CN112375124B - Novel proto-oncoprotein SET inhibitor, fusion polypeptide thereof and application thereof - Google Patents

Abstract

The invention belongs to the field of medicine, and specifically discloses a novel proto-oncoprotein SET inhibitor, its fusion polypeptide and application thereof; the amino acid sequences of the proto-oncoprotein SET inhibitor are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO. 3. The amino acid sequences of the corresponding fusion polypeptides are SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.5. It can inhibit the cell proliferation mediated by the SET protein at the level of 1 micromolar concentration, and achieve a good tumor inhibition effect in vitro and in vivo experiments, which has potential new drug development value.

Description

A novel proto-oncoprotein SET inhibitor and its fusion polypeptide and its application

technical field

The invention belongs to the field of medicine, and specifically relates to a proto-oncoprotein SET inhibitor fusion polypeptide CY-1, CY-2, CY-3 and application thereof, which has the function of inhibiting SET-mediated tumor cell proliferation and related functions.

technical background

Tumor has always been a serious medical problem, and tumor metastasis is the main reason for its high lethality. In 2018, there were 18.1 million new cancer cases and 9.6 million deaths worldwide. The medical community has been devoting itself to the study of effective means of tumor treatment. Numerous small molecule inhibitors and immunotherapy drugs have been scrambled to develop, which relatively prolongs the survival time of tumor patients.

SET protein is a multifunctional proto-oncoprotein, which is widely expressed in various cells. SET protein is involved in the regulation of cell cycle, cell movement, cell apoptosis and other cellular processes, and plays a key role in cell transcription regulation and DNA damage repair effect. High expression of SET protein is significantly correlated with tumorigenesis, tumor metastasis and poor prognosis. Breast cancer is one of the most common tumors in women, and its morbidity and mortality are increasing year after year. Estrogen receptor-positive breast cancer accounts for about 70%. Estrogen can bind to estrogen receptors and activate estrogen. The expression of hormone-responsive genes promotes the proliferation and metastasis of tumor cells, so the main treatment for estrogen receptor-positive breast cancer in clinic is to use estrogen competitive antagonists such as tamoxifen to inhibit estrogen receptor-mediated The response of estrogen gene, and then inhibit the proliferation and metastasis of tumor cells to slow down the progression of the disease. SET protein has been found to interact with estrogen receptors, but its regulatory function and mechanism of action in estrogen-induced gene transcription activation have been controversial. Overexpression is an important regulator of hormone receptor-mediated gene expression. Our study found for the first time that SET proteins promote estrogen-induced estrogen receptor α-mediated gene expression.

SET protein is a typical nucleosome assembly protein. In eukaryotes, each chromatin nucleosome is composed of a 147bp DNA chain, a histone octamer and a histone linker. Each histone octamer contains pairs of H2A, H2B, H3, H4 histones, and the linker histone H1 further compresses the nucleosome connection into compact chromatin, so chromosomal DNA is not directly accessible under normal conditions of. Adjustment of cellular replication and transcriptional status usually requires changes in chromatin structure first, so histone modifiers, chromatin remodeling complexes and histone chaperones play important regulatory roles in the process of adjusting chromatin DNA accessibility. As one of the important members of the nucleosome assembly protein family, SET can interact with core histones, participate in the remodeling of chromatin structure, promote the recognition of DNA by Pol II and initiate the initiation of gene transcription. At the same time, SET protein can also interact with histone linker H1 and participate in the removal of H1 to release the H1-mediated DNA transcriptional repression state. However, the epigenetic and molecular biological mechanisms of SET involved in transcriptional regulation still need to be further explored.

Contents of the invention

purpose of invention

The present invention provides a novel proto-oncoprotein SET polypeptide inhibitor sequence and fusion polypeptide sequence with potential medical and pharmaceutical value, the SET protein inhibitor CY-1, 2, 3 and its fusion polypeptide CPCY-1, 2, 3 It has a good effect on treating tumors in vivo and in vitro.

Technical solutions

A proto-oncoprotein SET inhibitor, characterized by: Xm-QYYLVPDM-Yn, wherein X or Y is any amino acid, m and n represent the number of amino acids, m≥0, n≥0. That is to say, on the basis of QYYLVPDM as the parent sequence, the C-terminal or N-terminal can be substituted by any amino acid greater than or equal to one.

Further options:

A SET protein inhibitor, characterized in that: its amino acid sequence is CY-1: SEQ ID NO.1, CY-2: SEQ ID NO.2, CY-3: SEQ ID NO.3.

A fusion polypeptide formed by a SET protein inhibitor and a penetrating peptide is characterized in that: its amino acid sequence is CPCY-1:SEQ ID NO.4, CPCY-2:SEQ ID NO.5, CPCY-3:SEQ ID NO. 6.

The amino acid sequence of the penetrating peptide is SEQ ID NO.7.

The application of the SET protein inhibitor and its fusion polypeptide CY-3 in the preparation of drugs for treating and suppressing tumors.

Said tumors include gastric cancer, lung cancer, liver cancer, breast cancer, colon cancer, glioma, melanoma, cervical cancer, pancreatic cancer, colon cancer, rectal cancer, ovarian cancer, prostate cancer or testicular cancer.

The SET polypeptide inhibitor sequence is

CY-1:QYYLVPDM;

CY-2:LQYYLVPMDMD;

CY-3:QYYLVPMDMDDE;

The sequence of the penetrating peptide is KETWWETWWTEWSQPKKKRKV;

CPCY-1:KETWWETWWTEWSQPKKKRKVQYYLVPDM;

CPCY-2:KETWWETWWTEWSQPKKKRKVLQYYLVPMDMD;

CPCY-3: KETWWETWWTEWSQPKKKRKVQYYLVPDMDDE.

Beneficial effect

1. As shown in Figures 1-3, the present invention found for the first time that SET protein can interact with histone variant H2A.Z through its acidic domain. In the presence of estrogen, SET protein can be recruited to estrogen receptors. However, after mutating its acidic domain, the mutant cannot bind to estrogen-responsive elements. At this time, the expression of estrogen receptor-positive breast cancer MCF-7 cells The estrogen response was markedly reduced. Based on this, the present invention designs a competitive small molecular polypeptide that blocks the interaction between SET protein and histone H2A.Z, inhibits the expression of estrogen response genes mediated by SET protein, and thus inhibits the proliferation and metastasis of tumor cells.

The present invention synthesizes polypeptide inhibitors CY-1, CY-2, and CY-3 through chemical methods. The polypeptide inhibitors can selectively inhibit the interaction between SET protein and histone H2A.Z, and reverse the up-regulation of SET protein The changes in the transcriptome of tumor cells reflect a good effect of inhibiting the proliferation of tumor cells, and can pass through the cell membrane and nuclear membrane to localize in the nucleus, inhibiting the estrogen response of the estrogen receptor positive cell MCF-7. Because CY-1 is mainly used as the target sequence for binding, theoretically as long as the CY-1 sequence is contained, or one or more amino acids are substituted at the C-terminus or N based on the CY-1 sequence, similar effects as above can be achieved, but the effect is different. .

At present, there is no inhibitor designed with the SET protein as the nucleosome assembly protein as the therapeutic target in the world. The present invention uses this target for drug design for the first time to obtain fusion polypeptides CPCY-1, CPCY-2, and CPCY-3. Utilize the penetrating peptide and CY-1, CY-2 or CY-3 to form a new fusion polypeptide, which increases the ability of CY-1, CY-2 or CY-3 to enter cells, so it shows good effect in inhibiting tumor progression It has a good development prospect. The present invention takes CPCY-3 as an example to carry out cytotoxicity, cell migration experiments, and cell localization experiments. CPCY-3 inhibits the response of estrogen receptor-positive breast cancer MCF-7 to estrogen, and finds that SET protein inhibitors can inhibit SET protein in vitro. Mediated cell proliferation, transcriptional regulation and other functions.

Description of drawings

Figure 1 The results of the interaction between SET protein and histone H2A.Z, in which Figure 1A shows that different amounts of H2A.Z protein can compete with H2A for binding to SET protein, and Figure 1B shows the structural domain diagram of SET protein and its mutant M1.

Fig. 2 The results of SET protein binding to estrogen action elements, in which Fig. 2A is a schematic diagram of estrogen response elements; Fig. 2B-2E are the estrogen response elements (ERE) 1 and 2 of SET protein and mutants before and after estrogen treatment respectively The binding results on the transcription start site TSS and the coding region (coding region).

Fig. 3 The result of interaction between CPCY-3 and histone H2A.Z, Fig. 3A is the predicted binding site of polypeptide CY-3 and SET protein, and Fig. 3B is the result of the predicted interaction between CY-3 and SET protein.

Fig. 4 Verification of nuclear localization of CPCY-3 cells.

Figure 5 The peptide inhibitor CPCY-3 inhibits the response of estrogen receptor positive breast cancer MCF-7 to estrogen, where Figure 5A-C is the mRNA QPCR of the estrogen response genes TFF1, GREB1, and PGR under the action of CPCY-3, respectively The result of the test.

Detailed ways

CY-1, CY-2, CY-3, CPCY-1, CPCY-2 and CPCY-3 involved in the present invention are synthesized by GenScript.

Example 1 Inhibitory Effects of SET Protein Inhibitors and Fusion Polypeptides on the Proliferation of Various Tumor Cells

MTT method was used to detect the inhibitory effect of SET protein inhibitors on the proliferation of various tumor cells, including melanoma cell B16F10, gastric cancer cell MGC-803, lung cancer cell A549, liver cancer cell Hep-G2, breast cancer cell MDA-MB-231, Breast cancer MCF-7, breast cancer MCF-10CA1A, colon cancer HCT-116, human glioma U87, cervical cancer Hela.

The tumor cells were cultured in an incubator at 37°C and 5% CO ₂ until the density was above 90% and collected by trypsinization. The cells were resuspended in the culture medium and counted under a microscope. The cell concentration was adjusted to 3.0× ¹⁰⁴ /mL, inoculate the cell suspension into a 96-well plate, 100 μL per well, and incubate overnight in a 37°C, 5% CO ₂ incubator. Dilute the SET protein inhibitor and the positive drug Taxol to respective predetermined concentrations with culture medium. After the cells were completely attached to the wall, each dilution was added to a 96-well plate, 100 μL per well. Add SET inhibitor as administration group, Taxol as positive control group, culture solution without any drug as blank control group, and incubate at 37°C, 5% CO ₂ incubator for 48 hours. Add 20 μL of 5 mg/mL MTT to each well of the 96-well plate and continue culturing for 4 hours. Aspirate the medium, add 100μL DMSO to each well to dissolve. Use a microplate reader to detect at 570nm, and measure the absorbance at the reference wavelength of 630nm, and calculate the growth inhibition rate (proliferation inhibition, PI), the formula is as follows:

PI(%)=1-Ntest/Ncontrol*100%,

Among them, N _test is the OD value of the test group, and N _control is the OD value of the blank control group.

Statistics:

The experiment was repeated 5 times independently, and the results obtained from the experiment were calculated as mean±SD, and statistical t-test was performed. P<0.05 was considered a significant difference, and P<0.01 was considered an extremely significant difference. The experimental results are shown in Table 1.

Table 1 IC50 values of CY-1, CY-2, CY-3, CPCY-1, CPCY-2, CPCY-3 against different tumors

Example 2 Three-dimensional transwell method to detect the activity of CPCY-3 in inhibiting the migration of human umbilical vein endothelial cells

Take CPCY-3 as an example for detection:

Human umbilical vein endothelial cells (HUVEC) are cultured in an endothelial cell culture medium containing 5% fetal bovine serum and 1×ECGS in an incubator at 37°C and 5% CO ₂ to a confluence of more than 90%, using the transwell method Detect the activity of the SET protein inhibitor in inhibiting endothelial cell migration. Endothelial cell HUVEC only use the 2nd to 8th passages. The specific operation is as follows:

(1) Dilute 10mg/mL Matrigel with DMEM medium at a rate of 1:4, spread on the transwell chamber membrane, and air-dry at room temperature;

(2) Digest the HUVEC cells cultivated to the logarithmic growth phase with 0.2% EDTA, collect, wash twice with PBS, resuspend with endothelial cell culture medium containing 0.1% BSA, count under a microscope, and adjust the cell concentration to 1×10 ⁵ pieces/mL;

(3) Prepare the test solution of each group and dilute it to 100 μL with the cell culture solution containing 0.1% BSA;

Grouped as follows:

Blank control group: cell culture medium without drugs;

SET protein fusion peptide group: CPCY-3 preparation 0.25-4μg/mL;

(4) Inoculate cells into transwell chambers, 100 μL per well, and add each group of test solutions into the chambers. Add 0.6 mL of endothelial cell culture medium containing 5% fetal bovine serum and 1×ECGS to the 24-well plate to stimulate cell migration, and incubate at 5% CO ₂ at 37°C for 24 hours;

(5) Discard the culture medium in the well, fix with 90% alcohol at room temperature for 30 minutes, stain with 0.1% crystal violet for 10 minutes at room temperature, rinse with clean water, gently wipe off the upper layer of non-migrated cells with a cotton swab, observe under a microscope and select four fields of view to take pictures Count, and calculate the migration inhibition rate (migration inhibition, MI) according to the formula:

MI(%)=1-Ntest/Ncontrol*100%,

Among them, N _test is the number of cell migration in the test group, and N _control is the number of cell migration in the blank control group.

Statistics:

The experiment was repeated 3 times independently, and the results obtained from the experiment were calculated as mean±SD, and statistical t-test was performed. P<0.05 was considered a significant difference, and P<0.01 was considered an extremely significant difference. The experimental results are shown in Table 2.

Table 2 The results of CPCY-3 inhibiting the migration of human umbilical vein endothelial cells detected by transwell method

Example 3 CPCY-3 Coupled FITC Cell Localization Verification

Human breast cancer cells MCF-7 were cultured in an incubator at 37°C and 5% CO ₂ to a density of over 90% and collected by trypsinization. The cells were resuspended in culture medium and counted under a microscope. The cell concentration was adjusted to 3.0 ×10 ⁴ cells/mL, inoculate cells into 24-well plates, 400ul per well, and culture overnight. After co-incubating FITC-CPCY-3 with the cells at a final concentration of 10ug/ml for 1 hour, the green fluorescent position was observed under a fluorescence microscope. The experimental results are shown in Figure 4, CPCY-3 can be clearly localized in the nucleus.

Example 4 CPCY-3 inhibits the response of estrogen receptor positive breast cancer MCF-7 to estrogen.

(1) Human breast cancer cells MCF-7 were cultured in an incubator at 37°C and 5% CO ₂ to a density above 90% and collected by trypsinization, resuspended cells in culture medium and counted under a microscope. Adjust to 3.0× ¹⁰ cells/mL, inoculate the cells into a 6-well plate, 1.5 ml per well, and culture overnight.

(2) The next day the cells were washed once with PBS, and replaced with phenol red-free DMEM medium to continue culturing for 24 hours.

(3) Repeat the second day's operation.

(4) On the fourth day, the cells were treated with estrogen (E2) at a final concentration of 100 nM under the action of 7.5 μg/mL and 15 μg/mL of CY-3, and the total RNA was extracted 24 hours later.

(5) Reverse transcription, detecting changes in estrogen response genes TFF1, GREB1, and PGR.

The experimental results are shown in A-C of Figure 5. After administration of CPCY-3, the expression of estrogen-responsive genes TFF1, GREB1, PGR, etc. was significantly reduced, which can significantly inhibit the response of estrogen receptor-positive breast cancer MCF-7 to estrogen.

sequence listing

<110> China Pharmaceutical University

<120> Proto-oncoprotein SET inhibitor and its fusion polypeptide and application thereof

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<170> SIPOSequenceListing 1.0

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<211> 8

<212> PRT

<213> CY-1(2 Ambystoma laterale x Ambystoma jeffersonianum)

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Gln Tyr Tyr Leu Val Pro Asp Met

1 5

<210> 2

<211> 10

<212> PRT

<213> CY-2(2 Ambystoma laterale x Ambystoma jeffersonianum)

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Leu Gln Tyr Tyr Leu Val Pro Asp Met Asp

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<210> 3

<211> 11

<212> PRT

<213> CY-3(2 Ambystoma laterale x Ambystoma jeffersonianum)

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Gln Tyr Tyr Leu Val Pro Asp Met Asp Asp Glu

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<210> 4

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<212> PRT

<213> CPCY-1(2 Ambystoma laterale x Ambystoma jeffersonianum)

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Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

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Lys Lys Arg Lys Val Gln Tyr Tyr Leu Val Pro Asp Met

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<210> 5

<211> 31

<212> PRT

<213> CPCY-2(2 Ambystoma laterale x Ambystoma jeffersonianum)

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Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

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Lys Lys Arg Lys Val Leu Gln Tyr Tyr Leu Val Pro Asp Met Asp

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Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

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Lys Lys Arg Lys Val Gln Tyr Tyr Leu Val Pro Asp Met Asp Asp Glu

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<213> Penetrating peptide (2 Ambystoma laterale x Ambystoma jeffersonianum)

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Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

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Claims (4)

1. An inhibitor of proto-oncoprotein SET, characterized by: the amino acid sequence is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

2. A fusion polypeptide, characterized in that: the amino acid sequence is SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

3. A kit comprising the protooncoprotein SET inhibitor of claim 1 or the fusion polypeptide of claim 2.

4. Use of the proto-oncoprotein SET inhibitor of claim 1 or the fusion polypeptide of claim 2 or the kit of claim 3 for the preparation of a medicament for the prevention and treatment of a tumor; the tumor is melanoma cell, gastric cancer cell, and breast cancer.

Images