Methods for identifying compounds useful in the treatment of cancer - Patents.com

Abstract

The present invention relates to a method for identifying compounds useful in the treatment of cancer based on assessing the ability of the compound to modulate the interaction between the AIF protein and the CHCHD4 protein.

Description

Technical Field

[0001] The present invention relates to a method for identifying a compound useful for the treatment of cancer based on evaluating the ability of a compound to modulate the interaction between AIF protein and CHCHD4 protein.

Background Art

[0002] During the carcinogenic process, the metabolism of cancer cells is initialized, promoting the growth of tumor cells, improving their repair ability, invasion ability, and resistance to anticancer treatment. Mitochondria play an important role in cell metabolism, especially through their ability to produce ATP, various metabolites and macromolecules, produce and detoxify reactive oxygen species, and regulate cell death. For this reason, targeting mitochondrial activity and affecting the metabolism of cancer cells is a promising approach in cancer treatment. However, the molecular mechanisms of mitochondria that can bring about therapeutic usefulness remain unclear.

[0003] To function, mitochondria must take up 1500 - 2000 proteins encoded by the nuclear genome through specific protein machinery. Thus, AIF (apoptosis-inducing factor) and CHCHD4 (coiled-coil helix coiled-coil helix domain-containing 4) are two proteins expressed in the intermembrane space, and once bound, they form an uptake mechanism for various proteins with cysteine motifs.

[0004] The AIF / CHCHD4 interaction is described in the paper Hangen et al., 2015 in vitro and in cellulo. In particular, this paper shows that the function of AIF protein in the biosynthesis of mitochondrial respiratory chain complexes is mediated by physical and functional interactions with CHCHD4.

Prior Art Documents

Non-Patent Documents

[0005] [Non-Patent Document 1] Hangen et al., 2015 in vitro and in cellulo. [Non-Patent Document 2] Eglen RM et al., The use of AlphaScreen technology in HTS: current status. Curr Chem Genomics. 2008;1:2 - 10 pages [Non-Patent Document 3] Ullman EF et al., Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc Natl Acad Sci U S A. 1994;91(12):5426 - 5430 pages [Non-Patent Document 4] 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 pages [Non-Patent Document 5] Seethala and Prabhavathi, "Homogeneous Assays: AlphaScreen, Handbook of Drug Screening," Marcel Dekkar Pub. 2001, 106 - 110 pages [Non-Patent Document 6] Goktug et al., Drug Discovery, 2013 doi10.5772 / 52508 [Summary of the Invention] [Means for Solving the Problems]

[0006] For the purpose of identifying novel compounds useful for the treatment of cancer, the inventors have developed an identification method based on evaluating the ability of compounds to inhibit the interaction between AIF protein and CHCHD4 protein. The inventors' hypothesis is that compounds that inhibit the formation of the AIF / CHCHD4 complex may be able to affect cancer cells whose survival and proliferation depend on mitochondrial activity.

[0007] The method developed by the inventors has proven to be particularly effective in enabling the identification of compounds with anti-cancer properties. Furthermore, since this method can be applied to high-throughput screening, it has the advantage of facilitating the identification of compounds potentially useful for the treatment of cancer.

[0008] Accordingly, one aspect of the present invention is a method for identifying a compound potentially useful for the treatment of cancer, comprising the step of evaluating the ability of a compound to inhibit the interaction between AIF protein and CHCHD4 protein, wherein if the compound inhibits the interaction, it is identified as potentially useful for the treatment of cancer.

[0009] In a specific embodiment, the method (a) contacting AIF protein and CHCHD4 protein in the presence and absence of the compound; (b) measuring the interaction between AIF protein and CHCHD4 protein in the presence and absence of the compound; (c) comparing the measured values of the interaction in the presence and absence of the compound and if the measured value of the interaction is lower in the presence of the compound than in the absence of the compound, the compound is identified as potentially useful for the treatment of cancer.

[0010] In a specific embodiment, the compound is identified as potentially useful for the treatment of cancer if it inhibits the interaction between AIF protein and CHCHD4 protein by at least 50%, 60%, 70%, 80%, or at least 90%.

[0011] The measurement of the interaction between the AIF protein and the CHCHD4 protein can be carried out using an amplified luminescence proximity homogeneous assay (ALPHA) or a surface plasmon resonance (SPR) assay. Furthermore, the method according to the present invention may include confirmation of the anti-cancer properties of the identified compound in a non-human animal model or cell model of cancer.

[0012] In a specific embodiment, the method comprises (i) determining the ability of a compound to inhibit the interaction between the AIF protein and the CHCHD4 protein using an amplified luminescence proximity homogeneous assay (ALPHA); (ii) determining the ability of a compound to inhibit the interaction between the AIF protein and the CHCHD4 protein using a surface plasmon resonance (SPR) assay; (iii) confirming the anti-cancer properties of the identified compound in a non-human animal model or cell model of cancer and comprising.

[0013] Another aspect of the present invention is a kit for identifying a compound potentially useful for the treatment of cancer, comprising - an AIF protein, - a CHCHD4 protein, - means suitable for measuring the interaction between the AIF protein and the CHCHD4 protein, - optionally, a buffer suitable for the experiment for measuring the interaction, - optionally, a compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein and relating to a kit characterized by comprising.

[0014] A means suitable for measuring the interaction between the AIF protein and the CHCHD4 protein can be a means suitable for an amplified luminescence proximity homogeneous assay (ALPHA). In a particular embodiment, a buffer suitable for an experiment measuring the interaction between the AIF protein and the CHCHD4 protein contains phosphate buffered saline (PBS) and bovine serum albumin (BSA). In a particular embodiment, a 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 any functional variant having at least 70%, 80%, 90%, or at least 99% identity to the sequence of SEQ ID NO: 4. Brief Description of the Drawings

[0015]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0016] 1- Identification method One aspect of the present invention is a method for identifying a compound potentially useful for the treatment of cancer, which includes the step of evaluating the ability of a compound to inhibit the interaction between an AIF protein and a CHCHD4 protein, and when the compound inhibits the interaction, it is identified as potentially useful for the treatment of cancer. One aspect of the present invention is, in particular, a method for screening a compound library to identify a compound potentially useful for the treatment of cancer, which includes the step of evaluating the ability of a compound in the compound library to inhibit the interaction between an AIF protein and a CHCHD4 protein, and when the compound inhibits the interaction, it is identified as potentially useful for the treatment of cancer.

[0017] In a specific embodiment, the method according to the present invention (a) contacting an AIF protein and a CHCHD4 protein in the presence and absence of a compound; (b) measuring the interaction between the AIF protein and the CHCHD4 protein in the presence and absence of the compound; (c) comparing the measured values of the interaction in the presence or absence of the compound; comprising If the measured value of the interaction is lower in the presence of the compound than in its absence, the compound is identified as potentially useful for the treatment of cancer.

[0018] AIF protein and CHCHD4 protein The physical and direct interaction between the AIF protein and the CHCHD4 protein was shown in the paper by Hangen et al., 2015.

[0019] The AIF (apoptosis-inducing factor) protein is a flavoprotein present in mitochondria and promotes apoptosis when released from mitochondria. In the context of the present invention, the AIF protein may be of any origin, preferably of animal origin, particularly mammalian origin, more preferably of human origin. In a particular embodiment, the AIF protein is the human native AIF protein with the NCBI sequence reference "NP_004199.1", or any functional variant thereof, for example, corresponding to AIF2 which is a specific brain isoform. In particular, the AIF protein according to the present invention may correspond to the native human polypeptide of the sequence of SEQ ID NO: 1, or any functional variant. The expression "functional variant" when referring to the AIF protein means any polypeptide derived from the structure of the AIF protein and retaining the ability to bind to the CHCHD4 protein, particularly the CHCHD4 protein of SEQ ID NO: 2. The functional variant may be a natural or synthetic variant such as a fragment, a deletion mutant, etc. Preferably, the functional variant of the AIF protein corresponds to a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or at least 95% sequence identity with the AIF protein of the sequence of SEQ ID NO: 1.

[0020] The CHCHD4 protein (also known as mitochondrial intermembrane space import and assembly protein 40, or MIA40) is a protein involved in the import of proteins in the mitochondrial intermembrane space. In the context of the present invention, the CHCHD4 protein may be of any origin, preferably of animal origin, particularly of mammalian origin, and more preferably of human origin. In a particular embodiment, the CHCHD4 protein corresponds to the human native CHCHD4 protein with an NCBI sequence reference of "NP_001091972.1". In particular, the CHCHD4 protein according to the present invention may correspond to the native human polypeptide of the sequence of SEQ ID NO: 2, or any functional variant. The expression "functional variant" when referring to the CHCHD4 protein means any polypeptide that is derived from the structure of the CHCHD4 protein and retains the ability to bind to the AIF protein, particularly the AIF protein of SEQ ID NO: 1. The functional variant may be a natural or synthetic variant such as a fragment, mutant, or deletion. Preferably, the functional variant of the CHCHD4 protein corresponds to a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or at least 95% sequence identity with the CHCHD4 protein of the sequence of SEQ ID NO: 2.

[0021] In a particular embodiment, the functional variant of the AIF protein is deleted or truncated as compared to the AIF protein of SEQ ID NO: 1. In particular, the functional variant of the AIF protein may correspond to a polypeptide lacking the transmembrane portion of the AIF protein. In a particular embodiment, the functional variant of the AIF protein corresponds to the polypeptide of the sequence of SEQ ID NO: 3, or a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or at least 95% sequence identity with the polypeptide of the sequence of SEQ ID NO: 3.

[0022] The AIF protein and the CHCHD4 protein can be modified as long as the interaction between the two proteins is not hindered. For example, due to the need for measurement experiments of their interaction, the AIF protein and / or the CHCHD4 protein can be fused to a fragment used as a tag. Any conventional tag may be used as long as the interaction between the two proteins is not hindered. In particular, any tag suitable for an amplified luminescence proximity homogeneous assay (ALPHA) or a surface plasmon resonance (SPR) assay can be used. More specifically, the AIF protein and / or the CHCHD4 protein can be fused to a histidine tag corresponding to a motif consisting of several histidine residues, or a GST tag corresponding to a glutathione S-transferase protein.

[0023] Test compound The compounds that can be identified by the method of the present invention can be compounds of various properties, structures and origins. The compounds that can be identified can be, in particular, biological compounds, chemical compounds, synthetic compounds, etc. They can be, in particular, compounds with the properties of nucleic acids, peptides, lipids, carbohydrates. This may also include libraries, in particular chemical libraries, protein libraries, peptide libraries, nucleic acid libraries or natural product libraries, etc.

[0024] Contact of the test compound with the AIF protein and the CHCHD4 protein The test compound can be brought into contact with the AIF protein and the CHCHD4 protein on any suitable support, and in particular on a plate, in a tube or flask, on a membrane, etc. In particular, the contact can be carried out in a multi-well plate, which enables a large number of and various assays to be performed in parallel. Typical supports include microtiter plates, and more specifically, easy-to-handle 96-well or 384-well (or more) plates.

[0025] The amount (or concentration) of the test compound can be adjusted by the user according to the type of the compound, the duration of the incubation period, etc. In particular, the concentration of the test compound may vary from 1 nM to 1 mM. Of course, it is also possible to test other concentrations without departing from the present invention. Furthermore, each compound can be tested in parallel at various concentrations. Similarly, the amount (or concentration) of AIF protein and CHCHD4 protein can be variable and can be adjusted by the user. In particular, the concentrations of AIF protein and CHCHD4 protein are adjusted so that optimal interaction between the two proteins is possible in the absence of the test compound.

[0026] The contact between the protein and the test compound can be maintained for, for example, several minutes to several hours or days, particularly 30 minutes to 72 hours, more specifically 1 to 5 hours.

[0027] The order of addition of AIF protein, CHCHD4 protein and the test compound when contacting them can be adjusted by those skilled in the art. In particular, the test compound can be pre-incubated with AIF protein for a certain period before contacting with CHCHD4 protein. Alternatively, the test compound can be pre-incubated with CHCHD4 protein for a certain period before contacting with AIF protein. The period during which pre-incubation with one or the other of the proteins is carried out can be several minutes, or several hours or days, more specifically 5 to 60 minutes, more specifically 5 to 30 minutes.

[0028] Measurement of the interaction between AIF protein and CHCHD4 protein The interaction between AIF protein and CHCHD4 protein in the presence or absence of the test compound can be measured according to any technique known to those skilled in the art that enables measurement or quantification of the interaction between the two proteins.

[0029] The term "interaction" means the pairing or physicochemical binding between two proteins. The interaction between two proteins can result from covalent and / or non-covalent bonds. Non-covalent bonds include, in particular, electrostatic, ionic, hydrogen, and hydrophobic bonds, as well as van der Waals forces.

[0030] The method according to the present invention includes the step of measuring the interaction between AIF protein and CHCHD4 protein, on the one hand, in the absence of a test compound, and on the other hand, in the presence of a test compound. The two measurements can be carried out continuously or simultaneously. Then, the measured values of the two interactions in the presence or absence of the test compound are compared. In a specific embodiment, the compound is identified as potentially useful for the treatment of cancer if it inhibits the interaction between AIF protein and 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 the interaction between AIF protein and CHCHD4 protein by at least 50%, 60%, 70%, 80%, or at least 90%. In a specific embodiment, the compound is identified as potentially useful for the treatment of cancer if it inhibits the interaction between AIF protein and CHCHD4 protein by at least 50%.

[0031] The measurement of the interaction between AIF protein and CHCHD4 protein, in the presence or absence of a test compound, can be carried out using any technique that enables the measurement or quantification of the interaction between the two proteins, such as amplified luminescence proximity homogeneous assay (ALPHA), surface plasmon resonance (SPR) assay, thermal shift assay (TSA) method, immunoprecipitation, isothermal titration calorimetry (ITC) assay, fluorescence resonance energy transfer (FRET) assay, fluorescence polarization assay, or ELISA assay. In a specific embodiment, the measurement of the interaction between AIF protein and CHCHD4 protein is carried out using an ALPHA or SPR assay.

[0032] Preferably, the technique for measuring the interaction between the AIF protein and the CHCHD4 protein can be applied to high-throughput screening.

[0033] In a particular embodiment, the measurement of the interaction between the AIF protein and the CHCHD4 protein is carried out using the amplified luminescent proximity homogeneous assay (ALPHA). The ALPHA technology is a very sensitive technology based on the detection of chemiluminescence, which enables the screening of a wide range of interactions and biological activities and is also suitable for high-throughput screening (see, for example, 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 immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc Natl Acad Sci U S A. 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, 106-110).

[0034] The ALPHA technology enables the measurement of the interaction of two molecules bioconjugated to donor beads and acceptor beads, also known by the trademark names AlphaScreen® and AlphaLISA®. Donor beads contain a photosensitive molecule such as phthalocyanine, which converts ambient oxygen to singlet oxygen upon excitation at 680 nm. Singlet oxygen can diffuse in solution up to approximately 200 nm. If acceptor beads are within this distance, energy is transferred from singlet oxygen to a thioredoxin derivative in the acceptor beads, resulting in emission at 520 - 620 nm (AlphaScreen®) or 615 nm (AlphaLISA®). When the donor beads are not in proximity to the acceptor beads, the singlet oxygen returns to the ground state and no emission signal is generated. 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 beads approach the acceptor beads, and as a result of the excitation of the donor beads, a quantifiable light signal is emitted on the acceptor bead side. The measurement of the light signal is performed using a reader compatible with the ALPHA technology, such as an EnVision® or EnSpire® plate reader. In a particular embodiment, the measurement of the interaction is performed using an AlphaScreen® assay.

[0035] Thus, it becomes possible to quantify the protein interaction in the sample based on the intensity of the optical signal. Therefore, when the measured value of the optical signal is lower in the presence of the compound than in the absence of the compound, the test compound is identified as potentially useful for the treatment of cancer. In a particular embodiment, the compound is identified as potentially useful for the treatment of cancer if it inhibits the optical signal measured in the absence of the test compound 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 the optical signal measured in the absence of the test compound by at least 50%, 60%, 70%, 80%, or at least 90%. In a particular embodiment, the compound is identified as potentially useful for the treatment of cancer if it inhibits the optical signal measured in the absence of the test compound by at least 50%.

[0036] In the context of the present invention, the AIF protein can be bound to donor beads, and the CHCHD4 protein can be bound to acceptor beads. Conversely, the AIF protein can be bound to acceptor beads, and the CHCHD4 protein can be bound to donor beads. In a particular embodiment, the donor beads and acceptor beads are coated with a molecule or functional group that enables them to bind to one or the other of the AIF protein or CHCHD4 protein.

[0037] The proteins can be fused to tags that enable them to bind to donor beads or acceptor beads. For example, the AIF protein or CHCHD4 protein can be fused to a histidine tag that enables it to bind to nickel-coated beads, or a GST tag that enables it to bind to glutathione-coated beads.

[0038] Thus, according to a particular embodiment, the method comprises (a') contacting an AIF protein, a CHCHD4 protein, donor beads, and acceptor beads in the presence or absence of a candidate compound; (b') exciting the donor beads at a given wavelength, preferably at a wavelength of approximately 680 nm; (c') measuring a light signal emitted by the acceptor beads, preferably a light signal having a wavelength of 520 - 620 nm; (d') comparing the measured values of the light signal in the presence and absence of the compound; comprising the donor beads are capable of binding to the AIF protein, the acceptor beads are capable of binding to the CHCHD4 protein, or the donor beads are capable of binding to CHCHD4, and the acceptor beads are capable of binding to the AIF protein; if the measured value of the light signal is lower in the presence of the compound than in the absence of the compound, the compound is identified as potentially useful for the treatment of cancer.

[0039] In another embodiment, the measured value of the interaction between the AIF protein and the CHCHD4 protein in the presence or absence of a test compound is measured using a surface plasmon resonance (SPR) assay such as a Biacore® assay. During the SPR experiment, one of the two proteins is bound to the surface (sensor chip), and the other is supplied to the surface via a continuous flow of buffer using a microfluidic system. The interaction between the proteins is monitored by surface plasmon resonance, which detects mass changes at the surface level.

[0040] In a specific embodiment, the steps of contacting (a), measuring the interaction (b), and comparing (c) of the method of the present invention as described above can be repeated. In particular, steps (a), (b), and (c) can be repeated using the same technique or different measurement techniques for measuring the interaction between the AIF protein and the CHCHD4 protein in each repetition. The advantage of repeating steps (a), (b), and (c) is to refine the screening method and confirm the ability of a compound to inhibit the interaction between the AIF protein and the CHCHD4 protein. For example, for each test compound, steps (a), (b), and (c) can be performed twice, three times, four times, or five times.

[0041] Furthermore, steps (a), (b), and (c) of the method of the present invention can be repeated using the same technique for measuring protein interactions, such as the ALPHA technique, but changing the concentration of the test compound for each repetition. By changing the concentration of the test compound, there is an advantage of specifying the inhibitory ability of the identified compound, for example, by determining the dose-effect relationship of the compound. By measuring the interaction between the AIF protein and the CHCHD4 protein by changing the concentration of the test compound, it becomes possible to determine, in particular, the median inhibitory concentration (IC50) of the compound. The IC50 indicates the concentration of the compound required to inhibit 50% of the interaction between the AIF protein and the CHCHD4 protein, and thus it is possible to evaluate the effectiveness of the compound with respect to the inhibition of the interaction.

[0042] In a specific embodiment, the method comprises - performing the above-described steps (a), (b), and (c) in which the interaction between the AIF protein and the CHCHD4 protein is measured using the ALPHA assay; and - repeating the above-described steps (a), (b), and (c) in which the interaction between the AIF protein and the CHCHD4 protein is measured using the SPR assay. and includes.

[0043] In a particular embodiment, the method comprises: - performing steps (a), (b), and (c) above, wherein the interaction between the AIF protein and the CHCHD4 protein is measured using an ALPHA assay in a high-throughput screening experiment using a library of test compounds; - selecting a compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein by at least 50%; - for each selected compound, determining the dose-effect by repeating steps (a), (b), and (c) above by measuring the interaction between the AIF protein and the CHCHD4 protein using an ALPHA assay while varying the concentration of the selected compound; - repeating steps (a), (b), and (c) above using the selected compound, wherein the interaction between the AIF protein and the CHCHD4 protein is measured using an SPR assay; in this order.

[0044] In a particular embodiment, the method of the invention comprises performing steps (a), (b), and (c) above, wherein the compound is a compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein. The use of a compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein can thus function as a positive control and enables the validation of the experiment.

[0045] In a particular embodiment, a 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. The term "functional variant" is herein understood to mean any variant of the sequence of SEQ ID NO: 4 that is capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein.

[0046] In a particular embodiment, compounds capable of inhibiting the interaction between AIF protein and CHCHD4 protein are selected from the group consisting of disulfiram, bromocriptine or its salts such as bromocriptine mesylate, its salts such as thioridazine or thioridazine hydrochloride, Chicago sky blue 6B, mitoxantrone or its salts such as mitoxantrone dihydrochloride, rifapentine, tetraethylenepentamine or its salts such as tetraethylenepentamine pentahydrochloride, nisoldipine, merbromin, triethylperazine or its salts such as triethylperazine dilactate, and benidipine or its salts such as benidipine hydrochloride.

[0047] In a particular embodiment, the method of the present invention includes a step of comparing the inhibition of the interaction between AIF protein and CHCHD4 protein obtained in the presence of a test compound with the inhibition of the interaction obtained in the presence of a compound capable of inhibiting the interaction between AIF protein and CHCHD4 protein. In a particular embodiment, when 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 a positive control, i.e., the N27 peptide, the compound is identified as potentially useful in the treatment of cancer. In particular, when the inhibition obtained with the compound corresponds to at least 50% of the inhibition obtained with a positive control, i.e., the N27 peptide, the compound is identified as potentially useful in the treatment of cancer.

[0048] Confirmation of the anti-cancer properties of the compound In a particular embodiment, the method of the present invention also includes a step of confirming the anti-cancer properties of a compound identified as capable of inhibiting the interaction between AIF protein and CCHD4 protein in a non-human animal model or cell model of cancer.

[0049] In a particular embodiment, the method of the present invention includes the steps of administering an identified compound in an animal model of cancer and then analyzing the anti-cancer properties of the compound. Any animal model of cancer can be used, but preferably it is a mammal. In particular, the animal may be a mouse, rat, pig, rabbit, chicken, or non-human primate.

[0050] In a particular embodiment, the method of the present invention includes the step of contacting an identified compound with a cancer cell model and then analyzing the cytotoxic properties of the compound. It may be a two-dimensional (2D) or three-dimensional (3D) cultured cell model of cancer. Any cancer cell model can be used, such as a cancer cell line. For example, the identified compound can be contacted with A549, MCF7, or HCT116 cancer cell lines.

[0051] 2-kit Another aspect of the present invention is a kit for identifying compounds potentially useful for the treatment of cancer, - an AIF protein, - a CHCHD4 protein, - means suitable for measuring the interaction between the AIF protein and the CHCHD4 protein characterized by comprising, a kit.

[0052] In the context of the present invention, the AIF protein may be of any origin, preferably of animal origin, particularly of mammalian origin, and more preferably of human origin. In a particular embodiment, the AIF protein corresponds to the human native AIF protein with the NCBI sequence reference "NP_004199.1", or any functional variant thereof. In particular, the AIF protein according to the present invention may correspond to the native human polypeptide of the sequence of SEQ ID NO: 1, or any functional variant, such as AIF2, a specific brain isoform. The expression "functional variant" when referring to the AIF protein means any polypeptide that is derived from the structure of the AIF protein and retains the ability to bind to the CHCHD4 protein, particularly the CHCHD4 protein of SEQ ID NO: 2. The functional variant may be a natural or synthetic variant such as a fragment, mutant, or deletion. Preferably, the functional variant of the AIF protein corresponds to a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or at least 95% sequence identity with the AIF protein of the sequence of SEQ ID NO: 1.

[0053] In the context of the present invention, the CHCHD4 protein may be of any origin, preferably of animal origin, particularly of mammalian origin, and more preferably of human origin. In a particular embodiment, the CHCHD4 protein corresponds to the human native CHCHD4 protein with the NCBI sequence reference "NP_001091972.1". In particular, the CHCHD4 protein according to the present invention may correspond to the native human polypeptide of the sequence of SEQ ID NO: 2, or any functional variant thereof. The expression "functional variant" when referring to the CHCHD4 protein means any polypeptide that is derived from the structure of the CHCHD4 protein and retains the ability to bind to the AIF protein, particularly the AIF protein of SEQ ID NO: 1. The functional variant may be a natural or synthetic variant such as a fragment, mutant, or deletion. Preferably, the functional variant of the CHCHD4 protein corresponds to a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or at least 95% sequence identity with the CHCHD4 protein of the sequence of SEQ ID NO: 2.

[0054] In a particular embodiment, the functional variant of the AIF protein is deleted or truncated as compared to the AIF protein of SEQ ID NO: 1. In particular, the functional variant of the AIF protein may correspond to a polypeptide lacking the transmembrane portion of the AIF protein. In a particular embodiment, the functional variant of the AIF protein corresponds to the polypeptide of the sequence of SEQ ID NO: 3.

[0055] The AIF protein and the CHCHD4 protein can be modified as long as the interaction between the two proteins is not hindered. For example, due to the need for an experiment to measure their interaction, the AIF protein and / or the CHCHD4 protein can be fused to a fragment that functions as a tag. Any conventional tag can be used as long as the interaction between the two proteins is not hindered. In particular, the AIF protein and / or the CHCHD4 protein can be fused to a histidine tag corresponding to a motif consisting of several histidine residues, or a GST tag corresponding to a glutathione S-transferase protein.

[0056] The expression "means suitable for measuring the interaction between the AIF protein and the CHCHD4 protein" is intended to mean any means necessary to carry out a technique for measuring the interaction between proteins, such as any reagent, compound, material, support or composition.

[0057] The means suitable for measuring the interaction between the AIF protein and the CHCHD4 protein may be any technique that enables the measurement or quantification of the interaction between the 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) method, 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.

[0058] Preferably, the means suitable for measuring the interaction between the AIF protein and the CHCHD4 protein can be applied to high-throughput screening.

[0059] In a particular embodiment, the kit comprises means suitable for an ALPHA assay and / or an SPR assay.

[0060] In a preferred embodiment, the kit comprises means suitable for measuring the interaction between the AIF protein and the CHCHD4 protein using an ALPHA assay. In particular, the kit comprises - the AIF protein, - the CHCHD4 protein, - donor beads capable of emitting reactive oxygen species such as singlet oxygen when excited at a given wavelength, preferably a wavelength of approximately 680 nm, - acceptor beads capable of emitting a light signal, preferably a light signal in the range of 520 nm to 620 nm, after reaction with reactive oxygen species such as singlet oxygen and the donor beads and the acceptor beads can also bind to one or the other of the AIF protein or the CHCHD4 protein.

[0061] In a particular embodiment, the donor beads and the acceptor beads are coated with a molecule or functional group that enables them to bind to one or the other of the AIF protein or the CHCHD4 protein. In particular, the donor beads and the acceptor beads can be coated with nickel, glutathione, streptavidin, protein A, protein G, or protein L, or a layer of antibody. In a particular embodiment, the donor beads are coated with nickel and the acceptor beads are coated with glutathione.

[0062] In certain embodiments, the donor beads included in the kit are pre-bound to either the AIF protein or the CHCHD4 protein. In certain embodiments, the acceptor beads included in the kit are pre-bound to either the AIF protein or the CHCHD4 protein.

[0063] In certain embodiments, the donor beads and acceptor beads are beads from a commercially available assay (PerkinElmer) such as Alphascreen® or Alphalisa®.

[0064] Optionally, the kit described above may include a buffer suitable for an experiment measuring the interaction between the AIF protein and the CHCHD4 protein. The term "buffer" is intended to mean any solution that contacts the AIF protein and the CHCHD4 protein, the test compound, and optionally, means suitable for measuring the interaction between the proteins. The buffer is chosen so as not to inhibit the interaction between the AIF protein and the CHCHD4 protein. Thus, the buffer can also function as a negative control during the implementation of the method of the present invention.

[0065] In certain embodiments, the kit includes a buffer suitable for an ALPHA assay.

[0066] In certain embodiments, the buffer includes phosphate buffered saline (PBS) and bovine serum albumin (BSA). In certain embodiments the buffer includes PBS and 0.1% BSA.

[0067] Optionally, the kit described above can include a compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein. A compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein can thus function as a positive control during the implementation of the method of the present invention and makes it possible to confirm an experiment measuring the interaction between the AIF protein and the CHCHD4 protein.

[0068] In a particular embodiment, a 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 to the sequence of SEQ ID NO: 4. By "functional variant" is meant any variant of the sequence of SEQ ID NO: 4 that is capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein.

[0069] In a particular embodiment, a compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein is selected from the group consisting of disulfiram, bromocriptine or its salts such as bromocriptine mesylate, its salts such as thioridazine or thioridazine hydrochloride, Chicago sky blue 6B, mitoxantrone or its salts such as mitoxantrone dihydrochloride, rifapentine, tetraethylenepentamine or its salts such as tetraethylenepentamine pentahydrochloride, nisoldipine, merbromin, thietylperazine or its salts such as thietylperazine dilactate, and benidipine or its salts such as benidipine hydrochloride.

[0070] The kit described above may also include any support suitable for an experiment to measure the interaction between the AIF protein and the CHCHD4 protein. For example, the support can be selected from plates, tubes, flasks, membranes, etc. In particular, the support may be a multiwell plate, which enables a large number of and various assays to be carried out in parallel. Typical supports include microtiter plates, and more specifically, easy-to-handle 96-well or 384-well (or more) plates.

[0071] One aspect of the present invention also relates to the use of the kit described above in a method for identifying compounds potentially useful for the treatment of cancer.

[0072] 4 - Composition Another aspect of the present invention relates to a pharmaceutical composition comprising a compound identified by the method described above in combination with a pharmaceutically acceptable carrier.

[0073] Accordingly, one aspect of the present invention relates to a pharmaceutical composition comprising a compound capable of inhibiting the interaction between AIF protein and CHCHD4 protein in combination with a pharmaceutically acceptable carrier.

[0074] The term "pharmaceutically acceptable carrier" should be understood to mean any substance other than the active ingredient in a pharmaceutical. Its addition is preferably intended to give the final product physicochemical and / or biochemical properties for oral, sublingual, respiratory, rectal, nasal, enteral, parenteral administration, or administration by intravenous, intraperitoneal, intramuscular, subcutaneous injection, or otherwise, while avoiding covalent chemical interactions with the active ingredient, or to promote certain viscosity or taste characteristics.

[0075] The pharmaceutical composition of the present invention may be in the form of simple tablets or dragees, sublingual tablets, gel capsules, orally disintegrating tablets, capsules, troches, injectable preparations, aerosols, nasal drops, suppositories, or creams, ointments, or skin gels.

[0076] 5 - Use Another aspect of the present invention relates to a compound identified by the method described above for its use as a medicament. Accordingly, one aspect of the present invention relates to a compound capable of inhibiting the interaction between AIF protein and CHCHD4 protein for its use as a medicament.

[0077] In particular, one aspect of the present invention relates to a compound identified by the method described above for its use in a method of treating cancer. Accordingly, one aspect of the present invention relates to a compound capable of inhibiting the interaction between AIF protein and CHCHD4 protein for its use in a method of treating cancer.

[0078] In a specific embodiment, the compounds capable of inhibiting the interaction between AIF protein and CHCHD4 protein are selected from the group consisting of Chicago Sky Blue 6B, rifapentine, nisoldipine, merbromin, thietylperazine or its salts such as thietylperazine malate, and its salts such as benidipine or benidipine hydrochloride.

[0079] Described herein is a method for treating cancer in a subject, which includes administering to the subject a compound capable of inhibiting the interaction between AIF protein and CHCHD4 protein, or a pharmaceutical composition containing the compound.

[0080] The treatment method may include administering a compound capable of inhibiting the interaction between AIF protein and CHCHD4 protein, or a pharmaceutical composition containing the compound, alone or in combination with any anti-cancer treatment such as radiotherapy, chemotherapy, and immunotherapy. In particular, the treatment method may include administering a compound capable of inhibiting the interaction between AIF protein and CHCHD4 protein in combination with the administration of a chemotherapeutic agent. The administration of a compound capable of inhibiting the interaction between AIF protein and CHCHD4 protein can be carried out before, simultaneously with, or after the administration of the chemotherapeutic agent. The term "chemotherapeutic agent" is intended to mean a chemical product that can be used to destroy cancer cells or slow down, stop, or reverse the growth of cancer cells.

[0081] In the context of the present invention, the terms "subject" or "patient" mean an animal, preferably a mammal, particularly a human, suffering from cancer, regardless of its age or gender. The term also includes domestic animals and experimental animals such as non-human primates, members of the Felidae, Canidae, Equidae, members of the Suidae, cattle, goats, sheep, rabbits, rats, and mice. Preferably, the patient to be treated is a human.

[0082] As used herein, the term "cancer" means any type of malignant tumor. The malignant tumor may correspond to a primary tumor or a secondary tumor (i.e., metastasis). Further, the tumor may correspond to a solid malignant tumor including, for example, carcinoma, adenocarcinoma, sarcoma, melanoma, mesothelioma, and granuloma, or a blood cell cancer such as leukemia, lymphoma, and myeloma. Cancer may correspond to, for example, skin cancer, lung cancer, bladder cancer, kidney cancer, gastrointestinal cancer (colon, pancreas, liver), ovarian cancer, brain cancer, cancer of the face and neck, etc.

[0083] The term "treatment" includes curative treatment and / or prophylactic treatment. Curative treatment refers to the reduction, improvement, stabilization, and / or removal of the symptoms of a disease, or otherwise the suppression of the progression of the symptoms of a disease. Prophylactic treatment refers to any one of the following effects: preventing or delaying the occurrence of a particular disorder, reducing the onset, risk of onset, incidence, or severity of a disorder, delaying the appearance of symptoms, and / or improving the survival of a patient.

[0084] Another aspect of the present invention relates to the use in academic research of a compound identified by the above-described method as a compound for interfering with mitochondrial uptake and / or cell metabolism. In a specific embodiment, compounds capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein are selected from the group consisting of disulfiram, bromocriptine or a salt thereof such as bromocriptine mesylate, a salt thereof such as thioridazine or thioridazine hydrochloride, Chicago sky blue 6B, mitoxantrone or a salt thereof such as mitoxantrone dihydrochloride, rifapentine, tetraethylenepentamine or a salt thereof such as tetraethylenepentamine pentahydrochloride, nisoldipine, merbromin, thietylperazine or a salt thereof such as thietylperazine dilactate, and benidipine or a salt thereof such as benidipine hydrochloride.

Examples

[0085] Materials and Methods Abbreviations AIF apoptosis-inducing factor BSA Bovine Serum Albumin CHCHD4 Coiled-Coil Helix Coiled-Coil Helix (CHCH) Domain Containing 4 IC50 Median Inhibitory Concentration CV Coefficient of Variation DMSO Dimethyl Sulfoxide GST Glutathione S-Transferase HIS Histidine HTS High-Throughput Screening MEF Mouse Embryonic Fibroblasts PBS Phosphate Buffered Saline RT Room Temperature RU Resonance Unit S / B Signal-to-Background Ratio SD Standard Deviation SPR Surface Plasmon Resonance TH Threshold

[0086] Protein Expression and Purification The AIF103-613 protein (AIF protein fragment 103-613) and the CHCHD4 protein were produced in BL21+DE3 bacteria (RIPL) via induction with 0.5 mM IPTG at 37 °C for 3 hours. The bacteria were transformed according to the recommendations of the supplier (Agilent). Subsequently, the proteins were purified. The total protein concentration was determined using a Bradford assay kit. Also, the purity of the expression was evaluated on polyacrylamide gels in the presence of SDS.

[0087] Identification of the Interaction between AIF Protein and CHCHD4 Protein and Compounds that Regulate this Interaction Using Alpha Assay Enabled the identification of compounds capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein and developed a method suitable for high-throughput screening. This method is - Use of the already produced AIF103-613 protein and CHCHD4 protein, and - Use of an AlphaScreen® kit (PerkinElmer) containing donor beads and acceptor beads capable of binding to AIF protein and CHCHD4 protein, and - Use of a positive control: N27 peptide (corresponding to the 27 amino acids at the N-terminal end of the CHCHD4 protein, as described by Hangen et al., 2015), and - Use of a negative control: 0.1% bovine serum albumin (BSA) solution in phosphate-buffered saline (PBS), and - Use of a library of test compounds: Prestwick Chemical Library® including.

[0088] The ALPHA assay was first performed in high throughput using a 384-well white microplate (Greiner, reference number 781075) with a total volume of 25 μl per well. Proteins, positive controls, and beads were diluted in 0.1% BSA / PBS solution. All dispensing was performed using an electronic multi-pipette, and then the plate was centrifuged (200 g, 1 minute). The plate was sealed and incubated in the dark at 23°C in a room.

[0089] A library of 1,280 small molecule compounds (Prestwick Chemical Library (registered trademark)) supplied by Prestwick Chemical was screened. This library contains compounds with various chemical and pharmacological properties that have already been approved by health agencies such as the FDA (U.S. Food and Drug Administration) or the EMEA (European Medicines Agency). Each compound in the library was diluted in a 0.1% DMSO solution and used at a concentration of 10 μM. First, 5 μl of a 10 μM compound, 5 μl of 3 μM N27 (positive control), or 5 μl of 0.1% BSA / PBS (negative control) was added to the wells. Second, 5 μl of the AIF103 - 613 protein was incubated with the compound for 20 minutes, and then 5 μl of CHCHD4 was added for 2 hours. Third, 10 μl of a mixture of 5 μg / ml donor beads and acceptor beads was incubated for 3 hours. Analysis of the results was performed using an EnSpire (registered trademark) multimode plate reader (PerkinElmer). The interaction between the AIF protein and the CHCHD4 protein was revealed by detection of the light signal by the plate reader.

[0090] Analysis of ALPHA assay data For each experiment measuring the interaction between the AIF protein and the CHCHD4 protein, three measurements were performed to confirm the quality of the experiment: the coefficient of variation (CV) of the control, the signal / background noise (S / B) ratio, and the Z' factor described below.

[0091] For each 384-well plate, 16 wells corresponded to the positive control and 16 wells corresponded to the negative control. The positive control corresponded to the measurement of the interaction between the AIF103 - 613 protein and the CHCHD4 protein in the presence of the N27 peptide (equivalent to the minimum signal). The negative control corresponded to the measurement of the interaction between the AIF103 - 613 protein and the CHCHD4 protein in the presence of the buffer (0.1% BSA / PBS) (equivalent to the maximum signal).

[0092] CV is the ratio of the standard deviation (SD) divided by the mean value of each control:

[0093]

Number

[0094] is defined as.

[0095] S / B is the ratio corresponding to the average value of the negative control divided by the average value of the positive control:

[0096]

Number

[0097] is.

[0098] The Z'-factor measures the difference between the positive and negative controls and evaluates the amplitude of the assay signal by taking into account the standard deviation (SD):

[0099]

Number

[0100] When CV < 15%, S / B > 10, and Z'-factor > 0.5, each series of measurements is confirmed [Goktug et al., Drug Discovery, 2013 doi10.5772 / 52508].

[0101] After high-throughput screening, the signal obtained in the presence of the compound is the following threshold TH: TH = average value (signal of negative control) - 5 × SD (signal of negative control) If less, the compound is selected.

[0102] For each compound already selected, the ALPHA assay is manually repeated using another batch of the powder of that compound.

[0103] Each compound is tested at 10 μM and 30 μM in order to select compounds that give signals located on the ascending part of the Hook bell due to the Hook effect specific to the ALPHAscreen technology, and thus compounds that are active at low doses from the perspective of drug development.

[0104] - The signal obtained in the presence of the compound is less than the above-mentioned threshold TH, - The compound is selected when the signal obtained at 30 μM is smaller than the signal obtained at 10 μM.

[0105] Furthermore, for each compound thus selected, the dose-effect relationship of the compound is determined. Thereby, it is possible to determine the median inhibitory concentration (IC50) of the compound.

[0106] Confirmation using an SPR assay of compounds that inhibit the interaction between AIF protein and CHCHD4 protein Some of the compounds identified using the above Alpha assay were tested by surface plasmon resonance (Biacore™ T100, GE Healthcare Life Sciences). The CHCHD4 protein was covalently immobilized on a dextran matrix containing a COOH-functional carboxyl group (Series S CM5® sensor chip, GE Healthcare Life Sciences, 29149603). To perform the screening, the compound (10 μM) was pre-incubated with the AIF protein in DPBS buffer (Sigma, D8577) at pH 7.4 for 20 minutes at room temperature. Next, the mixture of the compound and the AIF protein was injected at 30 μl per minute for 3 minutes at 25°C. The negative control was performed by injecting 1 μM of AIF protein alone (diluted in 0.1% DMSO / PBS). The positive control was performed by injecting a mixture of N27 peptide (3 μM) and AIF protein.

[0107] A compound is confirmed 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, i.e., the N27 peptide. Sensorgrams were analyzed using BIAevaluation software (version 2.0.4).

[0108] Effect of hits on cellular energy metabolism and their cytotoxicity The effect of the compound on the metabolism of non-small cell lung cancer cell line (A549) was tested using Seahorse technology (XFe96, Agilent), and cell death was evaluated by measuring the release of lactate dehydrogenase (LDH, Promega). Cells seeded in 96-well microplates (20,000 cells / well) were treated with the compound as a dose response (0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM) in serum-free DMEM medium for 3 hours, and then mitochondrial respiration (OXPHOS) and glycolysis were measured in real time. At the end of the experiment, the culture supernatant was removed to analyze cell death, and the cell number was determined by labeling the cells with DAPI and counting them using a fluorescence microscope (Zeiss). Subsequently, the results obtained by Seahorse were normalized using the cell number per well at the end of the experiment.

[0109] Results Protocol development and optimization for high-throughput screening The concentrations of AIF103-613 protein and CHCHD4 protein required to obtain optimal signals in Alphascreen® were evaluated. Figure 1 shows that the optimal signals obtained with AIF103-613 and CHCHD4 are dose-dependent. Subsequently, batches of proteins, peptides, and reagents were verified in preliminary experiments, and Z'>0.5 was obtained (not shown).

[0110] Summary of statistical values obtained by high-throughput screening in 384-well plates Subsequently, a Prestwick library (1280 molecules) was screened using four 384-well plates (plates #1 - #4).

[0111] Prior to the screening itself, all reagents (proteins, beads, buffers) and materials (plates, chips, pipettors) were subjected to quality control called a "prescreening validation assay", and a number of criteria were analyzed. As described above, when CV < 15%, S / B > 10, and Z'-factor > 0.5, each set of measurements is confirmed.

[0112] The statistical data obtained from high-throughput screening in 384-well plates are detailed in Table 1 below.

[0113]

Table 1

[0114] Identification of Compounds that Inhibit the Interaction between AIF Protein and CHCHD4 Protein 1) Identification of Compounds Using the ALPHA Assay As described above, a Prestwick compound library containing 1280 molecules was screened using four 384-well plates. Compounds were selected based on the signals obtained by Alphascreen® in the presence of the compound at a concentration of 10 μM. Specifically, if the signal obtained at 10 μM was below the threshold TH (TH = mean value (signal of negative control) - 5 × SD (signal of negative control)), the compound was selected. Out of the 1280 compounds analyzed, 148 compounds, i.e., 12% of the compound library, were selected (see Figure 2).

[0115] The 148 selected compounds were retested using the ALPHA assay performed manually as described above. Out of the 148 compounds tested, 11 compounds were finally selected. For the 11 selected compounds, - The signal obtained at 10 μM was below the threshold TH, - The signal obtained at 30 μM was less than the signal obtained at 10 μM.

[0116] The 11 compounds selected by the Alpha screen are listed in Table 2 below. The CAS numbers and chemical structures of each compound are reported in Table 2 below.

[0117]

Table 2A

[0118]

Table 2B

[0119] Some of these compounds have already shown anti-cancer properties, and thus confirm the screening method of the present invention. For example, the anti-cancer properties of thioridazine are described in the paper by Shen et al., 2017. Furthermore, mitoxantrone is sold as an anti-cancer agent (under the name Novantrone®) especially in the treatment of metastatic breast cancer, non-Hodgkin lymphoma, or other acute myeloid leukemia (AML).

[0120] The IC50 values of the identified 11 compounds obtained manually using the Alphascreen assay are listed in Table 3 below.

[0121]

Table 3

[0122] 2) Confirmation of the inhibition of the interaction between AIF protein and CHCHD4 protein using SPR assay The inhibition of the interaction between AIF protein and CHCHD4 protein by thioridazine and mitoxantrone was analyzed using a surface plasmon resonance assay (see Figure 3). If the inhibition obtained with the compound corresponds to at least 50% of the inhibition obtained with the positive control, i.e., the N27 peptide, the compound is considered to inhibit the interaction between AIF protein and CHCHD4 protein.

[0123] It was confirmed by surface plasmon resonance assay that thioridazine and mitoxantrone are capable of inhibiting the interaction between AIF protein and CHCHD4 protein.

[0124] 3) Confirmation of the cytotoxic properties of the compound The cytotoxic properties of a certain compound against non-small cell lung cancer cells (A549 human cell line) are shown in Table 4 below. The compound concentration shown in the "Toxicity" column represents the lowest dose tested at which a toxicity exceeding 20% was measured.

[0125]

Table 4

[0126] The table shows that treatment with the compound for 3 hours induces cytotoxicity in A549 cells in the tested concentration range (see the method described above). Furthermore, this experiment showed that the compound acts on energy metabolism, whether by regulating glycolysis or mitochondrial activity.

[0127] Therefore, these results confirm the anti-cancer properties of the compounds identified by the screening method. [References] TIFF0007679392000009.tif44170

Claims

1. 1. A method for identifying a compound potentially useful in the treatment of cancer, comprising the step of evaluating the ability of the compound to inhibit the interaction between an AIF protein and a CHCHD4 protein, wherein if the compound inhibits the interaction it is identified as potentially useful in the treatment of cancer.

2. (a) contacting an AIF protein and a CHCHD4 protein in the presence and absence of a compound; (b) measuring the interaction between the AIF protein and the CHCHD4 protein in the presence and absence of a compound; (c) comparing the measured interaction values ​​in the presence and absence of the compound; 2. The method of claim 1, wherein the compound is identified as potentially useful in the treatment of cancer if the measured interaction is lower in the presence of the compound than in the absence of the compound.

3. 3. The method according to claim 1 or 2, characterized in that a compound is identified as potentially useful for the treatment of cancer if it inhibits the interaction between the AIF protein and the CHCHD4 protein by at least 50%, 60%, 70%, 80%, or at least 90%.

4. 4. The method according to claim 2 or 3, characterized in that the measurement of the interaction between the AIF protein and the CHCHD4 protein is carried out using an amplified luminescent 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, preferably using an amplified luminescent proximity homogeneous assay (ALPHA) or a surface plasmon resonance (SPR) assay.

5. 5. The method of any one of claims 1 to 4, further comprising confirming the anti-cancer properties of the identified compounds in a non-human animal or cell model of cancer.

6. (i) determining the ability of a compound to inhibit the interaction between the AIF protein and the CHCHD4 protein using an amplified luminescent proximity homogeneous assay (ALPHA); (ii) determining the ability of the compound to inhibit the interaction between the AIF protein and the CHCHD4 protein using a surface plasmon resonance (SPR) assay; (iii) confirming the anti-cancer properties of the identified compounds in non-human animal or cell models of cancer; 6. The method according to any one of claims 1 to 5, comprising:

7. 1. A kit for identifying a compound potentially useful in the treatment of cancer, comprising: - AIF protein, - CHCHD4 protein, - suitable means for measuring the interaction between the AIF protein and the CHCHD4 protein, A kit comprising:

8. The kit of claim 7, further comprising a buffer solution.

9. The kit of claim 7 or 8, further comprising a compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein.

10. 10. The kit according to any one of claims 7 to 9, wherein the means suitable for measuring the interaction between the AIF protein and the CHCHD4 protein are means suitable for an Amplified Luminescent Proximity Homogeneous Assay (ALPHA).

11. 11. The kit of any one of claims 8 to 10, wherein the buffer comprises phosphate buffered saline (PBS) and bovine serum albumin (BSA).

12. A kit according to any one of claims 7 to 11, wherein the 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 to the sequence of SEQ ID NO:

4.

13. The kit according to any one of claims 7 to 12, wherein the compound capable of inhibiting the interaction between the AIF protein and the CHCHD4 protein is selected from the group consisting of disulfiram, bromocriptine mesylate, thioridazine hydrochloride, Chicago Sky Blue 6B, mitoxantrone dihydrochloride, rifapentine, tetraethylenepentamine pentahydrochloride, nisoldipine, merbromine, thiethylperazine dimalate, and benidipine hydrochloride.

14. A compound capable of inhibiting the interaction between AIF protein and CHCHD4 protein for use in the treatment of cancer, the compound being selected from the group consisting of Chicago Sky Blue 6B, rifapentine, nisoldipine, merbromine, thiethylperazine dimalate, and benidipine hydrochloride.

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