HK1113378A - Novel peptides which interact with anti-apoptotic members of the family of bcl-2 proteins and use thereof - Google Patents

Description

Novel peptides that interact with anti-apoptotic members of the BCL-2family of proteins and uses thereof

The present invention is in the field of searching for and identifying novel peptides that interact with anti-apoptotic members of the Bcl-2 protein family.

The present invention relates to methods of screening and identifying modulators of protein interactions between those novel peptides and anti-apoptotic members of the Bcl-2 protein family. Modulators isolated by this screening method may be used to modulate apoptotic and/or autophagic programmed cell death. These modulators are administered to patients during the treatment of cancer.

The present invention relates to five peptide motifs each capable of interacting with an anti-apoptotic member of the Bcl-2 protein family and to their use in causing programmed cell death in cancer patients.

In one aspect, programmed cell death includes apoptosis, and in another aspect autophagic death. Apoptosis is a more well-known phenomenon. This type of cell death includes morphological changes such as nuclear agglutination and DNA fragmentation, but also biochemical phenomena such as activation of caspases (which then degrade key structural components of the cell causing cell disassembly and death). The regulation of the apoptotic process is complex and involves the activation or inhibition of multiple intracellular signaling pathways. Autophagic death is the second less well-known mechanism of programmed cell death. At the cellular level, autophagy can be generalized into three phases: formation of autophagosomes (autophagosomes) is initiated, the autophagosomes mature into degradative vesicles which then fuse with lysosomes. Autophagic death thus involves a lysosomal degradation process characterized by accumulation of autophagic vesicles and is independent of caspase-type regulatory pathways.

Keeping the cells alive or causing their programmed death necessitates the regulation of the major signaling pathway involved, especially in the BCl-2 protein family.

The BCL-2 protein family is divided into three major classes. Anti-apoptotic proteins (e.g. Bcl-2, Bcl-X)_LAnd Bcl-W) have a high degree of homology in their four BH domains. Pro-apoptotic proteins fall into two categories: one class is multidomain proteins (e.g., BAX and BAK) and The other class is pro-apoptotic proteins (e.g., BID, NOXA, PUMA, BIK, BIM and BAD) characterized by The presence of a single homeodomain-BH 3 motif (Cory and Adams, The Bcl-2 family: ligands of The cellular life-or-death)switch Nature reviews, Vol.12, 9 months 2002).

The BH3 motif is an amphipathic alpha-helical region with relatively low sequence identity in the Bcl-2 protein family. In addition, the presence of the BH3 motif is required in proteins to allow interaction with anti-apoptotic members of the Bcl-2family of proteins. Indeed, the activity of anti-apoptotic members of the Bcl-2 protein family is regulated by pro-apoptotic gene products of the family, and both proteins assemble into heterodimers. When in that state, the anti-apoptotic members of the Bcl-2 protein family are inactive and therefore no longer have their anti-apoptotic activity. Furthermore, the specific interaction of the BH3 motif with anti-apoptotic members of the Bcl-2 protein family may be modified by modulators to cause programmed cell death in a specific manner.

The present invention therefore contemplates the screening and identification of novel peptides that interact with anti-apoptotic members of the Bcl-2 protein family, and the use of these novel peptides to screen and identify compounds that modify those interactions to obtain true candidate agents effective for down-regulation of the pathology involved in apoptosis, particularly cancer.

The initial two-hybrid system consisted of the detection of two recombinant proteins in yeast. The first protein (referred to as "bait") is a fusion protein containing a DNA binding domain (or BD) that binds upstream to protein a. The second protein is also a fusion protein, commonly referred to as a "prey," which contains an activation domain (or AD) that binds to protein B. Commonly used binding and activation domains are domains of Gal4 or e.coli (e.coli) Lex a. Proteins A and B are anti-apoptotic members of the Bcl-2 protein family and motifs obtained from cDNA libraries, respectively. The binding of proteins a and B by protein interaction allows the formation of a functional domain (BD-AD) by complementation that is capable of binding to the binding site (or BS) present upstream of the reporter gene and ensures transcription of the reporter gene.

However, this conventional two-hybrid system has its limitations. For example, it is well known that such screening methods can cause false positives and/or false negatives and require results obtained by biochemical validation. False positives obtained with the two-hybrid system are particularly frequent and demonstrate functional rather than structural interactions.

More efficient techniques allowing the minimization of false positives and/or false negatives and the use of recombinant haploid yeast containing "bait" and "prey" polypeptides are described in international patent application WO 99/42612 or patent US6,187,535. This system allows for the detection of a greater number of "prey" with a single "bait" in a more accurate, reproducible and sensitive manner than other conventional methods used in the art.

Using the two-hybrid system, the inventors have determined that there is a structural interaction between an anti-apoptotic member of the Bcl-2 protein family and the following peptides having the amino acid sequences of SEQ ID No.1 to SEQ ID No.5 of the present invention. This protein interaction between those partners is similar to that found in the regulation of apoptotic phenomena between anti-apoptotic and pro-apoptotic partners of the Bcl-2 protein family.

The initial peptides having the 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 identified in the present invention by a two hybrid system. Each of these peptides is capable of interacting with anti-apoptotic members of the Bcl-2 protein family in a highly specific manner. The specificity of this interaction is actually related to the sequence, three-dimensional structure and/or helicity of the original motif of the selected peptide.

In addition, these peptides correspond to Bcl-2, Bcl-X_LAnd/or precise domains of Bcl-W interactions and having typical structural criteria that allow the formation of homo-or heterodimers.

Peptide motifs_NH2-XXXXXXXXLXXXDXXXXXXXXXX-_COOH(SEQ ID NO.6) represents a consensus sequence of the peptide sequences SEQ ID NO.1 to SEQ ID NO.5 according to the present invention, wherein X represents any amino acid. This consensus sequence is similar to the Bcl-2 protein family in promoting cell apoptosisThe sequence of the BH3 motif present in apoptotic members.

The size of the peptides according to the invention does make them ideal candidates for the development of assays allowing an efficient screening of compounds capable of modulating the interaction of those peptides with anti-apoptotic members of the Bcl-2 protein family. A large number of assays for screening protein-protein interactions can be found in the literature but they often have limitations in terms of sensitivity and high throughput feasibility. Commonly used methods necessitate the use of complex tools (fusion proteins, recombinant proteins, etc.) that are quite incompatible with high throughput screening. They generate high levels of background noise very frequently and are low in confidence from a quantitative point of view: they provide a reduced reading window that does not allow optimal screening of the tested compounds.

As an alternative to the methods already available, a high efficiency Screening assay based on fluorescence polarization has been used in the present invention (Owicki et al, Journal of Biomolecular Screening, 5, 2000, 297-306). This technique allows, for example, the measurement of the interaction between a fluorophore labelled ligand and a receptor. The principle involves measuring the increase in polarization of fluorescence emitted by the ligand upon binding to its receptor as compared to the polarization emitted by the free ligand. The fluorescence polarization of the free ligand depends on its molecular weight, and the higher the molecular weight the greater the fluorescence polarization. Thus, it is difficult to reliably assess the difference in fluorescence polarization between free and bound ligands when tested with high molecular weight ligands having high levels of intrinsic fluorescence polarization. On the other hand, the use of ligands of minimum molecular weight enhances the differentiation and thus the accuracy of the process. Thus, the true activity of the compound can be better evaluated and high throughput screening can be performed.

The present invention relates to peptides that interact with anti-apoptotic members of the Bcl-2family of proteins. This peptide contains the following amino acid sequences and functional variants of those 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)；

"amino acid sequence" is understood to mean a peptide sequence which has been isolated from its natural environment, in particular isolated, chemically synthesized and/or purified, and, if possible, modified by genetic engineering.

"functional variants" are understood to be amino acid sequences of the above-mentioned peptides which contain conservative substitutions or conservative point mutations and have substantially the same properties as the peptides encoded by the sequences SEQ ID No.1 to SEQ ID No.5, respectively, or that is to say the ability to interact with anti-apoptotic members of the Bcl-2 protein family. Conservative substitutions or mutations of the amino acid sequences SEQ ID NO.1 to SEQ ID NO.5 are, for example, the following substitutions or mutations: alanine for glycine (G-A), leucine for valine (V-L), glutamic acid for aspartic acid (D-E), glutamine for asparagine (N-Q), lysine for arginine (R-K), leucine for tyrosine (Y-L), methionine for leucine (L-M), isoleucine for valine (V-I) and histidine for glutamine (Q-H).

The invention also relates to nucleic acid sequences encoding peptides of SEQ ID NO.1 to SEQ ID NO. 5. The 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, respectively, are as follows:

a)atggcgactgtcattcacaaccccctgaaagcgctcggggaccagttctacaaggaagccattgagcactgc(SEQ ID NO.7)；

b)gttatgacccaagaggttggccagctcctgcaagacatgggtgatgatgtataccagcagtaccggtcactt(SEQ ID NO.8)；

c)agacttaaacattcctgcctgctggctctgaagagagcagcggatctcctaggacagcgctcaagctctact(SEQ ID NO.9)；

d)gatatgtgggacactcgtatagccaaactccgagtgtctgctgacagctttgtgagacagcaggaggca(SEQ ID NO.10)；

e)gttgctacaagacgattaagtggcttcctgaggacacttgcagaccggctggagggcaccaaagaactgctt(SEQ ID NO.11)。

these nucleic acid sequences of the invention can be obtained by means of the genetic code starting from the corresponding amino acid sequences and variants thereof.

Variants of those nucleic acid sequences are, in particular:

-sequences which hybridize under stringent conditions with the nucleic acid sequences SEQ ID No.7 to SEQ ID No.11 or with sequences complementary thereto and which encode polypeptides having substantially the same properties as the peptides having the sequences SEQ ID No.1 to SEQ ID No.5, respectively, or

-a sequence of a mammalian species homologous to the sequence SEQ ID No.7 to SEQ ID No.11 isolated from human.

"stringent conditions" are understood to be conditions which allow specific hybridization of two single-stranded DNA sequences after washing at about 65 ℃ in, for example, 6 XSSC solution, 0.5% SDS, 5 XDenhard's solution and 100. mu.g of non-specific carrier DNA or any other solution of equal ionic strength and at 65 ℃ in at most 0.2 XSSC and 0.1% SDS or any other solution of equal ionic strength. The parameters defining the stringency conditions depend on the temperature (Tm) at which 50% of the paired strands separate. For sequences containing more than 30 bases, Tm is defined by the formula: tm ═ 81.5+0.41 (% G + C) +16.6 Log (cation concentration) -0.63 (% formamide) - (600/base number). For sequences less than 30 bases in length, Tm is defined by the formula: tm is 4(G + C) +2(a + T). The stringency conditions can therefore be adjusted by the person skilled in the art on the basis of the sequence size, GC content and any other parameters, in particular according to the methods described in Sambrook et al, 2001 (Molecular Cloning: A laboratory Manual, third edition, Cold Spring Harbor, laboratory Press, Cold Spring Harbor, N.Y.).

"sequences of mammalian species homologous to the sequences SEQ ID No.7 to SEQ ID No. 11" is understood as sequences which are structurally similar to the nucleotide sequences described and which encode individual polypeptides having essentially the same properties as in non-human mammalian species, in particular primates, rats and mice. The percentage identity of the two homologous sequences within the functional region is generally higher than 80%, preferably higher than 90%.

The invention also relates to a recombinant vector comprising the nucleic acid sequences SEQ ID NO.7 to SEQ ID NO.11 as claimed in the present invention. A vector is understood to be any type of vector which allows for the introduction of a nucleic acid sequence into a host cell and optionally for the expression of a polypeptide encoded by the nucleic acid sequence in the host cell.

Such vectors are, for example, plasmids, cosmids, bacterial artificial chromosomes or phages which contain sequences necessary for the expression of peptides having the amino acid sequences SEQ ID No.1 to SEQ ID No. 5.

The recombinant vector according to the invention preferably contains the sequences necessary for the expression of the peptides having the sequences SEQ ID No.1 to SEQ ID No.5 in a host cell. These sequences are, in particular, promoter sequences and also terminator sequences which are transcribed and translated in the host cell. The recombinant vector may also contain sequences encoding secretion signals which allow the release of the translated protein into the extracellular environment.

The invention also relates to host cells transformed with the recombinant vectors of the invention. In particular embodiments, those host cells are bacterial cells (e.g., E.coli and Streptococcus) or eukaryotic cells (e.g., yeast cells, filamentous fungal cells, insect cells and preferably mammalian cells).

Transformation of a suitable host cell with a recombinant vector containing a nucleic acid sequence of the invention allows expression of the respective claimed peptides SEQ ID No.1 to SEQ ID No. 5. The proteins expressed in those host cells can then be purified by various methods known to those skilled in the art and extensively described in the prior art. It is to be mentioned that purification is for example by precipitation with ammonium sulphate, by size exclusion chromatography and preferably by affinity chromatography.

Peptides having the amino acid sequence SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5 can also be custom synthesized by N esostem chemistry. Chemical synthesis of the peptide sequences SEQ ID NO.1 to SEQ ID NO.5 and functional variants thereof was performed by synthesis on a solid support with the Boc/benzyl strategy by means of an "Applied Biosystems 430A" peptide synthesizer. The synthesis is based on the assembly of the desired sequence on the resin and the subsequent deprotection of the N-and C-terminal functional groups. Taking the Boc/benzyl strategy as an example, it is necessary to introduce the amino acids Boc-L-Lys (Fmoc) -OH during peptide synthesis. After the complete sequence has been assembled, the amino function is deprotected and the peptide is cleaved from the resin in the presence of a strong acid.

The invention also relates to a pharmaceutical composition comprising as active principle a peptide having the amino acid sequence SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5 in combination with one or more pharmaceutically acceptable excipients.

In the present invention, an "excipient" of a pharmaceutical composition is understood to be any agent that ensures the transport of the active principle into the internal blood vessels of the patient to be treated. By "active ingredient" is understood any pair of substances which confers a pharmacodynamic or therapeutic property on the pharmaceutical composition.

Non-toxic, pharmaceutically acceptable excipients mentioned by way of example and meant to be limiting in any way are diluents, solvents, preservatives, wetting agents, emulsifiers, dispersing agents, binders, swelling agents, disintegrating agents, retarding agents, lubricants, light absorbing agents, suspending agents, coloring agents or flavoring agents.

The present invention not only relates to the pharmaceutical composition thus contemplated and as defined above, but also to the use of this composition in a method of causing programmed cell death, said method comprising administering to a patient, in particular a cancer patient, an effective amount of a pharmaceutical composition comprising one of the peptides of the invention.

The invention also relates to a method for identifying a modulator of the interaction between a peptide according to the invention and an anti-apoptotic member of the Bcl-2 protein family, comprising the steps of:

a) contacting the peptide with an anti-apoptotic member of the Bcl-2 protein family;

b) adding a test compound; and

c) measuring the activity of the test compound as a modulator of the protein interaction between the peptide and an anti-apoptotic member of the Bcl-2 protein family, and then comparing said measurement in the absence of the test compound.

Advantageously, the method of identifying a modulator of an interaction comprises the steps of:

a) fluorescently labeling the peptide according to claim 1;

b) incubating the peptide in the presence of a test compound;

c) adding an anti-apoptotic member of the Bcl-2 protein family;

d) measuring the fluorescence polarization;

e) the measurements with and without test compound were compared.

By "modulator" is understood any compound capable of enhancing, preventing or at least limiting a specific activity, such as a protein-protein interaction, an enzymatic activity or binding to a cellular receptor. According to the invention, the modulator is an inhibitor or indeed an activator of the protein interaction between a partner (being a peptide having the amino acid sequence SEQ ID No.1 to SEQ ID No.5) and an anti-apoptotic member of the Bcl-2 protein family.

The invention also relates to a method for identifying an inhibitor of the interaction between a peptide which is one of the peptides of the invention and an anti-apoptotic member of the Bcl-2 protein family, which reduces the fluorescence polarization compared to a control consisting of such an interaction in the absence of a modulator.

The invention also relates to a method for identifying an activator of the interaction between a peptide which is one of the peptides of the invention and an anti-apoptotic member of the Bcl-2 protein family, which enhances the fluorescence polarization compared to a control consisting of such an interaction in the absence of a modulator.

The fluorescent ligands (i.e. the fluorescent peptide motifs SEQ ID No.1 to SEQ ID No.5) have a rotation constant after binding to the anti-apoptotic partner of the Bcl-2 protein family which is lower than the corresponding free ligand, as a result of which the fluorescence emitted by the bound ligand becomes polarized. Thus, an increase in the fluorescence polarization emitted by the bound ligand compared to the free ligand is observed.

In a preferred embodiment, the fluorescent probe used in the screening and identification method according to the invention is Bodipy, Oregon Green, more preferably fluorescein.

More particularly, the anti-apoptotic member of the Bcl-2 protein family used as interaction partner in the screening and identification process of the invention may be the proteins Bcl-2, Bcl-X_LOr Bcl-W.

The anti-apoptotic members of the Bcl-2 protein family are advantageously fusion proteins. "fusion protein" is understood to mean the proteins Bcl-2, Bcl-X_LOr a fusion of a domain of Bcl-W to a domain of a protein (e.g., GST, glutathione S transferase).

The invention also relates to pharmaceutical compositions comprising as an active ingredient of the composition at least one modulator, activator or inhibitor identified according to the method of identifying modulators of the invention, in combination with one or more pharmaceutically acceptable excipients.

The present invention also relates to a method of inducing apoptotic (caspase-dependent) programmed cell death and/or autophagic (caspase-independent) programmed cell death by administering to a cancer patient an effective amount of the above composition.

The above pharmaceutical composition is suitable for the treatment of cancer by acting on apoptotic-type programmed cell death and/or autophagic-type programmed cell death.

The compositions according to the invention are in a form suitable for oral, parenteral, nasal, transdermal, rectal, lingual, ocular or respiratory administration, especially tablets, dragees, sublingual tablets, sachets, paquests, capsules, glossettes, lozenges, suppositories, creams, ointments, dermal gels and drinkable or injectable ampoules.

The invention is described without limitation by the following figures and examples:

FIG. 1 amino acid sequence SEQ ID NO.1 of Cerd4 peptide interacting with anti-apoptotic members of the Bcl-2 protein family.

FIG. 2 amino acid sequence of the Kiaa 1578 peptide interacting with anti-apoptotic members of the Bcl-2 protein family SEQ ID NO. 2.

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

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

FIG. 5 amino acid sequence of the Mina 53 peptide interacting with anti-apoptotic members of the Bcl-2 protein family SEQ ID NO. 5.

FIG. 6. consensus motif SEQ ID NO.6 of the peptide sequences SEQ ID NO.1 to SEQ ID NO.5 according to the present invention.

FIG. 7 determination of Ki of competing peptides with pro-apoptotic Bak peptide and anti-apoptotic member Bcl-X using fluorescence polarization_LThe amino acid sequences SEQ ID No.1 to SEQ ID No.5 according to the invention which are related to each other.

FIG. 8. summary of the 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 of the peptides depicted in FIGS. 1-5 by a two-hybrid System

Three human cDNA pools (placenta, brain, cell line CEMC7) were screened in yeast by the two-hybrid technique using a combination protocol as described by Legain et al (Nature Genetics, 1997, Vol. 16, 277-282) (US6,187,535).

1) Preparation of "bait" and "prey"

a) The "bait" used was:

Bcl-X fused to the LexA DNA binding domain_L(accession number Z23115) a C-terminal truncation (1-209);

a C-terminal truncation of Bcl-2 (accession XM _008738) fused to a LexA DNA binding domain (1-211).

In Saccharomyces cerevisiae strain L40 Δ gal4(MATa ade2, trpl-901, leu2-3, 112, LYS2-801, his3 Δ 200, LYS2(lexAop)₄-HIS3，ura3-52::URA3(lexAop)₈-LacZ，GAL4::Kan^R) Expressing these baits and pre-culturing to optical density OD at 30 ℃ in synthetic medium lacking tryptophan (DO-Trp)_600nmIs 0.1-0.5 (inclusive). 50 ml of a dilution (OD) of the preculture was added_600nm0.006) was incubated at 30 ℃ overnight.

b) The Saccharomyces cerevisiae strain YHGX13 (MTA. alpha. Gal 4. delta. Gal 80. delta. ade2-101: Kan was obtained by transformation^RHis3, Leu2-3-112, trp1-901, URA3-52URA3 UASGAL1-LacZ, Met) and then selection on medium lacking leucine (DO-Leu), wherein said strain contains a plasmid of the cDNA library expressed in fusion with the Gal4 transcriptional activation domain.

2) Bonding of

Binding was performed with a "bait"/"prey" ratio of 2.

A quantity of yeast "bait" cells (corresponding to OD) obtained in step 1) a)_600nm50 units of) Mixing with the yeast "prey" obtained in step 1) b). After centrifugation, the pellet was resuspended in YPGlu medium, plated onto YPGlu plates and incubated at 30 ℃ for 4 hours and 30 minutes. Selecting in DO-Leu-Trp-His medium, a yeast binding containing a "bait" and a "prey" capable of interacting with each other: the absence of leucine and tryptophan makes it possible to maintain the selection pressure allowing only yeasts containing both types of plasmids ("bait"/"prey") to grow; the absence of histidine in the medium makes it possible to select conjugated yeasts containing a "bait" plasmid and a "prey" plasmid capable of interacting with each other; the complementation made it possible to activate the HIS3 gene (encoding an enzyme involved in histidine biosynthesis) as a reporter gene.

3) Identification of Positive clones

Amplifying by PCR a "prey" fragment of a yeast colony selected according to the binding method described in paragraph 2) starting from a crude lysate of the colony with primers specific for a "prey" vector, wherein said primers are:

ABS1 5’-GCTTTGGAATCACTACAGG-3’(SEQ ID NO.12)；

ABS2 5’-CACGATGCACGTTGAAGTG-3’(SEQ ID NO.13)。

PCR product sequencing was then performed and the resulting sequences were identified by comparison with a database.

4) Identification of the peptides depicted in FIGS. 1-5

For each "bait" fragment tested, the two-hybrid system allowed the identification of a large number of "prey" fragments. This identification was performed by comparing the sequences of selected "prey" using a software program (e.g., BlastWun, available from the university of Washington website, http:// biobob.

Example 2: the peptides obtained in example 1 were verified against Bcl-2, Bcl-X_LAnd/or Bcl-W interaction

1) Measurement of Ki by fluorescence polarization

Determination of Ki by fluorescence polarization involves measuring the competing peptides having the amino acid sequences SEQ ID No.1 to SEQ ID No.5, respectively, for pro-apoptotic Bak peptides and anti-apoptotic members of the Bcl-2 protein family (e.g., Bcl-X)_L) The influence of interactions between them.

The following reagents were mixed in the order stated:

-a competing peptide at a final concentration of 1nM-100 μ Μ;

-fluorescent peptide ligand (Bak BH3 carboxyfluorescein) at a final concentration of 15 nM;

anti-apoptotic members of the Bcl-2 protein family (Bcl-X) at a final concentration of 100nM_L). These reagents were dissolved in interaction buffer (Na)₂HPO₄20mM pH 7.4, EDTA 1mM, NaCl 50mM and pluronic acid F-680.05%).

The mixture was then incubated at room temperature for 30 minutes and the fluorescence polarization was measured on a Fusion instrument (Packard) (excitation at 485nm and reading at 530 nm). Values are expressed in mP (units of fluorescence polarization).

These fluorescence polarization analyses demonstrated that the peptides having the amino acid sequences SEQ ID NO.1 to SEQ ID NO.5 are Bak and Bcl-X_LCompeting peptides for peptide interactions. The Ki values obtained during these fluorescence polarization tests were as follows:

Bcl-X_L/Bak/SEQ ID NO.1 Ki＝9Mm

Bcl-X_L/Bak/SEQ ID NO.2 Ki＝32μM

Bcl-X_L/Bak/SEQ ID NO.3 Ki＝1μM

Bcl-X_L/Bak/SEQ ID NO.4 Ki＝8μM

Bcl-X_L/Bak/SEQ ID NO.5 Ki＝20μM

2) determination of Ki for each mutant peptide motif (L- -A) by fluorescence polarization

Determination of Ki by fluorescence polarization involves measuring the mutation from leucine to alaninePeptides SEQ ID NO.1 to SEQ ID NO.5 of acids (L- -A) for pro-apoptotic Bak peptides and anti-apoptotic members of the Bcl-2 protein family (e.g., Bcl-X)_L) The influence of interactions between them.

The peptide sequences SEQ ID NO.1 to SEQ ID NO.5 of the mutant forms (L- -A) 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 measuring Ki using fluorescence polarization is the same as described above.

The results of the comparative fluorescence polarization analysis show that mutant (L-A) peptides SEQ ID NO.14 to SEQ ID NO.18 have improved the apoptosis-promoting Bak peptides and anti-apoptotic members of the Bcl-2 protein family (e.g.Bcl-X) compared to the peptides SEQ ID NO.1 to SEQ ID NO.5 according to the invention_L) The competition effect in the peptide interaction between the two is lost.

Example 3: can inhibit Bcl-2 and/or Bcl-X_LScreening test for Compounds interacting with the peptides obtained in example 1

The test compounds were dispersed on 384 well plates (corning flat Bottom) at a final concentration of 10 μ g/ml. One well was filled with an equal amount of buffer/solvent without test compound as a control. The fluorescein-labeled peptide obtained in example 1 was added to the wells to obtain a final concentration of 1-100 nM. Then adding fusion protein GST-Bcl-X_LGST-Bcl-2 or GST-Bcl-W so as to contain Na₂HPO₄20mM pH 7.4, EDTA 1mM, NaCl 50mM, and pluronic acid F-680.05% buffer to a final concentration of 0.1-1. mu.M. The fluorescence polarization was then measured by an En Vision instrument (PackardPerkin-Elmer). And no testA significant decrease in fluorescence polarization was noted in the test with the test compound compared to the fluorescence polarization obtained with the test compound (control wells), concluding that the compound has inhibitory activity. Conversely, a significant increase in fluorescence polarization in the test with the test compound compared to the control concluded that the compound has activator activity.

Claims (19)

1. A peptide that interacts with an anti-apoptotic member of the Bcl-2 protein family, characterized by comprising an amino acid sequence selected from the group consisting of seq id nos:

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)；

2. a nucleic acid sequence encoding a peptide according to claim 1.

3. A nucleic acid sequence according to claim 2, characterized in that it comprises a nucleotide sequence selected from the group consisting of:

a)atggcgactgtcattcacaaccccctgaaagcgctcggggaccagttctacaaggaagccattgagcactgc(SEQ ID NO.7)；

b)gttatgacccaagaggttggccagctcctgcaagacatgggtgatgatgtataccagcagtaccggtcactt(SEQ ID NO.8)；

c)agacttaaacattcctgcctgctggctctgaagagagcagcggatctcctaggacagcgctcaagctctact(SEQ ID NO.9)；

d)gatatgtgggacactcgtatagccaaactccgagtgtctgctgacagctttgtgagacagcaggaggca(SEQ ID NO.10)；

e)gttgctacaagacgattaagtggcttcctgaggacacttgcagaccggctggagggcaccaaagaactgctt(SEQ ID NO.11)。

4. recombinant vector, characterized in that it contains one of the nucleic acid sequences according to any one of claims 2 or 3.

5. A recombinant vector according to claim 4, characterized in that the vector is a plasmid, cosmid, bacterial artificial chromosome or phage containing the sequences necessary for the expression of the peptide according to claim 1.

6. Recombinant vector according to claim 5, characterized in that the sequences necessary for the expression of the peptide according to claim 1 comprise transcriptional and translational promoter sequences.

7. A host cell, characterized in that it has been transformed with a recombinant vector according to any one of claims 4 to 6.

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

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

10. A method of causing apoptosis comprising administering to a cancer patient an effective amount of a pharmaceutical composition according to claim 9.

11. Method for identifying a modulator of the interaction between a peptide according to claim 1 and an anti-apoptotic member of the Bcl-2 protein family, characterized in that it comprises the following steps:

a) contacting the peptide with an anti-apoptotic member of the Bcl-2 protein family;

b) adding a test compound; and

c) measuring the activity of the test compound as a modulator of the protein interaction between the peptide and an anti-apoptotic member of the Bcl-2 protein family, and then comparing said measurement in the absence of the test compound.

12. Method for identifying an interaction modulator according to claim 11, characterized in that it comprises the following steps:

a) fluorescently labeling the peptide according to claim 1;

b) incubating the peptide in the presence of a test compound;

c) adding an anti-apoptotic member of the Bcl-2 protein family;

d) measuring the fluorescence polarization;

e) the measurements with and without test compound were compared.

13. The method of claim 12, wherein the modulator of the interaction between the peptide and the anti-apoptotic member of the Bcl-2 protein family is an activator of increased fluorescence polarization.

14. The method of claim 12, wherein the modulator of the interaction between the peptide and the anti-apoptotic member of the Bcl-2 protein family is an inhibitor that reduces the polarization of fluorescence.

15. The method according to any one of claims 11 to 14, wherein the fluorescent probe is fluorescein.

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

17. A pharmaceutical composition comprising as active principle a modulator obtained with the method for identifying modulators according to any one of claims 11-16 in combination with one or more pharmaceutically acceptable excipients.

18. A method of causing apoptosis comprising administering to a cancer patient an effective amount of the pharmaceutical composition of claim 17.

19. The pharmaceutical composition according to any one of claims 9 or 17 for use in the treatment of cancer.