CN111394334B - Anti-tumor polypeptide for inhibiting EZH2 activity and application thereof - Google Patents

Abstract

The invention discloses an antitumor polypeptide for inhibiting EZH2 activity and application thereof. According to the invention, based on the research, the protein interaction between FBP1 and EZH2 is obtained, and the key peptide segment of the combination region of FBP1 and EZH2 can interfere with the function of EZH2, so that the effect of inhibiting the growth of tumor cells is achieved; and the identified corresponding polypeptide fragments and specific sites of interaction between FBP1 and EZH2 provide a design scheme of small molecular polypeptide with the effects of inhibiting the activity of EZH2 and further resisting tumors, and polypeptide molecules derived from FBP1 truncation are constructed, and all have the effect of targeting EZH2 and inhibiting tumors. The polypeptide has small molecular weight, is easy to penetrate cell membranes, can directly inhibit the activity of EZH2 methyltransferase, blocks the effect of EZH2 on promoting the growth of cancer cells, achieves the anti-tumor effect, and has good application value and prospect in the development of anti-tumor drugs of EZH2 high-expression solid tumors.

Description

Anti-tumor polypeptide for inhibiting EZH2 activity and application thereof

Technical Field

The invention belongs to the technical field of biological medicines. More particularly, it relates to an antitumor polypeptide inhibiting the activity of EZH2 and its application.

Background

A variety of cancers are associated with gene mutations, including some of the chromatin remodeling and histone methylation pathways. EZH2 is the active core subunit of polycomb inhibitory complex 2(PRC2), which also includes EED, SUZ12, and RbAp 46/48. The PRC2 complex mainly takes methyltransferase of EZH2 as a core to catalyze the trimethylation modification of the 27 th lysine of histone 3, thereby leading to chromatin compaction and gene silencing. Scientific research shows that the occurrence and development of various cancers are related to EZH2, and EZH2 shows obvious high expression in various malignant tumors such as breast cancer, prostate cancer, lung cancer, liver cancer, kidney cancer and the like. The results of the study indicate that EZH2 is essential for malignant proliferation of cancer cells, and exogenous overexpression of EZH2 in normal cells significantly promotes cell proliferation.

At present, various small molecule drugs have been used to achieve antitumor effect by reducing EZH2 activity, such as GSK126, UNC1999 and tazemetostat. Among them, the protocol of tazemetostat for the treatment of metastatic/locally advanced epithelioid sarcoma that is not amenable to surgical treatment has been qualified by the U.S. FDA priority, suggesting that epigenetic drugs represented by EZH2 inhibitors are becoming more and more important.

However, existing EZH2 inhibitors still have many drawbacks in practical clinical trials and are poorly responsive in many solid tumors. Therefore, the development of new EZH2 inhibitors becomes one of the major problems in the field of tumor drug research.

Disclosure of Invention

The invention aims to develop a novel EZH2 inhibitor to further develop an anti-tumor drug, in particular to an anti-tumor drug for solid tumors with high EZH2 expression.

The invention aims to provide an antitumor polypeptide capable of inhibiting the activity of EZH 2.

The invention also aims to provide application of the polypeptide in preparing anti-tumor medicaments.

Still another object of the present invention is to provide an antitumor agent comprising a polypeptide inhibiting EZH2 activity.

The above purpose of the invention is realized by the following technical scheme:

we have studied renal clear cell carcinoma, and found that the precondition for promoting the growth of EZH2 is the deletion of fructose-1, 6-bisphosphatase 1(FBP 1). FBP1 is a key enzyme in the gluconeogenic metabolic pathway and its expression is down-regulated in a variety of tumors. Experimental studies have shown that FBP1 can inhibit glycolytic metabolism, thereby preventing tumor cells from acquiring sufficient energy and metabolic intermediates to inhibit tumor cell growth. In addition, FBP1 can enter the nucleus to play a role in non-classical enzymatic activities, such as the inhibition of hypoxia inducible factor, Wnt/b-actenin signal and the activity of GTPase activation interacting protein 1, etc., which suggests that the metabolic enzyme FBP1 can inhibit tumor growth through various pathways. It was found that FBP1 directly affects PRC2 methyltransferase activity by binding to EZH 2. Therefore, the key peptide segment of the binding region of FBP1 and EZH2 is suggested to interfere the function of EZH2 and achieve the effect of inhibiting the growth of tumor cells. The invention can provide a new polypeptide molecule capable of inhibiting EZH2, and helps to develop a new anti-tumor drug.

Therefore, designing small molecular polypeptide according to key peptide fragments of FBP1 and EZH2 binding region can inhibit EZH2 activity, and further resist tumor.

The design principle of the small molecule polypeptide sequence is as follows:

M+X+LVAAGYALYGSATMLVLAMDCGVNCFMLDP，

wherein X is an additional 0-20 amino acids at the 5' end of the LVAAGYALYGSATMLVLAMDCGVNCFMLDP fragment in the binding region of FBP1 to EZH 2.

Namely, the shortest sequence of the anti-tumor polypeptide capable of inhibiting the activity of EZH2 developed by the invention is shown in SEQ ID NO. 1: MLVAAGYALYGSATMLVLAMDCGVNCFMLDP

Further, the polypeptide fragment can be further lengthened at the 5' end based on the above design principle, such as the polypeptide shown in SEQ ID NO. 2: MKSTDEPSEKDALQPGRNLVAAGYALYGSATMLVLAMDCGVNCFMLDP

The nucleic acid sequences of the polypeptides shown in SEQ ID NO.1 and SEQ ID NO.2 are shown in SEQ ID NO.3 and 4, respectively.

(SEQ ID NO.3) a nucleic acid sequence encoding a small molecule polypeptide AA2: ATGCTGGTGGCAGCCGGCTACGCACTGTATGGCAGTGCCACCATGCTGGTCCTTGCCATGGACTGTGGGGTCAACTGCTTCATGCTGGACCCG

(SEQ ID NO.4) a nucleic acid sequence encoding the small molecule polypeptide AA1: ATGAAATCAACTGATGAGCCTTCTGAGAAGGATGCTCTGCAACCAGGCCGGAACCTGGTGGCAGCCGGCTACGCACTGTATGGCAGTGCCACCATGCTGGTCCTTGCCATGGACTGTGGGGTCAACTGCTTCATGCTGGACCCG

The small molecular polypeptide can specifically inhibit the activity of epigenetic factor EZH2, block the cancer promotion effect of EZH2 in solid tumor, and achieve the effect of inhibiting the growth of tumor cells.

Specifically, the polypeptide activity verification scheme is as follows:

firstly, after EZH2 is knocked down in a renal clear cell carcinoma cell line, the content of H3K27me3 is obviously reduced, and meanwhile, the proliferation speed of renal clear cell carcinoma cells is also obviously reduced, which proves the necessity of EZH2 for the growth of renal carcinoma cells (figure 1). The potential interaction of FBP1 with EZH2 was then explored based on our previous study of gluconeogenic metabolic enzyme FBP 1. Specifically, the tagged FBP1 or EZH2 plasmids were transiently transfected in HEK-293T and then subjected to protein co-immunoprecipitation experiments. The results show that FBP1 does bind to EZH2 (fig. 2).

In order to analyze the interaction region between FBP1 and EZH2, 7 exons of FBP1 were deleted from cDNA by mutation technique to construct 7 FBP1 mutants lacking different exons. These FBP1 mutants were then transiently transfected into EZH2 expressing cells, respectively, and subjected to co-immunoprecipitation experiments. The results show that exon 4 of FBP1 binds to EZH 2. Through further structural analysis and mutation experiments, serine 169 at FBP1 is finally found to be a key site of the interaction. The FBP1S169A mutation could severely disrupt the interaction with EZH2 (fig. 3).

Based on the above studies, we speculate that the polypeptide fragment near S169 on FBP1 protein can inhibit the methyltransferase activity and exert the anti-tumor function by binding with EZH 2. Therefore, we cut out two polypeptide fragments (AA1 and AA2) with more complete secondary structure near the S169 residue according to the FBP1 structure, and constructed corresponding expression vectors respectively (FIG. 4). Then, the two polypeptides were expressed separately, and the effect of AA1 and AA2 on EZH2 activity was examined by quantitative PCR. The results showed that AA1 and AA2 had different degrees of significant inhibitory effect on EZH2 activity in two renal cancer cells (fig. 5).

The two polypeptide fragments (AA1 and AA2) are stably transfected into kidney cancer cells after being packaged by viruses respectively, and the inhibition effect of the two polypeptide fragments on the proliferation of tumor cells is detected. CCK-8 experiments show that the inhibitory effect of the polypeptides AA1 and AA2 on renal cancer cells is consistent with the inhibitory effect on the activity of EZH 2: both AA1 and AA2 very significantly inhibited the growth of RCC4 kidney cancer cells, whereas in RCC10 kidney cancer cells, AA2 exerted a more significant inhibitory effect (fig. 5). The results indicate that the two polypeptides have obvious inhibition effect on the proliferation of renal clear cell carcinoma.

Therefore, the application of the polypeptide in preparing the EZH2 inhibitor and preparing an anti-tumor medicament is within the protection scope of the invention.

In addition, the anti-tumor medicine containing the polypeptide also falls into the protection scope of the invention.

Specifically, the tumor is a solid tumor with high EZH2 expression. Such as renal cancer, liver cancer, breast cancer, lung cancer, colorectal cancer, etc.

The invention has the following beneficial effects:

based on the protein interaction between FBP1 and EZH2 obtained by research, FBP1 can interfere the combination of EZH2 and EED and influence the integrity of PRC2 complex, thereby reducing the activity of histone methyltransferase in PCR2 complex, and the identified corresponding polypeptide segment and specific site of interaction between FBP1 and EZH2, the invention provides a design scheme of small molecule polypeptide for inhibiting the activity of EZH2 and resisting tumors, and constructs polypeptide molecules derived from FBP1 truncated body, which all have the effect of targeting EZH2 and inhibiting tumors. The polypeptide has small molecular weight, is easy to penetrate cell membranes, can directly inhibit the activity of EZH2 methyltransferase, blocks the action of EZH2 on promoting the growth of cancer cells, achieves the effect of resisting solid tumors with high expression of EZH2 such as renal clear cell carcinoma and the like, and has good application value and prospect in the aspect of developing anti-tumor drugs of the solid tumors with high expression of EZH 2.

Drawings

Figure 1 shows that knockdown of EZH2 inhibits growth of renal clear cell carcinoma cells.

FIG. 2 shows the interaction of EZH2 with FBP1 protein.

FIG. 3 is the interaction site of EZH2 with FBP 1.

FIG. 4 shows the structure of the polypeptide and the vector of the encoding gene.

FIG. 5 is a graph showing inhibition of EZH2 activity following polypeptide expression.

FIG. 6 shows inhibition of renal tumor cell growth following polypeptide expression.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Various materials and reagents used in the present invention: HEK-293T, RCC4, RCC10 and UMRC2, cultured in DuLbecco's Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (Hyclone) and 1% Penicillin-Streptomycin Solution (Hyclone) at 37 ℃ in 5% carbon dioxide. Other reagents include PEI (Polyethylenimine, Linear, MW 25000, Polysciences Inc.), streptavidin-conjugated magnetic beads (Promega, Z5482), Cell Counting Kit-8 (Biyunyan), GSK126(MCE), FBP1(Abcam), EZH2(CST), HA (CST), Puromycin (Sigma), Q5 High-Fidelity DNA Polymerase (NEB), RNA extraction Kit (AXYGEN), reverse transcription Kit (TAKARA), qPCR detection Kit (all gold).

The various experimental procedures of the following examples are as follows:

(1) plasmid construction:

extracting mRNA of HEK-293T cells, performing reverse transcription to obtain cDNA, using the cDNA as a template, designing primers, amplifying EZH2 and FBP1 genes by using a PCR technology, carrying out enzyme digestion and connection on a carrier and an amplified fragment, transferring the carrier and the amplified fragment into an escherichia coli competent cell Stabl3, selecting positive clones on an agar plate containing ampicillin, and sending the positive clones to a company Limited in the Biotechnology engineering (Shanghai) for carrying out first-generation sequencing verification.

(2) Viral packaging and infection:

HEK-293T cells were seeded at 50% confluence on 10cm dishes and 24 hours after the cells were adherent, the cells were co-transfected with lentiviral expression plasmid (10. mu.g), viral packaging plasmid (psPAX2, 5. mu.g) and envelope plasmid (pMD2.G, 2. mu.g) using PEI. Following the manufacturer's recommendations, 48 hours post-transfection, virus supernatants were collected and filtered through 0.45 μm pore size. For viral infection, target cells were seeded into 6-well plates, and appropriate amounts of collected virus fluid and polybrene at a final concentration of 8 μ g/ml were added for co-culture for 24 hours, after which the cells were trypsinized into 10cm dishes and antibiotics were added to screen stably transfected cells for subsequent experiments.

(3) Real-time fluorescent quantitative PCR:

total RNA was extracted from cells using the RNA Midiprep kit (Axygen, AP-MN-MS-RNA-250) according to the manufacturer's instructions. cDNA was generated by reverse transcriptase PCR in the reverse transcription kit (Takara, RR036A) using RNA as template. Real-time quantitative PCR was performed using TB Green Premix Ex Taq qPCR kit (Transgen Biotech, AQ 101). Ribosomal component 18S was used as a housekeeping gene for normalization. The primer sequence was designed by itself or obtained from PrimerBank (https:// pga. mgh. harvard. edu/PrimerBank /).

(4) Protein immunoblotting:

after cells were lysed by adding a lysate of a protease inhibitor to RIPA buffer (1% Triton X-100or NP-40), the BCA protein was quantified, SDS loading buffer was added, and the cells were heated at 95 ℃ for 10 minutes, followed by SDS-PAGE of protein samples (10 to 50. mu.g) having the same mass. And transferring the protein on the PAGE gel to a PVDF membrane through a transfer membrane system after electrophoresis, and respectively carrying out primary antibody incubation and secondary antibody incubation. Antibodies used include: EZH2(Cell Signaling Technology,5246), FBP1(Abcam, ab109732), HA-Tag (Cell Signaling Technology, 3724).

(5) Co-immunoprecipitation:

after the lysis of cells was performed by adding the lysis solution of the protease inhibitor to the 1% NP-40 IP lysis solution, the protein amount in the lysis solution was measured by BCA protein assay. mu.L of lysate was left for input control, the rest of the same amount of protein lysate was added to streptavidin-bound magnetic beads (Promega, Z5482), incubated in a freezer at 4 ℃ for 4h with slow rotation, the beads were washed 4 times with IP lysate, and finally the proteins bound to the beads were eluted with 2 XSDS loading buffer for Western blotting.

(6) Cell proliferation assay:

cells were seeded into 96-well plates at a density of 1000 cells/well by counting and cultured overnight in an incubator. Fresh medium was changed every 3 days, and Cell proliferation was measured using Cell Counting Kit-8 (Biyun day).

(7) The statistical analysis method comprises the following steps:

the software analysis adopts SPSS, the experimental result is expressed by Mean value plus or minus standard error (Mean plus or minus SD), the difference comparison between groups divided into two groups in the experiment adopts Student's-T test, and the difference is statistically significant when P is less than 0.05. Wherein P < 0.05 is marked x; p < 0.01 is marked x; p < 0.001 marked x; n.s. indicates that the difference is not statistically significant.

Example 1 knockdown of EZH2 inhibits growth of renal clear cell carcinoma

1. Renal clear cell carcinoma cells UMRC2 and 786-O were infected with lentiviral particles carrying two EZH2 shRNAs. A part of cells are subjected to Western blotting detection by using an EZH2 antibody, and the other part of cells are inoculated into a 96-well plate after being screened by antibiotics, are placed into a cell culture box for culture and are subjected to a CCK-8 experiment for detecting cell growth. After 24 hours of incubation, a 96-well plate was taken for CCK-8 assay, and calibrated as the initial amount of inoculated cells. Thereafter, the cells in the 96-well plate were replaced with a new medium every 3 days, and one 96-well plate was taken every one to two days to examine the proliferation of the cells with CCK-8. CCK-8 is applied by diluting CCK-8 stock solution with culture medium at a ratio of 1:10, removing the culture medium from the plate, adding 100 μ L of the diluted solution, culturing at 37 deg.C for 2 hr, and detecting OD 450nm absorbance with microplate reader.

2. The results show that: EZH2 protein was knocked down by EZH2shRNA in two renal clear cell carcinoma cell lines (fig. 1A). The level of H3K27me3 decreased with knock-down of EZH 2. The 2 different sequences of EZH2shRNA all significantly inhibited the growth of renal clear cell carcinoma cells (fig. 1B), suggesting that EZH2 expression is essential for renal carcinoma cell growth.

Example 2 interaction of FBP1 with EZH2

1. Experiment one:

the method comprises the steps of firstly, stably infecting and expressing biotin ligase BirA in HEK-293T cells to obtain cells stably expressing BirA, then carrying out secondary infection by using EZH2(EZH2-Avi) virus packaged with a biotin label, and finally carrying out transient transfection on the cells by using a PEI transfection reagent to obtain FBP1 plasmid. 293T stably expressing BirA and transiently transfecting FBP1 is used as a control group cell, cells stably expressing BirA, EZH2-Avi and transiently transfecting FBP1 are used as an experimental group cell, the two groups of cells are cracked by using IP cell lysate, after quantification is carried out by a BCA protein quantification method, 50uL of cell lysate is used as an input control, the rest cell lysate is added to streptavidin magnetic beads according to the same protein quality for protein immune co-precipitation, and finally the obtained sample is used for western blot detection, wherein the result is shown in figure 2A. Another experiment performed simultaneously was the infection of biotin-tagged FBP1(FBP1-Avi) in BirA-expressing cells followed by transient transfection of the EZH2 plasmid with PEI transfection reagent. Then 293T stably expressing BirA and transiently transfecting EZH2 is used as a control group cell, cells stably expressing BirA, FBP1-Avi and transiently transfecting EZH2 are used as an experimental group cell, the two groups of cells are cracked by using IP cell lysate, after quantification is carried out by a BCA protein quantification method, 50uL of cell lysate is used as an input control, the rest cell lysate is added to streptavidin magnetic beads according to the same protein mass for protein immune coprecipitation, and finally the obtained sample is used for western blot detection, and the result is shown in figure 2B.

2. Experiment two:

in order to further explore a specific region of interaction between FBP1 and EZH2, 7 exons of FBP1 are respectively deleted by a PCR mutation technology to construct 7 FBP1 mutants deleting different exons. These FBP1 mutants were then transiently transfected into EZH2-Avi expressing cells, respectively, and protein co-immunoprecipitation was performed using streptavidin magnetic beads, and the results showed that FBP1 mutant lacking exon 4 had significantly reduced interaction with EZH2, as shown in fig. 3A. Then, the exon 4 of FBP1 and EZH2 protein were subjected to SPPIDER (http:// SPPIDER. cchmc. org /) interaction site analysis on the net, and the most probable serine 169 site was selected (S169) for subsequent study. Serine at position 169 of FBP1 was mutated to alanine by PCR technique (S169A). EZH2-avi was then stably transfected into BirA-containing HEK-293T cells, and FBP1 WT and S169A mutant plasmids were transiently transfected with PEI transfection reagents. After quantification by BCA protein quantification method, 50uL of cell lysate is taken as an input control, the rest of cell lysate is added to streptavidin magnetic beads according to the same protein mass for protein co-immunoprecipitation, and finally the obtained sample is used for Western blot detection, and the experimental result shows that the 169 th serine of FBP1 is an important site for interaction with EZH2, as shown in FIG. 3B.

Example 3 construction of antitumor Polypeptides that inhibit EZH2

Based on the experimental results of example 2, serine 169 of exon 4 of FBP1 was found to be an important binding site for EZH 2. Two polypeptides based on the EZH2-FBP1 interaction, namely MKS TDE PSE KDA LQP GRN LVA AGY ALY GSA TML VLA MDC GVN CFM LDP (named AA1) and MLV AAG YAL YGS ATM LVL AMD CGV NCF MLD P (named AA2), were designed based on the crystal structure of FBP1 (PDB: 5et6) (FIG. 4A). AA1 is the full amino acid of FBP1 exon 4, whereas AA2 contains only 3 beta-folded structural sequence amino acids. The nucleic acid coding sequence of the polypeptide fragment is then cloned into a plasmid by PCR techniques. The amplification primer sequences are respectively:

AA1 upstream: 5'-ctagTctagaatgctggtggcagccggctacg-3'

Downstream: 5'-cgcGGATCccgggtccagcatgaagca-3'

AA2 upstream: 5'-CTAGTctagaatgaaatcaactgatgagCCTTCTGAG-3'

Downstream: 5'-ATATAGGATCccgggtccagcatgaagcAG-3'

The vector map is shown in FIG. 4B.

The expression plasmids of the polypeptides are stably expressed in kidney cancer cells by a virus packaging and infection method, then RNAs of a control group and an experimental group are respectively extracted by an RNA extraction kit of AXYGEN company, then mRNA is subjected to reverse transcription by a reverse transcription kit of TAKARA, and finally the expression condition of the small molecular polypeptides is detected by quantitative PCR, wherein the detection delta CT values of AA1 and AA2 of RCC4 are respectively 5.25 and 5.00 (the detection delta CT values are corrected by ACTB for an internal reference gene). The Δ CT values detected at RCC4 for AA1 and AA2 were 5.96 and 6.04, respectively (corrected for the reference gene with ACTB). The primer sequences for detecting the expression amount are as follows:

an upstream primer: 5-ctggtggcagccggctacgCAC-3

A downstream primer: 5-CGTAGAATCGAGACCGAGGAGA-3

The results indicate that the polypeptides AA1 and AA2 are expressed in both kidney cancer cells.

Example 4 Activity of antitumor Polypeptides to inhibit EZH2

The reverse transcription products in example 3 were taken and the expression of the downstream target gene of EZH2 was detected by quantitative PCR to evaluate the effect of AA1 and AA2 on EZH2 activity. In RCC4 renal cancer cells, the inhibitory effect of AA1 on EZH2 activity was very significant (fig. 5A). In RCC10 renal carcinoma cells, AA2 inhibited EZH2 activity more than AA1 (fig. 5B).

Example 5 Activity of antitumor Polypeptides to inhibit EZH2

The two polypeptide fragments (AA1 and AA2) are stably transfected into renal cancer cells RCC4 and RCC10 after being packaged by viruses respectively, the renal cancer cells in logarithmic phase are taken and inoculated into a 96-well plate according to 1000 cells per well after being screened by antibiotics, each treatment is inoculated into 3 multiple wells, and the inoculated cells are placed in a cell culture box for culture. After 24 hours of incubation, a 96-well plate was taken for CCK-8 assay, and calibrated as the initial amount of inoculated cells. CCK-8 is applied by diluting CCK-8 stock solution with culture medium at a ratio of 1:10, removing the culture medium from the plate, adding 100 μ L of the diluted solution, culturing at 37 deg.C for 2 hr, and detecting OD 450nm absorbance with microplate reader. After that, the cells in the 96-well plate were replaced with a new medium every 3 days, and every two days, one 96-well plate was taken out to examine the proliferation of the cells with CCK-8. The inhibitory effect of the polypeptides AA1 and AA2 on renal cancer cells (fig. 6) was consistent with the inhibitory effect on EZH2 activity (fig. 5). Both AA1 and AA2 inhibited cell growth of RCC4 very significantly, while AA1 inhibited cell growth more strongly than AA2 (fig. 6A). In contrast, AA2 exerted a more pronounced inhibitory effect in RCC10 cells (fig. 6B). The above results suggest that both polypeptides have cell specificity for the effect of proliferation of renal clear cell carcinoma, but at least one of them has a significant inhibitory effect, and can serve as a starting point for the development of potent EZH2 inhibitors.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Zhongshan university

<120> antitumor polypeptide inhibiting EZH2 activity and application thereof

<130>

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 31

<212> PRT

<213> Small molecule polypeptide AA2 amino acid sequence

<400> 1

Met Leu Val Ala Ala Gly Tyr Ala Leu Tyr Gly Ser Ala Thr Met Leu

1 5 10 15

Val Leu Ala Met Asp Cys Gly Val Asn Cys Phe Met Leu Asp Pro

20 25 30

<210> 2

<211> 48

<212> PRT

<213> Small molecule polypeptide AA1 amino acid sequence

<400> 2

Met Lys Ser Thr Asp Glu Pro Ser Glu Lys Asp Ala Leu Gln Pro Gly

1 5 10 15

Arg Asn Leu Val Ala Ala Gly Tyr Ala Leu Tyr Gly Ser Ala Thr Met

20 25 30

Leu Val Leu Ala Met Asp Cys Gly Val Asn Cys Phe Met Leu Asp Pro

35 40 45

<210> 3

<211> 93

<212> DNA

<213> nucleic acid sequence encoding the small molecule polypeptide AA2

<400> 3

atgctggtgg cagccggcta cgcactgtat ggcagtgcca ccatgctggt ccttgccatg 60

gactgtgggg tcaactgctt catgctggac ccg 93

<210> 4

<211> 144

<212> DNA

<213> nucleic acid sequence encoding the small molecule polypeptide AA1

<400> 4

atgaaatcaa ctgatgagcc ttctgagaag gatgctctgc aaccaggccg gaacctggtg 60

gcagccggct acgcactgta tggcagtgcc accatgctgg tccttgccat ggactgtggg 120

gtcaactgct tcatgctgga cccg 144

Claims (6)

1. The antitumor polypeptide for inhibiting the activity of EZH2 is characterized in that the polypeptide is designed according to the key peptide segment of the binding region of FBP1 and EZH2, and the design principle is as follows: m + X + LVAAGYALYGSATMLVLAMDCGV NCFMLDP;

wherein X is an additional 0-20 amino acids at the 5' end of the LVAAGYALYGSATMLVLAMDCG VNCFMLDP fragment in the binding region of FBP1 to EZH 2;

the amino acid sequence of the obtained antitumor polypeptide is shown as SEQ ID NO.1 or SEQ ID NO. 2.

2. The anti-tumor polypeptide of claim 1, wherein the nucleic acid sequence encoding the anti-tumor polypeptide of SEQ ID No.1 is as shown in SEQ ID No. 3.

3. The anti-tumor polypeptide of claim 1, wherein the nucleic acid sequence encoding the anti-tumor polypeptide of SEQ ID No.2 is as shown in SEQ ID No. 4.

4. Use of the polypeptide of claim 1 in the preparation of an EZH2 inhibitor.

5. The use of the polypeptide of claim 1 in the preparation of an anti-tumor medicament, wherein the tumor is a renal cancer tumor with high expression of EZH 2.

6. An antitumor agent comprising the polypeptide of claim 1, wherein the tumor is a renal cancer tumor highly expressed by EZH 2.

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