CN114874307A

CN114874307A - PKM2 tetramer allosteric activation peptide and application thereof in reversing Warburg effect and chemosensitization in tumors

Info

Publication number: CN114874307A
Application number: CN202210428852.5A
Authority: CN
Inventors: 徐万海; 王璐; 王浩; 李莉莉; 侯大勇; 吕玉林; 付博; 杨光; 于家傲
Original assignee: Harbin Medical University
Current assignee: Harbin Medical University
Priority date: 2022-04-22
Filing date: 2022-04-22
Publication date: 2022-08-09
Anticipated expiration: 2042-04-22
Also published as: CN114874307B

Abstract

The invention discloses PKM2 tetramer allosteric activation peptide and application thereof in reversing Warburg effect and chemosensitization in tumors, and belongs 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 fluorescence signal motif with aggregation-induced emission function. The polypeptide can react with OGA enzyme over-expressed in a renal cell carcinoma region to remove N-acetylglucosamine, further expose PKM2 tetramer activator serine, promote the conversion of PKM2 from a dimer to a tetramer form, trigger the in-situ self-assembly function of a self-assembly motif, form a water-insoluble nano-complex, increase the in-situ retention time of serine and the AIE fluorescence signal intensity, and finally play the roles of reversing Warburg effect and increasing chemotherapy sensitivity. The invention provides a new direction for treating the renal cell carcinoma.

Description

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

Technical Field

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

Background

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

It has been shown that cancer metastasis and chemotherapy resistance are caused by abnormal nutrient metabolism and gene transcription. In tumor cells, the tumor cells are more prone to metabolize glucose oxidation to lactate even when oxygen is sufficient to support mitochondrial oxidative phosphorylation, a mode of metabolism known as the Warburg effect. Lactic acid produced by the Warburg effect is highly related to various biological functions of tumor proliferation, metastasis, angiogenesis and the like, and leads to malignant tumor progression. In addition, abnormal expression of the VHL gene of renal cancer also causes renal cancer to have a stronger Warburg effect than other tumors. Therefore, inhibition of the Warburg effect is a key strategy for kidney cancer treatment.

Pyruvate kinase M2(PKM2) is an important regulator of Warburg effect and transcriptional activation, playing a key role in tumor progression, metastasis and chemoresistance. PKM2 can interconvert between dimers and tetramers and is present in the form of dimers in a variety of cancers. The pyruvate kinase activity of the PKM2 dimer is lower than that of the tetramer, resulting in a stronger Warburg effect. Meanwhile, PKM2 tetramer is restricted in cytoplasm, and dimer can translocate to nucleus to participate in transcriptional activation, and finally chemotherapy resistance is induced. Serine, as a natural ligand for PKM2, has been shown to activate PKM2 tetramerization, but not to maintain the persistence of the allosteric effect of PKM 2. Therefore, sustained stimulation of PKM2 tetramerization is critical for tumor suppression. However, no report exists for the sustained maintenance of the PKM2 allosteric activator and the inhibition of tumor-related studies.

Disclosure of Invention

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

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a PKM2 tetramer allosteric activation peptide which comprises an N-acetylglucose coated serine motif, a self-assembly motif capable of forming a hydrophobic nano-complex and a fluorescence signal motif with an aggregation-induced luminescence function.

Preferably, the self-assembly motif that can form a complex with hydrophobicity is derived from the KLVFF peptide-based sequence in amyloid beta.

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 highly expressing OGA enzyme, and the PKM2 tetramer allosteric activation peptide has the effect of reversing the Warburg effect of the tumor.

Preferably, the tumors include renal cell carcinoma, acute myeloid leukemia, cholangiocarcinoma, and head and neck squamous cell carcinoma.

The invention also provides application of the PKM2 tetramer allosteric activation peptide in preparation of drugs for reversing Warburg effect and chemosensitization in tumors.

Preferably, the tumor is renal cell carcinoma.

The invention discloses the following technical effects:

the PKM2 tetramer allosteric activation Peptide (PAC) can react with OGA enzyme over-expressed in Renal Cell Carcinoma (RCC) region to remove protective N-acetylglucosamine (GLcNAc), so that PKM2 tetramer activator serine is exposed, the conversion of PKM2 dimer to tetramer form is promoted, and the in-situ self-assembly of KLVFF polypeptide is triggered to form water-insoluble nanofiber, which is helpful for enhancing the accumulation of serine and continuously stimulating the tetramerization of PKM2, and has the dual effects of reversing Warburg effect and chemotherapy sensitization. Experiments prove that the proliferation capacities of 786-O and ACHN of kidney cancer cells are remarkably inhibited by polypeptide PAC, wherein the migration capacities of 786-O and ACHN are inhibited by 71.9 +/-4.6 percent and 55.8 +/-3.5 percent respectively; the invasion capacity is respectively inhibited by 64.6 +/-3.5 percent and 64.7 +/-3.5 percent; meanwhile, PAC obviously 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 is generated in ACHN cells. Finally, PAC inhibited tumor growth and metastasis in vivo experiments in mice; on the other hand, CCK-8 experiments find that PAC has obvious killing effect on sunitinib drug-resistant cell lines (786O-R) and mouse in-vivo experiments prove that PAC has inhibition effect on kidney cancer drug-resistant cells. Therefore, the PKM2 tetramer allosteric activation peptide can play an anti-cancer role by inhibiting the Warburg effect of tumors and increasing the sensitivity of chemotherapy.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings 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 it is obvious for those skilled in the art to obtain other drawings without creative efforts.

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

fig. 2 is a graph demonstrating that polypeptide PAC can be destructured and self-assembled in aqueous OGA to form hydrophobic nanofibers: a: the PAC is subjected to transmission electron microscope inspection after being added into the OGA solution; b: PAC-C is subjected to transmission electron microscope inspection after being added into OGA solution; a scale: 50 nm;

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

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

FIG. 5 shows H & E staining of major organs of PAC-injected and PAC-C-injected mice to verify the biological safety of the material;

FIG. 6 is a graph of the effect of PAC and PAC-C on the migration and invasion capacity of renal cancer cells 786-O and ACHN; a: the effects of PAC and PAC-C on the migration and invasion capabilities of 786-O cells; b: the effects of PAC and PAC-C on the migratory and invasive capacity of ACHN cells;

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

FIG. 8 is a western blot experiment to examine the effect of the polypeptides PAC, PAC-C and PBS on tetramers and dimers of PKM2 in 786-O, ACHN cells; a: the effect of PAC, PAC-C and PBS on tetramers and dimers of PKM2 in 786-O cells; b: the effects of PAC, PAC-C and PBS on tetramers and dimers of PKM2 in ACHN cells;

FIG. 9 is a graph of 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: the effect of PAC and PAC-C on glucose consumption by ACHN cells;

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

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

FIG. 12 shows that the polypeptides PAC and PAC-C inhibit tumor progression in vivo; a: effect of PAC and PAC-C on mouse tumor volume; b: effects of PAC and PAC-C on mouse tumor metastasis; c: effects of PAC and PAC-C on mouse survival;

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

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

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description 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. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, 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 herein 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 present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

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

1. Preparation of PKM2 tetrameric allosteric activation peptide PAC ((s (GLcNAc) -K (TPA-1) LVFF), which can react with excess OGA enzymes in the Renal Cell Carcinoma (RCC) region to remove N-acetylglucosamine (GLcNAc) to expose serine, promote conversion of PKM2 from dimer to tetrameric form, and self-assemble in situ to form water-insoluble nanofibers, achieve long-lasting retention and generate fluorescence signals, the PKM2 tetrameric allosteric activation peptide consisting of the following three moieties:

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

2) an amino acid sequence that can self-assemble into β -sheet nanofibers derived from KLVFF in amyloid-beta (SEQ ID NO: 1) the peptide-based sequence can be self-assembled into water-insoluble nano fibers with a beta-sheet secondary structure due to the interaction of hydrogen bonds;

3) fluorescent signal motifs with aggregation-induced emission (AIE) function can generate fluorescence amplification signals.

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

Artificially synthesizing polypeptides PAC ((S (GlcNAc) -K (TPA-1) LVFF) and PAC-C (S (GlcNAc) -K (TPA-1) AAGG) from C end to N end by adopting a solid phase synthesis method, firstly swelling 200mg of resin by 10mL of DMF for at least 1 hour, then carrying out suction filtration and removing liquid, flushing the resin for 3 times by respectively using DCM and DMF, then adding 10mL of deprotection agent (20% hexahydropyridine in DMF solution) into a polypeptide solid phase synthesis tube, removing Fmoc protective groups, repeatedly carrying out suction filtration and removing liquid, flushing the resin by DCM and DMF for 3 times, detecting by using a ninhydrin method, preparing Kaiser detection reagent which consists of A, B, C reagents together, and preparing the method comprises the following steps of 1) A reagent: dissolving 0.5g ninhydrin in 20mL of ethanol; 2) and B, reagent: dissolving 0.4g ascorbic acid in 20mL ethanol; 3) and C, reagent: dissolving 80g of phenol in 20mL of ethanol; and (3) taking one drop of each of the three reagents into a centrifuge tube, then placing the centrifuge tube into boiling water for heating in a water bath for 1 minute, wherein if the resin turns purple black, the deprotection is successful, and if the resin does not turn purple black, the deprotection is repeated. Taking 10 times excess corresponding amino acid and HBTU, adding 4% N-methylmorpholine into DMF solution to prepare a coupling agent, adding the amino acid and HBTU into 10mL of the coupling agent to activate for 10 minutes, and placing on a shaking bed to react for at least 1 hour. And performing suction filtration again to remove liquid, alternately washing the resin for 3 times by using DCM and DMF, placing the resin into a centrifugal tube, dripping three reagents into the centrifugal tube, placing the centrifugal tube into boiling water to heat the resin for 1 minute, and if the resin does not change the color, indicating success, otherwise, repeating the steps. Repeating the deprotection-coupling reaction steps on the polypeptide resin obtained by the reaction until the final amino acid reaction is finished. Alternately washing with DMF and DCM for 3 times, washing with methanol for 3 times, and draining for 15-30 min; the cleavage was performed for two hours with trifluoroacetic acid in an ice-water bath. Suction filtering and blowing with nitrogen until the liquid volatilizes, adding ether, centrifuging at 10000rpm, washing twice with glacial ethyl ether, putting the finally obtained solid into a vacuum drying oven overnight and collecting.

The molecular structure pattern of the polypeptides PAC and PAC-C is shown in FIG. 1.

Example 2 polypeptide PAC undergoes an OGA enzymatic reaction to remove N-acetylglucosamine (GLcNAc) and undergoes allosteric, self-assembly into water-insoluble nanofibers after serine exposure.

1. Preparing phosphate solution and gradually dissolving OGA to the pH value of 6.5, adding PAC and PAC-C into the solution containing OGA enzyme to enable the final concentration of PAC and PAC-C to be 100 mu M, and observing PAC and PAC-C solution samples by using a transmission electron microscope after 2 hours.

The results are shown in fig. 2, and it can be seen from the results that the polypeptide PAC can be destructured and self-assembled in the solution containing OGA enzyme to form hydrophobic nanofibers, while PAC-C cannot be destructured and self-assembled in the solution containing OGA enzyme.

2. Preparing phosphate solution and gradually dissolving OGA to the pH value of 6.5, adding PAC into the solution containing OGA enzyme to enable the final concentration of PAC to be 100 mu M, after 1 hour of incubation, adding ThT solution to enable the concentration to be 20 mu M, continuing the incubation 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 reaction of PAC with OGA enzyme leads to allosteric behavior, resulting in a β -sheet structure, and that the ThT reagent fluoresces upon binding to the β -sheet structure.

Example 3 method of cell assay administration

Human-derived renal carcinoma 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 a DMSO solvent to prepare a polypeptide nano-material solution with the solution concentration of 10 mM. Experimental cells which are good in state and grow logarithmically are randomly divided into PAC (PAC), PAC-C and PBS (phosphate buffered saline) groups, the PAC-C and the 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 verified respectively.

Example 4 killing and Biosafety of the polypeptide PAC against 786-O, ACHN cells and HK-2 cells

786-O, ACHN cells and HK-2 cells grown logarithmically in good condition at 1X 10 per well were used ⁴ Adding each cell and 100 mu L of total volume into a 96-well plate, placing the 96-well plate into a cell incubator at 37 ℃, randomly dividing the cell into PAC and PAC-C groups after 24 hours, adding PAC and PAC solutions into a cell culture medium at the concentrations of 0, 10, 20, 50, 100 and 200 mu M respectively, replacing the fresh culture medium after 1 hour of co-culture, placing the cell culture medium in the cell incubator at 37 ℃, discarding the culture medium and adding prepared CCK-8 solution after 48 hours, placing the cell culture medium in the cell incubator at 37 ℃ for 4 hours, measuring absorbance, and respectively verifying the influence of PAC and PAC-C on the survival state of the cells.

Three groups of mice were given PAC, PAC-C and PBS, once every 48 hours, 5 times intravenously, and the systemic toxicity of the material was verified 48 hours after the 5 th injection, and H & E examination was performed on the major organs of the mice.

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

As shown in fig. 5, H & E staining examination showed no significant difference between the three groups of mice, indicating that PAC and PAC-C are biologically safe.

Example 5 Effect of PAC and PAC-C on the migration and invasion Capacity of renal cancer cells 786-O and ACHN;

to explore the migration and invasion abilities of PAC and PAC-C to tumor cellsThe effect of (1X 10) using 200. mu.L of fetal bovine serum-free medium ⁵ Cells 786-O and ACHN were seeded in a Transwell upper chamber (for the invasion experiment, matrigel was applied to the upper chamber in advance) containing PBS and PAC-C, PAC (50. mu.M, medium: DMSO 99.5:0.5v/v), and 700. mu.L of 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 20min, and the number of cells on the bottom surface was counted.

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

example 6 Effect of the 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 trypsinized to single cell suspensions, uniformly plated on confocal dedicated petri dishes, and then transferred to a cell culture chamber at 37 ℃ with 5% CO ₂ Until the cells adhere to the wall. After the cells were completely adherent, the fresh complete medium was replaced, and PAC-C were added for treatment to give final concentrations of PAC and PAC-C of 50 μ M, and fluorescence image acquisition was performed using a confocal laser scanning microscope (LSM700, carl zeiss, 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 of PAC is higher than that of the PAC-C group.

Example 7Western blot experiment to examine the Effect of the Polypeptides PAC, PAC-C and PBS on tetramer and dimer of PKM2 in 786-O, ACHN cells

786-O, ACHN cells are inoculated in a culture dish, after the cells are attached to the wall, PAC-C and PBS solution are added to make the final concentration of PAC and PAC-C50 mu M, after 1 hour of incubation, fresh culture medium is replaced, the cells are placed in a cell culture box at 37 ℃ for culture, after 48 hours, the cells are cracked by RIPA lysate to extract cell protein, the concentration is measured by a BCA kit, and then the tetramer and dimer level of PKM2 in the cells are detected by adopting a western blot experiment.

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

Example 8 Effect of Polypeptides PAC and PAC-C on glucose consumption by 786-O and ACHN cells

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

As shown in FIG. 9, the glucose consumption of PAC-treated 786-O cells decreased by 49.3. + -. 15%, and the glucose consumption of PAC-treated ACHN cells decreased by 47.1. + -. 1.7%.

Example 9 Effect of the Polypeptides PAC and PAC-C on extracellular lactate production in 786-O and ACHN cells

Will be 1 × 10 ⁵ 786-O and ACHN cells were seeded in 6-well plates. PAC, PAC-C and PBS were added to a final concentration of 50. mu.M, and the medium was collected from the cultured cells for lactic acid assay 24 hours after treating the cells. The lactic acid production level was determined using the lactic acid detection kit according to the methods of the instructions. The fluorescence intensity was measured under a fluorescent microplate reader.

As shown in FIG. 10, the production of lactate 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 Polypeptides PAC and PAC-C

Balb/c nude mice, 5X 10, were used in this experiment ⁶ cells of kidney cancer cells were inoculated to the right hip of the mouse. When the tumor volume reaches 50mm ³ PAC and PAC-C solutions (10mg/kg) were injected intravenously. Fluorescence imaging of mice was detected at 1, 4, 12, 24, 48, 72, 96 hours post-injection using a multispectral fluorescent in vivo mouse imaging system.

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

Example 11 Polypeptides PAC and PAC-C inhibit tumor progression in vivo

Balb/c nude mice, 5X 10, were used in this experiment ⁶ cells renal carcinoma cells were seeded in the right hip of the mice and the mice were randomly divided into two groups. When the tumor volume reaches 50mm ³ PAC and PAC-C solutions (10mg/kg) were administered intravenously 5 times every 2 days by intravenous injection. The first group measured tumor volume periodically and recorded survival time. The second group observed tumor metastasis.

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

Example 12 Effect of Polypeptides PAC, PAC-C and PBS on Activity on drug-resistant cells

Sunitinib-resistant 786O-R cells in good state and logarithmic growth were used at 1X 10 per well ⁴ Adding 100 mu L of each cell and the total volume into a 96-well plate, placing the 96-well plate into a cell incubator at 37 ℃, randomly dividing the cell into PAC and PAC-C groups after 24 hours, adding PAC and PAC-C solutions into a cell culture medium at the concentrations of 0, 10, 20, 50, 100 and 200 mu M, after 1 hour of co-culture, replacing the fresh culture medium, placing the cell culture medium in the cell incubator at 37 ℃, discarding the culture medium and adding prepared CCK-8 solution after 48 hours, placing the cell culture medium in the cell incubator 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 Effect of Polypeptides PAC and PAC-C on chemotherapy-resistant tumors in vivo

Balb/c nude mice, 5X 10, were used in this experiment ⁶ cells resistant renal carcinoma cells were inoculated in the right hip of the mice. When the tumor volume reaches 50mm ³ PAC and PAC-C solutions (10mg/kg) were administered intravenously 5 times every 2 days by intravenous injection. Tumor volumes were measured periodically and survival time 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-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> Harbin university of medicine

<120> PKM2 tetramer allosteric activation peptide and application thereof in reversing 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. A PKM2 tetramer allosteric activation peptide, characterized in that it comprises an N-acetylglucose-coated serine motif, a self-assembly motif that can form a hydrophobic nanocomplex, and a fluorescent signal motif with aggregation-induced emission function.

2. The PKM2 tetrameric allosteric activation peptide according to claim 1, characterized in that said self-assembly motif that can form a complex with hydrophobicity is derived from the KLVFF peptide-based sequence in amyloid beta.

3. The PKM2 tetrameric allosteric activation peptide according to claim 1, having the structural formula:

4. use of the PKM2 tetramer allosteric activation peptide according to any one of claims 1-3 for the preparation of an anti-tumor medicament.

5. The use according to claim 4, wherein the tumor is a tumor highly expressing OGA enzyme, and the PKM2 tetrameric allosteric activation peptide has the effect of reversing the Warburg effect of the tumor.

6. The use according to claim 5, wherein the tumors comprise renal cell carcinoma, acute myeloid leukemia, cholangiocarcinoma, and head and neck squamous cell carcinoma.

7. The use of the PKM2 tetrameric allosteric activation peptide according to any one of claims 1-3 for the preparation of a medicament for reversing Warburg effect and chemosensitization in a tumor.

8. The use according to claim 7, wherein the tumors comprise renal cell carcinoma, acute myeloid leukemia, cholangiocarcinoma, and head and neck squamous cell carcinoma.

9. The use of claim 8, wherein the tumor is renal cell carcinoma.