EP1054997A1

EP1054997A1 - Method for developing a pharmaceutical active ingredient

Info

Publication number: EP1054997A1
Application number: EP99911661A
Authority: EP
Inventors: Christof Antz; J. Peter Ruppersberg
Original assignee: Otogene AG
Current assignee: Otogene AG
Priority date: 1998-02-11
Filing date: 1999-02-10
Publication date: 2000-11-29
Also published as: DE19805334A1; AU3027499A; WO1999041407A1; JP2002503481A

Abstract

The invention relates to a method for developing an active ingredient capable of directly or indirectly influencing, preferably in an intracellular manner, the function of physiologically active peptides or proteins. The influence on the function of the peptides or proteins preferably takes the form of an inhibition of said function. According to the method, first a target peptide or target protein which is able to or does physiologically participate in a peptide-peptide or protein-protein interaction is identified and selected. Usually only the identification or selection or one or more fragments of said peptide or protein is necessary. Secondly an amino acid sequence is derived or screened which interacts with the target peptide or target protein or with fragments thereof with high affinity. The derived amino acid sequence interacts with the target peptide preferably at least as well as and especially better than with its physiological interaction partner.

Description

description

Process for the development of a pharmaceutical. Active ingredient

The invention relates to a method for the development of an active substance which, preferably intracellularly, is able, directly or indirectly, to influence the function of physiologically active peptides or proteins. The active substances developed should be applicable in particular with the aid of so-called gene therapy, and the function of the peptides / proteins should preferably be inhibited by this function.

So far, essentially two different approaches have been taken for gene therapy inhibition of gene products or the expression of these gene products. On the one hand, attempts have been made to effect the inhibition by means of short antisense oligonucleotides which are introduced into a cell in order to inhibit the so-called messenger RNA (mRNA) of a specific gene. This concept has the disadvantage that, on the one hand, a high concentration of the oligonucleotides has to be taken up by the cells and, on the other hand, a complete inhibition of the gene in question is usually not achieved. The other proposed way is to introduce so-called antisense genes, which are packaged in viral vectors, into the cell. These antisense genes lead to transcription of antisense RNA, which in turn inhibits the gene to be influenced. This second concept may have the advantage of being highly effective, but it is unlikely that the vector will spread, especially when applied locally. So-called peptide libraries are also known which contain a very large number, for example> 10 ⁸ recombinants. However, useful strategies are required to select one or more peptides or their coding nucleic acid sequences from such libraries. Basically already known screening methods, which use the so-called yeast two hybrid system, for example, can only reasonably be used if a certain preselection has been made via the amino acid sequence. With usually 20 usable amino acids, the variability with a chain length n of the sequence corresponds to a value of 20 ⁿ possibilities. In this context, it should also be mentioned that, more recently, proteins have also been designed by computer simulation (computer molecular modeling). In all procedures, the final structural analysis of a peptide-peptide interaction or protein-protein interaction can be carried out, for example, by nuclear magnetic resonance spectroscopy (NMR).

The object of the invention is to avoid the disadvantages of the prior art. The aim is to provide a process for developing an active substance defined above, in which an optimized amino acid sequence is obtained with comparatively less effort using systematic use of certain process steps. This amino acid sequence or the active ingredient resulting from it should preferably be suitable for inhibiting physiologically active peptides / proteins. Furthermore, it is the aim of the invention to influence, in particular inhibit, the function of such peptides / proteins by gene therapy. In this context, the simplest possible application of the active ingredient with high effectiveness is to be achieved. The amino acid sequence obtained by the method should act on the physiologically active target peptide / protein preferably at least as well as its physiological interaction partner.

The described objects are achieved by the method having the features of claim 1. Preferred embodiments are presented in the dependent claims 2 to 16. The wording of all claims is hereby incorporated by reference into the content of this description.

The method mentioned at the outset is essentially characterized by two method steps. On the one hand, a target peptide or protein that physiologically participates or can participate in a peptide-peptide or protein-protein interaction is identified and selected. Preferably, only one or more sections of this peptide / protein are identified or selected, the corresponding interactions then taking place on this section or these sections. On the other hand, an amino acid sequence is derived or screened, which interacts with the target peptide / target protein or its section or sections with high affinity. This interaction is preferably at least as good, in particular better than that of the target peptide / target protein / section with its physiological interaction partner.

The derivation of the amino acid sequence preferably comprises the selection of at least one starting structure of an amino acid sequence, this selection preferably at the beginning of the

Procedure or at the beginning of a process run. This has the advantage that an arbitrary amino acid sequence does not have to be assumed. The starting structure can be, in particular, the amino acid sequence of a physiological interaction partner of the target peptide / target protein or one of its subsections or an amino acid sequence derived with the aid of molecular modeling. If the derivation of the amino acid sequence takes place in several process runs, the selection of one of the two start structures determined in the above-mentioned ways is particularly appropriate during the first process run. In these cases, the (partially optimized) amino acid sequences resulting from the previous process run can then be used as the starting structure for the further process runs.

The method according to the invention is further characterized in that a number of degenerate nucleic acid sequences are determined and selected on the basis of the starting structure (s). These code for the corresponding variations of the amino acid sequence selected as the starting structure. This procedure provides an at least manageable number of sequences, the examination of which is practically possible with the aid of a screening method. By specifying the starting structure it is achieved that the nucleic acid sequence does not have to be completely degenerate, but can at least be planned within limits. The variability mentioned at the outset can thus be significantly reduced. Normally, selecting the amino acid sequence of a known physiological interaction partner as starting structure with low variability will be sufficient as in the case of an amino acid sequence resulting from molecular modeling as starting structure.

The degenerate nucleic acid sequence obtained (possibly also several) is then preferably a known one Subject to screening methods with the aid of which the interaction between target peptide / target protein / partial section and the amino acid sequences which correspond to the individual degenerate nucleic acid sequence can be determined. Because of this screening step, a certain number will appear

Amino acid sequences or their nucleic acid sequences selected in which this interaction is strongest or best.

The various screening methods that can be used in principle are known to the person skilled in the art. In particular, the so-called Yeast Two Hybrid method should be mentioned here, which has gained great importance in recent years. As literature, reference is made here to the publication by Fields et al., Nature 340, 245-247 (1989). According to the invention, a modification of this method is preferred, which is to be mentioned under the keyword FRET-Yeast-Two-Hybrid-Method. The essential features of this method are explained in connection with the example.

The nucleic acid sequences that show the best interaction and have been selected accordingly, i.e. in a conventional screening method, the corresponding yeast cells are usually sequenced after cloning. This sequencing then necessarily results in the associated amino acid sequences. The sequences obtained in this way are preferably compared with one another and possibly also with existing sequences in order to obtain similarities or common structural motifs that can be useful for a possible further optimization of the amino acid sequence.

The associated amino acid sequences / peptides / proteins are then synthesized for the selected sequenced nucleic acid molecules by customary methods. The peptides can also be expressed quantitatively in cells. The exact interaction structure of the amino acid sequences obtained in this way is then determined using NMR spectroscopy. As already stated, this interaction structure is formed between the target peptide / target protein / partial section and the synthesized amino acid sequence. For better comparability of the results, one or more of the partial sections of the target peptide / target protein can also be produced synthetically for this or another process step.

In a further development, the overall process, but in particular the derivation of the amino acid sequence or its individual process steps, can be carried out at least twice, preferably several times. As already shown, the amino acid sequence sought can be gradually optimized until either a sufficiently high affinity or even a better affinity than that of the physiological interaction partner is reached. The user of the process is free to choose the process runs at a specific time, i.e. terminate at a certain degree of optimization of the amino acid sequence. In this connection, it will make sense to carry out the process steps listed in deriving the amino acid sequence between three and twenty times, preferably between three and ten times, until an amino acid sequence of sufficient affinity is obtained.

It is further preferred in the invention if an amino acid sequence obtained (previously optimized or partially optimized) from earlier process steps with already good interaction / affinity is used in further process steps as a so-called petitor. This applies in particular to the implementation of the screening process at which then the further optimized sequences can be compared directly with the competitor.

Finally, the method according to the invention can be carried out in particular for the derivation of amino acid sequences for several subsections of a target peptide / target protein. The overall affinity and specificity of the derived structure can thus be further improved, as will be explained in the following. Here too, the required process steps for deriving the amino acid sequence per section are carried out with the abovementioned frequency of three to twenty times, preferably three to ten times.

According to the invention, the derived amino acid sequence usually comprises the smallest possible number of amino acids in order not to make the derivation of the amino acid sequence too complex. On the other hand, in most cases a minimum number of amino acids will be required to ensure the required interaction with the target protein / target peptide / section. It is therefore preferred that the deduced amino acid sequence comprise less than 30 amino acids, preferably less than 15 amino acids.

When identifying and selecting one or more

Sub-sections of the target peptide / target protein can in principle be taken into account any amino acid sequences which are involved in a corresponding physiological protein-protein or peptide-peptide interaction. However, so-called helix or β-sheet structures are particularly suitable here. If, as in many cases, there are several active sections within a target peptide / protein, these can therefore preferably be mentioned Structures are selected and derived for these amino acid sequences.

The essential features of the invention can accordingly be summarized as follows:

The invention is based on the identification and selection of a target peptide / target protein or one or more of its subsections which are suitable for a physiological interaction with other peptides, proteins and the like. come into question. The terms “peptide” and “protein” selected in the invention are intended to encompass in the broadest sense any amino acid sequence as known to the person skilled in the art. According to the invention, an amino acid sequence is derived from this identified and selected amino acid sequence that is capable of an interaction, preferably an interaction that is at least as good as that between the sequence presented and its physiological interaction partner.

Various methods are then used to derive the amino acid sequence sought, as already described. These can be classified under the keywords random peptide library screen, in particular yeast two-hybrid system, NMR-based drug design and computer

List molecular modeling. As shown, the starting point of the development is an already known peptide-peptide interaction, an interaction resulting from an earlier process or a purely computer-modeled structure. In other words, the background of the invention is therefore a combination of a largely random screening and a deterministic modeling strategy. The combination of these two techniques makes it possible to choose from the vast number of possible sequences to select a series of, for example, 10 particularly good sequences.

The advantages of the invention are first of all evident in the inhibition of the function of physiologically active peptides mentioned at the beginning. In comparison to an antisense strategy, which has hitherto permitted at most 60 to 80% inhibition, essentially complete blocking of the corresponding peptide-peptide interactions or of the corresponding receptor can be achieved by optimizing the amino acid sequence according to the invention. Because of its specific effect, the amino acid sequence derived according to the invention is referred to by the inventors as Intraactor ©. Because of the high effectiveness that can be achieved in this way, the situation can occur that on viral

In contrast to the methods of the prior art, vectors can be completely dispensed with. Accordingly, the half-life of a therapeutic effect is determined by the properties of the active ingredient and its target molecule and not by that of the gene therapy vehicle. In these

In some cases, the required concentration of the active ingredient or the amino acid sequence, which is usually in the nanomolar range, can already be achieved with a single copy of a corresponding DNA in a cell.

The invention further comprises an active substance, preferably an intracellular active substance, which is capable of influencing the function of physiologically active peptides or proteins directly or indirectly. According to the invention, this active ingredient results from the method according to the invention already described or from one of its embodiments. There is a direct influence by the active ingredient, for example, if the amino acid sequence derived by the method according to the invention is used, ie this 10

the desired interaction occurs "directly". An indirect influence is present, for example, when a nucleic acid molecule coding for the desired amino acid sequence is used as the active ingredient, which is introduced into the corresponding cell and which expresses the optimized amino acid sequence.

Accordingly, the active ingredient according to the invention is in particular an amino acid sequence obtainable by the method according to the invention or one of its embodiments. This amino acid sequence is in particular a peptide which preferably has fewer than 15 amino acids. In a preferred development, the active substance can contain at least two (optimized) amino acid sequences. In principle, these can exist independently of one another and, for example, interact with corresponding subsections of a target peptide. However, it is preferred if the at least two amino acid sequences are linked to one another via physiologically largely inactive molecular intermediate members. This is in particular a covalent connection between the amino acid sequences and the pontics. A further increase in affinity is achieved by using such "covalent linkers". If one of the amino acid sequences has found its associated interaction site, the probability that the second amino acid sequence or another will also find its corresponding binding site is significantly increased. The length of the molecular links between the amino acid sequences is the distance between the active sections in the target peptide or, under certain circumstances, also between different ones

Adjusted target peptides. This adaptation can take into account the biological conditions, so that the distance can possibly be greater than the actual distance. However, the latter is preferred. The linker 11

Molecules can be constructed after structural analysis of individual receptor areas (partial section) or the entire receptor (target peptide). If, as in the present case, the derivation of amino acid sequences is used, poly-glycine or poly-alanine * chains of corresponding length can be inserted in a simple manner.

Further active substances according to the invention which can be used are nucleic acid molecules, preferably recombined nucleic acid molecules, which code for at least one amino acid sequence obtainable by the method according to the invention. This can be a DNA molecule, cDNA molecule or RNA molecule. If molecular links are used as a linker between several amino acid sequences, the nucleic acid molecules additionally code for these linker chains.

In a further development, the active substance according to the invention can be in the form of a so-called vector or vehicle which carries at least one of the nucleic acid molecules already described which code for the amino acid sequence. Such vectors can be the usual viral and non-viral vectors as are known to the person skilled in the art. If a viral vector is used, a retro virus, an adenovirus or an adeno-associated virus can be used. In addition to non-viral vectors in which no viral DNA is involved, the packaging of the nucleic acid molecules in the so-called liposomes or lipoplexes should be emphasized. In principle, the use of non-viral vectors or liposomes / lipoplexes is preferred in order to avoid the known disadvantages of viral vectors, some of which are described at the beginning. According to the advantages of the invention, active ingredients can be made available with very high effectiveness, which in principle are viral without use 12

Vectors can be introduced into a cell. The advantage of the invention is quite fundamentally that active ingredients, in particular amino acid sequences with nanomolar affinity, can be made available.

Finally, the invention comprises a pharmaceutical composition or a medicament that contains at least one of the described active ingredients, i.e. an optimized amino acid sequence containing the coding nucleic acid molecule or a vehicle carrying the nucleic acid molecule, and a pharmaceutically acceptable carrier.

The described features and further features of the invention result from the following description of preferred embodiments in conjunction with the subclaims, examples and the drawings. The individual features can be implemented individually or in combination with one another.

The drawings show:

Fig. 1 shows the protein structure of a complex of the cyclin-dependent kinase CDK2, the cyclin A and their

Inhibitor p27 ^κi P ¹ ,

Fig. 2 shows the interaction between a section of cyclin A and a section of p27 ^Kl P ¹ and

Fig. 3 shows the flow of certain embodiments of the method according to the invention in a schematic representation. 13

example

Starting from the known structure of a protein complex, the possible uses of the invention are to be presented. The complex is selected from the cyclin-dependent kinase CDK2, cyclin A and its inhibitor p27 ^Kl P ¹ . This structure is disclosed in the publication by AA Russo et al in Nature, 382, 325-331 (1996) and is shown in FIG. 1.

The aim of using the method according to the invention is to inhibit or prevent an interaction of the p27 ^ ¹ P ^ - with the other two proteins. For this purpose, an amino acid sequence is to be derived which preferably interacts better than the p27 ^Kl P ¹ with the other two proteins, preferably the cyclin A.

In Fig. 1, the places where peptide-peptide interaction between p27 ^Kl P ¹ and CDK2 or Cyclin A takes place are highlighted in black. The binding / interaction denoted by a is a binding site which was initially identified as a binding site with high affinity. Further binding sites exist between an o ^ -helix of cyclin A and an o ^ -helix of p27 ^K; "-P ¹ (binding / interaction b), a β-sheet structure of CDK2 and a β-turn of p27 ^Kl P ¹ (binding / interaction c), a β-strand of CDK2 and a β-strand of p27 ^Kl P ¹ (binding / interaction d) In addition, another helix of p27 ^Kl P ¹ occupies the so-called ATP binding site.

Based on FIG. 1, the interaction of the binding / interaction b to derive an amino acid sequence is now to be examined 14

be picked out of the invention. The corresponding binding site is shown in Fig. 2. It shows the interaction between the two o ^ helices of cyclin A and p27 ^Kl P ¹ , the amino acids 38 to 49 being involved on the p27 ^Kl P ¹ side and theo ^ -5 helix on the cycline A side .

Fig. 2 shows that the fit between the two interacting structures is not perfect and therefore there will not be a very high affinity between the two interacting partners. The method according to the invention is now intended to replace one of the two helices, in particular the helix of p27 ^Kl P ^1, with an optimized peptide structure (amino acid sequence) which then binds the other helix, preferably that of cyclin A, with high affinity. This optimized peptide structure is then expressed intracellularly as a peptide of, for example, and preferably up to 15 amino acid length, by an artificially introduced gene. The high affinity of the peptide then interferes with the interaction of the physiological peptide partners and, with high probability, inhibition of the kinase by the p27 ^Kl P ^{1 is} prevented. Because of this approach, the concentration of the amino acid sequence in the cell should only be approximately the same as the concentration of p27 ^κi P ¹ , which can be estimated at 10 nM / 1 or approximately 1,000 to 10,000 molecules per cell. As described, a single copy of a DNA encoding the amino acid sequence per cell could be sufficient to achieve such a concentration.

3 schematically shows the process sequence or the process sequences in a preferred method according to the invention. 15

It is based either on the known interaction between two peptide / protein molecules (top left rectangle) or on the known protein structure of an intracellular receptor or target peptide (top right rectangle). On this basis, suitable subdomains (subsections) are selected in the present case, which are to be used as the basis for the derivation of the amino acid sequence sought. In the protein complex of FIG. 1, this corresponds to the selection of the binding / interaction at the binding site b.

If, as in the case of the protein complex of FIG. 1, the real structure of a physiological interaction partner of the target protein can be assumed, in this case the structure of p27 ^Kl P ¹ , then this structure represents the starting structure for the derivation of the amino acid sequence. It is then, as shown in Fig. 3, advantageous if both interaction partners are first synthesized as a peptide and the interaction is measured by means of NMR structure analysis. Usually, a complete synthesis of the entire peptides will not be required here, but it will suffice if the corresponding subsections of the peptide sequences which interact at the binding site b are synthesized.

If the NMR structure analysis does not show an optimal interaction (which is generally desirable so that a better-functioning amino acid sequence can be obtained at all), the starting structure is varied with the help of molecular modeling. The (spatial) peptide structure of at least one, preferably many different, predetermined amino acid sequences is determined in a manner known per se and their fit with the corresponding subsection of the target peptide is examined. Based on molecular modeling 16

a number of degenerate sequences are then designed, from which one or more are selected for a screening process. In the present case, the screening process is the FRET yeast two hybrid process. From about 10 ⁸ to 10 ⁹ cells with clones of different sequences, a number of the best cells, preferably about 10, are selected in which the interaction between the peptides, measured by fluorescence resonance energy transfer (FRET), is strongest or is best. The cells with the best interaction are cloned and the corresponding nucleic acid sequences or the associated amino acid sequences are determined by sequencing. If one now compares, as also shown in FIG. 3, whether similarities between the selected sequences are recognizable or whether there are other starting points for common structural motifs, this information can be used for the further implementation of the method. If there are indications of structural motifs that can be optimized, selected sequences are produced as peptides and their interaction structure is measured by means of NMR spectroscopy. If the interaction is not yet sufficient, either in comparison to the natural interaction partner of the target peptide or with regard to an affinity to be achieved, the process run beginning with the molecular modeling process step is started again in order to achieve improved affinities in this way. If an amino acid sequence with good interaction is already known from an earlier process run, this can be used as a competitor in the following process steps, in particular in the screening process, in order to identify the even better amino acid sequences in this way. As already described, the cycle of deriving the amino acid sequence shown in FIG. 3 usually has to be carried out three to twenty times, preferably three to ten times per receptor domain (partial 17

section of the target peptide) are run through in order to obtain amino acid sequences with sufficient affinity. This desired affinity is normally in the nanomolar range, as has also already been shown.

A procedure according to FIG. 3, which cannot start from the real structure of a physiological interaction partner as the starting structure, differs essentially only in that, after selection of the corresponding subdomain (partial section of the target peptide), an amino acid sequence is selected as the starting structure with the help of molecular modeling must become. It is in principle possible to also subject this (simulated) start structure to peptide synthesis and NMR structure analysis, as shown in FIG. 3 for the case of the physiological binding partner. However, this will not make sense in most cases, since the simulated start structure will most likely not show an optimal interaction. The simulated starting structure is therefore only used to plan a degenerate nucleic acid sequence, which is then used in the actual screening process. Molecular modeling, however, ensures that a complete degeneration of the nucleic acid sequence can be omitted, which can reduce the variability from values around 10 ²⁰ to about 10 ¹⁰ . The variability can be successively further reduced in the manner already described by further process runs.

The FRET yeast two hybrid method which can be used according to FIG. 3 is essentially based on the fact that the two peptides, the interaction of which is to be investigated with one another, are coupled to different fluorescent components. The absorption and emission spectra of the fluo- 18th

resected components such that fluorescence resonance energy transfer (FRET) occurs. This detection can also be carried out in a host cell such as a yeast cell using the known yeast two hybrid method, the fluorescence signal measured providing direct information about the affinity of the binding or interaction between the two peptide components. As in the present case, the fluorescence resonance energy transfer (FRET) can be carried out by known fluorometric methods, such as fluorescence-activated cell sorting (FACS) or by fluorescence microscopy. Fluorescent peptides such as the green fluorescent protein (GFP) and the blue fluorescent protein (BFP) can preferably be used as fluorescent components. The fluorescence is preferably excited by laser. In this context, reference can be made to the relevant prior art.

As can also be seen from FIG. 3, the final analysis of the protein structures and their interaction takes place with the aid of an NMR spectrometer. In principle, other detection methods can also be used, such as the X-ray structure analysis of a crystallized peptide or protein complex. Due to the fact that the analysis of the peptide structure with the NMR spectrometer requires a comparatively high expenditure of time, the corresponding process step occupies an important position in the overall process. As has already been started, the method according to the invention can be understood as a combination of a random screening and a deterministic modeling strategy. This procedure used here is based on the mathematical sequence of certain fitting algorithms, in which a random variation and a targeted change in the parameters also take place alternately 19

to achieve the best fit of an equation with numerous parameters to an experimental curve. Here too, as in the present case, the best or as good as possible (here: optimized. Amino acid sequence) must be selected from a comparatively large number of possibilities (here: possible amino acid sequences). It is important for this strategic approach that the deviation is calculated again and again in each process cycle, since both the experimental data points and the mathematical equation are exactly known. During this calculation, the influence of all variable parameters on this deviation is then tried out each time in order to plan the subsequent next phase of the variation (process cycle). Applied to the invention, this means that the measurement with the NMR spectrometer allows this control and determination of the deviation.

However, since, for example, with a 600 MHz spectrometer with completely unknown structures (after several so-called TOCSY and NOESY protocols have expired) approx. 80 to 120 hours of pure measurement time are required, the effort can best be achieved using the strategy described , in which only already optimized amino acid sequences are introduced into an NMR structure analysis. Even using this strategy, three to ten process runs and examination of four peptide domains (subsections) of a target peptide usually still require around 2,000 hours of measurement time for the development of an optimized amino acid sequence.

Claims

20 patent claims

1. A process for the development of an active substance which, preferably intracellularly, is able, directly or indirectly, to influence the function of physiologically active peptides or proteins, characterized by the process steps:

- Identification and selection of a target peptide or target protein, in particular at least a section of this peptide or protein, the target peptide / target protein or the section taking part or being able to participate physiologically in a protein-protein or peptide-peptide interaction;

Derivation or screening of an amino acid sequence with the target peptide / target protein or its section or sections with high

Affinity interacts, preferably at least as well as its physiological interaction partner.

2. The method according to claim 1, characterized in that the derivation of the amino acid sequence comprises the selection of at least one starting structure of an amino acid sequence, preferably begins with such a selection.

3. The method according to claim 2, characterized in that it is in the starting structure to the amino acid sequence of a physiological interaction partner of the target peptide / target protein or one of its subsections, and / or one of one with the help of 21

so-called molecular modeling computer-simulated protein structure derived amino acid sequence.

Method according to one of claims 2 or 3, characterized in that on the basis of the starting structure at least one, preferably a number, of degenerate nucleic acid sequences is determined and selected, which codes for variations in the amino acid sequence selected as the starting structure.

5. The method according to claim 4, characterized in that at least one degenerate nucleic acid sequence is subjected to a known screening method, with the aid of which the interaction between target peptide / target protein or its portion and the amino acid sequences which correspond to the degenerate nucleic acid sequence can be determined, and Amino acid sequences with good interaction can be selected.

6. The method according to claim 5, characterized in that the screening method is a so-called yeast two-hybrid method, preferably the so-called FRET yeast two-hybrid method.

7. The method according to any one of claims 4 to 6, characterized in that certain nucleic acid sequences are sequenced, preferably the screening method comprises a sequencing of the nucleic acid sequences with a comparatively good interaction.

Method according to Claim 7, characterized in that the nucleic acid sequences, preferably the cells which carry these nucleic acid sequences in a screening process, are cloned before the sequencing. 22

9. The method according to claim 7 or claim 8, characterized in that at least partially the amino acid sequences belonging to the sequenced nucleic acid molecules are synthesized or largely quantitatively expressed.

10. The method according to claim 9, characterized in that the interaction structure between target peptide / target protein or its portion and the synthesized amino acid sequence is measured by NMR spectroscopy.

11. The method according to any one of the preceding claims, characterized in that one or more

Process steps for optimizing the amino acid sequence are carried out at least twice, preferably several times.

12. The method according to claim 11, characterized in that at least one amino acid sequence obtained from previous process steps with good interaction is used in further process steps as a competitor.

13. The method according to any one of the preceding claims, characterized in that the method for deriving amino acid sequences for several sections of a target peptide / protein is carried out.

14. The method according to any one of the preceding claims, characterized in that the required process steps for deriving an amino acid sequence per 23

Section be run between three times and twenty times, preferably between three times and ten times.

15. The method according to any one of the preceding claims, characterized in that the derived. amino acid sequence comprises less than 30 amino acids, preferably less than 15 amino acids.

16. The method according to any one of the preceding claims, characterized in that a portion of the target peptide / target protein is a so-called c-helix or a so-called β-sheet structure.

17. Active ingredient which, preferably intracellularly, is able, directly or indirectly, to influence the function of physiologically active peptides or proteins, resulting from a method according to at least one of claims 1 to 16.

18. Active ingredient according to claim 17, characterized in that it is an amino acid sequence obtainable according to at least one of claims 1 to 16.

19. Active ingredient according to claim 18, characterized in that it is a peptide, preferably a peptide with less than 15 amino acids.

20. Active ingredient according to one of claims 17 to 19, characterized in that at least two amino acid sequences are connected to one another via physiologically largely inactive molecular links. 24

21. Active ingredient according to claim 20, characterized in that the length of the molecular intermediate members is adapted to the distance between active sections of one or more target peptides / target proteins, preferably corresponds to this distance.

22. Active ingredient according to claim 20 or claim 21, characterized in that the intermediate link is a poly-glycine or poly-alanine chain.

23. Active ingredient according to claim 17, characterized in that it is a nucleic acid molecule, preferably a recombined nucleic acid molecule, which codes for at least one amino acid sequence obtainable according to at least one of process claims 1 to 16.

24. Active ingredient according to claim 23, characterized in that the nucleic acid molecule is a DNA molecule, cDNA molecule or RNA molecule.

25. Active ingredient according to claim 17, characterized in that it is in the form of a so-called vector which carries a nucleic acid molecule according to one of claims 23 or 24.

26. Active ingredient according to claim 25, characterized in that the vector is a viral vector.

27. Active ingredient according to claim 26, characterized in that the virus is a retrovirus, an adenovirus or an adeno-associated virus. 25th

28. Active ingredient according to claim 25, characterized in that the vector is a non-viral vector.

29. Active ingredient according to claim 23 or claim 24, characterized in that it is a nucleic acid molecule packaged in a liposome or a lipoplex.

30. Pharmaceutical composition or medicament, characterized in that it contains at least one active ingredient according to one of claims 17 to 29 in an effective amount and a pharmaceutically acceptable carrier.