Method for identifying compounds useful in cancer treatment

Abstract

The invention relates to a method for identifying compounds useful for the treatment of cancer, based on the evaluation of the compound's ability to modulate the interaction between the AIF protein and the CHHCD4 protein.

Description

Title of the invention: Method for identifying compounds useful for the treatment of cancer Technical field

[0001] The invention relates to a method for identifying compounds useful for the treatment of cancer, based on the evaluation of the capacity of the compound to modulate the interaction between the AIF protein and the CHCHD4 protein. Technological background

[0002] During the process of carcinogenesis, the metabolism of cancer cells is reprogrammed to promote tumor cell growth, improve their repair and invasion capacities and their resistance to anticancer treatments. Mitochondria are major players in cellular metabolism via, among other things, their ability to produce ATP, produce different metabolites and macromolecules, produce and detoxify reactive oxygen species and modulate cell death. For this reason, targeting mitochondrial activity in order to affect cancer cell metabolism is an avenue of interest in the treatment of cancers. However, the mitochondrial molecular mechanisms likely to provide a therapeutic benefit remain to be determined.

[0003] To function, the mitochondria must import between 1500 and 2000 proteins encoded by the nuclear genome using specific protein machineries. Thus, AIF (Apoptosis Inducing Factor) and CHCHD4 (coiled-coil-helix-coiled-coil-helix domain containing 4) are two proteins expressed in the inter-membrane space which, once associated, form an import machinery for different proteins possessing cysteine ​​motifs.

[0004] The AIF / CHCHD4 interaction was described in the article Hangen et al. 2015 in vitro and in cellulo. This article demonstrates in particular that the function of the AIF protein in the biogenesis of mitochondrial respiratory chain complexes is mediated by its physical and functional interaction with CHCHD4. Summary of the invention

[0005] With the aim of identifying new compounds useful for the treatment of cancer, the inventors have developed an identification method, based on the evaluation of the capacity of a compound to inhibit the interaction between the AIF protein and the CHCHD4 protein. The inventors' hypothesis would be that a compound inhibiting the formation of the AIF / CHCHD4 complex would be capable of affecting cancer cells whose survival and proliferation depend on mitochondrial activity.

[0006] The method developed by the inventors has proven to be particularly effective since it has made it possible to identify compounds with anticancer properties. In addition, the present method has the advantage of being applicable to high-throughput screening, thus facilitating the identification of compounds potentially useful for the treatment of cancer.

[0007] One aspect of the invention therefore relates to a method for identifying a compound potentially useful for the treatment of cancer, characterized in that it comprises evaluating the capacity of said compound to inhibit the interaction between the AIF protein and the CHCHD4 protein, said compound being identified as potentially useful for the treatment of cancer if it inhibits said interaction.

[0008] In a particular embodiment, the method comprises:

[0009] (a) bringing the AIF protein and the CHCHD4 protein into contact in the presence and in the absence of said compound;

[0010] (b) measuring the interaction between the AIF protein and the CHCHD4 protein, in presence and absence of said compound; and

[0011] (c) comparing the measurement of said interaction in the presence and absence of said compound;

[0012] said compound being identified as potentially useful for the treatment of cancer if the extent of said interaction is lower in the presence of said compound than in the absence of said compound.

[0013] In a particular embodiment, the compound is identified as potentially useful for the treatment of cancer if it inhibits by at least 50%, 60%, 70%, 80%, or at least 90% the interaction between the AIF protein and the CHCHD4 protein.

[0014] The measurement of the interaction between the AIF protein and the CHCHD4 protein can be carried out by means of an amplified luminescence proximity homogeneous assay (ALPHA) or a surface plasmon resonance (SPR) assay. In addition, the method according to the invention can comprise the confirmation, in a non-human cellular or animal model of cancer, of the anticancer properties of the identified compound.

[0015] In a particular embodiment, the method comprises:

[0016] (i) determining the capacity of said compound to inhibit the interaction between the AIF protein and CHCHD4 protein, using an amplified luminescence proximity homogeneous assay (ALPHA);

[0017] (ii) determining the ability of said compound to inhibit the interaction between the AIF protein and the CHCHD4 protein, by means of a surface plasmon resonance (SPR) test; and

[0018] (iii) confirmation, in a non-human cellular or animal model of cancer, of the anticancer properties of the identified compound.

[0019] Another aspect of the invention relates to a kit for the identification of a compound potentially useful for the treatment of cancer, characterized in that it comprises:

[0020] - an AIF protein;

[0021] - a CHCHD4 protein;

[0022] - means suitable for measuring the interaction between the AIF protein and the protein CHCHD4;

[0023] - optionally a buffer adapted to the experiment of measuring said interaction; And

[0024] - optionally a compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein.

[0025] The means suitable for measuring the interaction between the AIF protein and the CHCHD4 protein may be means suitable for an amplified luminescence proximity homogeneous assay (ALPHA). In a particular embodiment, the buffer suitable for the experiment for measuring the interaction between the AIF protein and the CHCHD4 protein comprises phosphate buffered saline (PBS) and bovine serum albumin (BSA). In a particular embodiment, said compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein consists of the sequence of SEQ ID NO: 4 or any functional variant having at least 70%, 80%, 90%, or at least 99% identity with the sequence of SEQ ID NO: 4. Brief description of the figures

[0026] [Fig. 1] AIF103-613 and CHCHD4 titration by Alphascreen. Titration of CHCHD4 protein against AIF103-613 protein by ALPHAscreen technology leads to a bell-shaped response called the Hook effect.

[0027] [Fig.2] Inhibition of the interaction of AIF103-613 / CHCHD4. The Prestwick library consisting of 1280 compounds was screened using an Alphascreen assay at a concentration of 100M. Selected compounds are identified by white squares, black dots correspond to unselected compounds. The compound is selected if the raw signal obtained in the presence of the compound is lower than a threshold value TH (TH = mean (signal of the negative control) - 5 * SD (signal of the negative control). The Y axis corresponds to the normalized signal, i.e. the signal calculated as a percentage of the mean.

[0028] [Fig.3] SPR analysis of compounds identified by Alphascreen. (A) SPR validation of thioridrazine and mitoxantrone using the Alphascreen assay as inhibitors of the AIF103-613 / CHCHD4 interaction. Compounds at 10 pM were incubated for 20 min with AIF in DPBS at pH 7.4 at room temperature. Then, the mixture was injected for 3 min at a rate of 30 pL per min onto the CHCHD4 protein. Peptide N27 at 3 pM served as a positive control. (B) Surface plasmon resonance (SPR) sensorgrams, monitoring the binding of AIF103-613 to CHCHD4 in the presence of thioridazine hydrochloride or of mitoxantrone dihydrochloride. Thioridazine hydrochloride and mitoxantrone dihydrochloride were tested at increasing concentrations from 0.5 pM to 50 pM. Detailed description 1- Identification process

[0029] One aspect of the invention relates to a method for identifying a compound potentially useful for the treatment of cancer, characterized in that it comprises evaluating the ability of a compound to inhibit the interaction between the AIF protein and the CHCHD4 protein, said compound being identified as potentially useful for the treatment of cancer if it inhibits said interaction. One aspect of the invention relates in particular to a method for screening a library of compounds to identify a compound potentially useful for the treatment of cancer, characterized in that it comprises evaluating the ability of the compounds of said library of compounds to inhibit the interaction between the AIF protein and the CHCHD4 protein, said compounds being identified as potentially useful for the treatment of cancer if they inhibit said interaction.

[0030] In a particular embodiment, the method according to the invention comprises:

[0031] (a) bringing the AIF protein and the CHCHD4 protein into contact in the presence and in the absence of said compound;

[0032] (b) measuring the interaction between the AIF protein and the CHCHD4 protein, in presence and absence of said compound; and

[0033] (c) comparing the measurement of said interaction in the presence and absence of said compound;

[0034] said compound being identified as potentially useful for the treatment of cancer if the extent of said interaction is lower in the presence of said compound than in the absence of said compound.

[0035] AIF and CHCHD4 proteins

[0036] The physical and direct interaction of AIF and CHCHD4 proteins was demonstrated in the article by Hangen et al., 2015.

[0037] The AIF protein (for “Apoptosis Inducing Factor”) is a flavoprotein present in mitochondria and which promotes apoptosis when released from the mitochondria. In the context of the present invention, the AIF protein may be of any origin, preferably of animal origin, in particular a mammal, more preferably of human origin. In a particular embodiment, the AIF protein corresponds to the natural human AIF protein whose NCBI sequence reference is “NP_004199.1”, or any functional variant thereof such as AIF2 which is a brain-specific isoform. In particular, the AIF protein according to the invention may correspond to the natural human polypeptide of sequence SEQ ID NO: 1, or any functional variant. The expression “functional variant” which refers to the AIF protein designates any polypeptide derived from the structure of the AIF protein and retaining the capacity to bind to the CHCHD4 protein, in particular to the CHCHD4 protein of SEQ ID NO: 2. The functional variants may be natural or synthetic variants such as fragments, mutants or deletions. Preferably, the functional variant of the AIF protein corresponds to a polypeptide having a sequence identity of at least 50%, 60%, 70%, 80%, 90%, or at least 95% with the AIF protein of sequence SEQ ID NO: 1.

[0038] The CHCHD4 protein (also called "mitochondrial intermembrane space import and assembly protein 40" or "MIA40") is a protein that participates in protein import, into the intermembrane space of the mitochondria. In the context of the present invention, the CHCHD4 protein may be of any origin, preferably of animal origin, in particular a mammal, more preferably of human origin. In a particular embodiment, the CHCHD4 protein corresponds to the natural human CHCHD4 protein whose NCBI sequence reference is "NP_001091972.1". In particular, the CHCHD4 protein according to the invention may correspond to the natural human polypeptide of sequence SEQ ID NO: 2 or any functional variant.The expression "functional variant" which refers to the CHCHD4 protein designates any polypeptide derived from the structure of the CHCHD4 protein and retaining the capacity to bind to the AIF protein, in particular to the AIF protein of SEQ ID NO: 1. The functional variants may be natural or synthetic variants such as fragments, mutants or deletions. Preferably, the functional variant of the CHCHD4 protein corresponds to a polypeptide having a sequence identity of at least 50%, 60%, 70%, 80%, 90%, or at least 95% with the CHCHD4 protein of sequence SEQ ID NO: 2.

[0039] In a particular embodiment, the functional variant of the AIF protein is deleted or truncated relative to the AIF protein of SEQ ID NO: 1. In particular, the functional variant of the AIF protein may correspond to a polypeptide in which the transmembrane portion of the AIF protein has been deleted. In a particular embodiment, the functional variant of the AIF protein corresponds to a polypeptide of sequence SEQ ID NO: 3, or to a polypeptide having a sequence identity of at least 50%, 60%, 70%, 80%, 90%, or at least 95% with the polypeptide of sequence SEQ ID NO: 3.

[0040] The AIF and CHCHD4 proteins can be modified, provided that this does not prevent the interaction between the two proteins. For example, for the purposes of the experiment to measure their interaction, the AIF and / or CHCHD4 proteins can be fused to a fragment serving as a label (or "tag"). Any A conventional tag can be used, provided that it does not prevent the interaction between the two proteins. In particular, any tag suitable for an amplified luminescence proximity homogeneous assay (ALPHA) or a surface plasmon resonance (SPR) assay can be used. In particular, the AIF and / or CHCHD4 proteins can be fused to a Histidine tag corresponding to a motif consisting of several histidine residues, or to a GST tag corresponding to the glutathione-S-transferase protein.

[0041] Compound to be tested

[0042] The compounds that can be identified by the method of the invention can be compounds of varied nature, structure and origin. The compounds that can be identified can in particular be biological, chemical, synthetic compounds, etc. They can in particular be compounds of nucleic, peptide, lipid or carbohydrate nature. They can also be libraries, in particular chemical libraries, protein, peptide, nucleic acid or natural substance libraries, etc.

[0043] Contacting the compound to be tested with the AIF and CHCHD4 proteins

[0044] The compound to be tested can be brought into contact with the AIF protein and the CHCHD4 protein in any suitable support and in particular on a plate, in a tube or a flask, a membrane, etc. In particular, the contact can be carried out in a multi-well plate which makes it possible to carry out, in parallel, numerous and varied tests. Among the typical supports are microtitration plates and more particularly 96 or 384 well (or more) plates, which are easy to handle.

[0045] The amount (or concentration) of test compound can be adjusted by the user depending on the type of compound, the duration of the incubation period, etc. In particular, the concentration of the test compound can vary from InM to ImM. It is of course possible to test other concentrations without deviating from the present invention. Each compound can, moreover, be tested, in parallel, at different concentrations. Similarly, the amount (or concentration) of AIF and CHCHD4 proteins can vary and be adjusted by the user. In particular, the concentration of AIF and CHCHD4 proteins is adjusted to allow optimal interaction between the two proteins, in the absence of the test compound.

[0046] The contact between the proteins and the compound to be tested can be maintained for example between a few minutes and several hours or days, particularly between 30 minutes and 72 hours, more particularly between 1 and 5 hours.

[0047] The order of addition of the AIF protein, the CHCHD4 protein and the compound to be tested when they are brought into contact can be adapted by a person skilled in the art. In particular, the compound to be tested can be pre-incubated with the AIF protein, for a certain period of time, before being brought into contact with the CHCHD4 protein. Alternatively, the test compound may be pre-incubated with the CHCHD4 protein for a period of time before being brought into contact with the AIF protein. The duration of the pre-incubation with either protein may be a few minutes, or several hours or days, more particularly between 5 and 60 minutes, more particularly between 5 and 30 minutes.

[0048] Measurement of the interaction of AIF and CHCHD4 proteins

[0049] The interaction of the AIF and CHCHD4 proteins, in the presence or absence of the compound to be tested, can be measured according to any technique known to those skilled in the art making it possible to measure or quantify the interaction between two proteins.

[0050] The term "interaction" means the physicochemical association or bond between the two proteins. The interaction between two proteins can result from covalent and / or non-covalent bonds. Non-covalent bonds include electrostatic, ionic, hydrogen, hydrophobic bonds, as well as Van der Waals forces.

[0051] The method according to the invention comprises measuring the interaction of the AIF and CHCHD4 proteins, on the one hand in the absence of the compound to be tested, and on the other hand in the presence of the compound to be tested. The two measurements can be carried out consecutively or simultaneously. The two interaction measurements, in the presence or absence of the compound to be tested, are subsequently compared. In a particular embodiment, the compound is identified as potentially useful for the treatment of a cancer if it inhibits the interaction between the AIF protein and the CHCHD4 protein by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90%. Preferably, the compound is identified as potentially useful for the treatment of cancer if it inhibits by at least 50%, 60%, 70%, 80%, or at least 90% the interaction between the AIF protein and the CHCHD4 protein.In a particular embodiment, the compound is identified as potentially useful for the treatment of cancer if it inhibits by at least 50% the interaction between the AIF protein and the CHCHD4 protein.

[0052] The measurement of the interaction of the AIF and CHCHD4 proteins in the presence or absence of the test compound can be carried out using any technique for measuring or quantifying the interaction between two proteins, for example by means of an amplified luminescence proximity homogeneous assay (ALPHA), a surface plasmon resonance (SPR) assay, a thermal shift assay (TSA) method, an immunoprecipitation method, an isothermal titration calorimetry (ITC) assay, a fluorescence resonance energy transfer (FRET) assay, a fluorescence polarization assay or an ELISA assay. In a particular embodiment, the measurement of the interaction of the AIF and CHCHD4 proteins CHCHD4 is performed using an ALPHA or SPR test.

[0053] Preferably, the technique for measuring the interaction of AIF and CHCHD4 proteins is applicable to high-throughput screening.

[0054] In a particular embodiment, the measurement of the interaction between the AIF protein and the CHCHD4 protein is carried out using an amplified luminescence proximity homogeneous assay (ALPHA). The ALPHA technology is a highly sensitive technique based on chemiluminescence detection that allows screening of a wide range of biological interactions and activities and is also suitable for high-throughput screening (see, for example,

[0055] Eglen RM et al. The use of AlphaScreen technology in HTS: current status. Curr Chem Genomics. 2008;1:2-10 ; Ullman EF et al. Luminescent oxygen channeling im-munoassay: measurement of particle binding kinetics by chemiluminescence. Proc Natl Acad Sci USA. 1994;91(12):5426-5430 ; Yasgar A et al. AlphaScreen-Based Assays: Ultra-High-Throughput Screening for Small-Molecule Inhibitors of Challenging Enzymes and Protein-Protein Interactions. Methods Mol Biol. 2016;1439:77-98 ; Seethala and Prabhavathi, "Homogeneous Assays: AlphaScreen, Handbook of Drug Screening," Marcel Dekkar Pub. 2001, pp. 106-110.).

[0056] ALPHA technology, also known under the trade names AlphaScreen® and AlphaLISA®, measures the interaction of two molecules bioconjugated to donor beads and acceptor beads. The donor beads contain a photosensitive molecule, such as phthalocyanine, which converts ambient oxygen to singlet oxygen after excitation at 680 nm. Singlet oxygen can diffuse up to about 200 nm in solution. If an acceptor bead is within this distance, energy is transferred from the singlet oxygen to thioxene derivatives in the acceptor bead, leading to light emission at 520-620 nm (AlphaScreen®) or 615 nm (AlphaLISA®). If the donor bead is not near an acceptor bead, the singlet oxygen returns to the ground state and no luminescent signal is produced.In a protein / protein interaction assay, one protein is conjugated to the donor beads, and the other protein is conjugated to the acceptor beads. Thus, when the two proteins interact, the donor bead is brought into proximity to the acceptor bead, and excitation of the donor bead leads to the emission of a quantifiable light signal from the acceptor bead. The measurement of the light signal is performed using an ALPHA technology-compatible reader such as the EnVision® or EnSpire® plate reader. In a particular embodiment, the measurement of the interaction is performed using the AlphaScreen® assay.

[0057] The intensity of the light signal therefore makes it possible to quantify the interaction of proteins in a sample. Thus, the compound to be tested is identified as potentially useful for the treatment of cancer if the measurement of the light signal is lower in the presence of said compound than in the absence of said compound. In a particular embodiment, the compound is identified as potentially useful for the treatment of cancer if it inhibits by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90% the light signal measured in the absence of the compound to be tested. Preferably, the compound is identified as potentially useful for the treatment of cancer if it inhibits by at least 50%, 60%, 70%, 80%, or at least 90% the light signal measured in the absence of the compound to be tested. In a particular embodiment, the compound is identified as potentially useful for the treatment of cancer if it inhibits by at least 50% the light signal measured in the absence of the compound to be tested.

[0058] In the context of the present invention, the AIF protein can be conjugated to the donor bead and the CHCHD4 protein to the acceptor bead, and vice versa: the AIF protein can be conjugated to the acceptor bead and the CHCHD4 protein to the donor bead. In a particular embodiment, the donor beads and the acceptor beads are coated with molecules or functional groups allowing their conjugation with one or other of the AIF or CHCHD4 proteins.

[0059] Proteins can be fused to tags that allow their conjugation to donor or acceptor beads. For example, the AIF protein or the CHCHD4 protein can be fused to a histidine tag that allows its conjugation to a nickel-coated bead, or to a GST tag that allows its conjugation to a glutathione-coated bead.

[0060] Thus, according to a particular embodiment, the method comprises:

[0061] (a') bringing into contact an AIF protein, a CHCHD4 protein, a bead donor and an acceptor bead, in the presence or absence of a candidate compound

[0062] (b') the excitation of the donor bead at a given wavelength, preferably at a wavelength of about 680 nm;

[0063] (c') the measurement of the light signal emitted by the acceptor ball, the light signal preferably having a wavelength between 520 and 620 nm;

[0064] (d') the comparison of the measurement of the light signal in the presence and in the absence of the compound ;

[0065] wherein the donor bead is capable of conjugating to the AIF protein and the acceptor bead to the CHCHD4 protein, or

[0066] wherein the donor bead is capable of conjugating to the CHCHD4 protein and the acceptor bead to the AIF protein;

[0067] said compound being identified as potentially useful for the treatment of cancer if the measurement of the light signal is lower in the presence of said compound than in the absence of said compound.

[0068] In another embodiment, the measurement of the interaction of the AIF and CHCHD4 proteins in the presence or absence of the test compound is measured using a surface plasmon resonance (SPR) assay such as the Biacore® assay. During an SPR experiment, one of the two proteins is fixed to a surface (sensor chip), while the other is delivered to the surface via a continuous flow of buffer using a microfluidic system. The interaction of the proteins is monitored by surface plasmon resonance, which detects mass changes at the surface.

[0069] In a particular embodiment, the steps of contacting (a), measuring the interaction (b) and comparing (c) of the method of the invention as described above can be repeated. In particular, steps (a), (b), (c) can be repeated using, for each repetition, the same technique for measuring the interaction between the AIF and CHCHD4 proteins, or a different measurement technique. The repetition of steps (a), (b), (c) has the advantage of refining the screening method and confirming the ability of the compound to inhibit the interaction between the AIF and CHCHD4 proteins. For example, for each compound to be tested, steps (a), (b), (c) can be carried out in duplicate, triplicate, quadruplicate or quintuplicate.

[0070] Furthermore, steps (a), (b), (c) of the method of the invention can be repeated using the same technique for measuring protein interaction, for example ALPHA technology, but by varying, for each repetition, the concentration of the compound to be tested. Varying the concentration of the compound to be tested has the advantage of specifying the inhibition capacities of the identified compound, for example by determining the effect-dose relationship of the compound. Measuring the interaction of the AIF and CHCHD4 proteins, by varying the concentration of the compound to be tested, makes it possible in particular to determine the median inhibitory concentration (IC50) of said compound. The IC50 gives an indication of the concentration of the compound necessary to inhibit by 50% the interaction between the AIF and CHCHD4 proteins and thus makes it possible to evaluate the effectiveness of said compound in inhibiting the interaction.

[0071] In a particular embodiment, the method comprises:

[0072] - implementing steps (a), (b), (c) as described above, in which the interaction of AIF and CHCHD4 proteins is measured using an ALPHA assay; and

[0073] - repeating steps (a), (b), (c) as described above, in which The interaction of AIF and CHCHD4 proteins is measured using an SPR assay.

[0074] In a particular embodiment, the method comprises in this order:

[0075] - the implementation of steps (a), (b), (c) as described above, in which the interaction of AIF and CHCHD4 proteins is measured using an ALPHA assay by a high-throughput screening experiment using a library of test compounds;

[0076] - the selection of compounds capable of inhibiting by at least 50% the interaction of AIF and CHCHD4 proteins;

[0077] - the determination of the dose-effect for each selected compound comprising the re petition of steps (a), (b), (c) as described above by varying the concentration of the selected compound and measuring the interaction of AIF and CHCHD4 proteins using an ALPHA assay; and

[0078] - repeating steps (a), (b), (c) as described above with the compounds selected, in which the interaction of AIF and CHCHD4 proteins is measured using an SPR assay.

[0079] In a particular embodiment, the method of the invention comprises the implementation of steps (a), (b), (c) as described above, in which the compound is a compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein. The use of a compound to inhibit the interaction between the AIF protein and the CHCHD4 protein can thus serve as a positive control, making it possible to validate the experiment.

[0080] In a particular embodiment, said compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein consists of the sequence of SEQ ID NO: 4, or consists of the sequence of SEQ ID NO: 4 or any functional variant having at least 70%, 80%, 90%, or at least 99% identity with the sequence of SEQ ID NO: 4. By "functional variant" is meant here any variant of the sequence SEQ ID NO: 4 capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein.

[0081] In a particular embodiment, said compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein is selected from the group consisting of: disulfiram, bromocriptine or one of its salts such as bromocriptine mesylate, thioridazine or one of its salts such as thioridazine hydrochloride, Chicago sky blue 6B, mitoxantrone or one of its salts such as mi-toxantrone dihydrochloride, rifapentine, tetraethylenepentamine or one of its salts such as tetraethylenepentamine pentahydrochloride, nisoldipine, merbromine, thiethylperazine or one of its salts such as thiethylperazine dimalate and benidipine or one of its salts such as benidipine hydrochloride.

[0082] In a particular embodiment, the method of the invention comprises a step of comparing the inhibition of the interaction of the AIF and CHCHD4 proteins obtained in the presence of the compound to be tested and the inhibition of the interaction obtained in the presence of the compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein. In a particular embodiment, the compound is identified as potentially useful in the treatment of cancer if the inhibition obtained with the compound corresponds to at least 30%, 40%, 50%, 60%, 70%, 80% or at least 90% of the inhibition obtained with the positive control, namely with the peptide N27. In particular, the compound is identified as potentially useful in the treatment of cancer if the inhibition obtained with the compound corresponds to at least 50% of the inhibition obtained with the positive control, namely with the N27 peptide.

[0083] Confirmation of the anticancer properties of the compound

[0084] In a particular embodiment, the method of the invention further comprises a step of confirmation in a non-human cellular or animal model of cancer, of the anticancer properties of the compound identified as capable of inhibiting the interaction between the AIF and CHCHD4 proteins.

[0085] In a particular embodiment, the method of the invention comprises a step of administering the identified compound into an animal model of cancer, then a step of analyzing the anticancer properties of said compound. Any animal model of cancer can be used, the animal preferably being a mammal. In particular, the animal can be a mouse, a rat, a pig, a rabbit, a chicken, or a non-human primate.

[0086] In a particular embodiment, the method of the invention comprises a step of bringing the identified compound into contact with a cancer cell model, then a step of analyzing the cytotoxic properties of said compound. It may be a cell model cultured in two dimensions (2D) or three dimensions (3D). Any cancer cell model may be used, such as a cancer cell line. For example, the identified compound may be brought into contact with the cancer lines A549, MCF7, or HCT116. 2-Kit

[0087] Another aspect of the invention relates to a kit for the identification of a compound potentially useful for the treatment of cancer, characterized in that it comprises:

[0088] - an AIF protein;

[0089] - a CHCHD4 protein; and

[0090] - means suitable for measuring the interaction between the AIF protein and the protein CHCHD4.

[0091] In the context of the present invention, the AIF protein may be of any origin, preferably of animal origin, in particular a mammal, more preferably of human origin. In a particular embodiment, the AIF protein corresponds to the natural human AIF protein whose NCBI sequence reference is “NP_004199.1”, or any functional variant thereof. In particular, the AIF protein according to the invention may correspond to the natural human polypeptide of sequence SEQ ID NO: 1, or any functional variant, such as AIF2 which is a brain-specific isoform. The expression “functional variant” which refers to the AIF protein designates any polypeptide derived from the structure of the AIF protein and retaining the capacity to bind to the CHCHD4 protein, in particular to the CHCHD4 protein of SEQ ID NO: 2. The functional variants may be variants natural or synthetic such as fragments, mutants, or deleters. Preferably, the functional variant of the AIF protein corresponds to a polypeptide having a sequence identity of at least 50%, 60%, 70%, 80%, 90%, or at least 95% with the AIF protein of sequence SEQ ID NO: 1.

[0092] In the context of the present invention, the CHCHD4 protein may be of any origin, preferably of animal origin, in particular a mammal, more preferably of human origin. In a particular embodiment, the CHCHD4 protein corresponds to the natural human CHCHD4 protein whose NCBI sequence reference is “NP_001091972.1”. In particular, the CHCHD4 protein according to the invention may correspond to the natural human polypeptide of sequence SEQ ID NO: 2 or any functional variant. The expression “functional variant” which refers to the CHCHD4 protein designates any polypeptide derived from the structure of the CHCHD4 protein and retaining the capacity to bind to the AIF protein, in particular to the AIF protein of SEQ ID NO: 1. The functional variants may be natural or synthetic variants such as fragments, mutants or deletions.Preferably, the functional variant of the CHCHD4 protein corresponds to a polypeptide having a sequence identity of at least 50%, 60%, 70%, 80%, 90%, or at least 95% with the CHCHD4 protein of sequence SEQ ID NO: 2.

[0093] In a particular embodiment, the functional variant of the AIF protein is deleted or truncated relative to the AIF protein of SEQ ID NO: 1. In particular, the functional variant of the AIF protein may correspond to a polypeptide in which the transmembrane portion of the AIF protein has been deleted. In a particular embodiment, the functional variant of the AIF protein corresponds to a polypeptide of sequence SEQ ID NO: 3.

[0094] The AIF and CHCHD4 proteins may be modified, provided that this does not prevent the interaction between the two proteins. For example, for the purposes of the experiment to measure their interaction, the AIF and / or CHCHD4 proteins may be fused to a fragment serving as a label (or "tag"). Any conventional label may be used, provided that it does not prevent the interaction between the two proteins. In particular, the AIF and / or CHCHD4 proteins may be fused to a Histidine tag corresponding to a motif consisting of several histidine residues, or to a GST tag corresponding to the glutathione-S-transferase protein.

[0095] By "means suitable for measuring the interaction between the AIF protein and the CHCHD4 protein" is meant any means, for example any reagent, compound, material, support or composition, necessary for implementing the technique for measuring the interaction between said proteins.

[0096] Means suitable for measuring the interaction between the AIF protein and the protein CHCHD4, may be means suitable for any technique for measuring or quantifying the interaction between two proteins. For example, the kit may include means suitable for an amplified luminescence proximity homogeneous assay (ALPHA), a surface plasmon resonance (SPR) assay, a thermal shift assay (TSA), an immunoprecipitation method, an isothermal titration calorimetry (ITC) assay, a fluorescence resonance energy transfer (FRET) assay, a fluorescence polarization assay and / or an ELISA assay.

[0097] Preferably, the means adapted to the measurement of the interaction between the AIF protein and the CHCHD4 protein are applicable to high-throughput screening.

[0098] In a particular embodiment, the kit comprises means suitable for an ALPHA test and / or an SPR test.

[0099] In a preferred embodiment, the kit comprises means suitable for measuring the interaction of AIF and CHCHD4 proteins by an ALPHA test. In particular, the kit may comprise:

[0100] - an AIF protein;

[0101] - a CHCHD4 protein;

[0102] - donor beads capable of emitting reactive oxygen species, such as than singlet oxygens, when excited at a given wavelength, preferably about 680 nm; and

[0103] - acceptor beads capable of emitting a light signal, preferably between 520 nm and 620 nm, following reaction with reactive oxygen species, such as singlet oxygens;

[0104] the donor and acceptor beads being further capable of conjugating with one or other of the AIF or CHCHD4 proteins.

[0105] In a particular embodiment, the donor beads and the acceptor beads are coated with molecules or functional groups allowing their conjugation with one or other of the AIF or CHCHD4 proteins. In particular, the donor and acceptor beads may be coated with a layer of Nickel, Glutathione, streptavidin, protein A, G, L or antibodies. In a particular embodiment, the donor beads are coated with Nickel and the acceptor beads are coated with glutathione.

[0106] In a particular embodiment, the donor beads included in the kit are pre-conjugated to one or other of the AIF or CHCHD4 proteins. In a particular embodiment, the acceptor beads included in the kit are pre-conjugated to one or other of the AIF or CHCHD4 proteins.

[0107] In a particular embodiment, the donor and acceptor beads are beads from the commercial Alphascreen ® or Alphalisa® test (Perkinelmer).

[0108] Optionally, the kit as described above may comprise a buffer adapted to the experiment for measuring the interaction between the AIF protein and the CHCHD4 protein. By "buffer" is meant any solution in which the AIF and CHCHD4 proteins, the compound to be tested and possibly the means suitable for measuring the interaction of the proteins are brought into contact. The buffer is chosen so as not to inhibit the interaction between the AIF and CHCHD4 proteins. Thus, the buffer can also serve as a negative control when implementing the method of the invention.

[0109] In a particular embodiment, the kit comprises a buffer suitable for an ALPHA test.

[0110] In a particular embodiment, the buffer comprises phosphate buffered saline (PBS) and bovine serum albumin (BSA). In a particular embodiment, the buffer comprises PBS and 0.1% BSA.

[0111] Optionally, the kit as described above may comprise a compound capable to inhibit the interaction between the AIF protein and the CHCHD4 protein. The compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein can thus serve as a positive control, making it possible to validate the experiment measuring the interaction between the AIF and CHCHD4 proteins, when implementing the method of the invention.

[0112] In a particular embodiment, said compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein consists of the sequence of SEQ ID NO: 4. or any functional variant having at least 70%, 80%, 90%, or at least 99% identity with the sequence of SEQ ID NO: 4. By "functional variant" is meant here any variant of the sequence SEQ ID NO: 4 capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein.

[0113] In a particular embodiment, said compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein is selected from the group consisting of: disulfiram, bromocriptine or one of its salts such as bromocriptine mesylate, thioridazine or one of its salts such as thioridazine hydrochloride, Chicago sky blue 6B, mitoxantrone or one of its salts such as mi-toxantrone dihydrochloride, rifapentine, tetraethylenepentamine or one of its salts such as tetraethylenepentamine pentahydrochloride, nisoldipine, merbromine, thiethylperazine or one of its salts such as thiethylperazine dimalate and benidipine or one of its salts such as benidipine hydrochloride.

[0114] The kit as described above may further comprise any support suitable for the experiment of measuring the interaction between the AIF protein and the CHCHD4 protein. For example, the support may be chosen from a plate, a tube, a flask, a membrane, etc. In particular, the support may be a multi-well plate which makes it possible to conduct, in parallel, numerous and varied tests. Typical supports include microtitration plates and more particularly plates 96 or 384 wells (or more), easy to handle.

[0115] One aspect of the invention further relates to the use of the kit as described above, in a method of identifying a compound potentially useful for the treatment of cancer. 4- Composition

[0116] Another aspect of the invention relates to a pharmaceutical composition comprising a compound identified by the method as described above, in association with a pharmaceutically acceptable carrier.

[0117] Thus, one aspect of the invention relates to a pharmaceutical composition comprising a compound capable of inhibiting the interaction between the AIF and CHCHD4 proteins, in association with a pharmaceutically acceptable vehicle.

[0118] By pharmaceutically acceptable vehicle is meant any substance other than the active ingredient in a medicament. Its addition is intended to confer physicochemical and / or biochemical characteristics to promote administration by oral, sublingual, respiratory, rectal, nasal, intestinal, parenteral, intravenous, intraperitoneal, intramuscular, subcutaneous injection, or other particular consistency or taste characteristics, to the final product, preferably avoiding covalent chemical interactions with the active ingredients.

[0119] The pharmaceutical compositions of the invention may be in the form of simple or coated tablets, sublingual tablets, capsules, glossettes, capsules, lozenges, injectable preparations, aerosols, nasal drops, suppositories, creams, ointments or dermal gels. 5- Uses

[0120] Another aspect of the invention relates to a compound identified by the method as described above, for use as a medicament. Thus, one aspect of the invention relates to a compound capable of inhibiting the interaction between AIF and CHCHD4 proteins, for use as a medicament.

[0121] In particular, one aspect of the invention relates to a compound identified by the method as described above, for use in a method of treating cancer. Thus, one aspect of the invention relates to a compound capable of inhibiting the interaction between AIF and CHCHD4 proteins, for use in a method of treating cancer.

[0122] In a particular embodiment, the compound capable of inhibiting the interaction between the AIF and CHCHD4 proteins is selected from the group consisting of: chicago sky blue 6b, rifapentine, nisoldipine, merbromine, thiethylperazine or one of its salts such as thiethylperazine dimalate, and benidipine or one of its salts such as benidipine hydrochloride.

[0123] A method of treating cancer in a subject is thus described, comprising administering to said subject a compound capable of inhibiting the interaction between the AIF and CHCHD4 proteins or the pharmaceutical composition comprising said compound.

[0124] The method of treatment may comprise administering the compound capable of inhibiting the interaction between the AIF and CHCHD4 proteins or the pharmaceutical composition comprising said compound, alone or in combination with any anti-cancer treatment such as radiotherapy, chemotherapy and immunotherapy. In particular, the method of treatment may comprise administering said compound capable of inhibiting the interaction between the AIF and CHCHD4 proteins, in combination with the administration of a chemotherapeutic agent. The administration of the compound capable of inhibiting the interaction between the AIF and CHCHD4 proteins may be carried out prior to, simultaneously with, or subsequent to the administration of the chemotherapeutic agent. The term "chemotherapeutic agent" means a chemical product that can be used to destroy a cancer cell, or to slow, stop or reverse the growth of a cancer cell.

[0125] In the context of the invention, the term "subject" or "patient" means an animal, preferably a mammal, in particular a human being, regardless of age or sex, suffering from cancer. The term includes domestic animals as well as laboratory animals such as non-human primates, felines, canines, equines, porcines, bovines, goats, sheep, rabbits, rats and mice. Preferably, the patient to be treated is a human being.

[0126] As used herein, the term "cancer" means any type of malignant tumor. The malignant tumor may be a primary tumor or a secondary tumor (i.e., a metastasis). In addition, the tumor may be a solid malignant tumor, which includes, for example, carcinomas, adenocarcinomas, sarcomas, melanomas, mesotheliomas, blastomas, or a blood cell cancer such as leukemias, lymphomas, and myelomas. The cancer may, for example, be skin cancer, lung cancer, bladder cancer, kidney cancer, digestive cancer (colon, pancreas, liver), ovarian cancer, brain cancer, face and neck cancer, etc.

[0127] The term “treatment” includes curative and / or preventive treatment. Curative treatment refers to the reduction, improvement, stabilization and / or elimination of a symptom of the disease, or the inhibition of the progression of a symptom of the disease. Preventive treatment refers to any of the following effects: preventing or delaying the onset of a given disorder, reducing the development, risk of development, incidence or severity of a disorder, increasing the time to onset of symptoms and / or patient survival.

[0128] Another aspect of the invention relates to the use in academic research of a compound identified by the method as described above, for its use as a compound for disrupting mitochondrial import and / or cellular metabolism.In a particular embodiment, said compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein is selected from the group consisting of: disulfiram, bromocriptine or one of its salts such as bromocriptine mesylate, thioridazine or one of its salts such as thioridazine hydrochloride, Chicago sky blue 6B, mitoxantrone or one of its salts such as mi-toxantrone dihydrochloride, rifapentine, tetraethylenepentamine or one of its salts such as tetraethylenepentamine penta-hydrochloride, nisoldipine, merbromine, thiethylperazine or one of its salts such as thiethylperazine dimalate and benidipine or one of its salts such as benidipine hydrochloride. Examples

[0129] MATERIALS AND METHODS

[0130] English / French abbreviations:

[0131] AIF apoptosis-inducing factor

[0132] BSA bovine serum albumin

[0133] CHCHD4 Coiled-Coil-Helix-Coiled-Coil-Helix (CHCH) Domain 4

[0134] CI50 concentration inhibitrice médiane

[0135] CV coefficient de variation

[0136] DMSO dimethyl sulfoxide

[0137] GST Glutathione S-transferase

[0138] HIS histidine

[0139] HTS High Throughput Screening

[0140] MEF Mouse embryonic fibroblast

[0141] PBS phosphate buffer saline

[0142] RT room température

[0143] RU résonance unit

[0144] S / B signal over background ratio

[0145] SD standard deviation / déviation standard

[0146] SPR surface plasmon résonance

[0147] TH threshold / seuil Expression protéique et purification

[0148] The AIF103-613 (fragment 103-613 of the AIF protein) and CHCHD4 proteins were produced in BL21 + DE3 (RIPL) bacteria, via a 3-hour induction with 0.5 mM IPTG at 37°C. The bacteria were transformed according to the supplier's recommendations (Agilent). The proteins were then purified. The Total protein concentration was determined by a Bradford assay kit. Expression purity was also assessed on SDS-containing polyacrylamide gel.

[0149] Testing the interaction of AIE and CHCHD4 proteins and identifying compounds modulating this interaction using an Alpha test

[0150] A method for identifying compounds capable of inhibiting the interaction between AIF and CHCHD4 proteins and suitable for high-throughput screening has been developed. This method comprises the use of:

[0151] - previously produced AIF103-613 and CHCHD4 proteins;

[0152] - an AlphaScreen® kit (PerkinElmer) comprising donor beads and beads acceptors capable of conjugating to AIF and CHCHD4 proteins;

[0153] - a positive control: peptide N27 (corresponding to the 27 amino acids of the N-ter terminus of the CHCHD4 protein, as described in Hangen et al, 2015;

[0154] - a negative control: 0.1% bovine serum albumin (BSA) solution in phosphate buffered saline (PBS)

[0155] - a bank of compounds to be tested: the Prestwick chemical compound bank (Prestwick Chemical Library®).

[0156] The ALPHA assay was first performed at high throughput using 384-well white microplates (Greiner, reference 781075), using 25 pL of total volume per well. Proteins, positive control and beads were diluted in a 0.1% BSA / PBS solution. All distributions were performed with an electronic multipette, then the plates were centrifuged (200 g, 1 min). The plates were sealed and incubated in the dark at 23°C.

[0157] A library of 1,280 small molecules provided by Prestwick Chemicals (Prestwick Chemical Library®) was screened. This library includes compounds with diverse chemical and pharmacological properties, which are already approved by health agencies such as the FDA (Food and Drug Administration) or the EMEA (European Medicines Evaluation Agency). Each compound in the library was diluted in 0.1% DMSO solution and used at a concentration of 10 pM. First, 5 pL of 10 pM compound, 3 pM N27 (positive control) or 0.1% BSA / PBS (negative control) were added to the wells. Second, 5 pL of AIF103-613 proteins were incubated with the compounds for 20 min before adding 5 pL of CHCHD4 for 2 h. Third, 10 μL of a mixture of donor beads and acceptor beads at 5 μg / mL was incubated for 3 h. Analysis of the results was performed using an EnSpire® multi-mode plate reader (Perkin Elmer).The interaction of AIF and CHCHD4 proteins is revealed by detection of a light signal by the plate reader. Analysis of ALPHA test data

[0158] For each experiment measuring the interaction of AIF and CHCHD4 proteins, 3 measurements validating the quality of the experiment were carried out: the coefficient of variation of the controls (CV), the signal / background noise ratio (S / N) and the Z' factor as described below.

[0159] For each 384-well plate, 16 wells correspond to the positive control and 16 wells correspond to the negative control. The positive control corresponds to the measurement of the interaction of the AIF103-613 and CHCHD4 proteins in the presence of the N27 peptide (equivalent to the minimal signal). The negative control corresponds to the measurement of the interaction of the AIF103-613 and CHCHD4 proteins in the presence of the buffer (0.1% BSA / PBS) (equivalent to the maximal signal).

[0160] CV is defined as the ratio of the standard deviation (SD) divided by the mean of each control:

[0161] [Math.l] 51) Cst i / Mf dis contrôfe) ----- .---—--:----y— WG moyeîmt' stu concrète)

[0162] S / B is the ratio corresponding to the average of the negative controls, divided by the average of the positive controls:

[0163] [Math.2]

[0164] The Z' factor evaluates the amplitude of the signal of a test, by measuring the difference between the positive and negative controls, and taking into account the standard deviations (SD):

[0165] [Math.3] £ ---------------------------------------' ----------;--------------------------------—'--------

[0166] Each measurement series is validated if CV < 15%, S / N > 10 and the Z' factor > 0.5 [Goktug et al., Drug Discovery, 2013 doilO.5772 / 52508].

[0167] Following high-throughput screening, compounds are selected if the signal obtained in the presence of the compound is lower than the following TH threshold:

[0168] TH = mean (negative control signal) - 5 * SD (negative control signal)

[0169] For each previously selected compound, the ALPHA test is repeated manually using another batch of powder of the compounds.

[0170] Each compound is tested at 10pM and 30pM due to the Hook effect specific to ALPHAscreen technology and in order to select compounds giving a signal located in the ascending part of the Hook bell and therefore active at low doses from a drug development perspective.

[0171] The compound is selected if:

[0172] - the signal obtained in the presence of the compound is lower than the TH threshold as described above. above; and

[0173] - that the signal obtained at 30pM is lower than the signal obtained at 10pM.

[0174] In addition, for each compound thus selected, the effect-dose relationship of the compound is determined. This makes it possible to determine the median inhibitory concentration (IC50) of said compound.

[0175] Validation of compounds inhibiting the interaction of AIF and CHCHD4 proteins using an SPR test

[0176] Some of the compounds identified using the Alpha assay as described above were tested by surface plasmon resonance (Biacore™ T100, GE Healthcare Life Sciences). The CHCHD4 protein was covalently immobilized on a dextran matrix comprising COOH functional carboxyl groups (Series S CM5® sensor chip, GE Healthcare Life Sciences, 29149603). To perform the screening, the compounds (at 10 pM) were pre-incubated for 20 min with the AIF protein, at room temperature in a DPBS buffer at pH 7.4 (Sigma, D8577). Then, the mixture of the compound and the AIF protein was injected for 3 min at 30 pL per minute at 25°C. A negative control was performed by injecting the AIF protein alone at 1 pM (diluted in 0.1% DMSO / PBS). A positive control was performed by injecting a mixture of peptide at N27 (at 3 pM) and AIF protein.

[0177] A compound is validated if it is capable of significantly reducing the interaction between AIF103-613 and CHCHD4, i.e. if the inhibition obtained with the compound corresponds to at least 50% of the inhibition obtained with the positive control, namely with the peptide N27. The sensorgrams were analyzed using the BIAevaluation software (version 2.0.4).

[0178] RESULTS

[0179] Protocol development and optimization for high-throughput screening.

[0180] The concentrations of AIF103-613 and CHCHD4 proteins required to obtain an optimal signal in Alphascreen® were evaluated. [Fig.l] shows that the optimal signal obtained with AIF103-613 and CHCHD4 is dose-dependent. Then the batches of proteins, peptides and reagents were checked in a preliminary experiment and obtained a Z'>0.5 (not shown).

[0181] Summary of statistics obtained by high-throughput screening in 384-well plates

[0182] Then the Prestwick library (1280 molecules) was screened using 4 384 plates wells (Plate #1 to #4).

[0183] Before the screening itself, all reagents (proteins, buffer beads) and materials (plates, tips, pipettor) are subjected to a quality control called "pre-screening validation test" and a number of criteria are analyzed. As indicated above, each measurement series is validated if CV < 15%, S / N > 10 and the Z' factor > 0.5.

[0184] The statistical data obtained by high-throughput screening in 384-well plates are detailed in the following Table 1:

[0185] [Tables 1] Analysis criteria Pre-screening validation tests Plate "phqaT' 42 iage Instar #3 Plate 44 16 16 14 16 Number of wells wHîixOes positives es negattis Çtgùmks negatives | Movetme 22249 2 " 30459 19650 SD 2504 1303 1436 2170 "12 i CT CT) m 6- 5 •7 4 i. oiCTCT- positives Mvjcnnc 2123 1776 2242 2526 [S33 SD 290 146 i2» 26° Mi j cvcw 14 & c 11 *7 SB 12.4 H ) ' 12< n ZMaetw 0.65 0.79 0.74 0.86

[0186] Identification of compounds inhibiting the interaction between AIF and CHCHD4 proteins

[0187] 1) Identification of compounds using an ALPHA test

[0188] The Prestwick compound library, comprising 1280 molecules, was screened using 4 384-well plates, as described above. The compounds were selected on the basis of the signal obtained in Alphascreen® in the presence of the compound at a concentration of 100M. In particular, the compound is selected if the signal obtained at 100M is lower than a threshold value TH (TH = mean (signal of the negative control) - 5 * SD (signal of the negative control)). Of the 1280 compounds analyzed, 148 were selected, i.e. 12% of the compound library (see [Fig.2]).

[0189] The 148 selected compounds were again tested using a manually performed ALPHA test as described above. Of the 148 compounds tested, 11 compounds were finally selected. For the 11 selected compounds:

[0190] - the signal obtained at OpM is lower than the threshold value TH; and

[0191] - the signal obtained at 30pM is lower than the signal obtained at 10pM.

[0192] The following Table 2 lists the 11 compounds selected by ALPHA screen. The CAS numbers and chemical structures of each compound are reported in the following Table 2. Compound Name V CAS Chemical Structure 1 Disulfiram 97-77-8 h H=c' ? s V" H,C^ £ > OT Bromocriptine Mesylate 22260-51-1 . / "À T rvx 'il c „ HsC—S—OH f 7" II p—S 3 Thioridazine Hydrochloride 130-61-0 v..XÀ X i \ ..7 XX Xx >> 4 Chicago sky blue 6B 2610-05-1 ' pi-S . a:y fiwS ' / ' ? v / "'Xa

[0194] [Tables2] 5 Mitoxane dichlorhydrate finds 70476-82-3 -XK ..-X OH 0 K” x'z à A x ct ij OH O iX „.-x .GH X.-- 6 Recap 61379-65-5 OH i S V.H HC -1 OH :: i ■■ H;C CF '......A ' W rr ,1 XA..f A - K o % 9 1 . XX" < 7 cio" o \ k À-. / "Q XV b 7 ,0^ HC""OK < . •' one. 9 Merbromine 129-16-8 ..OH O(. .....O, / .X. ..,0" Br'' x8r JA 10 Thiethylperazme dimalate 52239-63-1 R o A x, a  .. .... y .0 ' ; ' y œ' O' * ' - ; ,. <y.àx ..-y ..s.. .a < .--a ,9' .... s- x,- 11 chlorhydrate de benidipine 91599-74-5 ya » y ? n h£. a .¼ v 0        v a- - ch-

[0196] The anti-cancer properties of some of these compounds have already been demonstrated, thus validating the screening method of the present invention. For example, the anti-cancer properties of thioridazine are described in the article by Shen et al., 2017. In addition, mitoxantrone is marketed as an anti-cancer agent (under the name Novantrone®), particularly in the treatment of metastatic breast cancer, non-Hodgkin's lymphoma or acute myeloid leukemia (AML).

[0197] The following table 3 lists the IC50 of the 11 identified compounds, obtained manually using the Alphascreen test: Compound IC50 AIF / OECHD4 Disuifiram 415 nM Bromocriptine mesylate 4.57 pM Thioridazine hydrochloride 24 pM Chicago sky bine 6B 1.44 pM Mitoxantrone dihydrochloride 1.27 pM Rifapentine 4.8 pM Tetraethylenepentamine pentahydrochloride 9 pM Nisoldipine 12 pM Merbromine 169 nM Thiethy dimalate Iperazine 10.6 pM Benidipine hydrochloride 8 pM

[0199] 2) Confirmation of the inhibition of the interaction of AIF and CHCHD4 proteins at using an SPR test

[0200] The inhibition of the interaction of AIF and CHCHD4 proteins by thioridazine and mitoxantrone was analyzed by means of a surface plasmon resonance test (see [Fig.3]). A compound is considered to inhibit the interaction of AIF and CHCHD4 proteins if the inhibition obtained with the compound corresponds to at least 50% of the inhibition obtained with the positive control, namely with the peptide N27.

[0201] Surface plasmon resonance assay confirmed that thioridazine and mitoxantrone are capable of inhibiting the interaction between AIF and CHCHD4 proteins. References

[0202] Hangen, E., et al., Interaction between AIF and CHCHD4 regulates respiratory chain biogenesis. Molecular cell, 2015. 58(6): p. 1001-1014.

[0203] Shen, J., et al., Thioridazine has potent antitumor effects on lung cancer stem-like cells. Oncology letters, 2017. 13(3): p. 1563-1568.

Claims

Claims

1. A compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein, for use in the treatment of cancer, wherein the compound is selected from the group consisting of: chicago sky blue 6B, rifapentine, nisoldipine, merbromine, and thiethylperazine or a salt thereof such as thiethylperazine dimalate.

2. A compound for use according to claim 1, wherein the compound is selected from the group consisting of: chicago sky blue 6B, rifapentine, nisoldipine, merbromine, and thiethylperazine dimalate.

3. A compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein, said compound being selected from the group consisting of: chicago sky blue 6B, rifapentine, nisoldipine, merbromine, and thiethylperazine or a salt thereof such as thiethylperazine dimalate; said compound being combined with a chemotherapeutic agent, for use in the treatment of cancer.

4. A compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein, said compound being selected from the group consisting of: chicago sky blue 6B, rifapentine, nisoldipine, merbromine, and thiethylperazine or a salt thereof such as thiethylperazine dimalate; said compound being combined with an immunotherapeutic agent, for use in the treatment of cancer.

5. A compound for use according to any preceding claim, wherein the cancer is a solid malignant tumor, in particular a carcinoma, an adenocarcinoma, a sarcoma, a melanoma, a mesothelioma, or a blastoma.

6. A compound for use according to any one of claims 1 to 4, wherein the cancer is a blood cell cancer, in particular leukemia, lymphoma or myeloma.

Images