CN109666064A - SALL4-RBBp4 complex blocks polypeptide and derivative antineoplastic polypeptide and its application - Google Patents
SALL4-RBBp4 complex blocks polypeptide and derivative antineoplastic polypeptide and its application Download PDFInfo
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- CN109666064A CN109666064A CN201811633492.2A CN201811633492A CN109666064A CN 109666064 A CN109666064 A CN 109666064A CN 201811633492 A CN201811633492 A CN 201811633492A CN 109666064 A CN109666064 A CN 109666064A
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- sall4
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- rbbp4
- antineoplastic
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- 150000001875 compounds Chemical class 0.000 claims abstract description 17
- 230000005764 inhibitory process Effects 0.000 claims abstract description 15
- 230000000259 anti-tumor effect Effects 0.000 claims abstract description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 10
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
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- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
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- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of polypeptide with targeted inhibition SALL4-RBBp4 compound Forming ability and antineoplastic polypeptide and its applications, the amino acid sequence of the polypeptide with targeted inhibition SALL4-RBBp4 compound Forming ability can be used for preparing anti-tumor drug as shown in SEQ ID NO:1；The antineoplastic polypeptide include targeted inhibition SALL4-RBBp4 compound formed structural domain and and wear spanning domain, as shown in SEQ ID NO:1, which can be used for preparing anti-tumor drug the domain amino acid sequence that the targeted inhibition SALL4-RBBp4 compound is formed.Antineoplastic polypeptide of the invention has significant anti-tumor activity, and the structure of cell-penetrating peptide domain of antineoplastic polypeptide itself is without cytotoxicity.Antineoplastic polypeptide of the invention does not have cytotoxicity to normal cell, can carry out adjuvant therapy treatment separately as anti-tumor drug or auxiliary other treatment mode.
Description
Technical field
The present invention relates to neoplasm targeted therapy fields, more specifically it relates to which a kind of have targeted inhibition SALL4-RBBp4 multiple
The polypeptide and a kind of antineoplastic polypeptide of conjunction object Forming ability and its application.
Background technique
Malignant tumour is the disease that second of research threatens human health after cardiovascular and cerebrovascular disease.According to world health group
Statistics is knitted, about 7,000,000 people of the annual whole world is because malignant tumour loses life, and tumor mortality number will persistently rise, it is contemplated that arrives
The whole world will be every year because the number of tumor mortality will be more than 13,000,000 the year two thousand thirty.
Currently, the treatment means for malignant tumour mainly have operative treatment, radiotherapy, chemotherapy, targeted therapy etc..Operation is cut
Except being one of most important means of oncotherapy, however the excision that can not perform the operation when many tumour discoveries.Radiation and chemotherapy can be shown
The progress for inhibiting tumour is write, however these treatment means often cause very big toxic side effect to body normal tissue.Tumour
Targeted therapy is specifically to select carcinogenic site to combine simultaneously using drug for explicitly carcinogenic shot design drug
It has an effect, keeps tumor cell specific dead.There is no corresponding carcinogenic target spot in usual normal tissue or cell, medicine is targeted
Object will not involve normal tissue or cell, thus targeted drug is significantly less than chemotherapeutics for the side effect of patient.In recent years
Targeted therapy is the hot spot means of oncotherapy, and designs and develops specific height, Small side effects, targeted drug significant in efficacy point
It is sub then be that neoplasm targeted therapy is successfully crucial.
Target polypeptide be a kind of molecular surface structures, amino acid according to target proteins form and surface charge etc. because
Element, simulated albumin-protein-interacting mode are designed the peptide molecule of optimization, can be with target proteins molecular specificity
Combination, thus influence target proteins normal function or realize targeted delivery function.Since such target polypeptide molecule has
There is high-affinity, highly selective, hypotoxicity, be readily synthesized, has become a kind of ideal targeted drug molecule, mesh
Previous conviction scholars have developed the novel targeted polypeptide drugs that can treat a variety of diseases also based on peptide molecule.
SALL4 is the core factor for maintaining stem cell versatility, specific expressed in fetal cell, in most of adults
Expression is lowered or is lacked in tissue.SALL4 and Oct4, Nanog and Sox2 form core transcription regulatory network, and driving embryo is dry thin
The self-renewing of born of the same parents.The transcriptional activity of SALL4 is to embryonic stem cell self-renewing and the transcription of differentiation associated gene is inhibited to have
There is double action.However, the study found that the expression of SALL4 activates again in some malignant tumours, and usually with prognosis
It is bad related.Research in liver cancer has shown that there are the excessive of SALL4 in tissue in up to 55% hepatocellular carcinoma (HCC) patient
Activation, and the transcriptional regulatory activity of SALL4 is exactly the crucial driving factors of hepatocellular carcinoma grade malignancy.Meanwhile research has shown that
SALL4 can be used as the biomarker of tumour ancestral cells.
Nucleosome remodeling deacetylase (NuRD) compound be chromatin remodeling attemperator, participate in embryonic stem cell and at
The silencing of many key regulatory genes in body cell.NuRD has two kinds of independent enzyme activity of ATPase and histone deacetylase,
Nucleosome is relocated by CDH3/4 ATPase subunit, so that histone deacetylase (HDAC1/2) subunit is close to target gene
Regulatory site in the genome simultaneously inhibits target gene.Retinoblastoma conjugated protein 4 (RBBP4) is the subunit of NURD.
It is a kind of protein containing WD40 repetitive sequence, the β propeller arrangement domain You Qiye composition.In NuRD, RBBp4 pass through by
Histone H 3 and H4 are integrated on the DNA newly replicated, play molecular chaperones in nucleosome assembling.The study found that SALL4
It plays a significant role in the recruitment building process of NuRD compound.In tumour cell, SALL4 passes through the phase interaction of RBBP4
Promote the progress of tumour to participate in the silencing of many tumor suppressor genes such as PTEN with NuRD compound is raised.Therefore, target is designed
The silencing shape of tumor suppressor gene is released by blocking the recruitment of NuRD to the peptide molecule for inhibiting SALL4-RBBp4 to interact
State is the excellent means for inhibiting tumour cell existence.
Efficient permeable membrane efficiency is one of the key condition that peptide molecule inhibits target spot intracellular, and cell-penetrating peptide is then fabulous
One of membrane carrier.Cell-penetrating peptide be one kind have carry positive charge small peptide, have very strong penetrating cell film ability, can directly into
Enter cytoplasm and nucleus, can be used as carrier and carry drug progress cell.The toxic side effect very little of cell-penetrating peptide normal tissue, because
It is the antitumor of a great prospect that this carries SALL4-RBBp4 target polypeptide molecule to carry out polypeptide drugs design using cell-penetrating peptide
The direction of targeted drug exploitation.
Summary of the invention
The present invention devises a kind of octapeptide small molecule of targeted inhibition SALL4-RBBp4 interaction, using cell-penetrating peptide and
Octapeptide small molecule carries out covalent linkage, obtains a kind of ten nonapeptide drug molecules, has efficient permeable membrane efficiency and antitumor work
Property.
The present invention provides a kind of polypeptide with targeted inhibition SALL4-RBBp4 compound Forming ability, amino acid
Sequence is as shown in SEQ ID NO:1.
The present invention also provides the polypeptide of above-mentioned targeting SALL4-RBBp4 compound answering in the preparation of antitumor drugs
With.
The present invention also provides a kind of antineoplastic polypeptides comprising the knot that targeted inhibition SALL4-RBBp4 compound is formed
Structure domain and and wear spanning domain, the domain amino acid sequence such as SEQ that the targeted inhibition SALL4-RBBp4 compound is formed
Shown in ID NO:1.
Preferably, the spanning domain amino acid sequence of wearing is as shown in SEQ ID NO:2.
Preferably, amino acid sequence is as shown in SEQ ID NO:3.
Preferably, the structure of cell-penetrating peptide is located at the N-terminal of the antineoplastic polypeptide, the targeted inhibition SALL4-RBBp4
The structural domain that compound is formed is located at the C-terminal of the antineoplastic polypeptide.
The present invention also provides above-mentioned antineoplastic polypeptide application in preparations of anti-tumor drugs.
It is an advantage of the current invention that antineoplastic polypeptide of the invention is established in kinds of tumor cells system, tumor cell line
Have significant anti-tumor activity in CDX mouse model and PDX mouse model, and the structure of cell-penetrating peptide domain sheet of antineoplastic polypeptide
Body does not have cytotoxicity.Antineoplastic polypeptide of the invention does not have cytotoxicity to normal cell, can be separately as antineoplastic
Object or auxiliary other treatment mode carry out adjuvant therapy treatment.
Detailed description of the invention
Fig. 1 is the relative quantitative assay system that SALL4 is expressed in the human normal cell line and tumour cell of different tissue sources
Meter figure.
Fig. 2 is the fluorescent microscopy images for wearing membrane efficiency and inner cellular localization detection of polypeptide.
Fig. 3 is polypeptide for the human normal cell line of different tissue sources and the cytotoxicity analysis statistical chart of tumour cell.
Fig. 4 is polypeptide through the statistical chart that antitumous effect is analyzed in the CDX model of huh7 cell construction is injected intraperitoneally,
A left side is tumor growth curve, and the right side is tumour weight in wet base.
Fig. 5 is polypeptide through the statistics that antitumous effect is analyzed in the CDX model of MGC-803 cell construction is injected intraperitoneally
Figure, a left side are tumor growth curve, and the right side is tumour weight in wet base.
Specific embodiment
It is explained further the present invention with reference to embodiments, but embodiment does not do any type of limit to the present invention
It is fixed.
Embodiment one: peptide molecule design and synthesis
SALL4 structure is predicted by bioinformatic analysis, in conjunction with the crystal analytic structure information of known RBBp4 albumen, is utilized
Computer molecular docking software analyzes the binding structural domain and critical amino acid residues of SALL4-RBBp4, then utilizes polypeptide-egg
The analysis of white molecular docking software, design high-affinity combination RBBp4 albumen and can block the more of SALL4-RBBp4 interaction
Peptide molecule, the polypeptid acid sequence through the selectively targeted combination RBBp4 of screening acquisition is as shown in SEQ ID NO:1, hereinafter referred to as
Targeting peptides.
A kind of cell-penetrating peptide is screened, amino acid sequence is as shown in SEQ ID NO:2, for guiding above-mentioned targeting peptides efficient
Enter endochylema and nucleus.A kind of natineoplaston, including cell-penetrating peptide sequence and targeting peptide sequence are designed, wherein cell-penetrating peptide is located at
N-terminal, targeting peptides are located at C-terminal, cell-penetrating peptide and targeting peptides by covalent linkage, and natineoplaston sequence is as shown in SEQ ID NO:3.
Above-mentioned cell-penetrating peptide and natineoplaston trust money Si Rui Biotechnology Co., Ltd are carried out using the method for synthesis in solid state
Peptide systhesis, purity >=95%, C-terminal carry out fluorescein isothiocynate (FITC) label, and wherein cell-penetrating peptide is as negative control.
The verifying of two: SALL4-RBBp4 expression specificity of embodiment
1. buying adult primary cell, including Human Cardiomyocytes, human chondrocytes, application on human skin capilary from PromoCell company
Endothelial cell, human mammary epithelial cell, human lung cancer cell A549, human liver cell, human pulmonary artery smooth muscle cells, people's skeletonization are thin
Born of the same parents, human keratinocytes utilize corresponding condition of culture culture cell.
2. tumour cells such as recovery culture hepG2, HuH7, Ishikawa, HT29, Caco2, MGC-803, MKN45.
3. extracting total serum IgE from above-mentioned cell using Trizol method, and it is reversed to cDNA.
4. the fluorescence quantification PCR primer of the consensus sequence design SALL4 gene for two transcripts of SALL4, sequence
It is as follows:；ACTB gene is reference gene, primer are as follows: F:5 '-GGCACTCTTCCAGCCTTCC-3 ', R:5 '-
GAGCCGCCGATCCACAC-3´。
5. carrying out fluorescence quantitative PCR detection to the expression of SALL4 in above-mentioned cell using SYBR Green method, △ △ is utilized
Ct method carries out relative quantitative assay, verifying to the expression of SALL4 in above-mentioned adult normal tissue primary cell and tumour cell
Expression specificity of the SALL4 in malignant cell.
Testing result is as shown in Figure 1.
Embodiment three: polypeptide wears film and cellular localization detection
1. recovery culture HuH7, HT29, Caco2, MGC-803, MKN45, people's liver primary cell.
2. above-mentioned 6 plants of cells are seeded to 96 orifice plates, every plant of 3 multiple holes of cell inoculation by suitable cell number.
3. after cell inoculation 6 hours, subject polypeptide, final concentration 30uM is added.
4. recording cell FITC fluorescence respectively for 24 hours the 0th, 0.5,2,4,8 of polypeptide processing the, polypeptide permeable membrane efficiency is judged
And inner cellular localization.
Testing result show polypeptide can efficient penetrating cell film, inner cellular localization is in endochylema and karyon.Wherein by MGC-803
For cell as cell is represented, permeable membrane and inner cellular localization testing result are as shown in Figure 2.
Example IV: polypeptide anti tumor activity in vitro detection
1. on recovery people's primary cell, including Human Cardiomyocytes, human chondrocytes, application on human skin microvascular endothelial cells, human milk gland
Chrotoplast, human lung cancer cell A549, human liver cell, human pulmonary artery smooth muscle cells, human osteoblast cell, application on human skin cutin are formed carefully
Born of the same parents utilize corresponding condition of culture culture cell.
2. tumour cells such as recovery culture hepG2, HuH7, Ishikawa, HT29, Caco2, MGC-803, MKN45.
3. above-mentioned cell is seeded to 96 orifice plates by 3000 holes cells/.
4. after cell inoculation 6 hours, subject polypeptide, final concentration 30uM is added.
5. for 24 hours, 48h is utilized respectively the activity that CCK8 detects each cell in the 0th, 4h, 8h of polypeptide processing, polypeptide is assessed
For the cytotoxicity of normal cell and tumour cell.
Testing result shows cell-penetrating peptide control to cell without significant toxicity.Antineoplastic polypeptide is to people's Normal primary cell without aobvious
Toxicity is write, and the tumour cell positive to SALL4 expression shows significant cytotoxicity.Testing result is as shown in Figure 3.
Embodiment five: anti-tumor activity detects in polypeptide body
1. the representative cell line verified using HuH7, MGC-803 cell as anti-tumor activity in polypeptide body, recovery cell, by phase
Condition of culture is answered, at 37 DEG C, 5% CO2Incubator culture.
2. 18-22g female BAl BIc/c nude mouse, adaptive feeding 1 week.Two batches are randomly divided into, wherein a batch inoculates
HuH7 cell (total number of cells 107, volume 100ul), another batch of inoculation MGC-803 cell (total number of cells 107, volume 100ul),
Inoculation position is at nude mice right hind back.HuH7 cell CDX model and MGC-803 cell CDX model are constructed respectively.
3. observation tumour growth situation and tumor size daily, (V=0.5 × a × b2, wherein a is indicated measurement gross tumor volume
Tumour major diameter, b indicate tumour minor axis)；As gross tumor volume >=50mm3, by HuH7 cell CDX model and MGC-803 cell CDX
Model is randomly divided into cell-penetrating peptide control intervention group and natineoplaston intervention group respectively.
4. cell-penetrating peptide compare intervention group using cell-penetrating peptide according to 15.6mg/kg dosage progress intraperitoneal injection, every 2 days
It is administered once, amounts to administration 5 times；Natineoplaston intervention group carries out abdominal cavity note according to the dosage of 26.1mg/kg using natineoplaston
Administration is penetrated, is administered once within every 2 days, administration 5 times is amounted to；Since injecting virus for the first time, every 3 days measurement gross tumor volumes, according to swollen
Knurl product size draws the growth curve of each group nude mouse tumor；Last time administration put to death experimental animal after 3 days, took out tumour
Tissue measures tumour weight in wet base；Polypeptide antitumous effect is assessed according to tumor growth curve and weight in wet base.
Testing result show cell-penetrating peptide control without significant anti-tumor activity, and natineoplaston then show it is significant antitumor
Effect, tumor growth curve and the comparison of tumour weight in wet base are as illustrated in figures 4-5.
Embodiment described above only describe the preferred embodiments of the invention, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those skilled in the art are to technical solution of the present invention institute
The various changes and improvements of work should all be fallen within the protection scope that claims of the present invention determines.
<110>Shanghai Rui Sai Bioisystech Co., Ltd
<120>SALL4-RBBp4 complex blocks polypeptide and derivative antineoplastic polypeptide and its application
<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 (7)
1. a kind of polypeptide with targeted inhibition SALL4-RBBp4 compound Forming ability, which is characterized in that amino acid sequence is such as
Shown in SEQ ID NO:1.
2. the polypeptide described in claim 1 with targeted inhibition SALL4-RBBp4 compound Forming ability prepare it is antitumor
Application in drug.
3. a kind of antineoplastic polypeptide, which is characterized in that the block function knot formed including targeted inhibition SALL4-RBBp4 compound
Structure domain and transmembrane ability structural domain, the block function domain amino acid that the targeted inhibition SALL4-RBBp4 compound is formed
Sequence is as shown in SEQ ID NO:1.
4. antineoplastic polypeptide according to claim 3, which is characterized in that the transmembrane ability domain amino acid sequence is such as
Shown in SEQ ID NO:2.
5. antineoplastic polypeptide according to claim 3, which is characterized in that the amino acid sequence of antineoplastic polypeptide such as SEQ ID
Shown in NO:3.
6. the antineoplastic polypeptide according to any one of claim 3, which is characterized in that the transmembrane ability structural domain is located at
The N-terminal of the antineoplastic polypeptide, the block function structural domain that the targeted inhibition SALL4-RBBp4 compound is formed are located at described
The C-terminal of antineoplastic polypeptide.
7. antineoplastic polypeptide application in preparation of anti-tumor drugs described in any one of claim 3-6.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113683703A (en) * | 2020-05-18 | 2021-11-23 | 中国人民解放军陆军军医大学第二附属医院 | hTERT target substance and application thereof |
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| 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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2018
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| CN103945860A (en) * | 2011-09-20 | 2014-07-23 | 布莱根妇女医院 | SALL4 and uses thereof |
| US20150080315A1 (en) * | 2011-09-20 | 2015-03-19 | Brigham And Women's Hospital | 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 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113683703A (en) * | 2020-05-18 | 2021-11-23 | 中国人民解放军陆军军医大学第二附属医院 | hTERT target substance and application thereof |
| CN113683703B (en) * | 2020-05-18 | 2023-08-15 | 中国人民解放军陆军军医大学第二附属医院 | hTERT target and application thereof |
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Address after: 201315 room 1701, 25 building, 3399 lane, Kang Xin Road, Pudong New Area, Shanghai. Patentee after: Shanghai Ruisai Bio-Technology Co.,Ltd. Address before: 201315 room 1701, 25 building, 3399 lane, Kang Xin Road, Pudong New Area, Shanghai. Patentee before: SHANGHAI R&S BIOTECHNOLOGY CO.,LTD. |