EP2475680A1

EP2475680A1 - Molecules capable of inducing cell death by targeting the mitochondria and applications thereof

Info

Publication number: EP2475680A1
Application number: EP10760064A
Authority: EP
Inventors: Abdel Aouacheria
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite Claude Bernard Lyon 1 UCBL
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite Claude Bernard Lyon 1 UCBL
Priority date: 2009-09-09
Filing date: 2010-09-08
Publication date: 2012-07-18
Also published as: CA2772748A1; WO2011030296A1; JP2013504559A; FR2949787A1; US20120289472A1

Abstract

The invention relates to the use of derivatives of the membrane insertion regions of proteins of the Bcl-2 family to induce cell death by targeting the mitochondria, characterised in that said derivatives are peptides belonging to regions α5 and/or α6 of the Bax protein or equivalent regions found in the other proteins of the Bcl-2 family.

Description

Molecules capable of inducing cell death by targeting the mitochondria and their

applications

The invention relates to the use of peptides isolated to induce cell death by targeting the mitochondria and their biological applications.

In addition to providing the essential energy for cell survival, the mitochondrion performs essential functions in the control of apoptosis in vertebrates.

The mitochondrial pathway of apoptosis is defined by a major event: the permeabilization of the outer mitochondrial membrane, which leads to the release into the cytoplasm of toxic proteins (such as cytochrome c) normally contained in the intermembrane space.

The integrity of this membrane is under the control of Bcl-2 family proteins. The proteins of this family, which are either pro-apoptotic (like Bax and Bid) or anti-apoptotic (like Bcl-2 and Bcl-xL), form homo- and heterodimers, all of these protein-protein interactions. participating in the regulation of apoptosis.

However, it is known that the interaction of Bcl-2 family proteins with intracellular membranes is crucial for their function. Thus, both pro- (such as Bax and Bid) and anti-apoptotic (such as Bcl-2 and Bcl-xL) proteins of the Bcl-2 family can form in vitro pores in artificial membranes. Experiments also indicate that composite channels formed at the level of the outer mitochondrial membrane by oligomers of the pro-apoptotic proteins Bax and Bak (alone or in combination with other proteins) would allow the passage of solutes such as cytochrome c.

Proteins homologous to Bcl-2 share a globular structure (reminiscent of certain bacterial membrane-forming bacterial toxins) with between 6 and 9 amphipathic alpha-helices. Several structural studies have shown that one of the crucial structural determinants for the function of these proteins is a supersecondary ("helix-elbow-helix") motif consisting of two anti-parallel "hairpin" alpha helices (alpha5- alpha6 in Bax), able to insert into the membranes biological and to form pores. The 3D structure of Bax is given on the Figurel. Each helix of the motif has a size of about 15-20 amino acids (order of magnitude for an alpha helix capable of crossing a lipid bilayer), separated by a bend formed of 3-4 residues. If available studies support the importance of this field for the insertion of Bcl-2 family proteins into artificial membranes and the formation of pores in model membrane systems (see for review Petros et al., Biochim Biophys Acta , 2004), they do not provide information on the mechanism of action of this motif at the mitochondrial level. Thus, the exact contribution of this motif to cellulosic and in vivo pore formation by Bcl-2 family proteins is still largely speculative.

It has been reported in the literature that peptides corresponding to the central membrane insertion helices (alpha5 and alpha6) of Bax were capable of inducing on their own the release of molecules contained in liposomes (Garcia-Saez et al., Biophys. 2005, Garcia-Saez et al, FEBS J, 2006, Garcia-Saez et al, Biophys J, 2007). In addition, a recent structural study has shown that a peptide corresponding to the Bax alpha5 helix forms a lipid pore (Qian et al., Proc Natl Acad Sci U S A, 2008). Although these studies shed light on the fundamental mechanisms of pore formation in artificial and acellular systems, there is no indication of the ability of the peptides used to induce cell death, and their effect on mitochondria has not been explored.

One study (Ishibashi et al., Biochem Biophys Res Commun, 1998) indicated that ectopic expression, by bacteria or mammalian cells, of constructs encoding Bax fragments including alpha5 and / or alpha6 helices exerted an toxic. The authors of the study report that Bax regions capable of killing mammalian cells are the same ones that kill bacteria, which are devoid of mitochondria. Based on this study, it is not possible to determine the mechanism of action of the expressed fragments, and in particular whether the cytotoxic activity observed with mammalian cells depends on a mitochondrial action. Notably, the study only reports on the use of recombinant plasmids, the effect of peptides corresponding to the alpha5 and / or alpha6 regions of Bax having not been tested.

It is likely that the membrane insertion regions of the Bcl-2 family proteins have been evolutionarily selected to integrate specifically into the mitochondrial membranes of animal cells. It is important to test experimentally if these regions, apart from the global context of the proteins which contain them, are capable of reaching the mitochondria, of interfering with their structure (such as for example their membranes) and / or their function (for example their transmembrane potential) and cause a cell death. To date, the ability of peptides derived from this membrane insertion motif to trigger an apoptotic process by acting on mitochondria alone is not supported by any experimental evidence. Such peptides could represent an alternative to the artificial cationic peptides used to trigger cell death (Ellerby et al., Nat Med, 1999), which are not very active and therefore not conducive to industrial development.

The work of the inventors has focused on the study of peptides from these regions to determine whether they alone can target mitochondrial membranes and cause cell death by apoptosis.

The results obtained identified minimal structural determinants governing the interaction of Bcl-2 family proteins with membranes. Advantageously, certain regions have proved of great interest in targeting the mitochondria and destabilizing the mitochondrial membranes. From these regions, bioactive peptides have been developed opening a pathway to new therapeutically usable molecules.

These new molecules have been designated by the term "poropeptides" (from the Greek word Poros ΠΟΡΟΣ: hole or sea passage, figuratively: an effective way around a difficulty), a poropeptide designating a peptide having the ability to form pores in membranes lipids or destabilize biological membranes such as mitochondrial membranes.

Poropeptides, designed from the membrane insertion regions of proteins of the Bcl-2 family, have thus been shown to be capable of inducing the apoptosis of harmful cells, for example tumor cells.

The purpose of the invention is therefore the use of peptides whose sequence derives from the membrane insertion regions of the Bcl-2 family proteins to induce cell death by targeting the mitochondria. It also aims to provide new peptide molecules developed from these regions.

The invention also aims to provide, with these different molecules, high efficiency means to restore an apoptosis process in cancer cells while minimizing the maximum impact on surrounding healthy cells. This targeting step may for example be based on the vectorization of peptides by coupling to a domiciliation molecule ensuring their selective targeting at the tumor site, sparing healthy tissue. Since all living cells in the body (healthy or pathological) have mitochondria, poropeptides inducing the breakdown of mitochondrial permeability can be used to induce the death of many cell types. With advances in therapeutic targeting techniques, it may be possible to selectively eliminate certain cell populations that are harmful to the body. The invention thus relates to the use of membrane insertion helix derivatives of Bcl-2 family proteins to induce cell death by targeting the mitochondria, characterized in that they are peptides corresponding to the a5 regions. and / or a6 of the Bax protein or equivalent regions present in the other proteins of the Bcl-2 family. The invention further relates to derivatives, variants, mutants or fragments of the aforementioned peptides.

Synthetic peptides derived from those defined above comprise, for example, means allowing cell penetration or recognition of a particular cell type.

Advantageous means correspond to a transduction motif allowing their internalization in the targeted cell. It is in particular a polyarginine unit, advantageously comprising 8 arginine residues. Other modifications correspond to deletions and / or substitutions of one or more amino acids. Thus, one or more Cysteine residues of said regions may be replaced by Serine or Glycine motifs.

The invention also relates to conjugates that contain a peptide derived from those defined above and an addressing molecule, such as recognizing molecules. the endothelium, to target a particular tissue or cell type, such as certain tumor cells or angiogenic endothelial cells.

The invention also relates to functional equivalents of the peptides defined above, such as peptides comprising modifications resulting from post-translational processes such as glycosylation or chemical modifications such as coupling with lipids, sugars, nucleotide sequences or d amino acids since these modifications do not modify the proapoptotic activity of said peptides according to the tests given in the experimental part below. Functional equivalents also include peptides of which one or more amino acids are D-conforming amino acids. The invention also encompasses retro-peptides and retro-inverso-peptides.

The invention is directed in particular to the peptides derived from regions a5 and a6, for example, the peptide of sequence SEQ ID No. 1: NH ₂ -NWGRVVALFYFASKLVLKALSTKVPELIR-COOH.

After internalization, these "mitochondrial-toxic" compounds are able to trigger apoptosis by destabilizing the mitochondrial membranes, especially the outer mitochondrial membrane. Advantageously, they induce the release of multiple toxic molecules contained in the inter-membrane space of mitochondria, in particular cytochrome c.

As illustrated by the examples, their cytotoxic activity has been demonstrated in cellulo. Thus, according to another aspect, the invention relates to the use of the peptides defined above as medicaments.

The pharmaceutical compositions of the invention are characterized in that they comprise a therapeutically effective amount of at least one peptide as defined above in association with a pharmaceutically acceptable vehicle.

The present invention also relates to a pharmaceutical composition comprising as active ingredient at least one peptide as defined previously associated in said composition with one or more pharmaceutically acceptable carriers, diluents or excipients.

The administration of a pharmaceutical composition according to the invention can be carried out according to the techniques known to those skilled in the art by any of the accepted modes of administration for the therapeutic agents. These methods include systemic, topical, oral or central administration, for example by surgery or intraocular administration. We can also mention the subcutaneous implantation of biodegradable implants.

The dosage for administration of the peptides according to the invention is chosen according to many factors including the type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the state of renal and hepatic function of the subject and the nature of the particular compound, or salt, used. The normally experienced person skilled in the art will determine and prescribe the effective amount to prevent, hinder or halt the progress of the medical condition to be treated.

Due to their direct effect on mitochondrial permeability, the above compounds are particularly advantageous for initiating an apoptotic process after internalization in tumor cells or neovascular tumor-irrigating cells.

Because of their ability to target mitochondria, the invention also relates to the use of the aforementioned peptides as mitochondrial targeting molecules for delivering a compound or polypeptide to the mitochondrial membranes to induce apoptosis.

They are generally useful in situations with increased neovascularization, such as rheumatoid arthritis or obesity. Indeed, sustained angiogenesis characterizes severe forms of arthritis and the maintenance of adipose mass during obesity. Pathological angiogenesis is also found in psoriasis and some forms of diabetes. According to another advantageous embodiment of said poropeptides, they are used to counter certain other types of harmful cells, such as immune cells that recognize the self and cause autoimmune diseases. The poropeptides as defined above have all the characteristics of antimicrobial peptides: amphipathic, cationic character, destabilization of lipid membranes. The present invention therefore also relates to the use of the peptides as defined above for the preparation of a molecule with antibiotic properties capable of killing resistant bacteria.

In general, therapeutic peptides are generally metabolized and eliminated more easily than conventional drugs, giving the patient a better chance of healing while minimizing the complications associated with standard therapies.

In addition, the poropeptides can be used, according to the invention, as adjuvants in addition to conventional drugs, thus reducing the doses and side effects of the latter. According to another aspect, the invention relates to cosmetic compositions containing in their active principle at least one peptide as defined above in combination with a vehicle acceptable in cosmetology.

Advantageously, the peptides of the invention have stabilizing properties for these compositions.

According to another aspect, the invention is further directed to agronomic or agri-food compositions containing in their active ingredient at least one peptide as defined above, in combination with a vehicle acceptable in agronomy and in the food field.

Other features and advantages of the invention are given in the examples which follow. In these examples, reference is made to Figures 1 to 11 which represent, respectively, - Figure 1, A) the "channel" domain of colicin A; B) the 3D structure of Bax; C) the α-helical hairpin motif of Bcl-2 family proteins (alpha5-alpha6 in Bax);

FIG. 2, constructs coding for fusions between GFP (green fluorescent protein) and the various regions of anchoring or membrane insertion of the pro-apoptotic protein Bax;

- Figure 3, analysis results of the expression of Bax regions by Western Blot;

- Figure 4, the cellular location of the GFP with respect to said regions;

- Figure 5, the death rate of cells expressing GFP or GFP fused to the peptides used according to the invention;

FIG. 6, the apoptosis rate of wild-type embryonic fibroblasts (MEFs) or deficient in Bax and Bak (MEF-DKO);

FIG. 7, the mitochondrial transmembrane potential of cells expressing GFP or GFP fused with the peptides used according to the invention;

- Figure 8, the helical wheel projection of a peptide of the invention;

9, the effect of a poropeptide of the invention on isolated mitochondria;

FIGS. 10 and 11, the effect of microinjection of peptide fragments of Bax on viability and embryonic (Fig. 9) and cellular morphology (Fig. 10); and

FIG. 12, the proapoptotic effect of a poropeptide of the invention.

Plasmid constructs comprising inserts corresponding to the nucleic acids encoding one or more Bax regions fused to GFP: amino acids 1-192 (whole Bax protein: α! -Α2-α3-α4-α5-α6-α6'-α7-α8 -α9), 20-37 (a1), 106-134 (a5), 130-150 (a6, 102-150 (a5-a6, 102-192 (a5-a6-a6'-a7-a8-a9 or a5 -a9, 169-192 (a9.

The nucleic acids coding for the various anchoring or membrane insertion regions present on the pro-apoptotic protein Bax were cloned into the pEGFP-C1 plasmid. The inserted sequences are given in Figure 2. A construct (GFP-KLAK) encoding a GFP-KLAKLAKKLAKLAK polypeptide was also made. Artificial peptides enriched with KLA sequences are known to target the negatively charged bacterial membrane and exhibit antibiotic activity.

Recombinant plasmids were verified by molecular sequencing. The analysis of the expression of Bax inserts carried out by Western Blot is reported in FIG. upper half corresponds to the Western Blot analysis, using an anti-GFP antibody, of HT 1080 fibrosarcoma cell lysates transfected transiently by the above plasmid constructs; the middle part gives the immunoblot obtained using an antibody recognizing the apoptotic cleavage fragment of PARP; the lower part gives the immunoblot obtained using an antibody recognizing the activated form of caspase-3.

The different constructs were transfected into human cells cultured in vitro (HT-1080 and SK-MEL-28), and the subcellular localization and cytotoxicity induced by GFP-fused Bax membrane insertion fragments was determined.

Figure 4 shows the subcellular localization of the complete GFP protein relative to that of inserts composed of Bax-fused membrane insertion fragments. MEF-DKO cells were co-transfected with the pEGFP-C1 plasmids encoding the various Bax inserts and the MitoDsRed vector. It is observed that peptides corresponding to or comprising the membrane insertion helices of Bax and / or the TM domain are sufficient to address GFP (in green) to mitochondrial membranes (marked in red with the MitoDsRed protein, comprising the mitochondrial targeting CoxVIII). Similar results were obtained using an antibody directed against the mitochondrial protein Hsp70 (bottom inserts). These confocal microscopy results indicate that short fragments of the Bax protein (particularly the regions corresponding to amino acids 106-134, 130-150 and 169-172) contain the determinants necessary for their specific mitochondrial targeting. Figure 5 shows the cellular toxicity induced by the expression of the different constructs. Figure 5A shows the death rate (apoptosis / necrosis) of HT-1080 cells expressing GFP alone or in fusion with different membrane insertion fragments of the Bax death protein. The cells were treated (+) or not (-) with the general caspase inhibitor zVAD.fmk (ΙΟΟμΜ). Cell death was quantified 24 h after transfection by counting the number of green cells (expressing GFP) under an epifluorescence microscope. The type of cell death (apoptosis and necrosis) was determined by membrane permeability to propidium iodide (necrosis) and nuclear morphology (condensed or fragmented nuclei) after staining with Hoechst 33342 (apoptosis). Green cells negative for propidium iodide and having a pyknotic nucleus were counted as being in apoptosis. The results represent the mean values (with standard deviations) of three experiments.

In the test used, the green cells (expressing the construction) presenting a pycnotic nucleus are apoptosis, those stained in red by propidium iodide are in necrosis.

It is observed that the constructs encoding the central helices of Bax (GFP-Bax-a5a6, GFP-Bax-a5 and GFP-Bax-a6) are highly cytotoxic and induce cell death mainly of the apoptotic type. The apoptotic character of cell death caused by the expression of membrane insertion fragments of Bax is confirmed by the cleavage of the PARP protein and the activation of caspase-3, demonstrated by the Western blot technique (FIG. .3) using specific antibodies, and blocking apoptosis with a broad-spectrum caspase inhibitor (zVAD.fmk) (Fig.5A). In addition, the onset of apoptotic cell death is demonstrated by Annexin-V labeling, which specifically recognizes phosphatidylserines extemalized by apoptotic cells (Fig. 5B). In this experiment, cell death was quantified using the Annexin V-Cyanine 3 assay by 24h flow cytometry after transient cell transfection with the plasmids of interest. It should be noted that the GFP-Bax-a5 and GFP-Bax-a6 constructs are more effective at inducing apoptosis than the GFP-KLA construct.

Figure 6 shows that the toxic proteins encoded by the inserts do not act via the endogenous Bax and Bak proapoptic proteins, since murine fibroblasts deficient in Bax and Bak (MEF-DKO) are not resistant to stress. induced by their expression (see also Fig.3). The graph (FIG. 6A) corresponds to kinetics of cell death (quantified by flow cytometry using the Annexin V-Cy3 assay) of wild-type MEF versus MEF-DKO murine fibroblasts. It is observed that the fibroblasts deficient for Bax and Bak are as sensitive to the induction of apoptosis by the expression of the GFP-Bax-alpha5 protein as to wild murine fibroblasts. At the same time, DKO MEF cells are resistant to staurosporine-induced apoptosis (Fig. 6B). These results indicate that the Bax-alpha5 region is capable of apoptotic killing of MEF-DKO cells, described as being resistant to a large number of apoptotic stimuli (Wei et al., Science, 2001). FIG. 7 shows that cells (HT-1080) expressing the polypeptides comprising the Bax cc5 and / or cc6 helices undergo a significant fall in their mitochondrial transmembrane potential (ΔΨιη), unlike the cells expressing GFP alone, suggesting the implication of a mitochondria-dependent pathway in the induction of apoptosis. Cells were stained with Mitotracker 24h after transfection and analyzed by flow cytometry (Fig. 7A). The histograms (FIG. 7B) show the average values of three flow cell cytometry quantification experiments (expressing GFP) with Mitotracker incorporation defects (ie a weak ΔΨιη). The results presented show that the regions of the pro-apoptic protein Bax corresponding to amino acids 106-104 and 130-150 are capable of targeting mitochondrial membranes and of inducing apoptotic cell suicide. These regions are alpha helix structured within the complete Bax protein (Suzuki et al., 2000, Cell, 103, 645-654) and have an amphipatic character favorable to the destabilization of biological membranes.

Poropeptide of sequence SEQ ID No. 1 It is an amphipatic peptide (see FIG. 8), longer than the a5 helix deduced from the 3D structure of the Bax protein in solution (Suzuki et al., 2000). ). It contains a serine residue incorporated in position 126 in place of the natural cysteine residue in order to limit the formation of disulfide bridges. · Characterization of its mode of action by incubation with isolated mitochondria.

The ex-cellulo activity of poropeptide-Bax (106-134) was tested by determining its ability to promote the release of cytochrome c from the inter-membrane space of isolated mitochondria. The results obtained are illustrated in Figure 9 (A).

The peptide is incubated with the mitochondria for 5, 15, 30 or 60 min, and the mitochondrial release of cytochrome c in the supernatant (SN) is measured by Western Blot. The mitochondrial fractions (Mito) are calibrated using an antibody directed against mitochondrial proteins HSP70 or ATPase (subunit 6). With the poropeptide-Bax (106-134), the cytochrome c is released from the inter-membrane space of the mitochondria at 10 μΜ and in 5 min of incubation. No release is observed when an artificial antimicrobial peptide (KLAK) of sequence SEQ ID No. 2 KLAKLAKKLAKLAK is used.

At 10 μΜ, which corresponds to the minimum concentration for which re-drop is observed with poropeptide-Bax (106-134), no release is observed for the Bax-BH3 peptide (Bax death domain Bax corresponding to the helix a2, located about thirty residues upstream of the helix a5). Cytochrome c is released from the mitochondrial inter-membrane space from 25μΜ using mitochondria isolated from HEK293T cells and never released with SK-MEL-28 cells. Poropeptide-Bax (106-134) is therefore more active than Bax-BH3 in releasing cytochrome c from the inter-membrane space of the mitochondria. In addition, it is more efficient since it allows a release from the first 5 minutes, while it is necessary to wait 30 minutes for the BH3 peptide in the HEK293T cells.

These experiments show that poropeptide-Bax [106-134], of sequence SEQ ID No. 1, has a strong ability to induce the permeabilization of mitochondrial membranes.

Figure 9B further shows that poropeptide-Bax (106-134) profoundly alters the organelle physiology, causing a physical process of swelling (left panel) and depolarization (right). Poropeptide-Bax (106-134) induces mitochondrial swelling (DD ₅₀ = 1.68 ± 0.39 μΜ) and the fall of their transmembrane potential (SD ₅₀ = 3.98 ± 0.57 μΜ). These two parameters were measured using mitochondria isolated from rat liver incubated in the presence of different concentrations of poropeptide-Bax (106-134) (NT = untreated).

These results obtained in vitro confirm the involvement of a mitochondria-dependent pathway in the induction of apoptosis by the cc5 region of Bax. · Measures of induced cytotoxicity

- Endogenously administering the peptide (by microinjection). Poropeptide-Bax [106-134] was microinjected in vivo. The zebrafîsh (Danio rerio) model was used because (i) the apoptosis machinery is conserved between human and fish cells; (ii) the egg corresponds to an integrated cellular system compared to isolated mitochondria; (ii) zebrafish eggs are easily injectable routinely.

The results obtained are illustrated in Figure 10:

(A) Zebrafis eggs were microinjected at stage 1-2 cell (s) with increasing concentrations of poropeptide-Bax [106-134], Bax-BH3 peptide or with ultracure water Cmock ' ). A concentration of 100 μl inside the microinjection capillary corresponds to approximately 6 × 10 ¹² peptide molecules per egg. The histograms represent the percentage of mortality 24 h after fertilization and the percentage of surviving embryos with severe malformations. The data presented are representative of 2 independent experiments giving the same results.

(B) Embryonic morphology 24 h after microinjection (100 μl peptide). Severe malformations are observed in the group of fish injected with Poropeptide-Bax [106-134] compared to the group injected with the peptide BH3. Poropeptide-Bax [106-134] was then mixed with a dextran-fluorescein solution (to allow visualization by epifluorescence microscopy), and then microinjected into SK-MEL-28 human melanoma cells. Cell viability was then monitored and quantified 12 hours after microinjection. Figure 11 gives the results obtained:

(A) Photomicrographs showing SK-MEL-28 human melanoma cells injected with fluorescent tracer Dextran-FITC alone Cmock ') or mixed with Poropeptide-Bax [106-134]. The cells were photographed 12 h after microinjection. The micro-injected cells with the poropeptide-Bax [106-134] exhibit morphological manifestations characteristic of apoptosis (cytoplasmic budding, retraction and cell rounding). Magnification x 800. (B) Cell viability after microinjection of poropeptide-Bax [106-134] or peptide Bax-BH3, KLAK or R8. The microinjected cells were visualized using the Dextran-FITC tracer. Cell death was estimated 12 hours after microinjection by counting the number of microinjected cells by peptide exhibiting morphological manifestations typical of apoptosis, compared to control (microinjection of ultrapure water with dextran-fluorescein added). ). The data presented are representative of 2 independent experiments giving the same results.

The results show that micro-injection of poropeptide-Bax [106-134] induces a substantial cell death, whereas micro-injected cells by Bax-BH3 or KLAK have no significant effect. Micro-injected cells control peptide (R8) SEQ ID NO: 3 sequence RRRRRRRR (a polyarginine intracellular transduction pattern) have viability rates comparable to the control. These data demonstrate that poropeptide-Bax [106-134] exerts dose-dependent cytotoxic effects after introduction into zebrafis eggs or tumor cells cultured in vitro, and is more effective in inducing cell death than in a pro-apoptotic peptide derived from the same protein (Bax-BH3) or other peptides known to be active at the membrane level (KLAK and R8). - Exogenously administered (by coupling with polyarginine transducer peptide).

The poropeptide of sequence SEQ ID No. 1, comprising an N-terminal polyarginine extension (R8) separated by a glycine residue, was used. A fluorescein isothiocyanate group (FITC) was added at the C-terminal position so as to allow its visualization by epifluorescence microscopy. The R8-Scr-FITC peptide is a "scramble" sequence of the sequence present in the R8-Bax [106-134] -FITC peptide, that is to say a control sequence in which the amino acids (their order in the sequence) were mixed.

Figure 12 shows the results obtained:

(A) HeLa cells were incubated for 6 h in the presence of 10 μΜ of FITC-R8-Bax [106-134] poropeptide or R8-Bax [Scr] control peptide (scramble version of the poropeptide), then observed under the epifluorescence microscope. The cells incubated in presence of the fluorescent peptides show a strong cytoplasmic labeling (visible from 1h of incubation). Scale 10 μιη.

(B) The poropeptide R8-Bax [106-134] induces a loss of cell viability dependent on the dose and the incubation time. HeLa cells were treated with increasing concentrations (5, 10, 25 and 50 μΜ) of R8-Bax peptide [106-134] or R8-Bax [106-134]. Cytotoxicity was determined by measuring the lactate dehydrogenase (LDH) cell release after 3, 6, or 24 hours of incubation (n = 4). The R8-Bax [Scr] peptide does not induce significant leakage of LDH at the tested concentrations. The results are given in means and standard deviations.

(C) The general caspase inhibitor zVAD.fmk reduces poropeptide-induced cell death R8-Bax [106-134] (25μΜ). Cytotoxicity was determined by measuring the cell release of lactate dehydrogenase (LDH) after incubation for 3, 6 or 24 hours in the presence of 100 μl of zVAD.fmk or buffer alone ('DMSO'). The results are given in means and standard deviations.

(D) Death rate (apoptosis / necrosis) of HeLa cells treated with poropeptide R8-Bax [106-134]. The type of cell death was determined 24 h after transfection by observation of cell permeability to propidium iodide (necrosis) or nuclear morphology after staining of DNA with Hoechst 33342 (apoptosis). Green cells (expressing GFP) were visualized by epifluorescence microscopy. About 300 cells were counted by experiment. The results are an average of three independent experiments (Γ standard deviation is also shown).

Thus, the two peptides (poropeptide FITC-R8-Bax [106-134] and scramble control peptide R8-Bax [Scr]) are rapidly internalized (in less than one hour) by HeLa cells in vitro culture (FIG. 12A). Unlike the control peptide, the poropeptide R8-Bax [106-134] -FITC inhibits HeLa cell viability in a dose-dependent and incubation-time manner (Fig. 12B). A pan-caspase inhibitor zVAD.fmk, added in vitro, significantly inhibited R8-Bax [106-134] -FITC-induced cell death (Fig. 12C), indicating the participation of caspases in its pro-apoptotic activity (Fig. 12D). (E) Apoptosis rate of wild-type fibroblastic cells (MEFs) or deficient in death proteins Bax and Bak (MEF DKO) treated with FITC-R8-Bax poropeptide [106- 134] or FITC-R8-control peptide Bax [Scr]. Apoptosis was quantified using the Annexin V-Cyanine 3 test by flow cytometry 6h or 24h after treatment, in the presence or absence of the caspase inhibitor zVAD.fmk. The results correspond to the percentage of apoptotic cells having internalized the fluorescent peptide in each condition.

Claims

1 - Use of derivatives of the membrane insertion helices of proteins of the Bcl-family

2 for inducing cell death by targeting the mitochondria, characterized in that they are peptides corresponding to the a5 and / or a6 regions of the Bax protein or to the equivalent regions present in the other proteins of the Bcl-2 family.

2 - The use according to claim 1, characterized in that said peptides are developed from the regions corresponding to the amino acids at position 106-134, 130-150 and 102-150 of the protein Bax.

3 - Peptides derived from the a5 and / or a6 regions of the Bax protein or equivalent regions present in other proteins of the Bcl-2 family.

4 - Peptides according to claim 5, characterized in that they comprise means for cell penetration or recognition of a particular cell type.

5 - Peptides according to claim 6, characterized in that said means correspond to a transduction domain such as a polyarginine motif.

6. Peptides according to claim 5, wherein one or more amino acids are deleted or substituted by another.

7 - Peptides according to claim 8, characterized in that one or more cysteine residues are replaced by Serine or Glycine residues.

Peptide having the sequence SEQ ID No. 1: NH ₂ -NWGRVVALFYFASKLVLKALSTKVPELIR-COOH.

9 - Pharmaceutical compositions, characterized in that they contain a therapeutically effective amount of at least one peptide as defined in any one of claims 3 to 8, in association with a pharmaceutically acceptable vehicle. 10 - Cosmetic compositions, characterized in that they contain in their active principle at least one peptide as defined in any one of claims 3 to 8, in combination with a cosmetically acceptable carrier. 11 - Agri-food or agronomic compositions comprising in their active principle at least one peptide as defined in any one of claims 3 to 8, in combination with a vehicle acceptable in agronomy and in the food field.