DE10112748A1 - Invention relating to HCV diseases - Google Patents

Abstract

The invention relates to the binding partners for the NS2 protein of human Hepatitis C Virus (HCV), to binding partners for a cellular J domain protein (Jiv; <u>J</u>-domain protein <u>i</u>nteracting with <u>v</u>iral protein), and to methods for identifying the binding partners which effect a competitive or allosteric inhibition of the bond between the HCV-NS2 protein and Jiv. The invention also relates to agents for treating HCV diseases that contain said binding partners, and to the use of the binding partners in the prevention, diagnosis and treatment of HCV diseases.

Description

The present invention relates to binding partner for the NS2 protein of the human hepatitis C virus (HCV), binding partner for a cellular J-domain protein (Jiv; J -domain protein i nteracting with v Iral protein), methods for Iden tification of the binding partners which cause a competitive or allosteric inhibition of the binding between the HCV NS2 protein and Jiv, agents for the treatment of HCV diseases which contain these binding partners, and the use of these binding partners for the prevention, diagnosis and treatment of HCV diseases.

description

Hepatitis C is a viral disease affecting the liver attacks, diseases and failure of this organ and last Lich can lead to death. 3% of the world population are already infected with the hepatitis C virus (HCV), 20 million in the western world alone. HCV is more common and more contagious than HIV, the rate of newly infected people is increasing annually. A vaccine is currently not available and their development bumps u. a. due to the high mutation rate of the viral genome at great difficulty. The current one Action against HCV currently includes a largely unspecified Specific medical therapy with interferon alpha alone or in combination with ribavirin (Wang and Heinz, 2000). al However, in many cases the treatment is unsuccessful because a large part of the HCV isolates are resistant to the inter feron treatment shows (Taylor, 2000). The treatment used Development strategy leads especially in the high dose required tion of interferon alpha to significant and undesirable Side reactions, the long-term effects of which are are known.

The hepacivirus genus (the only representative is the human He patitis C virus, HCV) is grouped with the Genera Flavivi rus and pestivirus to the Flaviviridae family (Heinz et al., 2000). So far they have not been able to continue with this family  classified GBV-A, GBV-B and GBV-C. The Genus Pestivirus includes a. the bovine viral virus Diarrhea (BVDV).

At the molecular level, the representatives of Genera Pesti virus and hepacivirus (HCV) within the Flavi family viridae a high degree of correspondence on (see below) (Rice, 1996). Therefore, pestiviruses, v. a. BVDV as a model system used for HCV. The need for a model system results from the fact that, in contrast to the Pestivirus system for HCV no, for drug screening usable, infectious cell culture system exists. So can Until now, infection attempts have only been possible in the primate system be carried out casually (Kolykhalov et al., 2000). Thereby is z. B. the determination of the titer of infectious viruses talked difficult about the development of antiviral substances zen decisively inhibits. The recently developed HCV While replicons can replicate their RNA in cell cultures, So far, however, this approach has not succeeded in creating new infectious diseases generate viruses (Lohmann et al., 1999). A targeted Search for substances that prevent the formation of infectious HCV So far, reducing virions has been extremely easy satisfactorily.

The replication of the members of Flaviviridae includes how the synthesis of all positive stranded RNA viruses at least one polyprotein that of cellular and viral proteases  co- and post-translational into the mature virus proteins is split (Rice, 1996). For viruses, their replication strategy involves the synthesis of a polyprotein comes to that correct sequence and the exact kinetics of the polyprotein pro cessation is of crucial importance with regard to the Vi soot increase in the cell. Antiviral strategies, e.g. B. against the human immunodeficiency virus (HIV), therefore aim often seek to interfere with polyprotein processing. The Success of this method shows u. a. the currently practiced HIV therapy with protease inhibitors (Flexner, 2000).

For pestiviruses and HCV, the processing takes place in the C- terminally located non-structural protein (NS) region of the Po lyproteins (NS2-NS3-NS4A-NS4B-NS5A-NS5B) in very similar Wise. The main protease in both polyproteins is egg ne serine protease in the NS3 protein, which is responsible for its full function Ability to use the NS4A protein as a cofactor (Rice, 1996). Inactivation of the NS3 protease results in both Vi soot systems to stop replication (Behrens et al., 1998; Kolykhalov et al., 2000). Another virus-encoded Protease has been described for NS2-3 from HCV, which the Cleavage of NS2-3 catalyzed (Grakoui et al., 1993; Hijika ta et al., 1993; Pieroni et al., 1997). Due to in vitro Studies have been postulated to be efficient for NS2-3 Cleavage at HCV previously unknown host factors an important play an important role (Pieroni et al., 1997). molecular biology  Studies at the HCV have shown that an inactivity the NS2-3 protease and RNA replication of the virus terbet (Kolykhalov et al., 2000). So the NS2-3 comes Cleavage and thus the activity of the NS2-3 protease in the case of the HCV is of essential importance. From virological View it can be assumed that a malfunction of the NS2-3 Spal viral replication.

With regard to pestiviruses, the existence of one of the HCV Enzyme activity corresponding to NS2-3 protease has been repeated several times required (Kümmerer and Meyers, 2000; Mendez et al., 1998; Tautz, et al .; 1996).

The investigations of the invention in BVDV system show that the BVDV NS2 protein a stable interaction with an unidentified cellular J-domain protein Jiv (J - domain protein i nteracting with v Iral protein; Fig. 2A) is one. In BVDV infected cells, the intracellular amount of the Jiv protein correlates directly with the extent of the NS2-3 cleavage. Interestingly, a Jiv fragment, which is designated according to the invention as Jiv90 and which comprises amino acids 533-622 of the bovine Jiv protein ( FIG. 2B), has the property of binding to NS2 of BVDV and stimulating NS2-3 cleavage. Jiv90 can be shortened by either 5 amino acids, either carboxy or amino, without losing the properties mentioned above; functional are thus Jiv or fragments of Jiv which comprise at least the amino acid 538-622 or 533-617.

Surprisingly, an interaction with the Jiv90 domain of the Jiv protein identified in the bovine system can also be demonstrated for the HCV NS2 protein (HCV-NS2). In the relevant region of the Jiv protein, amino acids 533-621 (numbers refer to the bovine sequence), bovine and human Jiv are identical (see FIG. 3).

In view of the findings obtained in the BVDV system is of that assume that the human ortholog of the bovine Jiv protein is one of the postulated host factors for HCV that stimulates the activity of the NS2-3 protease (Pieroni et al., 1997). From a virological point of view it can be assumed that ei inhibition of this interaction to reduce the Re of the HCV.

The object of the present invention is therefore the identifica tion of binding partners which bind the HCV-NS2 and inhibit Jiv competitively or allosterically, continue testing systems that lead to the finding of these binding partners, Ver application of these binding partners for prevention and diagnosis of HCV diseases and agents for the treatment of HCV Diseases that have at least one of these binding partners contain.

The interaction between Jiv and NS2 from pestiviruses, esp especially from BVDV, can serve as a model system. With  With the help of this model system, a correlation between inhibition of the NS2 / Jiv interaction and thereby determine the reduction in virus titer caused. In this System successfully tested substances / proteins / ligands can be adapted to the HCV system.

The present invention therefore relates to binding partner for the NS2 protein from HCV (HCV-NS2), which thereby are marked to bind HCV-NS2 to the Inhibit jiv protein competitively or allosterically.

The present invention also relates to the binding part ner for Jiv, who are characterized by the fact that they are the Binding of HCV-NS2 to Jiv competitive or allosteric hem men.

Binding is understood to mean any molecular interaction between Jiv and HCV-NS2, especially under physiological Conditions. These are usually classic protein Protein interactions, including electrostatic attraction hung, hydrogen bond, hydrophobic bond, van der Waals forces and metal complex-like coordinative bond belong. In addition to the aforementioned reversible molecular interactions are invented for binding binding partner according to the invention on Jiv or HCV-NS2 also crazy versible interactions, such as covalent bonds, in Be costume.  

Competitive inhibition means that according to the invention Connect with Jiv or HCV-NS2 to bind HCV-NS2 and Jiv compete, respectively. H. the bond of one hinders the other's bond.

Allosteric inhibition means that inventive bind binding partner to Jiv or HCV-NS2 and thereby Jiv or HCV-NS2 so that the bond between Jiv and HCV-NS2 is hindered.

Preferably, the HCV-NS2 side binding site is over which the binding partner binds to HCV-NS2, of at least ei part of the HCV-NS2-side binding site, via the HCV NS2 binds to Jiv, formed. The same applies to the Jiv side binding site in terms of binding between Jiv and HCV-NS2 or Jiv and binding partner.

Binding partners for the HCV-NS2 protein are preferred.

According to a preferred embodiment of the present invention, binding partners of HCV-NS2 bind to a part of the amino acid sequence of HCV BK (subtype 1b) 1 to 217 (Takamizawa et al., 1991) shown in FIG. 1A, to NS2 of other HCV subtypes or homologous Sequences of it.

Homologous sequences are understood to mean amino acid sequences which are derived from the amino acid sequence shown in FIG. 1A by single or multiple amino acid deletion, substitution, insertion and / or inversion. Homologous sequences are thus part of mutated HCV-NS2 proteins, provided that the amino acid sequence shown in FIG. 1A is defined as a wild-type sequence. By definition, homologous sequences of the amino acid sequence shown in FIG. 1A are essentially functionally equivalent to this. In particular, homologous HCV-NS2 sequences can bind to Jiv, whereby the binding affinity of a homologous HCV-NS2 sequence to Jiv can differ from the binding affinity of the sequence shown in FIG. 1A to Jiv.

According to the invention, the term “HCV-NS2” stands for the amino acid sequence shown in FIG. 1A as well as for NS2 of other HCV subtypes and homologous sequences thereof; The NS2 sequences of GBV-A, GBV-B and GBV-C are also included.

According to the invention, the term “Jiv” stands for the amino acid sequence of the bovine Jiv protein shown in FIG. 2A as well as for Jiv of other Euksryotes, in particular that of mammals and homologous sequences thereof.

Binding partners according to the invention preferably bind to Jiv or HCV-NS2 with higher affinity than Jiv on HCV-NS2 or HCV-NS2 to Jiv. A higher binding affinity has the intention share that lower concentrations of binding partner he are required to have a competitive or allosteric hem effect. On the other hand, a suitable inhibition also with binding partners of lower binding affinity be enough by having a higher concentration of bin application partner applies.  

The binding partners according to the invention can be Peptides, peptoids, organic or inorganic substances act. Peptides and peptoids are preferred.

Peptides are understood to be sequences of naturally occurring proteogenic amino acids linked to each other via peptide are linked. Sequences of peptoids, i. H. Peptide- similar compounds, in addition to naturally occurring, proteogenic amino acids also other amino acids and ami contain noic acid derivatives, for example certain stereo and diastereoisomers such as D-amino acids occur naturally de, non-proteogenic amino acids or chemically synthesized Compounds with properties that the skilled worker as Ami are known similar to no acid, such as beta-alanine, gamma-carboxy glutamic acid, hydroxyproline, norleucine, norvaline, ornithine, Phenylglycine, pyroglutamic acid, sarcosine, statin, or t- Leucine. Peptides are also understood to mean sequences whose N- and / or C-terminus can be modified at for example with acetylated N-terminus and / or amidated C-terminus. The same applies to chemically modifiable ones Amino acid side groups, in particular those in the orga African chemistry and especially in peptide chemistry protective groups to be used.

Suitable binding partners are also antibodies, in particular monoclonal and recombinant antibodies to at least bind part of the binding site between HCV-NS2 and Jiv.  Are particularly preferred in this context gene binding fragments of the hypervariable domains of such Antibodies, for example Fab, Fv, scFv fragments or fragments based on one or more CDRs.

With regard to the use of the binding according to the invention The partner in pharmaceutical products is the incorporation of Ami Non-acids that do not occur in nature are therefore advantageous clinging, as a result the stability against natural degradation processes is increased. Should a mammal as according to the invention a specific peptide is administered to the binding partner, for example, its half-life can be there in vivo by extending that you can at least part of the natural Chen L-amino acids replaced by D-isomers and thus to one corresponding peptoid.

Methods for producing according to the invention are more familiar to the person skilled in the art Peptides and peptoids are known. Solid phase are preferred Syntheses using functionalized resins, on wel the peptides to be synthesized can be linked. Wang resins for FMOC solid phase peptide synthesis or Merri field resins for BOC solid-phase peptide synthesis are common Examples. After a desired peptide has been built up it is then detached from the carrier, with or simultaneously afterwards there may also be a protective group can be split off. A possibly required Liche, subsequent purification, for example via  HPLC delivers the peptide in the desired purity for example by means of mass spectroscopy or NMR can be. The exact design of the procedure is aimed the peptide or peptoid to be synthesized and is based on professional knowledge.

The peptides and peptoids according to the invention can also be used be produced using molecular biological methods. To can, for example, the primary amino acid sequence of syn thetizing peptide into the corresponding cDNA or mRNA Sequence are implemented. The synthesized in a known way The nucleic acids can then be transferred to a suitable host cell introduced, expressed and then, if necessary, to the desired peptoids can be modified. additional Refinements of this method are also based on subject male knowledge.

Furthermore, libraries can randomized peptide genes are then used to isolate peptides the desired binding specificity is possible. Beispielswei one can randomize oligonucleotides into a suitable one Bring expression system, which the selection of invent oligopeptides according to the invention with different amino acids sequence allowed.

Methods for producing specific, monoclonal and recombinant antibodies and in particular Antibody fragments known.  

For example, monoclonal antibodies that are specific against the HCV-NS2 or Jiv side, at the bond between HCV-NS2 and Jiv involved binding site are directed can be obtained by means of conventional hybridoma technology by peptide fragments from the HCV-NS2 or Jiv-side bin used for immunization. Antibody fragments these monoclonal antibodies can in the usual way for example by proteolytic digestion.

The basis for the production of recombinant antibodies and Antibody fragments preferentially form antibody genes libraries using PCR from antibody-related geneti information can be created. As a genetic Information can be, for example, cBNA from defined cells lines, such as hybridomas or myelomas, cDNA from seropositive Donors or non-immunized donors, genomic DNA as a whole of the variable regions or randomized oli gonucleotides can be used for hypervariable regions in the order of the above examples for Bi libraries with increasing complexity.

Individual antibodies or antibodies can then be made from these fragments with the desired specificity, d. H. erfindungsge moderate binding partner, be prepared by the be apt gene cloned into a production vector and into pro caryotic systems such as E. coli, Bacillus subtilis, Strep tomyces lividans, or eukaryotic systems, such as plasmacytoma  or myeloma cells, COS cells, CHO cells, be agreed insect, plant or fungal cells, for example unicellular yeast cells, expressed. Even cell-free systems, especially when adding a cytotoxic fusion component to the binding partner according to the invention can be used come. Examples of this are reticulocyte extracts. Binding partners according to the invention bind to HCV-NS2 or Jiv, being competitive between HCV-NS2 and Jiv or inhibit allosterically. Numerous ver drive to the detection of such a bond.

As a rule, at least one participates in the binding Component, d. H. Binding partner, HCV-NS2 and / or Jiv, with Provide markings and / or immobilizing agents. A Marking can be chosen so that the proof is direct or indirectly using specific binding pairs can be led. These specific bond pairs can be of an immunological or non-immunological type. Immuno logical, specific binding pairs are usually based on Hapten / anti-hapten systems using specific anti-hapten antibody.

These include, for example, fluorescein / anti-fluorescein, Dinitrophenyl / anti-dinitrophenyl, biotin / anti-biotin, di goxigenin / anti-digoxigenin systems and the like. In nich Immunological binding pairs have both components  a natural affinity for each other, like biotin / - Streptavidin.

Markings or parts of markings, for example a Component of a specific binding pair, can be covalent be bound to the molecule to be detected. That’s why Specialist familiar with various methods and corresponding ge suitable reagents commercially available.

For example, biotin can be bound to amine, aldehyde, carboxyl and sulfhydryl groups using biotin-N-hydroxysuccinimide ester, biotin hydrazide, biotin maleimide or iodoacetylbiotin. Analog reagents are also available for fluorescein or digoxigenin. The detection is then carried out via a component which binds to the hapten and can be coupled, for example, to a radioactive isotope, such as [ ¹²⁵ I] or [ ³ H], an enzyme, and fluorogenic, chemiluminescent or electrochemical agents. Measurement methods for the detection of radioactivity, fluorescence, e.g. B. the EVOTEK approach to the detection of fluorescence correlation, fluorescence energy transfer, cf. z. B. WO 97/28261, or conventional fluorescence quenching, chemiluminescence or electrochemical processes are known to the person skilled in the art. Suitable enzymes are, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, luciferase, beta-lactamase, urease or lysozyme. The associated detection reactions, such as the reaction of o-phenylenediamine, 4-chloronaphthol or tetramethylbenzidine by horseradish peroxidases, are likewise well known to the person skilled in the art.

The detection and, if necessary, the quantification of a competitive or allosteric inhibition can be achieved using Ver urgent titration. For example, fluorescence labeled HCV-NS2 on the one hand and binding partner of others merged in a stopped flow device and the resulting increase in fluorescence was measured. In a two measurement is a mixture of fluorescence-labeled HCV NS2 and attachment partners on the one hand and Jiv on the other in a stopped flow device. The fluores zenz remains unchanged if the complex of HCV-NS2 and Binding partner is more stable than the complex of HCV-NS2 and Jiv.

The detection methods can also do little immobilization at least one binding component on a solid support leg hold. For example, synthetic supports are suitable Polymers such as polypropylene, polystyrene, substituted Po lystyrene, for example aminated or carboxylated Po lystyrene, polyacrylamides, polyamides, polyvinyl chlorides and like. Vessels such as microtiter chambers, Dip sticks, fibers or particles, for example magnetic Beads, act. When immobilizing largely take place the specific ones previously described as markers  Binding pairs application. As a rule, HCV-NS2 or Jiv immobilized while the binding partner is free to this in the can bind mobilized proteins.

Evidence of competitive or allosteric binding binding partner according to the invention to Jiv or HCV-NS2 also about structure-mapping processes, such as nuclear spin sonance or X-ray crystal diffraction. To do this Complexes from binding partners with HCV-NS2 or Jiv or Frag elements of which were examined spectroscopically. That way the binding area between a bin according to the invention partner and HCV-NS2 or Jiv. OF INVENTION According to the invention, this bond area at least partially overlaps wise with the bond area between HCV-NS2 and Jiv. Mass spectroscopic methods are especially for ra detection of bonds with small amounts of substance Advantage.

Computer-aided, qualitative and quantitative methods and ter using appropriate search algorithms or substance Databases, such as the DOCK program of the University of San Franzisko or QSAR methods, and structural requirements for HCV-NS2 and / or Jiv, such as crystal and NMR structural data, offer another way of proving your commitment.

Known systems for the detection of protein are also suitable in / protein interactions, such as the so-called GST pulldown Assay and especially yeast two hybrid approaches. The latter are based  on the modular structure of eukaryotic transcripts ons factors, the interaction of bin partner with HCV-NS2 or with Jiv for functional re constitution of a transcription factor whose DNA Binding and transactivation domain has previously been separated. Evidence of the interaction, i.e. H. the bond is at possible, for example, via auxotrophic markers or reporter genes, such as the LacZ gene from E. coli and the enzyma coupled to it table detection reactions.

Binding partners according to the invention, which are in gene libraries are deposited, can bind to HCV-NS2 or Jiv can be examined by making them surface bound, i.e. H. usually membrane-bound on the surface of an or ganism expressed. Bacts, for example, are suitable for this riophages, especially filamentous bacteriophages, bacteri s, such as E. coli, in the cell wall of which the binding partner induces can be core, or eukaryotic viruses, such as Baculo virus. Phage display and bacteria display are used for the search binding partner according to the invention preferred. eukaryotic Viruses that carry binding partners according to the invention have the advantage that they are used directly to inject the binding partners in target cells, especially HCV-infected or with HCV replicons transfected cells can be used. The binding of the surface-expressed binding partner HCV-NS2 or Jiv can then use the methods described above  be demonstrated based on the binding partner affinity based on HCV-NS2 or Jiv. In addition, this bin tion to separate binding partners according to the invention from whose peptides stored in the library are used the. The use of fluorescence and biotinylation for the Mar markings and separation using FACS, affinity chromatography phy or adsorption on solid, for example immobilized Carriers containing streptavidin, such as polystyrene vessels advantageous.

Combinations of those described above can also be used Method for detecting the binding of bin according to the invention partner to HCV-NS2 or Jiv. Beson Efficient attachment partners lead in several of these Procedure for a positive proof. Even the own optimized of a binding partner, d. H. advantageous be shaped by going through slight changes of the binding partner a pool of possible further binding provides partners with different properties from de a binding partner with improved properties through the repeat one or more of the above ten methods can be derived. Such an itera tive approach leads to particularly preferred execution men of the present invention.

According to one embodiment of the present invention, binding partners for HCV-NS2 are derived from the Jiv amino acid sequences 1 to 699 shown in FIG. 2A, preferably from the region designated as Jiv90, which comprises the amino acids 533 to 622 from Jiv. The amino acid sequence of a binding partner advantageously differs from the corresponding amino acid sequence shown in FIG. 2A in that at least one amino acid is exchanged, removed, added or modified in such a way that the binding partner has a higher affinity for binding to HCV-NS2 than Jiv self.

According to the invention, the term "Jiv" stands for the amino acid sequence shown in FIG. 2A as well as for homologous sequences thereof.

According to a further embodiment of the present invention, binding partners for Jiv are derived from the HCV-NS2 amino acid sequence 1 to 217 shown in FIG. 1A. The amino acid sequence of a binding partner advantageously differs from the corresponding HCV-NS2 amino acid sequence shown in FIG. 1 in that at least one amino acid is exchanged, removed, added or modified in such a way that the binding partner has a higher affinity for binding to Jiv exhibits as HCV-NS2 itself.

According to a further embodiment of the present invention, binding partners for Jiv are derived from the BVDV-NS2 amino acid sequence 1 to 444 shown in FIG. 1B. The amino acid sequence of a binding partner advantageously differs from the corresponding BVDV-NS2 amino acid sequence shown in FIG. 3 in that at least one amino acid is exchanged, removed, added or modified in such a way that the binding partner has a higher affinity for binding to Jiv as BVDV-NS2 itself.

According to the invention, the term "BVDV-NS2" stands for the amino acid sequence of the BVDV CP7ins shown in FIG. 1B - just as for homologous sequences thereof; these include the NS2 sequences of other pestiviruses.

The present invention also relates to a method for intra- and / or extracellular competitive or allosteric inhibition of the binding between HCV-NS2 and Jiv, which is characterized in that at least one

• a) Binding partner for HCV-NS2, which binds to Jiv HCV-NS2 competitively or allosterically inhibits and / or
• b) Binding partner for Jiv who binds HCV-NS2 Jiv competitively or allosterically inhibits with HCV-infected or cells transfected with HCV replicon RNA, preferably HuH7 cells, and / or with HCV-NS2 from body fluid, ins special blood that brings into contact.

This method can be carried out both in vitro and in vivo be performed. The binding partner can be directly in vitro in a cell infected with HCV or transfected with HCV replicons len and / or HCV-NS2 containing solution, for example a Tissue culture medium, to be given or initially in  a suitable liquid, for example a buffer, dissolved or dispersed and then added to the cell and / or medium containing HCV-NS2.

The in vitro application is also for the diagnosis of HCV Diseases of importance. So can the invention Binding partner for the specific detection of intra- and / or extracellular HCV-NS2 can be used. This cannot just found an HCV infection in general but also an HCV-NS2 dependent diagnosis can be made, the further qualitative and quantitative conclusions about the In fection, for example their stage. Bonds Diagnostic partners are usually based on the way described above marked and / or immobi lised so that they can be tested in usual tests based on the above written verification methods are used can. Handy kits for quick and reliable through management of the diagnostic procedure is sufficient Binding partner and at least part of the others, for Detection of binding to HCV-NS2 required reagents in an appropriate arrangement available, for example coated vessels for immobilization of binding partners bound HCV-NS2 and / or antibody for specific Er Identification of the binding partner / HCV-NS2-protein complex. Kits for serological detection of HCV-NS2 are preferred. This can contain the usual means for blood preparation,  how to determine common lysis buffers and enrichment agents ter cell fractions.

Pharmaceutical formulations are for in vivo use gene suitable, the one or more of the invention Binding partner together with a pharmaceutically acceptable Contain carriers and / or customary auxiliaries.

To have a competitive or allosteric inhibition of HCV NS2 To achieve jiv binding, dosages are preferred Binding partner chosen, the extracellular binding partner Concentrations of less than 100 ng / ml, preferably of less than 10 ng / ml and especially less than 1 ng / ml Result in body fluid.

Examples of suitable pharmaceutical formulations are fe medicinal forms, such as powders, powders, granules, tablets, Coated tablets, capsules, suppositories or vaginal dosage forms, semi-solid dosage forms, such as ointments, creams, hydrogels, pastes or plasters, as well as liquid pharmaceutical forms, such as solutions, Emulsions, especially oil-in-water emulsions, suspensions NEN, for example lotions, injection and infusion accessories horse riding, eye and ear drops. The disposition binding partner according to the invention by implanted Dispensing devices are possible, and Liposo can also be used men, microspheres or polymer matrices are used.

To pharmaceutically acceptable carriers or usual auxiliaries substances include, for example, antioxidants, anti-irritants,  Chelating agents, disinfectants, dispersants, Dra yawing aids, emulsifiers, emulsion stabilizers, opp optionally ethoxylated and / or propoxylated fatty alcohols, Fatty amines, fatty amine oxides, fatty acid alkylolamides, fatty acids ster, fatty acids, humectants, film formers, gel formers, Odor masking agents, taste correctives, resins, hy drocolloids, preservatives, solvents, solvent ver medium, wetting agent, neutralizing agent, permeation agent accelerators, pigments, protein derivatives and / or -Hydrolysates, quaternary ammonium compounds, refatting and superfatting agents, ointment, cream or oil base materials, Ointment bases, silicone derivatives, spreading aids, stabilizers lisators, sterilizers, suppository bases, suspenders medium, tablet excipients, such as binders, fillers, Lubricants, disintegrants or coatings, clays, fuels, Desiccants, opacifiers, thickeners, waxes, Plasticizers, white oils. Refinements of this type are based on professional knowledge and are for example in Fiedler, H. P., Lexicon of auxiliaries for pharmacy, cosmetics and an bordering areas, 4th edition, Aulendorf: ECV-Editio-Kantor- Verlag, 1996.

If the binding partner is a peptide, The method according to the invention can also consist in that a system capable of expressing this peptide, in which Usually a suitable vector system with HCV-infected or  cells transfected with HCV replicon RNA is brought. Suitable nucleic acids are preferably used structures in the HCV-infected or with HCV replicon RNA transfected cells, in which then the peptide is expressed. Various physical me methods are used, e.g. B. liposomes, virosomes or li gene-mediated gene transfers, or viruses such as retro, fla vi, adeno, adeno associated or herpes viruses. The specialist are measures for tissue-specific infection and / or ex known pression.

A peptide binding partner can also be direct or attached Carriers, for example liposomes, coupled into the virus infected cells.

Because of the competitive or allosteric inhibition of the Binding between HCV-NS2 and Jiv are those of the invention Binding partners and pharmaceutical agents that at least contain a binding partner according to the invention, and that Process according to the invention for intra- and / or extracellular ren competitive or allosteric inhibition of binding between HCV-NS2 and Jiv for prevention, diagnosis and / or treatment suitable for treatment of HCV diseases.  

legends

Fig. 1A protein sequence of the NS2 protein of the HCV BK strain (residues 1-217) is substantially identical to the published sequence of the clear HCV subtype 1B (Takamizewa et al., 1991)

Fig. 1B amino acid sequence of the NS2 protein of BVDV CP7ins- (amino acids 1-444) (Tautz et al., 1996)

Fig. 2A amino acid sequence of the bovine Jiv protein from Mardin Darby bovine Kidney (MDBK) cells

FIG. 2B amino acid sequence of the region according to the invention as designated Jiv90 of bovine Jiv (Jiv amino acids 533-622)

Fig. 3 Comparison of human and bovine Jiv90 amino acid sequence (amino acids 533-622)

Fig. 4 Regulation of NS2-3 cleavage of BVDV in MBDK _Tet-On / Jiv cells

Fig. 5 Detection of the interaction between BVDV NS2 and Jiv by co-precipitation of both proteins in the GST-pull down assay Fig. 6 Detection of the interaction between HCV NS2 and Jiv by co-precipitation of both proteins in the GST-pull down assay

Examples

The following examples are intended to explain the invention in more detail without restricting them.

example 1

The regulation of the NS2-3 cleavage of the BVDV in MBDK _Tet-On / Jiv cells was examined by means of Western blot ( FIG. 4).

The MBDK _Tet-on / Jiv cell line was manufactured using the Tet-On system. For this, the cDNA sequence coding for the open reading frame of the Jiv was ligated into the Tet-On expression plasmid pTRE. The resulting plasmid pTRE / Jiv was transfected together with an expression plasmid which mediates puromycin resistance into the cell line MBDK _Tet-On , which constitutively expresses the rtTA activator protein (Gos sen et al., 1995). The transfection was carried out by means of electro poration (Tautz et al., 1999). The cell lines were selected under 1 g / l G418 (Calbiochem, Frankfurt, Germany). In the MBDK _Tet-On / Jiv cell line, the addition of the inductor doxycycline (Dox) leads to a regulated overexpression of the complete bovine Jiv protein.

After the cells had been lysed, the proteins were electrophoretically separated in 8% polyacrylamide tricin SDS gels (Schägger and Jagow, 1997). After separation in the polyacrylamide Tricin SDS gel, the proteins were transferred to a nitrocellulose membrane (Optitran BA-S83 reinforced NC: Schleicher & Schuell, Düren, Germany) using a "semi-dry" electroblot apparatus (BIORAD, Munich). To block nonspecific binding sites, the membrane was washed for 1 h with 3% (w / v) skim milk powder in TPBS (0.05% Tween-20 in PBS [8 g / l NaCl; 0.2 g / l KCl; 1 , 44 g / l Na ₂ HPO ₄ ; 0.24 g / l KH ₂ PO ₄ ; pH 7.4]). Jiv was detected with a Jiv-specific rabbit serum (1: 10000 in TPBS), a secondary, peroxidase-coupled anti-rabbit antibody (1: 10000 in TPBS; Dianova, Hamburg) and the Renaissance We stern Blot Chemiluminescence Reagent Plus (NEN, Life Sciences, Boston, MA).

Due to the infection of the cells with the BVDV strain NCP7 there is expression of the NS2-3 protein which is in We sternblot (see above) by an NS3-specific monoclonal Antibodies (mAb 8.12.7; 1: 200 in TPBS; [Corapi et al., 1990]) a secondary, peroxidase-coupled anti-mouse antibody (1: 10000 in TPBS; Dianova, Hamburg) and the Renaissance We stern Blot Chemiluminescence Reagent Plus (NEN, Life Science ces) has been demonstrated

Results

In the MBDK _Tet-On / Jiv cell line, the addition of the inductor doxycycline (Dox) leads to a regulated overexpression of the complete bovine Jiv protein. The Dox-dependent increase in Jiv expression can be seen in the upper half of the Western blot shown. ( Fig. 4, upper part)

The infection of the cells with the BVDV strain NCP7 leads to the expression of the NS2-3 protein. With increasing Dox concentration, there is an increased Jiv expression (see above) and an increase in NS2-3 cleavage in these cells; the latter is shown in the increase in the amount of NS3 and a decrease in the amount of NS2-3 ( FIG. 4, lower part).

Example 2

Evidence of the interaction between BVDV-NS2 and Jiv by co-precipitation of both proteins in the GST pull-down assay ( FIG. 5)

The proteins were expressed in baby hamster kidney (BHK) cells which were infected with the vaccinia virus MVA-T7Pol (Sutter et al., 1995); the vaccinia virus MVA-T7Pol expresses the T7 RNA polymerase in these cells. The following expression constructs were used for expression (the open reading frames are symbolized by boxes; TM: pu tative transmembrane region; J: J domain; Jiv90: Jiv90 domain corresponds to amino acids 533-622 of the Jiv protein; GST: gluthation-S-transferase) :
pFlag-NS2: In the vector pCITE-2A, the NS2 gene of BVDV CP7ins- (amino acids 1137-1580) (Tautz et al., 1996) (see FIG. 1B) was controlled by the T7 RNA polymerase promoter (T7 promoter ) cloned. In order to allow a specific detection of NS2 and to facilitate immunoprecipitation, a sequence was cloned in front of the reading frame of the NS2 which codes for a so-called flag epitope.
pGST-Jiv: in the vector pCITE-2A the gene for the gluthation-S-transferase (GST) followed by the open reading frame of the bovine Jiv protein is cloned under the control of the T7 promoter.
pGST-Jiv90: in the vector pCITE-2A the gene for glutathon-S-transferase (GST) followed by codons 533-622 of the open reading frame of the bovine Jiv protein is cloned under the control of the T7 promoter.

Protein expression and immunoprecipitation

After transfection of the cells with the cDNA Kon structs there are expression of the coded proteins Flag- NS2, GST-Jiv or GST-Jiv90. By adding 70 µCi [35S] labeled methionine and cysteine (NEN, Life Sciences) per ml Medium, radioactive proteins are synthesized which can be demonstrated by immunoprecipitation (IP). For the IP were determined using the RIPA buffer [50 mN Tris-HCl (pN 8.0), 150 mN NaCl, 1% (v / v) NP-40, 1% (w / v) deoxycholate, 0.1% (W / V) SDS, 0.5 mN PefablocSC (Merck, Darmstadt, Ger many)] cell lysates under non-denaturing conditions manufactured. For the precipitation of the GST fusion protein GST-Jiv and GST-Jiv90 became a GST-specific monoclonal antibody  (mAk) used (Sigma) while to detect the Flag-NS2 uses a flag-epitope-specific mAb (Sigma) has been. Incubation was at 4 ° C for 1 h. According to Precise tation of the antibody / protein complexes by protein A Sepharose (Sigma) and wash three times with RIPA buffer the aggregates were dissolved by boiling in 10% polyacryla separated with Tricin SDS gels and for fluorography with Enlightning solution (NEN Life Science, Zavantem, Belgi um) prepared.

Results

The mAb against GST precipitated from cells in which GST-Jiv and Flag-NS2 were expressed, both GST-Jiv and Flag-NS2 ( FIG. 5, lower part lane 6); likewise the mAb directed against the flag epitope precipitates Flag-NS2 and GST-Jiv ( FIG. 5, lower part lane 5). Analogous results were obtained for cells in which Flag-NS2 and GST-Jiv90 were expressed ( FIG. 5, lower part lanes 9, 10). On the other hand, no flag NS2 could be precipitated from cells in which only flag NS2 was expressed with the GST-specific mAb ( FIG. 5, lower part lane 3). From cells in which only GST-Jiv or GST-Jiv90 was expressed, there was no precipitation of GST-Jiv or GST-Jiv90 with the flag-epitope-specific mAb ( FIG. 5, lower part lanes 8, 12).

These data demonstrate a specific interaction between Jiv or Jiv90 and the NS2 protein from BVDV (Flag-NS2).

Example 3 Detection of the interaction between HCV-NS2 and Jiv by co-precipitation of both proteins in the GST pull-down assay ( FIG. 6)

Protein expression and immunoprecipitation were carried out analogously to Example 2. The following expression constructs were used to express the NS2 protein or parts of the NS3 protein from HCV BK (the open reading frames are symbolized by boxes; see Example 2):
pFlag-HCV-NS2-3-His: the complete NS2 gene of HCV strain BK (217 codons) was cloned into pCITE-2A; the NS2 gene is followed by 213 codons of the NS3 coding sequence .; in front of the reading frame of the HCV-NS2, a sequence was cloned which codes for a so-called flag epitope. The NS3 fragment is followed by 3 vector-encoded amino acids, a hexa-His tag and a stop codon.

Protein expression and immunoprecipitation (see also example 2)

A was used to precipitate the GST fusion protein GST-Jiv90 GST-specific mAb used during the detection of Flag-HCV-NS2 and Flag-HCV-NS2-3-His specified a flag epitope shear mAb was used.  

Results

The mAb against GST precipitated from cells in which GST-Jiv90 and Flag-HCV-NS2-3-His were expressed, both GST-Jiv90 and Flag-HCV-NS2 and Flag-HCV-NS2-3-His ( FIG. 6, lower part lane 3); likewise the mAb directed against the flag epitope precipitates Flag-HCV-NS2 and Flag-HCV-NS2-3-His and GST-Jiv90 ( FIG. 6, lower part lane 4). In contrast, from cells in which only Flag-HCV-NS2-3-His was expressed, neither Flag-HCV-NS2 nor Flag-HCV-NS2-3-His could be precipitated with the GST-specific mAb ( FIG. 6, lower part lane 6 ). From cells in which only GST-Jiv90 was expressed, there was no precipitation of GST-Jiv90 with the flag epitope-specific mAb ( FIG. 6, lower part lane 1).

These data demonstrate a specific interaction between Jiv90 and the HCV-NS2 protein.  

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Claims (11)

1. binding partner for HCV-NS2, characterized in that it inhibits the binding of Jiv to HCV-NS2 competitively or allosterically. 2. attachment partner for Jiv, characterized in that he the binding of HCV-NS2 to Jiv competitive or allosteric inhibits. 3. Binding partner according to claim 1, characterized in that it binds to a part of the amino acid sequence shown in Fig. 1 1 to 217 or homologous sequences thereof. 4. binding partner according to any one of claims 1 to 3, characterized characterized that it is a peptide, a peptoid, a organic or inorganic substance. 5. A method for intra- and / or extracellular competitive or allosteric inhibition of the binding between HCV-NS2 and Jiv, characterized in that at least one

• a) binding partner for HCV-NS2, which competitively or allosterically inhibits the binding of Jiv to HCV-NS2 and / or
• b) Binding partner for Jiv, which competitively or allosterically inhibits the binding of HCV-NS2 to Jiv, in contact with HCV-infected / HCV replicon-RNA-transfected cells and / or HCV-NS2 from body fluid.

6. The method according to claim 5, characterized in that it the binding partner is a peptide whose  cDNA or mRNA in the HCV infected / HCV replicon RNA transfected cells is introduced and expressed. 7. The method according to claim 5, characterized in that it the binding partner is a peptide or peptoid delt, which is coupled to a carrier in the HCV-infected / HCV replicon RNA transfected cells are introduced. 8. Use of at least one binding partner of the claims 1 to 4 for the prevention, diagnosis and / or treatment of HCV Diseases. 9. Pharmaceutical composition containing at least one bin end partner of claims 1 to 4 in a pharmaceutical acceptable carrier and / or excipient. 10. Method for overexpression of Jiv protein, characterized by the following steps

• a) Construction of a Jiv coding cDNA sequence and insertion into an expression vector
• b) transfection of the vector into a host cell
• c) Cultivation of the transfected host cell and start of overexpression by adding an inductor
• d) extraction and purification of the recombinant protein produced.

11. Use of a protein according to claim 10 for Prevention, diagnosis and therapy of HCV diseases.