CN112375124B

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

Info

Publication number: CN112375124B
Application number: CN202011292810.0A
Authority: CN
Inventors: 郭长缨; 于丽婷; 顾亚茹
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-11-18
Filing date: 2020-11-18
Publication date: 2022-11-11
Anticipated expiration: 2040-11-18
Also published as: CN112375124A

Abstract

The invention belongs to the field of medicines, and particularly discloses a novel protooncoprotein SET inhibitor, a fusion polypeptide thereof and application thereof; the amino acid sequence of the proto-oncoprotein SET inhibitor is SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. The amino acid sequence of the corresponding fusion polypeptide is SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.5. Can inhibit cell proliferation mediated by SET protein at the level of 1 micromolar concentration, realizes good tumor inhibition effect in vitro and in vivo experiments, and has potential new drug development value.

Description

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

Technical Field

The invention belongs to the field of medicines, and particularly relates to proto-oncoprotein SET inhibitor fusion polypeptides CY-1, CY-2 and CY-3 and application thereof, which are polypeptides with tumor cell proliferation and related functions mediated by SET.

Technical Field

Tumors have been a serious medical problem, and tumor metastasis is a major cause of high mortality. In 2018, the cancer is newly developed 1810 thousands and the death is 960 ten thousands all over the world. The medical community is always dedicated to research on effective means of tumor treatment, and a plurality of small molecule inhibitors and immunotherapy drugs are controversially developed, so that the survival time of tumor patients is relatively prolonged.

The SET protein is a multifunctional proto-oncoprotein and is widely expressed in various cells, participates in various cell processes such as regulation of cell cycle, cell movement and apoptosis, and plays a key role in cell transcription regulation and DNA damage repair. High expression of SET proteins is clearly associated with tumorigenesis, tumor metastasis and poor prognosis. The incidence and mortality of breast cancer, one of the female high-grade tumors, show a trend of increasing year after year, wherein the estrogen receptor positive breast cancer accounts for about 70%, and estrogen can combine with estrogen receptor and start the expression of estrogen response gene, thereby promoting the proliferation and metastasis of tumor cells, so the main treatment means of clinical estrogen receptor positive breast cancer is to use estrogen competitive antagonist tamoxifen and the like to inhibit the response of estrogen gene mediated by estrogen receptor, thereby inhibiting the proliferation and metastasis of tumor cells to slow down the development of disease. SET protein was found to interact with estrogen receptor, but its regulatory function and mechanism of action in estrogen-induced transcriptional activation of genes has been controversial, and over-expression of SET protein in MCF-7 cells positive for estrogen receptor ER α is an important regulator of hormone receptor-mediated gene expression. Our studies found for the first time that the SET protein promotes estrogen-induced estrogen receptor alpha-mediated gene expression.

The SET protein is a typical nucleosome assembly protein, and 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, linker histone H1, which further compact the nucleosome junction into compact chromatin, so chromosomal DNA is not directly accessible under normal conditions. Modulation of cellular replication and transcription state usually requires a first change in chromatin structure, and thus histone modification factors, chromatin remodeling complexes and histone chaperones play important regulatory roles in the process of modulating chromatin DNA accessibility. SET, one of the important members of nucleosome assembly protein family, can interact with core histone to participate in chromatin structure remodeling, and promotes PolII to recognize DNA so as to start gene transcription initiation. Meanwhile, the SET protein can also interact with histone linker H1 to remove H1, thereby relieving the H1-mediated DNA transcription inhibition state. However, epigenetic and molecular biological mechanisms of SET's involvement in transcriptional regulation remain to be explored further.

Disclosure of Invention

Object of the Invention

The invention provides a brand new proto-oncoprotein SET polypeptide inhibitor sequence and fusion polypeptide sequence with potential medical and pharmaceutical values, and the SET protein inhibitor CY-1,2,3 and the fusion polypeptide CPCY-1,2, 3 have good effects on treating tumors in vivo and in vitro.

Technical scheme

An inhibitor of proto-oncoprotein SET, characterized by: xm-QYYLVPDM-Yn, wherein X or Y is any amino acid, m and n represent the number of amino acids, m is more than or equal to 0, n is more than or equal to 0. That is, based on the QYYLVPDM as the parent sequence, the C-terminal or N-terminal may be substituted with one or more arbitrary amino acids.

Further selection is carried out:

an inhibitor of SET protein, characterized by: the amino acid sequence is CY-1 SEQ ID NO.1, CY-2 SEQ ID NO.2, CY-3.

A SET protein inhibitor and a cell-penetrating peptide form a fusion polypeptide, which is characterized in that: the amino acid sequence of the polypeptide is as shown in SEQ ID NO.4, CPCY-2.

Wherein the amino acid sequence of the cell-penetrating peptide is SEQ ID NO.7.

The SET protein inhibitor and the fusion polypeptide CY-3 thereof are applied to the preparation of the drugs for treating and inhibiting tumors.

The tumor comprises 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 sequence of the SET polypeptide inhibitor is

CY-1:QYYLVPDM；

CY-2:LQYYLVPDMD；

CY-3:QYYLVPDMDDE；

The sequence of the cell-penetrating peptide is KETWWETWWTEWSQPKKKRKV;

CPCY-1:KETWWETWWTEWSQPKKKRKVQYYLVPDM；

CPCY-2:KETWWETWWTEWSQPKKKRKVLQYYLVPDMD；

CPCY-3:KETWWETWWTEWSQPKKKRKVQYYLVPDMDDE。

advantageous effects

1. As shown in fig. 1-3, the present inventors have found for the first time that SET protein can interact with histone variant h2a.z through its acidic domain, and in the presence of estrogen, SET protein can be recruited to estrogen receptor binding site to participate in the expression regulation of estrogen response gene, but after mutating its acidic domain, the mutant can not bind to estrogen response element, and at this time, the estrogen response of breast cancer MCF-7 cells positive to estrogen receptor is significantly reduced. Based on the above, the invention designs competitive micromolecular polypeptide for blocking the interaction between the SET protein and the histone H2A.Z, and inhibits the expression of estrogen response gene mediated by the SET protein, thereby inhibiting the proliferation and the metastasis of tumor cells.

The invention synthesizes polypeptide inhibitors CY-1, CY-2 and CY-3 by a chemical method, the polypeptide inhibitors can selectively inhibit the interaction of SET protein and histone H2A.Z, reverse the change of tumor cell transcriptome mediated by SET protein up-regulation, embody good effect of inhibiting tumor cell proliferation, can pass through a cell membrane and a nuclear membrane to be positioned in a cell nucleus, and inhibit the estrogen response of estrogen receptor positive cells MCF-7. As CY-1 is mainly used as a target sequence for combination, theoretically, the similar effect can be achieved by only containing a CY-1 sequence or substituting more than one amino acid by C end or N based on the CY-1 sequence, but the effect is different.

At present, the SET protein serving as a nucleosome assembly protein is not used internationally as a therapeutic target for designing an inhibitor, the invention firstly utilizes the target for drug design to obtain fusion polypeptides CPCY-1, CPCY-2 and CPCY-3, and the cell-penetrating peptide and CY-1, CY-2 or CY-3 are utilized to form a new fusion polypeptide, so that the ability of CY-1, CY-2 or CY-3 to enter cells is increased, and the inhibitor has good effect on inhibiting tumor development and good development prospect. The invention takes CPCY-3 as an example to carry out cytotoxicity, cell migration experiment and cell localization experiment, and finds that the CPCY-3 inhibits the response of estrogen receptor positive breast cancer MCF-7 to estrogen, and the SET protein inhibitor has the functions of inhibiting SET protein-mediated cell proliferation, transcriptional regulation and the like in vitro.

Drawings

Figure 1SET protein and histone H2a.z interaction results, wherein figure 1A is a domain representation of different amounts of H2a.z protein that can compete with H2A binding to SET protein, and figure 1B is a domain representation of SET protein and its mutant M1.

FIG. 2 is a graph of the results of binding of SET protein to an estrogen responsive element, wherein FIG. 2A is a schematic of the estrogen responsive element; FIGS. 2B-2E show the binding results of SET protein and mutants on Estrogen Response Elements (EREs) 1 and 2, TSS and coding region (coding region), respectively, before and after estrogen treatment.

FIG. 3 shows the result of the interaction between CPCY-3 and Histone protein H2A. Z, FIG. 3A shows the predicted binding site of polypeptide CY-3 and SET protein, and FIG. 3B shows the predicted interaction between CY-3 and SET protein.

FIG. 4 verification of CPCY-3 Nuclear localization.

FIG. 5 shows that the polypeptide inhibitor CPCY-3 inhibits the response of the estrogen receptor positive breast cancer MCF-7 to estrogen, wherein FIGS. 5A-C show the results of mRNA QPCR detection of the estrogen responsive genes TFF1, GREB1, PGR under the action of CPCY-3, respectively.

Detailed Description

The invention relates to a method for synthesizing CY-1, CY-2, CY-3, CPCY-1, CPCY-2 and CPCY-3 by Kinseri.

Example 1 inhibition of proliferation of various tumor cells by SET protein inhibitors and fusion polypeptides

The MTT method is adopted to detect the activity inhibition effect of the SET protein inhibitor on the proliferation of various tumor cells, including melanoma cells B16F10, gastric cancer cells MGC-803, lung cancer cells A549, liver cancer cells Hep-G2, breast cancer cells MDA-MB-231, breast cancer MCF-7, breast cancer MCF-10CA1A, colon cancer cells HCT-116, human brain glioma U87 and cervical cancer cell Hela.

Tumor cells are treated at 37 deg.C and 5% CO ₂ The cells were collected by trypsinization when cultured in the incubator (2) to a density of 90% or more, and the cells were resuspended in the culture medium and counted under a microscope to adjust the cell concentration to 3.0X 10 ⁴ Cell suspension/mL, seeded into 96-well plates at 100. Mu.L per well and at 37 ℃ 5% CO ₂ The culture was carried out overnight in an incubator. The SET protein inhibitor and the positive drug Taxol were diluted to each predetermined concentration with the culture broth. After the cells were fully attached to the wall, each dilution was added to a 96-well plate at 100. Mu.L per well. Adding SET inhibitor as administration group, taxol as positive control group, and culture solution without any drug as blank control group, at 37 deg.C, 5% ₂ Incubate for 48 hours. 20 μ L of 5mg/mL MTT was added to each well of the 96-well plate and incubation was continued for 4 hours. The medium was aspirated off and dissolved by adding 100. Mu.L DMSO per well. The measurement was carried out with a microplate reader at 570nm, the absorbance was measured at a reference wavelength of 630nm, and the growth inhibition ratio (proliferation inhibition,PI), the formula is as follows:

PI(％)＝1-Ntest/Ncontrol*100％，

wherein N is _test To test the OD value of the group, N _control The OD value of the blank control group.

And (3) data statistics:

the test is independently repeated for 5 times, mean plus or minus SD is calculated according to the result obtained by the test, statistical t-test is carried out, the significance difference is when P is less than 0.05, and the significance difference is when P is less than 0.01. The results are shown in Table 1.

TABLE 1 CY-1, CY-2, CY-3, CPCY-1, CPCY-2, CPCY-3 IC50 values for different tumors

Example 2 three-dimensional transwell assay for the Activity of CPCY-3 to inhibit migration of human umbilical vein endothelial cells

Taking CPCY-3 as an example for detection:

human Umbilical Vein Endothelial Cells (HUVEC) were treated with 5% CO at 37 ℃ in a culture medium containing 5% fetal bovine serum and 1 × ECGS ₂ When the mixture is cultured in an incubator to a confluency of more than 90%, a transwell method is adopted to detect the activity of the SET protein inhibitor for inhibiting the migration of endothelial cells, and the endothelial cells HUVEC only use 2-8 generations, and the specific operation is as follows:

(1) Diluted with 10mg/mL Matrigel in DMEM medium at 1;

(2) HUVEC cells cultured to logarithmic growth phase were digested with 0.2% EDTA, collected, washed twice with PBS, resuspended in endothelial cell culture medium containing 0.1% BSA, counted under microscope, and cell concentration adjusted to 1X 10 ⁵ Per mL;

(3) Preparing test solutions for each group, and diluting to 100. Mu.L with a cell culture solution containing 0.1% BSA;

the grouping is as follows:

blank control group: is cell culture fluid without medicine;

SET protein fusion polypeptide group: preparing 0.25-4 mu g/mL of CPCY-3;

(4) Cells were seeded into transwell chambers at 100 μ L per well and groups of test solutions were added to the chambers. Adding 0.6mL of endothelial cell culture medium containing 5% fetal bovine serum and 1 × ECGS to a 24-well plate to stimulate cell migration by 5% CO ₂ Incubating for 24h at 37 ℃;

(5) Discarding culture solution in the hole, fixing with 90% alcohol at normal temperature for 30min, dyeing with 0.1% crystal violet at normal temperature for 10min, rinsing with clear water, slightly wiping off non-migrated cells on the upper layer with a cotton swab, observing under a microscope and selecting four visual fields for photographing and counting, and calculating the migration inhibition rate (MI) according to a formula:

MI(％)＝1-Ntest/Ncontrol*100％，

wherein N is _test To test the number of cells migrated in the group, N _control Cell migration number for the blank control group.

And (3) data statistics:

the test is independently repeated for 3 times, mean plus or minus SD is calculated according to the result obtained by the test, statistical t-test is carried out, the significance difference is that P is less than 0.05, and the extreme significance difference is that P is less than 0.01. The results are shown in Table 2.

TABLE 2 transwell method for detecting the result of CPCY-3 inhibiting migration of human umbilical vein endothelial cells

Example 3CPCY-3 conjugated FITC cell localization validation

Human breast cancer cells MCF-7 at 37 ℃ 5% CO ₂ The cells were collected by trypsinization when cultured in the incubator (2) to a density of 90% or more, and the cells were resuspended in the culture medium and counted under a microscope to adjust the cell concentration to 3.0X 10 ⁴ Cells per mL were seeded into 24-well plates at 400ul per well and cultured overnight. FITC-CPCY-3 was incubated with cells at a final concentration of 10ug/ml for 1 hourThereafter, the green fluorescence position was observed under a fluorescence microscope. The experimental result is shown in figure 4, CPCY-3 can be obviously positioned in the cell nucleus.

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

(1) Human breast cancer cells MCF-7 at 37 ℃ 5% CO ₂ The cells were collected by trypsinization after culturing in the incubator of (1) until the density became 90% or more, and the cells were resuspended in a culture medium and counted under a microscope to adjust the cell concentration to 3.0X 10 ⁴ Cells per mL were seeded into 6-well plates, 1.5mL per well, and cultured overnight.

(2) The next day the cells were washed once with PBS and cultured for 24 hours in DMEM medium without phenol red.

(3) The operation was repeated the next day.

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

(5) Reverse transcription, and detecting the change of the estrogen response gene TFF1, GREB1 and PGR.

The experimental result is shown in A-C of figure 5, which can obviously reduce the expressions of the estrogen response genes TFF1, GREB1, PGR and the like after being combined with CPCY-3, and can obviously inhibit the response condition of the breast cancer MCF-7 with positive estrogen receptor to estrogen.

Sequence listing

<110> university of Chinese pharmacy

<120> proto-oncoprotein SET inhibitor, fusion polypeptide thereof and application thereof

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<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

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

<400> 1

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)

<400> 2

Leu Gln Tyr Tyr Leu Val Pro Asp Met Asp

1 5 10

<210> 3

<211> 11

<212> PRT

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

<400> 3

Gln Tyr Tyr Leu Val Pro Asp Met Asp Asp Glu

1 5 10

<210> 4

<211> 29

<212> PRT

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

<400> 4

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

1 5 10 15

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

20 25

<210> 5

<211> 31

<212> PRT

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

<400> 5

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

1 5 10 15

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

20 25 30

<210> 6

<211> 32

<212> PRT

<213> CPCY-3(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

1 5 10 15

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

20 25 30

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<211> 21

<212> PRT

<213> transmembrane peptide (2 Ambystoma latex x Ambystoma jeffersonia)

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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

20

Claims

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.