 481 IEKTKEEKHQIKKK 340 0 IEKLNENKHQIEKK 418 O IEKLEEKFHQIKKE 483 IEKTKEEKHQLEKE 341 0 IEKLNENKHQIKKE 419 O IEKLEEKFHQLEKE 485 IEKTKEEKHQLEKK 342 0 IEKLNENKHQLEKE 421 O IEKLEEKLHQIEKE 489 IEKTKEEKHQLKKE 343 0 IEKLNENKHQLEKK 422 O IEKLEEKLHQIEKK 490 1EKTKEEKHQLKKK 344 0 IEKLNENKHQLKKE 423 O IEKLEEKLHQIKKE 491 IEKTKEEEHQIEKE 345 0 1EKLNENEHQIEKE 425 O 1EKLEEKLHQLEKE 493 IEKTKEEEHQIEKK 346 0 IEKLNENEHQIEKK 426 O IEKLEEKLHQLEKK 494 IEKTKEEEHQIKKE 347 0 IEKLNENEHQIKKE 427 O IEKLEEKLHQLKKE 495 IEKTKEEEHQIKKK 348 0 IEKLNENEHQIKKK 428 O IEKLEEKKHQIEKE 497 IEKEKEEEHQLEKE 349 0 IEKLNENEHQLEKE 429 O IEKLEEKKHQIEKK 498 IEKTKEEEHQLEKK 350 0 IEKLNENEHQLEKK 430 O IEKLEEKKHQIKKE 499 IEKTKEEEHQLKKE 351 0 IEKLNENEHQLKKE 431 O IEKLEEKKHQIKKK 500 IEKTKEEEHQLKKK 352 0 IEK LNENEHQLKKK 432 O IEKLEEKKHQLEKE 501 IEKTKEENHQIEKE 353 0 IEKLNENNHQIEKE 433 O IEKLEEKKHQLEKK 502 IEKTKEENHQIEKK 354 0 IEKLNENNHQIKKE 435 O IEKLEEKKHQLKKE 503 IEKTKEENHQIKKE 355 0 IEKLNENNHQLEKE 437 O IEKLEEKEHQIEKE 505 IEKTKEENHQIKKK 356 0 IEKLNENNHQLEKK 438 O IEKLEEKEHQIEKK 506 IEKTKEENHQLEKE 357 0 1EKLNENNHQLKKE 439 O lEKLEEKEHQIKKE 507 IEKTKEENHQLEKK 358 0 1EKLNEEFHQIEKE 441 O IEKLEEKEHQiKKK 508 IEKTKEENHQLKKE 359 0 IEKLNEEFHQIEKK 442 O IEKLEEKEHQLEKE 509 IEKTKEENHQLKKK 360 0 IEKLNEEFHQ1KKE 443 O IEKLEEKEHQLEKK 510 IEKLNEKFHQIEKE 361 0 IEKLNEEFHQLEKE 445 O IEKLEEKEHQLKKE 511 IEKLNEKFHQLEKE 365 0 IEKLNEEFHQLEKK 446 O IEKLEEKEHQLKKK 512 1EKLNEKLHQ1EKE 369 0 (EKLNEEFHQLKKE 447 O IEKLEEKNHQIEKE 513 IEKLNEKLHQIKKE 371 0 IEKLNEELHQIEKE 449 O IEKLEEKNHQIEKK IEKLNEKLHQLEKE 514 373 0 450 O IEKLNEELHQIEKK IEKLEEKNHQIKKE IEKLNEKLHQLEKK 515 374 0 451 O IEKLNEELHQ1KKE iEKLEEKNHQIKKK IEKLNEKLHQLKKE 516 375 0 452 O IEKLNEELHQIKKK IEKLEEKNHQLEKE IEKLNEKKHQIEKE 517 377 0 453 O IEKLNEELHQLEKE IEKLEE KNHQLEKK 518 IEKLNEKKHQIEKK 378 0 IEKLNEELHQLEKK 454 O IEKLEEKNHQLKKE 519 IEKLNEKKHQLEKE 381 0 IEKLNEELHQLKKE 455 O IEKLEEKNHQLKKK 520 IEKLNEKKHQLEKK 382 0 IEKLNEELHQLKKK 456 O IEKLEENFHQIEKE 521 IEKLNEKKHQLKKE 383 0 IEKLNEEKHQIEKE 457 O IEKLEENFHQIEKK 522 IEKLNEKEHQIEKE 385 0 IEKLNEEKHQIEKK 458 O IEKLEENFHQLEKE 525 IEKLNEKEHQIEKK 386 0 IEKLNEEKHQ1KKE 459 O IEKLEENFHQLEKK 526 IEKLNEKEHQIKKE 387 0 IEKLNEEKHQLEKE 461 O IEKLEENFHQLKKE 527 IEKLNEKEHQIKKK 388 0 IEKLNEEKHQLEKK 462 O IEKLEENLHQIEKE 529 IEKLNEKEHQLEKE 389 0 IEKLNEEKHQLKKE 463 O IEKLEENLHQIEKK 530 IEKLNEKEHQLEKK 390 0 IEKLNEEKHQLKKK 464 O IEKLEENLHQIKKE 531 lEKLNEKEHQLKKE 391 0 IEKLNEEEHQIEKE 465 O IEKLEENLHQIKKK 532 IEKLNEKEHQLKKK 392 0 IEKLNEEEHQIEKK 466 O IEKLEENLHQLEKE 533 IEKLNEKNHQIEKE 393 0 1EKLNEEEHQIKKE 467 O IEKLEENLHQLEKK 534 IEKLNENKNHQIKKE 395 0 IEKLNEEEHQIKKK 468 O LEBLEENLHQLKKE 535 IEKLENKNHQLEKE 397 0 IEKLNEEEHQLEKE 469 O IEKLEENKHQIEKE 537 IEKLNEKNHQLEKK 398 0 IEKLNEEEHQLEKK 470 O IEKLEENKHQIEKK 538 IEKLNEKNHQL KKE 399 0 IEKLNEEEHQLKKE 471 O IEKLEENKHQ1KKE 539 IEKLNENFHQIEKE 401 0 IEKLNEEEHQLKKK 472 O IEKLEENKHQIKKK 540 IEKLNENFHQIEKK 402 0 IEKLNEENHQIEKE 473 O IEKLEENKHQLEKE 541 IEKLNENFHQLEKE 405 0 IEKLNEENHQIEKK 474 O IEKLEENKHQLEKK 542 IEKLNENLHQIEKE 409 0 IEKLNEENHQIKKE 475 O lEKLEENKHQLKKE 543 IEKLNENLHQIEKK 410 0 IEKLNEENHQ1KKK 476 O lEKLEENEHQIEKE 545 IEKLNENLHQIKKE 411 0 IEKLENENHQLEKE 477 O IEKLEENEHQIEKK 546 O IEKLEENEHQIKKE 547 0 IEKLEEELHQLKKE 575 0 IEKLKEKLHQIKKE 611

O IEKLEENEHQIKKK 548 0 IEKLEEELHQLKKK 576 0 IEKLKEKLHQLEKE 613

O IEKLEENEHQLEKE 549 0 IEKLEEEKHQIEKE 577 0 IEKLKEKLHQLKKE 615

O IEKLEENEHQLEKK 550 0 IEKLEEEKHQIEKK 578 0 IEKLKEKKHQIEKE 617

O IEKLEENEHQLKKE 551 0 IEKLEEEKHQIKKE 579 0 IEKLKEKKHQIEKK 618

O IEKLEENEHQLKKK 552 0 IEKLEEEKHQIKKK 580 0 IEKLKEKKHQLEKE 621

O IEKLEENNHQIEKE 553 0 IEKLEEEKHQLEKE 581 0 IEKLKEKKHQLEKK 622

O 1EKLEENNHQIEKK 554 0 IEKLEEEKHQLKKE 583 0 IEKLKEKKHQLKKE 623

O IEKLEENNHQIKKE 555 0 IEKLEEEKHQLKKK 584 0 IEKLKEKEHQIEKE 625

O IEKLEENNHQIKKK 556 0 IEKLEEEEHQIEKE 585 0 IEKLKEKEHQIEKK 626

O IEKLEENNHQLEKE 557 0 IEKLEEEEHQIEKK 586 0 IEKLKEKEHQIKKE 627

O IEKLEENNHQLEKK 558 0 IEKLEEEEHQIKKE 587 0 IEKLKEKEHQIKKK 628

O IEKLEENNHQLKKE 559 0 IEKLEEEEHQIKKK 588 0 1EKLKEKEHQLEKK 630

O IEKLEENNHQLKKK 560 0 IEKLEEEEHQLEKE 589 0 IEKLKEKEHQLKKE 631

O IEKLEEEFHQIEKE 561 0 IEKLEEEEHQLEKK 590 0 IEKLKEKEHQLKKK 632

O IEKLEEEFHQIEKK 562 0 IEKLEEEEHQLKKE 591 0 IEKLKEKNHQIEKE 633

O IEKLEEEFHQIKKE 563 0 IEKLEEEEHQLKKK 592 0 IEKLKEKNHQIKKE 635

O IEKLEEEFHQIKKK 564 0 IEKLEEENHQIEKE 593 O IEKLKEKNHQLEKE 637

O IEKLEEEFHQLEKE 565 0 IEKLEEENHQIEKK 594 0 IEKLKEKNHQLEKK 638

O IEKLEEEFHQLEKK 566 0 IEKLEEENHQIKKE 595 0 IEKLKENFHQIEKE 641

O 1EKLEEEFHQLKKE 567 0 IEKLEEENHQIKKK 596 0 IEKLKENFHQLEKE 645

O IEKLEEELHQIEKE 569 0 IEKLEEENHQLEKE 597 0 IEKLKENLHQIEKK 650

O IEKLEEELHQIEKK 570 0 IEKLEEENHQLEKK 598 0 IEKLKENLHQ1KKE 651

O IEKLEEELHQIKKE 571 0 IEKLEEENHQLKKK 600 0 IEKLKENLHQLKKE 655

O IEKLEEELHQIKKK 572 0 IEKLKEKFHQIEKE 601 0 IEKLKENKHQIEKE 657

O IEKLEEELHQLEKE 573 0 LEBKEFHQLEKE 605

O IEKLEEELHQLEKK 574 0 IEKLKEKLHQIEKK 610

Table 3:

YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNW

TSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

YTSLIHSLIEESQNQQEKNEQELLELDKWASLWN

TSLIHSLIEESQNQQEKNEQELLELDKWASLWNW

SLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

YTSLIHSLIEESQNQQEKNEQELLELDKWASLW

TSLIHSLIEESQNQQEKNEQELLELDKWASLWN

SLIHSLIEESQNQQEKNEQELLELDKWASLWNW

LIHSLIEESQNQQEKNEQELLELDKWASLWNWF

TSLIHSLIEESQNQQEKNEQELLELDKWASLW

LIHSLIEESQNQQEKNEQELLELDKWASLWNW

IHSLIEESQNQQEKNEQELLELDKWASLWNWF

IHSLIEESQNQQEKNEQELLELDKWASLWNW

HSLIEESQNQQEKNEQELLELDKWASLWNWF

LIHSLIEESQNQQEKNEQELLELDKWASLW

IHSLIEESQNQQEKNEQELLELDKWASLWN

HSLIEESQNQQEKNEQELLELDKWASLWNW

SLIEESQNQQEKNEQELLELDKWASLWNWF

IHSLIEESQNQQEKNEQELLELDKWASLW

HSLIEESQNQQEKNEQELLELDKWASLWN

SLIEESQNQQEKNEQELLELDKWASLWNW

LIEESQNQQEKNEQELLELDKWASLWNWF

HSLIEESQNQQEKNEQELLELDKWASLW

SLIEESQNQQEKNEQELLELDKWASLWN

LIEESQNQQEKNEQELLELDKWASLWNW

IEESQNQQEKNEQELLELDKWASLWNWF

SLIEESQNQQEKNEQELLELDKWASLW

LIEESQNQQEKNEQELLELDKWASLWN IEESQNQQEKNEQELLELDKWASLWNW

EESQNQQEKNEQELLELDKWASLWNWF

LIEESQNQQEKNEQELLELDKWASLW

IEESQNQQEKNEQELLELDKWASLWN

EESQNQQEKNEQELLELDKWASLWNW

ESQNQQEKNEQELLELDKWASLWNWF

IEESQNQQEKNEQELLELDKWASLW

EESQNQQEKNEQELLELDKWASLWN

ESQNQQEKNEQELLELDKWASLWNW

SQNQQEKNEQELLELDKWASLWNWF

EESQNQQEKNEQELLELDKWASLW

ESQNQQEKNEQELLELDKWASLWN

SQNQQEKNEQELLELDKWASLWNW

QNQQEKNEQELLELDKWASLWNWF

ESQNQQEKNEQELLELDKWASLW

QNQQEKNEQELLELDKWASLWNW

NQQEKNEQELLELDKWASLWNWF

SQNQQEKNEQELLELDKWASLW

QNQQEKNEQELLELDKWASLWN

NQQEKNEQELLELDKWASLWNW

QQEKNEQELLELDKWASLWNWF

QNQQEKNEQELLELDKWASLW

NQQEKNEQELLELDKWASLWN

QQEKNEQELLELDKWASLWNW

QEKNEQELLELDKWASLWNWF

NQQEKNEQELLELDKWASLW

QQEKNEQELLELDKWASLWN

QEKNEQELLELDKWASLWNW

EKNEQELLELDKWASLWNWF

QQEKNEQELLELDKWASLW

QEKNEQELLELDKWASLWN

EKNEQELLELDKWASLWNW

KNEQELLELDKWASLWNWF

QEKNEQELLELDKWASLW

EKNEQELLELDKWASLWN

KNEQELLELDKWASLWNW

NEQELLELDKWASLWNWF

EKNEQELLELDKWASLW

KNEQELLELDKWASLWN

NEQELLELDKWASLWNW

EQELLELDKWASLWNWF

KNEQELLELDKWASLW

NEQELLELDKWASLWN

EQELLELDKWASLWNW

QELLELDKWASLWNWF

NEQELLELDKWASLW

EQELLELDKWASLWN

QELLELDKWASLWNW

ELLELDKWASLWNWF

EQELLELDKWASLW

QELLELDKWASLWN

ELLELDKWASLWNW

LLELDKWASLWNWF

EQELLELDKWASL

QELLELDKWASLW

ELLELDKWASLWN

LLELDKWASLWNW

LELDKWASLWNWF

EQELLELDKWAS

QELLELDKWASL

ELLELDKWASLW

LLELDKWASLWN LELDKWASLWNW ELDKWASLWNWF

EQELLELDKWA

QELLELDKWAS

ELLELDKWASL

LLELDKWASLW

LELDKWASLWN

ELDKWASLWNW

LDKWASLWNWF

EQELLELDKW

QELLELDKWA

ELLELDKWAS

LLELDKWASL

LELDKWASLW

ELDKWASLWN

LDKWASLWNW

DKWASLWNWF

EQELLELDK

QELLELDKW

ELLELDKWA

LLELDKWAS

LELDKWASL

ELDKWASLW

LDKWASLWN

DKWASLWNW

KWASLWNWF

EQELLELD

QELLELDK

ELLELDKW

LLELDKWA

LELDKWAS

ELDKWASL

LDKWASLW

DKWASLWN

KWASLWNW

WASLWNWF

EQELLEL

QELLELD

ELLELDK

LLELDKW

LELDKWA

ELDKWAS

LDKWASL

DKWASLW

KWASLWN

WASLWNW

ASLWNWF

Table 4:

NFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGS

AGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGS

LEAAASALQGNVTSIHSLLDEGKQSLTKLAAAWGGS

AAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGS

FAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLA

AAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGG

AASAIQGNVTSIHSLLDEGKQSLTKLAAAWGG

FAGIEAAASAIQGNVTSIHSLLDEGKQSLT

GIEAAASAIQGNVTSIHSLLDEGKQSLTKL

AAASAI ^Q GNVTSIHSLLDEGKQSLTKLAAA

ASAIQGNVTSIHSLLDEGKQSLTKLAAAWG AAASAIQGNVTSIHSLLDEGKQSLTKLAA

AIQGNVTSIHSLLDEGKQSLTKLAAAWGG

AASAIQGNVTSIHSLLDEGKQSLTKLAA

QGNVTSIHSLLDEGKQSLTKLAAAWGGS

FAGIEAAASAIQGNVTSIHSLLDEGKQ

AIQGNVTSIHSLLDEGKQSLTKLAAAW

NFAGIEAAASAIQGNVTSIHSLLDEG

QGNVTSIHSLLDEGKQSLTKLAAAWG

NVTSIHSLLDEGKQSLTKLAAAWGGS

AGIEAAASAIQGNVTSIHSLLDEGK

SAIQGNVTSIHSLLDEGKQSLTKLA

NVTSIHSLLDEGKQSLTKLAAAWGG

IEAAASAIQGNVTSIHSLLDEGKQ

SAIQGNVTSIHSLLDEGKQSLTKL

AIQGNVTSIHSLLDEGKQSLTKLA

IQGNVTSIHSLLDEGKQSLTKLAA

QGNVTSIHSLLDEGKQSLTKLAAA

VTSIHSLLDEGKQSLTKLAAAWGG

EAAASAIQGNVTSIHSLLDEGKQ

AIQGNVTSIHSLLDEGKQSLTKL

GNVTSIHSLLDEGKQSLTKLAAA

TSIHSLLDEGKQSLTKLAAAWGG

GIEAAASAIQGNVTSIHSLLDE

IEAAASAIQGNVTSIHSLLDEG

EAAASAIQGNVTSIHSLLDEGK

SAIQGNVTSIHSLLDEGKQSLT

TSIHSLLDEGKQSLTKLAAAWG

EAAASAIQGNVTSIHSLLDEG

ASAIQGNVTSIHSLLDEGKQS

GNVTSIHSLLDEGKQSLTKLA

NVTSIHSLLDEGKQSLTKLAA

VTSIHSLLDEGKQSLTKLAAA

HSLLDEGKQSLTKLAAAWGGS

NFAGIEAAASAIQGNVTSIH

EAAASAIQGNVTSIHSLLDE

AAASAIQGNVTSIHSLLDEG

NVTSIHSLLDEGKQSLTKLA

SIHSLLDEGKQSLTKLAAAW

FAGIEAAASAIQGNVTSIH

GIEAAASAIQGNVTSIHSL

ASAIQGNVTSIHSLLDEGK

GNVTSIHSLLDEGKQSLTK

NFAGIEAAASAIQGNVTS

QGNVTSIHSLLDEGKQSL

VTSIHSLLDEGKQSLTKL

TSIHSLLDEGKQSLTKLA

HSLLDEGKQSLTKLAAAW

SLLDEGKQSLTKLAAAWG

AGIEAAASAIQGNVTSI

NVTSIHSLLDEGKQSLT

IHSLLDEGKQSLTKLAA

LLDEGKQSLTKLAAAWG

LDEGKQSLTKLAAAWGG

DEGKQSLTKLAAAWGGS

NFAGIEAAASAIQGNV

FAGIEAAASAIQGNVT

AGIEAAASAIQGNVTS

AASAIQGNVTSIHSLL

HSLLDEGKQSLTKLAA

DEGKQSLTKLAAAWGG

AGIEAAASAIQGNVT

GIEAAASAIQGNVTS IEAAASAIQGNVTSI

EAAASAIQGNVTSIH

IQGNVTSIHSLLDEG

DEGKQSLTKLAAAWG

EGKQSLTKLAAAWGG

FAGIEAAASAIQGN

EAAASAIQGNVTSI

AAASAIQGNVTSIH

AASAIQGNVTSIHS

ASAIQGNVTSIHSL

NVTSIHSLLDEGKQ

DEGKQSLTKLAAAW

KQSLTKLAAAWGGS

NFAGIEAAASAIQ

ASAIQGNVTSIHS

SAIQGNVTSIHSL

GKQSLTKLAAAWG

NFAGIEAAASAI

FAGIEAAASAIQ

GIEAAASAIQGN

TSIHSLLDEGKQ

SLLDEGKQSLTK

KQSLTKLAAAWG

NFAGIEAAASA

FAGIEAAASAI

GIEAAASAIQG

TSIHSLLDEGK

SLLDEGKQSLT

DEGKQSLTKLA

KQSLTKLAAAW

LTKLAAAWGGS

FAGIEAAASA

AGIEAAASAI

IEAAASAIQG

VTSIHSLLDE

HSLLDEGKQS

LDEGKQSLTK

SLTKLAAAWG

TKLAAAWGGS

NFAGIEAAA

AGIEAAASA

GNVTSIHSL

IHSLLDEG

SLLDEGKQS

KQSLTKLAA

SLTKLAAAW

KLAAAWGGS

AIQGNVTS

Table 5:

DELERRIRELEARIK

FELERRIRELEARIK

LELERRIRELEARIK

DELEFRIREPFARIK

DELERRIREYFARIK

DELERRIRELFARIK

DELEIRIRELFARIK

FELERRIREPEARIK

LELERRIREPEARIK

DELERRIREYEARIK DELERRIREYEARIK

LELERRIREYEARIK

DELERRIREYEARWK

DFLERRIREYEARIK

DELEFRIRELEARIK

DELEIRIRELEARIK

FELERRIREPEARWK

DELERRIRELEARWK

Legend with detailed description of the illustrations:

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 α-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. Into a "hole" of the opposite helix in which it is surrounded by the residues 1d ^' , 1g', 2a 'and 2d' Analogously, the residues 1d, 1g, 2a and 2d enclose the opposing residue 2a ^' (not A similar "button" is also formed by the leucine in 2d, surrounded by 2a ', 2d ^' , 3a ^' and 2e'. The rectangular markings in the upper part of the illustration show, by way of example, the two layers of the hydrophobic contact surfaces. One consists of the residues of the positions e ^' , d, d ^' and e, the other contains g, a ^' , a, and g ^' . The arrow shows the axis of double rotational symmetry, which coincides with the axis of the superhelix.

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

Seq ID no. 3. Shown are the association signals of the individual peptides (dots 1-54).

Figure 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:

IEKTNEKFHQIEKE (output sequence) abcdef gabcdef g (heptad positions) E LE EE LK K (inserted residues) K K NK L L

N N

after incubation with Farbstff-labeled peptide of the sequence Seq ID no. Third

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 # δ after incubation with dye-labeled sequence of Seq ID No.3.

Figure 8: Binding signals of library (3) after incubation with dye-labeled sequence ββDELERRIRELEARIK. The following sequences showed binding: QDLEKYVEDTKIDL ^*

DLEKYVEDTKIDLW * LEKYVEDTKIDLWS * EKYVEDTKIDLWSY * KYVEDTKIDLWSYN * YVEDTKIDLWSYNA *

VEDTKIDLWSYNAE *

EDTKIDLWSYNAEL *

DTKIDLWSYNAELL *

TKIDLWSYNAELLV *

KIDLWSYNAELLVA *

IDLWSYNAELLVAL *

DLWSYNAELLVALE *

LWSYNAELLVALEN *

WSYNAELLVALENQ *

SYNAELLVALENQH *

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.

sequences

Seq ID No.1 (Hemagglutinin - Influenza A virus (H3N6)):

MKTIIVLSCFFCLAFSQNPSENNNNTATLCLGHHAVPNGTIVKTITDDQIEVTNATELVQ SSSTGKICNNPHRILDGRDCTLMDALLGDPHCDVFQDETWDLYVERSNAFSNCYPYDVPD YASLRSLVASSGTLEFITEGFTWTGVTQNGGSGACKRGPTNGFFSRLNWLTKSGSAYPLL NVTMPNNDNFDKLYVWGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVR GQSGRISIYWTIVKPGDILVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITP NGSIPNDKPFQNVNKITYGACPKYVKQSTLKLATGMRNVPEKQTRGLFGAIAGFIENGWE GMIDGWYGFRHQNSEGTGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEG RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENΆEDMGN GCFKIYHKCDNACIESIRNGTYDHDIYRDEALNNRFQIKGVELKSGYKDWILWISFAISC FLLCVVLLGFIMWACQRGNIRCNICI

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):

MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACV PTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVIKLTPLCVSLKCTDLKNDTNTNSSSGRMIM EKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGF AILKCNNKTFNGTGPCTINVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCT RPNINNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEI VTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCS SNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGALFLG FLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIW GCSGKLICTTAVPWNASWSNKSLEQIWNHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWN WFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSI RLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIA VAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL Seq ID No.5 (esat-6):

MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLΛRT ISEAGQAMASTEGNVTGMFA

SEQ ID NO: 6 (β = β-alanine):

Seq ID No.7:

NFAGIEAAASAIQGNVTS IHSLLDEGKQSLTKLAAAWGGS

references

Behncken SN, Billestrup N, Brown R, Amstrup J, Conway-Campbell B, Waters MJ. Growth hormone (GH) -independent dimerization of GH receptor by a leucine zipper results in constitutive activation. J Biol Chem 275 17000-7 (2000),

PA, Hughson FM, Skehel JJ, Wiley DC. Structure of influenza haemagglutinin atthe pH of membrane fusion. Nature 371 37-43 (1994).

Burkhard P, Stetefeld J, Strelkov SV. Coiled coils: a highly versatile protein folding motif.

Trends Cell Biol 11 82-88 (2001).

Chan DC, Kim PS. HIV entry and is inhibition. Ce // 93681-684 (1998).

Chen J, Skehel JJ, Wiley DC. W and C-terminal residues combine in thefusion-pH influenza hemagglutinin HA2 subunit to form an N cap that terminates the triple-stranded coiled coil. Proc Natl

Acad Sci USA 968967-8972 (1999).

Contegno F, Cioce M, Pelicci PG, Minucci S. Targeting protein inactivation through an oligomerization chain reaction. Proc Natl Acad Be U S A 99 1865-9 (2002).

Crick FH. The packing of α-helices: simple coiled coils. Acta Crystallogr 6 689-698 (1953).

Gentz R, Rauscher FJ, Abate C, Curran T. Parallel association of Fos and Jun leucine zippers juxtapose DNA binding domains. Science 243 1695-9 (1989).

Ghosh I, Hamilton AD, Regan L Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. JAm Chem Soc 122 5658-9

(2000).

Goldenberg DM. Advancing role of radiolabelled antibodies in the therapy of cancer. Cancer

Immunol Immunother 52 281-96 (2003).

Goodwin DA, Meares CF. Advances in pretargeting biotechnology. Biotechnol Adv 19435-50

(2001). • John M, Briand JP, Granger-Schnarr M, Schnarr M. Two pairs of oppositely charged amino acids from Jun and Fos confer heterodimerization to GGN4 leucine zipper. JS / o / Chem 269 16247-53 (1994).

• Katz BZ, Krylov D, Aota S, Olive M, Vinson C, Yamada KM. Green fluorescent protein labeling of cytoskeletal structures-novel targeting approach based on leucine zippers. Biotechniques 25 298-304 (1998).

• Knox SJ, Goris ML, Tempero M, Weiden PL, Gentner L, Breitz H, Adams GP, Axworthy D, Gaffigan S, Bryan K, Fisher DR, Colcher D, Horak ID, Weiner LM. Phase II trial of yttrium-90-DOTA-biotin pretargeted by NR-LU-10 antibody / streptavidin in patients with metastatic colon cancer. Clin Cancer Res 6 406-14 (2000).

• High School WH, Johnson PF, McKnight SL. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240 1759-64 (1988).

• Lau SY, Taneja AK, Hodges RS. Synthesis of a model protein of defined secondary and quaternary structure. Effect of chain length on the stabilization and formation of two-stranded alpha-helical coiled-coils. J BbI Chem 259 13253-61 (1984).

• Lupas A. Coiled coils: new structures and new functions. Trends Biochem. 21: 375-82 (1996).

• Mason JM, Arndt KM. Coiled coil domains: stability, speeificity, and biological implications. Chembiochem 5 170-6 (2004).

• McGaughey GB, Barbato G, Bianchi E, Freidinger RM, Garsky VM, WM Hurni, Joyce JG, Liang X ₁ Miller MD, Pessi A, Shiver JW, Bogusky MJ. Progress towards the development of an HIV-1 gp41-directed vaccine. CurrHIV Res 2 193-204 (2004).

• Moll JR, Ruvinov SB, Pastan I ₁ Vinson C. Designed heterodimerizing leucine zippers with a ranger of pls and capabilities up to 10 ^"15 M. Protein Be 649-55 (2001).

• O'Shea EK, Klemm JD, Kim PS, Alber T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254 539-44 (1991).

• O'Shea EK, Rutkowski R, Kim PS. Mechanism of speeificity in the Fos-Jun oncoprotein heterodimer. Ce // 68 699-708 (1992).

• O'Shea EK, Rutkowski R, Kim PS. Preferential heterodimer formation by isolated leucine zippers from fos and jun. Science 245 646-8 (1989).

• Petka WA, Harden JL, McGrath KP, Wirtz D, Tirrell DA. Reversible hydrogels from self-assembling artificial proteins. Science 281 389-92 (1998).

• Scott CA, Garcia KC, Carbone FR, Wilson IA, Teyton L. RoIe of chain pairing for the production of functional soluble IA major histocompatibility complex class II molecules. J Exp Med 183 2087-95 (1996).

• Tang A, Wang C, Stewart RJ, Kopecek J. The coiled coils in the design of protein-based construets: hybrid hydrogels and epitope displays. J Control Release 72 57-70 (2001).

Töpert F, Knaute T, Guffler S, Pires JR, Matzdorf T, Oschkinat H, Schneider-Mergener J. Combining SPOT synthesis. Angew Chem Int Ed Engl 42 1136-40 (2003). • Wang C, Kopecek J. ₁ Stewart RJ. Hybrid hydrogels cross-linked by genetically engineered coiled-coil block proteins. Biomacromolecules 2 912-20 (2001).

• Wang C, Stewart RJ, Kopecek J. Hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains. Nature 397 417-20 (1999).

All the features illustrated in the foregoing description, the appended claims and the drawings may be relevant to the realization of the invention in its various forms both individually and in any combination.

Claims

claims

1. Peptides for association with natural or derived alpha-helical coiled-coil sequences of the general formula (I)

(abcdefg) _n (I),

in which a-g denote amino acid residues of which a and / or d are hydrophobic and e and / or g are charged and n is a number from 1 to 3 or parts thereof consisting of a minimum of 2 and a maximum of 15 amino acid residues.

2. Peptide according to claim 1, characterized in that it is formed from a minimum of 7 and a maximum of 14 amino acid residues.

3. A peptide according to any one of claims 1 to 2, characterized in that on and d _n either both hydrophobic or _{n a} hydrophilic and d _{n is} hydrophobic or d _{n is} hydrophilic and a _n hydrophobic.

4. Peptide according to one of claims 1 to 3, characterized in that its sequence with a sequence portion of the sequence

(a (hydrophobic) bcd (hydrophobic) efg) i (a (hydrophilic) bcd (hydrophobic) ^β fg) 2 (a (hydrophobic) bcd (hydrophobic) efg) 3 or

(a (hydrophobic) bcd (hydrophobic) efg) i (a (hydrophobic) bcd (hydrophilic) efg) 2 (a (hydrophobic) bcd (hydrophobic) efg) 3 or

(a (hydrophobic) bcd (hydrophilic) efg) i (a (hydrophobic) bcd (hydrophobic) efg) 2 (a (hydrophobic) bcd (hydrophobic) efg) 3 or

(a (hydrophobic) bcd (hydrophobic) efg) i (a (hydrophobic) bcd (hydrophobic) efg) 2 (a (hydrophilic) bcd (hydrophobic) efg) 3 is consistent with or derived from.

5. A peptide according to any one of claims 1 to 4, characterized in that en and g _{n are} both charged, or e _{n is} charged and g _{n is} not charged, or g _{n is} charged and e _{n is} not charged.

6. Peptide according to one of claims 1 to 5, characterized in that its sequence with a sequence portion of the sequence

(abcde (g _e | aden) f9 (loaded)) i (abcde (geiaden) fg (not loaded)) 2 (abcde (geiaden) f9 (loaded)) 3 Or (abcde (loaded) fg (loaded)) i ( abcde ( _n i _ch loaded) fg (loaded)) 2 (abcde (loaded) fg (loaded)) 3 θder

(abcde (loaded) fg (not loaded)) i (abcde (charged) fg (charged)) 2 (abcde (charged) fg (charged)) 3 Or (abcde (geiaden) fg (charged)) i (abcde (geiaden ) fg (charged)) 2 (abcde (not charged) f9 (charged)) 3 matches or is derived from it.

7. A peptide according to any one of claims 1 to 6, characterized in that two contiguous amino acid residues are ga and / or de.

8. A peptide according to any one of claims 1 to 7, characterized in that a _n and d _{n are} both formed as leucine valine or isoleucine, or a _n as leucine, valine or isoleucine residue and d _n as the residue with non-branched side chain is formed, or d _{n is formed} as a leucine, valine or isoleucine residue and a _n as a residue with non-branched side chain.

9. A peptide according to any one of claims 1 to 8, characterized in that e _n and g _{n are} both formed as aspartic, glutamic, lysine or arginine, or e _n as aspartic, glutamic, lysine or arginine and g _n is formed as an uncharged residue or g _{n is formed} as an aspartic acid, glutamic acid, lysine or arginine residue and e _n as an uncharged residue.

10. A peptide according to any one of claims 1 to 9, characterized in that it consists essentially of naturally occurring amino acid residues, which may be modified, and / or their D analogues is formed.

11. A peptide according to any one of claims 1 to 10, characterized in that it is formed with a free or acetylated N-terminus and / or a free or amidated C-terminus.

12. A peptide according to any one of claims 1 to 11, characterized in that its sequence with a sequence portion of the sequence IQDLEKYVEDTKIDLWSYNAE or

VEDTKIDLWSYNAELLVALEN or LWSYNAELLVALENQHTI or NGKLNRVIEKTNEKFHQIEKE or IEKTNEKFHQIEKEFSEVEGR or FHQIEKEFSEVEGRIQDLEKY or QAAIDQINGKLNRVIEKTNEK or STQAAIDQINGKLNRVIEK is consistent with or derived from.

13. A peptide according to claim 12, characterized in that its sequence AAIDQINGKLNRVI or

KLNRVIEKTNEKFH or EKTNEKFHQIEKEF or FSEVEGRIQDLEKY or EKYVEDtKIDLWSY or KYVEDTKIDLWSYN or YVEDTKIDLWSYNA or DTKIDLWSYNAELL or SYNAELLVALENQH or IEKLKEKFHQLKKK or IEKLEEKFHQLKKK or IEKLNENFHQLKKK.

14. Peptide according to one of claims 1 to 11, characterized in that it consists of 15 amino acid residues with the sequence DELERRIREL and further 5 residues at the D-terminus.

15. A peptide according to claim 14, characterized in that the five further radicals are the sequence RARTK, RARSK, NARRK or QARRK.

16. A peptide according to any one of claims 1 to 11, characterized in that its sequence consists of a sequence portion of the sequence DELERRIRELEARIK and has a maximum length of 14 amine acid residues.

17. A peptide according to claim 16, characterized in that it has the sequence LERRIRELEARIK or RRIRELEARIK.

18. Peptide selected from the group of peptides according to Tables 3-5.

19. Use of the peptides according to one of claims 1 to 18 for the association with at least one coiled-coil sequence of a eukaryote, prokaryote, archaebacterium or a virus or a system which is at least partially composed of these or with sequences derived therefrom, characterized in that a peptide sequence is selected which coincides completely or substantially with a sequence segment of a protein from the proteome of the eukaryote, prokaryote, archaebacterium or virus.

20. Use of the peptides or of a peptide according to claim 19 for the association with coiled-coil sequences of a vertebrate, a bacterium, a fungus or a virion or sequences derived therefrom, characterized in that a peptide sequence is selected which corresponds to a sequence segment of a sequence Protein from the proteome of vertebrate, bacterium, fungus or virion completely or largely coincides.

21. Use of the peptides or a peptide according to any one of claims 19 to 20 for the association with coiled-coil sequences of a signal transduction protein, a transcription factor, a metabolite protein or a cell wall or transmembrane protein or sequences derived therefrom, characterized in that a peptide sequence selected which is linked to a sequence segment of a signal transduction protein, a transcription factor, a metabolic protein or a cell wall or transmembrane protein completely or largely matches.

22. Use of the peptides or a peptide according to any one of claims 19 to

21 for the association with coiled-coil sequences of a bZip transcription factor, a bacterial M or CA protein or a viral fusion protein or sequences derived therefrom, characterized in that a peptide sequence is selected which corresponds to a sequence section of a bZip transcription factor, a bacterial M or CA protein or a viral fusion protein completely or largely matches.

23. Use of the peptides or a peptide according to any one of claims 19 to

22 for association with hemagglutinin from influenza, gp41 from HIV or SIV, TM from Visna, GP2 from Ebola virus, Env TM protein from MMVL, F protein from SV5 or RSV, GP from Marburg virus or derived therefrom Sequences, characterized in that a peptide sequence is selected which completely or substantially matches a sequence segment from gp41, TM, GP2, Env-TM protein, F-protein or GP.

24. Use according to any one of claims 19-23, characterized in that the peptide sequence with a sequence section of the sequence Seq ID No. [1] (hemagglutinin) or the sequence Seq ID No. [4] (gp41) or the sequence Seq ID No. [5] (esat-6) completely or largely coincides.

25. Use according to any one of claims 19-24, characterized in that the peptide sequence having a sequence portion of the sequence of Seq ID No. [2] (coiled-coil sequence of hemagglutinin) or a sequence portion of the sequence of Seq ID No. [ 6] (coiled-coil sequence of gp41) or a sequence portion of the sequence of Seq ID No. [7] (coiled-coil sequence of esat-6) completely or substantially coincides.

26. Use of a peptide selected from the group of peptides according to Table 3 for the manufacture of a medicament for the treatment, prevention or alleviating AIDS in humans, wherein the peptide is capable of inhibiting the formation of the homotrimeric structure of gp41, preferably by association with gp41.

27. Use of a peptide selected from the group of peptides according to Table 4 for the manufacture of a medicament for the treatment, prevention or alleviation of tuberculosis in humans, which peptide is capable of forming the heterotetrameric structure of the Esat -6 / Cfp-10 complex, preferably by association with Esat-6 and / or Cfp-10.

28. Use of a peptide selected from the group of peptides according to Table 5 or DELERRIRELEARIK for the manufacture of a medicament for the treatment, prevention or alleviation of influenza in humans, wherein the peptide is preferably capable of forming the homotrimeric structure of hemagglutinin, preferably by association with hemagglutinin.

29. A means for recognition, labeling and structural alteration of coiled-coil sequences with more than 15 amino acid residues, or of coiled-coil structures with more than 30 amino acid residues, characterized in that it comprises a peptide of the sequence according to one of claims 1 18 and a marker associated with the peptide.

30. A composition according to claim 29, characterized in that the marker is chemically, electrostatically and / or connected via hydrophobic interactions with the peptide.

31. A composition according to any one of claims 29 to 30, characterized in that the connection between the substance and the marker is covalent.

32. Composition according to one of claims 29 to 31, characterized in that the marker is formed as a chemical element, molecule and / or ion or is composed of these.

33. Composition according to one of claims 29 to 32, characterized in that the marker as a carrier material, linker, 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 or structure derived therefrom.

34. Composition according to one of claims 29 to 33, characterized in that the marker is formed as solid phase carrier, cross-linker, fluorescent dye, cysteine, antibody and / or enzyme or structure derived therefrom.

35. Composition according to one of claims 29 to 34, characterized in that the marker is formed as a humanized and / or bispecific antibody or as a humanized and / or bispecific antibody fragment.

36. Means for the production of drugs for the detection, labeling, treatment and / or prevention of a disease or infection associated with gene products comprising a coiled-coil sequence, characterized in that it comprises at least one peptide according to any one of Claims 1-28 or at least one means according to one of claims 29 to 36 includes.

37. A pharmaceutical composition comprising at least one peptide selected from the group of peptides according to Tables 3-5 and optionally a pharmaceutically acceptable carrier and / or diluent and / or excipient.

38. A method for identifying alpha-helical coiled-coil sequences, characterized in that a carrier surface comprising at least one surface-bound peptide according to one of claims 1 to 28 or at least one surface-bound agent according to one of claims 29 to 36, is contacted with a test solution, and an association of a surface-bound peptide or agent with a coiled-coil Sequence from the test solution is made visible optically, radiographically, scintigraphically or with the help of a biosensor.

39. The method according to claim 38, wherein at least one peptide bound to the carrier surface or at least one agent bound to the carrier surface is prepared by conventional solid-phase synthesis on the carrier surface, wherein optionally at least one mixture with different amino acid components has been used.

40. The method according to any one of claims 38 to 39, characterized in that at least two surface-bound peptides or agents are used whose peptide sequences with the sequence portion of the natural or derived therefrom coiled-coil sequence completely or largely match and the peptide sequences with at least one, preferably several, more preferably all but one, amino acid residue overlap in their sequence.

41. The method according to any one of claims 38 to 40, characterized in that at least two surface-bound peptides or agents are used, which were obtained by synthesis of N- and / or C-terminal truncated sequences of a peptide sequence.

A method for the treatment, prevention or alleviation of AIDS in humans which comprises administering to a human in need of a therapeutically suitable amount of at least one peptide selected from the group of peptides according to Table 3.

43. A method for the treatment, prevention or alleviation of human tuberculosis comprising administering to a human in need of a therapeutically appropriate amount of at least one peptide selected from the group of peptides according to Table 4.

44. A method for the treatment, prevention or alleviation of influenza in humans comprising administering a therapeutically suitable amount at least one peptide selected from the group of peptides according to Table 5 or DELERRIRELEARIK to a human in need of the same.

45. Use of at least one peptide of the sequence according to one of claims 1 to 28 or at least one agent according to one of claims 29 to 36 for the detection, labeling and treatment of or action on alpha-helical coiled-coil structures or coiled-coil structures. Sequences, characterized in that the peptide or the agent is brought into contact with a biological or biotechnological system.

46. Use according to claim 45, characterized in that the biological or biotechnological system is formed as an organism, cell tissue, cell, cell constituent, virus, DNA, RNA, cDNA, mRNA, cRNA, protein and / or peptide or structures derived therefrom, or this contains.

47. Use according to any one of claims 45 to 46, characterized in that at least two peptides or agents are part of or form a peptide library.

48. Use according to one of claims 45 to 47, characterized in that the biological or biotechnological system is formed as a test solution.

49. Use according to one of claims 45 to 48, characterized in that the biological or biotechnological system is formed as body fluid or contains.

50. Use according to one of claims 45 to 49, characterized in that the at least one peptide or the at least one agent is immobilized on a solid phase carrier and / or is free in solution.

51. Use according to any one of claims 45 to 50, characterized in that the at least one peptide or the at least one means is localized on a solid phase carrier immobilized.

52. Use according to one of claims 45 to 51, characterized in that the solid phase support is formed with or comprises an at least partially planar surface.

53. Use according to one of claims 45 to 52, characterized in that the solid phase carrier is at least partially formed from glass, plastic or a membrane.

54. Use according to claim 53, characterized in that the membrane surface is formed essentially of cellulose, nitrocellulose or PVDF.

55. Use according to any one of claims 47 to 54, characterized in that at least two immobilized peptides or agents are part of or form a microarray.

56. Use according to one of claims 47 to 55, characterized in that the at least two immobilized peptides or agents are part of a biochip.

57. Use according to one of claims 45 to 56, characterized in that the surface is brought into contact with a solution from or with a biological or biotechnological system according to one of claims 46 to 49 and the components possibly associated with the solution on the Surface be made visible.

58. Use according to any one of claims 45 to 57, characterized in that for visualization dyeing reagents, proteins or peptides are used.

59. Use according to one of claims 45 to 58, characterized in that a solution with at least one detection reagent is used for visualization.

60. Use according to claim 59, characterized in that the detection reagent is formed as a labeled antibody, labeled streptavidin or labeled peptide with a coiled-coil sequence or structures derived therefrom.

61. Use according to any one of claims 59 to 60, characterized in that the detection reagent is formed as an agent according to any one of claims 29-36.

62. Use according to one of claims 59 to 61, characterized in that the visualization by means of signals emanating from the marker or the marker are read out with the aid of an optically, radiographically or scintigraphically sensitive apparatus.

63. Test kit for the detection and labeling of natural or derived coiled coil sequences, characterized in that it comprises a support surface according to any one of claims 51 to 57 with at least one attached peptide according to any one of claims 1 to 28 or at least one attached thereto means according to any one of claims 29 to 36 and a detection reagent according to any one of claims 59 to 61.