KR100467198B1 - Peptides capable of binding to the GAP protein SH3 region, nucleotide sequences encoding them, and pharmaceutical compositions containing them - Google Patents

Abstract

Peptides capable of interacting with the GAP protein SH3 region, nucleic acid sequences encoding them, and pharmaceutical compositions containing the same are described.

Description

Peptides bindable to the GAP protein SH3 region, nucleotide sequences encoding them, and pharmaceutical compositions containing them

The present invention relates to novel peptides and nucleotide sequences and their pharmaceutical use. In particular, the present invention relates to a peptide capable of binding to the SH3 region of a GAP protein.

The product of the Ras gene, commonly termed p21 protein, plays a major role in the control of cell division in all eukaryotic microorganisms that have been investigated. Certain specific modifications of these proteins cause them to lose their normal control and lead them to tumorigenicity. Thus, the majority of human tumors are associated with the presence of modified Ras genes. Likewise, overexpression of this p21 protein can lead to deregulation of cell proliferation. In total, p21 protein is involved in 30% of human cancers.

Thus, understanding the precise role of these p21 proteins is one of the main purposes of research in the field of oncology.

The models currently available for explaining the function of the p21 protein are based on the similarity they share with the G transducing protein. In cells, there is an equilibrium between an active p21 protein that binds GTP and an inactive form with bound GDP. In dormant cells where no p21 protein is required, most of these proteins are in GDP form. When a cell is stimulated, the nucleotide exchange factor GEF becomes more active, promoting the removal of GDP and its replacement by GTP. The protein then takes an active form that allows it to recognize and stimulate the GAP protein, the "GTPase-activated protein", its agonist, which is bound to almost all other proteins. The p21-GTP-GAP complex then interacts with one or more other proteins, possibly leading to the transmission of a signal that will elicit a biological response by the cell. The binding of p21-GTP and GAP simultaneously triggers the hydrolysis of GTP and the return of p21 to its inactive form.

In the case of oncogenic p21 proteins, the mutations they deliver prevent the return to the inactive state. In this latter case, the equilibrium is displaced towards the active type of p21.

This complex equilibrium between the active and inactive forms of p21 is responsible for the intrinsic factors of p21 proteins (relative affinity for GDP and GTP, nucleotide exchange rates, etc.) and for their activity, particularly those that regulate GAP proteins. By date and time.

GAP proteins are cytosolic proteins present in all eukaryotic organisms and thus possessing properties that strongly accelerate the hydrolysis of GTP that binds to normal p21 proteins (Trehey and McCormick (1987)). The protein has two regions that are involved in separate functions. Its carboxy terminus delivers catalytic activity that binds to p21 protein and increases its GTPase activity. Downstream of the amino terminal portion at the other end thereof is juxtaposed with regions SH2 and SH3 which may participate in interaction with other proteins.

Currently, two kinds of proteins are known that interact with GAP proteins. These proteins are proteins named p62 and p190 having molecular weights of 62 kDa and 190 kDa, respectively. Since these two kinds of proteins are immunoprecipitated by antibodies to different epitopes of GAP, they obviously form specific complexes with GAP. In particular, it is known that p62 protein interacts with GAP protein in the SH2 region.

As far as the SH3 region is concerned, its presence in a variety of proteins, especially the Cγ phospholipase (PLC-γ), the p85 subunit of 3-phosphatidylinositol kinase and the grb-2 protein, all of which are involved in the transduction of Ras p21 signals, This region is particularly important for the indication of protein / protein interactions and thus implies the function of the corresponding protein and / or its intracellular positioning. In special cases, such as GAP proteins, this SH3 region can therefore also participate in the transduction of Ras signals. It is clear that understanding the precise role of these SH3 regions will be particularly valuable at the therapeutic level.

1: Effect of G3BP overexpression in NIH 3T3 fibroblasts on CAT activity.

2: Effect of G3BP overexpression in NIH 3T3 fibroblasts on the formation of lesions induced by Src and Ras tumor genes.

Materials and methods

General cloning techniques

Preliminary extraction of plasmid DNA, centrifugation of plasmid DNA in a cesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification by electrolysis of DNA fragments, extraction of proteins with phenol or phenol / chloroform, in physiological saline medium Conventional methods used in molecular biology, such as precipitation of DNA with ethanol or isopropanol, transformation into E. coli, and the like, are well known to those skilled in the art and described in Maniatis T. et al., “Molecular Cloning, a Laboratory Manual ", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982; Ausubel F.M. et al., (eds), "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1987.

Restriction enzymes are supplied by New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham and used according to the vendor's recommendations.

Plasmid pGEX 2T is commercially available.

So-called PCR [polymerase-catalyst chain reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullis K.B. et Faloona F.A., Meth. Enzym. 155 (1987) 335-350] Enzymatic amplification of DNA fragments by the technique is carried out using a "DNA heat cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications.

Nucleotide sequences can be obtained using Sanger et al., Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467.

In general, the normal stringent conditions for hybridization experiments are as follows: Hybridization: 3 x SCC in the presence of 5 x Denhart's at 65 ° C; Wash: 0.5 × SSC at 65 ° C.

It is an object of the present invention to strictly explain the contribution to the transduction of the Ras signal in the SH3 region.

Thus, Applicant demonstrated the presence of a protein that can attach to the GAP protein by directly binding to the SH3 region.

More specifically, the present invention is based on the identification, isolation and characterization of proteins that can interact with the SH3 region of GAP proteins. In addition, the present invention is based on the structural characterization of these proteins by identifying the corresponding peptide sequences.

In particular, polypeptides of the invention capable of interacting with the SH3 region of a GAP protein include peptide sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 9, or It includes all or part of the derivative.

In the sense of the present invention, the term derivative refers to other molecules obtained and retained by the genetic and / or chemical modifications of the polypeptides according to the invention. Genetic modifications and / or chemical modifications are other mutations of one or more residues. By substitutions, deletions, additions and / or modifications. Such derivatives may be used for different purposes, e.g., in particular, to increase the affinity of the peptide for the interaction site, to improve the level produced, to increase the protease-tolerance, to increase the therapeutic efficiency or to reduce side effects, or to novel pharmacokinetics. It can be produced for characterization and / or biological characterization.

It may also refer to fragments of the aforementioned sequences of derivatives of these fragments. Such fragments can be produced in different ways. In particular, they can be chemically synthesized based on the sequences given in FIG. 1 using peptide synthesizers known to those skilled in the art. They can also be genetically synthesized by expressing in the cell host a nucleotide sequence encoding a rare peptide. In this case, the nucleotide sequence can be chemically prepared using an oligonucleotide synthesizer, based on the peptide sequence and the genetic code given in the present application. Nucleotide sequences can also be prepared by screening DNA libraries by enzymatic restriction, ligation, cloning, or the like, or by probes developed on the basis of SEQ ID NO: 1 according to techniques known to those skilled in the art using the sequences given in the present application. Can be.

According to one aspect of the invention, the claimed peptide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 4.

Preferably, the polypeptide according to the invention has a molecular weight on the order of 68 kDa.

According to one aspect of the invention, the polypeptide is a polypeptide of human origin comprising all or part of SEQ ID NO: 5 or SEQ ID NO: 9 or one or all of its derivatives as described above.

The polypeptide is in particular a polypeptide comprising SEQ ID NO: 5.

More preferably, the polypeptide is one of the polypeptides represented by SEQ ID NO: 9 or derivatives thereof.

Unexpectedly, such proteins do not possess homology with other p68 proteins already identified as binding to the SH3 region of Src. Its tyrosine motif is not phosphorylated in growth cells or cells in mitotic state.

Analysis of protein sequence number 9 revealed that the protein, attached to the SR + H3 region of the GAP protein, is named G3BP and belongs to the hnRNP (heterologous nuclear ribonucleoprotein) family. Specifically, G3BP is a 466 amino acid protein with an apparent molecular weight of 68 kDa and contains several regions characteristic of proteins that bind to RNA:

-RNP2 and RNP1 region (amino acids 342-347)

-4 RGG box (amino acids 435-449)

Acidic auxiliary regions (amino acids 144-221).

The invention also extends to peptides that can antagonize the interaction between G3BP and the SH3 region of GAP. The activity of these peptides can be demonstrated in competition tests (see Examples 2 and 3) or in tests involving interference of signals transduced by Ras protein.

The invention also relates to polyclonal or monoclonal antibodies or antibody fragments against the aforementioned polypeptides. Such antibodies can be produced by methods known to those skilled in the art. In particular, these antibodies immunize an animal against a polypeptide whose sequence is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 9 or other fragments or derivatives of these sequences The sample can be prepared by collecting blood and then isolating the antibody. These antibodies can also be produced by preparing hybridomas according to techniques known to those skilled in the art.

The antibody or antibody fragment of the invention can then be used to modulate the activation state of the Ras gene product.

Moreover, these antibodies can also be used to detect and / or analyze peptides according to the invention present in biological samples and thus provide information on the activation status of Ras gene products.

The invention also extends to antagonists, ie other peptides capable of blocking the interaction of the polypeptides according to the invention with the SH3 region of the GAP protein. Such peptides can be demonstrated in competition tests (see Examples 2 and 3) or in tests in which Ras activity is inhibited.

Thus, the present invention allows the production of polypeptides derived from the sequences identified above and also antibodies against the corresponding proteins with corresponding biological properties in view of such polypeptides or pharmaceutical use.

The present invention also provides non-peptide compounds, or compounds that are not wholly peptides, which can be used pharmaceutically. Thus, based on the active protein motifs described in this application, it is possible to construct molecules that inhibit the p21 protein-dependent signal pathway and are virtually not entirely peptide and suitable for pharmaceutical use. In this regard, the present invention is directed to pharmacologically active non-peptide molecules, by determining the structural elements of the polypeptide that are important for its activity and regenerating these elements using non-peptide constructs or substantially non-peptide constructs, Or to the use of a polypeptide of the invention as described above, in order to prepare a pharmacologically active molecule that is in fact not entirely a peptide. The invention also relates to pharmaceutical compositions comprising one or more molecules prepared in this manner.

The invention also relates to other nucleic acid sequences encoding polypeptides capable of binding to the SH3 region of a GAP protein. More preferably, the present invention hybridizes with (a) all or part of SEQ ID NO: 6, or SEQ ID NO: 10 or one of its complementary chains, (b) sequence (a) and encodes a polypeptide according to the present invention. And (c) sequences derived from sequences (a) and (b) by degeneracy of the genetic code.

Preferably, it comprises the sequence of SEQ ID NO: 6 and more preferably is represented by SEQ ID NO: 10.

Different nucleotide sequences of the invention may or may not be of artificial origin. These may be genomic sequences, cDNA or RNA sequences, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained, for example, by screening DNA libraries (cDNA libraries or genomic DNA libraries) using probes designed on the basis of the sequences set forth above. Such libraries can be prepared from cells of different origin using conventional molecular biology techniques known to those skilled in the art. Nucleotide sequences of the present invention may also be prepared, in particular, by chemical synthesis according to the phosphoramidite method, or by mixing methods involving chemical or enzymatic modification of the sequences obtained by screening libraries.

Such nucleotide sequences according to the invention can be used to produce polypeptides of the invention, for the construction of antisense sequences that can be used for gene therapy, or to detect and diagnose the activity of GAP proteins present in biological samples by hybridization experiments. Or to separate homologous sequences from other cell sources.

In order to produce the polypeptides of the present invention, the nucleic acid sequences defined above are generally placed under the control of signals that allow them to be expressed in the host cell. The choice of such signals (promoter, terminator, secretory leader sequence, etc.) may vary depending on the host cell used. Preferably, such nucleotide sequences of the present invention form a vector moiety or integration that can self replicate. In particular, self-replicating vectors can be prepared using sequences that autonomously replicate in selected hosts. Integration vectors can be prepared, for example, using sequences that are homologous to specific regions of the host genome and allow the vectors to be integrated by homologous recombination.

Host cells that can be used to produce the polypeptides of the invention can be eukaryotic or prokaryotic hosts. Examples of suitable eukaryotic hosts are animal cells, yeasts or fungi. In particular, yeast that may be mentioned are yeasts of Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula. . Animal cells that may be cited are cells such as COS, CHO, C127, and the like. Particularly mentioned fungi are Aspergillus spp. Or Trichoderma spp. The following bacteria are preferably used as prokaryotic cell hosts: E. coli, Bacillus or Streptomyces.

Nucleic acid sequences according to the invention can also be used in the construction of antisense nucleic acids that can be used as medicaments. Inhibition of expression of certain oncogenes by antisense nucleic acids has proven to be a useful strategy in understanding the role of these oncogenes and a particularly promising solution to achieving anticancer treatment. Antisense oligonucleotides are small oligonucleotides that are complementary to the coding strand of the gene of interest and for this reason can specifically hybridize with the transcribed mRNA and thereby inhibit their translation into proteins. Such oligonucleotides may be constituted by all or part of a nucleic acid sequence as defined above. In general, they are sequences or fragments thereof that are complementary to the sequences encoding the peptides according to the invention. Such oligonucleotides can be obtained from the aforementioned sequences by cleavage or the like, or by chemical synthesis.

The present invention also relates to nucleotide sequences capable of hybridizing with the aforementioned nucleotide sequences encoding the polypeptides of the invention, or the corresponding mRNAs, whether or not they are synthetic sequences. Such probes can be used as diagnostic means in vitro. Such probes must be labeled in advance, and different techniques for performing this are known to those skilled in the art. Hybridization conditions under which these probes can be used are conventional stringent conditions. Such probes can be used for the detection and isolation of homologous nucleic acid sequences encoding polypeptides of the invention using other cell sources. Such probes include, in particular, probes represented by SEQ ID NO: 7 and SEQ ID NO: 8 used in Example 3 below.

The invention also relates to a pharmaceutical composition comprising at least one polypeptide as described above as an active ingredient.

The invention also relates to a pharmaceutical composition comprising at least one antibody and / or antibody fragment as described above as an active ingredient, and a pharmaceutical composition comprising at least one antisense oligonucleotide as described above as an active ingredient.

In addition, the present invention relates to pharmaceutical compositions wherein the polypeptides, antibodies and oligonucleotides described above are attached to one another or attached to other active ingredients.

The pharmaceutical compositions according to the invention can be used to modulate the activation of p21 protein and thus to modulate the proliferation of certain cell types. In particular, these pharmaceutical compositions are for the treatment of cancer. Thus, many cancers have been associated with the presence of oncogenic Ras proteins. Cancers that contain the mutated Ras gene most frequently include pancreatic adenocarcinoma, in particular 90% containing the Ki-Ras oncogene, in which the twelfth codon is mutated, and in which the twelfth codon is mutated [Almoguera et al. al., Cell 53 (1988) 549], colon adenocarcinoma and thyroid cancer (50%), or lung and myeloid leukemia (30%, Bos, JL Cancer Res. 49 (1989) 4682).

The invention also relates to the use of the aforementioned molecules to modulate, ie inhibit, the activity of p21 protein. In particular, the present invention relates to the use of all, or fragments, of G3BP to interfere with signals transduced by Ras gene products. Protein fragments homologous to hnRNP are advantageously used to inhibit G3BP from binding to its target RNA. Sequences identical or complementary to these target RNAs can also be used to interfere with signals transduced by the G3BP protein.

The invention also provides a method of detecting expression and / or overexpression of G3BP protein in a biological sample. Such methods include, for example, contacting such a sample with an antibody or antibody fragment according to the invention, detecting the antigen / antibody complex, and then comparing the results obtained with a standard sample. In this method, the antibody may be in suspension or may be immobilized on a support beforehand. This method also includes contacting a sample with a nucleotide probe according to the invention, detecting the hybrid obtained and then comparing it with the results obtained in the case of a standard sample.

The present invention can be used in a variety of ways in the therapeutic field: the polypeptides, antibodies and nucleotide sequences of the present invention can modulate the activity of the Ras gene, thus allowing them to intervene in the development of cancer. Due to the fact that they are strongly expressed in skeletal hepatic muscle cells, these peptides intervene and also intervene in pathologies associated with signaling defects such as, for example, diabetes. Another aspect of the invention is the use of a DNA or RNA nucleotide sequence capable of interacting with a claimed polypeptide. Such sequences can be prepared using the methods described in international application WO 91/19813.

The invention can also be used in connection with the diagnosis and classification of cancer.

Other advantages of the present invention will become apparent upon reading the following examples and drawings, which are for illustrative purposes only and are not intended to limit the invention.

Example  One

Preparation of Glutathione-S-Transferase SH3 Fusion Proteins

DNA sequences encoding the SH3 region (residues 275-350) and c-Src (residues 84-148) protein of the GAP were expressed in P.C.R. Amplify by technique and clone between the BamHI and EcoRI restriction sites in the expression vector pGEX2T. The transformed bacteria are cultured, induced with IPTG (1-thio-β-D-galactopyranoside) and lysed by sonication. GST SH3 fusion protein is purified by affinity chromatography on glutathione agarose beads (Pharmacia LKB biotech) and then eluted with 10 mM reduced glutathione.

Example 2

1) Preparation of Cell Lysate

ER22 cells (hamster fibroblasts overexpressing human EGF receptors) or NIH 3T3 cells expressing C-Src pp60 (F-527) were treated with antibiotics G418 (200 μg / ml) and 2 mM / L glutamine (5GIBCO-BRL). Incubate at 37 ° C under 5% CO ₂ on DMEM medium supplemented with 10% fetal calf serum.

In each assay, ER22 cells are incubated in 100 mm dishes until they adhere and then incubated for 18 hours without serum. Sodium orthovanadate is added to a final concentration of 100 μM and continued incubation for 30 minutes. EGF is then added directly to the medium to a final concentration of 80 nM over 10 minutes at 37 ° C.

For certain assays, mitotic cells are harvested by treatment with nocodazole 0.4 μg / (SIGMA) / ml for 18 hours. They were quickly washed with phosphate-based physiological saline buffer, re-cooled in ice and then rinsed with 1 ml of lysis buffer, HNTG (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X 100, 10 over 30 minutes at 4 ° C). In glycerol, 1 mM MgCl ₂ , 1 mM EGTA, phosphatase inhibitors (1 mM Na ₃ VO ₄ , 10 mM Na ₄ P ₂ O ₇ and 10 mM NaF) and protease inhibitors (1 μg / ml leupetin, 1 μg / Ml trypsin inhibitor, 1 μg / ml pepstatin A, 2 μg / ml aprotinine, 10 μg / ml benzamidine, 1 mM phenylmethanesulfonyl fluoride, 1 μg / ml antipain and 1 μg / ml chymostatin) Solubilize in the presence of

The lysate is clarified by centrifugation at 15,000 rpm for 10 minutes. Protein concentration is then measured (Biorad microtest).

2) direct bonding test

Whole cell lysate (200 μg) and immunoprecipitated protein were separated on 7.5% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and then transferred to polyvinylidene difluoride membrane (PVDF, Millipore Corp.) do. Non-specific binding with the filter is blocked with 2% skim milk in PBS containing 0.05% Tween 20 for 2 hours at room temperature. The filter is incubated for 12 hours at 4 ° C. with GST protein and GST-SH3 protein in blocking buffer. After washing with PBS-0.05% Tween 20, the bound protein was anti-GST monoclonal antibody (0.25 μg / ml) (Hybridolab Pasteur Inst.), Anti-mouse antibody conjugated to alkaline phosphatase and 5-bromo- It is detected by continuous incubation with 4-chloro-3-indolyl phosphate toluidium nitroblue tetrazolium salt (PROMEGA).

3) Competition test

Synthetic peptides corresponding to sequences (275-305), (299-326), (317-326) and 9320-350 of GAP SH3 or corresponding to the putative sequence of dynamin (RRAPAVPPARPGS) that bind to the SH3 region were subjected to FMOC chemistry. On Applied Biosystems 431 A instrument.

Purified p68 protein is isolated by electrophoresis on sodium dodecyl cellulose polyacrylamide gel and transferred to PVDF membrane by electrophoresis. The membrane is incubated with increasing amounts of peptide. Peptide poly-L-proline (SIGMA) is used at 500 μM in the control reaction. After incubation for 1 hour, protein GST-GAP-SH3 (2 μg / ml) is added to each reaction medium. The filtrate is probed with anti-GST monoclonal antibodies as described above.

4) Immunoprecipitation and Immune Transfer

Soluble cell lysate (3 mg) is clarified with 50 μl Protein A Sepharose CL-4B (Pharmacia Biotech) at 4 ° C. for 2 hours. Clarified cell lysates are incubated with anti-phosphotyrosine monoclonal antibody (monoclonal antibody 4G10-Upstate Biotechnology Incorporated) at 4 ° C. for 4 hours. 50 μl of Protein A Sepharose is then added to the complex and the incubation is continued at 4 ° C. overnight. Immunoprecipitates are washed three times with HNTG buffer and dissolved in a sample of SDS buffer (100 μl). The complex is then separated by SDS-PAGE and transferred to PVDF membranes by electrolysis.

The membrane is incubated with phosphotyrosine monoclonal antibody in TBS (10 mM Tris, pH 7.4, 150 mM NaCl, 3% bovine serum albumin) and also with a second antibody conjugated with alkaline phosphatase. Subsequently, a substrate for alkaline phosphatase is added for proper color development.

5) Purification and Analysis of Sequences

Approximately 5.10 ⁹ ER22 cells are lysed in 200 ml of HNTG buffer. Lysates are centrifuged at 15,000 g for 15 minutes and diluted 5 times in HNG buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EGTA, phosphatase inhibitors and protease inhibitors). Lysates are incubated overnight with 6 ml of high speed S-Sepharose equilibrated in the same buffer. The complex is transferred to a column (IBF- 2.5 x 1.3 cm). The column is washed with its 10-fold volume of buffer and the bound protein is then eluted with a linear gradient of 0-1 M NaCl 60 mL in the same buffer at an elution rate of 60 mL / h. Fractions with binding activity measured under the conditions of Example 2.2 (0.15 to 0.37 M NaCl) were collected, diluted 10-fold in HNG buffer, and then to Heparin-Sepharose Cl-6B (Pharmacia LKB) pre-equilibrated with the same buffer. Load at an elution rate of 24 ml / h. After washing with HNG buffer, the column is eluted with a 24 ml gradient of 0-1 M NaCl. The active fractions from S fast flow chromatography were collected and diluted four times in MES buffer (100 mM MES, pH 6.8, 1 mM MgSO ₄ , 1 mM EGTA) and pre-equilibrated with agarose ATP column (prepared with MES buffer containing 50 mM NaCl). 3 ml) (Sigma No. A9264) at a dissolution rate of 6 ml / h. The column is then washed with 20 ml MES buffer containing 50 mM NaCl and eluted at a rate of 30 ml / h with a linear gradient of 50 mM to 2 M NaCl in MES buffer. p68 protein elutes between 0.3 M and 0.4 M NaCl. The active fractions are collected, dialyzed to 20 mM NH ₄ HCO ₃ , pH 8.3 and concentrated. After drying, the proteins are resuspended in SDS buffer and separated by electrophoresis on polyacrylamide gels. The gel is stained with Coomassie Blue and the 68 kDa molecular weight band corresponding to the SH ₃ binding activity is recovered. It is washed with the following solution for 1 hour: water, water / methanol (90/10), water / CH ₃ CN (80/20) and water / CH ₃ CN (50/50).

The gel band containing the purified p68 protein is then divided into small fragments and dried under SPEED / VAC (SAVANT). 400 μl of solution containing 25 mM Tris, pH 8.5, 1 mM EDTA, 0.05% SDS and 5 μg Lys-c endoproteinase (Boehringer Mannheim) is added and the whole is incubated overnight at 37 ° C. The hydrolyzate is injected into a reversed phase HPLC column (Vydac C18: 2.1 × 250 mm). The column was eluted in a straight gradient of 0-35% B (A: H ²⁰ + 0.07% TFA, and B: CH ₃ CN + 0.07% TFA) within 150 minutes at a rate of 0.2 ml / min and retention time 113.7, Elution peaks observed at 117.7 and 133.7 minutes are collected and sequenced directly using the Applied Biosystems 477A microsequencer. The corresponding peptide sequences obtained in this manner are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. These sequences show no homology with the proteins referenced in the protein database (PIR 33. Swiss-Prot 33 (intelligenetics)).

To determine whether p68 protein is tyrosine-phosphorylated in mitotic or non-synchronized cells, the protein in lysate derived from ER22 cells cultured in 10% fetal calf serum or from nocodazole treated cells Immunoprecipitate with phosphotyrosine antibodies, transfer to the membrane and then immunodetect with an anti-phosphotyrosine antibody or incubate with GST-GAP-SH3 or GST-Src-SH3 under the conditions described in Examples 2.2 and 2.4. As a control, the protein of NIH 3T3 cells transformed with the c-Src activated allele (c-Src Y527F) is tested under the same conditions as described for the ER22 protein. The results obtained demonstrate that the protein is tyrosine-phosphorylated, especially in the 60-70 kDa region. GST-GAP-SH3 or GST-Src-SH3 probes were used to detect no binding to the phosphorylated p68 protein in ER22 cells. In NIH3T3 cells (c-Src-Y527F), the GST-Src-SH3 probe binds to a tyrosine-phosphorylated protein of 68 kDa molecular weight while the GST-GAP-SH3 probe does not interact with any protein.

Competitive experiments to confirm functional involvement of the protein in the Ras signaling pathway are performed under the conditions described in Example 2.3. The results obtained indicate that peptides derived from GAP can block Ras-induced disruption of Xenopus egg distribution (GVBD); GAP SH3 peptides (299-326) and (317-326) also demonstrate that they can block the interaction between G3BP protein and GAP.

These results clearly confirm that the blocking or blocking ability of the protein activity according to the present invention constitutes a particularly promising new method in the treatment of cancer.

Example 3

Isolation of Human Sequence SEQ ID NO: 6 from Human Placental cDNA Library

SEQ ID NOs: 7 and 8, which are sequences based on the peptide sequences SEQ ID NO: 1 and SEQ ID NO: 3, are used as probes. DNA from the Clonetech HL1008B library is prepared and used for the PCR reaction. Thirty amplification cycles are performed under the following conditions: denaturation at 94 ° C. for 1 minute, annealing at 35 to 40 ° C. for 1 minute, and extension at 72 ° C. for 1 minute. This reaction produces a 1.3 kb DNA fragment in which part of the sequence is given in SEQ ID NO: 6. Northern blot analysis demonstrated that the corresponding RNA (3.3 kb) was ubiquitous and very strongly expressed in adult skeletal muscle.

Example 4

Isolation from Human Placental cDNA Library of SEQ ID NO: 10 Sequence Encoding Human p68

Two oligonucleotides (SEQ ID NO: 7) and (SEQ ID NO: 8) are used as primers for amplifying cDNA in human placental libraries. PCR is carried out using an Perkin Elmer Amplitaq at an annealing temperature of 35 ° C., followed by an additional 1 minute at 72 ° C. in the presence of 10% formamide. Applicants amplified the 1164 bp cDNA fragment. After cloning directly into a pMOSblue vector (Amersham) containing the post--20 sequences, the fragments are sequenced using fluorescent probes. This PCR fragment is then used as a probe for screening 10 ⁶ phages from the human placenta λ gt11 cDNA library (Clonetech). The probe is synthesized with the Amersham Rediprime system and the filter is incubated for 16 hours at 45 ° C. in a hybridization buffer containing the probe (6 × SSC, 5 × Denhardt's, 100 μg / ml salmon sperm, 0.25% SDS). The filter is then washed in 2 x SSC, 0.05% SDS for 20 minutes at hybridization temperature for 1 hour at room temperature. Eight positive clones are identified. Two of them are purified to digest their cDNA with EcoRI to cleave the insert. They are cloned into M13mp18 / EcoRI for sequencing. Sequence analysis is performed on fragments obtained by progressive deletion of cDNA with exonuclease III (Nested Deletion Kit, Pharmacia Biotech). The size difference of fragments is analyzed by PCR amplification between -20 primers before and after the M13 vector, and different size populations are selected for sequencing. By assembling the different sequences obtained, it becomes possible to reconstruct the entire open reading frame of p68.

Example 5

Overexpression of G3BP in NIH 3T3 Fibroblasts

NIH 3T3 fibroblasts are transfected with the reporter gene, the reporter gene of chloramphenicol acetyl transferase, which is under the control of the Ras response element derived from the polyom virus enhancer. These elements are stimulated 15-30 fold when cells are transfected with expression vectors carrying the cDNAs of the Src and Ras oncogenes. This stimulus is modified when G3BP protein is expressed after cotransfection with an expression vector containing cDNA corresponding to the open reading frame of the protein. G3BP dose-dependently inhibits CAT activity stimulated by Src and by oncogenic forms of Ras. These observations are shown in FIG.

In the same way, expression of G3BP protein prevents the formation of lesions induced by Src and Ras oncogenes. 2 provides the basis for this observation.

These experiments show the ability of G3BP to counteract the proliferative effect of signals transduced by normal or oncogenic Ras proteins.

Sequence list

(1) General Information:

(i) Applicant:

Full Name: LongFran Laura Societe Anonym

(B) Street name: Avenue Lamont Along 20

(C) City name: Anthony

(E) Country: France

(F) Zip code: 92165

(ii) Name of the invention: Peptide capable of binding to the SH3 region of the GAP protein

(iii) SEQ ID NO: 10

(iv) computer readable form:

(A) Medium type: Tape

(B) Computer: IBM PC compatible model

(C) operating system: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0, Version # 1.25 (OEB)

(2) Information about sequence number 1:

(i) Sequence features:

(A) Length: 19 amino acids

(B) Type: amino acid

Phase: Linear

(ii) Molecular Type: Protein

(vi) First source: hamster

(xi) Sequence description: SEQ ID NO: 1:

(2) Information about sequence number 2:

(i) Sequence features:

(A) Length: 15 amino acids

(B) Type: amino acid

Phase: Linear

(ii) Molecular Type: Protein

(vi) First source: hamster

(ix) Characteristics:

(D) Other information: The first Xaa represents an alanine motif or threonine motif and the second Xaa represents another amino acid.

(ix) Sequence description: SEQ ID NO: 2:

(2) Information about sequence number 3:

(i) Sequence features:

(A) Length: 17 amino acids

(B) Type: amino acid

Phase: Linear

(ii) Molecular Type: Protein

(vi) First source: hamster

(xi) Sequence description: SEQ ID NO: 3:

(2) Information about sequence number 4:

(i) Sequence features:

(A) Length: 19 amino acids

(B) Type: amino acid

Phase: Linear

(ii) Molecular Type: Protein

(vi) First source: hamster

(xi) Sequence description: SEQ ID NO: 4:

(2) Information about sequence number 5:

(i) Sequence features:

(A) Length: 68 amino acids

(B) Type: amino acid

Phase: Linear

(ii) Molecular Type: Protein

(vi) Initial Source: Person

(xi) Sequence description: SEQ ID NO: 5:

(2) Information about sequence number 6:

(i) Sequence feature: SEQ ID NO: 6

(A) Length: 204 base pairs

(B) Type: nucleic acid

(C) chain type: double strand

Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iii) anti-sense: none

(vi) Initial Sources:

 (A) organism: human

(xi) Sequence description: SEQ ID NO: 6:

(2) Information about sequence number 7:

(i) Sequence feature: SEQ ID NO: 7

(A) Length: 32 base pairs

(B) Type: nucleic acid

(C) Main print type: Japanese print

Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iii) anti-sense: none

(vi) Initial Sources:

(A) organism: human

(xi) Sequence description: SEQ ID NO: 7:

(2) Information about sequence number 8:

(i) Sequence feature: SEQ ID NO: 8

(A) Length: 26 base pairs

(B) Type: nucleic acid

(C) Main print type: Japanese print

Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(iii) anti-sense: none

(vi) Initial Sources:

(A) organism: human

(xi) Sequence description: SEQ ID NO: 8:

(2) Information about sequence number 9:

(i) Sequence features:

(A) Length: 466 amino acids

(B) Type: amino acid

(C) Main print type: Japanese print

Phase: Linear

(ii) Molecular Type: Peptide

(xi) Sequence description: SEQ ID NO: 9:

(2) Information about sequence number 10:

(i) Sequence features:

(A) Length: 2129 base pairs

(B) Type: nucleotide

(C) Main print type: Japanese print

Phase: Linear

(ii) Molecular Type: cDNA

(iii) Hypothesis: None

(xi) SEQ ID NO: 10:

Claims (25)

An isolated polypeptide that binds to the SH3 region of a GAP protein, Any one peptide sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 9, Or a polypeptide comprising SEQ ID NO: 1. The polypeptide of claim 1 comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a combination thereof. The polypeptide of claim 1 comprising SEQ ID NO: 5 or SEQ ID NO: 9. The polypeptide of claim 1 which contains the sequence of SEQ ID NO: 9. The polypeptide of claim 1 having a molecular weight of 68 kDa. The polypeptide according to any one of claims 1 to 5, which is of human origin. 7. A polypeptide according to any one of claims 1 to 6 comprising at least one tyrosine motif which is not phosphorylated in mitotic cells or growth cells. An antibody or antibody fragment against a polypeptide according to any one of claims 1 to 5. The antibody or antibody fragment of claim 8, wherein the antibody or antibody fragment is directed to any one of the peptide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 9. 9. 9. An antibody or antibody fragment according to claim 8 having an inhibitory activity of interaction between the GAP protein and the polypeptide according to claim 1. An isolated nucleotide sequence encoding a polypeptide according to any one of claims 1 to 5. The method of claim 11, (a) the sequence of SEQ ID NO: 6, SEQ ID NO: 10 or their complementary strand, (b) a sequence that hybridizes with sequence (a) and encodes a polypeptide according to claim 1, or (c) a nucleotide sequence comprising sequences derived from sequences (a) and (b) according to the degeneracy of the genetic code. 12. The nucleotide sequence of claim 11, comprising the sequence of SEQ ID NO: 6. 12. The nucleotide sequence of claim 11 comprising the sequence of SEQ ID NO: 10. Antigen nucleic acid capable of at least partially inhibiting the production of a polypeptide according to any one of claims 1 to 5. A composition comprising the nucleotide sequence according to claim 35 for the diagnosis of cancer by detecting the expression of a polypeptide according to any one of claims 1 to 5 or for the detection of a genetic abnormality. A method of constructing a non-peptide compound or a non-peptide compound that is capable of interacting with a GAP protein, in its entirety, 1. A peptide comprising a peptide sequence selected from the sequences of SEQ ID NOs: 1, 2, 3, 4, 5 and 9 as defined in claim 1 important for its activity and capable of interacting with the SH3 region of a GAP protein. Determining the structural elements of the polypeptide according to any one of claims 5 to 6, Regenerating said element using a construct that is substantially entirely non-peptide or a non-peptide construct. A pharmaceutical composition for treating cancer comprising at least one polypeptide according to any one of claims 1 to 5 as an active ingredient. The pharmaceutical composition of claim 18 for modulating the activity of p21 protein. A pharmaceutical composition for treating cancer comprising at least one antibody or antibody fragment according to claim 8 as an active ingredient. A pharmaceutical composition for treating cancer comprising at least one antigen nucleic acid according to claim 15 as an active ingredient. A composition for diagnosing cancer by detecting the expression or overexpression of a polypeptide according to claim 1 which is amplified, mutated or rearranged in a biological sample comprising the antibody or antibody fragment according to claim 10. A composition for diagnosing cancer by detecting the expression or overexpression of a polypeptide according to claim 1 in a biological sample comprising a nucleotide sequence according to claim 11. A composition for diagnosing cancer by classification of a cancer or disease associated with a signaling defect, comprising the antibody or antibody fragment according to claim 8. A composition for treating cancer comprising an RNA or DMA nucleotide sequence capable of interacting with a polypeptide according to any one of claims 1 to 5.

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