EP1844067A2

EP1844067A2 - Novel peptides which interact with anti-apoptotic members of the family of bcl-2 proteins and use thereof

Info

Publication number: EP1844067A2
Application number: EP06709202A
Authority: EP
Inventors: Olivier Geneste; John Hickman; Jean-Christophe Rain
Original assignee: Hybrigenics SA; Laboratoires Servier SAS
Current assignee: Hybrigenics SA; Laboratoires Servier SAS
Priority date: 2005-02-01
Filing date: 2006-01-31
Publication date: 2007-10-17
Also published as: JP2008528048A; US7927813B2; FR2881430A1; WO2006082304A3; CN101111517A; FR2881430B1; JP4713597B2; US20090325883A1; CA2596674A1; WO2006082304A2

Abstract

The invention relates to the research and identification of novel peptides which interact with anti-apoptotic members of the family of Bcl-2 proteins by means of the double-hybrid system. More specifically, the invention relates to a method of screening and identifying modulators of the protein interaction between said novel peptides and the anti-apoptotic members of the family of Bcl-2 proteins. The modulators identified on the basis of said method are administered to cancer patients in order to induce apoptotic- and/or autophagic-type programmed cell death.

Description

NOVEL PEPTIDES INTERACTING WITH ANTI-APOPTOTIC MEMBERS OF THE BCL-2 PROTEIN FAMILY

AND USES

The present invention is part of the research and identification of novel peptides interacting with the anti-apoptotic members of the Bcl-2 family of proteins.

The invention relates to a method for screening and identifying modulators of the protein interaction between these novel peptides and the anti-apoptotic members of the Bcl-2 family of proteins. Modulators isolated by this screening method are useful for regulating programmed apoptotic and / or autophagic cell death. These modulators are administered to patients during cancer treatments.

The invention relates to five peptide motifs capable of interacting respectively with an anti-apoptotic member of the Bcl-2 family of proteins and their uses for inducing programmed cell death in cancer patients.

The programmed cell death is composed, on the one hand, of apoptosis and, on the other hand, of autophagic death. Apoptosis is the best known phenomenon. This type of cell death involves morphological changes, such as core condensation, DNA fragmentation, as well as biochemical phenomena, such as the activation of caspases that will degrade key structural components of the cell to induce it. his disassembly and his death. The regulation of the apoptosis process is complex and involves activation or repression of several intracellular signaling pathways.

Autophagic death is a second less known mechanism of programmed cell death. At the cellular level, autophagy can be summed up in three stages: the formation of an initial autophagic vacuole (autophagosome) and the maturation of the autophagosome into a degradative vacuole and its fusion with the lysosome. Autophagic death therefore involves processes of lysosomal degradation, characterized by the accumulation of autophagic vacuoles, independent of a caspase-type regulatory pathway.

Maintaining a cell alive or programming its death requires the regulation of a major signaling pathway involving in particular proteins of the Bcl-2 family.

Proteins of the Bcl-2 family are divided into three main classes.

Anti-apoptotic proteins, such as Bcl-2, BCI-XL and BcI-W, show strong homology in their four BH domains. The pro-apoptotic proteins are divided into two categories, on the one hand, multidomain proteins such as BAX and BAK and, on the other hand, pro-apoptotic proteins such as BID, NOXA, PUMA, BIK, BIM and BAD. characterized by the presence of a single domain of homology, the BH3 motif (Cory and Adams, The Bcl-2 family: regulators of the cellular life-or-death switch Nature reviews vol.2 september 2002).

The BH3 motif is an amphiphilic helical α region whose sequence identity is relatively low in the BcI-2 family of proteins. In addition, the presence of the BH3 motif in a protein is required to allow interaction with the anti-apoptotic members of the Bcl-2 family of proteins. Indeed, the activity of an anti-apoptotic member of the Bcl-2 family of proteins is regulated by the product of the pro-apoptotic genes of said family, the two proteins assembling into heterodimers. Under this state the anti-apoptotic member of the Bcl-2 family of proteins is inactive and therefore no longer has its anti-apoptotic activity. In addition, the specific interaction of the BH3 motif with the anti- Apoptotic family of Bcl-2 proteins can be modified by modulators to specifically induce programmed cell death.

The invention therefore proposes to screen and identify new peptides interacting with the anti-apoptotic members of the protein family

Bcl-2 and use as such these new peptides to screen and identify molecules capable of modifying these interactions to obtain real drug candidates effective in pathologies involving deregulation of apoptosis, including cancers.

The dual hybrid system initially consists of a yeast test between two recombinant proteins. The first protein, called "bait", is a fusion protein containing a DNA binding domain (BD) linked upstream of a protein A. The second protein is also a fusion protein, commonly called "prey", containing an activation domain (activation domain or

AD) bound to a protein B. The binding and activation domains commonly used are those of Gal4 or E. CoIi Lex A. The proteins A and B are respectively an anti-apoptotic member of the family of Bcl-2 proteins and a pattern from a cDNA library. The association of proteins A and B by protein interaction allows the formation, by complementation, of a functional domain (BD-AD) capable of binding to the binding site (BS or BS) present upstream of a reporter gene and to ensure the transcription of said reporter gene.

However, this conventional double hybrid method has its limitations. It is, for example, well known that such screenings can lead to false positives and / or false negatives and biochemical confirmations of the results obtained are necessary. False positives obtained through the dual hybrid system are particularly common and are the origin of evidence of functional and non-structural interactions.

A more efficient technique for minimizing false positives and / or negatives is described in International Patent Application WO 99/42612 or US Patent 6,187,535 and uses recombinant haploid yeasts containing the "bait" and "prey" polypeptides. This system allows the detection of a greater number of "preys" from a single "bait", more precisely, more reproducible and more sensitive than the other conventional methods used in the field.

The inventors have established, on the basis of the double hybrid system, the existence of a structural interaction between the anti-apoptotic members of the Bcl-2 protein family and the following peptides of amino acid sequences SEQ ID NO. 1 to SEQ ID No. 5 according to the invention. This protein interaction between these partners is similar to that existing in the regulation of the apoptotic phenomenon between the anti- and pro-apoptotic partners of the Bcl-2 protein family.

Original peptides of peptide sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5 have been demonstrated in the context of the invention by the system. double hybrid. Each of these peptides is able to interact very specifically with the anti-apoptotic members of the Bcl-2 family of proteins. Indeed, this interaction specificity is related to the sequence, the three-dimensional structure and / or the helicity of said selected peptide. In addition, these peptides correspond to the precise domain of interaction with Bcl-2, BCI-XL and / or BcI-W and possess the typical structural criteria for the formation of homo- or heterodimers. The following peptide unit NH ₂ - XXXXXXXXLXXXXDXXXXXXXXXX -COOH (SEQ ID NO.6) in which X corresponds to any amino acid, corresponds to a consensus sequence of the peptide sequences SEQ ID NO.1 to SEQ ID No. 5 according to the invention. This consensus sequence is similar to that of the BH3 motifs present in the pro-apoptotic members of the Bcl-2 family of proteins.

Finally, the size of the peptides according to the invention make them ideal candidates for the elaboration of tests allowing the highly efficient screening of molecules capable of modulating the interactions between these peptides and an anti-apoptotic member of the Bcl-2 protein family. . Numerous screening assays for protein-protein interaction modulators are found in the literature, but often have limitations on their sensitivity and high-throughput feasibility. The commonly used methods require the implementation of complex tools (fusion proteins, recombinant proteins, etc.) that are not very compatible with high throughput screening. They generate most often a significant background noise and are unreliable from a quantitative point of view: they have a reduced reading window that does not allow optimal screening of the molecules tested.

As an alternative to the methods already available, a highly efficient screening test based on fluorescence polarization has been implemented in the present invention (Owicki et al., Journal of Biomolecular Screening, 5, 2000, 297-306). This technique makes it possible, for example, to measure the interaction between a labeled ligand with a fluorophore and a receptor. The principle consists in measuring an increase in the fluorescence polarization emitted by the ligand fixed to its receptor compared to that emitted by the free ligand. The fluorescence polarization of the free ligand is dependent on its molecular weight and will be all the more important as the molecular weight will be high. Thus when this test is performed with a high molecular weight ligand, having a strong intrinsic fluorescence polarization, it will be difficult to reliably assess the difference in fluorescence polarization between the free ligand and the ligand fixed. The use of a ligand with a minimum molecular weight will, on the contrary, exacerbate this difference, and consequently increase the accuracy of the method. It will thus be possible to better evaluate the real activity of a molecule, and to perform high throughput screens.

The present invention relates to a peptide interacting with the anti-apoptotic members of the Bcl-2 family of proteins. This peptide comprises the following amino acid sequences: a) MATVIHNPLKALGDQFYKEAIEHC (SEQ ID NO.1); b) VMTQEVGQLLQDMGDDVYQQYRSL (SEQ ID NO.2); c) RLKHSCLLALKRAADLLGQRSSST (SEQ ID NO.3); d) DMWDTRIAKLRVSADSFVRQQEA (SEQ ID NO.4); e) VATRRLSGFLRTLADRLEGTKELL (SEQ ID NO.5); f) XXXXXXXXLXXXXDXXXXXXXXXX (SEQ ID NO.6); and functional variants of these amino acid sequences.

By "amino acid sequence" is to be understood a peptide sequence isolated from the natural context. These include isolated sequences, synthesized chemically and / or purified and possibly modified by genetic engineering.

The term "functional variants" is intended to mean the amino acid sequences of the peptides described above comprising conservative substitutions or conservative point mutations and having essentially the same properties as the peptides, respectively, encoded by the SEQ sequences.

ID NO.1 to SEQ ID NO.5, being the ability to interact with an anti-apoptotic member of the Bcl-2 protein family. Conservative substitutions or mutations of amino acid sequences SEQ ID NO.1 to SEQ ID

NO.5 are, for example, the following: glycine by alanine (GA), valine by leucine (VL); aspartic acid by glutamic acid (DE), asparagine by glutamine (NQ), arginine by lysine (R - K), tyrosine by leucine (YL), leucine by methionine (LM), valine by isoleucine (VI) and glutamine by histidine (QH).

The invention also relates to the nucleic acid sequences encoding the peptides of SEQ ID NO.1 to SEQ ID NO.5. These nucleotide sequences corresponding to the peptide sequences SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 are respectively: a) atggcgactgtcattcacaaccccctgaaagcgctcggggaccagttctacaaggaa gccattgagcactgc (SEQ ID NO. 7); b) gttatgacccaagaggttggccagctcctgcaagacatgggtgatgatgtataccagcag taccggtcactt (SEQ ID NO.8); c) agacttaaacattcctgcctgctggctctgaagagagcagcggatctcctaggacagcgc tcaagctctact (SEQ ID NO.9); d) gatatgtgggacactcgtatagccaaactccgagtgtctgctgacagctttgtgagacag caggaggca (SEQ ID NO.10); g) gttgctacaagacgattaagtggcttcctgaggacacttgcagaccggctggagggcacc aaagaactgctt (SEQ ID NO.11).

These nucleic acid sequences according to the invention can be obtained according to the genetic code from the corresponding amino acid sequences and their variants.

The "variants" of these nucleic acid sequences are in particular: sequences capable of hybridizing under stringent conditions with the nucleic acid sequences SEQ ID No. 7 to SEQ ID No. 11 or sequences complementary to those and encoding polypeptides having respectively substantially the same properties as the peptides of SEQ ID NO.1 to SEQ ID NO.5, or, sequences of a mammalian species homologous to sequences SEQ ID No. 7 to SEQ ID No. 11 isolated in humans.

By "stringent conditions" is meant the conditions which allow the specific hybridization of two single-stranded DNA sequences at about 65 ° C., for example in a solution of 6 × SSC, 0.5% SDS, 5 ×

Denhardt's solution and 100 μg nonspecific carrier DNA or any other solution of equivalent ionic strength and after washing at 65 ° C, for example in a solution of at most 0.2 x SSC and 0.1% SDS or any another solution of equivalent ionic strength. The parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm). For sequences with more than 30 bases, Tm is defined as: Tm = 81, 5 + 0.41 (% G + C) + 16.6Log (cation concentration) - 0.63 (% formamide) - ( 600 / number of bases). For sequences of length less than 30 bases, Tm is defined by the relation: Tm = 4 (G + C) + 2 (A + T). The stringency conditions can therefore be adapted by those skilled in the art depending on the size of the sequence, the GC content and any other parameter, in particular according to the protocols described in Sambrook et al., 2001 (Molecular Cloning: A laboratory Manual, 3rd Ed., Spring Harbor Co, Spring Press, Spring Harbor Co, New York).

The term "sequences of a species of mammals homologous to the sequences SEQ ID No. 7 to SEQ ID NO.11", structural sequences similar to said nucleotide sequences and coding respectively for polypeptides having essentially the same properties in species of non-human mammals, including primates, rats or mice. The percentage identity between two homologous sequences in the functional regions is generally greater than 80%, preferably greater than 90%. The invention also relates to a recombinant vector comprising one of the nucleic acid sequences, SEQ ID No. 7 to SEQ ID No. 11 as claimed according to the invention. By vector, it is necessary to understand any type of vector allowing the introduction of the nucleic acid sequence into the host cell and possibly the expression of the polypeptide encoded by the nucleic acid sequence in this host cell. Such a vector is for example a plasmid, a cosmid, an artificial bacterial chromosome or a bacteriophage comprising the sequences necessary for the expression of the peptides of amino acid sequences SEQ ID NO.1 to SEQ ID NO.5.

Preferably, the recombinant vector according to the invention comprises the sequences necessary for the expression of the peptides of SEQ ID NO.1 to SEQ ID NO.5 sequences in the host cell. These sequences include sequences promoting transcription and translation in the host cell as well as terminator sequences. The recombinant vector may also include sequences encoding secretion signals allowing the release of translated proteins into the extracellular environment.

The invention also relates to the host cells transformed with a recombinant vector according to the invention. In a particular embodiment, these host cells are bacterial cells such as Escherichia coli, for example streptococci or eukaryotic cells such as yeast cells, filamentous fungi, insect cells and preferably mammalian cells. .

The transformation of appropriate host cells with a recombinant vector comprising the nucleic acid sequences according to the invention makes it possible respectively to express the claimed peptides SEQ ID NO.1 to SEQ ID NO.5. In a second step, it is possible to purify the peptides expressed in these host cells from different methods known to those skilled in the art and extensively described in the state of the art. Examples include ammonium sulfate precipitation purifications by size exclusion chromatography and preferably by affinity chromatography.

The peptides of amino acid sequence SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, can also be synthesized chemically, by contract, by Neosystem. The chemical synthesis of the peptide sequences SEQ ID No. 1 to SEQ ID No. 5 and their functional variants is carried out by solid-state synthesis according to the Boc / benzyl strategy using an "Applied Biosystems 430A" peptide synthesizer. . The synthesis is based on the development of the desired sequence on resin, followed by deprotection of the N- and C-terminal amino functions. In Boc / benzyl strategy, it is necessary to introduce the amino acid Boc-L-Lys (Fmoc) -OH during the synthesis of the peptide. After developing the entire sequence, the amino function is deprotected and the peptide cut off from the resin in the presence of a strong acid.

The invention also relates to a pharmaceutical composition comprising a peptide of amino acid sequences SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. active ingredient in combination with one or more pharmaceutically acceptable excipients.

In the context of the invention, the term "excipients" of a pharmaceutical composition any agent ensuring the transport of the active ingredient in the internal routes in the patient treated. By "active principle" is meant any substance responsible for the pharmacodynamic or therapeutic properties of the pharmaceutical composition. Among the non-toxic, pharmaceutically acceptable excipients, mention may be made, by way of indication and without limitation, of diluents, solvents, preservatives, wetting agents, emulsifiers, dispersing agents, binders, blowing agents, disintegrating agents, retardants, lubricants, absorbents, suspending agents, dyes or flavoring agents.

The present invention relates not only to the pharmaceutical composition as such and defined above, but also to the use of this composition in a method of inducing programmed cell death. This induction method comprises the administration to the patient, in particular of cancers, of an effective quantity of pharmaceutical composition comprising one of the peptides according to the invention.

The subject of the invention is also a method for identifying modulators of the interaction between a peptide according to the invention and an anti-apoptotic member of the Bcl-2 family of proteins comprising the following steps:

a) bringing said peptide and said anti-apoptotic member of the BcI-2 protein family into contact; b) adding the test compound; and c) measuring the activity of the test compound as a modulator of the protein interaction between the peptide and the anti-apoptotic member of the Bcl-2 family of proteins and then comparing said measurement in the absence of the compound to test. Advantageously, the method for identifying modulators of the interaction comprises the following steps:

a) the fluorescent labeling of a peptide according to claim 1; b) incubating said peptide in the presence of the test compound; c) adding an anti-apoptotic member of the Bcl-2 protein family; d) measuring the fluorescence polarization; e) comparing the measurement with or without the test compound.

By "modulator" is meant any compound capable of increasing, preventing or at least limiting a specific activity such as a protein-protein interaction, an enzymatic activity, an attachment to cellular receptors. According to the present invention, the modulators are inhibitors or activators of the protein interaction between the peptide partners of amino acid sequences SEQ ID NO.1 to SEQ ID NO.5 and the anti-apoptotic members of the Bcl-2 proteins.

The invention also relates to a method for identifying an inhibitor of the interaction between one of the peptides according to the invention and an anti-apoptotic member of the Bcl-2 family of proteins capable of decreasing the fluorescence polarization with respect to a control consisting of this interaction in the absence of a modulator.

The invention also relates to a method for identifying an activator of the interaction between one of the peptides according to the invention and an anti-apoptotic member of the Bcl-2 family of proteins capable of increasing the fluorescence polarization by compared to a control consisting of this interaction in the absence of a modulator. The fluorescent ligand, ie one of the peptide motifs SEQ ID NO.1 to SEQ ID No. 5 fluorescent, after binding with the anti-apoptotic partner of the Bcl-2 protein family, has a lower rotation constant than the corresponding free ligand. and thus the fluorescence emitted by the bound ligand becomes polarized. As a result, there is an increase in the polarization of the fluorescence emitted by the ligand bound versus free ligand.

In a preferred embodiment, the fluorescence probe used in the screening and identification method according to the invention is bodipy, oregon green and more preferably fluorescein. More particularly, the anti-apoptotic member of the protein family

Bcl-2, acting as a partner of the interaction in the screening and identification process according to the invention, may be the Bcl-2 protein, BCI-XL or BcI-W.

Advantageously, the anti-apoptotic member of the Bcl-2 family of proteins is a fusion protein. By "fusion protein" is meant the fusion between a domain of the Bcl-2 protein, Bcl-X _L or BcI-W and a protein domain such as GST (Glutathione-S-transferase).

The invention also relates to a pharmaceutical composition comprising at least one modulator, activator or inhibitor, identified from the modulator identification method according to the invention, as the active principle of said composition in combination with one or more pharmaceutically acceptable excipients. acceptable.

The invention also relates to a method of inducing programmed apoptotic (caspase-dependent) and / or autophagic (independent caspase) programmed cell death from the administration of an effective amount of composition defined above to a patient suffering from cancers. The pharmaceutical compositions as described above can be used in the treatment of cancers, by action on programmed apoptotic and / or autophagic cell death.

The compositions according to the invention are in a form suitable for oral, parenteral, nasal, percutaneous, rectal, perlingual, ocular or respiratory administration and in particular simple or coated tablets, sublingual tablets, sachets, packets, capsules, glossettes, tablets, suppositories, creams, ointments, dermal gels, and ampoules drinkable or injectable.

The present invention is illustrated without being limited by the following figures and examples:

- Figure 1: amino acid sequence SEQ ID NO.1 of the Cerd4 peptide interacting with the anti-apoptotic members of the Bcl-2 family of proteins. - Figure 2: amino acid sequence SEQ ID NO.2 of the Kiaa peptide

1578 interacting with the anti-apoptotic members of the Bcl-2 protein family.

3: amino acid sequence SEQ ID No. 3 of the Genematch peptide interacting with the anti-apoptotic members of the Bcl-2 protein family.

FIG. 4: amino acid sequence SEQ ID NO. 4 of the Loc 51569 peptide interacting with the anti-apoptotic members of the Bcl-2 protein family.

5: Amino acid sequence SEQ ID No. 5 of the peptide Mina53 interacting with the anti-apoptotic members of the Bcl-2 protein family.

FIG. 6: SEQ ID NO.6 consensus pattern of the peptide sequences SEQ ID NO.1 to SEQ ID No. 5 according to the invention. FIG. 7: determination of Ki in fluorescence polarization of the peptides competing with amino acid sequences SEQ ID No. 1 to SEQ ID No. 5 according to the invention, on the interaction between the pro-apoptotic peptide Bak and the member anti-apoptotic BCI-XL.

- Figure 8: Summary table of amino acid sequences SEQ ID NO.1 to SEQ ID NO.5 and their respective nucleic acid sequences SEQ ID NO.7 to SEQ ID NO.11.

EXAMPLE 1 Identification by the Double Hybrid System of the Peptides Described in Figures 1 to 5

Three human cDNA libraries were screened (placenta, brain, CEMC7 cell line) by the yeast double hybrid technique (Fields et al.) Using the conjugation protocol described by Legrain et al. in Nature Genetics, 1997, vol.16, 277-282 (US 6,187,535).

1) Preparation of "bait" and "prey"

a) The "baits" used are:

BCI-XL (accession number Z23115) deleted from its C-terminus (1-209) and fused to the LexA DNA binding domain;

Bcl-2 (accession number XM_008738) deleted from its C-terminus (1-211) and fused to the LexA DNA binding domain. These baits are expressed in the yeasts Saccharomyces cerevisiae strain L40Δgal4 (MATa ade2, trp1-901, leu2-3, 112, Iys2-8O1, his3Δ200, LYS2 (lexAop) ₄ -HIS3, ura3-52 :: URA3 (lexAop) ₈ - LacZ, GAL4 :: Kan ^R ) and placed in preculture at 30 ⁰ C in a synthetic medium free of tryptophan (DO-Trp) until an optical density OD ₆ oonm of between 0.1 and 0 is obtained, 5. Fifty ml of a dilution of this preculture (OD ₆ oonm = 0.006) are incubated at 30 ^° C. overnight. b) A Saccharomyces cerevisiae yeast strain YHGX13 strain (MATα Gal4Δ GalδOΔ ade2-101 :: Kan ^R , his3, leu2-3-112, trp1-901, ura3-52 URA3 :: UASGAL1-LacZ, Met) containing the plasmids Expressing the cDNA libraries fused to the Gal4 transcriptional activation domain is obtained by transformation after selection on a culture medium lacking leucine (OD-Leu). These yeasts are aliquoted and stored at -80 ^° C.

2) Conjugation

The conjugation is carried out with a ratio "bait" / "prey" equal to 2. A quantity of yeast cells "baits" obtained in stage 1) a) corresponding to 50 units ODβoonm is mixed with yeasts "prey" obtained in stage 1 ) b). After centrifugation, the pellet is resuspended in YPGIu medium, spread on YPGIu culture dishes and incubated for 4 hours 30 to 30 ^° C. The selection of conjugated yeasts containing a "bait" and a "prey" capable of interacting sets is performed on a medium DO-

Leu-Trp-His: the absence of leucine and tryptophan makes it possible to maintain a selection pressure that allows only the yeasts containing both types of plasmids ("bait" / "prey") to grow; the absence of histidine in the medium makes it possible to select the conjugated yeasts containing a "bait" plasmid and a "prey" plasmid capable of interacting together: the complementation as described makes it possible to activate the HIS3 gene, as a gene reporter coding for an enzyme involved in the biosynthesis of histidine.

3) Identification of positive clones

The "prey" fragments of a colony of yeasts selected according to the conjugation method described in paragraph 2) are amplified by PCR at from a crude lysate of this colony, using primers specific for the vector "prey":

ABS 1 5'-GCTTTGGAATCACTACAGG-3 '(SEQ ID NO.12);

ABS2 5'-CACGATGCACGTTGAAGTG-3 '(SEQ ID NO.13). The PCR products are then sequenced and the sequences obtained are identified by comparison with databases.

4) Identification of the peptides described in FIGS. 1 to 5

For each "bait" fragment tested, the double hybrid system makes it possible to identify several "prey" fragments. This identification is done by comparing sequences of "prey" selected from a computer program such as Blastwun available on the website of the University of Washington: http://bioweb.pasteur.fr/seqanal/interfaces/blastwu .html.

EXAMPLE 2 Validation of the Interaction Between the Peptides Obtained in Example 1 and Bcl-2, BcI-Xi and / or BcI-W

1) Determination of Ki in Fluorescence Polarization

The determination of Ki in fluorescence polarization consists in measuring the effect of the competing peptides of respective amino acid sequences SEQ ID NO. 1 to SEQ ID NO. 5 on the interaction between the pro-apoptotic peptide Bak and the anti members. -apoptotic family of Bcl-2 proteins such as BCI-XL.

The following reagents are mixed in the order specified:

competitor peptide at a final concentration of 1 nM to 100 μM; fluorescent ligand peptide (Bak BH3 carboxyfluoresceine) at a final concentration of 15 nM;

the anti-apoptotic member of the Bcl-2 protein family at a final concentration of 100 nM for BCI-XL.

These reagents are in solution in the interaction buffer (20 mM Na ₂ HPO 4 pH 7.4, 1 mM EDTA, 50 mM NaCl and 0.05% pluronic acid F-68). The mixture is then incubated for 30 minutes at room temperature and the fluorescence polarization determined on the Fusion apparatus (Packard) (excitation at 485 nm and reading at 530 nm). The values are given in mP (fluorescence polarization unit).

These fluorescence polarization analyzes have demonstrated that the peptides of amino acid sequences SEQ ID NO.1 to SEQ ID NO.5 are peptides that compete in the peptide interaction between Bak and BCI-XL. The Ki obtained during these fluorescence polarization tests are as follows:

BCI-XL / Bak / SEQ ID No. 1 Ki = 9 μM

BCI-XL / Bak / SEQ ID No. 2 Ki = 32 μM

BCI-XL / Bak / SEQ ID No. 3 Ki = 1 μM

BCI-XL / Bak / SEQ ID No. 4 Ki = 8 μM BCI-XL / Bak / SEQ ID NO.5 Ki = 20 μM

2) Determination of Ki in Fluorescence Polarization with the Mutated Peptide Patterns (L-A)

The determination of Ki in fluorescence polarization consists of measuring the competitive effect of the peptides SEQ ID NO.1 to SEQ ID NO.5 mutated, from leucine to alanine (LA), on the interaction between the pro-apoptotic peptide BAK and the anti-apoptotic members of the Bcl-2 family of proteins such as Bcl-X _L. The peptide sequences SEQ ID NO.1 to SEQ ID NO.5 mutated (LA) are as follows:

MATVIHNPAKALGDQFYKEAIEHC (SEQ ID NO.14);

VMTQEVGQLAQDMGDDVYQQYRSL (SEQ ID NO.15); RLKHSCLLAAKRAADLLGQRSSST (SEQ ID NO.16);

DMWDTRIAKARVSADSFVRQQEA (SEQ ID NO.17);

VATRRLSGFARTLADRLEGTKELL (SEQ ID NO.18).

The protocol for determining Ki in fluorescence polarization is identical to the protocol described above. By comparison of the fluorescence polarization analysis results, a loss of competitive effect of the mutated peptides (LA) SEQ ID No. 14 to SEQ ID NO. 18 compared with the peptides SEQ ID NO. 1 to SEQ ID is observed. NO.5 according to the invention in the peptide interaction between the pro-apoptotic peptide Bak and the anti-apoptotic members of the Bcl-2 family of proteins such as Bcl-X _L.

EXAMPLE 3 Screening Assay for Molecules Capable of Inhibiting the Interaction Between Bcl-2 and / or BcI-Xi and the Peptides Obtained in Example 1

The products to be tested are distributed in 384-well plates (Corning Fiat Bottom) at a final concentration of 10 μg / ml. A well is filled with an equivalent amount of buffer / solvent without test compound and will constitute the control. The peptides obtained in Example 1 labeled with fluorescein are added to the wells so as to obtain a final concentration ranging from 1 to 100 nM. The GST-BCI-XL or GST-Bcl-2 or GST-BcI-W fusion protein is then added so as to obtain a final concentration of 0.1 to 1 μM in a buffer containing

20 mM Na ₂ HPO ₄ pH 7.4, 1 mM EDTA, 50 mM NaCl, acid pluronic F-68 0.05%. The fluorescence polarization is then measured by the device In Vision (Packard Perkin-Elmer). A significant decrease in the fluorescence polarization recorded in the test carried out with the test compound compared to that obtained without the test compound (control well) makes it possible to conclude that the molecule has an inhibitory activity. Conversely, a significant increase in the fluorescence polarization in the test with the test product compared to the control makes it possible to conclude that the activating activity of the molecule.

Claims

A peptide interacting with the anti-apoptotic members of the Bcl-2 protein family, characterized in that it comprises the following amino acid sequences: a) MATVIHNPLKALGDQFYKEAIEHC (SEQ ID NO.1); b) VMTQEVGQLLQDMGDDVYQQYRSL (SEQ ID NO.2); c) RLKHSCLLALKRAADLLGQRSSST (SEQ ID NO.3); d) DMWDTRIAKLRVSADSFVRQQEA (SEQ ID NO.4); e) VATRRLSGFLRTLADRLEGTKELL (SEQ ID NO.5); f) XXXXXXXXLXXXXDXXXXXXXXXX (SEQ ID NO.6); and functional variants of said amino acid sequences.

Nucleic acid sequence encoding a peptide according to claim 1.

3. Sequence of nucleic acids according to claim 2, characterized in that it comprises the following nucleotide sequences:

a) atggcgactgtcattcacaaccccctgaaagcgctcggggaccagttctacaaggaa gccattgagcactgc (SEQ ID NO.7); b) gttatgacccaagaggttggccagctcctgcaagacatgggtgatgatgtataccagcag taccggtcactt (SEQ ID NO.8); c) agacttaaacattcctgcctgctggctctgaagagagcagcggatctcctaggacagcgc tcaagctctact (SEQ ID NO.9); d) gatatgtgggacactcgtatagccaaactccgagtgtctgctgacagctttgtgagacag caggaggca (SEQ ID NO.10); e) gttgctacaagacgattaagtggcttcctgaggacacttgcagaccggctggagggcacc aaagaactgctt (SEQ ID NO.11).

4. Recombinant vector characterized in that it comprises one of the nucleic acid sequences according to one of claims 2 or 3.

5. Recombinant vector according to claim 4, characterized in that the vector is a plasmid, a cosmid, an artificial bacterial chromosome or a bacteriophage comprising the sequences necessary for the expression of a peptide according to claim 1.

Recombinant vector according to claim 5, characterized in that the sequences necessary for the expression of a peptide according to claim 1 comprise a promoter sequence for transcription and translation.

7. Host cell characterized in that it is transformed by a recombinant vector according to any one of claims 4 to 6.

8. Host cell according to claim 7, characterized in that said host cell is a bacterial or eukaryotic cell.

A pharmaceutical composition comprising a peptide according to claim 1 as an active ingredient in combination with one or more pharmaceutically acceptable excipients.

A method of inducing programmed cell death comprising administering to the cancer patient an effective amount of a pharmaceutical composition according to claim 9.

11. A method of identifying modulators of the interaction between a peptide according to claim 1 and an anti-apoptotic member of the Bcl-2 family of proteins, characterized in that it comprises the following steps: a) bringing said peptide into contact with said anti-apoptotic member of the Bcl-2 family of proteins; b) adding the test compound; and c) measuring the activity of the test compound as a modulator of the protein interaction between the peptide and the anti-apoptotic member of the Bcl-2 family of proteins and then comparing said measurement in the absence of the compound to test.

12. A method of identifying modulators of the interaction according to claim 11, characterized in that it comprises the following steps: a) the fluorescent labeling of a peptide according to claim 1; b) incubating said peptide in the presence of the test compound; c) adding an anti-apoptotic member of the Bcl-2 protein family; d) measuring the fluorescence polarization; e) comparing the measurement with or without the test compound.

13. The method of claim 12, characterized in that the modulator of the interaction between the peptide and an anti-apoptotic member of the Bcl-2 family of proteins is a fluorescence polarization increasing activator.

14. Method according to claim 12, characterized in that the modulator of the interaction between the peptide and an anti-apoptotic member of the Bcl-2 protein family is an inhibitor decreasing the fluorescence polarization.

15. Method according to one of claims 11 to 14, characterized in that the fluorescence probe is fluorescein.

16. Method according to one of claims 11 to 15, characterized in that the anti-apoptotic member of the Bcl-2 protein family is the Bcl-2 protein, Bcl-X _L or BcI-W.

17. A pharmaceutical composition comprising a modulator obtained from the method of identifying modulators according to one of claims 11 to 16, as an active ingredient in combination with one or more pharmaceutically acceptable excipients.

A method of inducing programmed cell death comprising administering to the cancer patient an effective amount of a pharmaceutical composition according to claim 17.

19. Pharmaceutical composition according to one of claims 9 or 17 for use in the treatment of cancers.