Lacto-dehydrogenase activity polypeptide inhibitors for use in cancer therapy

Abstract

The present invention relates to polypeptides comprising the amino acid sequence of formula (I): GX1MMX2LQHGSX3X4X5QTP. These polypeptides modulate the activity of the naturally occurring tetrameric lactate dehydrogenase LDH-1 by inhibiting the tetramerization of its subunits. The present invention also relates to the therapeutic use of these polypeptides, in particular as medicaments for the prevention and / or treatment of cancer.

Description

[Technical Field]

[0001] Field of Invention This invention relates to polypeptides that regulate the activity of natural tetrameric lactate dehydrogenase as activators for cancer therapy. More specifically, this invention relates to polypeptides that inhibit the tetramerization of lactate dehydrogenase subunits. [Background technology]

[0002] Dysregulation of glucose metabolism is a common characteristic of most cancer cells. The high glycolytic flow rate in cancer cells has two origins: an adaptation to hypoxia (anaerobic glycolysis) and an adaptation to high growth rates (aerobic glycolysis, also known as the "Warburg effect"). This higher glycolytic flow rate provides cancer cells with the energy and biomass essential for maintaining anabolic growth. At the end of the glycolysis pathway, the reduction of pyruvate to lactate occurs, catalyzed by lactate dehydrogenase (LDH).

[0003] Lactate has long been considered merely a byproduct of glycolysis, but given the numerous benefits it provides to tumor growth, lactate is now seen as potentially serving the purpose of accelerating glycolysis in cancer. Today, increased lactate production is recognized to actually promote several phenomena, including angiogenesis, aggression, commensalism, inflammation, and redox homeostasis. Lactate metabolism further establishes metabolic symbiosis between oxidative cancer cells that preferentially use lactate as fuel over glucose and glycolytic cancer cells that rapidly convert glucose to lactate. LDH-mediated oxidation of lactate to pyruvate further promotes lysosomal acidification and autophagy.

[0004] LDH is pyruvate and NADH lactate and NAD +LDH is a crucial enzyme at the heart of this adaptive metabolism because it catalyzes the termination of lactate biosynthesis through interconversion. LDH functions as an obligate tetramer composed of homozygous or heterozygous associations of two isoenzymes, LDH-H (encoded by the LDHB gene) and LDH-M (encoded by the LDHA gene). These two isoenzymes exhibit extremely high homology and identity. The two LDH homotetramers, LDH-1 (LDH-H4) and LDH-5 (LDH-M4), are the most extensively studied forms of LDH and are attractive targets for cancer therapy.

[0005] Initially, due to its widespread potential importance in cancer pathogenesis, intensive efforts were focused on selective LDH-5 inhibition. However, more recent reports on the involvement of LDH-1 in cancer pathogenesis have provided clues to elucidating LDH-1 inhibition. First, it was reported that LDH-1 interacts with lysosomal vesicular ATP-degrading enzymes, thereby regulating autophagy and being essential for metabolic reprogramming via p53 and RAS mutations (Brisson et al.; Lactate Dehydrogenase B Controls Lysosome Activity and Autophagy in Cancer. Cancer Cell 2016,30,418-431; Smith et al.; Addiction to Coupling of the Warburg Effect with Glutamine Catabolism in Cancer Cells. Cell Rep. 2016,17(3),821-836). Next, the LDHB gene was identified as essential for triple-negative breast cancer (McCleland et al.; An Integrated Genomic Screen Identifies LDHB as an Essential Gene for Triple-Negative Breast Cancer. Cancer Res. 2012, 72(22), 5812-5823). Finally, it was shown that one LDH isoenzyme can complement the genetic disruption of the other enzyme to maintain the Warburg phenotype (Zdralevic et al.; Disrupting the 'Warburg Effect' Re-Routes Cancer Cells to OXPHOS Offering a Vulnerability Point via 'Ferroptosis'-Induced Cell Death. Adv. Biol. Regul. 2018, 68, 55-63). In summary, these studies support the idea that dual LDH inhibitors may provide additional therapeutic effects beyond selective isoenzyme inhibition.

[0006] Due to the therapeutic importance of LDH inhibition, the development of potent bipolar or selective active-site LDH inhibitors has progressed. However, despite intensive efforts, translating pharmacological LDH inhibition into in vivo activity has been challenging. Indeed, LDH is generally recognized as a poorly druggable target, and several reasons can explain this. Firstly, LDH active-site inhibitors are difficult to achieve selectivity with compared to other dehydrogenases, particularly due to their common NAD-binding domain. For example, gossypol derivatives, one of the first reported LDH inhibitors, showed significant inhibition compared to other dehydrogenases. Secondly, the catalytic site of LDH exhibits suboptimal physicochemical properties, accompanied by high solvent exposure and hydrophilicity, resulting in poor absorption, distribution, metabolism, and excretion (ADOME) properties for most LDH active-site inhibitors. Finally, an inherent difficulty in achieving therapeutic LDH inhibition stems from its high intracellular concentration; in fact, LDH is highly concentrated in cancer cells, with protein concentrations reported to be in the μM range. Due to this high cell concentration, cell-based inhibition cannot often be observed below the μM threshold, even with more potent nanomolar concentration inhibitors that reach micromolar concentrations in tumors.

[0007] These various challenges to LDH inhibition necessitate the development of novel strategies targeting this enzyme family, which possesses high therapeutic potential. For this purpose, tool compounds capable of targeting the boundaries of LDH oligomers, rather than their active sites, have recently been developed. Targeting the oligomeric state of proteins offers several advantages compared to active-site targeting and is therefore an unexplored strategy that could overcome existing problems faced by LDH orthosteric inhibitors. Targeting LDH self-assembly can indeed lead to the identification of novel and potentially more druggable allosteric sites. Notably, LDH subunits can form homo- and hetero-tetramers, and the tetramer boundary is shared between two different isoenzymes. Targeting LDH tetramers can therefore produce molecules that disrupt both LDH-1 and LDH-5, which is consistent with current pan-LDH inhibition strategies. Furthermore, protein self-assembly disruptors can induce protein misfolding and degradation. Therefore, targeting the LDH oligomeric state reduces its intracellular concentration, resulting in quasi-stoichiometric inhibition and thus higher efficacy.

[0008] To this end, we developed and characterized a dimerized model of LDH-H (LDH-Htr) by cleaving its N-terminal tetramerization domain (Thabault et al.; Interrogating the Lactate Dehydrogenase Tetramerization Site Using (Stapled) Peptides. J.Med.Chem. 2020, 63(9), 4628-4643). This model allowed us to investigate the LDH tetramer boundary, previously leading to the identification of a primary allosteric site and the generation of linear and cyclic polypeptides based on LDHA and LDHB subunits that act as LDH tetramerization inhibitors for treating cancer (see, e.g., International Publication No. 2020221899). Another example is the in silico design and production of peptides that mimic the N-terminal domain of LDHA to prevent interaction between the N-terminal and C-terminal regions of the enzyme, thereby inhibiting its tetramerization (Jafary et al., Novel Peptide Inhibitors for Lactate Dehydrogenase A (LDHA): a Survey to Inhibit LDHA Activity via Disruption of Protein-Protein Interaction, Scientific Reports, 2019). Novel LDH inhibitors are the subject of this invention. [Overview of the project]

[0009] A first aspect of the present invention is a polypeptide that inhibits tetramerization of LDH subunits, comprising formula (I): GX1MMX2LQHGSX3X4X5QTP(I)(Sequence ID 5) The formula includes the amino acid sequence, -X1 represents amino acid residue E, D, or A; -X2 represents amino acid residue D, E, or A; -X3 represents amino acid residues L, A, V, I, F, W, or Y; -X4 represents amino acid residue F, A, L, V, I, W, or Y; -X5 represents amino acid residues L, A, V, I, F, W, or Y. The polypeptide comprises 16 to 200 amino acid residues. The amino acid sequence described above relates to a polypeptide other than SEQ ID NO: 87.

[0010] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 51.

[0011] In certain embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 28.

[0012] A further aspect of the present invention relates to nucleic acids encoding polypeptides according to the present invention.

[0013] Another aspect of the present invention relates to a nucleic acid vector comprising at least one nucleic acid according to the present invention.

[0014] In one embodiment, the present invention relates to a pharmaceutical composition comprising (i) at least one polypeptide, at least one nucleic acid, or at least one nucleic acid vector according to the present invention, and (ii) at least one pharmaceutically acceptable vehicle.

[0015] Further aspects of the present invention relate to (i) a kit comprising at least one polypeptide, at least one nucleic acid, at least one nucleic acid vector, or at least one pharmaceutical composition according to the present invention, and (ii) at least one means for administering the polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition.

[0016] In some embodiments, the kit further comprises an anticancer agent.

[0017] Further aspects of the present invention relate to polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention for use as pharmaceuticals.

[0018] In certain embodiments, the polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention are for use in the prevention and / or treatment of cancer.

[0019] The present invention also relates to the use of polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention for inhibiting lactate dehydrogenase tetramerization.

[0020] In some embodiments, the lactate dehydrogenase subunit is the LDH-1 subunit and / or the LDH-5 subunit.

[0021] In certain embodiments, the lactate dehydrogenase subunit is the LDH-1 subunit.

[0022] In another aspect, the present invention relates to a method for preventing and / or treating cancer in an individual in need thereof, comprising at least the step of administering to the individual a therapeutically effective amount of the polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition of the present invention.

[0023] definition In this invention, the following terms have the meanings set forth below.

[0024] - The word "approximately" before a number means ±10% of the value of that number.

[0025] - "Amino acid substitution" refers to the exchange of one amino acid in a polypeptide with another amino acid. In one embodiment, an amino acid is exchanged for another amino acid having similar structural and / or chemical properties, for example, by conservative amino acid substitution. Conservative amino acid substitution may be carried out based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine (A), leucine (L), isoleucine (I), valine (V), proline (P), phenylalanine (F), tryptophan (W), and methionine (M); polar neutral amino acids include glycine (G), serine (S), threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N), and glutamine (Q); positively charged (basic) amino acids include arginine (R), lysine (K), and histidine (H); and negatively charged (acidic) amino acids include aspartic acid (D) and glutamic acid (E). Non-conservative substitutions inevitably involve the exchange of a member of one of these classes with another. For example, amino acid substitution can also result in the exchange of one amino acid for another amino acid having different structural and / or chemical properties, e.g., the exchange of an amino acid with one group (e.g., polar) for another amino acid with a different group (e.g., basic). Amino acid substitutions can be produced using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for altering the side chain groups of amino acids by non-genetic engineering methods such as chemical modification are also intended to be useful.

[0026] - A "polypeptide" refers to any peptide or protein containing two or more amino acids linked together by peptide bonds or modified peptide bonds, i.e., by peptide isodistributors. "Polypeptide" can mean both short chains, commonly called peptides, oligopeptides, or oligomers, and longer chains, commonly called proteins. Polypeptides may contain amino acids other than the 20 genetically coding amino acids.

[0027] - “Nucleic acid” or “polynucleotide” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. “Nucleic acid” or “polynucleotide” includes, but is not limited to, single-stranded and double-stranded DNA, DNA which is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA which is a mixture of single-stranded and double-stranded regions, single-stranded, or more typically double-stranded, or hybrid molecules containing DNA and RNA which may be a mixture of short and double-stranded regions. In addition, “nucleic acid” or “polynucleotide” refers to RNA or DNA, or a triple-stranded region containing both RNA and DNA. The term “nucleic acid” or “polynucleotide” also includes DNA or RNA which contains one or more modified bases, and DNA or RNA which has a backbone modified for stability or other reasons. “Modified” bases include, for example, tritylated bases and rare bases such as inosine. DNA and RNA undergo a wide variety of modifications; therefore, “nucleic acids” or “polynucleotides” encompass polynucleotides in chemically, enzymatically, or metabolically modified forms typically found in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotides” also often encompass relatively short polynucleotides called oligonucleotides.

[0028] - "Preventing cancer" is intended to mean avoiding the occurrence of the adverse effects or symptoms of at least one type of cancer.

[0029] - "Treatment," "treatment," or "alleviation" of cancer refers to both therapeutic treatments and preventive or protective measures, the purpose of which is to prevent or delay (weaken) cancer. Persons requiring treatment include those who already have cancer, as well as those who are prone to developing cancer or who should be prevented from developing cancer. An individual or mammal is successfully "treated" with respect to cancer if, after being administered a therapeutic dose of the polypeptide according to the present invention, the individual exhibits one or more observable and / or measurable reductions or absences of: a reduction in the number of cancer cells; a reduction in the percentage of total cells that are cancer-like; and / or some degree of alleviation of one or more cancer-related symptoms; a reduction in morbidity and mortality, and an improvement in quality of life issues. The above parameters for assessing the success of cancer treatment and alleviation are readily measurable by standard procedures familiar to physicians.

[0030] - “Therapeutic dose” is intended to mean a level or amount of a drug that, without causing significant undesirable or harmful side effects to the target, aims to (1) delay or prevent the onset of cancer, (2) slow or halt the progression, exacerbation or worsening of one or more symptoms of cancer, (3) improve the symptoms of cancer, (4) reduce the severity or incidence of cancer, or (5) prevent cancer formation. In one embodiment, the therapeutic dose is administered before the onset of cancer formation for a prophylactic or preventive effect.

[0031] - “Individual” refers to an animal, preferably a mammal, more preferably a human. In one embodiment, the individual is male. In another embodiment, the individual is female. In one embodiment, the individual may be a “patient” i.e., a warm-blooded animal, more preferably a human, who is waiting to receive medical treatment, is receiving medical treatment, has been / is / will be subject to medical treatment, or is being monitored for the development of cancer. In one embodiment, the individual is an adult (e.g., a subject over 18 years of age). In another embodiment, the individual is a child (e.g., a subject under 18 years of age). [Modes for carrying out the invention]

[0032] This invention relates to a polypeptide that modulates the activity of at least one isoform of a natural tetrameric lactate dehydrogenase. The inventors hereby report the existence of a newly identified allosteric site that enables the development of a novel polypeptide family that functions as an LDH inhibitor.

[0033] "Lactate dehydrogenase" or "LDH" is a enzyme that combines NADH and NAD. + This refers to a tetrameric enzyme capable of catalyzing the interconversion of pyruvate and lactate, which involves the mutual conversion of these two substances. To date, five isoforms of lactate dehydrogenase, namely LDH-1, LDH-2, LDH-3, LDH-4, and LDH-5, have been identified, and these describe specific combinations of two subunits, namely the LDH-A subunit and the LDH-B subunit.

[0034] In the context of the present invention, "regulating" means that the polypeptide of the present invention has a biological effect of significantly upregulating or downregulating the biological activity of any one of the five isoforms of lactate dehydrogenase, namely LDH-1, LDH-2, LDH-3, LDH-4, and LDH-5, and / or one or more subunits, namely the LDH-H subunit and the LDH-M subunit.

[0035] "Natural" means that the lactate dehydrogenase (LDH) sequences referred to in this application are of natural origin, for example, of any species. Furthermore, such natural sequences of lactate dehydrogenase can be isolated from nature or produced by recombinant means or by synthetic means from the subunits LDH-H and / or LDH-M.

[0036] In some embodiments, the LDH-M subunit is represented by the amino acid sequence of SEQ ID NO: 1, and the LDH-H subunit is represented by the amino acid sequence of SEQ ID NO: 2.

[0037] In certain embodiments, the polypeptides of the present invention inhibit the activity of at least one isoform or at least one subunit of a natural tetrameric lactate dehydrogenase. In certain embodiments, the present invention relates to polypeptide inhibitors of the activity of at least one isoform or at least one subunit of a natural tetrameric lactate dehydrogenase.

[0038] "Inhibitor" or "inhibiting" means that the polypeptide of the present invention has a biological effect of inhibiting, significantly reducing, or downregulating the biological activity of any one of the five isoforms of lactate dehydrogenase. In certain embodiments, the polypeptide of the present invention can inhibit the activity of innate lactate dehydrogenase by up to about 10%, preferably up to about 25%, preferably up to about 50%, preferably up to about 75%, 80%, 90%, 95%, and more preferably up to about 96%, 97%, 98%, 99%, or 100%.

[0039] In one embodiment, the polypeptide of the present invention inhibits the tetramerization of lactate dehydrogenase subunits.

[0040] In some embodiments, the polypeptides of the present invention inhibit the tetramerization of at least one of the four LDH-M subunits so as to inhibit the activity of isoform LDH-5.

[0041] In some embodiments, the polypeptides of the present invention inhibit the tetramerization of at least one of the three LDH-M subunits and / or LDH-H subunits so as to inhibit the activity of isoform LDH-4.

[0042] In some embodiments, the polypeptides of the present invention inhibit the tetramerization of at least one of the two LDH-M subunits and / or at least one of the two LDH-H subunits, thereby inhibiting the activity of isoform LDH-3.

[0043] In some embodiments, the polypeptides of the present invention inhibit the tetramerization of at least one LDH-M subunit and / or three LDH-H subunits so as to inhibit the activity of isoform LDH-2.

[0044] In some embodiments, the polypeptides of the present invention inhibit the tetramerization of at least one of the four LDH-H subunits so as to inhibit the activity of isoform LDH-1.

[0045] It goes without saying that inhibition of lactate dehydrogenase subunit tetramerization can be evaluated by any suitable means available at the most advanced level of technology, in particular by any suitable biochemical or biophysical method.

[0046] Exemplary methods include biochemical methods such as affinity electrophoresis, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation, tandem affinity purification, intrinsic tryptophan fluorescence, size exclusion chromatography, fractionation centrifugation techniques, and cross-linking (SDS-PAGE) electrophoresis; or biophysical methods such as biacore, two-plane polarization interferometry (DPI), dynamic light scattering (DLS), microscale thermophoresis (MST), NMR WaterLOGSY, saturated migration difference (STD) spectroscopy, Carr-Purcell-Meiboom-Gill (CPMG) pulse sequencing, and / or static light scattering (SLS), surface plasmon resonance (SPR).

[0047] In some embodiments, inhibition of tetramerization of at least one lactate dehydrogenase subunit can be evaluated by the ability of the polypeptide according to the present invention to bind to one or more LDH subunits lacking 20 N-terminal amino acid residues, namely cleaved LDH-M or LDH-Mtr, and cleaved LDH-H or LDH-Htr.

[0048] In some embodiments, the polypeptide of the present invention does not bind to the 20 N-terminal amino acid residues of LDH-H and / or LDH-M. In some embodiments, the polypeptide of the present invention does not bind to the 15 N-terminal amino acid residues of LDH-H and / or LDH-M.

[0049] In some embodiments, the polypeptide is bound to at least one amino acid at the 62, 65, 71, 72, or 73 position of LDH-H, where the amino acid position is defined with respect to SEQ ID NO: 2.

[0050] In certain embodiments, LDH-Mtr is represented by the amino acid sequence of SEQ ID NO: 3. In some embodiments, LDH-Htr is represented by the amino acid sequence of SEQ ID NO: 4.

[0051] In some embodiments, when the MST method is performed, significant binding of the polypeptide according to the present invention to LDH-Mtr (SEQ ID NO: 3) or LDH-Htr (SEQ ID NO: 4), preferably to LDH-Htr (SEQ ID NO: 4), may result in a dissociation constant (Kd) ranging from 1 μM to 5 mM, preferably from about 50 μM to about 3.5 mM.

[0052] Within the scope of the present invention, the expression "about 1 μM to about 5 mM" includes 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, and 5 mM.

[0053] One aspect of the present invention is formula (I): GX1MMX2LQHGSX3X4X5QTP(I)(Sequence ID 5) A polypeptide comprising or derived from the amino acid sequence of the formula, -X1 represents amino acid residue E, D, or A; -X2 represents amino acid residue D, E, or A; -X3 represents amino acid residues L, A, V, I, F, W, or Y; -X4 represents amino acid residue F, A, L, V, I, W, or Y; -X5 refers to a polypeptide representing amino acid residues L, A, V, I, F, W, or Y.

[0054] In certain embodiments, the polypeptide according to the present invention modulates the activity of at least one isoform of a natural tetrameric lactate dehydrogenase. In some embodiments, the polypeptide according to the present invention inhibits the tetramerization of lactate dehydrogenase subunits.

[0055] A further aspect of the present invention is a polypeptide that inhibits tetramerization of LDH subunits, comprising formula (I): GX1MMX2LQHGSX3X4X5QTP(I)(Sequence ID 5) The formula includes the amino acid sequence, -X1 represents amino acid residue E, D, or A; -X2 represents amino acid residue D, E, or A; -X3 represents amino acid residues L, A, V, I, F, W, or Y; -X4 represents amino acid residue F, A, L, V, I, W, or Y; -X5 refers to a polypeptide representing amino acid residues L, A, V, I, F, W, or Y.

[0056] In a particular embodiment, the polypeptide is of formula (I): GX1MMX2LQHGSX3X4X5QTP(I)(Sequence ID 5) It consists of the amino acid sequence, in the formula, -X1 represents amino acid residue E, D, or A; -X2 represents amino acid residue D, E, or A; -X3 represents amino acid residues L, A, V, I, F, W, or Y; -X4 represents amino acid residue F, A, L, V, I, W, or Y; -X5 represents amino acid residues L, A, V, I, F, W, or Y.

[0057] In a particular embodiment, the polypeptide is of formula (I): GX1MMX2LQHGSX3X4X5QTP(I)(Sequence ID 5) The formula includes the amino acid sequence, -X1 represents amino acid residue E or A; -X2 represents amino acid residue D or A; -X3 represents amino acid residue L or A; -X4 represents amino acid residue F or A; -X5 represents amino acid residue L or A.

[0058] In a particular embodiment, the polypeptide is of formula (I): GX1MMX2LQHGSX3X4X5QTP(I)(Sequence ID 5) It consists of the amino acid sequence, in the formula, -X1 represents amino acid residue E or A; -X2 represents amino acid residue D or A; -X3 represents amino acid residue L or A; -X4 represents amino acid residue F or A; -X5 represents amino acid residue L or A.

[0059] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 51, SEQ ID NOs: 53, 56, and SEQ ID NOs: 62 to 64.

[0060] In a particular embodiment, the polypeptide consists of an amino acid sequence selected from the group comprising SEQ ID NOs: 6 to 51, SEQ ID NOs: 53, 56, and SEQ ID NOs: 62 to 64.

[0061] In some embodiments, the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 51.

[0062] In some embodiments, the polypeptide comprises or consists of the amino acid sequence GEMMDLQHGSLFLQTP (SEQ ID NO: 6). In practice, the polypeptide of the amino acid sequence GEMMDLQHGSLFLQTP (SEQ ID NO: 6) is called polypeptide GP-16.

[0063] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 28.

[0064] In some embodiments, the polypeptide consists of an amino acid sequence selected from the group comprising SEQ ID NOs: 6 to 28.

[0065] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, SEQ ID NOs: 8, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 17, and SEQ ID NOs: 23.

[0066] In some embodiments, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, SEQ ID NOs: 8, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 17, and SEQ ID NOs: 23.

[0067] In certain embodiments, the polypeptide comprises or consists of the amino acid sequence LEDKLKGEMMDLQHGSLFLQTP (SEQ ID NO: 29). In practice, the polypeptide of the amino acid sequence LEDKLKGEMMDLQHGSLFLQTP (SEQ ID NO: 29) is called polypeptide LP-22.

[0068] In some embodiments, the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 51.

[0069] In certain embodiments, the polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 33, and 34, 40, and 46.

[0070] In some embodiments, the polypeptide contains or consists of LDH-H (also called LDH-1), i.e., the amino acid sequence of SEQ ID NO: 2, in which case one amino acid residue is substituted according to: E62A (SEQ ID NO: 53), D65A (SEQ ID NO: 56), L71A (SEQ ID NO: 62), F72A (SEQ ID NO: 63), or L73A (SEQ ID NO: 64), with the position of each substitution calculated relative to the first amino acid residue at the N-terminus of SEQ ID NO: 2.

[0071] In certain embodiments, the N-terminal amino acid residue of the polypeptide according to the present invention is acetylated. In some embodiments, the N-terminal G amino acid residue of the polypeptide sequence numbers 6 to 28 is acetylated.

[0072] In certain embodiments, the C-terminal amino acid residue of the polypeptide according to the present invention is amidated. In some embodiments, the C-terminal P amino acid residue of the polypeptides of sequence numbers 6 to 51 is amidated.

[0073] In some embodiments, the amino acid sequence of the polypeptide is not SEQ ID NO: 87. In some embodiments, the amino acid sequence of the polypeptide does not include SEQ ID NO: 87. In some embodiments, the polypeptide does not consist of an amino acid sequence having 100, 99, 98, 97, 96, 95, 90, 85, 80, or 75% identity with SEQ ID NO: 87. In some embodiments, the polypeptide does not consist of an amino acid sequence having 100, 99, 98, 97, 96, 95, 90, 85, 80, or 75% identity with SEQ ID NO: 87.

[0074] In some embodiments, polypeptides are not detection reagents for single organ-specific proteins.

[0075] In certain embodiments, the C-terminal amino acid residue of the polypeptide according to the present invention is further N-alkylamidated or N-arylamidated.

[0076] In some embodiments, the -OH group of the free -COOH group of the last amino acid residue at the C-terminus of the polypeptide is replaced with a group selected from -O-alkyl groups, -O-aryl groups, -NH2 groups, -N-alkylamine groups, -N-arylamine groups or -N-alkyl / aryl groups.

[0077] Non-limiting examples of suitable alkyl groups include C1-C 12 alkyl. Non-limiting examples of aryl groups include phenyl, xylyl, or naphthyl groups, which may be substituted with one or more atoms or groups selected from O, N, -OH, -NH2, C1-C 12 alkyl groups, and halogens (F, Cl, Br, I). Non-limiting examples of -N-alkylamine groups include -NR 1 R 2 groups, wherein R 1 and R 2 represent H or a C1-C 12 alkyl group. Non-limiting examples of -N-arylamine groups include -NHR 3 , wherein R 3 represents a phenyl, tolyl, xylyl, or naphthyl group, which may be substituted with one or more atoms or groups selected from O, N, -OH, -NH2, C1-C 12 alkyl groups, and halogens (F, Cl, Br, I). Non-limiting examples of -N-alkyl / arylamine groups include -NR 4 R 5 , wherein R 4 represents C1-C 12 alkyl, and R 5 represents a phenyl, tolyl, xylyl, or naphthyl group, which may be substituted with one or more atoms or groups selected from O, N, -OH, -NH2, C1-C 12 alkyl groups, and halogens (F, Cl, Br, I).

[0078] In practice, replacement of the -OH group of the free -COOH group can be performed according to any suitable method known in the prior art, or a method modified therefrom.

[0079] In some embodiments, the lactate dehydrogenase subunit is the lactate dehydrogenase H(LDH-H) subunit (also known as LDH-1).

[0080] In some embodiments, the lactate dehydrogenase subunit is the lactate dehydrogenase M(LDH-M) subunit (also known as LDH-5).

[0081] In certain embodiments, the polypeptide of the present invention can prevent the formation of a functional tetramer of the LDH-H subunit (corresponding to isoform LDH-1) by interacting with amino acid residues L166, A169, R170, P183, K246, W251, A252, and L255 of the full-length LDH-H subunit of the sequence of SEQ ID NO: 2.

[0082] The present invention also relates to derivatives of polypeptides as defined herein.

[0083] In fact, the present invention also encompasses any polypeptide different from the polypeptides specifically disclosed herein, e.g., the polypeptide of the amino acid sequence of SEQ ID NO: 5, through one or more substitutions, deletions, additions, and / or insertions. Such derivatives may be of natural origin or may be produced by synthesis, for example, by modifying one or more of the polypeptide sequences of the present invention, evaluating the inhibitory activity of one or more of the polypeptides of the present invention, and / or by using any of the many techniques well known in the art.

[0084] The structure of the polypeptide of the present invention may be modified, and functional molecules encoding derivative polypeptides having desirable properties can still be obtained. If it is desirable to modify the amino acid sequence of the polypeptide according to the present invention in order to produce equivalents, or even improved variants or portions, those skilled in the art will typically modify one or more codons of the coding nucleic acid (e.g., DNA) sequence.

[0085] For example, a specific amino acid residue in the protein structure may be substituted with another amino acid residue without recognizing its ability to bind to other polypeptides (e.g., polypeptide LDH-Htr of sequence number 4). Since the binding ability and properties of a protein define its biological functional activity, specific amino acid sequence substitutions may be made to the protein sequence and, of course, its underlying DNA coding sequence, and nevertheless, proteins with similar properties can be obtained.

[0086] Accordingly, it is intended that various changes may be made to the polypeptide sequence of the present invention, or to the corresponding nucleic acid sequence (e.g., DNA sequence) encoding the polypeptide, without recognizably losing their inhibitory activity. Often, variants of the polypeptide according to the present invention include one or more conservative substitutions. A “conservative substitution” is a substitution in which an amino acid residue is substituted for another amino acid residue having similar properties, and which a person skilled in the art of peptide chemistry would predict will not substantially change the secondary structure and hydroxyl properties of the polypeptide.

[0087] Therefore, as outlined above, amino acid substitutions are usually based on the relative similarity of amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions considering various aforementioned characteristics are well known to those skilled in the art and include amino acid residues R and K; amino acid residues D and E; amino acid residues S and T; amino acid residues Q and N; and amino acid residues A, V, L, and I.

[0088] Further amino acid substitutions may be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the amino acid residues. For example, negatively charged amino acid residues include amino acid residues D and E; positively charged amino acid residues include amino acid residues K and R; and amino acid residues with uncharged head groups having similar hydrophilic values ​​include amino acid residues A, L, I and V; amino acid residues G and A; amino acid residues N and Q; and amino acid residues S, T, F and Y. Other groups of amino acid residues that may represent conservative changes include (1) amino acid residues A, P, G, E, D, Q, N, S, T; (2) amino acid residues C, S, Y, T; (3) amino acid residues V, I, L, M, A, F; (4) amino acid residues K, R, H; and (5) amino acid residues F, Y, W, H.

[0089] Derivatives of the polypeptide according to the present invention may similarly or otherwise involve non-conservative modifications. In another embodiment, the derivative differs from the polypeptide sequence by substitution, deletion, or addition of five or fewer amino acid residues. The derivative may similarly (or otherwise) be modified, for example, by deletion or addition of amino acid residues that have little effect on the inhibitory ability of the polypeptide according to the present invention.

[0090] In another specific embodiment, the polypeptide of the present invention comprises all or part of a lactate dehydrogenase subunit, more specifically, a tetramerized domain of a lactate dehydrogenase M (LDH-M) or lactate dehydrogenase H (LDH-H) subunit.

[0091] In some embodiments, the polypeptide according to the present invention comprises at least 16 amino acid residues. Within the scope of the present invention, the expression “at least 16 amino acid residues” encompasses 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more amino acid residues.

[0092] In certain embodiments, the polypeptide of the present invention comprises 16 to 200 amino acid residues, preferably 16 to 150 amino acid residues, more preferably 16 to 125 amino acid residues. In some embodiments, the polypeptide of the present invention comprises 16 to 100 amino acid residues, preferably 16 to 75 amino acid residues, 16 to 50 amino acid residues, or 16 to 40 amino acid residues. In certain embodiments, the polypeptide of the present invention comprises 16 to 30 amino acid residues, 16 to 22 amino acid residues, or 16 to 20 amino acid residues.

[0093] In certain embodiments, the polypeptide of the present invention comprises 22 to 200 amino acid residues, preferably 22 to 150 amino acid residues, more preferably 22 to 125 amino acid residues. In some embodiments, the polypeptide of the present invention comprises 22 to 100 amino acid residues, preferably 22 to 75 amino acid residues, 22 to 50 amino acid residues, or 22 to 40 amino acid residues. In certain embodiments, the polypeptide of the present invention comprises 22 to 30 amino acid residues, or 22 to 25 amino acid residues.

[0094] In one embodiment, the polypeptide of the present invention contains up to 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 90, 80, 70, 60, 50, 40, and 30 amino acid residues. In a particular embodiment, the polypeptide of the present invention contains up to 22 amino acid residues, preferably up to 16 amino acid residues.

[0095] In one embodiment, the polypeptide of the present invention contains up to 332 or 334 amino acid residues. In one embodiment, the polypeptide of the present invention contains 16 to 332 amino acid residues. In one embodiment, the polypeptide of the present invention contains 16 to 334 amino acid residues.

[0096] In one embodiment, the polypeptide of the present invention contains up to 312 or 314 amino acid residues. In another embodiment, the polypeptide of the present invention contains fewer than 312 or 314 amino acid residues.

[0097] In some embodiments, the amino acid sequence of the polypeptide according to the present invention is not SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0098] In certain embodiments, the polypeptide according to the present invention further comprises at least one additional amino acid sequence, hereafter referred to as a “tagged polypeptide.” As used herein, the term “tagged polypeptide” refers to a polypeptide that enables the polypeptide of the present invention to be specifically labeled by an epitope so as to be detected or purified, or to be targeted to a particular cell, a particular tissue or a particular organ, i.e., a particular body part of interest. In the embodiments described above, the polypeptide further comprises at least one tagged polypeptide.

[0099] Furthermore, in certain embodiments, the tagged polypeptide further enables the polypeptide of the present invention to target the cytoplasm, nucleus, or organelles of target cells, and more preferably cancer cells.

[0100] In certain embodiments of the present invention, the tag polypeptide is short enough so as not to interfere with the inhibitory activity of the polypeptide of the present invention. Exemplarily, a suitable tag polypeptide typically has at least 6 amino acid residues, preferably about 8 to about 50 amino acid residues, and more preferably about 10 to about 20 amino acid residues.

[0101] The tag polypeptides for use in the present invention may be tag polypeptides that provide epitopes to which anti-tag antibodies can selectively bind, or which enable the easy purification of the polypeptides of the present invention by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.

[0102] Various tag polypeptides are known in the art. Examples include polyhistidine (polyhis) or polyhistidine-glycine (polyhis-gly) tags; influenza HA tag polypeptides, MIC tags, herpes simplex virus glycoprotein D (gD) tags, flag peptides; KT3 epitope peptides; α-tubulin epitope peptides; and T7 gene 10 protein peptide tags.

[0103] The polypeptides according to the present invention may also be modified to be readily detectable, for example, by biotinylation or by incorporating any detectable label known in the art, such as radiolabeling, fluorescent labeling or enzymatic labeling. Accordingly, in certain embodiments, the polypeptides of the present invention may further include any amino acid sequence (e.g., His tag, biotin tag or streptavidin tag) that enables easier purification or detection of the polypeptide.

[0104] Accordingly, in certain embodiments, the polypeptide according to the present invention may further comprise at least one tagged polypeptide in a cell membrane permeable peptide (CPP), also known as a protein transduction domain that facilitates entry into cells. As is well known in the art, cell membrane permeable peptides are typically short-chain peptides of up to 30 amino acid residues that have a positive net charge and act in a receptor-independent and energy-dependent manner.

[0105] Therefore, the polypeptide according to the present invention may contain one or more cell membrane-permeable peptides. In this case, the cell membrane-permeable peptides may be cleavable within the cell. Examples of CPPs include CPPs selected from the group consisting of hydrophilic and amphiphilic CPPs. Hydrophilic CPPs are usually peptides mainly composed of hydrophilic amino acids rich in amino acid residues R and K.

[0106] Non-specific examples of hydrophilic CPPs include: Antennapedia penetratin (RQIKWFQNRRMKWKK, Sequence ID 68), TAT(YGRKKRRQRRR, Sequence ID 69), SynB1(RGGRLSYSRRRFSTSTGR, Sequence ID 70), SynB3(RRLSYSRRRF, Sequence ID 71), PTD-4 (PIRRRKKLRRLK, Sequence ID 72), PTD-5 (RRQRRTSKLMKR, Sequence ID 73), FHV Coat-(35-49)(RRRRNRTRRNRRRVR, Sequence ID 74), BMV Gag(7-25)(KMTRAQRRAAARRNRWTAR, Sequence ID 75), HTLV-II Rex-(4-16)(TRRQRTRRARRNR, SEQ ID NO: 76), D-Tat(GRKKRRQRRRPPQ, Sequence ID 77), and R9-Tat(GRRRRRRRRRPPQ, Sequence ID 78).

[0107] Amphiphilic CPPs are typically peptides rich in the amino acid residue K. Non-exclusive examples of amphiphilic CPPs include antimicrobial peptides such as MAPs or transportans. Transportan (GWTLNSAGYLLGKINLKALAALAKKIL, Sequence ID 79), MAP(KLALKLALKLALALKLA, SEQ ID NO: 80), SBP(MGLGLHLLVLAAALQGAWSQPKKKRKV, Sequence ID 81), FBP(GALFLGWLGAAGSTMGAWSQPKKKRKV, Sequence ID 82), MPG(GALFLGFLGAAGSTMGAWSQPKKKRKV, Sequence ID 83), MPG (ΔNLS) (GALFLGFLGAAGSTMGAWSQPKSKRKV, Sequence ID 84) Pep-1 (KETWWETWWTEWSQPKKKRKV, Sequence ID 85), and Pep-2(KETWFETWFTEWSQPKKKRKV, Sequence ID 86).

[0108] Antennapedia-derived penetratin and TAT peptides, or their derivatives, are widely used tools, in particular, for the delivery of cargo molecules such as peptides, proteins, and oligonucleotides into cells (Fischer et al.; Cellular Delivery of Impermeable Effector Molecules in the Form of Conjugates with Peptides Capable of Mediating Membrane Translocation; Bioconjugate Chem. 2001, 12, 6, 825-841). In another embodiment, the polypeptides of the present invention also include cell membrane-permeable peptides such as the polypeptides disclosed in International Publication Nos. 2011 / 157713 and 2011 / 157715 (Hoffmann La Roche®), or their derivatives.

[0109] In certain embodiments of the present invention, the polypeptide according to the present invention is linked to at least one cell membrane-permeable peptide (CPP) by a linker. In the sense of the present invention, “linker” means a portion comprising a single covalent bond or a series of stable covalent bonds, which often incorporates 1 to 40 multivalent atoms selected from the group consisting of C, N, O, S, and P, and covalently affixes a binding functional group or a physiologically active group to the ligand of the present invention. The number of multivalent atoms in the linker may be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, or 30 or a larger number up to 40. The linker may be linear or nonlinear, and some linkers may have a pendant side chain or a pendant functional group (or both).

[0110] The polypeptides of the present invention can be prepared by culturing cells transformed with or transfected with a vector containing a nucleic acid encoding a desired polypeptide, or by alternative methods well known to those skilled in the art, such as direct peptide synthesis using solid-phase technology or in vitro protein synthesis.

[0111] The present invention also relates to nucleic acids encoding polypeptides according to the present invention.

[0112] In some embodiments, the nucleic acid includes a DNA nucleic acid sequence.

[0113] The present invention also relates to a nucleic acid vector comprising at least one nucleic acid according to the present invention.

[0114] Within the scope of the present invention, the expression “at least one nucleic acid” is intended to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleic acids.

[0115] In some embodiments, the vector enables the controlled expression of the at least one polypeptide. As used herein, the expression “controlled expression” is intended to refer to expression that is controlled temporally and / or spatially. In other words, controlled expression of the polypeptide according to the present invention may occur, for example, in a specific part of the body, such as a particular organ, and / or over a specific period of time.

[0116] In certain embodiments, the vector is a viral vector selected from the group including viruses, preferably adenoviruses, adeno-associated viruses (AAVs), alphaviruses, herpesviruses, lentiviruses, non-embedded lentiviruses, retroviruses, vaccinia viruses, and baculoviruses.

[0117] In some embodiments, the polypeptides, nucleic acids, or nucleic acid vectors according to the present invention may be included in a delivery molecule, particularly in combination with other natural or synthetic compounds such as lipids, proteins, peptides, or polymers.

[0118] Within the scope of the present invention, the delivery particles are intended to supply, or "deliver," the polypeptide, nucleic acid, or nucleic acid vector according to the present invention to a target cell, tissue, or organ.

[0119] In some embodiments, the delivery particles may be in the form of lipoplexes containing cationic lipids; lipid nanoemulsifiers; solid lipid nanoparticles; peptide-based particles; polymer-based particles containing natural and / or synthetic polymers in particular; and mixtures thereof.

[0120] In some embodiments, the polymer-based particles may include synthetic polymers, particularly polyethyleneimine (PEI), dendrimers, poly(DL-lactide) (PLA), poly(DL-lactide-co-glycoside) (PLGA), polymethacrylates, and polyphosphoesters.

[0121] In some embodiments, the delivery particle further comprises on its surface one or more ligands suitable for directing polypeptides, nucleic acids, or nucleic acid vectors to target cells, tissues, or organs.

[0122] Another aspect of the present invention relates to a pharmaceutical composition comprising (i) at least one polypeptide, at least one nucleic acid, or at least one nucleic acid vector according to the present invention, and (ii) at least one pharmaceutically acceptable vehicle.

[0123] In some embodiments, the present invention relates to a pharmaceutical composition comprising (i) at least one polypeptide according to the present invention, and (ii) at least one pharmaceutically acceptable vehicle.

[0124] In some embodiments, a pharmaceutically acceptable vehicle is selected from the group comprising or consisting of solvents, diluents, carriers, excipients, dispersions, coatings, antimicrobial agents, antifungal agents, isotonic agents, absorption retarders, and any combination thereof. The carrier, diluent, solvent, or excipient must be compatible with the polypeptide or its derivatives and “acceptable” in the sense that it is not harmful when administered to an organism. Typically, the vehicle does not produce adverse reactions, allergic reactions, or other undesirable reactions when administered to an organism, preferably a human organism.

[0125] For specific purposes of human administration, pharmaceutical compositions must meet the sterility, pyrogenicity, general safety, and purity standards required by regulatory authorities such as the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA).

[0126] In some embodiments, the carrier may be sterile, pyrogenic water or saline solution (e.g., physiological saline). Suitable excipients include mannitol, dextrose, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate.

[0127] Acceptable carriers, solvents, diluents, or excipients for therapeutic use are well known in the pharmaceutical technology field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (ARGennaro ed. 1985). The selection of suitable pharmaceutical carriers, solvents, excipients, or diluents can be made with respect to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may contain, or in addition to, any suitable binders, lubricants, suspending agents, coatings, or solubilizers as carriers, excipients, solvents, or diluents. Preservatives, stabilizers, colorants, and even flavoring agents may be supplied to the pharmaceutical composition.

[0128] The formulations may conveniently exist as unit dosage forms and can be prepared by any method and good practice well known in the pharmaceutical art. Such methods include the step of mixing a polypeptide with a carrier constituting one or more auxiliary components.

[0129] Formulations according to the present invention suitable for oral administration may be presented as separate units, each containing a predetermined amount of the polypeptide according to the present invention, such as capsules, cachets, or tablets; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-oil liquid emulsions. The polypeptide of the present invention may also be presented as a bolus, lick, or paste.

[0130] Formulations suitable for parenteral administration may include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the target recipient; as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. Formulations may be presented in unit dose or multi-dose containers, such as sealed ampoules and vials, or may be stored in a freeze-dried state, requiring only the addition of a sterile liquid carrier, such as distilled water for injection, immediately before use. Immediate injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations for use in the present invention may further contain other conventional agents in the art, depending on the type of formulation; for example, formulations suitable for oral administration may contain flavoring agents.

[0131] The present invention further relates to a drug comprising at least one polypeptide, nucleic acid, vector, or delivery particle according to the present invention.

[0132] Further aspects of the present invention relate to (i) a kit comprising at least one polypeptide, at least one nucleic acid, at least one nucleic acid vector, or at least one pharmaceutical composition according to the present invention, and (ii) at least one means for administering the polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition.

[0133] In certain embodiments, the means for administering polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention may include syringes, sheaths, catheters, cups, spatulas, and the like.

[0134] In some embodiments, the kit further comprises an anticancer agent.

[0135] Anticancer drugs are publicly known thanks to the latest technology. Non-exclusive examples of anticancer drugs include acalabrutinib, alectinib, alemtuzumab, anastrozole, avapritinib, avelumab, bellinostat, bevacizumab, bleomycin, blinatumomab, bosutinib, brigatinib, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, copanlisib, cytarabine, daunorubicin, decitabine, dexamethasone, docetaxel, doxorubicin, encorafenib, erdafitinib, etoposide, everolimus, and Examples include xemestane, fludarabine, 5-fluorouracil, gemcitabine, ifosfamide, imatinib mesylate, leuprolide, lomustine, mechloretamine, melphalan, methotrexate, mitomycin, nelarabine, paclitaxel, pamidronic acid, panobinostat, pralatrexate, prednisolone, ofatumumab, rituximab, temozolomide, topotecan, tocitumomab, trastuzumab, vandetanib, vincristine, vorinostat, and zanubrutinib.

[0136] In certain embodiments, the anticancer agent is administered simultaneously or sequentially with a polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition according to the present invention.

[0137] One aspect of the present invention relates to a polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition according to the present invention for use as a pharmaceutical.

[0138] In some further embodiments, the present invention also relates to the use of polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention for the manufacture or preparation of pharmaceuticals.

[0139] One aspect of the present invention relates to polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions for use in the prevention and / or treatment of cancer.

[0140] In some embodiments, the cancer to be prevented / treated is characterized by metabolic reprogramming. In some embodiments, the cancer to be prevented / treated is characterized by cancer cells having a high glycolysis flow rate. In some embodiments, the cancer to be prevented / treated is characterized by cancer cells having a high lactate production rate.

[0141] The present invention also relates to a method for preventing and / or treating cancer in an individual in need thereof, comprising at least the step of administering to the individual a therapeutically effective amount of a polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition according to the present invention.

[0142] The present invention also relates to polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention for use in inhibiting the growth of cancer cells.

[0143] The present invention also relates to a method for inhibiting the growth of cancer cells in an individual in need thereof, comprising at least the step of administering to the individual a therapeutically effective amount of a polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition according to the present invention.

[0144] The present invention also relates to polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention for use in improving the overall survival of individuals with cancer.

[0145] The present invention also relates to a method for improving the overall survival of an individual with cancer, comprising at least the step of administering to the individual a therapeutically effective amount of a polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition according to the present invention.

[0146] The present invention also relates to polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention for use in improving the prognosis of individuals with cancer.

[0147] The present invention also relates to a method for improving the prognosis of an individual having cancer, comprising at least the step of administering to the individual a therapeutically effective amount of a polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition according to the present invention.

[0148] As used herein, “cancer” is intended to mean the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all precancerous and cancer-like cells and tissues.

[0149] Within the scope of this invention, the terms “cancer” and “cancer-like” are intended to refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth or proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include breast cancer, prostate cancer, colon cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancers.

[0150] In another aspect, the present invention further relates to polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention for use in the prevention and / or treatment of cancers involving oxidative cancer-like cells and / or glycolytic cancer-like cells.

[0151] A further aspect of the present invention relates to polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions for use in the prevention and / or treatment of cancer in individuals requiring it.

[0152] The present invention also relates to polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention for use in inhibiting the growth of cancer cells in individuals requiring such inhibition.

[0153] As used herein, “individual” means a mammal or a non-mammal, and preferably a human being.

[0154] In some embodiments, the non-human animal may be selected from a group of useful or pet animals, including dogs, cats, rats, mice, monkeys, cattle, sheep, goats, pigs, and horses.

[0155] In some embodiments, the "individual needing it" has been diagnosed with cancer and / or metastasis. In certain embodiments, the individual is susceptible to developing cancer and / or metastasis. In some embodiments, the "individual needing it" is at risk of developing cancer and / or metastasis. In certain embodiments, the "individual needing it" has already been treated for cancer and / or metastasis.

[0156] In some embodiments, individuals to be treated with the polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition according to the present invention may be further administered with additional anticancer agents.

[0157] For example, when preventing or treating breast cancer, additional therapeutic agents may be drugs known to prevent or treat breast cancer. Similarly, when preventing or treating uterine cancer, additional therapeutic agents may be drugs known to prevent or treat uterine cancer.

[0158] Exemplary examples include any anticancer agent known in the art. Examples of further anticancer agents include doxorubicin, doxorubicin, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, such as paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (toxotereDD, Rhone-Poulenc). Examples include Rorer, Antony, France, toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamycin (see U.S. Patent No. 4,675,187), melphalan, and other related nitrogen mustards. Furthermore, this definition includes hormonal agents that act to modulate or inhibit the action of hormones on tumors, such as tamoxifen and onapristone.

[0159] Further anticancer agents may be administered simultaneously with the polypeptide of the present invention (i.e., co-administration as a co-formulation, if necessary) or at a different time from the polypeptide (i.e., sequential administration in which the further therapeutic agent is administered before or after the polypeptide is administered). Further anticancer agents may be administered in the same manner as the polypeptide of the present invention, or by using the usual route of administration for the further anticancer agent.

[0160] The present invention further relates to polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention for use in blocking basal-level autophagy in individuals requiring it.

[0161] The present invention further relates to a method for blocking basal-level autophagy in an individual requiring such blocking, comprising at least the step of administering to the individual a therapeutically effective amount of a polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition according to the present invention.

[0162] As used herein, the term “basal-level autophagy” is intended to refer to macroautophagy activity during cell growth in normal media containing amino acids and serum, which appears to be highly active in many cell types and animal tissues.

[0163] Some aspects of the present invention relate to the use of polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention for inhibiting the tetramerization of lactate dehydrogenase subunits.

[0164] In some embodiments, the lactate dehydrogenase subunit is the LDH-1 subunit and / or the LDH-5 subunit.

[0165] In fact, isoform LDH-1 is composed of four LDH-H subunits, so LDH-1 subunits are called LDH-H subunits, while isoform LDH-5 is composed of four LDH-M subunits, so LDH-5 subunits are called LDH-M subunits.

[0166] In some embodiments, the lactate dehydrogenase subunit is the LDH-1 subunit.

[0167] In some embodiments, polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention are administered orally, parenterally, topically, by inhalation spray, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term administration includes subcutaneous, intravenous, intramuscular, intraocular, intra-articular, intra-sacral, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques.

[0168] In preferred embodiments, the polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition of the present invention is administered parenterally, subcutaneously, intravenously, or via an implanted reservoir.

[0169] In some embodiments, the polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions of the present invention are in a form suitable for injection, for example, intraocular, intramuscular, subcutaneous, intradermal, transdermal, or intravenous injection or infusion.

[0170] Examples of forms suitable for injection include, but are not limited to, solutions such as sterile aqueous solutions, dispersions, emulsions, and suspensions, and solid forms suitable for use in preparing solutions or suspensions when adding liquids before use, such as powders and liposomes.

[0171] The treatment may consist of a single dose or multiple doses over a certain period of time. The polypeptides, nucleic acids, nucleic acid vectors, or pharmaceutical compositions according to the present invention may be formulated into sustained-release formulations to provide sustained release over a long period of time, such as at least 2, 4, 6, or 8 weeks. Preferably, the sustained release is provided over at least 4 weeks.

[0172] In certain embodiments, the effective amount of polypeptide, nucleic acid, nucleic acid vector, or pharmaceutical composition to be administered may depend on various parameters, including the substance selected for administration, whether the administration is a single dose or multiple doses, and parameters of the subject, such as the age, health status, physique, weight, sex, and severity of the cancer being treated.

[0173] In some embodiments, the polypeptide according to the present invention is administered in a therapeutically effective dose to a subject in need.

[0174] "Therapeutic dose" means the level or amount of polypeptide or pharmaceutical composition necessary and sufficient to slow or halt the progression, exacerbation or worsening of one or more symptoms of cancer; or to alleviate the symptoms of cancer; or to cure cancer, without causing significant undesirable or harmful side effects to an individual.

[0175] In certain embodiments, the effective amount of the polypeptide according to the present invention may be in the range of about 0.001 mg to about 3,000 mg per dose unit, preferably about 0.05 mg to about 1,000 mg per dose unit.

[0176] Within the scope of this invention, approximately 0.001 mg to approximately 3,000 mg refers to approximately 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, and 0.2 mg per dose unit. , 0.3mg, 0.4mg, 0.5mg, 0.6mg, 0.7mg, 0.8mg, 0.9mg, 1mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 20mg, 30 mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg , 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1,000mg, 1,100mg, 1,150mg, 1,200mg, 1,250mg, 1, 300mg, 1,350mg, 1,400mg, 1,450mg, 1,500mg, 1,550mg, 1,600mg, 1,650mg, 1,700mg, 1,750mg, 1,800mg, 1,850m Includes g, 1,900 mg, 1,950 mg, 2,000 mg, 2,100 mg, 2,150 mg, 2,200 mg, 2,250 mg, 2,300 mg, 2,350 mg, 2,400 mg, 2,450 mg, 2,500 mg, 2,550 mg, 2,600 mg, 2,650 mg, 2,700 mg, 2,750 mg, 2,800 mg, 2,850 mg, 2,900 mg, 2,950 mg and 3,000 mg.

[0177] In certain embodiments, the polypeptide according to the present invention should be administered at a dose level sufficient to deliver the body weight of the subject per day, approximately 0.001 mg / kg to approximately 100 mg / kg, approximately 0.01 mg / kg to approximately 50 mg / kg, preferably approximately 0.1 mg / kg to approximately 40 mg / kg, preferably approximately 0.5 mg / kg to approximately 30 mg / kg, approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, and more preferably approximately 1 mg / kg to approximately 25 mg / kg.

[0178] In some embodiments, the effective amount of nucleic acid or nucleic acid vector to be administered is approximately 1 × 10⁻⁶ 5 ~Approx. 1×10 15 This may be within the range of copies / dosage units.

[0179] Within the scope of the present invention, approximately 1 × 10 5 ~Approx. 1×10 15 The copy / dosage unit is 1 × 10⁻⁶ 5 , 2×10 5 , 3 x 10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1 x 10 6 , 2×10 6 , 3 x 10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1 x 10 7 , 2×10 7 , 3 x 10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1 x 10 8 , 2×10 8 , 3 x 10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1 x 10 9 , 2×10 9 , 3 x 10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×109 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , 9×10 13 , 1×10 14 , 2×10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , 9×10 14 and 1×10 15 comprising copies / dosage units.

[0180] Sequences used herein

Table 1

[0181] [Figure 1A-E] This is a series of schemes showing interaction mappings of the LDH-H tetramer boundary, highlighting two major clusters. A: X-ray crystal structure of LDH-1 (LDH-H4) as a dimer of dimers with two dimers (subunits A, C and B, D). B: Model of dimer LDH-Htr. C: Model of dimer LDH-H interacting with a single LDH-H subunit (PDB ID: 1I0Z) used to highlight the LDH tetramer boundary. D: Mapping of interactions between LDH-H subunit C and the LDH-1 tetramer boundary (dimer BD) using MOE software. The X and Y axes represent the number of residues in dimer BD and subunit C, respectively. This mapping identifies two interaction clusters, cluster A and B. E: Representation of different domains of natural LDH-1 (uniplot P07195). The number of residues is on the same scale as the X axis in Figure 1D. [Figure 2A-E]This is a series of graphs showing that LDH-Htr behaves as a weak tetramer. A: Superposition of size exclusion chromatograms of LDH-Htr and LDH-1 using a Superdex200 10 / 300 GL column. B: Evaluation of LDH-Htr self-interaction using microscale thermophoresis (MST) at 20 seconds "on-time" (n=3) (Kd=1.25 μM [0.96~1.62 μM]). C: Evaluation of the effect of its subunit concentration on the melting temperature of LDH-Htr using nanoscale differential scanning fluorescence (nanoDSF) (n=3). Tm1 and Tm2 refer to the two transition temperatures observed for the LDH-Htr denaturation pattern. D: NanoDSF profiles of LDH-Htr at various concentrations (n=3). RFU: Relative fluorescence units. Mass photometry of E:LDH-Htr is shown, along with the calculated molecular weight of the complex in solution and their relative intensities indicated on the peaks (theoretical Mw of the dimer is 73.2 kDa). [Figure 3A-D] This is a series of graphs showing that polypeptide LP-22 interacts with LDH at the LDH tetramer boundary, destabilizing tetrameric LDH and stabilizing dimeric LDH. A: WaterLOGSY spectra of the interaction between LP-22 (400 μM) and 15 μM dimeric LDH-Htr (upper curve) and tetrameric LDH-1 (lower curve). B: MST binding curve between polypeptide LP-22 and LDH-Htr. Binding curves were extracted from MST output figures at 1.5 seconds MST on-time (n=3). C: NanoDSF modification of dimeric LDH-Htr (15 μM) in the presence (curve 2) and absence (curve 1) of polypeptide LP-22 (500 μM) (ΔTm=2.8℃ n=3). D: Shows ΔTm (°C) of tetramer LDH-5 (300 nM) as a function of polypeptide LP-22 concentration (EC50 = 47 μM [32~68 μM], n = 3). [Figure 4A-D]A series of schemes and graphs showing that end-removal of polypeptide LP-22N results in polypeptide GP-16 with a similar interaction profile. A: (Upper panel) Comparison of differences in WaterLOGSY and 1H NMR spectra between polypeptide LP-22 (upper) and polypeptide GP-16 (lower) in the presence of 15 μM LDH-Htr. Signals appearing in the 1H spectrum but not in WaterLOGSY correspond to non-interacting residues. The polypeptide LP-22 spectrum clearly shows that several lysine, glutamic acid, aspartic acid, and leucine residues do not interact with LDH-Htr. These non-interacting signals are no longer present on the polypeptide GP-16 spectrum (lower). (Lower panel) Amino acid sequence of polypeptide LP-22 is shown. Dark residues correspond to non-interacting residues according to ΔG calculations and WaterLOGSY analysis. B: Calculation of the contribution of polypeptide LP-22 residues to the total free energy of binding using MOE software. C: Shows the MST binding curve between polypeptide GP-16 and LDH-Htr. The binding curve was extracted from the MST output figure at 1.5 seconds MST on time (n=3). D: Shows the difference in melting temperature (ΔTm, °C) of tetramer LDH-5 (300 nM) as a function of polypeptide GP-16 concentration (EC50 = 262 μ<[142~383 μM], n=3). [Figure 5A-D] This is a series of graphs showing that mutations in cluster B1 elucidate the residues crucial for LDH tetramerization. Mass photometry was performed on LDH-1(A) and LDH-H variants E62A(B), L71A(C), and F72A(D), and the results are shown along with the experimental molecular weights of the complexes in solution and their relative intensities. The theoretical molecular weight of the tetramer is 155 kDa; the theoretical molecular weight of the dimer is 78 kDa. [Figure 6A-D]This series of graphs demonstrates how the use of orthogonal analysis clarifies the effects of important mutations on the stability of the LDH-H tetramer. A: Tryptophan fluorescence spectra of LDH-1 (curve 1) and various LDH-H variants: LDH-HE62A (curve 2), LDH-HL71A (curve 3), and LDH-HF72A (curve 4). λexec=286nm (n=6). B: NanoDSF profiles of LDH-Htr (curve 1) and LDH-HD65A (curve 2) (n=6). C: NanoDSF profiles of LDH-HL66A (curve 1) and LDH-HL73A (curve 2) (n=6). D: Fluorescence intensity of tetramers LDH-HL66A (curve 1) and LDH-HL73A (curve 2) at 50 μg / mL (1.3 μM) upon addition of guanidium-HCl (n=6). [Figure 7A-C] This is a series of graphs showing structural models of the interaction between cluster B1 hotspots and cluster B2. A: Shows the interaction between the sequence corresponding to polypeptide GP-16 and cluster B2. The surface corresponds to the molecular surface of LDH-H cluster B2. B: Focuses on the hydrophobic hotspot of cluster B1, where the interaction is formed by L71, F72, and L73 and cluster B2. C: Focuses on the hydrophobic hotspot of cluster B1, where the interaction is formed by D65 and E62 and cluster B2. This figure was isolated from the LDH-1 crystal structure and further minimized using MOE software (PDB ID: 1I0Z).

[0182] Examples The present invention will be further explained by the following examples.

[0183] 1. Materials and Methods 1.1-Chemicals and Peptides All reagents were purchased from different chemical manufacturers and used without further purification. Peptides were purchased from Genecust® (https: / / www.genecust.com). Structural integrity and severity grade (>95%) were evaluated by high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Polypeptide GP-16 was amidated and acetylated at its C and N termini, respectively, while polypeptide LP-22 was amidated only at its C termini.

[0184] 1.2-Production and Purification of Human LDH Protein The hLDH-H nucleotide sequences used for the production of full-length, truncated, and variant LDH-H proteins, and for insertion into the pET-28a expression vector, were ordered from Genecust®. Ndel and Bpu1102I restriction enzyme sites were used for sequence insertion, enabling the addition of an N-terminal 6-His tag. Protein production and purification were carried out according to the previous description by Thabault et al. (Interrogating the Lactate Dehydrogenase Tetramerization Site Using (Stapled) Peptides. J. Med. Chem. 2020, 63(9), 4628-4643). The recombinant plasmid was then transformed in the host bacterium E. coli Rosetta (DE3). Transformants were cultured at 37°C in Lysogeny Broth (LB) medium supplemented with 50 μg / mL kanamycin and 34 μg / mL chloramphenicol until an optical density of 0.6 was achieved. LDH expression was induced at 20°C for 20 hours by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Subsequently, cells were collected by centrifugation at 4°C for 25 minutes at 5,000 rpm (rotor 11150, Sigma®). The pellet was suspended in lysis buffer (50 mM Tris-HCl, pH 8.5, 10 mM MgCl2, 300 mM NaCl, 5 mM imidazole, and 10% glycerol), then pulverized by sonication, and subsequently centrifuged at 4°C at 10,000 rpm (rotor 12165-H, Sigma®) for 30 minutes. The insoluble fraction was discarded, and 1 μL of β-mercaptoethanol was added per 1 mL of the soluble fraction. Recombinant proteins were purified using a 1 mL His-Trap FF-crude column (GE Healthcare®) according to the manufacturer's instructions. Finally, protein concentrations were measured using the Bradford method with a protein assay kit (BioRad®), and sample homogeneity was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Coomassie brilliant blue as the staining agent.

[0185] The amino acid residue position of the LDH-H variant is calculated relative to the first amino acid residue at the N-terminus of LDH-H (also referred to herein as LDH-1; SEQ ID NO: 2).

[0186] 1.3-Nuclear Magnetic Resonance 1 Human LDH-H (full-length and cleaved)-6His protein was expressed for D NMR and purified from E. coli as described above. All experiments were performed on an Ascend Avance III 600 MHz system with a broadband cryoprobe, as described by Thabault et al. (see above).

[0187] For WaterLOGSY NMR studies, samples were prepared in 50 mM sodium phosphate buffer, pH 7.6, and 10% D2O containing 100 mM NaCl. LDH concentrations ranged from 5–20 μM monomers. Ligand binding was detected using the WaterLOGSY ephogsygpno.2 avance version sequence with a 1-second mixing time. Water signal suppression was achieved using an excitation sculpting scheme, and protein background signals were suppressed using a 50 ms spinlock. For spectral experiments of polypeptide LP-22 (SEQ ID NO: 29) and polypeptide GP-16 (SEQ ID NO: 6), 4096 scans were collected at 277 K, yielding 16 K free-induced decays (FIDs).

[0188] 1.4 - In Silico Evaluation The free binding energies and mappings of interactions at the LDH boundary were calculated using Molecular Operating Environment (MOE) software (ChemComp®) along with the LDH-1 crystal structure (PDB entry 1I0Z). The tetrameric LDH-1 was generated from the PDB crystal structure using the MOE bioassembly tool, following the previously described procedure (Thabault et al.; see above). A cleaved dimeric version of LDH-1 (LDH-Htr; SEQ ID NO: 4) was generated from the tetrameric complex by removing LDH-H subunits B and D, as well as the N-terminal domains of subunits A and C. Minimization was performed before free energy calculations and interaction mapping.

[0189] 1.5-Size Elimination Experiment The sample was loaded onto an equilibrated 200 10 / 300GL column and run at 0.75 mL / min using an AKTApure® system (GE Healthcare®) with 50 mM sodium phosphate, pH 7.6, and 100 mM NaCl as the mobile phase buffer. LDH-Htr was diluted to 15 μM in assay buffer according to the previously described procedure (Thabault et al. (see above)). The final injection volume was 500 μL. Molecular weight was determined using gel-filtered standards (BioRad®) in the same assay buffer, according to the manufacturer's instructions.

[0190] 1.6-Nano Differential Scanning Fluorescence Quantification Experiment NanoDSF was performed according to the previous description by Thabault et al. (see above).

[0191] a) Evaluation of variants Solutions of proteins (LDH-H, LDH-Htr, or variants) stored in 50 mM sodium phosphate, 100 mM NaCl, and 20% glycerol, pH 7.6, were evaluated using a Tycho NT.6 instrument (NanoTemper Technologies®) at concentrations ranging from 25 to 65 μM. Following standard manufacturer procedures, the sample was poured into a capillary tube and heated to 95°C for 3 minutes, while simultaneously tracking fluorescence emission at 330 and 350 nm. The melting temperature was extracted from the derivative of the 350 / 330 nm fluorescence ratio as the temperature rose.

[0192] b) Evaluation of peptides Solutions of peptide-containing proteins (LDH-1, LDH-5, or LDH-Htr) were evaluated using a Tycho NT.6 instrument (NanoTemper Technologies®). Evaluation was performed in 50 mM sodium phosphate and 100 mM NaCl, pH 7.6 buffer. Samples were poured into capillaries and processed as described above, following standard manufacturer procedures.

[0193] 1.7-Microscale Thermophoresis MST measurements were performed using a NanoTemper® Monolith NT.115 analyzer (NanoTemper Technologies®) with Red-dye-NHS fluorescent labeling. LDH-Htr, purified to homogenization, was labeled with Monilith Red-dye-NHS second-generation labeling dye (NanoTemper Technologies®) according to the provided protocol. Measurements were performed using standard treatment capillaries (NanoTemper Technologies®) in 50 mM sodium phosphate, pH 7.6, and 100 mM NaCl containing 0.01% Tween 20. The final protein concentration during the assay was 100 nM. The ligand was titrated at a 1:1 dilution according to the manufacturer's recommended conditions. The experiment was repeated three times using 40% LED output, high MST output, laser on-time of 20 seconds, and laser off-time of 3 seconds. The thermophoresis patterns of polypeptides were evaluated, and Kd was extracted from the raw data in 1.5-second MST on-time, according to the manufacturer's instructions.

[0194] 1.8-mass photometry Protein landing was recorded using a Refeyn OneMP (Refeyn Ltd., UK) mass photometry system by directly adding 1 μL of protein stock solution (1 μM) to a 16 μL droplet of filtered PBS solution. A 60-second video (6,000 frames) was acquired using AcquireMP (Refeyn Ltd., v2.1.1) software with standard settings. Data was analyzed using DiscoverMP (Refeyn Ltd, v2.1.1) with default settings. Contrast-to-mass (C2M) calibration was performed prior to the experiment using a mixture of proteins with molecular weights of 66 kDa, 146 kDa, 480 kDa, and 1048 kDa.

[0195] 1.9 - Spectrophotometric Experiment All spectrophotometric experiments were performed using a Spectramax m2e spectrophotometer (Molecular Devices) in opaque 96-well plates, following the previous description by Thabault et al. (see above).

[0196] Internal fluorescence assay: Using an excitation wavelength of 286 nm, the complete tryptophan fluorescence spectrum was recorded, and the emission spectrum at 320–400 nm at room temperature was recorded. The raw fluorescence of each experiment was subtracted from the corresponding protein-free control experiment. Experiments were carried out in 50 mM sodium phosphate and 100 mM NaCl, pH 7.6 buffer. For dissociation in subunits, the test protein (1.3 μM) was contacted with gradually increasing amounts of guanidium-HCl ranging from 0.3 M to 2 M, and the fluorescence spectrum was then recorded.

[0197] 1.10-Statistics All quantitative data are expressed as mean ± SEM. Error bars may be smaller than their abbreviations. n refers to the total number of repeated experiments. Data were analyzed using GraphPad Prism 7.0 software.

[0198] 2.Results 2.1-LDH-1 tetramer boundary in silico mapping identifies novel interaction clusters. The LDH tetramer state is a "dimer of a dimer." According to the X-ray structure, the orientation of the three different subunits can explain the LDH dimer structure. Indeed, cleavage of the LDH N-terminal domain results in a dimer (LDH-Htr; Sequence ID No. 4) (Thabault et al. (see above)). We hypothesized that only the association of dimers AC and BD in the tetramer can explain the role of this N-terminal domain in stabilizing the tetramer state (Figure 1A-C). Based on this hypothesis, we first mapped the interactions formed by one subunit and an LDH dimer (AC or BD) using Molecular Operating Environment (MOE) software. The mapping of these contact points clearly showed two clusters: A (A1 and A2) and B (B1 and B2) (Figure 1D). Clusters A1 and A2 corresponded to the LDH N-terminal tetramerization domain (Figure 1E) and its associated tetramerization site, respectively, as previously described by Thabault et al. (see above). Clusters B1 and B2 coincided with 22 previously unreported amino acid α-helices and their interaction sites. Interestingly, the sequence corresponding to cluster B1 was highly conserved in vertebrates. Overall, clusters A1 and B1 corresponded to a continuous epitope interacting with discontinuous oligomerization sites A2 and B2.

[0199] 2,2-LDH-Htr behaves as a weak tetramer via cluster B. Next, we aimed to confirm this interaction model and the predicted axis of symmetry of the LDH dimer using the dimeric LDH (LDH-Htr; SEQ ID NO: 4) model described by Thabault et al. (see above). Interaction mapping revealed that LDH-Htr lacks cluster A1 but still possesses clusters A2, B1, and B2. Therefore, it was inferred that LDH-Htr can still self-interact via clusters at high concentrations. Comparison of elution profiles between LDH-Htr and LDH-1 by size exclusion chromatography (SEC) suggested that LDH-Htr may indeed be in equilibrium between tetramer and dimer (Figure 2A). Consistently, evaluation of LDH-Htr self-interaction by microscale thermophoresis (MST) revealed that at high concentrations, the dimeric protein interacts with itself (Figure 2B, Kd = 1.25 μM [0.96~1.62 μM]). According to this model, this interaction could only be a result of LDH-Htr dimerization via cluster B. Monitoring this interaction using MST therefore provided useful information regarding the overall potency of cluster B. Next, we evaluated whether LDH-Htr self-association stabilizes the protein complex, given that protein oligomerization often leads to its stabilization. Using nanoscale differential scanning fluorescence (nanoDSF), we evaluated the LDH-Htr denaturation profile and revealed that the protein exhibits concentration-dependent destabilization (Figure 2C) and conformational changes (Figure 2D). We also evaluated the state of the LDH-Htr oligomer using mass photometry (MP). MP is a recent technique that enables single-molecule detection and mass measurement in solution based on light scattering (Young et al.; Quantitative Mass Imaging of Single Biological Macromolecules. Science 2018, 360(6387), 423-427). MP analysis of LDH-Htr revealed the equilibrium between dimers and tetramers in solution (Figure 2E). In summary, these results demonstrate that cleavage of the LDH N-terminal domain does not completely prevent protein tetramerization.The ability of these cleavage dimers to interact and form weak tetramers validated the in silico model and provided useful information about this novel tetramer boundary.

[0200] 2. Identification of peptide ligands at the 2.3-LDH tetramer boundary. Next, to identify peptides that target the LDH tetramer boundary, we began to further characterize the serial epitope B1. As discussed above, cluster B1 corresponds to a 22-amino acid peptide that folds into a long, "twisted" α-helix ending in a short loop. Therefore, we decided to test the interaction between the "cluster B1" derived polypeptide (named LP-22, LEDKLKGEMMDLQHGSLFLQTP (SEQ ID NO: 29)) and the LDH-H tetramer boundary. To that end, we performed a series of biophysical evaluations using nuclear magnetic resonance (NMR) WaterLOGSY, MST, and nanoDSF experiments.

[0201] Notably, WaterLOGSY experiments demonstrated that polypeptide LP-22 undergoes saturation transfer with the dimer LDH-Htr but not with the tetramer LDH-1, thus demonstrating that it interacts at the LDH tetramer boundary (Figure 3A). MST further confirmed this interaction with LDH-Htr using an estimated Kd of 156 μM (Figure 3B). Thermal shift experiments using nanoDSF revealed stabilization of dimeric cleaved LDH-Htr with polypeptide LP-22 (ΔTm = 2.8°C at 500 μM) (Figure 3C), consistent with interactions occurring at the exposed oligomer boundary. Conversely, polypeptide LP-22 destabilized the tetramer LDH in a concentration-dependent manner (Figure 3D), with an EC50 of 47 μM [32~68 μM]. These results are consistent with observations that ligands interacting at oligomer boundaries often induce thermal destabilization. The LDH-5 tetramer was more destabilized than LDH-1, consistent with the difference in stability between these two protein complexes that we previously reported in Thabault et al. (see above). Interestingly, a recent report of a selective LDH-5 inhibitor revealed an inhibitor that interacts at the LDH-5 tetramer boundary near cluster B (Friberg et al.; Structural Evidence for Isoform-Selective Allosteric Inhibition of Lactate Dehydrogenase A.ACS Omega 2020, 5(22), 13034-13041). This inhibitor is highly selective for LDH-5, which is consistent with the lower stability of the LDH-5 tetramer complex. Such a selectivity profile is also consistent with the higher destabilizing effect that polypeptide LP-22 exhibits against LDH-5 compared to LDH-1. Consistent with our previous report on LDH tetramer disruptors (Thabault et al. (see above)), LDH destabilization was also dependent on protein concentration, as increasing doses of the subunit reversed the effect. Overall, these results consistently demonstrate that polypeptide LP-22 interacts with the LDH tetramer boundary and destabilizes the tetrameric enzyme.

[0202] 2.4 Biophysical and computer experiments identify essential binding sites of polypeptide LP-22. Next, WaterLOGSY of polypeptide LP-22 and 1 The 1H-NMR spectra were compared. Since WaterLOGSY is a ligand-based NMR spectroscopy method that depends on protein-ligand saturation transfer, the polypeptide LP-22 residue, which does not interact with proteins, does not have a WaterLOGSY spectrum. Polypeptide LP-22 1 Careful comparison between H and the WaterLOGSY spectrum reveals characteristic features in the lysine, glutamate, aspartate, and leucine aliphatic regions that have not undergone saturation transfer. 1 The H chemical shift region was clearly shown (Figure 4A). Calculations of the contribution of each residue to the peptide-binding free energy suggested that the N-terminal residue LEDKLK of polypeptide LP-22, which is rich in those specific amino acids, does not account for much of the LP-22 binding energy (Figure 4B). Removal of these six N-terminal residues yielded polypeptide GP-16 (GEMMDLQHGSLFLQTP; SEQ ID NO: 6), which is a peptide with a similar WaterLOGSY spectrum (Figure 4A), thus suggesting that the two polypeptides interact in a very similar manner. MST showed a slightly weaker interaction with dimer LDH, with Kd = 240 μM (Figure 4C). Consistently, nanoDSF confirmed that polypeptide GP-16 can still destabilize tetramer LDH in a concentration-dependent manner (EC50 = 262 μM [142~383 μM]) (Figure 4D).

[0203] Detection of hotspots at the boundary of 2,5-polypeptide GP-16 and LDH-H tetramer. Computer and biophysical data suggested that the polypeptide GP-16 sequence (SEQ ID NO: 6) represents an essential binding region at the LDH tetramer boundary. To confirm this hypothesis, the contribution of each residue in cluster B1 to the stability of the LDH-H oligomer state was investigated. For this purpose, an alanine scan of the LDH-1 sequence (SEQ ID NO: 2) corresponding to polypeptide GP-16 (SEQ ID NO: 6) was performed. Subsequently, 16 corresponding LDH-H recombinant alanine variants were designed, produced, and purified, and their thermal and chemical stability, as well as their performance by microparticles (Table 2, Figures 5A-D). [Table 2]

[0204] The reported values ​​are mean ± SEM for melting temperature and EC50 (n=6), and mean ± SD for Mw (where the presented value is obtained from one measurement using MP and replicated at least three times with similar results). The reported molecular weight corresponds to the major oligomeric state of the protein. The amino acid residue position is calculated relative to the first amino acid residue at the N-terminus of LDH-1 (SEQ ID NO: 2).

[0205] MP results showed that among the various alanine single-point mutations, three significantly affected the state of the LDH-1 oligomer. Variants E62A (SEQ ID NO: 53) and F72A (SEQ ID NO: 63) behaved primarily as dimers in solution, while variant L71A (SEQ ID NO: 62) behaved as a mixture of tetramers and dimers (Figures 5B-D). Consistently, nanoDSF experiments showed that LDH-HF72A (SEQ ID NO: 63) and LDH-HE62A (SEQ ID NO: 53) exhibited similar denaturation patterns to the dimer LDH-Htr (SEQ ID NO: 4), with lower initial 350 / 330 nm ratios and a red shift instead of the blue shift typically observed in tetramer LDH variants (Table 2). Interestingly, LDH-HL71A (SEQ ID NO: 62) similarly showed lower initial ratios and a red shift, but its Tm was 10°C higher than LDH-Htr. This mixed profile is consistent with the mixture of dimers and tetramers that appear to be present in solution for this variant. Comparison of the tryptophan fluorescence spectra of these variants with LDH-1 showed a decrease in fluorescence intensity characteristic of the dimerized form of LDH in variants E62A and F72A, but not in L71A (Figure 6A).

[0206] Mutations at two other hotspots previously suggested by in silico analysis, L73 (SEQ ID NO: 64) and D65 (SEQ ID NO: 56), resulted in tetrameric variants exhibiting significant stability reduction when assessed by thermal and chemical denaturation (Table 2). Consistent with the predicted reduction in tetrameric stability, dilution experiments of the D65A variant (SEQ ID NO: 56) resulted in concentration-dependent destabilization of the protein and the appearance of a second unfolding event (Figure 6B). Interestingly, variants L66A (SEQ ID NO: 57) and L73A (SEQ ID NO: 64) showed similar stability by nanoDSF, with Tm values ​​of 61.1 and 62.0°C, respectively. However, chemical denaturation experiments showed significant differences between these two variants, with EC50 values ​​of 0.630 and 0.348 M, respectively (Figures 6C-D and Table 2). MP experiments confirmed the existence of equilibrium between dimers and tetramers for L73A (SEQ ID NO: 64), but not for L66A (SEQ ID NO: 57). Consistent with in silico calculations and available crystallographic data, these results confirm that mutant L73A reduces the stability of the LDH-1 oligomer. In contrast, mutant L66A appears to have a different effect on protein stability, for example by disrupting its hydrophobic core. These results further highlight the importance of using orthogonal methods when evaluating the effects of mutations on protein stability. Other mutations resulted in tetrameric proteins exhibiting moderate to low variability in their chemical and thermal stability compared to wild-type LDH-1, clearly indicating the lower importance of these residues to the oligomeric state of the protein (Table 2).

[0207] Overall, the differing stabilities of the mutants consistently matched the in silico prediction of the interaction ΔG, clearly indicating novel molecular determinants at the LDH tetramer boundary (Figure 4B). The cluster B1 hotspot was composed of two negatively charged amino acids, E62 and D65, and three consecutive hydrophobic residues: L71, F72, and L73. Based on the crystal structure (PDB ID: 1I0Z), E62 and D65 are involved in a hydrogen bonding network with water and adjacent residues R170, K246, A252, and W251. L71, F72, and L73 undergo hydrophobic interactions with each other and with residues L166, A169, P183, A252, and L255 (Figures 7A-C). Interestingly, cluster B1 consists of polar and nonpolar hotspots, which contrasts with the purely lipophilic hotspots we previously identified in the LDH tetramerization arm (Thabault et al. (see above)).

[0208] 3. Conclusion and Discussion Over the past few years, intensive efforts have been made to develop LDH inhibitors. Unfortunately, the polarity of the LDH active site and the high intracellular concentration of the enzyme have made it difficult to develop LDH inhibitors that exhibit potent and sustained in vivo inhibition. Recently, new advances in the development of ligands that target the boundaries of LDH oligomers have provided a new avenue for LDH inhibition (Thabault et al. (see above); Jafary et al. (Novel Peptide Inhibitors for Lactate Dehydrogenase A (LDHA): A Survey to Inhibit LDHA Activity via Disruption of Protein-Protein Interaction. Sci.Rep. 2019, 9(4686)); Friberg et al. (Structural Evidence for Isoform-Selective Allosteric Inhibition of Lactate Dehydrogenase A. ACS Omega 2020, 5(22), 13034-13041)). Targeting protein self-assembly is a novel concept in drug design that may offer several advantages compared to classical orthosteric inhibition. Firstly, targeting the LDH oligomer boundary may lead to the elucidation of novel allosteric sites and compounds exhibiting improved drug-like characteristics compared to LDH active site inhibitors. Secondly, molecules interacting at the protein homomer boundary may lead to its destabilization and degradation, potentially providing compounds with quasi-stoichiometric effects. Here, we report the identification and characterization of a novel LDH tetramer boundary and its essential residues using a combination of MP, nanoDSF, and chemical stability experiments. Furthermore, we report the identification of a peptide ligand family that targets the LDH tetramer boundary, destabilizing tetrameric LDH and stabilizing dimeric LDH-Htr. In summary, this study provides a useful pharmacological tool for structurally characterizing the molecular determinants of the LDH tetramer boundary and providing compounds that target the oligomeric state of LDH.

Claims

1. A polypeptide that inhibits tetramerization of the LDH subunit, comprising formula (I): GX １ MMX ２ LQHGSX ３ X ４ X ５ QTP (I) (array number 5) The formula includes the amino acid sequence, -X １ represents amino acid residue E or A; -X ２ represents amino acid residue D or A; -X ３ represents amino acid residue L or A; -X ４ represents amino acid residue F or A; -X ５ This represents an amino acid residue L or A. The polypeptide consists of 16 to 100 amino acid residues. The aforementioned amino acid sequence is not that of sequence number 87, but a polypeptide.

2. The polypeptide according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 51.

3. The polypeptide according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 28.

4. A nucleic acid encoding the polypeptide described in claim 1.

5. A nucleic acid vector comprising at least one nucleic acid as described in claim 4.

6. (i) at least one polypeptide, at least one nucleic acid encoding the polypeptide, or at least one nucleic acid vector containing the nucleic acid, and (ii) at least one pharmaceutically acceptable vehicle, a pharmaceutical composition comprising The polypeptide consists of 16 to 100 amino acid residues and has formula (I): GX １ MMX ２ LQHGSX ３ X ４ X ５ QTP (I) (array number 5) The formula includes the amino acid sequence, -X １ represents amino acid residue E or A; -X ２ represents amino acid residue D or A; -X ３ represents amino acid residue L or A; -X ４ represents an amino acid residue F or A; -X ５ This represents amino acid residue L or A. Pharmaceutical composition.

7. (i) at least one polypeptide, at least one nucleic acid encoding the polypeptide, at least one nucleic acid vector comprising the nucleic acid, or at least one pharmaceutical composition comprising the polypeptide, the nucleic acid, or the nucleic acid vector, and (ii) at least one means for administering the polypeptide, the nucleic acid, the nucleic acid vector, or the pharmaceutical composition, The polypeptide consists of 16 to 100 amino acid residues and has formula (I): GX １ MMX ２ LQHGSX ３ X ４ X ５ QTP (I) (array number 5) The formula includes the amino acid sequence, -X １ represents amino acid residue E or A; -X ２ represents amino acid residue D or A; -X ３ represents amino acid residue L or A; -X ４ represents an amino acid residue F or A; -X ５ This represents amino acid residue L or A. kit.

8. The kit according to claim 7, further comprising an anticancer agent.

9. The use of a polypeptide, a nucleic acid encoding the polypeptide, a nucleic acid vector containing the nucleic acid, or a pharmaceutical composition containing the polypeptide, the nucleic acid, or the nucleic acid vector in the manufacture of a pharmaceutical, The polypeptide consists of 16 to 100 amino acid residues and has formula (I): GX １ MMX ２ LQHGSX ３ X ４ X ５ QTP (I) (array number 5) The formula includes the amino acid sequence, -X １ represents amino acid residue E or A; -X ２ represents amino acid residue D or A; -X ３ represents amino acid residue L or A; -X ４ represents an amino acid residue F or A; -X ５ This represents amino acid residue L or A. use.

10. The use according to claim 9 in the manufacture of a pharmaceutical product for preventing and / or treating cancer.

11. The use according to claim 9 in the manufacture of a pharmaceutical product for inhibiting the tetramerization of lactate dehydrogenase subunits.

12. The use according to claim 11, wherein the lactate dehydrogenase subunit is the LDH-1 subunit and / or the LDH-5 subunit.

13. The use according to claim 12, wherein the lactate dehydrogenase subunit is the LDH-1 subunit.

14. (i) at least one polypeptide, at least one nucleic acid encoding the polypeptide, or at least one nucleic acid vector comprising the nucleic acid, and (ii) at least one pharmaceutically acceptable vehicle, a pharmaceutical composition for use as a pharmaceutical, The polypeptide consists of 16 to 100 amino acid residues and has formula (I): GX １ MMX ２ LQHGSX ３ X ４ X ５ QTP (I) (array number 5) The formula includes the amino acid sequence, -X １ represents amino acid residue E or A; -X ２ represents amino acid residue D or A; -X ３ represents amino acid residue L or A; -X ４ represents an amino acid residue F or A; -X ５ This represents amino acid residue L or A. Pharmaceutical composition.

15. The pharmaceutical composition according to claim 14 for use in the treatment or prevention of cancer.

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