WO1999057136A2

WO1999057136A2 - Binding partner and method for the competitive inhibition of binding between nef protein and calmodulin, agent containing said binding partner and it use in hiv-related diseases

Info

Publication number: WO1999057136A2
Application number: PCT/EP1999/003105
Authority: WO
Inventors: Markus Schott; Jacqueline Schorr; Christof Antz
Original assignee: Markus Schott; Jacqueline Schorr; Christof Antz
Priority date: 1998-05-06
Filing date: 1999-05-06
Publication date: 1999-11-11
Also published as: DE19820224A1; WO1999057136A3; AU4037899A

Abstract

The invention relates to binding partners for NEF protein or calmodulin which competitively inhibit the binding between calmodulin and NEF protein, as well as to a method for the intra- and/or extracellular competitive inhibition of binding between NEF protein and calmodulin. According to the method, at least one of the two binding partners is brought into contact with HIV-infected cells. The invention further relates to pharmaceutical preparations containing at least one of these binding partners and to the use of the binding partners in the prevention, diagnosis and/or treatment of HIV-related diseases.

Description

Binding partner and method for competitive inhibition of the binding between NEF protein and calmodulin, as well as means and use in HIV diseases

The present invention relates to binding partners for NEF protein, binding partners for calmodulin, methods for competitive inhibition of the binding between NEF protein and calmodulin using these binding partners, agents for the treatment of HIV achievements that contain these binding partners, and the use of these binding partners for the prevention, diagnosis and treatment of HIV diseases.

Around 30 million people worldwide are infected with HIV and the rate of newly infected people increases every year. Reliable vaccines are currently unavailable and may never be developed due to the high mutation rate of the viral coat proteins. The current approach to HIV comprises two main strategies: on the one hand, pure preventive measures ("sa er sex") and, on the other hand, a medical combination therapy consisting of substances, all of which have different modulatory effects on the virus-own reverse transcriptase, integrase and proteinase. Combination therapy has been a very successful therapy so far, however, the medication (a cocktail of proteinase, integrase and reverse transcriptase inhibitors) must be taken permanently and in large amounts to lead to a significant reduction in viral replication. Since these drugs have a strong non-specific cytotoxic activity, the high dosage leads to significant and undesirable side reactions, the long-term effects of which are still completely unknown.

The virus-encoded NEF protein plays a key role in AIDS. NEF (Negative factor) is a 27-35 kDa protein that is N-terminally myristoylated and found in HIV-1, -2 and SIV. The NEF protein, whose RNA makes up 80% of the total early mRNA, is already formed in the early phase of virus replication in relatively large amounts compared to the also early proteins REV and TAT. While REV and TAT are essential for both gene expression and HIV replication in culture, the NEF protein has no influence on HIV expression (AIDS research, April 1995, 171).

A variety of effects in vitro are attributed to the NEF protein. For example, NEF is said to induce internalization and degradation of CD4 receptors after HIV infection of T helper cells. In "Brian R. Cullen, Current Biology 1996, Vol. 6: 1557-1559" it is suggested that NEF specifically activate a PAH-dependent signal cascade. According to "Bregino et al, Proc. Natl. Acad. Sci. USA, April 1997, Vol. 94: pp. 3178-3182", interleukin 10 is to be induced by recombinant HIV-1 NEF protein. Involvement of signaling pathways is also considered for this effect, since the effect on the production of interleukin 10 is influenced by W-7, an inhibitor of calcium / calmodulin-dependent phosphodiesterase signaling pathways.

Various targets are proposed as interaction partners for NEF. In the above-mentioned publications by Cullen and Bregino et al it is assumed that the NEF protein binds to two serine / threonine kinases, p62 and p72, or to a surface receptor or to calcium channels.

The paramount importance of NEF continues to emerge from studies on so-called long-term survivors, i.e. People whose HIV test is positive, but who are symptom-free even after 10 or more years and who have not developed the full AIDS picture: these people often show a defect in the nef gene.

It is presumably due to the insufficient elucidation of the molecular mechanisms that underlie the effects of NEF that no therapy targeting NEF has been achieved so far of HIV diseases could be made available. This is the object of the present invention.

Surprisingly, it has been found that NEF protein and calmodulin bind to one another and treatment of HIV diseases is possible by inhibiting the binding between NEF protein and calmodulin.

The present invention therefore relates to binding partners for NEF protein, which are characterized in that they competitively inhibit the binding of calmodulin to NEF protein.

The present invention also relates to binding partners for calmodulin, which are characterized in that they competitively inhibit the binding of NEF protein to calmodulin.

Binding means any molecular interaction between calmodulin and NEF protein, preferably in the presence of calcium ions and in particular under physiological conditions. These are usually classic protein-protein interactions, which include electrostatic attraction, hydrogen bonding, hydrophobic bonds, van der Waals forces or metal complex-like coordinative bonds. In addition to the reversible molecular interactions mentioned above, irreversible interactions, such as covalent bonds, can also be considered for the binding of binding partners according to the invention to calmodulin or NEF protein.

Competitive inhibition means that binding partners according to the invention compete with calmodulin or NEF protein for binding to NEF protein or calmodulin, i.e. the bond of one hinders the bond of the other.

The NEF-side binding site, via which the binding partner binds to NEF protein, is preferably attached to the NEF protein by at least a part of the NEF-side binding site Calmodulin binds, is formed. The same applies to the binding site on the calmodulin with regard to the binding between calmodulin and NEF protein or calmodulin and binding partner.

Binding partners for NEF protein are preferred.

According to a preferred embodiment of the present invention, binding partners for NEF protein bind to a part of the amino acid sequence 1 to 206 shown in FIG. 1, or homologous sequences thereof (S. Wain-Hobson et al, Cell 1985, 40, pp. 9-17) .

According to a particularly preferred embodiment of the present invention, binding partners for NEF protein bind to a part of the amino acid sequence 1 to 80 shown in FIG. 1, or homologous sequences thereof, the binding to a part of the amino acids 1 to 57 or to a part of the amino acids 1 to 57 and part of the amino acids 58 to 80 can be done.

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

According to the invention, the term “NEF protein” stands for the amino acid sequence shown in FIG. 1 as well as for homologous sequences thereof. Binding partners according to the invention preferably bind to calmodulin or NEF protein with higher affinity than calmodulin to NEF protein or NEF protein to calmodulin. A higher binding affinity has the advantage that lower concentrations of binding partner are required to cause competitive inhibition. On the other hand, a suitable inhibition can also be achieved with binding partners of lower binding affinity by using a higher concentration of binding partners.

According to a preferred embodiment of the present invention, the dissociation constant for the binding of a binding partner to NEF protein or calmodulin is less than 1 μM, preferably less than 0.1 μM and in particular less than 10 nM.

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

Peptides are understood to be sequences of naturally occurring, proteogenic amino acids which are linked to one another via peptide binding. Sequences of peptoids, i.e. Peptide-like compounds can contain, in addition to naturally occurring, proteogenic amino acids, further amino acids and amino acid derivatives, for example certain stereo and diastereoisomers, such as D-amino acids, naturally occurring, non-proteogenic amino acids or chemically synthesized compounds with properties that are known to those skilled in the art as amino acids are similarly known, such as ß-alanine, 7-

Carboxyglutamic acid, hydroxyproline, norleucine, norvaline, ornithine, phenylglycine, pyroglutamic acid, sarcosine, statin, or t-leucine. Peptides are also understood to mean those sequences whose N- and / or C-terminus can be modified, for example with an acetylated N-terminus and / or amidated C-terminus. The same naturally also applies to chemically modifiable amino acid side groups, in particular those familiar in organic chemistry and especially in Peptide chemistry can be used protective groups.

Suitable binding partners are also antibodies, in particular monoclonal and recombinant antibodies, which bind to at least part of the binding site between NEF protein and calmodulin. In this context, antigen-binding fragments of the hypervariable domains of such antibodies, for example Fab, Fv, scFv fragments or fragments based on one or more CDRs, are very particularly preferred.

With regard to the use according to the invention of the binding partners in pharmaceutical compositions, the incorporation of amino acids which do not occur in nature is advantageous in that it increases the stability towards natural degradation processes. If a particular peptide is to be administered to a mammal as the binding partner according to the invention, its half-life can be extended in vivo, for example, by replacing at least some of the natural L-amino acids with D-isomers and thus obtaining a corresponding peptoid.

Methods for producing peptides and peptoids according to the invention are known to the person skilled in the art. To be favoured

Solid phase syntheses using functionalized resins to which the peptides to be synthesized can be linked. Wang resins for FMOC solid phase peptide synthesis or Merrifield resins for BOC solid phase peptide synthesis are common examples. After the desired peptide has been built up, it is then detached from the support, it being possible for protective groups which may be present to be split off simultaneously or subsequently. Any subsequent purification required, for example via HPLC, provides the peptide in the desired purity, which can be determined, for example, by mass spectroscopy or NMR. The exact design of the method depends on the peptide to be synthesized or peptoid and is based on professional knowledge.

In addition, the peptides and peptoids according to the invention can also be produced using molecular biological methods. For example, the primary

Amino acid sequence of the peptide to be synthesized can be converted into the corresponding c-DNA or m-RNA sequence. The nucleic acids can be synthesized in a known manner and then introduced into a suitable host cell, expressed and, if necessary, subsequently modified to the desired peptoids. Further refinements of this process are also based on expert knowledge.

Another possibility is to create libraries of randomized peptide genes, from which peptides with the desired binding specificity can then be isolated. For example, randomized oligonucleotides can be inserted into a suitable expression system which allows the selection of oligopeptides of different amino acid sequences according to the invention.

The production of specific, monoclonal and recombinant antibodies and in particular antibody fragments is also familiar to the person skilled in the art.

For example, monoclonal antibodies that are specifically directed against the NEF or calmodulin-side binding site involved in the binding between NEF protein and calmodulin can be raised by means of conventional hybridoma technology, by removing peptide fragments from the NEF or calmodulin-side binding site used for immunization. Antibody fragments of these monoclonal antibodies can be obtained in the usual way, for example by digestion.

The basis for the production of recombinant antibodies and antibody fragments is preferably formed by antibody gene libraries, which can be of different complexity. Using PCR, for example, create such libraries based on antibody-related genetic information. For example, cDNA from defined cell 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 oligonucleotides for hypervariable regions can be used as genetic information, which results in libraries in the order of the examples mentioned above leads to increasing complexity. Individual antibodies or antibody fragments with the desired one can then be obtained from these

Specificity, i.e. binding partners according to the invention can be produced by cloning the relevant gene into a production vector and in prokaryotic systems such as E. coli, Bacillus subtilis, Streptomyces lividans, or eukaryotic systems such as plasmacytoma or myelo a-

Cells, COS-2ellen, CHO cells, certain insect, plant or fungal cells, for example unicellular yeast cells, expressed. Cell-free systems can also be used, in particular if a cytotoxic fusion component is added to the binding partner according to the invention. Examples of this are reticulocyte extracts.

Binding partners according to the invention bind to NEF protein or calmodulin, where they competitively inhibit the binding between NEF protein and calmodulin. To prove such

A number of methods are known to those skilled in the art of binding.

As a rule, at least one component involved in the binding, i.e. Binding partner, NEF protein and / or calmodulin, provided with labels and / or immobilizing agents. A label can be selected so that the detection can be carried out directly or indirectly using specific binding pairs.

Such specific binding pairs can be immunological or non-immunological. Immunological, specific binding pairs are usually based on hapten / anti-hapten systems using specific anti-hapten antibodies. These include, for example, fluorescein / anti-fluorescein, dinitrophenyl / anti-dinitrophenyl, biotin / anti-biotin, digoxigenin / anti-digoxigenin systems and the like. In non-immunological binding pairs, both components have a natural affinity for one another, such as biotin / streptavidin.

Markings or parts of markings, for example a component of a specific binding pair, can be covalently bound to the molecule to be detected. Various methods are known to the person skilled in the art and in many cases suitable reagents are also commercially available. For example, biotin can be bound to A in, aldehyde, carboxyl and sulfhydryl groups using biotin-N-hydroxysuccinimide ester, biotin hydrazide, biotin maleimide or iodoacetylbiotin. Analog reagents are also for fluorescein or

Digoxigenin available. The detection is then carried out via a component which binds to the hapten and which 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. the EVOTEK approach for the detection of fluorescence correlation, fluorescence energy transfer, cf. e.g. 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, β-galactosidase, glucose oxidase, luciferase, jS-lactamase, urease or lysozy. The associated detection reactions, such as the reaction of o-phenylenediamine, 4-chloronaphthol or tetramethylbenzidine by horseradish peroxidases, are also well known to the person skilled in the art.

The detection and, if necessary, the quantification of a competitive inhibition can be done by displacement titration. For example, fluorescence-labeled NEF protein on the one hand and binding partner on the other hand are combined in a stopped flow device and the resulting increase in fluorescence is measured. In a second measurement, a Mixture of fluorescence-labeled NEF protein and binding partner on the one hand and calmodulin on the other hand combined in a stopped flow device. The fluorescence remains unchanged when the complex of NEF protein and binding partner is more stable than the complex of NEF protein and calmodulin.

The detection methods can also include immobilization of at least one binding component on a solid support. Suitable are, for example, carriers made of synthetic polymers, such as polypropylene, polystyrene, substituted polystyrene, for example aminated or carboxylated polystyrene, polyacrylamides, polyamides, polyvinyl chlorides and the like. Vessels such as microtitre cameras, dipsticks, fibers or particles, for example magnetic beads, can be involved. The specific binding pairs previously described as markers are largely used in the immobilization. As a rule, NEF protein or calmodulin is immobilized, while the binding partner can bind freely to these immobilized proteins.

The competitive binding of binding partners according to the invention to calmodulin or NEF protein can also be demonstrated by means of structure-imaging methods, such as nuclear magnetic resonance or X-ray crystal diffraction. For this purpose, complexes from binding partners with NEF or calmodulin or fragments thereof are examined spectroscopically. In this way, the binding area between a binding partner according to the invention and NEF or calmodulin can be mapped. According to the invention, this binding area overlaps at least partially with the binding area between NEF protein and calmodulin.

Mass spectroscopic methods are particularly advantageous for the rapid detection of bonds with small amounts of substance.

Computer-aided, qualitative and quantitative methods under Using suitable search algorithms or substance databases, such as the DOCK program of the University of San Francisco or QSAR methods, and structural specifications for NEF protein and / or calmodulin, such as crystal and NMR structural data, offer a further possibility for the detection of binding.

Known systems are also suitable for the detection of protein / protein interactions, such as the so-called GST pull-down assay and in particular yeast-two-hybrid approaches. The latter are based on the modular structure of eukaryotic

Transcription factors, wherein according to the invention the interaction of binding partner with NEF protein or with calmodulin leads to the functional reconstitution of a transcription factor whose DNA binding and transactivation domain has been previously separated. The interaction, i.e. the binding, can be detected, for example, via auxotrophic markers or reporter genes, such as the LacZ gene from E. coli and the enzymatic detection reaction coupled to it.

Binding partners according to the invention, which are stored in gene libraries, can be examined for their binding to NEF protein or calmodulin by surface-binding, i.e. usually expressed in membrane-bound form on the surface of an organism. Bacteriophages, in particular filamentous ones, are suitable for this

Bacteriophages, bacteria, such as E. coli, in the cell wall of which the binding partner can be anchored, or eukaryotic viruses, such as baculoviruses. Phage display and bacteria display are preferred for screening binding partners according to the invention. Eukaryotic viruses, the invention

Binding partners have the advantage that they can be used directly for introducing the binding partner into target cells, in particular HIV-infected cells.

The binding of the surface-expressed binding partners to NEF protein or calmodulin can then be demonstrated using the methods described above, based on the binding partner affinity for NEF protein or calmodulin based. In addition, this binding can be used to separate the binding partners according to the invention from other peptides stored in the library. The use of fluorescence and biotinylation for labeling and separation by means of FACS, affinity chromatography or adsorption on solid supports, for example immobilized streptavidin, such as polystyrene vessels, is advantageous.

Combinations of the methods described above for detecting the binding of the invention can also be used

Binding partners to NEF protein or calmodulin are carried out. Particularly efficient binding partners lead to positive evidence in several of these methods. The properties of a binding partner can also be optimized, i.e. can be advantageously designed by making a pool of possible further binding partners with different properties available from slight changes in the binding partner, from which binding partners with improved properties can be derived by performing one or more of the above-mentioned methods again. Such an iterative procedure leads to particularly preferred embodiments of the present invention.

According to one embodiment of the present invention, binding partners for NEF protein are derived from the calmodulin amino acid sequences 1 to 148 and 149 shown in FIGS. 2 and 3 (D. Marshak et al., Biochemistry 1984, 23, pp. 2891-2899 ). Fragments thereof with 3 to 30, preferably 4 to 20 and in particular 5 to 15 amino acids are preferred. The amino acid sequence of a binding partner advantageously differs from the corresponding 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 NEF protein than calmodulin itself.

According to the invention, the term "calmodulin" stands for the amino acid sequence shown in FIGS. 2 and 3 as well as for homologous sequences thereof.

According to a further embodiment of the present invention, binding partners for calmodulin are derived from the amino acid sequence 1 to 206 shown in FIG. 1 and in particular 1 to 80. Fragments thereof with 3 to 30, preferably 4 to 20 and in particular 5 to 15 amino acids are preferred. The amino acid sequence of a binding partner advantageously differs from the corresponding 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 calmodulin than NEF protein itself.

The present invention also relates to a method for intra- and / or extracellular competitive inhibition of the binding between NEF protein and calmodulin, which is characterized in that at least one i) binding partner for NEF protein, which binds calmodulin to NEF -Protein competitively inhibits and / or ii) binding partner for calmodulin, which competitively inhibits the binding of NEF to calmodulin, with HIV-infected cells, preferably T lymphocytes and macrophages, and / or with NEF protein from body fluid, in particular blood, in Brings contact.

This method can be carried out both in vitro and in vivo. In vitro, the binding partner can be added directly to a solution containing the HIV-infected cells and / or NEF protein, for example a tissue culture medium, or first of all itself dissolved or dispersed in a suitable liquid, for example a buffer, and then added to the cell and / or medium containing NEF protein.

In vitro use is also of great importance for the diagnosis of HIV diseases. The binding partners according to the invention can thus be used for the specific detection of intra- and / or extracellular NEF protein can be used. As a result, not only can an HIV infection be determined in general, but an NEF-dependent diagnosis can also be made, which enables further qualitative and qualitative conclusions to be drawn about the infection, for example its stage. Binding partners for diagnostic use are generally marked and / or immobilized in the manner described above, so that they can be used in conventional tests based on the detection methods described above. Handy kits for quick and reliable

Carrying out the diagnostic method provides sufficient binding partners and at least some of the further reagents required for recognizing the binding to NEF protein in a suitable arrangement, for example coated vessels for immobilizing binding partner-bound Nef protein and / or antibodies for specific recognition of the blinding partner / NEF protein complex. Kits for serological detection of NEF protein are preferred. These can include the usual means for blood processing, such as common lysis buffers and means for enriching certain cell fractions.

Pharmaceutical formulations which contain one or more of the binding partners according to the invention together with a pharmaceutically acceptable carrier and / or customary auxiliaries are suitable for use in vivo.

To effect a potent inhibition of the NEF-calmodulin binding, doses of binding partner are preferably selected which result in extracellular binding partner concentrations of less than 100 ng / ml, preferably less than 10 ng / ml and in particular less than 1 ng / ml of body fluid.

Examples of suitable pharmaceutical formulations are solid dosage forms, such as powder, powder, granules, tablets, dragees, capsules, suppositories or vaginal dosage forms, semi-solid dosage forms, such as ointments, creams, hydrogels, pastes or Plasters and liquid pharmaceutical forms, such as solutions, emulsions, in particular oil-in-water emulsions, suspensions, for example lotions, injection and infusion preparations, eye and ear drops. Implanted delivery devices can also be used for the administration of binding partners according to the invention. Liposomes, micro-spheres or polymer matrices can also be used.

Pharmaceutically acceptable carriers or customary auxiliary substances include, for example antioxidants, anti-irritants, chelating agents, disinfectants, dispersants, coating aids, emulsifiers, Emulionsstabilisatoren, optionally ethoxylated and / or propoxylated fatty alcohols, fatty amines, fatty amine oxides, fatty acid alkylolamides, fatty acid ester, fatty acids, humectants, film formers, gelling agents, odor masking agents , Taste correctives, resins, hydrocolloids, preservatives, solvents, solubilizers, wetting agents, neutralizing agents, permeation accelerators, pigments, protein derivatives and / or hydrolyzates, quaternary ammonium compounds, refatting and overfatting agents, ointment, cream or oil bases, ointment bases, Silicone derivatives, spreading aids, stabilizers, sterilizers, suppository bases, suspending agents, tablet auxiliaries, such as binders, fillers, lubricants, disintegrants or coatings, clay e, fuels, desiccants, opacifiers, thickeners, waxes, plasticizers, white oils. A design in this regard is based on professional knowledge, as is shown, for example, in Fiedler, H.P., Lexicon of auxiliaries for pharmacy, cosmetics and related 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 bringing a system capable of expressing this peptide, usually a suitable Vekor system, into contact with HIV-infected cells. Suitable ones are preferred Nucleic acid constructs are introduced into the HIV-infected cells, in which the peptide is then expressed. Various physical methods can be used for this purpose, for example liposome, virosome- or ligand-mediated gene transfers, or viruses such as retro, adeno, adeno-associated or herpes viruses. Measures for tissue-specific infection and / or expression are known to the person skilled in the art.

A peptide binding partner can also be introduced into the virus-infected cells directly or coupled to carriers, for example liposomes.

Because of the competitive inhibition of the binding between NEF protein and calmodulin, those according to the invention

Binding partners and pharmaceutical agents which contain at least one binding partner according to the invention and the method according to the invention for intra- and / or extracellular competitive inhibition of the binding between NEF protein and calmodulin are suitable for the prevention, diagnosis and / or treatment of HIV diseases.

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

example 1

The binding between NEF protein and calmodulin was examined using a protein overlay.

The calmodulin used for this was obtained in a known manner by expressing and purifying the calmodulin gene from Dictyostelium discoideum in Escherichia coli (E. coli).

The following NEF samples were examined:

a) The nef gene of the HIV-1 strain pNL4-3 was expressed and purified in E. coli (Wolber V. et al (1992) Eur. J. Biochem. 205: 1115-1121). The protein thus obtained is called NEF-full.

b) A NEF fragment truncated by 57 amino acids N-terminally (NEF-core; Freund J. et al (1994) Eur. J. Biochem. 223: 589-593) was also expressed in E. coli and successively by means of DEAE- Ion exchange chromatography, ammonium sulfate precipitation (40%) and gel filtration cleaned.

c) The 57 amino acid N-terminal fragment (NEF anchor) was produced synthetically (Freund et al, ibid).

1 μl of a solution containing NEF-full, NEF-core or NEF-Anker (2 mg / ml) was dropped onto a nitrocellulose membrane (Schleicher & Schuell). In order to block non-specific binding sites, the membrane was incubated for 1 hour at room temperature with 5% milk powder in TBS + 0.2% CaCl ₂ or 5 M EGTA. After 3 washes of 5 min each with TBS + 0.2% CaCl ₂ or 5 mM EGTA, the membrane was incubated with 5 μg calmodulin in the presence of 0.2% CaCl ₂ or 5 mM EGTA at room temperature. A NEF- Protein-treated membrane, but which was not incubated with calmodulin in order to detect non-specific binding of the anti-calmodulin antibody. Bound calmodulin was detected after washing the membrane three times in TBS with antibody. The first antibody used is polyclonal anti-calmodulin from Rabbit, which was used 1: 1000 in 3% milk powder in TBS. The incubation was at room temperature. The second antibody, monoclonal Goat anti-Rabbit from BioRad was also used 1: 1000. The immune complex was detected with Sigma almost DAB tablets.

Results:

Both NEF-full and the NEF anchor showed binding to calmodulin in the presence of CaCl ₂ . Binding could be weakened (inhibited) by the presence of EGTA. As a control experiment, a membrane treated with NEF-full and NEF-core was incubated without calmodulin in order to ensure that the antibody directed specifically against calmodulin did not also recognize NEF.

Example 2

The binding between NEF protein and calmodulin was examined by means of fluorescence measurement.

For the fluorescence measurements, both pyrene-labeled and unlabeled NEF (full and core), as well as dansyl-labeled and unlabeled calmodulin were used. The measurements were carried out at 25 ° C.

Pyrene marking:

NEF solutions (filling and core) were incubated with a two-fold excess of pyrenmaleimide (dissolved in DMF) at room temperature for 1 h and then separated from excess reagent on a PD 10 column. The dansyl-labeled calmodulin was purchased from Sigma.

The fluorescence emission spectra for pyrene-labeled NEF were recorded from 360 nm to 450 nm, at one

Excitation wavelength of 344 nm. The slot widths were 0.5 nm for the excitation and 2.0 nm for the emission.

The fluorescence emission spectra for dansyl-labeled calmodulin were recorded from 450 nm to 550 nm, with an excitation wavelength of 340 nm.

A 1 μM solution of the marked sample was placed in a quartz cuvette and the emission spectrum was recorded. The sample was then titrated with the unlabelled protein and the course of fluorescence measured.

Results:

FIG. 4 shows the fluorescence emission spectrum of pyrene-labeled NEF-full alone (curve 1), in the presence of calmodulin (curve 2) and after adding 5 M EGTA to the NEF-calmodulin complex (curve 3).

FIG. 5 shows the fluorescence emission spectrum of pyrene-labeled NEF-core alone (curve 1) and in the presence of calmodulin (curve 2).

FIG. 6 shows the fluorescence emission spectrum of dansyl-labeled calmodulin alone (curves 1 and 2) and after addition of NEF-full (curve 3).

The addition of calmodulin to labeled NEF-full (FIG. 4) caused a significant increase in fluorescence, which indicates an interaction of the two proteins. This interaction could be inhibited by adding 5 mM EGTA. Calmodulin could also cause an increase in fluorescence in NEF-core (Fig. 5), which also with an excess of EGTA (5 mM) could be inhibited.

The addition of BSA (Bovine serum albumin) or CaCl ₂ to the labeled NEF did not show this effect of the change in fluorescence. The addition of NEF-full to labeled calmodulin (Fig. 6) also showed an increase in fluorescence and a slight blue shift.

Example 3

The binding of NEF protein to calmodulin was examined using a stopped flow.

The same protein samples as in Example 2 were used for the stopped-flow measurements. The measurements were made at 25 ° C

The excitation wavelength for the pyrene labeling (Dansyl labeling) was 344 nm (340 nm), the emission was measured with an edge filter at wavelengths above 370 nm (389 nm).

In order to determine the rate constant of the association k _on , a 1 μM solution of the labeled sample was filled into one syringe of the stopped-flow apparatus and a protein solution with a concentration of 1 μM to -40 (90) μM into the other syringe.

For an exact determination of the rate constant k _{o ££} , a solution of 1 μM labeled NEF sample + 8 μM unlabeled CaM sample was placed in one syringe and a -40 μM unlabeled NEF sample in the other syringe.

The same procedure was followed with the dansyl-labeled calmodulin sample. Results:

The experiments of Examples 1 and 2 were confirmed using the stopped flow. An increase in fluorescence was observed in each case when calmodulin was added to labeled NEF (fill or core). However, only a change in fluorescence with NEF-full was achieved with dansyl-labeled calmodulin.

Claims

P a t e n t a n s r u c h e

Binding partner for NEF protein, characterized in that it competitively inhibits the binding of calmodulin to NEF protein.

Binding partner for calmodulin, characterized in that it competitively inhibits the binding of NEF protein to calmodulin.

3. Binding partner according to claim 1, characterized in that it binds to a part of the amino acid sequence shown in Figure 1 1 to 206, preferably 1 to 80, or homologous sequences thereof.

4. Binding partner according to one of claims 1 to 3, characterized in that the dissociation constant for its binding to NEF protein or calmodulin is less than 1 μM.

5. Binding partner according to one of claims 1 to 4, characterized in that it is a peptide, a peptoid, an organic or inorganic substance.

6. A method for intra- and / or extracellular competitive inhibition of the binding between NEF protein and calmodulin, characterized in that at least one i) binding partner for NEF protein, which competitively inhibits the binding of calmodulin to NEF protein and / or ii) binding partner for calmodulin, which competitively inhibits the binding of NEF to calmodulin, in contact with HIV-infected cells and / or NEF protein from body fluid.

7. The method according to claim 6, characterized in that the binding partner is a peptide whose cDNA or mRNA is introduced and expressed in the HIV-infected cells.

8. The method according to claim 6, characterized in that the binding partner is a peptide or peptoid which is coupled to a carrier and is introduced into the HIV-infected cells.

9. A pharmaceutical composition containing at least one binding partner of claims 1 to 5 in a pharmaceutically acceptable carrier and / or excipient.

10. Use of at least one binding partner of claims 1 to 5 for the prevention, diagnosis and / or treatment of HIV diseases.