CN117186178B - Polypeptide and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of biochemistry, and discloses a polypeptide which comprises the following components: (I) An amino acid sequence shown in any one of SEQ ID No.1, SEQ ID No.4 and SEQ ID No. 5; or (II), an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I); or, (III) an amino acid sequence having 80% or more similarity to the amino acid sequence as set forth in (I) or (II); or, (IV) a membrane-penetrating polypeptide obtained by combining the polypeptide shown in (I), (II) or (III) with a membrane-penetrating peptide; or, (V) stapling the polypeptides of (I), (II) or (IV) to obtain a stapled peptide. It has obvious methylation and METTL3 expression inhibiting effects.

Description

A polypeptide and preparation method thereof

Technical Field

The invention relates to the biochemical field, in particular to a polypeptide derivative and a preparation method thereof.

Background Art

Prostate cancer (PCa) is one of the most common malignant tumors in men and the second leading cause of death in men. According to statistics, the incidence and mortality of prostate cancer are increasing year by year, with more than 300,000 deaths from PCa each year, accounting for 6.6%. At present, anti-androgen therapy (ADT) is one of the commonly used treatments for PCa besides surgery. It inhibits the growth of cancer cells by interfering with androgen/androgen receptor signaling pathways through androgen synthesis inhibitors (abiraterone) or androgen receptor inhibitors (including bicalutamide, flutamide and enzalutamide, etc.). ADT treatment is more effective in the early stages of cancer, but it is easy to develop drug resistance. Usually, after 3 to 5 years of treatment, it eventually progresses to castration-resistant prostate cancer (CRPC), and the 5-year survival rate of patients is only 25.4%. At present, the FDA has not approved any effective drug for the treatment of CRPC prostate cancer, and the approved prostate cancer drugs are not effective for CRPC prostate cancer and can only prolong the patient's life for a few months. Therefore, in-depth research on new drugs for PCa is very urgent and necessary.

website:

https://wenku.baidu.com/view/8349065a940590c69ec3d5bbfd0a79563d1ed41d.html records polypeptide DNA methyltransferase inhibitors-DNA methylation inhibitors, which disclose 5-azacytidine, PEP515, bovine lactoferrin, HDAC cyclic peptide inhibitors, etc.

It can be seen that a large number of research units have begun to study peptides as methylation inhibitors.

The polypeptides suitable for methylation inhibition are not limited to the above.

Therefore, the technical problem solved in this case is: how to develop new peptides that have inhibitory effects on methylase activity and m6A modification.

Summary of the invention

The object of the present invention is to provide a polypeptide having a variety of selectable forms and having a relatively obvious RNA methylation and METTL3 expression inhibitory effect.

Meanwhile, the invention also discloses a preparation method of the polypeptide.

Unless otherwise specified in the present invention: mM represents millimole/liter, μM represents micromole/liter, and nM represents nanomole/liter;

To achieve the above object, the present invention provides the following technical solution: a polypeptide having:

(I), the amino acid sequence shown in SEQ ID No.1;

The sequence of SEQ ID No. 1 is: ELGRECLNLW;

The sequence of SEQ ID No. 4 is: QLQRIIRTGRTGHWLNHG;

SEQ ID No.5:FGRPHNVQ

The above-mentioned SEQ ID No. 4 & 5 polypeptide sequences are the remaining two polypeptide screening sequences that can inhibit METTL3.

or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same function as the amino acid sequence described in (I);

(III) an amino acid sequence having more than 80% similarity to the amino acid sequence described in (I) or (II);

For example: SEQ ID No.6: GRAMELGRECLNLWGYER;

The sequence is ELGRECLNLW, each extending 4 amino acids forward and backward;

For example, SEQ ID No. 2: RCMELGRECLNLW;

The amino acid A in the sequence SEQ ID No.7 is mutated to C;

For example, SEQ ID No. 7: RAM ELGRECLNLW;

The sequence is ELGRECLNLW extended forward by 3 amino acids; it is a truncated peptide of the METTL3 protein like the M3 peptide, and has similar functions to the M3 peptide. This part is the functional region of the target peptide drug, which can bind to the METTL3 and METTL14 complex proteins and exert its effect;

When it is used, it can be combined with various membrane-penetrating peptide structures, such as R9, to obtain R9-MPF13, whose amino acid sequence is: SEQ ID No. 8: RRRRRRRRRRAMELGRECLNLW;

It can also be further modified on the basis of R9-MPF13 by replacing some amino acids in the functional region. Among them, the enhancing mutations are concentrated at the 11th, 12th, and 18th positions of the amino acid sequence, such as the polypeptides R9-RKF, R9-RKY, R9-RKM, R9-RKL, R9-RRL, and R9-RKWL. This type of mutation can enhance the anti-proliferative effect of the polypeptide; the weakening mutations are concentrated at the 14th and 22nd positions of the amino acid sequence, such as the polypeptides R9-RA and R9-RE. This type of mutation can weaken the anti-proliferative effect of the polypeptide. The polypeptide R9-PSRKWL is a stapled peptide precursor obtained by mutating the 13th and 20th amino acids of the amino acid sequence to cysteine on the basis of R9-RKWL. R9-SRKWL is a stapled peptide obtained by stapling R9-PSRKWL on the 13th and 20th cysteines of the polypeptide through 4,4-bis(bromomethyl)biphenyl connection.

The polypeptide sequences are listed in Table 1 below:

Table 1 Sequence Listing

(Note: The peptide is N-C terminus from left to right)

or (IV), a cell-penetrating polypeptide obtained by combining the polypeptide shown in (I), (II) or (III) with a cell-penetrating peptide; or (V), a stapled peptide obtained by stapling the polypeptide shown in (I), (II) or (IV).

The sequence shown in SEQ ID No.1 has at least the following variations:

1. Able to combine with self-assembling peptides, such as NapFFKY and GYYF, KLVFFAE (core sequence in amyloid protein Aβ), to develop new peptide drugs.

2. A stapled peptide obtained by stapling a polypeptide having an amino acid sequence as shown in any one of SEQ ID No.1, SEQ ID No.4, and SEQ ID No.5, or a stapled peptide obtained by stapling a polypeptide having an amino acid sequence as shown in SEQ ID No.1 in which at least one amino acid is replaced with a non-natural amino acid having a reactive side chain.

For SEQ ID No. 1, any form of stapled peptide construction can be performed on the sequence through other linking molecules, such as: replacing E at position 1 and E at position 5, N at position 8, and R, E, and N with reactive side chain non-natural amino acids.

3. Add or replace with non-natural amino acids, such as changing the amino acid from L type to D type.

4. Modify peptide drugs through chemical modification, such as PEG modification to increase drug circulation time; fatty acid (such as C12 or C18) modification to increase stability and utilization.

5. Deliver the polypeptide drug by combining it with a drug carrier, such as liposomes, microspheres, micelles and hydrogels, to prepare a new drug dosage form.

In the above polypeptide, the cell-penetrating peptide is CPPsite 2.0

(https://webs.iiitd.edu.in/raghava/cppsite/stats1.php) can retrieve the cell-penetrating peptides, such as several classic cell-penetrating peptides: R9 cell-penetrating peptide, TAT cell-penetrating peptide (GRKKRRQRRRPPQ), Penetratin cell-penetrating peptide (RQIKIWFQNRRMKWKK) MAP (KLALKLALKALKAALKLA), MelittinGIGAVLKVLTTGLPALISWIKRKRQQ.

In the above polypeptide, the polypeptide has an amino acid sequence as shown in SEQ ID No.2.

The sequence of SEQ ID No. 2 is: RCMELGRECLNLW;

In the above-mentioned polypeptide, the second and ninth cysteines of the polypeptide with the amino acid sequence shown in SEQ ID No. 2 are stapled by 4,4-bis(bromomethyl)biphenyl linkage to obtain a stapled peptide named RSM3.

In the above polypeptide, the membrane-penetrating polypeptide has an amino acid sequence as shown in SEQ ID No.3.

The sequence of SEQ ID No. 3 is: RRRRRRRRRCMELGRECLNLW;

In the above polypeptide, the 10th and 17th cysteines of the polypeptide with the amino acid sequence shown in SEQ ID No. 3 are connected through 4,4-bis(bromomethyl)biphenyl to construct the i+7 stapled peptide.

At the same time, the present invention also discloses a method for preparing the polypeptide as described above, using AM resin, calculating, weighing and dissolving the required amino acids and putting them into a synthesis bottle, setting up the synthesis according to the operation of the fully automatic synthesis instrument, taking out the AM resin, cutting and precipitating to obtain the polypeptide, and then purifying it.

Compared with the prior art, the present invention has the following beneficial effects:

The polypeptide inhibitor designed by the present invention has the function of inhibiting RNA methyltransferase METTL3, and enables the polypeptide drug to enter cells to exert its efficacy by binding to the cell-penetrating peptide R9.

In addition, by synthesizing stapled peptides on the original sequence of the polypeptide, the structural stability of the polypeptide is increased, and the activity and efficacy of the polypeptide drug are improved.

This peptide drug can specifically bind to METTL3 and inhibit the activity of its RNA methyltransferase, thereby reducing the RNA methylation level.

The polypeptide can significantly inhibit the growth and proliferation of SaoS2 (human osteosarcoma cells), HepG2 (human hepatoblastoma cells), DU145 (human prostate cancer cells), HCT116 (colorectal cancer cell line), A375 (human malignant melanoma), Hela (human precervical cancer cells), A549 (lung cancer human alveolar basal epithelial cells), K562 (human chronic myeloid leukemia cells), and OVCAR-3 (ovarian adenocarcinoma cells).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram of the polypeptide sequence and molecular structure provided in Example 1 of the present invention;

FIG2 is a diagram showing the steps for synthesizing the stapled peptide provided in Example 2 of the present invention;

FIG3 is a diagram showing the RNA methylation inhibition effect on prostate cancer cells provided by Example 3 of the present invention;

FIG4 is a graph showing the results of inhibiting the expression of METTL3 in prostate cancer cells provided in Example 4 of the present invention;

FIG5A is a graph showing the growth inhibition results of prostate cancer cells provided by Example 5 of the present invention;

FIG. 5B shows the effect of inhibiting the growth of prostate cancer cells provided by Example 5 of the present invention.

FIG6 is a graph showing the results of inhibiting the growth of prostate cancer cells provided by Example 6 of the present invention;

FIG7A shows the indexes of white blood cells in the blood of mice after the polypeptides R9, RM3 and RSM3 of the present invention were injected into the body;

FIG7B shows the platelet index in the blood of mice after the polypeptides R9, RM3 and RSM3 of the present invention were injected into the body;

FIG7C shows the indexes of red blood cells in the blood of mice after the polypeptides R9, RM3 and RSM3 of the present invention were injected into the body;

FIG7D is an index of hemoglobin in the blood of mice after the polypeptides R9, RM3 and RSM3 of the present invention were injected into the body;

FIG7E shows the changes in the body weight of tumor model mice after the polypeptides R9, RM3 and RSM3 of the present invention were injected into the mice;

FIG7F shows the changes in tumor volume after the polypeptides R9, RM3 and RSM3 of the present invention were injected into tumor model mice;

FIG7G shows the changes in tumor mass after the polypeptides R9, RM3 and RSM3 of the present invention were injected into tumor model mice;

FIG7H shows the changes in tumor photos after the polypeptides R9, RM3 and RSM3 of the present invention were injected into tumor model mice;

FIG8A is a data diagram showing the survival rate of prostate cancer cell DU145 treated with various polypeptides of Example 5 of the present invention. In FIG8A , the horizontal axis represents the polypeptide concentration, and the horizontal axis represents the cell survival rate;

FIG8B is a table showing the inhibitory concentrations of various polypeptides of Example 5 of the present invention on prostate cancer cells DU145, wherein the vertical coordinate IC50 is the half inhibitory concentration;

FIG9A is a data diagram showing the survival rate of various cancer cells treated with the polypeptide R9-RKL of Example 5 of the present invention. In FIG9A , the horizontal axis represents the polypeptide concentration, and the horizontal axis represents the cell survival rate;

FIG9B is a table showing the inhibitory concentrations of the polypeptide R9-RKL of Example 5 of the present invention on various cancer cells, wherein the vertical axis IC50 is the half inhibitory concentration.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1

1.1 The peptide drug molecules were synthesized by solid phase synthesis, and the amino acid sequences were M3-1

(RCMELGRECLNLW) and RM3(RRRRRRRRRCMELGRECLNLW) (refer to Figure 1).

Using AM resin, the required amino acids are weighed and dissolved, and then placed in a synthesis bottle. Synthesis is set up according to the operation of the fully automatic synthesis instrument. The crude peptide is obtained after the resin is removed, cut, and precipitated. The corresponding peptide drug lyophilized powder can be obtained by purification and freeze-drying using a high-performance liquid chromatography.

The importance of M3 lies in that: M3 is the core active polypeptide. M3 can bind to the proteins of METTL3 and METTL14 in vitro, but without the membrane-penetrating peptide, this sequence cannot enter the cell to exert its effect. Therefore, RM3 has more application value in the actual treatment process.

1.2 The peptide drug molecules were synthesized by solid phase synthesis. The peptide drug molecules were R9-MPF13 (R9-Mettl3'speptide fragment) and its mutant derivative peptides. The derivative peptides were products obtained by replacing certain amino acids in R9-MPF13. The amino acid sequences were referenced in Table 1 and Table 2 below. Table 2 marked the amino acid mutation and replacement methods by bold and diagonal lines.

Using AM resin, the required amino acids are weighed and dissolved, and then placed in a synthesis bottle. Synthesis is set up according to the operation of the fully automatic synthesis instrument. The crude peptide is obtained after the resin is removed, cut, and precipitated. The corresponding peptide drug lyophilized powder can be obtained by purification and freeze-drying using a high-performance liquid chromatography.

The target amino acid sequence consists of two parts. The first part is the membrane-penetrating peptide R9 sequence, which provides a membrane-penetrating effect for the polypeptide drug and ensures that the functional sequence can enter the cell to exert its effect. The second part is the functional peptide MPF13 and its derivative polypeptides, which are the functional regions of the target polypeptide drug and can bind to and exert their effects on the METTL3 and METTL14 complex proteins.

1.3 The peptide drug molecule R9-RKL (R9-RKMELGRELLNLW) was synthesized by solid phase synthesis using AM resin. The required amino acids were weighed and dissolved and placed in a synthesis bottle. The synthesis was set up according to the operation of the fully automatic synthesis instrument. The crude peptide was obtained after the resin was removed, cut, and precipitated. The corresponding peptide drug lyophilized powder was obtained by purification and freeze-drying using a high performance liquid chromatography.

Table 2 Sequence Listing

Sequence number Sequence Name 1～9 10 11 12 13 14 15 16 17 18 19 20 twenty one twenty two 8 R9-MPF13 R9 R A M E L G R E C L N L W 9 R9-RKF R9 R K M E L G R E F L N L W 10 R9-RKY R9 R K M E L G R E Y L N L W 11 R9-RKM R9 R K M E L G R E M L N L W 12 R9-RKL R9 R K M E L G R E L L N L W 13 R9-RRL R9 R R W E L G R E L L N L W 14 R9-RKWL R9 R K M E L G R E L L N L W 15 R9-RA R9 R A M E L G R E C L N L A 16 R9-RE R9 R A M E E G R E C L N L W 17 R9-PSRKWL R9 R K W C L G R E L L C L W 18 R9-SRKWL R9 R K W C' L G R E L L C' L W

(Note: The polypeptide is N-C terminus from left to right).

Example 2

Synthesis of stapled peptides SM3 and RSM3:

Referring to Figure 2, the purified RM3 and M3 peptide freeze-dried powders were weighed and reacted separately, and the peptides were dissolved in a 1:1 acetonitrile/30mM ammonium bicarbonate mixed solution, and the final concentration of the peptides was 1mM. Then 1.5mM bhp solution was added and stirred at room temperature for 3h. The synthesized peptide products were subjected to HPLC analysis and mass spectrometry detection. Finally, the stapled peptide powder was obtained after purification by high performance liquid chromatography and freeze drying.

The stapled positions of RM3 and M3 are shown in Figure 2 , where cysteine (Cys) at position 10 and cysteine at position 17 are stapled.

Example 3

Inhibition of RNA methylation in prostate cancer cells.

Determination of RNA methylation in cancer cells: Peptides R9, RM3, and RSM3 were prepared into 50mM stock solutions with sterile pure water, and then added to 6-well plates with PC3 and DU145 cells pre-plated with 2mL per well, with three replicates for each concentration. After 12 hours, total RNA of the cells was extracted, and the RNA was added to the NC membrane prepared in advance for drying and fixation, and then incubated with primary and secondary antibodies for RNA methylation at one time, and finally exposed with ultra-sensitive ECL chemiluminescent reagent. Referring to Figure 3, the results show that RM3 and RSM3 can significantly inhibit RNA methylation;

Referring to FIG3 , the results show that RSM3 is better in terms of stability and methylation inhibition effect because it has been stapled.

In Figure 3, MB is methylene blue staining, and m ⁶ A is methylation content. The amount of RNA loaded in the 500ng and 3000ng experiments.

Example 4

It has an inhibitory effect on the expression of METTL3 in prostate cancer cells.

Referring to Figure 4, a certain amount of peptide was weighed and dissolved into a 50mM stock solution. Peptides R9, RM3, and RSM3 were prepared into a 50mM stock solution with sterile pure water. After gradient dilution, they were added to a 6-well plate with PC3 and DU145 cells pre-plated. The final concentration of the peptide was 20uM, 2mL per well, and three replicates were used for each concentration. After 24 hours of treatment, the cells were lysed to extract cell proteins, and the proteins were separated by electrophoresis and transferred to a PVDF membrane, incubated with the primary antibody and secondary antibody of METTL3, and finally exposed for imaging.

Referring to FIG. 4 , the results showed that both RM3 and RSM3 were able to inhibit the expression of METTL3.

In FIG. 4 , GAPDH is an internal reference in the immunoblotting experiment (WB experiment).

Example 5

It has an inhibitory effect on the proliferation of prostate cancer cells PC3 and DU145.

5.1 Referring to Figure 5A, peptides R9, RM3 and RSM3 were prepared into 50mM stock solutions with sterile pure water, and then gradiently diluted with basal culture medium. The diluted peptide solutions were added to 96-well plates pre-plated with PC3 and DU145 cells, 100μL per well, and three replicates for each concentration. After 24h of treatment, the OD value at 450nm was measured with a CCK-8 kit, and the results were calculated and analyzed.

Figure 5B: In the clone formation experiment, the grown PC3 and DU145 cells were digested with 0.25% trypsin and the cells were counted. 300 cells were added to each well of the six-well plate, and 2 ml of complete culture was added to continue culturing for 24 hours. M3, R9, RM3, and RSM3 were added to each well and continued to be cultured for 7 days. The medium was discarded, and the cells were washed, stained with crystal violet, and then microscopically photographed to count the number of cells.

The results showed that RSM3 and RM3 had a good effect in inhibiting the growth of prostate cancer cells, and the inhibitory effect of RSM3 was better than that of RM3.

5.2 Some of the peptides in Table 1 were prepared into 10mM stock solutions with sterile pure water, and then gradiently diluted with basal culture medium. The diluted peptide solutions were added to 96-well plates pre-plated with DU145 cells, 100μL per well, and four replicates for each concentration. After 48h of treatment, the modified MTT kit was used to treat and measure the OD value at 580nm, and the results were calculated and analyzed.

Referring to Figures 8A and 8B, the results show that all polypeptides have a significant inhibitory effect on the proliferation of prostate cancer cells DU145; among them, the three mutated polypeptides R9-RKL, R9-RKWL, and R9-PSRKWL in Table 2 have a better inhibitory effect, indicating that this type of mutation enhances the effect of the functional peptide, showing that the mutation of amino acid A at position 12 to K and the mutation of amino acid C at position 18 to L in the polypeptide sequence are better mutations.

FIG8A is a data diagram showing the survival rate of various polypeptides in Example 5 of the present invention acting on prostate cancer cell DU145. In FIG8A , the horizontal axis is the polypeptide concentration and the horizontal axis is the cell survival rate.

FIG8B shows the _IC50 (half inhibitory concentration) of various polypeptides of Example 5 of the present invention on prostate cancer cell DU145. In FIG8B , the abscissa is the polypeptide name, and the ordinate is _IC50 .

5.3 Referring to FIG. 9A and FIG. 9B , the inhibitory effect of R9-RKL on different cancer cell lines was verified using the better-performing R9-RKL;

The validated cell objects are: SaoS2 (human osteosarcoma cells), HepG2 (human hepatoblastoma cells), DU145 (human prostate cancer cells), HCT116 (colorectal cancer cell line), A375 (human malignant melanoma), Hela (human precervical cancer cells), A549 (lung cancer human alveolar basal epithelial cells), K562 (human chronic myeloid leukemia cells), OVCAR-3 (ovarian adenocarcinoma cells).

The experimental method is: the peptide is prepared into a 10mM stock solution with sterile pure water, and after gradient dilution with basal culture medium, the diluted peptide solution is added to a 96-well plate with various cells pre-plated, 100μL per well, and four replicates for each concentration. After 48h of treatment, the modified MTT kit is used to treat and measure the OD value at 580nm, and the results are calculated and analyzed.

FIG9A is a data diagram showing the survival rate of various cells acted upon by the polypeptide R9-RKL of Example 5 of the present invention. In FIG9A , the abscissa is the polypeptide concentration, and the ordinate is the cell survival rate;

FIG9B is a table showing the inhibitory concentrations of the polypeptide R9-RKL of Example 5 of the present invention on various cells, wherein the vertical coordinate IC50 is the half inhibitory concentration.

As can be seen from Figures 9A and 9B, R9-RKL has a good growth inhibitory effect on various cancer cell lines such as SaoS2, HepG2, DU145, HCT116, A375, Hela, A549, K562, and OVCAR-3, and its _IC50 is lower than 11 μM.

Example 6

The other two screened sequences NO.4&5M1 and M2, after being connected with the cell-penetrating peptide R9, had a certain growth inhibitory effect on prostate cancer cells PC3 and DU145.

Referring to Figure 6, the above two polypeptides were prepared into a 50mM stock solution with sterile pure water, and after gradient dilution with basal culture medium, the diluted polypeptide solution was added to a 96-well plate pre-plated with PC3 and DU145 cells, 100 μL per well, and three replicates for each concentration. After 24 hours of treatment, the OD value was measured at 450nm using CCK-8, and the obtained value was calculated.

In FIG6 , the horizontal axis represents the polypeptide concentration, and the vertical axis represents the cell survival rate.

The results showed that M1 and M2 had a certain effect of inhibiting cell growth after combining with the cell-penetrating peptide. However, the efficacy needs to be further optimized and has certain research and application value.

Example 7

Referring to Figure 7, (A) In vivo drug safety assessment: C57BL/C mice raised for 7 weeks were intraperitoneally injected with a dose of 50 mg/kg (R9, RM3, RSM3) once. After 24 hours, the mice were sacrificed and blood was taken to measure indicators such as white blood cells, platelets, red blood cells, and hemoglobin, and the toxicity of the drugs to normal animals was analyzed.

(B) In vivo anti-tumor experiment: We will use a prostate tumor-bearing mouse model. The specific steps to construct the animal model are as follows: PC3 prostate cancer cells with high expression of METTL3 are cultured to the logarithmic growth phase, digested, collected and prepared into a cell suspension with a concentration of 1×10 ⁷ cells/mL. Take 4-6 week-old male and female BALB/c nude mice, inject 100μL of cell suspension into the back of the hind limbs to establish a tumor model. After the diameter of the mouse tumor grows to about 0.5cm, it is randomly divided into groups according to the experimental requirements: PBS, R9, RM3, RSM3, etc. Peritumoral administration, once every two days, for two consecutive weeks. After administration, the growth of the mice was observed for six consecutive weeks, the changes in tumor volume and weight of the mice were measured, the survival status of the mice was monitored, and the tumor inhibition rate and survival rate were analyzed.

In FIG. 7 , FIG. 7A to FIG. 7D are blood index data

Figure 7E-H shows the body weight, tumor volume changes, tumor mass and pictures of tumor model mice.

From the results of Figures 7A to 7H, we can see that:

Referring to FIG7A , for white blood cells (WBC) in the blood, the peptide inhibitors RM3 and RSM3 both slightly increased the index, but were within the normal range, compared with the cell-penetrating peptide R9 and the control group PBS;

Referring to Figure 7B , in terms of platelets (PLT), the peptide inhibitors RM3 and RSM3 had no significant effect on this index, compared with the cell-penetrating peptide R9 and the control group PBS;

Referring to Figure 7C , in terms of red blood cells (RBC), the peptide inhibitors RM3 and RSM3 had no significant effect on this index, compared with the cell-penetrating peptide R9 and the control group PBS;

Referring to Figure 7D , in terms of hemoglobin (HMGB), the peptide inhibitors RM3 and RSM3 had some reducing effects on this index, but both were within the normal range;

Referring to FIG7E , in terms of body weight changes, the PBS group, the cell-penetrating peptide R9, and the peptide inhibitor groups RM3 and RSM3 had no significant effect on the body weight of mice, indicating that these treatment groups had no obvious toxic side effects;

Referring to FIG. 7F , in terms of tumor volume changes, peptide inhibitors RM3 and RSM3 can significantly inhibit the increase in tumor volume, indicating that the peptide inhibitors can effectively inhibit tumor growth;

Referring to Figure 7G, the tumor mass was used to further determine the therapeutic effect of the peptide. The mass of the tumor mass of mice treated with peptide inhibitors RM3 and RSM3 was significantly lower than that of the cell-penetrating peptide R9 group and the PBS treatment group. This result further shows that RM3 and RSM3 have good therapeutic effects and can inhibit tumor growth;

Referring to Figure 7H , the changes in tumor volume can confirm the results of Figures 7F and 7G ;

The above analysis shows that METTL3 peptide inhibitors have the effect of inhibiting the growth of prostate tumors, among which the stapled peptide inhibitor RSM3 is more effective.

It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims (10)

1. A polypeptide is characterized in that the polypeptide is obtained by connecting a polypeptide with an amino acid sequence shown as SEQ ID No.2 and a penetrating peptide.

2. The polypeptide of claim 1, wherein the transmembrane peptide is one of an R9 transmembrane peptide, TAT transmembrane peptide, iggd, PENETRATIN transmembrane peptide.

3. The polypeptide of claim 1, wherein the polypeptide is stapled; and the 2 nd cysteine and the 9 th cysteine in the polypeptide are connected through 4, 4-bis (bromomethyl) biphenyl for stapling to obtain the stapling peptide.

4. A polypeptide is characterized in that the polypeptide is obtained by connecting a polypeptide with an amino acid sequence shown in any one of SEQ ID No.4 and SEQ ID No.5 with a membrane penetrating peptide.

5. The polypeptide of claim 4, wherein the transmembrane peptide is one of an R9 transmembrane peptide, TAT transmembrane peptide, iggd, PENETRATIN transmembrane peptide.

6. A polypeptide is characterized in that the polypeptide is obtained by connecting a polypeptide with an amino acid sequence shown as SEQ ID No.7 with a membrane penetrating peptide.

7. The polypeptide of claim 6, wherein the transmembrane peptide is one of an R9 transmembrane peptide, TAT transmembrane peptide, iggd, PENETRATIN transmembrane peptide.

8. The polypeptide is characterized in that the amino acid sequence of the polypeptide is shown as any one of SEQ ID No.9 and SEQ ID No. 12-SEQ ID No. 17.

9. The polypeptide according to claim 8, wherein the polypeptide is a stapled peptide obtained by stapling the 13 th and 20 th cysteines of the polypeptide shown in SEQ ID No.17 by 4, 4-bis (bromomethyl) biphenyl ligation.

10. A method for preparing a polypeptide according to any one of claims 1 to 9, wherein the desired amino acid is obtained by dissolving the amino acid in AM resin, placing the amino acid in a synthesis bottle, performing setting synthesis according to the operation of a fully automatic synthesis apparatus, taking out the AM resin, cutting, precipitating, and purifying.