CN114874307B - PKM2 tetramer allosteric activating peptide and application thereof in reversing Warburg effect and chemosensitization in tumors - Google Patents

Abstract

The invention discloses a PKM2 tetramer allosteric activating peptide and application thereof in reversing the Warburg effect and chemosensitization in tumors, belonging to the field of biomedicine. The PKM2 tetramer allosteric activation peptide comprises an N-acetylglucose coated serine motif, a self-assembly motif capable of forming a hydrophobic nano-complex and a fluorescent signal motif with aggregation-induced emission function. The polypeptide can react with the OGA enzyme overexpressed in the renal cell carcinoma area to remove N-acetylglucosamine, so as to expose PKM2 tetramer activator serine, promote PKM2 to convert from dimer to tetramer form, trigger the in-situ self-assembly function of self-assembly motif, form water-insoluble nano-composite, increase in-situ retention time of serine and AIE fluorescence signal intensity, and finally play roles in reversing the Warburg effect and increasing chemotherapy sensitivity. The invention provides a new direction for treating renal cell carcinoma.

Description

PKM2 tetramer allosteric activating peptide and application thereof in reversing Warburg effect and chemosensitization in tumors

Technical Field

The invention relates to the biomedical field, in particular to PKM2 tetramer allosteric activating peptide and application thereof in reversing the Warburg effect and chemosensitization in tumors.

Background

Renal cancer (Renal Cell Carcinoma, RCC) is one of the 10 most common malignancies, and is also the malignancy with the highest mortality rate of the urinary system. The traditional Chinese medicine composition has the characteristics of high progress speed, easiness in early metastasis and insensitivity to treatment means such as chemotherapy and radiotherapy, and is considered as a main reason of treatment failure. More than 30% of renal cancer patients have metastasized at the time of diagnosis and have missed the best surgical opportunity. For chemotherapy, even patients who initially respond to treatment develop resistance within 10-14 months. These factors lead to high mortality rates of renal cell carcinoma, which are a serious threat to human life health.

Studies have shown that metastasis and chemotherapy resistance of cancer are caused by abnormal nutritional metabolism and gene transcription. Among tumor cells, even in cases where oxygen is sufficient to support mitochondrial oxidative phosphorylation, tumor cells are more prone to metabolize glucose to lactic acid, a metabolic mode known as the Warburg effect. Lactic acid produced by the Warburg effect is highly correlated with a variety of biological functions such as tumor proliferation, metastasis, angiogenesis, etc., leading to malignant progression of the tumor. Furthermore, the abnormal expression of the VHL gene due to kidney cancer also results in kidney cancer having a stronger Warburg effect than other tumors. Thus, inhibition of the Warburg effect is a key strategy for the treatment of renal cancer.

Pyruvate kinase M2 (PKM 2) is an important regulator of the Warburg effect and transcriptional activation, playing a key role in tumor progression, metastasis and chemotherapy resistance. PKM2 can be interconvertible between dimers and tetramers and exist in the form of dimers in a variety of cancers. The pyruvate kinase activity of PKM2 dimers is lower than that of tetramers, resulting in a stronger Warburg effect. At the same time, PKM2 tetramers are restricted to the cytoplasm, whereas dimers can translocate into the nucleus to participate in transcriptional activation, ultimately inducing chemotherapy resistance. Serine as a natural ligand for PKM2 has been shown to activate PKM2 tetramerization but not to maintain the persistence of PKM2 allosteric effects. Thus, continuous stimulation of PKM2 tetramerization is critical for tumor inhibition. However, there is no report on studies on the sustained maintenance of PKM2 allosteric activators and thus the inhibition of tumors.

Disclosure of Invention

The invention aims to provide PKM2 tetramer allosteric activating peptide and application thereof in reversing the Warburg effect and chemotherapy sensitization in tumors, so as to solve the problems in the prior art, and the polypeptide realizes the function of converting PKM2 dimer into tetrameric form in renal cell carcinoma, realizes serine long-acting retention through in-situ self-assembly, and has the dual effects of continuously reversing the Warburg effect and chemotherapy drug resistance.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a PKM2 tetramer allosteric activating peptide which comprises a serine motif coated by N-acetylglucose, a self-assembly motif capable of forming a hydrophobic nano-composite and a fluorescent signal motif with aggregation-induced emission function.

Preferably, the self-assembling motif capable of forming a hydrophobic complex is derived from KLVFF peptide-based sequences in beta amyloid.

Preferably, the structural formula is as follows:

the invention also provides application of the PKM2 tetramer allosteric activation peptide in preparation of antitumor drugs.

Preferably, the tumor is a tumor that highly expresses the OGA enzyme and the PKM2 tetrameric allosteric activation peptide has the effect of reversing the tumor Warburg effect.

Preferably, the neoplasm includes renal cell carcinoma, acute myeloid leukemia, cholangiocarcinoma, and head and neck squamous cell carcinoma.

The invention also provides application of the PKM2 tetramer allosteric activating peptide in preparing medicaments for reversing the Warburg effect and chemosensitization in tumors.

Preferably, the neoplasm includes renal cell carcinoma, acute myeloid leukemia, cholangiocarcinoma, and head and neck squamous cell carcinoma.

Preferably, the tumor is a renal cell carcinoma.

The invention discloses the following technical effects:

the PKM2 tetramer allosteric activating Peptide (PAC) can be reacted with the kidney cell carcinoma (RCC) region over-expressed OGA enzyme to remove protective N-acetylglucosamine (GLcNAc), so that PKM2 tetramer activator serine is exposed, PKM2 dimer is promoted to be converted into tetramer form, in-situ self-assembly of KLVFF polypeptide is triggered, water-insoluble nano fibers are formed, the accumulation of serine is enhanced, the tetramerization of PKM2 is continuously stimulated, and the dual effects of reversing the Warburg effect and chemosensitization are achieved. Experiments prove that the proliferation capacity of the kidney cancer cells 786-O and ACHN is obviously inhibited by the polypeptide PAC, wherein the migration capacity of 786-O and ACHN is respectively inhibited by 71.9+/-4.6% and 55.8+/-3.5%; the invasive abilities were inhibited by 64.6±3.5% and 64.7±3.5%, respectively; meanwhile, PAC significantly inhibits the Warburg effect of tumor cells, so that the glucose consumption of 786-O cells is reduced by 49.3+/-15%, the lactic acid yield is reduced by 64.8+/-20.2%, and the same change occurs in ACHN cells. Finally, PAC inhibited tumor growth and metastasis in vivo experiments in mice; on the other hand, the CCK-8 experiment finds that PAC has obvious killing effect on sunitinib drug-resistant cell line (786O-R) and the in-vivo experiment of mice proves that PAC has inhibiting effect on kidney cancer drug-resistant cells. Thus, the PKM2 tetrameric allosteric activation peptide of the present invention can exert an anticancer effect by inhibiting the Warburg effect of tumors and increasing chemosensitivity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a molecular structural formula of a polypeptide PAC and a control polypeptide PAC-C; a: pattern diagram of molecular structure of PAC, b: a molecular structure pattern diagram of PAC-C;

fig. 2 is a graph demonstrating the allosteric, self-assembly of polypeptide PAC in aqueous OGA solution to form hydrophobic nanofibers: a: PAC is subjected to transmission electron microscopy after addition to OGA solution; b: PAC-C was subjected to transmission electron microscopy after addition to the OGA solution; ruler: 50nm;

FIG. 3 shows the fluorescence intensity of thioflavin T (ThT) after addition of polypeptide PAC to OGA solution for co-incubation;

FIG. 4 shows the killing effect of the polypeptides PAC and PAC-C on kidney cancer cells 786-O, ACHN and HK2 (human renal cortex proximal tubular epithelial cells); a: PAC-C killing of 786-O cells; b: the killing effect of PAC and PAC-C on ACHN cells; c: the killing effect of PAC and PAC-C on HK2 cells;

FIG. 5 is a major organ H & E staining of PAC and PAC-C injected mice, verifying material biosafety;

FIG. 6 is a graph showing the effect of PAC and PAC-C on the ability of renal cancer cells 786-O and ACHN to migrate and invade; a: effects of PAC and PAC-C on migration and invasiveness of 786-O cells; b: effects of PAC and PAC-C on migration and invasiveness of ACHN cells;

FIG. 7 shows the effect of PAC-C polypeptide on retention of 786-O, ACHN cells; a: PAC-C effect on retention of 786-O cells; b: PAC and PAC-C effect on retention of ACHN cells; ruler: 20 μm;

FIG. 8 shows the effect of western blot assay to detect the tetramer and dimer of polypeptide PAC, PAC-C and PBS on PKM2 in 786-O, ACHN cells; a: PAC, PAC-C and PBS effects on tetramers and dimers of PKM2 in 786-O cells; b: PAC, PAC-C and PBS effects on tetramers and dimers of PKM2 in ACHN cells;

FIG. 9 is the effect of the polypeptides PAC and PAC-C on glucose consumption by 786-O and ACHN cells; a: effects of PAC and PAC-C on glucose consumption by 786-O cells; b: effects of PAC and PAC-C on glucose consumption by ACHN cells;

FIG. 10 is the effect of the polypeptides PAC and PAC-C on extracellular lactate production by 786-O and ACHN cells; a: effect of PAC and PAC-C on extracellular lactate production by 786-O cells; b: effects of PAC and PAC-C on extracellular lactate production by ACHN cells;

FIG. 11 shows the in vivo distribution of the polypeptide PAC-C;

FIG. 12 shows the inhibition of tumor progression by polypeptide PAC-C in vivo; a: effects of PAC and PAC-C on tumor volume in mice; b: effects of PAC and PAC-C on tumor metastasis in mice; c: influence of PAC on survival of mice by PAC-C;

FIG. 13 is the effect of polypeptides PAC, PAC-C and PBS on the activity of drug resistant cells;

FIG. 14 is the effect of polypeptide PAC-C on chemotherapy resistant tumors in vivo; a: effects of PAC and PAC-C on tumor volume in mice; b: PAC and PAC-C effect on survival of mice.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

EXAMPLE 1 preparation and molecular Structure of PKM2 tetrameric allosteric activation peptide

1. Preparation of PKM2 tetramer allosteric activating peptide PAC ((S (GlcNAc) -K (TPA-1) LVFF), which can react with excessive OGA enzyme in Renal Cell Carcinoma (RCC) region to remove N-acetamido glucose (GLcNAc) to expose serine, promote conversion of PKM2 from dimer to tetramer form, and self-assemble in situ to form water insoluble nano fiber, realize long-acting retention and generate fluorescent signal, wherein the PKM2 tetramer allosteric activating peptide consists of the following three parts:

1) An N-acetylglucosamine (GLcNAc) coated serine functional motif, the amino acid sequence of which is shown in the formula I, can react with OGA enzyme in a Renal Cell Carcinoma (RCC) region to remove the N-acetylglucosamine (GLcNAc) and further expose serine, and promote the conversion of PKM2 from a dimer form to a tetramer form;

i is a kind of

2) The amino acid sequence of the β -sheet nanofiber, which is self-assembled, is derived from KLVFF in β -amyloid (SEQ ID NO: 1) The peptide-based sequence can self-assemble into the water-insoluble nano fiber with the beta-sheet secondary structure due to the interaction of hydrogen bonds;

3) Fluorescent signal motifs with aggregation-induced emission (AIE) can produce fluorescent amplified signals.

2. A control polypeptide of non-allosteric self-assembly PAC-C (S (GlcNAc) -K (TPA-1) AAGG).

The polypeptides PAC ((S (GlcNAc) -K (TPA-1) LVFF) and PAC-C (S (GlcNAc) -K (TPA-1) AAGG) were synthesized artificially from C-to N-terminal by solid phase synthesis, comprising the steps of first swelling 200 mg resin with 10mL DMF for at least 1 hour, then suction filtering and removing the liquid, washing the resin 3 times with DCM and DMF, respectively, then adding 10mL of deprotecting agent (DMF solution containing 20% piperidine) to the polypeptide solid phase synthesis tube to remove Fmoc protecting group, repeatedly suction filtering and removing the liquid, DCM and DMF washing resin 3 times, and detecting by using ninhydrin method, equipped with Kaiser detection reagent consisting of A, B, C three reagents together, equipped with method 1) reagent A: 20 Dissolving 0.5. 0.5 g ninhydrin in mL ethanol; 2) And (2) reagent B: 20 Dissolving 0.4. 0.4 g ascorbic acid in mL ethanol; 3) And C, reagent: 20 Dissolving 80 g phenol in mL ethanol; one drop of each of the three reagents was placed in a centrifuge tube, then the centrifuge tube was heated in a boiling water bath for 1 minute, if the resin turned purple black, the deprotection was successful, and if the resin did not turn purple black, the deprotection was repeated. Taking 10 times excess corresponding amino acid and HBTU, adding 4% of azomethylmorpholine into DMF solution to prepare coupling agent, adding the amino acid and HBTU into 10mL coupling agent to activate for 10 min, and placing on a shaking table to react for at least 1 hr. Filtering again, removing liquid, alternately washing resin with DCM and DMF for 3 times, placing the resin in a centrifuge tube, dripping three reagents into the centrifuge tube, placing the centrifuge tube into boiling water for heating in a water bath for 1 minute, if the resin does not change color, indicating success, otherwise repeating the steps. Repeating the step of deprotection-coupling reaction on the polypeptide resin obtained by the reaction until the final amino acid reaction is completed. Alternately washing 3 times by using DMF and DCM, fully washing 3 times by using methanol, and then draining for 15-30min; under ice water bath, trifluoroacetic acid was used for cleavage for two hours. Suction filtration was performed and nitrogen was blown to evaporate the liquid, diethyl ether was added, centrifugation was performed at 10000 rpm, and the resulting solid was collected after being placed in a vacuum oven overnight after washing twice with glacial diethyl ether.

The molecular structure pattern of the polypeptide PAC and PAC-C is shown in figure 1.

Example 2 polypeptide PAC allosteric, self-assembled into water insoluble nanofibers after removal of N-acetylglucosamine (GLcNAc) exposing serine by OGA enzymatic reaction.

1. Phosphate solution is prepared, OGA is gradually dissolved to pH of 6.5, PAC and PAC-C are added into the solution containing the OGA enzyme, the final concentration of PAC and PAC-C is 100 mu M, and after 2 hours, a transmission electron microscope is used for observing PAC and PAC-C solution samples.

The results are shown in FIG. 2, from which it can be seen that the polypeptide PAC is allosteric, self-assembled in the solution containing the OGA enzyme to form hydrophobic nanofibers, whereas PAC-C is not allosteric and self-assembled in the solution containing the OGA enzyme.

2. Preparing a phosphate solution, gradually dissolving OGA to pH of 6.5, adding PAC into the solution containing the OGA enzyme to enable the final concentration of PAC to be 100 mu M, adding the ThT solution to the concentration of 20 mu M after co-incubation for 1 hour, continuously incubating for 30 minutes, and measuring the fluorescence intensity by using a fluorescence microplate reader.

The results are shown in FIG. 3, from which it can be seen that the PAC undergoes allosteric behaviour upon reaction with the OGA enzyme, producing a beta-sheet structure, and that the ThT reagent fluoresces upon binding to the beta-sheet structure.

Example 3 cell experiment administration method

Human-derived renal cancer cells 786-O and ACHN cells with high OGA enzyme expression and normal renal cortex proximal tubular epithelial cells (HK-2 cells) are selected as control cells. PAC and PAC-C polypeptide are dissolved in DMSO solvent to prepare polypeptide nano material solution with solution concentration of 10 mM. Experimental cells with good logarithmic growth state are randomly divided into PAC, PAC-C and PBS (phosphate buffer solution) groups, the PAC, PAC-C and PBS are added into a culture medium according to experimental concentration, and the influence of PAC, PAC-C and PBS solutions on the survival state of the cells is respectively verified.

EXAMPLE 4 killing of polypeptide PAC on 786-O, ACHN cells and HK-2 cells and biosafety

786-O, ACHN cells grown logarithmically well and HK-2 cells were used at 1X 10 per well ⁴ Adding 100 μl total volume of individual cells into 96-well plate, placing into 37 ℃ cell incubator, randomly dividing into PAC and PAC-C groups after 24 hr, adding PAC and PAC solutions into cell culture medium at 0, 10, 20, 50, 100, 200 μM respectively, co-culturing for 1 hr, replacing fresh culture medium, placing into 37 ℃ cell incubator, discarding culture medium after 48 hr, and adding prepared CCAnd (3) placing the K-8 solution in a cell incubator at 37 ℃ for 4 hours, measuring absorbance, and respectively verifying the influence of PAC and PAC-C on the survival state of cells.

PAC, PAC-C and PBS were administered to three groups of mice, once every 48 hours, 5 times intravenously, and the systemic toxicity of the material was verified 48 hours after the 5 th injection, and the major organs of the mice were taken for H & E examination.

The results are shown in FIG. 4, from which it can be seen that PAC has a killing effect on 786-O, ACHN cells at a concentration of 50. Mu.M, while having no toxic or side effects on the normal cell line HK-2; whereas PAC-C (50. Mu.M) has a killing effect on 786-O, ACHN cells, but its killing effect is much smaller than that of PAC group.

As shown in FIG. 5, the H & E staining examination results showed that there were no significant differences among the three groups of mice, indicating that PAC and PAC-C were biosafety.

Example 5 effect of PAC and PAC-C on migration and invasiveness of renal cancer cells 786-O and ACHN;

to investigate the effect of PAC and PAC-C on tumor cell migration and invasiveness, 1X 10 cultures were grown in 200. Mu.L of fetal bovine serum-free medium ⁵ The cells/well 786-O and ACHN were seeded in a Transwell upper chamber containing PBS, PAC-C, PAC (50. Mu.M, medium: DMSO=99.5:0.5 v/v) (the challenge experiment required prior to matrigel coating in the upper chamber), 700. Mu.L medium containing 10% fetal bovine serum was added to the lower chamber and incubated at 37℃for 24 hours. Cells on the bottom surface of the upper chamber were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.05% crystal violet for 20 minutes, and the bottom surface cell count was counted.

As shown in FIG. 6, the migration ability of renal cancer cells 786-O was inhibited by PAC by 71.9.+ -. 4.6%; the invasion capacity is inhibited by 64.6+/-3.5%; the ACHN migration ability of the kidney cancer cells is inhibited by 55.8+/-3.5% by PAC; the invasion capacity is inhibited by 64.7+/-3.5%;

EXAMPLE 6 influence of polypeptide PAC on the retention of 786-O and ACHN cells

To investigate the retention of PAC and PAC-C in tumor cells 786-O, ACHN and HK-2 cells were digested with pancreatin to single cell suspensions and then evenly inoculated into confocal dedicated dishes followed by transfer to a cell incubator at 37℃with 5% CO ₂ Is cultured until the cells adhere to the wall. After the cells are completely attached, the fresh complete culture solution is replaced, PAC and PAC-C are added for treatment, the final concentration of PAC and PAC-C is 50 mu M, and fluorescence image acquisition is carried out by using a confocal laser scanning microscope (LSM 700, calzeiss, germany) at 1, 4, 8, 12 and 24 hours respectively.

The results are shown in FIG. 7, from which it is seen that the long-term retention capacity of PAC is higher than that of PAC-C group.

EXAMPLE 7 western blot experiments to examine the effects of the polypeptides PAC, PAC-C and PBS on the tetramers and dimers of PKM2 in 786-O, ACHN cells

786-O, ACHN cells are inoculated in a culture dish, PAC-C and PBS solutions are added after the cells are attached to the culture dish, PAC and PAC-C final concentrations are 50 mu M, fresh culture media are replaced after the cells are incubated for 1 hour, the cells are placed in a 37 ℃ cell incubator for culturing, after 48 hours, RIPA lysate is used for lysing the cells to extract cell proteins, and after the concentration is measured through a BCA kit, the level of tetramers and dimers of PKM2 in the cells is detected by adopting a western blot experiment.

The results are shown in fig. 8, from which it can be seen that PAC significantly inhibits dimerization of PKM 2.

EXAMPLE 8 Effect of the polypeptides PAC and PAC-C on glucose consumption by 786-O and ACHN cells

Will be 1X 10 ⁵ Each 786-O and ACHN cells was seeded in 6-well plates. PAC, PAC-C and PBS were added to give final PAC and PAC-C concentrations of 50. Mu.M, and after 24 hours of cell processing, the medium was collected from the cultured cells for glucose determination. Glucose levels were determined by using a glucose determination kit. The glucose consumption was calculated by subtracting the measured glucose concentration in the medium from the original glucose concentration.

As shown in FIG. 9, the PAC treatment 786-O cells showed a 49.3.+ -. 15% decrease in glucose consumption and the PAC treatment ACHN cells showed a 47.1.+ -. 1.7% decrease in glucose consumption.

Example 9 Effect of the polypeptides PAC and PAC-C on extracellular lactic acid production by 786-O and ACHN cells

Will be 1X 10 ⁵ Each 786-O and ACHN cells was seeded in 6-well plates. Adding PAC, PAC-C and PBS to a final concentration of 50. Mu.M, and medium was collected from the cultured cells 24 hours after cell treatment for lactate determination. Lactic acid production levels were determined using a lactic acid detection kit according to the instructions. The fluorescence intensity was measured under a fluorescence microplate reader.

As a result, as shown in FIG. 10, lactate production was reduced by 64.8.+ -. 20.2% in PAC-treated 786-O cells and 60.5.+ -. 1.4% in PAC-treated ACHN cells.

EXAMPLE 10 in vivo distribution of the polypeptide PAC and PAC-C

The experiment adopts Balb/c nude mice, 5 multiplied by 10 ⁶ cells of renal cancer cells were inoculated into the right buttocks of the mice. When the tumor volume reaches 50mm ³ PAC, PAC-C solutions (10 mg/kg) were intravenously injected. Fluorescence imaging of mice was detected using a multispectral fluorescence in vivo small animal imaging system 1, 4, 12, 24, 48, 72, 96 hours post injection.

The results are shown in fig. 11, and it can be seen from the results that PAC is mainly distributed in tumor sites, indicating that it has good targeting.

EXAMPLE 11 polypeptide PAC and PAC-C inhibit tumor progression in vivo

The experiment adopts Balb/c nude mice, 5 multiplied by 10 ⁶ cells of renal cancer were inoculated on the right buttocks of mice and the mice were randomly divided into two groups. When the tumor volume reaches 50mm ³ PAC, PAC-C solutions (10 mg/kg) were administered intravenously 5 times every 2 days. The first group periodically measures tumor volume and records time to live. The second group observed tumor metastasis.

The results are shown in FIG. 12, from which it can be seen that the polypeptide PAC can inhibit proliferation and metastasis of tumor in vivo, increasing survival time of mice.

EXAMPLE 12 influence of the Polypeptides PAC, PAC-C and PBS on the Activity of drug-resistant cells

Sunitidine resistant 786O-R cells grown log well were used at 1X 10 cells per well ⁴ Adding 100 μL of individual cells and total volume into 96-well plate, placing into 37 deg.C cell incubator, randomly dividing into PAC and PAC-C groups after 24 hr, concentrating PAC and PAC-C solution with concentration of 0, 10, 20, 50, 100, 200 μMAdding the strain into a cell culture medium, co-culturing for 1 hour, replacing a fresh culture medium, placing the culture medium in a cell culture box at 37 ℃ for 48 hours, discarding the culture medium, adding the prepared CCK-8 solution, placing the culture medium in the cell culture box at 37 ℃ for 4 hours, measuring absorbance, and respectively verifying the influence of PAC and PAC-C on the survival state of drug-resistant cells.

The results are shown in FIG. 13, from which it can be seen that PAC has a killing effect on sunitinib resistant cells 786O-R.

EXAMPLE 13 influence of the polypeptide PAC-C on chemotherapy-resistant tumors in vivo

The experiment adopts Balb/c nude mice, 5 multiplied by 10 ⁶ cells of drug resistant kidney cancer cells were inoculated on the right buttocks of mice. When the tumor volume reaches 50mm ³ PAC, PAC-C solutions (10 mg/kg) were administered intravenously 5 times every 2 days. Tumor volumes were measured periodically and time to live was recorded.

The results are shown in FIG. 14, from which it can be seen that PAC has an inhibitory effect on kidney cancer resistant cells in vivo and can increase the survival time of cell-resistant mice.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Sequence listing

<110> university of Harbin medical science

<120> PKM2 tetramer allosteric activating peptides and their use in reversing the Warburg effect and chemosensitization in tumors

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Lys Leu Val Phe Phe

1 5

Claims (1)

1. The application of PKM2 tetramer allosteric activation peptide in preparing antitumor drugs is characterized in that the PKM2 tetramer allosteric activation peptide consists of a serine motif coated by N-acetylglucosamine, a self-assembly motif capable of forming a hydrophobic nano-composite and a fluorescent signal motif with aggregation-induced emission function;

   the self-assembling motif that can form a hydrophobic complex is derived from KLVFF peptide-based sequences in amyloid beta;

   the structural formula of the PKM2 tetramer allosteric activating peptide is shown as follows:

   

   ；

   the tumor is a tumor with high expression of OGA enzyme, and the tumor is renal cell carcinoma; the PKM2 tetrameric allosteric activation peptide has the effect of reversing the tumor Warburg effect.

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