Polypeptide for inhibiting eEF1A2 protein lactylation

Abstract

The invention relates to a polypeptide for inhibiting eEF1A2 protein lactylation. Specifically, the invention provides a polypeptide or a pharmaceutically acceptable salt thereof, and the polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1. The polypeptide provided by the invention can inhibit the combination of KAT8 and eEF1A2 protein in tumor cells, inhibit eEF1A2 lactylation and inhibit the synthesis of protein, so that the polypeptide has an excellent treatment effect on tumors.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a polypeptide for inhibiting the lactylation of eEF1A2 protein. Background Art

[0002] Tumors pose a serious threat to human health and survival. Recent statistics show that cancer deaths account for more than cerebrovascular disease, now the leading cause of death. As an oncogene, eEF1A2 plays a crucial role in the elongation step of protein translation and is highly expressed in various tumors, including colorectal and ovarian cancers. Post-translational protein modification promotes tumor progression by regulating eEF1A2 function. Among these, eEF1A2 lactylation plays a key role in the development of colorectal cancer. Inhibiting eEF1A2 lactylation can effectively suppress tumors. KAT8 is the "writer" of eEF1A2 lactylation. Therefore, developing a drug to inhibit eEF1A2 lactylation is crucial for tumor treatment.

[0003] Therefore, there is a need in the art to develop a drug that effectively inhibits the lactylation of eEF1A2 protein. Summary of the Invention

[0004] The object of the present invention is to provide a polypeptide that can effectively inhibit the lactylation of eEF1A2, thereby having an excellent therapeutic effect on tumors.

[0005] In a first aspect, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1.

[0006] Preferably, the polypeptide further comprises the amino acid sequence PFVYLI or HHHHHHFRRG.

[0007] Preferably, the two ends of the polypeptide are connected with PFVYLI or HHHHHHFRRG amino acid sequences.

[0008] Preferably, the N-terminus of the polypeptide is linked to the PFVYLI or HHHHHHFRRG amino acid sequence.

[0009] Preferably, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6.

[0010] Preferably, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 4 or SEQ ID NO: 6.

[0011] Preferably, the polypeptide is an isolated polypeptide.

[0012] Preferably, the amino acid sequence shown in SEQ ID NO: 1 is the active portion of the biological activity of the polypeptide.

[0013] Preferably, the biological activity is selected from one or more of the following groups: (i) preventing and / or treating tumors; (ii) inhibiting the binding of KAT8 to eEF1A2; (iii) inhibiting eEF1A2 lactylation; and / or (iv) inhibiting protein synthesis.

[0014] Preferably, the KAT8 comprises KAT8 of tumor cells.

[0015] Preferably, the eEF1A2 comprises eEF1A2 of tumor cells.

[0016] Preferably, the tumor comprises colorectal cancer.

[0017] Preferably, the tumor is selected from the group consisting of colorectal cancer, colon cancer, rectal cancer, or a combination thereof.

[0018] Preferably, the tumor comprises an adenocarcinoma.

[0019] Preferably, the colon cancer comprises colon adenocarcinoma.

[0020] Preferably, the rectal cancer includes rectal adenocarcinoma.

[0021] Preferably, the colorectal cancer includes colorectal adenocarcinoma.

[0022] Preferably, the tumor cells of the tumor include HCT116 cells and / or SW480 cells.

[0023] Preferably, the tumor comprises a human tumor.

[0024] Preferably, the inhibiting the binding between KAT8 and eEF1A2 comprises inhibiting the binding between KAT8 protein and eEF1A2 protein.

[0025] Preferably, the inhibiting eEF1A2 lactylation comprises inhibiting eEF1A2 protein lactylation.

[0026] In a second aspect, the present invention provides a fusion protein comprising:

[0027] (a) the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof;

[0028] (b) A peptide segment fused to the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0029] Preferably, the peptide segment comprises a carrier protein.

[0030] Preferably, the carrier protein is selected from the group consisting of human serum albumin (HSA), transferrin, or a combination thereof.

[0031] Preferably, the peptide segment is modified.

[0032] Preferably, the modification comprises polyethylene glycol (PEG) modification.

[0033] In a third aspect, the present invention provides an isolated nucleic acid encoding the polypeptide or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention.

[0034] In a fourth aspect, the present invention provides a pharmaceutical composition comprising:

[0035] (a) the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof; and

[0036] (b) a pharmaceutically acceptable carrier.

[0037] Preferably, the dosage form of the pharmaceutical composition is a solid preparation, a liquid preparation or a semisolid preparation.

[0038] Preferably, the pharmaceutical composition is in the form of an injection preparation or an oral preparation.

[0039] Preferably, the dosage form of the injection preparation is an intravascular injection preparation.

[0040] Preferably, the content of the polypeptide or a pharmaceutically acceptable salt thereof is 0.001-99.9wt%, preferably 0.1-99wt%, more preferably 1-99wt%, more preferably 1-90wt%, more preferably 10-90wt%, more preferably 20-80wt%, more preferably 30-70wt%, more preferably 20-40wt%, based on the weight of the pharmaceutical composition.

[0041] In a fifth aspect, the present invention provides a use of the polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention for preparing a composition or preparation, wherein the composition or preparation is used for one or more uses selected from the following groups: (i) preventing and / or treating tumors; (ii) inhibiting the binding of KAT8 to eEF1A2; (iii) inhibiting eEF1A2 lactylation; and / or (iv) inhibiting protein synthesis.

[0042] Preferably, the KAT8 comprises KAT8 of tumor cells.

[0043] Preferably, the eEF1A2 comprises eEF1A2 of tumor cells.

[0044] Preferably, the tumor comprises colorectal cancer.

[0045] Preferably, the tumor is selected from the group consisting of colorectal cancer, colon cancer, rectal cancer, or a combination thereof.

[0046] Preferably, the tumor comprises an adenocarcinoma.

[0047] Preferably, the colon cancer comprises colon adenocarcinoma.

[0048] Preferably, the rectal cancer includes rectal adenocarcinoma.

[0049] Preferably, the colorectal cancer includes colorectal adenocarcinoma.

[0050] Preferably, the tumor cells of the tumor include HCT116 cells and / or SW480 cells.

[0051] Preferably, the tumor comprises a human tumor.

[0052] Preferably, the composition or formulation is administered to a human.

[0053] Preferably, the inhibiting the binding between KAT8 and eEF1A2 comprises inhibiting the binding between KAT8 protein and eEF1A2 protein.

[0054] Preferably, the inhibiting eEF1A2 lactylation comprises inhibiting eEF1A2 protein lactylation.

[0055] Preferably, the composition or preparation is a pharmaceutical composition or preparation.

[0056] Preferably, the composition or preparation further comprises a pharmaceutically acceptable carrier.

[0057] Preferably, the dosage form of the composition or preparation is a solid preparation, a liquid preparation or a semisolid preparation.

[0058] Preferably, the composition or preparation is in the form of an injection preparation or an oral preparation.

[0059] Preferably, the dosage form of the injection preparation is an intravascular injection preparation.

[0060] In a sixth aspect, the present invention provides an in vitro method for inhibiting tumor cells, inhibiting the binding of KAT8 to eEF1A2, inhibiting eEF1A2 lactation, and / or inhibiting protein synthesis, the method comprising contacting cells with the polypeptide or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention, thereby inhibiting tumor cells, inhibiting the binding of KAT8 to eEF1A2, inhibiting eEF1A2 lactation, and / or inhibiting protein synthesis.

[0061] Preferably, the cells comprise tumor cells.

[0062] Preferably, the methods include non-therapeutic and non-diagnostic methods.

[0063] Preferably, said contacting comprises contacting in an in vitro culture medium.

[0064] Preferably, the KAT8 comprises KAT8 of tumor cells.

[0065] Preferably, the eEF1A2 comprises eEF1A2 of tumor cells.

[0066] Preferably, the tumor is as described above in the fifth aspect of the present invention.

[0067] Preferably, the inhibiting the binding between KAT8 and eEF1A2 comprises inhibiting the binding between KAT8 protein and eEF1A2 protein.

[0068] Preferably, the inhibiting eEF1A2 lactylation comprises inhibiting eEF1A2 protein lactylation.

[0069] In the seventh aspect of the present invention, a method is provided for (i) preventing and / or treating tumors; (ii) inhibiting the binding of KAT8 to eEF1A2; (iii) inhibiting eEF1A2 lactation; and / or (iv) inhibiting protein synthesis, the method comprising administering to a subject in need thereof the polypeptide as described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof, thereby (i) preventing and / or treating tumors; (ii) inhibiting the binding of KAT8 to eEF1A2; (iii) inhibiting eEF1A2 lactation; and / or (iv) inhibiting protein synthesis.

[0070] Preferably, the subject includes humans and non-human mammals.

[0071] Preferably, the non-human mammal comprises a cow, a horse, a sheep, a dog, a cat or a mouse.

[0072] Preferably, the KAT8 comprises KAT8 of tumor cells.

[0073] Preferably, the eEF1A2 comprises eEF1A2 of tumor cells.

[0074] Preferably, the tumor is as described above in the fifth aspect of the present invention.

[0075] Preferably, the inhibiting the binding between KAT8 and eEF1A2 comprises inhibiting the binding between KAT8 protein and eEF1A2 protein.

[0076] Preferably, the inhibiting eEF1A2 lactylation comprises inhibiting eEF1A2 protein lactylation.

[0077] Preferably, the administration is injection administration or oral administration.

[0078] Preferably, the injection administration is intravascular injection administration.

[0079] Within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below can be combined with each other to form new or preferred technical solutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 This is the ESI-MS spectrum of the control peptide (CPP).

[0081] Figure 2 This is the ESI-MS spectrum of peptide 1 (Pep1).

[0082] Figure 3 This is the ESI-MS image of peptide 2 (Pep2).

[0083] Figure 4 This is the ESI-MS image of peptide 3 (Pep3).

[0084] Figure 5 This is the ESI-MS spectrum of peptide 4 (GZ-Pep).

[0085] Figure 6 The interaction between eEF1A2 and KAT8 was detected by Co-IP in HCT116 cells treated with control peptide (CPP), peptide 1 (Pep1), peptide 2 (Pep2), or peptide 3 (Pep3) and in HCT116 cells of the untreated negative control group (Con).

[0086] Figure 7 The interaction between eEF1A2 and KAT8 was detected by Co-IP in SW480 cells treated with control peptide (CPP), peptide 1 (Pep1), peptide 2 (Pep2), or peptide 3 (Pep3) and in untreated negative control group (Con).

[0087] Figure 8 IP (Immunoprecipitation) was used to detect the eEF1A2 protein and its lactylation levels in HCT116 cells of the untreated negative control group (Con) and HCT116 cells treated with polypeptide 2 (Pep2).

[0088] Figure 9 IP (Immunoprecipitation) was used to detect the eEF1A2 protein and its lactylation levels in SW480 cells of the untreated negative control group (Con) and SW480 cells treated with polypeptide 2 (Pep2).

[0089] Figure 10The AHA labeling method was used to determine the level of nascent proteins in HCT116 or SW480 cells after or without Pep2 treatment, where “-” represents no Pep2 treatment and “+” represents Pep2 treatment.

[0090] Figure 11 This is the docking result of polypeptide 1 (Pep1) and KAT8 enzyme molecule.

[0091] Figure 12 This is the docking result of polypeptide 2 (Pep2) and KAT8 enzyme molecule.

[0092] Figure 13 This is the docking result of peptide 3 (Pep3) and KAT8 enzyme molecule.

[0093] Figure 14 The interaction between eEF1A2 and KAT8 was detected by Co-IP in HCT116 cells treated with Pep4 (GZ-Pep) and in the untreated negative control group (Con).

[0094] Figure 15 The interaction between eEF1A2 and KAT8 was detected by Co-IP in SW480 cells treated with Pep4 (GZ-Pep) and in the untreated negative control group (Con).

[0095] Figure 16 The eEF1A2 protein and its lactylation levels were detected by IP in HCT116 cells of the untreated negative control group (Con) and HCT116 cells treated with Pep4 (GZ-Pep).

[0096] Figure 17 The eEF1A2 protein and its lactylation levels were detected by IP in SW480 cells of the untreated negative control group (Con) and SW480 cells treated with Pep4 (GZ-Pep).

[0097] Figure 18 The AHA labeling method was used to determine the levels of nascent proteins in HCT116 or SW480 cells with or without Pep4 (GZ-Pep) treatment. “-” represents no Pep4 (GZ-Pep) treatment; “+” represents Pep4 (GZ-Pep) treatment.

[0098] Figure 19 This is the docking result of Pep4 (GZ-Pep) and KAT8 enzyme molecule. DETAILED DESCRIPTION

[0099] The present invention has discovered a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, which can inhibit the binding of KAT8 and eEF1A2 protein in tumor cells, inhibit eEF1A2 lactylation and inhibit protein synthesis, thereby having an excellent therapeutic effect on tumors.

[0100] the term

[0101] As used herein, the terms "include," "comprise," and "contain" are used interchangeably to encompass not only open definitions but also semi-closed and closed definitions. In other words, the terms encompass "consisting of," "consisting essentially of."

[0102] As used herein, the term "eEF1A2" refers to eukaryotic translation elongation factor 1α2.

[0103] As used herein, the term "KAT8" refers to lysine acetyltransferase 8.

[0104] In the present invention, the writing direction of the amino acid sequence is from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus).

[0105] In the present invention, the term "prevention" refers to a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease.

[0106] "Treatment" as used herein includes delaying and stopping the progression of a disease, or eliminating the disease, and does not require 100% inhibition, elimination, or reversal. In some embodiments, the polypeptides of the present invention reduce, inhibit, and / or reverse tumors by, for example, at least about 30%, at least about 50%, or at least about 80%, or 100%, compared to the levels observed in the absence of the polypeptides of the present invention.

[0107] In the present invention, the amino acid sequence shown in SEQ ID NO: 1 is PGKPMCVE.

[0108] In the present invention, the amino acid sequence shown in SEQ ID NO: 2 is PFVYLI.

[0109] In the present invention, the amino acid sequence shown in SEQ ID NO: 3 is PFVYLIEVPGKPM.

[0110] In the present invention, the amino acid sequence shown in SEQ ID NO: 4 is PFVYLIPGKPMCVE.

[0111] In the present invention, the amino acid sequence shown in SEQ ID NO: 5 is PFVYLIMVPGKPMC.

[0112] In the present invention, the amino acid sequence shown in SEQ ID NO: 6 is HHHHHHFRRGPGKPMCVE.

[0113] In the present invention, the amino acid sequence shown in SEQ ID NO: 7 is HHHHHHFRRG.

[0114] peptides

[0115] The present invention provides a polypeptide comprising the amino acid sequence of PGKPMCVE (SEQ ID NO: 1).

[0116] The polypeptide of the present invention may further comprise an amino acid sequence of PFVYLI (SEQ ID NO: 2) or HHHHHHFRRG (SEQ ID NO: 7), for example, the N-terminus of the polypeptide is linked to an amino acid sequence of PFVYLI (SEQ ID NO: 2) or HHHHHHFRRG (SEQ ID NO: 7).

[0117] In a preferred example of the present invention, the polypeptide comprises the amino acid sequence of PFVYLIPGKPMCVE (SEQ ID NO: 4) or HHHHHHFRRGPGKPMCVE (SEQ ID NO: 6).

[0118] Typically, the amino acid sequence of the polypeptide is shown in SEQ ID NO:4 or SEQ ID NO:6.

[0119] Specifically, the polypeptide is as described above in the first aspect of the present invention.

[0120] use

[0121] The present invention also provides a use of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof for preparing a composition or preparation, wherein the composition or preparation is used for one or more uses selected from the following groups: (i) preventing and / or treating tumors; (ii) inhibiting the binding of KAT8 to eEF1A2; (iii) inhibiting eEF1A2 lactylation; and / or (iv) inhibiting protein synthesis.

[0122] Specifically, the use is as described above in the fifth aspect of the present invention.

[0123] Composition

[0124] The composition or preparation of the present invention is preferably a pharmaceutical composition or preparation. The pharmaceutical composition or preparation of the present invention may further comprise a pharmaceutically acceptable carrier.

[0125] As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid, semisolid, liquid, or gel fillers suitable for human or animal use and possessing sufficient purity and sufficiently low toxicity. "Compatibility" refers to the ability of the components of the composition to be compatible with the active ingredient and their intermixing without significantly reducing the efficacy of the drug.

[0126] It should be understood that in the present invention, the pharmaceutically acceptable carrier is not particularly limited and can be selected from commonly used materials in the art, or prepared by conventional methods, or purchased from the market. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffers, chelating agents, thickeners, pH regulators, transdermal enhancers, colorants, flavorings, stabilizers, antioxidants, preservatives, antibacterial agents, pyrogen-free water, etc.

[0127] In the present invention, the dosage form of the composition or preparation includes but is not limited to oral preparations or injection preparations.

[0128] The pharmaceutical preparation should be compatible with the mode of administration, preferably oral administration or injection. When used, a therapeutically effective amount of the drug is administered to the desired subject (e.g., a human or non-human mammal). As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients of the drug used, the severity of the disease, and the combination with other drugs, all of which are within the skill of skilled physicians / researchers.

[0129] The main excellent technical effects of the present invention include:

[0130] The present invention develops a polypeptide that can inhibit the binding of KAT8 and eEF1A2 proteins in tumor cells, inhibit eEF1A2 lactylation and inhibit protein synthesis, thereby having an excellent therapeutic effect on tumors.

[0131] It should be understood that the following specific examples are based on the present technical solution and provide detailed implementation methods and specific operating processes, but the scope of protection of the present invention is not limited to these examples.

[0132] Example 1

[0133] This Example 1 provides the preparation and efficacy of a control peptide (CPP), polypeptide 1 (Pep1), polypeptide 2 (Pep2), polypeptide 3 (Pep3), and polypeptide 4 (GZ-Pep), as detailed below:

[0134] 1. Peptide Synthesis

[0135] The amino acid sequences of control peptide (CPP), polypeptide 1 (Pep1), polypeptide 2 (Pep2), polypeptide 3 (Pep3), and polypeptide 4 (GZ-Pep) are shown below:

[0136] Control peptide (CPP): Its amino acid sequence is PFVYLI (SEQ ID NO: 2).

[0137] Polypeptide 1 (Pep1): Its amino acid sequence is PFVYLIEVPGKPM (SEQ ID NO: 3).

[0138] Polypeptide 2 (Pep2): Its amino acid sequence is PFVYLIPGKPMCVE (SEQ ID NO: 4).

[0139] Polypeptide 3 (Pep3): Its amino acid sequence is PFVYLIMVPGKPMC (SEQ ID NO: 5).

[0140] Polypeptide 4 (GZ-Pep): its amino acid sequence is HHHHHHFRRGPGKPMCVE (SEQ ID NO: 6).

[0141] 1. Preparation before the experiment

[0142] 1.1 Weigh 1 mmoL of target amino acid into a 15 mL centrifuge tube and add 15 mL of DMF to dissolve completely.

[0143] 1.2 Activator preparation:

[0144] 0.3M HBTU solution: Accurately weigh 21.6 mg of HBTU and add 200 mL of DMF to make up to volume.

[0145] 0.6M DIEA solution: Accurately measure 9.9 mL of DIEA and add 90.1 mL of DMF to make up to volume.

[0146] According to the ratio of amino acid: HBTU: DIEA = 1:1:2.5 mmol, the activator was added to the above amino acid solution and mixed evenly before the reaction.

[0147] 1.3 Deprotection agent: Accurately measure 100 mL of piperidine, add DMF to make up to 500 mL and mix well for later use.

[0148] 1.4 Cutting fluid: Accurately measure 9.50 mL, 0.50 mL, and 0.50 mL of trifluoroacetic acid, triisopropylsilane, and ultrapure water, respectively, mix well, and set aside.

[0149] 2. Synthesis steps

[0150] Take the synthetic control peptide (sequence: PFVYLI) as an example.

[0151] Resin swelling: Weigh 0.3 g of resin into a 50 mL centrifuge tube, add 20 mL of DCM, and swell for 30 minutes.

[0152] Deprotection: rinse with DMF three times, rinse with DCM three times, add 10 mL of DMF solution containing 20% piperidine and react for 15 min, repeat twice to remove the Fmoc protecting group on the resin.

[0153] Condensation reaction: Add Fmoc-pro(tBu)-OH and the reaction reagent HBTU and DIEA and react for 2 hours. After the reaction, air dry the reaction solution and remove the Fmoc protecting group with 20% piperidine to replace Fmoc-Phe-OH. Repeat the above reaction and add the remaining amino acids in sequence to obtain the amino acid sequence PFVYLI. After the reaction is completed, add 10 mL of DMF for three washes and then remove the Fmoc protecting group from the amino acid with 20% piperidine.

[0154] Resin cleavage: Add 10 mL of cleavage solution to the completed amino acid sequence and shake on a shaker for 2 hours to cleave the resin and remove the side chain protecting groups.

[0155] Vacuum drying: After cutting, collect the reaction solution and slowly drip it into pre-chilled ether. Centrifuge at 8000 rpm at 4°C for 10 minutes, remove the supernatant, and repeat the washing process three times with ether. Place the sample in a vacuum drying oven and dry it at room temperature to obtain the crude product.

[0156] The peptide purification conditions are as follows:

[0157] Model: Waters 2707 Preparative HPLC

[0158] Chromatographic column: Absolue C18 (250×20 mm); mobile phase: 0.1% TFA water (A)-acetonitrile (B);

[0159] Elution gradient: 1-23 min, 1-40% (B); 23-27 min, 40-1% (B); 27-30 min, 1-1% (B);

[0160] Flow rate: 5 mL / min; detection wavelength: 220 nm; injection volume: 2 mL; column temperature: 30°C.

[0161] After purification, the solution corresponding to the highest peak in the chromatogram was collected using a centrifuge tube. The organic solvent in the solution was removed using a rotary evaporator, and the solution was frozen overnight at -80°C and lyophilized using a freeze dryer to obtain the purified peptide sample. It was then stored at -20°C for short-term storage.

[0162] Peptide 1 (Pep1), peptide 2 (Pep2), peptide 3 (Pep3) and peptide 4 (GZ-Pep) were synthesized in the same way.

[0163] ESI-MS (Electrospray Ionization Mass Spectrometry) was used to measure the control peptide (CPP), peptide 1 (Pep1), peptide 2 (Pep2), peptide 3 (Pep3) and peptide 4 (GZ-Pep). The results are as follows: Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 and Figure 5 As shown, from Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 and Figure 5 As can be seen in the figure, the control peptide (CPP), polypeptide 1 (Pep1), polypeptide 2 (Pep2), polypeptide 3 (Pep3) and polypeptide 4 (GZ-Pep) were successfully synthesized.

[0164] 2. Co-IP (co-immunoprecipitation) experiments were performed to determine the effects of control peptide (CPP), peptide 1 (Pep1), peptide 2 (Pep2), and peptide 3 (Pep3) on the interaction between eEF1A2 and KAT8 in HCT116 and SW480 cells

[0165] Experimental methods:

[0166] Human colorectal cancer cells HCT116 and SW480 were cultured in DMEM medium supplemented with 1% double-stranded antibody and 10% bovine serum albumin at 37°C and 5% CO2. HCT116 and SW480 cells in the logarithmic growth phase were seeded into multiwell plates and cultured in a cell culture incubator for 24 hours. The culture medium was removed and the cells were washed three times with PBS. A DMEM basal medium solution containing 50 μM of a control peptide (CPP), peptide 1 (Pep1), peptide 2 (Pep2), or peptide 3 (Pep3) was added to each well, and a DMEM basal medium solution was added to the negative control well. After incubation at 37°C and 5% CO2 for 48 h, the supernatant was removed. Co-IP experiments were performed to determine the interaction between eEF1A2 and KAT8 in HCT116 or SW480 cells treated with the control peptide (CPP), peptide 1 (Pep1), peptide 2 (Pep2), or peptide 3 (Pep3) and in HCT116 or SW480 cells in the untreated negative control group. The method is as follows:

[0167] 1. Wash HCT116 or SW480 cells treated with control peptide (CPP), peptide 1 (Pep1), peptide 2 (Pep2), or peptide 3 (Pep3) and untreated negative control HCT116 or SW480 cells twice with 1× PBS. Prepare lysis buffer (RIPA: protease inhibitor: phospholipase inhibitor = 100:1:1) and add 500 μl. After scraping the cells, transfer the cells to a 1.5 ml EP tube (on ice). Lyse on ice for 40 min. Centrifuge at 12,000 rpm at 4°C for 15 min, and aspirate the supernatant into a new EP tube.

[0168] 2. Remove approximately 100 μl of the sample and add 25 μl of 5X loading buffer (4:1 ratio). After pipetting and mixing, boil in a 100°C metal bath for 12 minutes. Cool on ice for approximately 1 minute and then store at -20°C. This will serve as the input group (for subsequent western blot exposures of A, B, and internal reference).

[0169] 3. Add the remaining protein supernatant (about 400 μl) to the stock solution of Antibody A (or Antibody B) at a ratio of 1:50-1:200 (depending on the antibody titer), mix well, and incubate in a 4° inverted mixer overnight.

[0170] 4. Magnetic bead preparation: Vortex the rProtein A / G Plus MaqPoly Beads magnetic beads and invert the bottle until there is no black precipitate at the bottom. Transfer 50μl of rProtein A / G plus MaqPoly Beads to a new EP tube for each group, centrifuge at 2000 rpm for 2 minutes, aspirate the supernatant and retain the solid. Add 200μl of washing solution, mix well, centrifuge at 2000 rpm for 2 minutes, aspirate the supernatant and retain the solid. Repeat twice. Add 3% BSA (prepared in PBS) to each tube and block for 1 hour on a 4° inverted mixer (can be placed on an ice box and shaker for blocking). Centrifuge to remove the supernatant, add 200μl of washing solution, mix well, centrifuge at 2000 rpm for 2 minutes, aspirate the supernatant and collect the magnetic beads.

[0171] 5. Add the antibody-antigen complex to the prepared magnetic beads and mix. Incubate at 4°C overnight. Centrifuge and remove the supernatant. Add 1 ml of washing buffer and pipette gently to mix. Centrifuge and discard the supernatant. Repeat the washing process twice.

[0172] 6. Add 80 μl 2X SDS-PAGE Loading Buffer, blow evenly, and heat at 95°C for 15 min.

[0173] 7. Perform WB (Western blotting) experiments, incubate with antibodies, and expose.

[0174] Experimental results:

[0175] Co-IP (Co-Immunoprecipitation) experiments were performed to determine the interaction between eEF1A2 and KAT8 in HCT116 or SW480 cells treated with control peptide (CPP), peptide 1 (Pep1), peptide 2 (Pep2), or peptide 3 (Pep3) and in HCT116 or SW480 cells in the untreated negative control group (Con). Figure 6 and Figure 7 shown.

[0176] from Figure 6 and Figure 7As can be seen, the Co-IP experiment verified that both the Input group and the IP group had bands for eEF1A2 and KAT8 proteins, proving that KAT8 interacted with eEF1A2 in HCT116 and SW480 cells. The normal group, control peptide (CPP), and polypeptide 1 (Pep1) had no effect on the interaction between eEF1A2 and KAT8 proteins in HCT116 and SW480 cells. Peptide 3 (Pep3) had a certain effect on the binding of KAT8 and eEF1A2 proteins in HCT116 and SW480 cells, while polypeptide 2 (Pep2) had a significant inhibitory effect on the binding of KAT8 and eEF1A2 proteins in HCT116 and SW480 cells.

[0177] IP (Immunoprecipitation) experiments verified that after the addition of peptide 2 (Pep2) to HCT116 or SW480 cells, there was no significant change in the eEF1A2 protein band in the peptide 2 (Pep2) treatment group and the untreated negative control group (Con). However, the eEF1A2 lactylation level in HCT116 and SW480 cell lines decreased after the addition of peptide 2 (Pep2), indicating that the intracellular eEF1A2 protein lactylation level was reduced (e.g. Figure 8 and Figure 9 Therefore, polypeptide 2 (Pep2) can inhibit the lactylation of eEF1A2, thereby preventing and treating colorectal cancer.

[0178] 3. AHA labeling method for the determination of nascent proteins

[0179] Experimental methods:

[0180] Human colorectal cancer cells HCT116 and SW480 were cultured in DMEM medium containing 1% double-stranded antibody and 10% bovine serum albumin at 37°C and 5% CO2. HCT116 and SW480 cells in the logarithmic growth phase were seeded into multiwell plates and incubated in a cell culture incubator for 24 hours. The culture medium was removed and the cells were washed three times with PBS. A DMEM basal medium solution containing 50 μM peptide 2 (Pep2) was added to each well. Negative control wells were incubated with DMEM basal medium. After incubation at 37°C and 5% CO2 for 48 hours, the supernatant was removed, the cells were washed three times with phosphate-buffered saline (PBS), and methionine (Met)-free medium was added to starve the cells of Met for 1 hour. Cells were then labeled with 4 mM AHA (azidohomoalanine, a Met analog). AHA is incorporated into proteins during mRNA translation in HCT116 and SW480 cells.

[0181] 1. Lyse cells: After AHA labeling, remove the culture medium and wash three times with PBS. Prepare lysis buffer (RIPA: protease inhibitor: phospholipase inhibitor = 100:1:1) and add 500 μl to each large dish. After scraping the cells, transfer the cells to a 1.5 ml EP tube (perform on ice). Lyse on ice for 40 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes and aspirate the supernatant into a new EP tube.

[0182] 2. Prepare the bioorthogonal reaction solution. For every 1ml of PBS (pH 7.8), add the following:

[0183] a. Add 1 μl of 200 mM THPTA stock solution and vortex at high speed for 10 seconds;

[0184] b. Add 1 μl of 0.5 M sodium ascorbate stock solution and vortex at high speed for 10 seconds;

[0185] c. Add 0.5-1 μl of 2 mM alkyne tag with a fluorescent group and vortex at high speed for 10 seconds;

[0186] d. Add 1 μl of 200 mM CuSO4 stock solution and vortex at high speed for 10 seconds.

[0187] 3. Bioorthogonal reaction. Add an appropriate amount of bioorthogonal reaction solution and incubate at 4°C overnight. The protein suspension obtained at this time can be detected by Western Blot or enzyme-linked immunosorbent assay (ELISA)

[0188] 4. (1) Prepare the sample. Take 15 μl of biotin-labeled protein sample and add 15 μl of 2×SDS protein loading buffer, mix well, boil for 5 minutes, and then ice-bath for 5 minutes. (2) Prepare the gel loading instrument and install the device correctly. (3) Prepare 4 mL of 12% separation gel buffer, mix well, add to the installed device, and solidify after 40 minutes. (4) Prepare 1 mL of 5% stacking gel buffer, mix well, add to the device, and slowly insert the comb to prevent bubbles from forming. After solidification for 30 minutes, slowly remove the comb. (5) Load the gel plate into the electrophoresis tank and pour in the electrophoresis buffer. (6) Take 20 μl of sample, install the device and perform electrophoresis. First, run at 80V for 20 minutes (the band runs to the boundary between the separation gel and the stacking gel), then switch to 120V for 1.5 hours (pay attention to observe the band to prevent it from running out of the gel plate). (7) When the band reaches the appropriate position, turn off the electrophoresis apparatus, disassemble the device, and remove the gel plate. (8) Draw the gel and place it in a suitable culture dish for subsequent Western Blot analysis. If the protein sample is fluorescently labeled, place the gel block in a fluorescence scanner for laser scanning.

[0189] 5. Western Blot detection of newly generated proteins (1) After trimming the gel blocks obtained above, transfer them to a membrane using a wet method. (2) PVDF membrane treatment. Place the cut PVDF membrane (same size as the gel blocks) in methanol for activation for 15 seconds, remove it and place it in water to wash off the methanol for later use. (3) Soak the gel, filter paper, PVDF membrane, etc. in transfer buffer and lay them out in order. Anode - extra-thick filter paper - PVDF membrane - gel - extra-thick filter paper - cathode. Gently remove bubbles after each layer. (4) Install the device and connect the electroporator, 100V at room temperature for 80 minutes. (5) After the transfer is completed, turn off the power and remove the PVDF membrane. Add TBST and wash the membrane 3 times, 10 minutes each time. (6) Add 5% skim milk powder and block at room temperature for 2 hours, then wash the membrane 3 times with TBST. (7) Add the primary antibody (HRP labeled with streptavidin diluted at 1:3000), incubate at room temperature for 2 hours, and wash the membrane 3 times with TBST. (8) ECL staining: if a color band appears on the PVDF membrane, it is positive; if not, it is negative.

[0190] Experimental results:

[0191] AHA labeling was used to determine the expression of newly synthesized proteins in HCT116 or SW480 cells treated with or without Pep2. Figure 10 shown.

[0192] from Figure 10 It can be seen that after peptide 2 (Pep2) treated HCT116 or SW480 cells, the new protein in the two cell lines decreased, indicating that peptide 2 (Pep2) affected its activity by downregulating the lactylation level of eEF1A2, thereby inhibiting protein synthesis in colorectal cancer cells, thereby inhibiting colorectal cancer.

[0193] 4. Molecular docking detection of the interaction between peptide and KAT8 enzyme

[0194] Experimental methods:

[0195] 1. Protein and ligand preparation

[0196] The Protein Data Bank (PDB) is a database of crystal structures of biological macromolecules. Proteins encoded by core genes were retrieved and downloaded from the PDB. Peptide structures were simulated using Alphafold2, and the files were converted to PDB format using OpenBabel 2.3.2.

[0197] 2. Molecular docking

[0198] The protein was pretreated using Pymol software to remove ligands, water, and small molecules. The receptor and ligand were imported into AutoDock Vina for unified hydrogenation and charge addition, and then exported to Pdbqt format. After charge optimization and ligand flexibility, the docking parameters were adjusted, docking was performed, and the optimal conformation with the lowest binding energy was selected for visualization using Pymol.

[0199] Experimental results:

[0200] The molecular docking results are as follows Figure 11 、 Figure 12 and Figure 13 As shown, from Figure 11 、 Figure 12 and Figure 13 As can be seen, Peptide 2 (Pep2) interacts well with the KAT8 enzyme, with multiple hydrogen bonds between them. Furthermore, the lysine in Peptide 2 (Pep2) has hydrogen bonds with the KAT8 enzyme, while the lysine on Peptide 1 (Pep1) and Peptide 3 (Pep3) has no hydrogen bonds with the KAT8 enzyme, or has fewer hydrogen bonds. Therefore, compared with Peptide 1 (Pep1) and Peptide 3 (Pep3), Peptide 2 (Pep2) can significantly and effectively compete with eEF1A2 for binding to the KAT8 enzyme, thereby inhibiting eEF1A2 lactylation and suppressing colorectal cancer.

[0201] 5. In vitro efficacy study of Pep4 (GZ-Pep)

[0202] 5.1 Co-IP assay to determine the effect of Pep4 (GZ-Pep) on the interaction between eEF1A2 and KAT8 in HCT116 and SW480 cells

[0203] The experimental method is the same as above. Co-IP (Co-Immunoprecipitation) experiment was used to determine the interaction between eEF1A2 and KAT8 in HCT116 or SW480 cells treated with Pep4 (GZ-Pep) and in HCT116 or SW480 cells in the untreated negative control group (Con). Figure 14 and Figure 15 Co-IP experiments verified that both the input and IP groups showed bands for eEF1A2 and KAT8 proteins, demonstrating that KAT8 interacts with eEF1A2 in HCT116 and SW480 cells. The normal group had no effect on the interaction between eEF1A2 and KAT8 proteins in HCT116 and SW480 cells. Pep4 (GZ-Pep) significantly inhibited the binding of KAT8 and eEF1A2 in HCT116 and SW480 cells.

[0204] 5.2 IP assay to determine the effect of Pep4 (GZ-Pep) on eEF1A2 lactylation in HCT116 and SW480 cells

[0205] The experimental method was the same as above. IP (Immunoprecipitation) experiments verified that after Pep4 (GZ-Pep) was added to HCT116 or SW480 cells, there was no significant change in the eEF1A2 protein band in the Pep4 (GZ-Pep) treatment group and the untreated negative control group (Con). However, after the addition of Pep4 (GZ-Pep), the eEF1A2 lactylation level in HCT116 and SW480 cell lines decreased, indicating that the intracellular eEF1A2 protein lactylation level was reduced (e.g. Figure 16 and Figure 17 Therefore, Pep4 (GZ-Pep) can inhibit eEF1A2 lactylation, thereby preventing and treating colorectal cancer.

[0206] 5.3 AHA labeling method for the determination of nascent proteins

[0207] The experimental method was the same as above. The AHA labeling method was used to measure the expression of newly synthesized proteins in HCT116 or SW480 cells after or without Pep4 (GZ-Pep) treatment. Figure 18 As shown. Figure 18 As can be seen, after Pep4 (GZ-Pep) treated HCT116 or SW480 cells, the new protein in the two cell lines decreased, indicating that Pep4 (GZ-Pep) affected its activity by downregulating the lactylation level of eEF1A2, thereby inhibiting protein synthesis in colorectal cancer cells, thereby inhibiting colorectal cancer.

[0208] 5.4 Molecular docking detection of the interaction between peptide and KAT8 enzyme

[0209] The experimental method is the same as above, and the molecular docking results are as follows Figure 19 As shown, from Figure 19 It can be seen that after modification, Pep4 (GZ-Pep) binds well to the KAT8 enzyme. There are multiple hydrogen bonding forces between Pep4 (GZ-Pep) and the KAT8 enzyme, and the lysine in Pep4 (GZ-Pep) has hydrogen bonding forces with the KAT8 enzyme, indicating that Pep4 (GZ-Pep) can significantly and effectively compete with eEF1A2 for binding to the KAT8 enzyme, thereby inhibiting eEF1A2 lactylation and inhibiting colorectal cancer.

[0210] The above is an implementation scheme of the present invention designed for a case. It should be pointed out that for ordinary technicians in this technical field, several improvements can be made without departing from the principles of the present invention, and these improvements should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The polypeptide comprises the amino acid sequence shown in SEQ ID NO:

1.

2. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, wherein: The polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO:

6.

3. A fusion protein, characterized in that The fusion protein comprises: (a) the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof; (b) A peptide segment fused to the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof.

4. An isolated nucleic acid, characterized in that The nucleic acid encodes the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a) the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier.

6. A use of the polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Used for preparing a composition or preparation for one or more uses selected from the following groups: (i) preventing and / or treating tumors; (ii) inhibiting the binding of KAT8 to eEF1A2; (iii) inhibiting eEF1A2 lactylation; and / or (iv) inhibiting protein synthesis.

7. The use according to claim 6, characterized in that The tumor is selected from the group consisting of colorectal cancer, colon cancer, rectal cancer, or a combination thereof.

8. The use according to claim 6, characterized in that The tumor cells of the tumor include HCT116 cells and / or SW480 cells.

9. A method for inhibiting tumor cells, inhibiting the binding of KAT8 to eEF1A2, inhibiting the lactylation of eEF1A2, and / or inhibiting protein synthesis in vitro, characterized in that: The method comprises contacting cells with the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof, thereby inhibiting tumor cells, inhibiting the binding of KAT8 to eEF1A2, inhibiting eEF1A2 lactylation, and / or inhibiting protein synthesis.

10. A method for (i) preventing and / or treating tumors; (ii) inhibiting the binding of KAT8 to eEF1A2; (iii) inhibiting eEF1A2 lactylation; and / or (iv) inhibiting protein synthesis, characterized in that: The method comprises administering the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof, thereby (i) preventing and / or treating tumors; (ii) inhibiting the binding of KAT8 to eEF1A2; (iii) inhibiting eEF1A2 lactylation; and / or (iv) inhibiting protein synthesis.

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