CN109666064B - SALL4-RBBp4 compound blocking polypeptide and derivative antitumor polypeptide and application thereof - Google Patents
SALL4-RBBp4 compound blocking polypeptide and derivative antitumor polypeptide and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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- A—HUMAN NECESSITIES
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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Abstract
The invention relates to a polypeptide with the ability of targeted inhibition of SALL4-RBBp4 complex formation, an antitumor polypeptide and application thereof, wherein the amino acid sequence of the polypeptide with the ability of targeted inhibition of SALL4-RBBp4 complex formation is shown in SEQ ID NO. 1, and the polypeptide can be used for preparing antitumor drugs; the anti-tumor polypeptide comprises a structural domain for targeted inhibition of SALL4-RBBp4 complex formation and a transmembrane structural domain, the amino acid sequence of the structural domain for targeted inhibition of SALL4-RBBp4 complex formation is shown in SEQ ID NO:1, and the anti-tumor polypeptide can be used for preparing anti-tumor drugs. The anti-tumor polypeptide has obvious anti-tumor activity, and the cell-penetrating peptide structure domain of the anti-tumor polypeptide has no cytotoxicity. The anti-tumor polypeptide has no cytotoxicity to normal cells, and can be independently used as an anti-tumor medicament or used for assisting other treatment modes to perform auxiliary tumor treatment.
Description
Technical Field
The invention relates to the field of tumor targeted therapy, in particular to a polypeptide with the ability of targeted inhibition of SALL4-RBBp4 complex formation, an anti-tumor polypeptide and application thereof.
Background
Malignant tumors are a disease that, following cardiovascular and cerebrovascular diseases, threatens human health in the second study. According to the world health organization, about 700 million people per year worldwide lose lives due to malignant tumors, and the number of tumor deaths will continue to rise, and more than 1300 million people per year worldwide by 2030 is expected.
At present, the treatment means aiming at malignant tumors mainly comprise surgical treatment, radiotherapy, chemotherapy, targeted treatment and the like. Surgical resection is one of the most important means for tumor treatment, however, many tumors cannot be removed by surgery when they are found. Radiotherapy and chemotherapy can significantly inhibit the progression of tumors, however, these treatments often cause significant toxic and side effects on normal tissues of the body. The tumor targeted therapy is to design a drug aiming at a definite carcinogenic target, and the drug is utilized to specifically select a carcinogenic site to combine and act so as to lead tumor cells to be specifically dead. Usually, no corresponding carcinogenic target exists in normal tissues or cells, and the targeted drug does not spread to the normal tissues or cells, so that the side effect of the targeted drug on patients is obviously smaller than that of chemotherapeutic drugs. In recent years, targeted therapy is a hotspot means of tumor therapy, and the design and development of targeted drug molecules with high specificity, small side effect and remarkable curative effect is the key to the success of tumor targeted therapy.
The targeted polypeptide is a polypeptide molecule which is designed and optimized by simulating a protein-protein interaction mode according to factors such as a molecular surface structure, amino acid composition, surface charge and the like of a target protein, and can be specifically combined with the target protein molecule, so that the normal function of the target protein is influenced or the targeted delivery function is realized. Because the targeted polypeptide molecules have the advantages of high affinity, high selectivity, low toxicity, easy synthesis and the like, the targeted polypeptide molecules become ideal targeted drug molecules, and scientists develop novel targeted polypeptide drugs capable of treating various diseases based on the polypeptide molecules.
SALL4 is a core factor that maintains stem cell pluripotency, is specifically expressed in fetal cells, and is down-regulated or deleted in most adult tissues. SALL4 forms a core transcriptional regulatory network with Oct4, Nanog and Sox2, driving the self-renewal of embryonic stem cells. The transcriptional activity of SALL4 has dual roles in embryonic stem cell self-renewal and in inhibiting the transcription of differentiation-related genes. However, it has been found that in some malignancies the expression of SALL4 is reactivated and often associated with a poor prognosis. Studies in liver cancer have demonstrated that up to 55% of hepatocellular carcinoma (HCC) patients have hyperactivation of SALL4 in tissues, while transcriptional regulatory activity of SALL4 is a key driver of the malignancy of hepatocellular carcinomas. Meanwhile, the study proves that SALL4 can be used as a biomarker of tumor stem/progenitor cells.
Nucleosome remodeling deacetylase (NuRD) complexes are regulators of chromatin remodeling and are involved in the silencing of many key regulatory genes in embryonic stem cells and adult cells. NuRD has two independent enzyme activities of ATPase and histone deacetylase, and nucleosomes are relocated by CDH3/4 ATPase subunits so that histone deacetylase (HDAC 1/2) subunits approach the regulatory site of a target gene on a genome and inhibit the target gene. Retinoblastoma binding protein 4 (RBBP 4) is a subunit of NURD. It is a protein containing the WD40 repeat sequence, consisting of a heptalobal beta-propeller domain. In NuRD, RBBp4 functions as a chaperone in nucleosome assembly by binding histones H3 and H4 to newly replicated DNA. It was found that SALL4 plays an important role in the recruitment and organization of NuRD complexes. In tumor cells, SALL4 recruits NuRD complex through RBBP4 interaction, thereby participating in the silencing of numerous tumor suppressor genes, such as PTEN, and promoting tumor progression. Therefore, designing a polypeptide molecule targeted to inhibit the SALL4-RBBp4 interaction, through blocking the recruitment of NuRD, relieves the silent state of the cancer suppressor gene, and is an excellent way to inhibit the survival of tumor cells.
Efficient membrane penetration efficiency is one of the key conditions for polypeptide molecules to inhibit intracellular targets, and the membrane-penetrating peptide is one of the excellent membrane-penetrating carriers. The cell-penetrating peptide is a short peptide with positive charge, has strong capability of penetrating cell membranes, can directly enter cytoplasm and cell nucleus, and can be used as a carrier to carry drugs to carry cells. The cell-penetrating peptide has little toxic and side effect on normal tissues, so that the polypeptide drug design by utilizing the cell-penetrating peptide to carry SALL4-RBBp4 targeted polypeptide molecules is a promising direction for developing antitumor targeted drugs.
Disclosure of Invention
The invention designs an octapeptide small molecule for targeted inhibition of SALL4-RBBp4 interaction, and utilizes the cell-penetrating peptide and the octapeptide small molecule to carry out covalent bond linkage to obtain a nineteen-peptide drug molecule, which has high-efficiency membrane penetration efficiency and antitumor activity.
The invention provides a polypeptide with the ability of targeted inhibition of SALL4-RBBp4 complex formation, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.
The invention also provides application of the polypeptide targeting the SALL4-RBBp4 complex in preparation of antitumor drugs.
The invention also provides an antitumor polypeptide which comprises a structural domain for targeted inhibition of SALL4-RBBp4 complex formation and a transmembrane domain, wherein the amino acid sequence of the structural domain for targeted inhibition of SALL4-RBBp4 complex formation is shown as SEQ ID NO. 1.
Preferably, the amino acid sequence of the transmembrane domain is shown as SEQ ID NO. 2.
Preferably, the amino acid sequence is shown in SEQ ID NO 3.
Preferably, the penetrating peptide structure is positioned at the N terminal of the anti-tumor polypeptide, and the domain for targeted inhibition of the formation of the SALL4-RBBp4 complex is positioned at the C terminal of the anti-tumor polypeptide.
The invention also provides the application of the anti-tumor polypeptide in preparing anti-tumor medicaments.
The invention has the advantages that the anti-tumor polypeptide has obvious anti-tumor activity in a CDX mouse model and a PDX mouse model established by a plurality of tumor cell lines and tumor cell lines, and the transmembrane peptide structural domain of the anti-tumor polypeptide has no cytotoxicity. The anti-tumor polypeptide has no cytotoxicity to normal cells, and can be independently used as an anti-tumor medicament or used for assisting other treatment modes to perform auxiliary tumor treatment.
Drawings
FIG. 1 is a statistical plot of the relative quantification of SALL4 expression in human normal and tumor cells of different tissue origins.
FIG. 2 is a fluorescent microscope photograph of the transmembrane efficiency and intracellular localization detection of polypeptides.
FIG. 3 is a statistic chart of cytotoxicity analysis of polypeptides on human normal cells and tumor cells of different tissue origins.
FIG. 4 is a statistical chart of the analysis of the anti-tumor effect of polypeptides in vivo by intraperitoneal injection into CDX models constructed from huh7 cells, with the left being the tumor growth curve and the right being the tumor wet weight.
FIG. 5 is a statistical chart of the analysis of the anti-tumor effect of polypeptides in vivo by intraperitoneal injection into a CDX model constructed from MGC-803 cells, with the left being the tumor growth curve and the right being the tumor wet weight.
Detailed Description
The present invention is further explained with reference to the following examples, which are not intended to limit the present invention in any way.
The first embodiment is as follows: polypeptide molecule design and synthesis
The structure of SALL4 is predicted through bioinformatics analysis, crystal analysis structure information of known RBBp4 protein is combined, computer molecular docking software is used for analyzing a binding domain and key amino acid residues of SALL4-RBBp4, polypeptide molecules capable of binding with high affinity to RBBp4 protein and blocking SALL4-RBBp4 interaction are designed through polypeptide-protein molecular docking software analysis, and polypeptide amino acid sequences specifically targeting to bind to RBBp4 are obtained through screening and are shown as SEQ ID NO:1 and are called targeting peptides in the following.
The amino acid sequence of the screened cell-penetrating peptide is shown as SEQ ID NO. 2, and the cell-penetrating peptide can be used for guiding the targeted peptide to enter cytoplasm and cell nucleus efficiently. Designing an anti-tumor peptide, which comprises a cell-penetrating peptide sequence and a targeting peptide sequence, wherein the cell-penetrating peptide is positioned at the N end, the targeting peptide is positioned at the C end, the cell-penetrating peptide and the targeting peptide are linked through a covalent bond, and the anti-tumor peptide sequence is shown as SEQ ID NO 3.
The cell-penetrating peptide and the anti-tumor peptide are consigned to Kingsler Biotechnology Limited to perform polypeptide synthesis by a solid-phase synthesis method, the purity is more than or equal to 95 percent, Fluorescein Isothiocyanate (FITC) labeling is performed at the C terminal, and the cell-penetrating peptide is used as a negative control.
Example two: SALL4-RBBp4 expression specificity verification
1. Adult primary cells including human cardiac myocytes, human chondrocytes, human skin microvascular endothelial cells, human mammary epithelial cells, human lung fibroblasts, human hepatocytes, human pulmonary artery smooth muscle cells, human osteoblasts, human skin keratinocytes were purchased from PromoCell corporation and cultured using the corresponding culture conditions.
2. And (3) recovering and culturing the tumor cells such as hepG2, HuH7, Ishikawa, HT29, Caco2, MGC-803, MKN45 and the like.
3. Total RNA was extracted from the above cells by Trizol method and inverted to cDNA.
4. Fluorescent quantitative PCR primers for the SALL4 gene were designed against the consensus sequence of the two transcripts of SALL4, as follows: (ii) a The ACTB gene is used as an internal reference gene, and the primers are as follows: f: 5 '-GGCACTCTTCCAGCCTTCC-3', R: 5 '-GAGCCGCCGATCCACAC-3'.
5. And performing fluorescent quantitative PCR detection on the expression of SALL4 in the cells by using a SYBR Green method, and performing relative quantitative analysis on the expression of SALL4 in the primary cells of the adult normal tissues and the tumor cells by using a delta-delta Ct method to verify the expression specificity of SALL4 in the malignant tumor cells.
The results of the detection are shown in FIG. 1.
Example three: polypeptide transmembrane and cell localization assay
1. Recovering and culturing HuH7, HT29, Caco2, MGC-803, MKN45 and primary human liver cells.
2. The 6 cells were inoculated into a 96-well plate at an appropriate cell number, and 3 wells were inoculated per cell.
3. 6 hours after cell inoculation, the test polypeptide was added to a final concentration of 30 uM.
4. Cell FITC fluorescence was recorded at 0, 0.5, 2, 4, 8, 24h of polypeptide treatment, and polypeptide transmembrane efficiency and intracellular localization were assessed.
The detection result shows that the polypeptide can efficiently penetrate cell membranes and is positioned in cytoplasm and nucleus. The results of detection of transmembrane and intracellular localization using MGC-803 cells as representative cells are shown in FIG. 2.
Example four: polypeptide in vitro antitumor activity detection
1. Reviving human primary cells including human cardiac muscle cells, human cartilage cells, human skin microvascular endothelial cells, human mammary epithelial cells, human lung fibroblasts, human liver cells, human pulmonary artery smooth muscle cells, human osteoblasts and human skin keratinocytes, and culturing the cells by using corresponding culture conditions.
2. And (3) recovering and culturing the tumor cells such as hepG2, HuH7, Ishikawa, HT29, Caco2, MGC-803, MKN45 and the like.
3. The cells were seeded at 3000 cells/well in 96-well plates.
4. 6 hours after cell inoculation, the test polypeptide was added to a final concentration of 30 uM.
5. The activity of each cell was measured at 0, 4h, 8h, 24h, and 48h of the polypeptide treatment using CCK8, respectively, to evaluate the cytotoxicity of the polypeptide against normal cells and tumor cells.
The detection result shows that the cell-penetrating peptide control has no obvious toxicity to cells. The antitumor polypeptide has no obvious toxicity to normal primary human cells, and shows obvious cytotoxicity to tumor cells with positive SALL4 expression. The results of the detection are shown in FIG. 3.
Example five: polypeptide in vivo antitumor activity detection
1. HuH7 and MGC-803 cells are taken as representative cell lines for verifying the in vivo anti-tumor activity of the polypeptide, the cells are recovered and cultured at 37 ℃ and 5% CO according to corresponding culture conditions2Culturing in an incubator.
2.18-22 g female BALB/c nude mice, acclimatized for 1 week. Randomly divided into two batches, one of which was inoculated subcutaneously with HuH7 cells (total cell number 10)7100ul volume), another batch was seeded with MGC-803 cells (total number of cells 10)7Volume 100ul), the inoculation site was on the back of the right hind limb of nude mice. A CDX model of HuH7 cells and a CDX model of MGC-803 cells were constructed separately.
3. Tumor growth and tumor size were observed daily and tumor volume was determined (V =0.5 × a × b2, where a represents the tumor major diameter and b represents the tumor minor diameter); when the tumor volume is more than or equal to 50mm3, the HuH7 cell CDX model and the MGC-803 cell CDX model are respectively randomly divided into a cell-penetrating peptide control stem prediction group and an anti-tumor peptide stem prediction group.
4. The cell-penetrating peptide control intervention group carries out intraperitoneal injection administration by using the cell-penetrating peptide according to the dose of 15.6mg/kg, the administration is carried out once every 2 days, and the administration is carried out for 5 times in total; the anti-tumor peptide intervention group carries out intraperitoneal injection administration by using anti-tumor peptide according to the dose of 26.1mg/kg, the administration is carried out once every 2 days, and the administration is carried out for 5 times in total; measuring the tumor volume every 3 days from the first virus injection, and drawing the growth curve of the tumors of each group of nude mice according to the tumor volume; the experimental animals were sacrificed 3 days after the last administration, tumor tissues were taken out, and tumor wet weights were measured; and evaluating the anti-tumor effect of the polypeptide according to the tumor growth curve and the wet weight.
The detection result shows that the cell-penetrating peptide control has no obvious anti-tumor activity, while the anti-tumor peptide shows obvious anti-tumor effect, and the tumor growth curve and the wet weight of the tumor are shown in the figures 4-5.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
<110> Shanghai Ruishi Biotechnology Ltd
<120> SALL4-RBBp4 compound blocking polypeptide and derivative antitumor polypeptide and application thereof
<130> AJ181881
<160> 3
<210> 1
<211> 8
<212> PRT
<213> Artificial sequence
<400> 1
Lys Phe Ala Lys Phe Gln Trp Ile
1 5
<210> 2
<211> 11
<212> PRT
<213> Artificial sequence
<400> 2
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
<210> 3
<211> 19
<212> PRT
<213> Artificial sequence
<400> 3
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys Phe Ala Lys
1 5 10 15
Phe Gln Trp Ile
16
Claims (5)
1. A polypeptide with the capability of targeted inhibition of SALL4-RBBp4 complex formation is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. An antitumor polypeptide, which is characterized by comprising a blocking functional domain and a transmembrane functional domain for targeted inhibition of SALL4-RBBp4 complex formation, wherein the amino acid sequence of the blocking functional domain for targeted inhibition of SALL4-RBBp4 complex formation is shown as SEQ ID NO. 1.
3. The anti-tumor polypeptide of claim 2, wherein the amino acid sequence of the anti-tumor polypeptide is shown in SEQ ID NO. 3.
4. The anti-tumor polypeptide of claim 2, wherein the transmembrane functional domain is located at the N-terminus of the anti-tumor polypeptide, and the blocking functional domain that targets the inhibition of the formation of the SALL4-RBBp4 complex is located at the C-terminus of the anti-tumor polypeptide.
5. The use of the antitumor polypeptide of claim 3 in the preparation of a liver cancer antitumor drug and a gastric cancer antitumor drug.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103945860A (en) * | 2011-09-20 | 2014-07-23 | 布莱根妇女医院 | SALL4 and uses thereof |
| CN106699850A (en) * | 2017-02-22 | 2017-05-24 | 华中科技大学同济医学院附属协和医院 | RBBP4 targeting polypeptide and anti-tumor polypeptide, and applications thereof |
| WO2017190032A2 (en) * | 2016-04-28 | 2017-11-02 | National University Of Singapore | Therapeutic sall4 peptide |
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|---|---|---|---|---|
| CN103945860A (en) * | 2011-09-20 | 2014-07-23 | 布莱根妇女医院 | SALL4 and uses thereof |
| WO2017190032A2 (en) * | 2016-04-28 | 2017-11-02 | National University Of Singapore | Therapeutic sall4 peptide |
| CN106699850A (en) * | 2017-02-22 | 2017-05-24 | 华中科技大学同济医学院附属协和医院 | RBBP4 targeting polypeptide and anti-tumor polypeptide, and applications thereof |
Non-Patent Citations (2)
| Title |
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| "SALL4在肿瘤中的作用及临床应用价值";张鹏等;《临床检验杂志》;20180228;第36卷(第2期);第130-132页 * |
| "Towards elucidating the stability, dynamics and architecture of the nucleosome remodeling and deacetylase complex by using quantitative interaction proteomics";Susan L Kloet等;《FEBS J》;20140911;第282卷(第9期);第1774-1785页 * |
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