CN110229233B - SLFN11 truncated peptide with sensitization effect, application and pharmaceutical composition thereof - Google Patents
SLFN11 truncated peptide with sensitization effect, application and pharmaceutical composition thereof Download PDFInfo
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- CN110229233B CN110229233B CN201910434838.4A CN201910434838A CN110229233B CN 110229233 B CN110229233 B CN 110229233B CN 201910434838 A CN201910434838 A CN 201910434838A CN 110229233 B CN110229233 B CN 110229233B
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- A—HUMAN NECESSITIES
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- 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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- 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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Abstract
The invention discloses an SLFN11 truncated peptide with a sensitization effect, and application and a pharmaceutical composition thereof, wherein an amino acid sequence of the truncated peptide is shown in SEQ.ID.NO. 1. The truncated peptide is obtained by modifying a hydrophilic region peptide segment of SLFN11 with Tat penetrating peptide. The SLFN11 truncated peptide can penetrate a cell membrane to enter the cell, improve the level of the SLFN11 of tumor cells, and enhance the sensitivity of the tumor cells to radiotherapy and chemotherapy, so that the SLFN11 truncated peptide can be used as a chemosensitizer for treating tumors.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an SLFN11 truncated peptide with a sensitization effect, and also relates to application and a pharmaceutical composition of the SLFN11 truncated peptide.
Background
The morbidity and mortality of cancer is second only to cardiovascular diseases, placing a heavy economic and mental burden on society and families. Worldwide, there are approximately 1410 million new cancer cases per year. With the acceleration of global aging, cancer will become a major health problem for humans, a world disease that is seriously life-threatening.
Chemotherapy is one of the most effective means for treating cancer at present. However, long-term or high-volume use of chemotherapeutic drugs in cancer cells may result in drug resistance, and chemotherapy may even enhance the ability of residual cancer cells to invade and metastasize. Meanwhile, large dose chemotherapy brings great side effects to the body, so that it is necessary to increase the sensitivity of chemotherapy and reduce the dose of chemotherapy.
The sleep gene protein (SLFN) family plays a key role in development, immune response and cell proliferation. The SLFN11 protein is localized in the nucleus of the cell, and is one of the members of SLFN protein family expressed only in mammals. The level of SLFN11 expression is positively correlated with the sensitivity of the tumor cells to DNA damaging agents. After DNA damage, replication protein A1 (RPA 1) and damaged DNA are combined to promote DNA recombination repair. In SLFN11 high expression tumor cells, SLFN11 can combine with RPA1 to form heterodimer, so that RPA1 is dissociated from DNA, and recombination repair of damaged DNA is inhibited, thereby promoting apoptosis and death. High levels of SLFN11 not only increase the sensitivity of tumor cells to type I/II topoisomerase inhibitors, DNA synthesis inhibitors, and alkylating agents, but also allow cells to assume a more sensitive phenotype for both IR and UV damage. Therefore, exogenous SLFN11 can increase SLFN11 level of tumor cells, and enhance sensitivity of tumor cells to radiotherapy and chemotherapy, thereby being used as chemosensitizer for treating tumors.
Because the protein plays a role generally represented in a plurality of specific functional key regions, the peptide segments of the specific functional regions are cut out to form functional truncation peptides which can show stronger biological activity than the parent protein. These truncated peptides have the following characteristics: small molecular weight, high expression amount, fully exhibiting active structure domain, etc. and exposing some amino acids from the original internal structure to make the truncated peptide segment play new role.
However, exogenous truncated peptides are difficult to enter the cell interior from the cell membrane. Research proves that a Protein Transduction Domain (PTD) derived from a trans-activator of transcription (TAT) of human immunodeficiency virus type I (HIV-I) can efficiently, quickly and safely introduce different biological macromolecules (such as nucleic acid, polypeptide, protein and the like) with the molecular weight of 15-120kD into cells, and the introduced biological macromolecules still have biological activity and hardly have toxicity to host cells. The discovery of the TAT protein transduction domain brings new eosin for protein therapy, and shows great application value.
Disclosure of Invention
The invention aims to provide a SLFN11 truncated peptide with sensitization, which solves the problem that the prior exogenous SLFN11 is difficult to enter the cell.
Another object of the present invention is to provide the use of the SLFN11 truncated peptide.
The third object of the present invention is to provide a pharmaceutical composition.
The technical scheme adopted by the invention is that the amino acid sequence of the SLFN11 truncated peptide with the sensitization function is shown as SEQ ID NO. 1.
The invention adopts another technical scheme that the application of the SLFN11 truncated peptide with the sensitization effect and the application of the SLFN11 truncated peptide in preparing the anti-proliferative active drug for breast cancer cells.
The present invention is also characterized in that,
the application of SLFN11 truncated peptide in preparing breast cancer chemoradiotherapy medicine.
The third technical scheme adopted by the invention is that the pharmaceutical composition comprises the SLFN11 truncated peptide and a pharmaceutically acceptable carrier.
The SLFN11 truncation peptide with the sensitization function has the beneficial effects that the SLFN11 truncation peptide with the sensitization function is obtained by modifying the amino acid hydrophilic segment of the SLFN11 by utilizing the Tat penetrating peptide, so that the SLFN11 truncation peptide can penetrate through a cell membrane to enter the interior of a cell, the level of the SLFN11 of a tumor cell is improved, the sensitivity of the tumor cell to radiotherapy and chemotherapy is enhanced, and the SLFN11 truncation peptide can be used as a chemotherapy sensitizer for treating tumors.
Drawings
FIG. 1 shows the sensitization of truncated peptides to CPT antiproliferative activity by MTT assay;
FIG. 2 shows the sensitivity of Tat-SLFN11t-2 to CPT antiproliferative activity measured by the RTCA method.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
The sequence of the SLFN11 truncation peptide with sensitization is shown in SEQ ID No.1, and a synthetic polypeptide with transmembrane activity is designed by selecting a hydrophilic section of 634-661 amino acids of SLFN11, and can directly interact with RPA 1.
1. Design of polypeptides
The study shows that SLFN11 acts with RPA1 through peptide segment formed by 580-901 amino acids. Therefore, the hydrophilic region of 580-901 is selected to design and synthesize a peptide segment with transmembrane activity, which can directly interact with RPA1 to play the role of anti-tumor sensitization.
The hydrophilicity and hydrophobicity of the SLFN11 protein are analyzed by using a Kyteand Doolitte algorithm of online analysis software ProtScale, a 580-901 sequence of the SLFN11 is input, 8 hydrophilic region peptide sections less than 0.5 are selected, the sequences are shown in table 1, in order to promote the truncated peptides to penetrate cell membranes in later experiments, tat penetrating peptides (Trans-Activator of transcription) are added to the N ends of the selected 8 truncated peptides for modification, and the sequences of the 8 truncated peptides SLFN11t are shown in table 2.
The Tat penetrating peptide adopted by the invention is a Protein Transduction Domain (PTD) of trans-activator of transcription (TAT) of human immunodeficiency virus I (HIV-I), and the sequence of the Tat penetrating peptide is as follows: YGRKKRRQRRR.
2. Determination of the Activity of a polypeptide
Human breast cancer cells (MCF-7) are reported in the literature as SLFN11 low expressing cell lines. Thus, the results of testing the sensitization of each of the 8 truncated peptides SLFN11t to the antiproliferative activity of Camptothecin (CPT) by MTT assay were shown in FIG. 1, in which the model group was 5. Mu.M CPT and the administration group was 5. Mu.M MCPT and 50. Mu.M of different truncated peptides SLFN11t, by testing the sensitization of each of the 8 truncated peptides SLFN11t to the antiproliferative activity of Camptothecin (CPT) in MCF-7 cells. As can be seen from the figure, the truncated peptides Tat-SLFN11t-2 and Tat-SLFN11t-4 were most active, with sensitization effects on CPT of 19.48% and 19.88%, respectively. Thus, the best active Tat-SLFN11t-2 and Tat-SLFN11t-4 were selected for further study at a later stage.
The cell real-time monitor is used for detecting the sensitization of Tat-SLFN11t-2 and Tat-SLFN11t-4 to CPT, the model group is added with 5 mu M CPT, the administration group is simultaneously added with 5 mu MCPT and 0.5 mu M, 5 mu M and 50 mu M truncated peptides respectively, and the figure 2 is an RTCA method for detecting the sensitization of Tat-SLFN11t-2 to CPT antiproliferative activity. The vertical axis is the Cell Index (CI), and the CI is proportional to the number of cells. As can be seen from the figure, tat-SLFN11t-2 significantly enhanced the antiproliferative activity of CPT on MCF-7 cells.
The sequences of the synthetic truncated peptide Tat-SLFN11t-2 and Tat-SLFN11t-2 are as follows:
YGRKKRRQRRR-GG-QPLRNFISDRNICARKETRKLLRENFEH (same as SEQ. ID. NO. 1).
1. Weighing the dichloro resin, putting the dichloro resin into a reactor, adding DCM (dichloromethane) to swell for 0.5 to 1h, and drying by suction.
2. Adding Fmoc (9-fluorenylmethoxycarbonyl) protected tyrosine with the molar weight of 1 to 3 times that of the dichloro resin, DIEA (N, N-diisopropylethylamine) with the molar weight of 1 to 10 times that of the dichloro resin, a mixed solution of DMF and DCM with the volume of 20 to 30 times that of the dichloro resin (the volume ratio of DMF to DCM is 1 to 1: 3), carrying out bubbling reaction for 0.5 to 1h by using nitrogen, pumping out the reaction solution, and washing by using DMF and methanol respectively.
3. And adding a piperidine DMF solution with the mass fraction of 6% and the molar weight which is 5-10 times of that of the dichloro resin to remove the Fmoc, washing with DMF, and detecting the resin with ninhydrin to be positive to show that the Fmoc is completely removed.
4. And (3) sequentially adding amino acids in the sequence in the modes of the steps 2 and 3 until the reaction of the last amino acid is finished.
5. Taking the dichloro resin out of the reactor, weighing the dichloro resin, pouring the dichloro resin into a flask, adding a TFA cutting solution with the mass fraction of 95%, oscillating the mixture to react for 1 to 2 hours, splitting the polypeptide from the resin carrier, and removing a side chain protecting group of the amino acid. Collecting the filtrate, adding ether into the filtrate, centrifuging, and washing to obtain the crude product of the sequence.
6. Analysis purification and mass spectrum detection: checking the correctness of the sequence molecular weight by an ESI ion source mass spectrometer; the crude product was purified to a purity of 95% or more by HPLC.
7. Collecting the purified target polypeptide, freezing in a freeze dryer, and drying to obtain white powder.
8. After freeze-drying the sample, HPLC and MS analysis were performed again, and the mobile phase was water and acetonitrile solvent containing 0.1% TFA, and the detection wavelength was 220nm.
Performing HPLC liquid chromatography detection on the obtained sample, wherein the purity of the sample is 95.14%; MS analysis and detection shows that MS (ESI, m/z) 573.09[ M +9H ]] 9+ The result shows that the synthetic sample is the target truncated peptide Tat-SLFN11t-2.
<110> Sian medical college
<120> SLFN11 truncated peptide with sensitization effect, application and pharmaceutical composition thereof
<160> 1
<210> 1
<211> 41
<212> PRT
<213> Artificial Synthesis
<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 (4)
1. An SLFN11 truncated peptide with sensitization, which is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.
2. A pharmaceutical composition comprising the SLFN11 truncated peptide of claim 1, and a pharmaceutically acceptable carrier.
3. The use of the SLFN11 truncated peptide with sensitizing effect of claim 1 in the preparation of a medicament having anti-proliferative activity on breast cancer cells.
4. The use of the SLFN11 truncated peptide with sensitizing effect of claim 1 in the preparation of a breast cancer chemoradiotherapy medicament.
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| Application Number | Priority Date | Filing Date | Title |
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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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| Application Number | Priority Date | Filing Date | Title |
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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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| CN110229233A CN110229233A (en) | 2019-09-13 |
| CN110229233B true CN110229233B (en) | 2022-10-04 |
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Citations (2)
| 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 |
| WO2018237034A1 (en) * | 2017-06-20 | 2018-12-27 | Nantomics, Llc | Quantifying slfn11 protein for optimal cancer therapy |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016004043A1 (en) * | 2014-06-30 | 2016-01-07 | Blend Therapeutics, Inc. | Targeted conjugates and particles and formulations thereof |
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2019
- 2019-05-23 CN CN201910434838.4A patent/CN110229233B/en active Active
Patent Citations (2)
| 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 |
| WO2018237034A1 (en) * | 2017-06-20 | 2018-12-27 | Nantomics, Llc | Quantifying slfn11 protein for optimal cancer therapy |
Non-Patent Citations (3)
| Title |
|---|
| "Putative DNA/RNA helicase Schlafen-11 (SLFN11) sensitizes cancer cells to DNA-damaging agents";Gabriele Zoppoli et al.;《PNAS》;20120911;第109卷(第37期);第15030-15035页 * |
| "SLFN11 is a general target for enhancing the sensitivity of cancer to chemotherapy (DNA-damaging agents)";Jing Luan et al.;《Journal of Drug Targeting》;20190516;第1-29页 * |
| "TAT蛋白及其转导技术在中枢神经系统研究中的应用";李榕 等;《神经解剖学杂志》;20111231;第27卷(第4期);第466-468页 * |
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