CN112375124A

CN112375124A - Novel proto-oncoprotein SET inhibitor, fusion polypeptide thereof and application thereof

Info

Publication number: CN112375124A
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: 2021-02-19
Anticipated expiration: 2040-11-18
Also published as: CN112375124B

Abstract

The invention belongs to the field of medicines, and particularly discloses a novel proto-oncoprotein 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 the SET protein 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 receptors, but its regulatory function and mechanism of action in estrogen-induced transcriptional activation of genes has been controversial, and its overexpression in estrogen receptor ER α positive MCF-7 cells 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 comprises pairs of H2A, H2B, H3, H4 histones, and linker histones H1 that further compact the nucleosome junction into compact chromatin, so that 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, as one of the important members of nucleosome assembly protein family, can interact with core histone to participate in chromatin structure remodeling, and promotes the recognition of DNA by PolII so as to start gene transcription initiation. Meanwhile, the SET protein can also interact with histone linker H1, and participate in removing H1 so as to relieve the H1-mediated DNA transcription inhibition state. However, epigenetic and molecular biological mechanisms of SET involvement in transcriptional regulation remain to be further explored.

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, and 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, and CY-3: SEQ ID NO. 3.

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

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, prostatic 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 to 3, the present inventors have found for the first time that the SET protein can interact with histone variant h2a.z through its acidic domain, and in the presence of estrogen, the SET protein can be recruited to an estrogen receptor binding site to participate in the expression regulation of estrogen response genes, but after mutating its acidic domain, the mutant cannot bind to an estrogen response element, and at this time, the estrogen response of estrogen receptor positive breast cancer MCF-7 cells 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 inhibitor 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 an estrogen receptor positive cell MCF-7. Because CY-1 is mainly used as a target sequence for combination, theoretically, the similar effect can be achieved by only containing the CY-1 sequence or substituting more than one amino acid by C end or N based on the CY-1 sequence, but the action 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 CPCY-3 inhibits the response of estrogen receptor positive breast cancer MCF-7 to estrogen, and finds that the SET protein inhibitor has the functions of inhibiting SET protein-mediated cell proliferation, transcriptional regulation and the like in vitro.

Drawings

Figure 1 interaction results of SET protein and histone H2a.z, wherein a is a domain representation of varying amounts of h2a.z protein that can compete with H2A for binding to SET protein, and B is a domain representation of SET protein and its mutant M1.

FIG. 2 is a graph showing the results of binding of SET protein to an estrogen responsive element, wherein Panel A is a schematic of the estrogen responsive element; FIGS. B-E 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, wherein A is the predicted binding site of polypeptide CY-3 and SET protein, and B is the predicted interaction between CY-3 and SET protein.

FIG. 4 verification of the nuclear localization of CPCY-3.

FIG. 5 shows that the polypeptide inhibitor CPCY-3 inhibits the response of the estrogen receptor positive breast cancer MCF-7 to estrogen, wherein A-C are the results of mRNAQ PCR detection of estrogen response genes TFF1 and GREB1, and PGR under the action of CPCY-3.

Detailed Description

The CY-1, CY-2, CY-3, CPCY-1, CPCY-2 and CPCY-3 related by the invention are synthesized by Kinsley.

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 a plurality of 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 cell HCT-116, human brain glioma U87 and cervical cancer cell Hela.

Tumor cells were treated at 37 ℃ with 5% CO₂The 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 10⁴Cell suspension was seeded into 96-well plates at 100. mu.L per well at 37 ℃ in 5% CO₂The 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 adherent, each dilution was added to a 96-well plate at 100 μ L per well. The SET inhibitor is added as a dosing group, Taxol is used as a positive control group, a culture solution without any drug is used as a blank control group, and the temperature is 37 ℃ and the CO content is 5 percent₂Incubate for 48 hours. mu.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 100 μ l of LDMSO was added per well to dissolve. Detecting at 570nm by using a microplate reader, measuring an absorbance value at a reference wavelength of 630nm, and calculating a growth inhibition ratio (PI), wherein the formula is as follows:

wherein N is_testTo test the OD value of the group, N_controlThe 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 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 1.

TABLE 1 IC50 values of CY-1, CY-2, CY-3, CPCY-1, CPCY-2, CPCY-3 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 cultured in endothelial cell culture medium containing 5% fetal bovine serum and 1 × ECGS at 37 deg.C with 5% CO₂When the mixture is cultured in an incubator to reach a confluency of more than 90 percent, a transwell method is adopted to detect the activity of the SET protein inhibitor for inhibiting the migration of endothelial cells, and the HUVEC of the endothelial cells only use the 2 nd to 8 th generations, and the specific operation is as follows:

(1) diluting with 10mg/mL Matrigel DMEM medium at a ratio of 1:4, coating on a transwell cell membrane, and air-drying at room temperature;

(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 a microscope, and adjusted to a cell concentration of 1X 10⁵Per mL;

(3) preparing test solutions for each group, and diluting the test solutions to 100 mu L by using 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: CPCY-3 is prepared into 0.25-4 mug/mL;

(4) cells were seeded into transwell chambers at 100 μ L per well and groups of test solutions were added to the chambers. The 24-well plate was supplemented with 0.6mL of endothelial cell culture medium containing 5% fetal bovine serum and 1 × ECGS to stimulate cell migration in 5% CO₂Incubating for 24h at 37 ℃;

(5) discarding culture solution in the hole, fixing with 90% alcohol at normal temperature for 30min, staining 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 fields to shoot and count, and calculating Migration Inhibition (MI) according to a formula:

wherein N is_testTo test the number of cells migrated in the group, N_controlCell 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 results of the transwell method for detecting the inhibition of CPCY-3 on migration of human umbilical vein endothelial cells

Example 3CPCY-3 conjugated FITC cell localization validation

Human breast cancer cells MCF-7 at 37 deg.C with 5% CO₂The 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 10⁴Cells per mL were seeded into 24-well plates at 400ul per well and cultured overnight. After incubating FITC-CPCY-3 with the cells at a final concentration of 10ug/ml for 1 hour, the green fluorescence position was observed under a fluorescence microscope. The experimental result is shown in figure 4, and 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 deg.C with 5% CO₂The 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 10⁴Cells 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) Repeating the operation on 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 results are shown in A-C of figure 5, and the expressions of estrogen response genes TFF1, GREB1, PGR and the like are obviously reduced after the gene is combined with CPCY-3, so that the response condition of the breast cancer MCF-7 positive to the estrogen receptor can be obviously inhibited.

Sequence listing

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Claims

1. 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, and n is more than or equal to 0.

2. The inhibitor of proto-oncoprotein SET of claim 1, wherein: the amino acid sequence is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.

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

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

5. Use of the proto-oncoprotein SET inhibitor according to claim 1 or the fusion polypeptide according to claim 3 or the kit according to claim 3 for the preparation of a medicament for the prevention and treatment of tumors.

6. Use according to claim 5, characterized in that: 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, prostatic cancer or testicular cancer.