CN110229233A - A kind of SLFN11 truncated peptide and its application and pharmaceutical composition with sensitization - Google Patents
A kind of SLFN11 truncated peptide and its application and pharmaceutical composition with sensitization Download PDFInfo
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- CN110229233A CN110229233A CN201910434838.4A CN201910434838A CN110229233A CN 110229233 A CN110229233 A CN 110229233A CN 201910434838 A CN201910434838 A CN 201910434838A CN 110229233 A CN110229233 A CN 110229233A
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- Prior art keywords
- slfn11
- peptide
- truncated peptide
- truncated
- sensitization
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- 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
Abstract
The SLFN11 truncated peptide and its application and pharmaceutical composition that the invention discloses a kind of with sensitization, amino acid sequence is as shown in SEQ.ID.NO.1.The truncated peptide is by carrying out modification acquisition using Tat penetrating peptide to the hydrophilic area SLFN11 peptide fragment.SLFN11 truncated peptide of the present invention can pass through cell membrane and enter cell interior, improve the level of the SLFN11 of tumour cell, enhance tumour cell to the sensibility of chemicotherapy, so as to be used for the treatment of tumour as chemotherapeutic sensitizer.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of SLFN11 truncated peptide with sensitization, this
Invention further relates to the application and pharmaceutical composition of above-mentioned SLFN11 truncated peptide.
Background technique
The morbidity and mortality of cancer are only second to cardiovascular disease, bring heavy economy and essence to society and family
Mind burden.In the world, about 14,100,000 new cases of cancer every year.With the acceleration of global aging, cancer will
As one main health problem of the mankind, a kind of world's disease seriously threatening human life.
Chemotherapy is one of most effective means of current treating cancer.However, long-term or largely thin using chemotherapeutics cancer
There may be drug resistance, chemotherapy can even enhance the ability of invasion and the transfer of residual cancer cell by born of the same parents.High-dose chemotherapy is to body simultaneously
The dosage that the sensibility brought very big side effect, therefore increase chemotherapy reduces chemotherapy simultaneously is very necessary.
Sleep gene protein (Schlafen family, SLFN) family is developing, and plays in immune response and cell Proliferation
Key effect.SLFN11 albumen is located in nucleus, is one of the SLFN protein family member only expressed in mammals.
The expression and tumour cell of SLFN11 is positively correlated to the sensibility of DNA damage agent.After DNA is damaged, replication protein
A1 (replication protein A1, RPA1) and damage dna, which combine, promotes DNA recombinantal repair.It is highly expressed in SLFN11
In tumour cell, and SLFN11 can form heterodimer in conjunction with RPA1, and RPA1 is caused to dissociate from DNA, inhibit damage dna weight
Group is repaired, and then promotes Apoptosis and death.High-level SLFN11 can not only promote tumour cell to I/II type topoisomerase
Enzyme inhibitor, the sensitivity of DNA synthetic inhibitor and alkylating agent, can also allow cell to both damages of IR and UV processing be also in
Reveal more sensitive phenotype.Therefore, the level of the SLFN11 of tumour cell can be improved in external source SLFN11, enhances tumour cell
To the sensibility of chemicotherapy, to be used for the treatment of tumour as chemotherapeutic sensitizer.
Since protein exhibits effect generally shows some specific function key areas, by these specific functional areas peptide fragments
It is cut into form function truncated peptide, can show compared with its stronger bioactivity of female albumen.These truncated peptides have following spy
The features such as point: molecular weight is small, and expression quantity is high, and active structure domain can sufficiently show, simultaneously as certain amino acid are out of script
It is exposed in portion's structure, and truncation peptide fragment is made to play new effect.
However, exogenous truncated peptide, which is difficult to cell membrane, enters cell interior.Research confirms that one kind is exempted from from human I type
The egg of the transcriptional transactivator (trans-activator oftranscription, TAT) of epidemic disease defective virus (HIV-I)
White transduction domain (protein transduction domain, PTD), can be by molecular weight in the different biological big of 15-120kD
Molecule (such as nucleic acid, more skins, protein) is efficient, quickly and safely imports cell, and still has bioactivity after importing, and
To host cell almost without toxicity.The protein therapeutic that is found to be in TAT protein transduction domain brings new dawn, shows huge
Big application value.
Summary of the invention
The object of the present invention is to provide a kind of SLFN11 truncated peptide with sensitization, solves existing external source SLFN11
It is difficult to the problem of cell membrane enters cell interior.
It is a further object of the present invention to provide the applications of above-mentioned SLFN11 truncated peptide.
Third object of the present invention is to provide a kind of pharmaceutical compositions.
The technical scheme adopted by the invention is that a kind of SLFN11 truncated peptide its amino acid sequence with sensitization is such as
Shown in SEQ.ID.NO.1.
The features of the present invention also characterized in that
SLFN11 truncated peptide is by carrying out modification acquisition using Tat penetrating peptide to the hydrophilic area SLFN11 peptide fragment.
The hydrophilic area SLFN11 peptide fragment is the 634-661 bit sequence of SLFN11.
It is a kind of application of the SLFN11 truncated peptide with sensitization that the present invention, which uses another technical solution,
SLFN11 truncated peptide is to the application in cellular antiproliferative active medicine.
The features of the present invention also characterized in that
Application of the SLFN11 truncated peptide in tumor chemoradiotherapy drug.
Third technical solution of the present invention is a kind of pharmaceutical composition, wherein containing SLFN11 truncated peptide and
Pharmaceutically acceptable carrier.
Beneficial effects of the present invention are that a kind of SLFN11 truncated peptide with sensitization of the present invention passes through to SLFN11's
Amino acid pro pool section, carries out modification acquisition using Tat penetrating peptide, can pass through cell membrane and enter cell interior, improve
The level of the SLFN11 of tumour cell enhances tumour cell to the sensibility of chemicotherapy, so as to use as chemotherapeutic sensitizer
In the treatment of tumour.
Detailed description of the invention
Fig. 1 is that mtt assay detects truncated peptide to the sensitization of CPT antiproliferative activity；
Fig. 2 is that RTCA method detects Tat-SLFN11t-2 to the sensitization of CPT antiproliferative activity.
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
A kind of SLFN11 truncated peptide with sensitization of the present invention, sequence are to choose as shown in SEQ.ID.NO.1
The hydrophilic section of SLFN11 634-661 amino acids designs having for synthesis and wears the polypeptide of film activity, which can be with
RPA1 direct interaction.
One, the design of polypeptide
Studies have shown that SLFN11 is had an effect by the peptide fragment that its 580-901 amino acids is formed with RPA1.Therefore,
The hydrophilic section of 580-901 is chosen, design synthesis has the peptide fragment for wearing film activity, it can rise with RPA1 direct interaction
To the effect of antitumor enhanced sensitivity.
Utilize the hydrophily of the Kyteand Doolitte algorithm analysis SLFN11 albumen of on-line analysis software Pro tScale
And hydrophobicity, the 580-901 sequence of SLFN11 is inputted, chooses 8 hydrophilic area peptide fragments of < 0.5, sequence is as shown in table 1, is
Promotion truncated peptide penetrating cell film in later experiments adds Tat penetrating peptide (Trans- in the N-terminal for 8 truncated peptides selected
Activator ofTranscription) it is modified, 8 respectively obtained truncated peptide SLFN11t, sequence such as 2 institute of table
Show.
The used Tat penetrating peptide of the present invention is the transcriptional transactivator of human I type immunodeficiency virus (HIV-I)
Protein transduction domain (the protein transduction of (trans-activator oftranscription, TAT)
Domain, PTD), sequence are as follows: YGRKKRRQRRR.
1 hydrophilic area peptide section sequence of table
| Coding | Sequence |
| 585-600 | FSRSLRKNRELFVHGL |
| 634-661 | QPLRNFISDRNICRAETRKTFLRENFEH |
| 669-694 | EAQNFRTEDGDWYGKAKSITRRAKGG |
| 707-734 | TSHLDCSGLPPLSDQYPREELTRIVRNA |
| 735-754 | DPIAKYLQKEMQVIRSNPSF |
| 764-783 | FPEAEWSQGVQGTLRIKKYL |
| 795-805 | TCRRFFDRGYS |
| 816-840 | AKEVEHYKYELLKAMRKKRVVQLSD |
Peptide section sequence after the modification of 2 Tat penetrating peptide of table
| Number | Sequence (N → C) |
| TAT-S11-1 | YGRKKRRQRRR-GG-FSRSLRKNRELFVHGL |
| TAT-S11-2 | YGRKKRRQRRR-GG-QPLRNFISDRNICRAETRKTFLRENFEH |
| TAT-S11-3 | YGRKKRRQRRR-GG-EAQNFRTEDGDWYGKAKSITRRAKGG |
| TAT-S11-4 | YGRKKRRQRRR-GG-TSHLDCSGLPPLSDQYPREELTRIVRNA |
| TAT-S11-5 | YGRKKRRQRRR-GG-DPIAKYLQKEMQVIRSNPSF |
| TAT-S11-6 | YGRKKRRQRRR-GG-FPEAEWSQGVQGTLRIKKYL |
| TAT-S11-7 | YGRKKRRQRRR-GG-TCRRFFDRGYS |
| TAT-S11-8 | YGRKKRRQRRR-GG-AKEVEHYKYELLKAMRKKRVVQLSD |
Two, the determination of activity of polypeptide
Document report human breast cancer cell (MCF-7) is SLFN11 down-regulated express cell strain.Therefore, by MCF-7 cell
Middle 8 tested respectively truncated peptide SLFN11t makees the enhanced sensitivity of camptothecine (Camptothecin, CPT) antiproliferative activity
With, the sensitization of each truncated peptide is detected using mtt assay, result as shown in Figure 1, in figure model group be added 5 μM of CPT,
Administration group is while 5 μM CPT and 50 μM different truncated peptide SLFN11t is added.It can be seen from the figure that truncated peptide Tat-
The activity of SLFN11t-2 and Tat-SLFN11t-4 is best, and the sensitization for CPT is respectively 19.48% and 19.88%.Cause
This, chooses activity best Tat-SLFN11t-2 and Tat-SLFN11t-4 and further studies as the later period.
By cell real-time monitor detect Tat-SLFN11t-2 and Tat-SLFN11t-4 for CPT sensitization,
Model group is that 5 μM of CPT are added, and administration group is respectively while 5 μM CPT and 0.5 μM, 5 μM, 50 μM of truncated peptides are added, and Fig. 2 is
RTCA method detects Tat-SLFN11t-2 to the sensitization of CPT antiproliferative activity.The longitudinal axis be cell index (cell index,
CI), CI value is directly proportional to cell quantity.It can be seen from the figure that Tat-SLFN11t-2 can significantly increase CPT for MCF-7
The antiproliferative activity of cell.
Synthesize truncated peptide Tat-SLFN11t-2, the sequence of Tat-SLFN11t-2 specifically:
YGRKKRRQRRR-GG-QPLRNFISDRNICRAETRKTFLRENFEH (same to SEQ.ID.NO.1).
1. weighing dichloro resin to be put into reactor, DCM (methylene chloride) is added and is swollen 0.5~1h, drains.
2. the tyrosine that 1~3 times of the Fmoc (9-fluorenylmethyloxycarbonyl) that mole is dichloro resin mole is protected is added,
Mole is 1~10 times of DIEA (n,N-diisopropylethylamine) of dichloro resin mole, 20~30 times of dichloro resin volumes
DMF and DCM mixed solution (DMF and DCM volume ratio be 1:1~1:3), with nitrogen 0.5~1h of blistering reaction, take out reaction
Liquid uses DMF, methanol cleaning respectively.
3. the Piperidine/DMF solution removal that 5~10 times of the mass fraction that mole is dichloro resin mole is 6% is added
Fmoc is cleaned using DMF, and resin ninhydrin test positive shows that Fmoc is completely removed.
4. amino acid in sequence is sequentially added according to the mode of step 2,3, until the reaction of the last one amino acid is completed.
5. weight is taken out and weighed from reactor by dichloro resin, pour into flask, it is 95% that mass fraction, which is added,
Polypeptide is cleaved from resin carrier, and removes the Side chain protective group of amino acid by TFA cutting liquid, 1~2h of concussion reaction
Group.Filtrate is collected, ether is then added into filtrate, is centrifuged, the crude product of the sequence can be obtained in cleaning.
6. analysis purification and Mass Spectrometer Method: detecting the correctness of the acid molecules amount with ESI ion-source mass spectrometer；Use HPLC
Crude product is purified to 95% or more purity by liquid chromatograph.
It is freezed 7. collecting purified target polypeptides and being put into freeze dryer, is dried to white powder.
8. carry out HPLC and MS analysis detection again after sample freeze-drying, mobile phase is that water containing 0.1%TFA and acetonitrile are molten
Agent, Detection wavelength 220nm.
HPLC liquid chromatographic detection, purity 95.14% are carried out to obtained sample；MS analysis detection shows MS
(ESI,m/z):573.09[M+9H]9+, show that synthetic sample is target truncated peptide Tat-SLFN11t-2.
<110>Xi'an Medical University
<120>a kind of SLFN11 truncated peptide and its application and pharmaceutical composition with sensitization
<160> 1
<210> 1
<211> 41
<212> PRT
<213>artificial synthesized
<400> SEQ.ID.NO.1
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Gln Pro Leu
1 5 10 15
Arg Asn Phe Ile Ser Asp Arg Asn Ile Cys Arg Ala Glu Thr Arg Lys
20 25 30
Thr Phe Leu Arg Glu Asn Phe Glu His
35 40
Claims (6)
1. a kind of SLFN11 truncated peptide with sensitization, which is characterized in that its amino acid sequence such as SEQ.ID.NO.1 institute
Show.
2. a kind of SLFN11 truncated peptide with sensitization according to claim 1, which is characterized in that the SLFN11
Truncated peptide is by carrying out modification acquisition using Tat penetrating peptide to the hydrophilic area SLFN11 peptide fragment.
3. a kind of SLFN11 truncated peptide with sensitization according to claim 2, which is characterized in that the SLFN11
Hydrophilic area peptide fragment is the 634-661 bit sequence of SLFN11.
4. a kind of pharmaceutical composition, which is characterized in that wherein contain SLFN11 truncated peptide of any of claims 1-3
And pharmaceutically acceptable carrier.
5. a kind of application of SLFN11 truncated peptide with sensitization according to claim 1-3, feature
It is, the SLFN11 truncated peptide is to the application in cellular antiproliferative active medicine.
6. a kind of application of the SLFN11 truncated peptide with sensitization according to claim 5, which is characterized in that described
Application of the SLFN11 truncated peptide in tumor chemoradiotherapy drug.
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| CN201910434838.4A CN110229233B (en) | 2019-05-23 | 2019-05-23 | SLFN11 truncated peptide with sensitization effect, application and pharmaceutical composition thereof |
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|---|---|---|---|
| CN201910434838.4A CN110229233B (en) | 2019-05-23 | 2019-05-23 | SLFN11 truncated peptide with sensitization effect, application and pharmaceutical composition thereof |
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| CN110229233B CN110229233B (en) | 2022-10-04 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1311215A (en) * | 2000-03-02 | 2001-09-05 | 上海博德基因开发有限公司 | New polypeptide-human immune response factor 64 and polynucleotide for coding said polypeptide |
| US20170151339A1 (en) * | 2014-06-30 | 2017-06-01 | Tarveda Therapeutics, Inc. | Targeted conjugates and particles and formulations thereof |
| WO2018237034A1 (en) * | 2017-06-20 | 2018-12-27 | Nantomics, Llc | Quantifying slfn11 protein for optimal cancer therapy |
-
2019
- 2019-05-23 CN CN201910434838.4A patent/CN110229233B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1311215A (en) * | 2000-03-02 | 2001-09-05 | 上海博德基因开发有限公司 | New polypeptide-human immune response factor 64 and polynucleotide for coding said polypeptide |
| US20170151339A1 (en) * | 2014-06-30 | 2017-06-01 | Tarveda Therapeutics, Inc. | Targeted conjugates and particles and formulations thereof |
| WO2018237034A1 (en) * | 2017-06-20 | 2018-12-27 | Nantomics, Llc | Quantifying slfn11 protein for optimal cancer therapy |
Non-Patent Citations (3)
| Title |
|---|
| GABRIELE ZOPPOLI ET AL.: ""Putative DNA/RNA helicase Schlafen-11 (SLFN11) sensitizes cancer cells to DNA-damaging agents"", 《PNAS》 * |
| JING LUAN ET AL.: ""SLFN11 is a general target for enhancing the sensitivity of cancer to chemotherapy (DNA-damaging agents)"", 《JOURNAL OF DRUG TARGETING》 * |
| 李榕 等: ""TAT蛋白及其转导技术在中枢神经系统研究中的应用"", 《神经解剖学杂志》 * |
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| Publication number | Publication date |
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| CN110229233B (en) | 2022-10-04 |
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