WO2002016574A2

WO2002016574A2 - Method for identifying peptides that can be specifically cleaved and the use of peptide sequences of this type

Info

Publication number: WO2002016574A2
Application number: PCT/EP2001/009102
Authority: WO
Inventors: Ralph Reimholz; Frank PLÖGER
Original assignee: Phylos, Inc.
Priority date: 2000-08-22
Filing date: 2001-08-07
Publication date: 2002-02-28
Also published as: EP1314039A2; CA2420065A1; DE10041238A1; WO2002016574A3; JP2004507240A; AU2001283985A1

Abstract

The invention relates to methods for detecting and identifying peptides that can be specifically cleaved, using a defined amino acid sequence based on a library of nucleic acid peptide fusion molecules. According to said methods, the variable part of the peptides is coded by the respective nucleic acid fused thereto, using proteolytically active solutions. The invention also relates to the use of said peptides for diagnosis.

Description

Methods for the identification of specifically cleavable peptides and the use of such peptide sequences

description

The invention relates to methods for finding and identifying specifically cleavable peptides with a defined amino acid sequence using proteolytically acting solutions and the use of these peptides for the targeted release of chemical active substances and for diagnosis.

A method for the detection of specifically cleaved peptides using a specific protein construct which fluoresces upon proteolytic cleavage of the construct is described in US Pat. No. 5981200. The method is based on the fluorescence resonance energy technique (FRET). An advantage of this method is that it can also be used for the in vivo detection of protein cleavage events. On the other hand, it is disadvantageous that only specific peptides can be labeled or, in the case of an examination of different labeled ones

Proteins an exact assignment of the cleavage event to the respective sequence would be associated with great effort. Another disadvantage of this method is that relatively large amounts of labeled substrates have to be used in order to obtain a detectable signal.

Another method that can be used for the selective identification of enzyme substrates is the phage display (eg laid down in WO 97/47314). A disadvantage of this method is that the phage display peptide libraries have a smaller number of possible different sequences than other surface-represented peptide libraries and that the subsequent one

Working up the phage particles and determining the identified substrates is relatively complex. The object of the present invention is to develop a method for finding and identifying specifically proteolytically cleavable peptides and to provide substances which allow a targeted release of chemical active substances.

The object is achieved by a method for identifying specifically proteolytically cleavable peptides, which comprises the following method steps: a) incubation of a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide with a proteolytically active sample, b) isolation of the proteolytically cleaved components the fusion molecules, c) determining the sequence of the nucleic acid component of the isolated

Fusion molecules.

The method according to the invention is based on the use of fusion molecules which have a phenotypic peptide part and a genotypic part

Contain nucleic acid part, which has a sequence coding the peptide part. The peptide part is linked to the nucleic acid part via a suitable linker. A protein acceptor, e.g. B. Puromycin used, which is covalently bound to the nucleic acid part. The linker can contain other components such as e.g. B. include a non-coding nucleic acid sequence, preferably a poly-A sequence. In a preferred embodiment, the nucleic acid part contains further regions which are constant in sequence and which lie on one or both sides of the coding region. These constant nucleic acid regions can e.g. B. serve as primer binding sites for performing a PCR or as restriction enzyme interfaces. The coding region of the

The nucleic acid part of the fusion molecules contains a variable part which codes for at least two amino acids, preferably for at least four, particularly preferably for seven to twelve amino acids. For this purpose, further constant coding nucleotide sequences can occur. The constant nucleic acid sequences can encode peptide sections which e.g. B. allow a simple fixation of the peptide part of the fusion molecule to a solid surface or generate the interesting structural elements. Such structural elements are e.g. B. epitopes for antibodies, tags for cleaning or for the detection of the constructs or elements that determine certain tertiary structures. The preparation of such fusion molecules can, for. B. according to WO98 / 31700. A system is described there in which a protein acceptor, for example a puromycin, is bound to the nucleic acid, preferably RNA, via a suitable linker. This ensures that shortly before the in vitro translation of the RNA into the corresponding protein ends, the synthesized protein can be covalently bound to its coding RNA and thus characterized in more detail. Comparable systems that can be used for the present invention are described, for example, in DE19646372C1, WO98 / 16636, US 5843701 or WO94 / 13623.

In a preferred embodiment of the method according to the invention, the fusion molecules are fixed to a surface (support) via their peptide part. For the purposes of the present invention, the term “carrier” is understood to mean material which is in solid or gel-like form. Examples of suitable carrier materials are ceramic, metal, in particular semiconductors, noble metal,

Glasses, plastics, crystalline materials or thin layers of the carrier, in particular the materials mentioned, or (bio) molecular filaments such as cellulose, framework proteins. Not only flat materials but also particles come into question as carriers, such as e.g. B. protein-loaded materials for column chromatography or beads made of organic polymers. The carrier is generally covalent, quasi-covalent, supramolecular or physical.

The peptide part of the fusion molecules can e.g. B. via biotin - streptavidin bond to the support, but also specific domains of the peptide part of the fusion molecules, such as. B. metal-binding domains (z. B. His-Tag), terminals

Cysteine residues or domains that contain epitopes recognizable by specific antibodies can mediate such a fixation.

Preferred in the method according to the invention are libraries whose fusion molecules have all possible permutations with regard to the variable part of the

Contain peptide sequence. Libraries of such fusion proteins that can still be handled practically have a size of approximately 10 ¹³ different sequences and are thus 10 ³ -10 ⁴ times (e.g. phage display) or 10 ⁶ -10 ⁷ times larger than other known peptide libraries (classic, chemically synthesized Libraries) larger. In order to cover all permutations, a variable peptide part of less than 11 amino acids is preferred; variable peptide parts of seven or eight amino acids are particularly preferred. Of course, libraries can also be created which contain longer variable peptide parts in their fusion molecules, but then a complete coverage of the sequence diversity is no longer possible. Since the previously known, specific peptide bond-cleaving proteases, a

Recognition sequence of at least four amino acids are preferred libraries with a variable peptide part of at least four amino acids are preferred.

The library of fusion molecules is exposed to a proteolytically active sample either in solution or preferably fixed to a support. Tissue-specific extracts of animal, human or vegetable origin, extracts of certain cell compartments, such as e.g. B. cytosolic extracts, subcellular extracts, extracts of membrane components, extracellular extracts or mixtures thereof

Extracts in question. Of particular interest are extracts from pathological tissue, such as. B. from carcinomas. However, extracts which represent all or part of the proteolytic activity of an organism, in particular viruses, microorganisms such as bacteria or protists, can also be used. Known protease solutions can also be used as a proteolytically active sample, preliminary sample or comparative sample.

The library is incubated with the proteolytically active sample preferably under physiological conditions, preferably at temperatures between 0 ° C. and 45 ° C. For this, the extracts to be examined for their proteolytic activity can be used directly, or the extracts are in a suitable solvent, such as. B. a physiological saline solution and used for incubation.

During the incubation, the variable peptide components of the

Fusion molecules cleaved by the proteases contained in the sample. The result is a specific proteolytic cleavage pattern that is characteristic of the sample extract used. The split-off parts of the fusion molecules are encoded by the nucleic acid part. After incubation of the library with the proteolytically active to be examined

The proteolytically cleaved fusion molecule parts are isolated and the sample

Sequence of the coding nucleic acid part determined. So that’s through the

Sample cleavable peptide sequences identified.

The isolation of the cut fusion proteins after incubation of the library and proteolytically active extract in solution can be carried out as follows. For isolation of the nucleic acid regions of the cut from the uncut fusion proteins, e.g. a constant structural feature, e.g. a known epitope for an antibody, in which that part of the peptide is used which is no longer connected to the rest of the fusion molecule, including the nucleic acid part, by the proteolytic cleavage. For this purpose, an affinity chromatography using z. B. said antibody. The nucleic acid part of the cut fusion proteins cannot bind, is separated from the nucleic acids of the uncut fusion proteins, and is therefore in the

Eluate included. Other structural features such as e.g. B. His-Tag or Strep-Tag can be used for separation. The fusion molecules remaining on the affinity matrix can be eluted and used further in solution or advantageously directly in the fixed state.

The cut fusion proteins can be isolated using different methods after incubation of the fixed library with extract.

In a preferred embodiment of the method according to the invention, after incubation of the fixed library with the extract, the nucleic acid part of the cut fusion molecules is obtained directly in the eluate.

In the simplest case, the sequences of the cleaved peptides can be identified directly by PCR amplification of the nucleic acid parts of the cleaved fusion proteins contained in the eluate with subsequent cloning and sequencing. However, it is also possible to first purify the cleaved fusion proteins from other ones present in the eluate by further affinity chromatography using specific structures in the peptide or nucleic acid part of the cleaved fusion proteins Components. After elution of the cleaved fusion proteins from the chromatographic material, e.g. B. by incubation with a KOH solution, the sequence of the cleaved peptides can be carried out again by means of PCR, cloning and sequencing.

In addition, a number of other chromatographic or electrophoretic methods for isolating the proteolytically cleaved fusion molecule parts are possible. For this purpose, it is advantageous if the fusion molecules are marked or modified for easier identification. So z. B. the nucleic acid part of the fusion molecules z. B. fluorescently labeled or radioactively labeled. Magnetic labeling of the nucleic acid part of the fusion molecules is particularly preferred; it can also be used in conjunction with a fluorescent label or a radioactive label. The magnetic isolation of the split-off parts of the fusion molecule can be carried out very easily and selectively. The proteolytic pattern is evaluated e.g. B. via a

Hybridization of the isolated nucleic acid sequences on a biochip as z. B. is available from Affymetrix or Nanogen. In this way, the isolated mixture of split-off fusion molecule parts containing different nucleic acid sequences can be analyzed directly.

When preparing the extracts to be examined, care must be taken to isolate the proteases in their active form. At the same time, however, DNA-degrading nucleases should be deactivated as far as possible.

The proteolytically active sample can be processed mechanically as follows: If necessary, the entire tissue is cleaned by external washing in cold buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 150 mM NaCl, 0.5 mM DTT ; Dignam, JD, Preparation of Extracts from Higher Eukaryotes. From: Deutscher, MP (ed.) Guide to Protein Purification. Methods in Enzymology, Vol 182, Academic Press, (1990). Page 194-202). The tissue is crushed in the cold buffer and, if necessary, components such as. B. connective tissue, skin, blood vessels removed. The tissue pieces can be homogenized with Kobayashi et al. (Kobayashi, H., Fujishiro, S., Terao, T., Cancer Res. (1994) 54, 6539-6548): 0.01 M HEPES buffer, pH 7.2, 0.25 M sucrose, 0.5% Triton X. -100 in volume ratio 1: 2 to 1: 5 (w / w) offset and in a suitable

Tissue homogenizer (see below) can be processed into a homogenate under ice cooling.

The method must be adapted to the desired tissue type. suitable

Tissue homogenizers are e.g. E.g .: - Mixer: e.g. Liver can be easily made into porridge in a mixer, then rubbed through a tightly woven nylon net. It creates a fine one

Tissue homogenate.

- 'Potter' homogenizer: rotating glass rod that is inserted tightly into a suitable vessel so that the tissue is rubbed between the vessel wall and the glass rod. Constant cooling must be ensured.

- Dispersing machines: homogenizers with rotating knives in one shaft.

The homogenized tissue is mixed with further homogenization buffer and shaken at 4 - 8 ° C for up to 45 min. Then that will

Centrifuge tissue homogenate at 4 ° C with 16,000 g and the supernatant directly or, for. B. further purified by column chromatography or dialysis and used for analysis according to the inventive method.

If frozen material is used instead of fresh tissue, the method should be modified. Do not simply let frozen tissue thaw, otherwise proteases released from lysosomes will inactivate the enzymes. The tissue is preferably pulverized with liquid nitrogen in a mortar with constant cooling and the powder freeze-dried in a lyophilizer overnight. The dry powder can then be used again.

In the following, a suitable processing of brain tissue and its separation into core fraction, cytosolic fraction, membrane fraction and cytoskeleton fraction is given as an example. The extracts obtained can be used directly or with the addition of other auxiliaries as a proteolytically active sample, preliminary sample or

Comparative sample can be used.

Brains, e.g. B. from mice, from several preparations (approx. 50 pieces) are placed in approx. 25 ml solution buffer 1 (LP1) (20 mM Tris-HCl pH 7.5; 150 mM NaCl, 1 mM EDTA, 1 mM EGTA) and homogenized with the Ultraturrax for 1 min at maximum speed. Alternatively, the glass potter is used for less material.

To isolate a core fraction, the homogenate can be centrifuged for 10 min at 1000 x g at 4 ° C. The supernatant is collected for further preparations. The centrifugate (nuclear pellet) is resuspended in 12 ml LP1 and centrifuged as before. The nuclear pellet is resuspended in LP2 (20mM Tris-HCl pH 7.5; 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% NP-40).

The collected supernatants can be used to isolate and purify a cytosolic fraction. They are centrifuged at 100,000 x g, 1 h, 4 ° C. The supernatant forms the extract containing cytosolic proteins.

To isolate the membrane fraction, the pellet is made from the cytosolic

Separation washed three times in LP1 and centrifuged at 100,000 x g, 1 h, 4 ° C. After the third wash step, the pellet is resuspended in 15 ml LP2 and solubilized overnight. The mixture is then centrifuged at 100,000 x g, 1 h, 4 ° C. The supernatant forms the membrane fraction.

To obtain a cytoskeleton fraction, the pellet from the membrane fraction separation is washed three times in LP2 and centrifuged at 100,000 x g, 1 h, 4 ° C. After the third washing step, the pellet is resuspended in LP3 (12.5 mM Tris-HCl pH 7.5; 0.4% SDS) and solubilized at 95 ° C. Final centrifugation at 20,800 x g, 15 min RT. The supernatant contains the

Zytoskelettfraktion.

To obtain proteolytically active extracts from cells and tissues that contain secreted proteases, z. B. human cells or cell lines in the cell culture as a monolayer. The cells are washed three times with cell culture medium. The medium is then replaced by fresh medium. The secretory proteases are secreted into the medium at 37 ° C. The suitable secretion period is 1 to 3 hours. The same procedure can be used with tissue pieces or tissue sections. To obtain tissue fluid from tissue associations, for. B. organs or tissues are carefully shredded with scissors and scalpel. The

Tissue pieces are washed several times with tissue buffer. The tissue pieces or small organs are incubated in physiological buffer or medium at 37 ° C for 1-3 hours. After the incubation, the tissue pieces are left in one

Sediment 15 ml pointed vessel, the medium is removed and can be examined further.

Alternatively, the tissues can be transferred to centrifugation tubes and gently centrifuged. The supernatant contains the proteases of the interstitial fluid from the spaces between the tissues.

Alternatively, protein extracts can also be obtained by the following method: Comminution of the tissue in ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCI, 1 mM EGTA, 1 mM PMSF, 1 mM Na ₃ VO ₄ , 1 mM NaF and 1 μg / ml aprotonin, leupeptin and pepstatin), freeze the shredded tissue and then crush it. To homogenize the tissue, RIPA buffer is added in a volume ratio of 1 to 2 and the batch is then thawed. After 15 min the lysate is centrifuged at 13,000 xg, 4 ° C for 10 min. The lysate can be aliquoted, snap frozen and stored in liquid nitrogen.

In a further modified method, the proteolytic activity of an unknown sample compared to a comparison sample can be determined. The method for the differential determination of the proteolytic activity comprises the following steps: a) incubation of a library of fusion molecules, containing a peptide and a nucleic acid encoding the peptide, with a proteolytically active sample, b) incubation of the same library of fusion molecules in at least one parallel approach , with at least one proteolytically active comparison sample, c) isolation of the proteolytically cleaved components of the fusion molecules, d) creation of a differential nucleic acid bank e) determination of the sequence of the nucleic acids determined.

The differential determination of the proteolytically cleaved nucleic acid parts of the fusion molecules can, for. B. by applying an excess single-stranded nucleic acid parts isolated from the comparison sample are carried out on a suitable support, saturation of the free binding sites of the support and subsequent hybridization with single-stranded nucleic acid parts isolated from the sample. The separable, non-hybridized nucleic acid parts represent only those cut in the sample

Peptide sequences.

In a further method according to the invention, the proteolytic activity of a sample against at least one pre-sample is determined. The differential proteolytic activity can be determined by successively incubating the fusion molecule library with the preliminary sample and sample. The method comprises the following process steps: a) incubation of a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide with at least one proteolytically active preliminary sample, b) removal of the split-off components of the fusion molecules, c) incubation of the library thus prepared with a proteolytically active Sample, d) isolation of the proteolytically cleaved components of the fusion molecules, e) determination of the sequence of the nucleic acid component of the separated fusion molecules.

In preferred embodiments of all three method variants according to the invention, the nucleic acid parts of the proteolytically cleaved and isolated fusion molecule parts are reproduced by means of PCR. For this purpose, fusion molecules are used, the nucleic acid part of which is on both sides of the coding

Nucleic acid sequence have constant nucleic acid sequences as primer binding sites.

In order to greatly increase both the amount of fusion molecule which is cut in the investigated extract and the specificity of the selection step (cleavage in the proteolytic active extract), the isolated nucleic acid of the cleaved fusion proteins is preferably amplified by means of PCR and transcribed in vitro in the method according to the invention. Starting from the RNA produced in this way, methods described above are used again new library of fusion proteins was prepared and incubated with the same extract containing proteolytic activities. This cycle can be repeated several times.

In a further variant of the method according to the invention, PCR, in vitro transcription and / or in vitro translation can be carried out in one or more of the described cycles with a higher than normal error rate. This increases the available peptide sequences to be tested (in vitro protein evolution).

Likewise, the variant of the directed in vitro protein evolution makes it possible to change known cleavage sequences quickly and easily in such a way that other kinetic properties with regard to the cutting proteases result.

The proteolytically active sample can contain other additives, such as. B. specifically acting protease inhibitors or nuclease inhibitors or non-specific RNA and / or DNA to saturate possible nucleases. In particular, if the sample extracts used contain non-specific cutting lysosomal or endosomal proteases (acidic peptidases, such as aspartate peptidases), the addition of one which acts specifically against these proteases is necessary

Inhibitors such as B. the addition of pepstatin A, recommended. The specific inhibition of exoproteases can also be advantageous in order to prevent the breakdown of the proteins contained in the sample, comparative sample or preliminary sample, in particular those of other proteases and their peptide cofactors. The fixed fusion molecules themselves do not have to be protected against exoproteases, since there are no free N- or C-terminal peptide ends. The breakdown of the peptide portion of the proteolytically cleaved fusion molecules is irrelevant for the further course of the process.

Removal of nuclease activities from tissue extracts to protect the

Nucleic acid parts of the fusion molecules can be obtained in the freshly prepared tissue homogenate already during tissue processing and the subsequent steps by treatment with RNAse or DNAse inhibitors. In a further embodiment, the nuclease inhibitors be covalently coupled to particles or suitable column materials. Nucleases that bind to surface-coupled nuclease inhibitors are thus separated from the remaining tissue homogenate. An alternative to inhibiting the

Nucleases, is the protection of the nucleic acid content of the fusion molecules by auxiliary substances. The auxiliary substances surround the DNA and RNA, form a complex and thus protect them from nucleases (Katayose, S. (1998) J. Pharm. Sei., 87: 160-163). These auxiliaries are in particular polyethyleneimine or PEG-PLL, but can also be proteins or chaparones. Protection against nucleases is also conceivable by using artificial nucleic acids in the fusion molecule, such as. B. PNA. The nucleic acids can also be chemically modified to increase stability, e.g. B. by methylation.

The incubation times of library and extract depend on the proteolytic activity of the sample used. In an expanded embodiment, the methods according to the invention are carried out repeatedly with variation of the incubation times. This has the advantage that kinetic statements regarding the sample and its peptide substrates, which contain the library of fusion molecules, can be made. Peptide substrates that are proteolytically cleaved at short incubation times have a high rate constant compared to proteolytically active components of the sample.

Another simple way of being able to make kinetic statements about the proteolytic activity of the sample is either by varying the concentration of the fusion molecules of the library which form the peptide substrates, or preferably by varying the concentration of the sample intended for incubation.

A great advantage of the method according to the invention is that the proteolytic activity of the sample examined is phenomenological, that is to say without complete

Knowledge of the proteolytic active components of the sample can be described. When comparing different samples, it is irrelevant whether a different proteolytic activity is due to the presence of different proteases or to a direct mutation of a protease, which is rarely the case, or for a malregulation of a protease gene or a protease inhibitor or

activator goes back. Changes in the proteolytic activity which are induced by extracellular signals can also be detected using the present methods. It is known that e.g. B. by binding ligands, such as. B. hormones on transmembrane receptors the kinase and / or phosphatase activity in

A cell's cytosol can be altered. The degree of phosphorylation plays a decisive role in the regulation of enzyme activities in the cells and thus also has a decisive influence on the protease activity. A deregulated intracellular phosphatase and, coupled to it, the protease activity plays e.g. B. play a crucial role in the development of cancer.

Above all, the differential determination of specifically cleavable peptides with regard to extracts from healthy and pathological tissue, as well as from healthy and pathogen-infected tissue is advantageous. It is also of particular interest to find specifically cleavable peptides when comparing individual organisms.

With the method according to the invention, sequences of peptides can furthermore be determined quickly and easily, which are cut, in particular specifically cut, by the examined sample. On the other hand, sequences can also be found which are not subject to proteolytic cleavage by the examined sample.

In addition to the identification of specifically proteolytically cleavable peptide sequences, the methods described above can also be used to find inhibitors or activators with a specific effect, without the proteases to be inhibited having to be known in individual cases. For this purpose, a potential inhibitor or activator is simply added to the sample solution and the differential proteolytic activity between the sample and samples containing potential inhibitors or activators is determined. The number of split is reduced

Sequences are inhibitors; if the number of cleaved sequences increases, they are activators. In a preferred embodiment, a preselected library of fusion molecules is used to identify inhibitors

Selection cycles with the proteolytically active sample was obtained. Alternatively, the specifically cleavable peptides identified with the methods according to the invention can also be used directly for screening inhibitors, such as, for. B. in a FRET

Assay.

This provides a quick and easy method to screen for

Protease inhibitors are available.

The rapid and simple screening of protease inhibitors also offers the possibility of combining many effective inhibitors in a mixture of active substances in order to prevent the occurrence of resistance.

Another object of the present invention are proteolytically cleavable substances, which are those obtainable by one of the methods described

Contain peptide sequence.

The specific proteolytically cleavable peptide sequences can be used for the synthesis of inhibitors with a specific action. For this, z. B. chemical modifications to the peptide sequence or one or more α-amino acids of the peptide are exchanged for β-amino acids or L-amino acids for D-amino acids in order to prevent proteolysis (see e.g. Werder M. & Hauser H (1999) Helvetica Chimica Acta, 82, 1774-1783). The specific proteolytically cleavable peptides that can be identified with the present methods can be used for the construction of drug delivery systems in order to achieve a selective release of active substance at the target site. It can be at the destination z. B. to a certain organ, tissue, a certain cell type, a subcellular compartment or to sick versus healthy tissue or to microbially infected versus unaffected tissues or cells.

In the case of an active ingredient which can be activated by protease cleavage, the specifically proteolytically cleavable substances can be used as covalently bound inhibitors. The specific proteolytically cleavable peptides can also be used as Linkers can be used to target a drug with a ligand, such as.

B. a specific antibody or a specific receptor ligand. However, it is also possible to construct drug delivery systems which are closed with specifically proteolytically cleavable substances (Minko T. et al., (2000) Int. J. Cancer, 86: 108-117).

The variant of the directed in vitro protein evolution makes it possible to change known peptide cleavage sequences so that other cleavage kinetics result. The time profile of the active ingredient release can thus be controlled.

With the help of the specifically proteolytically cleavable peptides, diseases can be combated more efficiently by means of a target-controlled release of active substances, in which proteolytic activities have changed at the desired site of action compared to the healthy state or which are due to an incorrect regulation or mutation of specific proteases or their activators or inhibitors , Examples of diseases with disturbed protease activity are e.g. B. asthma,

Osteoporosis, cancer, such as leukemia, breast cancer, colon cancer, stroke, neuronal diseases, such as Alzheimer's, arthritis, pancreatitis, high blood pressure, thrombosis, colds or schistosmiasis.

The targeted release of active ingredients can minimize the side effects of these active ingredients and the amount of active ingredient to be used can be reduced due to the changed distribution profiles in the organism. Pharmaceutical active ingredients are particularly interesting. B. against bacteria, fungi, viruses or against diseased tissue cells, but also herbicides, fungicides or insecticides can be used.

Another application of the method according to the invention is in use as a diagnostic assay. The assay can e.g. B. on a comparison of the profile of the proteolytic activity of extracts against a library of fusion molecules defined in their composition between diseased tissue, e.g. B. from cancer cells, and based on a sample to be examined. However, preference is given to assays which are based on peptides which can only be proteolytically cleaved by pathological tissue compared to a comparison or preliminary sample. In the case of such a selective assay, the separated parts of the Fusion molecules provided sequencing step are omitted. In this case, the presence of a specific disease-specific protease activity can be identified using a hybridization probe. Such a method can be highly integrated by using many cleavable peptides identified as disease-specific markers in such an assay.

Alternatively, the specifically cleavable peptides identified with the methods according to the invention can also be used directly for the screening of inhibitors, such as, for. B. in a FRET assay. The specifically cleavable sequences can also be used in vivo as a substrate for FRET assays. Another use of the peptides which can be specifically cleaved from a proteolytically active sample is for isolating the protease which cleaves this peptide sequence (see, for example, Li Y.M. (2000) Nature, 405: 689-694).

An applicable method for isolating serine proteases is e.g. B. described in US 5843701. Serine proteases are protein enzymes that catalyze the hydrolysis of peptide bonds within proteins. The target protein is often selectively cut due to the specific peptide linkage within the substrate. The serine proteases include, among others

Tissue plasmino activator, trγpsin, elastase, chymotrypsin, thrombin and plasmin. Many stages of the disease can be treated with serine protease inhibitors, such as blood clotting disorders. Elastase inhibitors can reduce the clinical progression of emphysema. To isolate proteases, the specific proteolytically cleavable peptides determined according to the methods described above can be used e.g. B. coupled with standard methods on column material and an affinity chromatography of the proteolytically active samples can be carried out on it. The buffer used during the binding cycles must not be denaturing so that the protease is not inactivated. The proteases interacting with the peptides can

Can also be bound to the fixed peptides using standard crosslinking methods. For this purpose, it is also possible to use cleavage sequences containing β-amino acids which are specifically recognized by proteases but cannot be cleaved. A few exemplary embodiments are given below to illustrate the invention:

Example 1: Preparation of fusion molecules containing a peptide with known ones

Protease interface and a nucleic acid encoding the peptide.

General:

Two types of fusion molecules were made. On the one hand, fusion molecules that contain a peptide sequence that is specifically cleaved by the protease thrombin (“thrombin fusion molecules”). On the other hand, fusion molecules which contain a peptide sequence which is specifically cleaved by the matrix metalloprotease-3 (MMP-3) ("MMP-3 fusion molecules"). The production of such fusion molecules is e.g. B. described in WO98 / 31700.

The fusion molecules produced in this way have protease interfaces for MMP-3 or for thrombin.

The MMP-3 protease cuts between Glu and Leu, the thrombin protease cuts between Arg and Ser. The peptide sequences used and the corresponding nucleotide sequence are given below.

Protease cleavage sites:

MMP-3 interface (Nagase, H., 1995, Methods Enzymol. 248, 449-470) Protein (AS): Arg-Pro-Lys-Pro-Val-Glu- | Leu-Trp-Arg-Lys (Seq. ID No. 1)

DNA (bp): AGA CCA AAA CCC GTT GAG CTC TGG AGA AAG (Seq. ID No. 2)

Thrombin interface (Le Bonniec, BF, 1996, Biochemistry 35, 7114-7122) Protein (AS): Val-Pro-Arg- | Ser-Phe-Arg (Seq. ID No. 3) DNA (bp): GTT CCA AGA AGC TTC AGG (Seq. ID No. 4) 1.1 Production of the MMP-3 and thrombin template DNA via PCR

In a 50 μl reaction mixture, 250 ng DNA template, 25 pmol 5 ^′ and 3 ^′ primer, 10 mM dNTPs, 5 μl 10 × PCR buffer and 5 U / μl Taq DNA polymerase were combined (buffer and enzyme from Promega, Madison, UNITED STATES). The PCR was carried out under the following conditions in the Biometra T gradient cycler: 1 min 94 ° C., 21 × (0.5 min 94 ° C., 0.5 min 55 ° C., 0.5 min 72 ° C.).

Primer and template: 5 ^' Strep-Tag Primer (90 bp):

5 ^' -taa tac gac tca cta tag gga caa tta cta ttt aca att aca atg tgg tcc cac ccc cag ttc gag aag agt ggc tca agc tca gga tca-3 ^' (Seq. ID No. 5)

3 ^' MMP-3 primer (44 bp):

5 ^' -ttt taa ata gcg gat gct act agg cta gac cca gag cta ccc ga-3 ^' (Seq. ID No. 6) 3 ^' thrombin primer (44 bp):

5 ^' -ttt taa ata gcg gat gct act agg cta gac cca gag gat cct ga-3 ^' (Seq. ID No. 7)

MMP-3 template:

5 ^' -agen ggc tca agc tca gga tca gga tct ggt aga cca aaa ccc gtt gag ctc tgg aga aag cac cat cac cat cac cat gga agt ggc tcg ggt agc tct ggg tct agc-3 ^' (Seq. ID No. 8) Thrombin template:

5 ^' -ag ggc tca agc tca gga tca gga tct ggt gtt cca aga agc ttc agg cac cat cac cat cac cat gga agt ggc tca gga tcc tct ggg tct agc-3 ^' (Seq. ID No. 9)

Fig. 1 shows the PCR products on 2% agarose-ethidium bromide gel. Lane 1: 50 bp DNA marker, Lane 2: MMP-3 DNA, Lane 3: Thrombin DNA,

Lane 4: 100 bp DNA marker

As FIG. 1 shows, the MMP-3 DNA and the thrombin DNA could be successfully amplified. The PCR products have the expected molecular weight of 199 bp for the MMP-3 DNA and 89 bp for the thrombin DNA.

1.2 In vitro transcription of the MMP-3 and thrombin DNA 100 pmol DNA was used in the transcription reaction (Ribomax T7 /

Promega, Madison, USA). In a 500 μl mixture, the DNA was incubated with 5 × T7 buffer, rNTPs and T7 RNA polymerase for 4 hours at 37 ° C.

The RNA was purified by means of phenol-chloroform extraction and on a 6%

Urea gel (Novex Gel / Invetrogen, Groningen, the Netherlands) was analyzed.

2 shows the RNA obtained after transcription on 6% urea gel. Lane 1: MMP-3 RNA, Lane 2: thrombin RNA

As can be seen from FIG. 2, RNA can be detected as expected after the transcription reaction. The RNA shows no degradation.

1.3 Ligation of the RNA with Puromycin Linker via UV Crosslinking

In a 120 μl mixture 3 nmol RNA, 4.5 nmol puromycin (Pu) linker (PEG polyethylene glycol, Pso = Psoralen) with the sequence

5 ^* - (Pso) 2 ^{^} OMe -U AGC GGA UGC AAA AAA AAA AAA AAA AAA PEG PEG CC- (Pu) -3 ^' (nucleotides 1 to 28 are given by Seq. ID No. 10)

(Intreractiva / Ulm), 12 μl 10x ligase buffer (1 M NaCl, 250 mM Tris pH 7.5) combined and incubated for 5 minutes at 85 ° C. and then for 10 minutes at room temperature. UV crosslinking was carried out at 366 nm for 15 minutes (UV hand lamp from LTF-Labortechnik, 12 W)

The ligation efficiency was checked on a 5% urea-agarose gel.

3 shows the analysis of the ligation reaction on a 5% urea TBE gel. Lane 1: MMP-3 RNA, Lane 2: MMP-3 RNA with puromycin linker, Lane 3: thrombin RNA, Lane 4: thrombin RNA with puromycin linker. (The signal L comes from the dye marker.) After ligation of the puromycin linker to the RNA, a clear increase in molecular weight can be seen, which indicates the successful linkage of the thrombin RNA and the MMP-3 RNA with the puromycin.

1.4 In vitro translation

8 μl of linked RNA were mixed with 200 μl of reticulocyte lysate, (Promega, Madison, USA L416X), 5 μl of ³⁵ S-methionine (Hartmann, 10.8 μM, specific activity: the specific activity is 72181 dpm / pmol), 6 μl amino acid mix (without methionine 1 mM, Promega) and 80 μl H ₂ O mixed and then incubated at 30 ° C for 30 minutes. After adding 130 μl 2 M KCI and 75 μl MgC, the mixture was incubated again at 30 ° C. for 30 minutes.

The resulting fusion molecules were purified with oligo dT-cellulose (Amersham Pharmacia, Freiburg, Germany) (removal of all components of the in vitro translation approach that do not contain any polyA RNA sequences).

1.5 Reverse transcriptase response

The purified 200 μl fusion molecules were mixed with 2.5 μl 100 μM reverse primer and incubated for 5 minutes at 80 ° C. After cooling the

Reaction mixture on ice, 50 ul 5x beach buffer, 20 ul dNTP ^'s (each 10 mM), 2.5 ul RT-Superscript II (all Promega, Madison, USA) were added and incubated at 42 ° C for 40 minutes

Example 2:

Proteolytic cleavage of the fusion molecules with thrombin and MMP-3 in solution

The fusion molecules produced were incubated with the corresponding proteases and the reaction products were analyzed (FIG. 4). 10 nM MMP-3 (Sigma, Deisenhofen, Germany) was added to 5 pmol fusion molecule containing the MMP-3 interface and added in MMP-3 buffer (50 mM Tris pH 7, 150 mM NaCl, 10 mM CaCl ₂ ) Incubated 37 ° C in a total volume of 15 ul for 45 minutes. 1 U thrombin was converted into 5 pmol fusion molecule containing the thrombin interface (1 NIH Unit = 0.324 μg, Sigma, Deisenhofen, Germany) and in

Thrombin buffer (50 mM Tris pH 8, 150 mM NaCl) at 37 ° C in one

Total volume of 15 ul incubated for 45 minutes. The analysis of the

Starting fusion molecules and the fusion molecules after proteolytic cleavage were carried out using a phosphoimager according to SDS-PAGE (FIG. 4)

4 shows the digestion of the fusion molecules with thrombin and MMP-3 in solution: phosphoimager image after 4-20% Tris-glycine SDS-PAGE.

Lane 1 "MMP-3 fusion molecules" before digestion; Lane 2 "Thrombin fusion molecules" before digestion; Lane 3 "thrombin fusion molecules" + thrombin Lane 4 "thrombin fusion molecules" before digestion Lane 5 "MMP-3 fusion molecules" + MMP-3 lane 6 "MMP-3 fusion molecules" before digestion. (Lanes i show fusion molecules, lanes ii show peptidic

By-products and bands iii originate from N-terminal fusion molecule fragments.)

The “MMP-3 or thrombin fusion molecules” produced were proteolytically cleaved by the protease MMP-3 or the protease thrombin.

Example 3

Isolation and detection of the proteolytically cleaved "thrombin fusion molecules" and amplification of the nucleic acid content

For the isolation and identification of specifically proteolytically cleaved fusion molecules, three successive process steps were carried out. 5 schematically shows the method steps according to this example for the identification of specifically cleavable peptides:

Step 1. Coupling of the Fusion Molecules to Streptactin-Sepharose (Carrier Material) via an N-Terminal Strep Tag Step 2. Proteolytic cleavage of the "thrombin fusion molecules" on

Carrier material with thrombin.

Step 3. Isolation of the cleaved C-terminal fusion molecule fragments and

Detection of the fragments after PCR amplification of the nucleic acid content. Before the analysis, the isolated fragments can optionally be purified via their His tag using affinity chromatography on nickel particles.

3.1 Coupling of "Thrombin Fusion Molecules" to Streptactin Sepharose

50 μl “thrombin fusion molecules” (corresponding to approx. 5 pmol) were mixed with 50 μl

Streptactin-Sepharose, washed 5 times in Streptactin buffer (150 mM NaCl, 100 mM Tris pH 8) and then incubated for 2 hours at 4 ° C. Unbound fusion molecules were removed by washing five times with Streptactin buffer.

3.2 Proteolytic digestion of the "thrombin fusion molecules" with thrombin

The fusion molecules bound to Sterptactin-Sepharose were resuspended in 80 μl water and incubated with 10 μl 10 × thrombin buffer (1.5 M NaCl, 500 mM Tris pH 8) and 10 μl 100 U / μl thrombin at 37 ° C. for 45 minutes. As a control, the same approach was carried out without thrombin.

3.2.1 Isolation and Detection of the Bound "Thrombin Fusion Molecules"

After the incubation period, the reaction mixture was centrifuged (1 minute at 3000 rpm). The peletted streptactin-Sepharose, containing the N-terminal fragment of the cleaved fusion molecules, was washed three times with streptactin buffer and then resuspended in 80 μl water.

The "Thrombin Fusion Molecules" Bound to Streptactin-Sepharose

(Control batch) or the bound N-terminal fragments after incubation with thrombin were separated by SDS-PAGE and visualized by means of phosphoimaging (FIG. 6).

6 shows “thrombin fusion molecules” or fragments of the fusion molecules bound to streptactin-Sepharose. 15 μl of the resuspended streptactin Sepharose was mixed with 5 μl Lämmli application buffer each, heated for 5 min at 82 ° C. and separated using 4-20% Tris-Glycine SDS-PAGE. The gel was then dried at 80 ° C. for 2 hours and visualized using phosphoimaging. Lane 1: "Thrombin fusion molecules" after incubation with thrombin. Lane 2: "Thrombin fusion molecules" without incubation with thrombin (control).

(Band i shows the intact fusion molecule, band ii comes from peptide by-products, and band iii comes from the N-terminal fusion molecule fragment.)

After incubation of the “thrombin fusion molecules” with thrombin, only the N-terminal fragments (labeled with ³⁵ S-methionine) are bound to streptactin-Sepharose.

3.2.2 Detection of the nucleic acid fractions of the "thrombin fusion molecules" by means of PCR analysis

After the streptactin-Sepharose had been separated off, 15 μl of the supernatant or 15 μl of the resuspended streptactin-Sepharose were used for the PCR. In addition to the 15 μl samples, the 50 μl PCR batches also contained 5 μl 5 ^′ and 3 ^′ thrombin primers (5 pmol / μl), 10 mM dNTPs, 5 μl 10 × PCR buffer and 5 U / μl Taq DNA

Polymerase (buffer and enzyme from Promega, Madison, USA). The amplification was carried out under the following conditions: 1 min 94 ° C, 21 x (0.5 min 94 ° C, 0.5 min 55 ° C, 0.5 min 72 ° C). The PCR products obtained are shown in FIG. 7.

7 shows the PCR products of the “thrombin fusion molecules” after 2% agarose

Gel electrophoresis. In lanes 1 and 2, the products can be seen as black signals on a light background, in lanes 3 and 4, due to a different imaging technique, as white signals on a dark background. Lane 1: "Thrombin fusion molecules" + thrombin; supernatant. Lane 2: "Thrombin fusion molecules" without thrombin (control); Got over.

Lane 3: "Thrombin fusion molecules" + thrombin; bound to Streptactin-Sepharose. Lane 4: "Thrombin fusion molecules" without thrombin; bound to streptactin-sepharose. Consequently, it can be shown that "thrombin fusion molecules" are cut by the incubation with thrombin. After separation of the N-terminal fragment which contains no nucleic acid part, the C-terminal part remains in the supernatant Lane 1, Fig. 7) a strong PCR

Amplificate, in contrast, only a very weak PCR amplificate was obtained in the pellet (eg lane 3, FIG. 7). In the control reaction, the “thrombin fusion molecules” were not incubated with thrombin. As a result, the fusion molecules remain bound to the streptactin-Sepharose and a strong PCR amplificate is obtained in the pellet (eg lane 4, FIG. 7) during the PCR -Amplicate of the supernatant (e.g.

Lane 2, Fig. 7) is only very weak.

Claims

claims:

1. A method for identifying specific proteolytically cleavable peptides comprising the method steps: a) incubation of a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide with a proteolytically active sample, b) isolation of the proteolytically cleaved components of the

Fusion molecules, c) determination of the sequence of the nucleic acid component of the separated fusion molecules.

2. A method for identifying specific proteolytically cleavable peptides comprising the method steps: a) incubation of a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide with a proteolytically active sample, b) incubation of the same library of fusion molecules in a parallel approach, with at least one proteolytically active comparison sample, c) isolation of the proteolytically cleaved components of the fusion molecules, d) creation of a differential nucleic acid bank e) determination of the sequence of the nucleic acids determined.

3. A method for the identification of specifically proteolytically cleavable peptides comprising the method steps: a) incubation of a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide with at least one proteolytically active preliminary sample, b) separation of the split-off components of the fusion molecules, c) Incubation of the library prepared in this way with a proteolytically active

Sample, d) isolation of the proteolytically cleaved components of the fusion molecules, e) determination of the sequence of the nucleic acid component of the separated

Fusion molecules.

4. The method according to any one of the preceding claims, characterized in that a library of fusion molecules containing a peptide and a nucleic acid encoding the peptide, the one or both sides of known

Flanked nucleotide sequences is used.

5. The method according to any one of the preceding claims, characterized in that a library of fusion molecules consisting of a peptide which is linked via a puromycin to a poly-A nucleic acid sequence to which the region encoding the peptide and one or more known nucleic acid sequences follow becomes.

6. The method according to any one of the preceding claims, characterized in that fusion molecules containing peptides from at least 4 variables

Amino acids can be used.

7. The method according to any one of the preceding claims, characterized in that fusion molecules containing peptides from 7 to 1 1 variable amino acids are used.

8. The method as claimed in one of the preceding claims, characterized in that fusion molecules are used which contain peptides which, in addition to the variable sequence region, have at least one constant sequence region.

9. The method according to any one of the preceding claims, characterized in that fusion molecules with a labeled nucleic acid component and / or peptide component are used.

10. The method according to claim 9, characterized in that fusion molecules with a magnetic label, a radioactive label, a fluorescent label of the nucleic acid component and / or peptide component or a label with a specific known

Nucleic acid sequence and / or a known specific amino acid sequence can be used.

11. The method according to any one of the preceding claims, characterized in that the fusion molecules of the library are fixed on a solid surface.

12. The method according to any one of the preceding claims, characterized in that the fusion molecules of the library are fixed on solid particles.

13. The method according to any one of the preceding claims, characterized in that the known nucleic acid sequences are cut with restriction enzymes and only the cut or cut nucleic acid part is used further.

14. The method according to any one of claims 4 to 13, characterized in that after isolation of the fusion molecule components cut from the proteolytically active sample, a PCR is carried out to amplify the nucleic acid components.

15. The method according to claim 14, characterized in that the amplified nucleic acid is used to create a new library of fusion molecules and with this library a process cycle according to claims 1 to 3 is carried out again.

16. The method according to claim 14 for in vitro protein evolution, characterized in that the PCR, the in vitro transcription and / or the in vitro translation takes place with an increased error rate.

17. The method according to any one of the preceding claims, characterized in that a whole cell extract, a cytosolic extract, a membrane extract, an extracellular extract, a subcellular extract, a combination of these extracts or a solution of known proteases is used as a proteolytically active sample, preliminary sample and comparative sample ,

18. The method according to any one of the preceding claims, characterized in that disease-specific and / or tissue-specific and / or oganspecific and / or organism-specific extracts are used as a sample, preliminary sample or comparative sample.

19. The method according to any one of the preceding claims, characterized in that the proteolytically active sample and / or pre-sample are used in the method under physiological conditions.

20. The method according to any one of the preceding claims, characterized in that further auxiliaries are added to the sample and / or preliminary sample.

21. The method according to claim 20, characterized in that specifically acting protease inhibitors and / or nuclease inhibitors are added as auxiliaries.

22. The method according to any one of the preceding claims, characterized in that potential protease inhibitors or activators are added to the sample.

23. The method according to any one of the preceding claims, characterized in that the method steps are repeated or carried out repeatedly with different incubation times and / or different sample concentrations.

24. Use of the nucleic acid sequences determined according to the method of claims 1 to 23 for the production of specifically proteolytically cleavable substances.

25. Specifically proteolytically cleavable substances containing a peptide obtainable by a process according to one of claims 1 to 23.

26. Specifically proteolytically cleavable substance according to claim 25 containing a chemical agent.

27. Specifically proteolytically cleavable substance according to claim 26, characterized in that the chemical agent can be activated by proteolytic release.

28. Specifically proteolytically cleavable substance according to one of claims 25 to 27, characterized in that further auxiliaries and additives are added to the substance.

29. Use of the specifically proteolytically cleavable substances according to claims 25 to 28 for the manufacture of medicaments for asthma, osteoporosis, cancer, stroke, neuronal diseases, arthritis, pancreatitis, high blood pressure, thrombosis, colds or schistosmiasis.

30. Diagnostic markers containing proteolytically cleavable peptides obtainable by a process according to one of claims 1 to 23.

31. A method for the detection of specifically acting proteases according to claims 1 to 23 containing diagnostic markers according to claim 30.

32. Use of substances containing specific proteolytically cleavable peptides determined by a method according to claims 1 to 23 for the identification of the proteases cleaving these peptides.

33. Use of specifically proteolytically cleavable peptides determined according to a method of claims 1 to 23 for the identification of protease inhibitors.

4. Use of specific proteolytically cleavable peptide sequences determined according to a method of claims 1 to 23 for the production of inhibitors.