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WO2017067510A1 - 一种多肽及其在制备治疗和预防肿瘤的药物中的应用 - Google Patents

一种多肽及其在制备治疗和预防肿瘤的药物中的应用 Download PDF

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WO2017067510A1
WO2017067510A1 PCT/CN2016/102897 CN2016102897W WO2017067510A1 WO 2017067510 A1 WO2017067510 A1 WO 2017067510A1 CN 2016102897 W CN2016102897 W CN 2016102897W WO 2017067510 A1 WO2017067510 A1 WO 2017067510A1
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polypeptide
trb3
cancer
seq
cells
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PCT/CN2016/102897
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French (fr)
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胡卓伟
花芳
李珂
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胡卓伟
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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
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a polypeptide and the use thereof in preparing medicine for treating and preventing tumor.
  • TRB3 (Tribbles Homologue 3) is a member of the Tribbles homologous protein family and is involved in the regulation of cell proliferation, migration and morphogenesis during development. As a member of the pseudo-kinase protein family, TRB3 has a adaptor-like function and is involved in the assembly of various protein complexes. A number of studies suggest that TRB3 interacts with a variety of transcription factors, ubiquitin ligase, type II BMP receptors, and MAPK, PI3K signaling pathway member proteins, involved in glycolipid metabolism, adipocyte differentiation, apoptosis, and Regulatory regulation.
  • TRB3 is highly expressed in a variety of tumor cell lines and human tumor tissues, and plays an important role in the development of tumors.
  • targeting the interaction between TRB3 and p62 is a potential target for the treatment of tumors. Therefore, research and development of substances that block the interaction between TRB3 and P62 protein have a good prospect of inhibiting tumorigenesis and development.
  • Protein-protein interactions play important roles in many biological processes, such as cell proliferation, growth, differentiation, and programmed death. Many potential therapeutic targets in human disease are primarily protein-protein interactions. In the process of protein-protein interaction, alpha helix and beta sheet secondary structures are the major interface elements involved in PPIs. In recent years, the use of chemical synthesis to obtain highly active, highly selective synthetic peptide drugs has become a new research hotspot. However, the binding ability of the polypeptide to the working protein is very weak, and the ordinary linear polypeptide cannot penetrate the cell membrane and is easily hydrolyzed by the protease. Therefore, there are still many shortcomings in peptide-targeted drugs. From the above, it is known that a highly active, highly selective synthetic polypeptide drug that targets the interaction between TRB3 and p62 is desired.
  • the technical problem to be solved by the present invention is to provide a polypeptide which specifically binds to TRB3 and its current situation in view of the current lack of a highly active and highly selective synthetic polypeptide drug targeting the interaction between TRB3 and p62.
  • the inventors of the present invention have conducted intensive studies and repeated experiments and found that the specific binding ability and biostability of the polypeptide A2 (amino acid sequence see SEQ ID No. 8) which interacts with TRB3 and TR62 are both specific for binding to TRB3. Relatively low. It is directly related to the alpha-helical conformation required for polypeptide A2 to not stably form an activity in solution.
  • the inventors conducted targeted studies and experiments and found that if the amino acid residue at a specific position in the polypeptide A2 is replaced with an unnatural amino acid to which the side chain can be linked, such as S-pentene alanine (S5),
  • S5 S-pentene alanine
  • the modified polypeptide has a stable ⁇ -helix secondary structure, which makes the modified polypeptide have high affinity, anti-enzymatic stability and cell penetrating property, ie, improve its ⁇ -helical stability, TRB3 binding ability and metabolic stability. Sexuality inhibits proliferation and metastasis of various tumor cells. It has been experimentally verified that the modified polypeptide can be applied to the preparation of a medicament for treating and preventing tumors. Based on the research work of the inventors, the present invention provides the following technical solutions.
  • One of the technical solutions provided by the present invention is: a polypeptide which specifically binds to TRB3, and the amino acid sequence of the polypeptide is replaced by two or more amino acids in the amino acid sequence shown in SEQ ID No. 8 of the Sequence Listing. Shown as unnatural amino acids to which the side chains can be attached.
  • the unnatural amino acid to which the other side chains are linked is a non-natural amino acid conventional in the art, preferably S-pentene alanine (S5).
  • the number of the substituted amino acids is two and the positions of the substituted amino acids are the ith position and the i+3 position, respectively, or the ith position and the i+th position. 4 bits, where 1 ⁇ i ⁇ 7, and i is a positive integer.
  • amino acid sequence of the polypeptide is as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6. And any one of SEQ ID No. 7.
  • amino acid sequence represented by the above SEQ ID No. 1 - SEQ ID No. 7 can be appropriately substituted, deleted or added as long as the modified amino acid sequence can still specifically bind to TRB3 and maintain the pre-engineering It can be active.
  • the second technical solution provided by the present invention is: the use of a polypeptide which specifically binds to TRB3 in the preparation of a medicament for treating and/or preventing tumors.
  • the tumor is a conventional tumor in the art.
  • liver cancer is a conventional liver cancer in the field, preferably a primary liver cancer or a secondary liver cancer.
  • the lung cancer is a conventional lung cancer in the art, preferably small cell lung cancer or non-small cell lung cancer.
  • the breast cancer is a conventional breast cancer in the art, preferably a non-invasive breast cancer, an early invasive breast cancer, an invasive special type breast cancer or an invasive non-special type breast cancer.
  • the intestinal cancer is a conventional intestinal cancer in the art, preferably colon cancer or rectal cancer.
  • the leukemia is a conventional leukemia in the art, preferably a lymphocytic leukemia or a non-lymphocytic leukemia.
  • the prevention is a routine prevention in the art, preferably when there is a possible tumor factor, preventing or lowering after use Low tumor production.
  • the treatment is conventional in the art, preferably to reduce the extent of the tumor, or to cure the tumor to normalize it, or to slow the progression of the tumor.
  • the third aspect of the present invention provides an antitumor pharmaceutical composition comprising the polypeptide which specifically binds to TRB3 as an active ingredient.
  • the active ingredient refers to a compound having a function of preventing or treating tumors.
  • the polypeptide which specifically binds to TRB3 may be used alone as an active ingredient or as an active ingredient together with other compounds having antitumor activity.
  • the administration route of the pharmaceutical composition of the present invention is preferably administered by injection or orally.
  • the administration by injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection.
  • the pharmaceutical composition is various dosage forms conventional in the art, preferably in the form of a solid, semi-solid or liquid, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule or a granule. , injection or infusion, etc.
  • the pharmaceutical compositions of the present invention further comprise one or more pharmaceutically acceptable carriers.
  • the pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutically acceptable carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably include pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above protein and 0.01-99.99% of a pharmaceutically acceptable carrier, the percentage being the mass percentage of the pharmaceutical composition.
  • the pharmaceutical composition is administered in an amount effective to reduce or delay the progression of a disease, degenerative or damaging condition.
  • the effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the results sought.
  • the reagents and starting materials used in the present invention are commercially available.
  • the positive progress of the present invention is that the polypeptide of the present invention is capable of specifically binding to TRB3, blocking the interaction of the TRB3 and P62 proteins, and is therefore useful in the preparation of a medicament for treating and preventing tumors.
  • the prepared drug has the advantages of remarkable curative effect, less toxic side effects and safe use in treating tumor diseases.
  • Figure 1 shows the surface plasmon resonance method for verifying the binding ability of polypeptides A2, S1, S2, S3, S4, S5, S6 and S7 to TRB3 protein.
  • Figure 1 (A) is the kinetic curve of the polypeptide A2 and TRB3 protein;
  • Figure 1 (B) is the binding kinetics curve of the polypeptide S1 and TRB3 protein;
  • Figure 1 (C) is the binding kinetics curve of the polypeptide S2 and TRB3 protein;
  • 1(D) is the binding kinetics curve of polypeptide S3 and TRB3 protein;
  • Figure 1 (E) is the polypeptide S4 and TRB3 protein Binding kinetic curve;
  • Figure 1 (F) is the binding kinetics curve of peptide S5 and TRB3 protein;
  • Figure 1 (G) is the binding kinetics curve of peptide S6 and TRB3 protein;
  • Figure 1 (H) is the binding dynamics of peptide S7 and TRB3 protein Learning curve.
  • Figure 2 is a graph showing the interaction of peptides A2, S1, S2, S3, S4, S5, S6 and S7 with TRB3 and p62 proteins.
  • A shows that A2, S1, S2 and S3 interfere with the interaction of TRB3 with P62 protein;
  • B shows that S4, S5, S6 and S7 interfere with the interaction of TRB3 with P62 protein.
  • “One” indicates the input, that is, the amount of protein of TRB3 protein and P62 protein in the cell lysate;
  • two indicates the output, that is, the amount of protein of TRB3 protein and P62 protein after precipitation by P62 antibody.
  • Figure 3 is a graph showing the results of inhibition of HepG2 growth of hepatoma cells by polypeptides S1, S2, S3, S4, S5, S6 and S7.
  • the abscissa is the administration time in days.
  • the ordinate is the number of cells in 10,000.
  • the control is polypeptide A2.
  • Figure 4 is a graph showing the results of inhibition of growth of lung cancer cells A549 by polypeptides S1, S2, S3, S4, S5, S6 and S7.
  • the abscissa is the administration time in days.
  • the ordinate is the number of cells in 10,000.
  • the control is polypeptide A2.
  • Figure 5 is a graph showing the results of inhibition of growth of breast cancer cells MDA-MB-231 by polypeptides S1, S2, S3, S4, S5, S6 and S7.
  • the abscissa is the administration time in days.
  • the ordinate is the number of cells in 10,000.
  • the control is polypeptide A2.
  • Figure 6 is a graph showing the results of inhibition of growth of intestinal cancer cells HCT-8 by polypeptides S1, S2, S3, S4, S5, S6 and S7.
  • the abscissa is the administration time in days.
  • the ordinate is the number of cells in 10,000.
  • the control is polypeptide A2.
  • Figure 7 is a graph showing the results of inhibition of K562 growth of leukemia cells by polypeptides S1, S2, S3, S4, S5, S6 and S7.
  • the abscissa is the administration time in days.
  • the ordinate is the number of cells in 10,000.
  • the control is polypeptide A2.
  • Figure 8 is a graph showing the results of inhibition of HepG2 migration of hepatoma cells by polypeptides A2, S1, S2, S3, S4, S5, S6 and S7.
  • the ordinate is the repair area ratio after cell scratching, and the unit is a percentage.
  • the control is polypeptide A2. *** indicates p ⁇ 0.001.
  • Figure 9 is a graph showing the results of inhibition of migration of lung cancer cells A549 by polypeptides A2, S1, S2, S3, S4, S5, S6 and S7.
  • the ordinate is the repair area ratio after cell scratching, and the unit is a percentage.
  • the control is polypeptide A2. *** indicates p ⁇ 0.001.
  • Figure 10 is a graph showing the results of inhibition of migration of breast cancer cells MDA-MB-231 by polypeptides A2, S1, S2, S3, S4, S5, S6 and S7.
  • the ordinate is the repair area ratio after cell scratching, and the unit is a percentage.
  • the control is polypeptide A2. *** indicates p ⁇ 0.001.
  • Figure 11 is a graph showing the results of inhibition of HCT-8 migration of intestinal cancer cells by polypeptides A2, S1, S2, S3, S4, S5, S6 and S7.
  • the ordinate is the repair area ratio after cell scratching, and the unit is a percentage.
  • the control is polypeptide A2. *** indicates p ⁇ 0.001.
  • Figure 12 is a graph showing the results of inhibition of K562 clone formation in leukemia cells by polypeptides A2, S1, S2, S3, S4, S5, S6 and S7.
  • the ordinate is the number of clone formations, and the unit is one.
  • the control is polypeptide A2. *** indicates p ⁇ 0.001.
  • the PBS solution used in the examples refers to a phosphate buffer solution having a concentration of 0.1 M and a pH of 7.2.
  • the room temperature in the examples is room temperature conventional in the art, preferably 15-30 °C.
  • polypeptide A2 The amino acid sequence of polypeptide A2 is shown in SEQ ID No. 8 of the Sequence Listing. Peptide A2 was synthesized and purified by Beijing Saibaisheng Gene Technology Co., Ltd.
  • S5 Two unnatural amino acids S-pentene alanine (S5) were introduced for solid phase polypeptide chain synthesis.
  • the target polypeptide is obtained by cyclization of olefin metathesis reaction (RCM) using hydrazine as a catalyst.
  • RCM olefin metathesis reaction
  • the target polypeptide is cleaved from the resin for purification.
  • the steps of synthesizing and purifying the above solid phase polypeptide chain are completed by the company of the Chinese peptide biochemical company.
  • two S-pentenylalanines are inserted at the i-th, i+3 or i, i+4 positions in the amino acid sequence of the polypeptide A2, thereby obtaining modified sequences of different sequences (amino acid sequence see sequence listing) SEQ ID No. 1 - SEQ ID No. 7), the specific insertion sites are as follows:
  • the surface plasmon resonance experiment was carried out in a surface plasmon resonator Biacore T200, and the procedure was carried out in accordance with the instructions of the plasma resonator Biacore T200. Specific steps are as follows:
  • TRB3 protein purchased from RD
  • CM5 chip purchased from GE
  • unbound protein was eluted at a flow rate of 10 ⁇ L/min, and the surface of the chip was equilibrated for 2 hours.
  • ammonia For specific steps of base coupling, elution and equilibration, refer to the relevant instructions of GE's CM5 chip.
  • Figures 1 (A)-(H) and Table 1 demonstrate that the affinity of the peptides S1, S2, S3, S4, S5, S6 and S7 with the TRB3 protein is significantly higher than the affinity of the polypeptide A2 with the TRB3 protein.
  • the alpha helix ratio of the polypeptide was measured by a circular dichroism (purchased from Jasco, Japan).
  • the polypeptides A2, S1, S2, S3, S4, S5, S6 and S7 prepared in Example 1 were dissolved in a PBS solution, and the concentration of the upper chromatograph was adjusted to 1 mg/mL. The results are shown in Table 2. .
  • Table 2 shows that the alpha helix rates of the polypeptides S1, S2, S3, S4, S5, S6 and S7 are significantly higher than those of the polypeptide A2.
  • the alpha helix ratio refers to the percentage of the number of peptides that maintain the alpha helix of the secondary structure as a percentage of the total number of peptides.
  • Example 4 Method of co-immunoprecipitation to verify that the polypeptide inhibits the binding of protein p62 to TRB3 at the cellular level.
  • the immunoprecipitation related reagents are as follows:
  • Lysate A 0.6057 g Tris base, 1.7532 g NaCl, 0.1017 g MgCl 2 ⁇ 6H 2 O, 0.0742 g EDTA, 10 mL glycerol and 10 mL 10% (v/v) NP40, deionized water to 150 mL, adjusted with HCl The pH was adjusted to 7.6, and the volume was adjusted to 191 mL. The mixture was thoroughly mixed, filtered through a 0.45 ⁇ m filter, and stored at 4 ° C.
  • Lysate B solution 200 ⁇ L of 2 M ⁇ -glycerophosphate, 4 mL of 2.5 M NaF, 2 mL of 100 mM PMSF, 200 ⁇ L of 1 M DTT, and 1 mg/mL of Leu, Pep, and Apr, respectively, in a total volume of 9 mL, and stored at -20 °C.
  • 2M ⁇ -glycerophosphate, 2.5 M NaF, 100 mM PMSF, 1 M DTT, and 1 mg/mL of Leu, Pep, and Apr were all stored in the form of a mother liquor, except that the mother liquor of PMSF was prepared by using methanol as a solvent, and the others were all in water.
  • the lysate B solution When in use, the lysate B solution is thawed, and the lysate B solution is added to the lysate A solution according to the lysate B solution: the lysate A solution at a volume ratio of 1:100 and mixed.
  • Immunoprecipitation wash 1% (v/v) NP40, 150 mM NaCl, 20 mM Hepes, pH 7.5 10% (v/v) glycerol and 1 mM EDTA.
  • Protein A/G Plus-Agarose was purchased from Santacruz, USA.
  • Hepatoma HepG2 cells purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
  • 1 mg/mL of the polypeptides A1, S1, S2, S3, and S4 prepared in Example 1 were added.
  • S5, S6 or S7 cells were harvested after incubation for 12 hours in a 37 ° C incubator.
  • step 3 Take 200 ⁇ g of each group of proteins obtained in step 2, add 2 ⁇ g of P62 antibody (purchased from Sigma), add 10 ⁇ L of Protein A/G Plus-Agarose (purchased from Santa Cruze), fully resuspend, and shake slowly at 4 °C. overnight. Centrifuge at 4 ° C, 3000 rpm for 5 min, carefully aspirate the supernatant. 0.5 mL of the immunoprecipitated washing solution was added, mixed, and allowed to stand for 1 min in an ice bath, and then centrifuged at 4 ° C, 3000 rpm for 30 seconds, and the supernatant was carefully aspirated. The washing was repeated 5 times and allowed to stand for 5 min before the last centrifugation.
  • liver cancer cells HepG2 in logarithmic growth phase purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
  • lung cancer cell A549 purchasedd from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
  • colon cancer cell HCT-8 purchasedd from Chinese medicine
  • breast cancer cell MDA-MB-231 purchasedd from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
  • leukemia cell K562 purchasedd from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
  • Step 2 Add 1 mL of the cell suspension prepared in Step 1 to a 12-well plate for culture (the medium used for HepG2, A549, HCT-8 and MDA-MB-231 cells is DMEM medium, and the medium used for K562 cells is 1640).
  • the medium was purchased from Invitrogen; the culture temperature was 37 ° C, the medium volume was 1 mL), and after 12 hours, it was replaced with a new medium, and 1 ⁇ g/mL of the polypeptides S1, S2, and S3 prepared in Example 1 were added. , S4, S5, S6 and S7. Pass once every other day and count. A growth curve was drawn after 12 days of culture. The experimental results are shown in Figure 3-7.
  • Figures 3-7 illustrate that polypeptides S1, S2, S3, S4, S5, S6, and S7 are more capable of inhibiting tumor cell growth than A2.
  • the polypeptide S1 inhibits the growth of lung cancer cells by up to 4 times relative to the polypeptide A2
  • the polypeptide S2 inhibits the growth of liver cancer and colon cancer cells by 3 times relative to the polypeptide A2
  • the polypeptide S3 is compared with the polypeptide A2 to the lung cancer cells.
  • the inhibition level of growth is up to 3 times
  • the inhibition level of polypeptide S4 relative to polypeptide A2 is up to 3 times
  • the inhibition level of polypeptide S5 relative to polypeptide A2 is up to 3 times
  • the polypeptide S6 is relative to
  • the level of inhibition of leukemia cell growth by polypeptide A2 is up to 3-fold
  • the inhibition of growth of breast cancer cells by polypeptide S7 relative to polypeptide A2 is up to 3-fold.
  • Example 6 Cell Scratch Test Validates Polypeptides A1, S1, S2, S3, S4, S5, S6 and S7 to Inhibit Healing of Tumor Cells after Scratch
  • tumor cells were added to each well, and the cells were adhered to the cells in a DMEM medium at 37 ° C overnight.
  • the tumor cells are hepatocellular carcinoma cells HepG2, lung cancer cells A549, colon cancer cells HCT-8, and breast cancer cells MDA-MB-231 in logarithmic growth phase.
  • the cells were washed 3 times with PBS, the delineated cells were removed, and a new medium was added while adding 1 ⁇ g/mL of the polypeptides A1, S1, S2, S3, S4, S5, S6 and S7 prepared in Example 1.
  • Top layer agar Add 4.6 mL of cell suspension to 0.6 mL of 50 ° C 5% (w/w) agar, mix well, and add a 24-well plate with a layer of agar, 0.8 mL per well. Solidified at room temperature. The number of cells per well is 100. The cell suspension is prepared by diluting leukemia cell K562 with 1640 culture solution to adjust the concentration to 132 cells/mL.
  • the cells obtained in the step 2 were incubated with the 1640 culture solution in a 37 ° C incubator for 3 weeks, and the number of clone formation was calculated.
  • Example 8 Tumor subcutaneous growth experiments confirmed that polypeptides A2, S1, S2, S3, S4, S5, S6 and S7 inhibited the growth of tumor cells in mice.
  • Experimental consumables and reagents sterilized EP tube 1.5mL, 15mL centrifuge tube, pipette tip, strainer (100 mesh), absorbent cotton ball, tweezers, alcohol cotton ball, sterile 1mL syringe, 500mL beaker (sterilization, Use pre-UV), PBS (filter), trypsin, serum.
  • mice and grouping 80 male nude mice aged 4-6 weeks (purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.), randomly divided into 8 groups: A2, S1, S2, S3, S4, S5, S6 And S7 group, 10 in each group.
  • the tumor cells are hepatocellular carcinoma cells HepG2, lung cancer cells A549, colon cancer cells HCT-8, and breast cancer cells MDA-MB-231 in logarithmic growth phase.
  • the suspended leukemia cell K562 was directly collected into a 15 mL centrifuge tube and centrifuged at 1200 rpm for 5 min. Discard the supernatant, resuspend in PBS, pass through a 100 mesh filter once; count the cells and adjust the final concentration of the cells to 2.5 ⁇ 10 7 /mL.
  • Tumor cell inoculation 5 ⁇ 10 6 tumor cells (200 ⁇ l of cell suspension) were inoculated subcutaneously into the left upper abdomen of nude mice.
  • the polypeptide was treated with a peptide one week after subcutaneous injection of tumor cells (5 mg/kg body weight twice a week), and the vernier caliper recorded the tumor size.

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Abstract

本发明公开了一种特异性结合TRB3的多肽及其在制备治疗和预防肿瘤的药物中的应用。所述多肽的氨基酸序列如将序列表SEQ ID No.8所示的氨基酸序列中的两个或两个以上的氨基酸替换为使其他侧链相连的非天然氨基酸所示。所述多肽能够特异性地与TRB3结合，从而阻断TRB3和P62蛋白的相互作用，因此应用于治疗和预防肿瘤的药物的制备中。所制备的药物在治疗肿瘤疾病中，具有疗效显著，毒副作用少，使用安全的优点。

Description

一种多肽及其在制备治疗和预防肿瘤的药物中的应用
本申请要求申请日为2015年10月22日的中国专利申请CN2015106951467的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物技术领域，具体涉及一种多肽及其在制备治疗和预防肿瘤的药物中的应用。
背景技术
TRB3(Tribbles Homologue 3)是Tribbles同源蛋白家族成员之一，参与调节发育过程中细胞的增殖、迁移及形态形成。TRB3作为假激酶蛋白家族成员，具有接头蛋白样的功能，参与多种蛋白复合体的组装。多项研究认为，TRB3可以与多种转录因子、泛素连接酶、细胞膜上II型BMP受体以及MAPK、PI3K信号通路成员蛋白发生相互作用，参与糖脂代谢、脂肪细胞分化、凋亡和应激等的调控。近来，多种证据表明，TRB3在多种肿瘤细胞系和人肿瘤组织中呈现高表达，并且在肿瘤的发展过程中发挥重要的促进作用。研究发现，TRB3通过与自噬货车蛋白p62发生相互作用，抑制细胞的自噬活性，促进肿瘤细胞的增殖和转移。由此可见，靶向TRB3与p62之间的相互作用是治疗肿瘤的一个潜在靶点。因此，研究和开发阻断TRB3与P62蛋白相互作用的物质，具有很好的抑制肿瘤发生和发展的成药前景。
蛋白-蛋白相互作用(PPIs)在许多生物过程中扮演着重要的角色，例如细胞的增殖、生长、分化及程序性死亡。人类疾病中许多潜在的治疗靶标主要是蛋白-蛋白相互作用。在蛋白-蛋白相互作用的过程中，α螺旋和β折叠二级结构是参与PPIs的主要接触面单元。近年来，用化学合成方式得到高活性、高选择性的合成多肽类药物已经成为新的研究热点。然而，多肽与作用蛋白的结合能力非常弱，普通的线性多肽不能透过细胞膜且易被蛋白酶水解。因此，多肽类靶点药物目前还有很多不足。由上可知，亟待获得靶向TRB3与p62之间的相互作用的，高活性、高选择性的合成多肽类药物。
发明内容
本发明所要解决的技术问题是针对目前缺乏靶向TRB3与p62之间的相互作用的，高活性、高选择性的合成多肽类药物的现状，提供一种特异性结合TRB3的多肽及其在 制备治疗和预防肿瘤的药物中的应用。
本发明的发明人经过深入的研究和反复的试验发现，靶向TRB3与p62蛋白相互作用的多肽A2(氨基酸序列参见序列表SEQ ID No.8)与TRB3的特异性结合能力和生物稳定性都比较低。而其与多肽A2在溶液中不能稳定形成活性所需的α螺旋构象直接相关。由此，发明人进行了针对性的研究和试验，发现如果将多肽A2中特定位置的氨基酸残基替换为侧链可以相连的非天然氨基酸，如S-戊烯丙氨酸(S5)，则改造后的多肽具有稳定的α螺旋的二级结构，使改造后的多肽具有极高的亲和力、抗酶解稳定性以及细胞穿膜性，即提高其α螺旋稳定性、TRB3结合能力和代谢稳定性，抑制多种肿瘤细胞增殖和转移。经实验验证，改造后的多肽能够应用于制备治疗和预防肿瘤的药物中。基于发明人的研究工作，本发明提供下述的技术方案。
本发明提供的技术方案之一是：一种特异性结合TRB3的多肽，所述多肽的氨基酸序列如将序列表SEQ ID No.8所示的氨基酸序列中的两个或两个以上的氨基酸替换为侧链可相连的非天然氨基酸所示。
所述的使其他侧链相连的非天然氨基酸为本领域常规的非天然氨基酸，较佳的为S-戊烯丙氨酸(S5)。
较佳的，所述的多肽中，所述替换的氨基酸的数目为两个且所述替换的氨基酸的位置分别为第i位和第i+3位，或者，为第i位和第i+4位，其中1≤i≤7，i为正整数。
更佳的，所述的多肽的氨基酸序列如将序列表SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6和SEQ ID No.7中任一项所示。
其中，上述SEQ ID No.1-SEQ ID No.7所示的氨基酸序列中可进行适当地氨基酸替换、缺失或添加，只要使改造后的氨基酸序列仍然能够与TRB3特异性结合并且保持改造前的活性即可。
本发明提供的技术方案之二是：一种特异性结合TRB3的多肽在制备治疗和/或预防肿瘤的药物中的应用。
所述的肿瘤为本领域常规的肿瘤。较佳地是肝癌、肺癌、乳腺癌、肠癌或白血病。其中，所述肝癌为本领域常规的肝癌，较佳的为原发性肝癌或继发性肝癌。所述肺癌为本领域常规的肺癌，较佳的为小细胞肺癌或非小细胞肺癌。所述乳腺癌为本领域常规的乳腺癌，较佳的为非浸润性乳腺癌、早期浸润性乳腺癌、浸润性特殊类型乳腺癌或浸润性非特殊类型乳腺癌。所述肠癌为本领域常规的肠癌，较佳的为结肠癌或直肠癌。所述白血病为本领域常规的白血病，较佳的为淋巴细胞型白血病或非淋巴细胞型白血病。
所述预防是本领域常规的预防，较佳的指存在可能的肿瘤因素时，使用后防止或降 低肿瘤的产生。所述治疗是本领域常规的治疗，较佳的指减轻肿瘤的程度，或者治愈肿瘤使之正常化，或者减缓肿瘤的进程。
本发明提供的技术方案之三是：一种抗肿瘤的药物组合物，其含有所述的特异性结合TRB3的多肽作为活性成分。
所述的活性成分是指具有预防或治疗肿瘤功能的化合物。在所述药物组合物中，所述特异性结合TRB3的多肽可以单独作为活性成分或和其他具有抗肿瘤活性的化合物一起作为活性成分。
本发明所述的药物组合物的给药途径较佳的为注射给药或口服给药。所述注射给药较佳的包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳的为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳的为片剂、胶囊、颗粒剂、注射剂或输注剂等。
较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01-99.99％的上述蛋白质和0.01-99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明的多肽能够特异性地与TRB3结合，阻断TRB3和P62蛋白的相互作用，从而应用于治疗和预防肿瘤的药物的制备中。所制备的药物在治疗肿瘤疾病中，具有疗效显著，毒副作用少，使用安全的优点。
附图说明
图1为表面等离子共振方法验证多肽A2、S1、S2、S3、S4、S5、S6和S7与TRB3蛋白的结合能力。其中图1(A)为多肽A2与TRB3蛋白的动力学曲线；图1(B)为多肽S1与TRB3蛋白结合动力学曲线；图1(C)为多肽S2与TRB3蛋白结合动力学曲线；图1(D)为多肽S3与TRB3蛋白结合动力学曲线；图1(E)为多肽S4与TRB3蛋白 结合动力学曲线；图1(F)为多肽S5与TRB3蛋白结合动力学曲线；图1(G)为多肽S6与TRB3蛋白结合动力学曲线；图1(H)为多肽S7与TRB3蛋白结合动力学曲线。图1中的横坐标为反应时间，单位为秒。纵坐标为反应芯片表面与多肽的反应强度，单位为RU。
图2为多肽A2、S1、S2、S3、S4、S5、S6和S7干扰TRB3与p62蛋白相互作用图谱。其中A显示A2、S1、S2和S3干扰TRB3与P62蛋白的相互作用；其中B显示S4、S5、S6和S7干扰TRB3与P62蛋白的相互作用。“一”表示输入，即细胞裂解液内TRB3蛋白与P62蛋白的蛋白量；“二”表示输出，即经过P62抗体沉淀之后所含TRB3蛋白与P62蛋白的蛋白量。
图3为多肽S1、S2、S3、S4、S5、S6和S7抑制肝癌细胞HepG2生长结果图。横坐标为给药时间，单位为天。纵坐标为细胞数量，单位为万。对照为多肽A2。
图4为多肽S1、S2、S3、S4、S5、S6和S7抑制肺癌细胞A549生长结果图。横坐标为给药时间，单位为天。纵坐标为细胞数量，单位为万。对照为多肽A2。
图5为多肽S1、S2、S3、S4、S5、S6和S7抑制乳腺癌细胞MDA-MB-231生长结果图。横坐标为给药时间，单位为天。纵坐标为细胞数量，单位为万。对照为多肽A2。
图6为多肽S1、S2、S3、S4、S5、S6和S7抑制肠癌细胞HCT-8生长结果图。横坐标为给药时间，单位为天。纵坐标为细胞数量，单位为万。对照为多肽A2。
图7为多肽S1、S2、S3、S4、S5、S6和S7抑制白血病细胞K562生长结果图。横坐标为给药时间，单位为天。纵坐标为细胞数量，单位为万。对照为多肽A2。
图8为多肽A2、S1、S2、S3、S4、S5、S6和S7抑制肝癌细胞HepG2迁移结果图。纵坐标为细胞划痕后修复面积比，单位为百分数。对照为多肽A2。***表示p<0.001。
图9为多肽A2、S1、S2、S3、S4、S5、S6和S7抑制肺癌细胞A549迁移结果图。纵坐标为细胞划痕后修复面积比，单位为百分数。对照为多肽A2。***表示p<0.001。
图10为多肽A2、S1、S2、S3、S4、S5、S6和S7抑制乳腺癌细胞MDA-MB-231迁移结果图。纵坐标为细胞划痕后修复面积比，单位为百分数。对照为多肽A2。***表示p<0.001。
图11为多肽A2、S1、S2、S3、S4、S5、S6和S7抑制肠癌细胞HCT-8迁移结果图。纵坐标为细胞划痕后修复面积比，单位为百分数。对照为多肽A2。***表示p<0.001。
图12为多肽A2、S1、S2、S3、S4、S5、S6和S7抑制白血病细胞K562克隆形成结果图。纵坐标为克隆形成数，单位为个。对照为多肽A2。***表示p<0.001。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
若无特别说明，实施例中所用的PBS溶液，指浓度为0.1M、pH值为7.2的磷酸盐缓冲液。
实施例中的室温为本领域常规的室温，较佳的为15-30℃。
实验结果用均值±标准误表示，经参数或者非参数方差检验，经比较p<0.05认为有显著性差异，p<0.01认为有极其显著性差异。
实施例1多肽的合成
多肽A2的氨基酸序列参见序列表SEQ ID No.8。多肽A2由北京赛百盛基因技术有限公司合成并纯化。
引入两个非天然氨基酸S-戊烯丙氨酸(S5)进行固相多肽链合成。固相多肽链合成完成后采用钌作为催化剂进行烯烃复分解反应(RCM)环化即得目标多肽。最后将目标多肽从树脂上切割下来进行纯化。上述固相多肽链合成及纯化的步骤由中肽生化有限公司公司完成。其中，两个S-戊烯丙氨酸插入在多肽A2氨基酸序列中的第i、i+3位或者i、i+4位，由此得到不同序列的改造后的多肽(氨基酸序列参见序列表SEQ ID No.1-SEQ ID No.7)，其具体插入位点如下所示：
S1：S5-Gly-Trp-S5-Thr-Arg-Leu-Leu-Gln-Thr-Lys；插入位点第i、i+3位，i＝1；
S2：Gly-S5-Trp-Leu-Thr-S5-Leu-Leu-Gln-Thr-Lys；插入位点第i、i+4位，i＝2；
S3：Gly-Gly-S5-Leu-Thr-Arg-S5-Leu-Gln-Thr-Lys；插入位点第i、i+4位，i＝3；
S4：Gly-Gly-Trp-S5-Thr-Arg-Leu-S5-Gln-Thr-Lys；插入位点第i、i+4位，i＝4；
S5：Gly-Gly-Trp-Leu-S5-Arg-Leu-Leu-S5-Thr-Lys；插入位点第i、i+4位，i＝5；
S6：Gly-Gly-Trp-Leu-Thr-S5-Leu-Leu-Gln-S5-Lys；插入位点第i、i+4位，i＝6；
S7：Gly-Gly-Trp-Leu-Thr-Arg-S5-Leu-Gln-Thr-S5；插入位点第i、i+4位，i＝7。
实施例2用表面等离子共振的方法检测多肽与TRB3蛋白的结合能力
表面等离子共振实验在表面等离子共振仪Biacore T200中进行，操作步骤按照等离子共振仪Biacore T200的说明书进行。具体步骤如下：
1.将纯化的TRB3蛋白(购自RD公司)通过氨基偶联到CM5芯片上(购自GE公司)，按10μL/min的流速洗脱除去未结合的蛋白，并且平衡芯片表面2小时。其中，氨 基偶联、洗脱和平衡的具体步骤参见GE公司CM5芯片的相关说明书。
2.自动进样250μL不同浓度(800、400、200、50、12.5、6.25和3.125nM)的实施例1所制备的S1-S7和A2多肽片段，整个表面等离子共振实验在25℃进行。所使用的缓冲液为HBS-EP缓冲液[0.01M HEPES、0.15M NaCl、3mM EDTA和0.005％(w/w)表面活性剂]。用Biacore T200自带分析软件模拟不同浓度多肽与TRB3的结合曲线，结果如图1(A)-(H)和表1所示。图1(A)-(H)和表1说明，肽段S1、S2、S3、S4、S5、S6和S7与TRB3蛋白的亲和力明显高于多肽A2与TRB3蛋白的亲和力。
表1多肽S1-S7和A2与TRB3蛋白的亲和力测试
多肽名称 与TRB3蛋白的亲和力常数(KD)
A2(对照) 5.68×10-8M
S1 2×10-8M
S2 1.2×10-8M
S3 3.2×10-8M
S4 2.5×10-8M
S5 1.09×10-8M
S6 2.76×10-8M
S7 1.2×10-8M
实施例3圆二色谱法检测多肽的α螺旋率
用圆二色谱仪(购自日本Jasco公司)检测多肽的α螺旋率。将实施例1所制备的多肽A2、S1、S2、S3、S4、S5、S6和S7溶解到PBS溶液中，将圆二色谱仪的上机浓度调整为1mg/mL，结果如表2所示。表2说明，多肽S1、S2、S3、S4、S5、S6和S7的α螺旋率明显高于多肽A2，由于多肽的α螺旋二级结构介导多肽与TRB3蛋白结合，因此，多肽S1-S7的α螺旋率的提高与其和TRB3蛋白结合能力的增加，以及多种肿瘤细胞的增殖和转移有关。其中，α螺旋率指保持二级结构α螺旋的肽段数量占总肽段数量的百分比。
表2圆二色谱法测定多肽α螺旋率
多肽名称 A2 S1 S2 S3 S4 S5 S6 S7
α螺旋率 0.87％ 42.1％ 39.6％ 50.2％ 47.8％ 46.0％ 41.6％ 45.0％
实施例4免疫共沉淀的方法验证多肽在细胞水平抑制蛋白p62与TRB3的结合
其中，免疫共沉淀有关试剂如下：
裂解液A液：0.6057g Tris碱、1.7532g NaCl、0.1017g MgCl2·6H2O、0.0742g EDTA、 10mL甘油和10mL 10％(v/v)NP40，加去离子水至150mL，用HCl调pH值至7.6，定容至191mL，充分混匀，0.45μm滤膜过滤，4℃储存。
裂解液B液：200μL 2Mβ-磷酸甘油、4mL 2.5M NaF、2mL 100mM PMSF、200μL 1M DTT和1mg/mL的Leu、Pep及Apr各200μL，总体积9mL，于-20℃储存。其中，2Mβ-磷酸甘油、2.5M NaF、100mM PMSF、1M DTT和1mg/mL的Leu、Pep及Apr均以母液形式储存，除PMSF的母液以甲醇为溶剂配制以外，其余均以水为溶剂。使用时，将裂解液B液解冻，按裂解液B液:裂解液A液1:100体积比将裂解液B液加入裂解液A液中并混匀。
免疫共沉淀洗液：1％(v/v)NP40、150mM NaCl、20mM Hepes、pH 7.5 10％(v/v)甘油和1mM EDTA。
Protein A/G Plus-Agarose购自美国Santacruz公司。
具体操作步骤如下：
1.将肝癌HepG2细胞(购自中国医学科学院基础医学研究所)铺90mm2培养皿，待细胞贴壁后分别加入1mg/mL的实施例1制得的多肽A1、S1、S2、S3、S4、S5、S6或S7，在37℃孵育箱中孵育12小时后收集细胞。
2.以100:1的体积比将裂解液A液和裂解液B液配置成10mL裂解液，加550μl裂解液裂解步骤1收集的细胞，收获细胞中总蛋白，将各组蛋白调整至相同浓度。每组蛋白各取200μg，作为对照。
3.分别取200μg步骤2所得的各组蛋白，加入2μg P62抗体(购自Sigma公司)，同时加入10μL Protein A/G Plus-Agarose(购自Santa Cruze公司)充分重悬，4℃缓慢旋转摇动过夜。4℃、3000rpm离心5min，小心吸除上清。加入0.5mL免疫共沉淀洗液，混匀，冰浴静置1min后4℃、3000rpm离心30秒，小心吸除上清。重复洗涤5次，最后一次离心前静置5min。小心吸除上清，加入30μL 2×SDS凝胶加样缓冲液，混匀，95℃变性3min，迅速转移至冰浴冷却。12000rpm室温离心2min，上清即为沉淀的蛋白样品，取部分或全部进行SDS-聚丙烯酰胺凝胶电泳。结果如图2所示。图2的结果说明，多肽S1、S2、S3、S4、S5、S6和S7干扰TRB3/p62蛋白相互作用能力明显高于多肽A2。
实施例5细胞计数实验验证多肽S1、S2、S3、S4、S5、S6和S7可以抑制肿瘤细胞的生长
具体操作步骤如下：
1.收集对数生长期的肝癌细胞HepG2(购自中国医学科学院基础医学研究所)、肺癌细胞A549(购自中国医学科学院基础医学研究所)、结肠癌细胞HCT-8(购自中国医学 科学院基础医学研究所)、乳腺癌细胞MDA-MB-231(购自中国医学科学院基础医学研究所)和白血病细胞K562(购自中国医学科学院基础医学研究所)，调整细胞浓度，制成15万个/mL的细胞悬液。
2.将1mL步骤1所制得的细胞悬液加入12孔板进行培养(其中HepG2、A549、HCT-8和MDA-MB-231细胞所用培养基为DMEM培养基，K562细胞所用培养基为1640培养基，均购自Invitrogen公司；培养温度为37℃，培养基体积为1mL)，12小时后换成新的培养基，并且加入1μg/mL实施例1所制得的多肽S1、S2、S3、S4、S5、S6和S7。每隔一天进行传代一次，并进行计数。培养12天后绘制出生长曲线。实验结果见图3-7。图3-7说明多肽S1、S2、S3、S4、S5、S6和S7相比于A2更能够抑制肿瘤细胞的生长。其中，多肽S1相对于多肽A2对肺癌细胞的生长的抑制水平高达4倍，多肽S2相对于多肽A2对肝癌和结肠癌细胞的生长的抑制水平高达3倍，多肽S3相对于多肽A2对肺癌细胞的生长的抑制水平高达3倍，多肽S4相对于多肽A2对乳腺癌细胞的生长的抑制水平高达3倍，多肽S5相对于多肽A2对肺癌细胞的生长的抑制水平高达3倍，多肽S6相对于多肽A2对白血病细胞的生长的抑制水平高达3倍，多肽S7相对于多肽A2对乳腺癌细胞的生长的抑制水平高达3倍。
实施例6细胞划痕实验验证多肽A1、S1、S2、S3、S4、S5、S6和S7抑制肿瘤细胞划痕后的愈合
具体操作步骤如下：
1.先用记号笔在6孔板背后，用直尺比着划横线，横穿过孔。
2.在每个孔中分别加入5×105个肿瘤细胞，在DMEM培养基中37℃孵育箱培养过夜后细胞贴壁。该肿瘤细胞为对数生长期的肝癌细胞HepG2、肺癌细胞A549、结肠癌细胞HCT-8和乳腺癌细胞MDA-MB-231。
3.第二天用枪头比着直尺，尽量垂直于背后的横线进行划痕，枪头要垂直。
4.用PBS洗细胞3次，去除划下的细胞，加入新的培养基，同时加入1μg/mL实施例1所制备的多肽A1、S1、S2、S3、S4、S5、S6和S7。
5.然后放入37℃5％(v/v)CO2培养箱培养，24小时后取样拍照。实验结果见图8-11和表3-6。表3-6的结果说明，损伤修复面积比越大说明肿瘤细胞的迁移能力越强，细胞划痕后愈合能力越强。因此多肽S1、S2、S3、S4、S5、S6和S7可以降低肿瘤细胞划痕后的愈合能力。
表3多肽S1-S7和A2抑制肝癌细胞HepG2迁徙
多肽名称 损伤修复面积比
A2(对照) 79.4±1.05
S1 22.6±0.33
S2 18.7±0.51
S3 19.8±0.69
S4 12.1±0.47
S5 13.3±0.31
S6 29.1±0.41
S7 20.0±0.35
表4多肽S1-S7和A2抑制肺癌细胞A549迁徙
多肽名称 损伤修复面积比
A2(对照) 92.3±0.85
S1 28.7±0.83
S2 10.3±0.91
S3 24.5±0.90
S4 12.4±0.88
S5 24.4±0.92
S6 10.8±0.84
S7 10.3±0.66
表5多肽S1-S7和A2抑制结肠癌细胞HCT-8迁徙
多肽名称 损伤修复面积比
A2(对照) 67.7±2.33
S1 24.5±1.97
S2 18.2±2.30
S3 12.7±2.37
S4 12.6±2.29
S5 12.5±2.60
S6 12.1±1.92
S7 11.2±1.01
表6多肽S1-S7和A2抑制乳腺癌细胞MDA-MB-231迁徙
多肽名称 损伤修复面积比
A2(对照) 91.3±3.22
S1 36.7±2.91
S2 20.3±2.11
S3 10.5±1.92
S4 21.7±3.35
S5 20.4±4.47
S6 10.4±1.96
S7 17.9±1.05
实施例7克隆形成实验验证多肽A1、S1、S2、S3、S4、S5、S6和S7抑制白血病细胞的克隆形成
其操作步骤如下：
1.铺下层琼脂：5％(w/w)琼脂沸水浴至完全融化，冷却至50℃，加9倍体积37℃预温的1640培养液(购自Invitrogen公司)，混匀，加入24孔板，每孔0.8mL，室温凝固备用。
2.铺上层琼脂：9.4mL细胞悬液中加入0.6mL 50℃5％(w/w)琼脂，混匀，加入已铺好下层琼脂的24孔板，每孔0.8mL。室温凝固。每孔细胞数100个。其中，细胞悬液的制备方法为：将白血病细胞K562用1640培养液稀释，调整浓度为132个细胞/mL。
3.将步骤2所得的细胞用1640培养液在37℃孵育箱中孵育培养3周，计算克隆形成数量。
其结果如图12和表7所示。表7的结果说明，多肽S1-S7相对于多肽A2对白血病细胞克隆形成的抑制水平显著提高。
表7多肽S1-S7和A2抑制白血病细胞的克隆形成
多肽名称 克隆形成数(个)
A2(对照) 110±2.94
S1 53±5.7
S2 47±2.3
S3 27±5.5
S4 30±5.4
S5 34±6.1
S6 29±5.3
S7 19±2.1
实施例8肿瘤皮下生长实验验证多肽A2、S1、S2、S3、S4、S5、S6和S7抑制肿瘤细胞在小鼠体内的生长
操作步骤如下：
1.实验耗材及试剂：灭菌EP管1.5mL，15mL离心管，枪头，滤网(100目)，脱脂棉球，镊子数把，酒精棉球，无菌1mL注射器，500mL烧杯(灭菌，用前照紫外)，PBS(过滤)，胰酶，血清。
2.实验动物及分组：4-6周龄雄性裸鼠80只(购自北京维通利华实验动物有限公司)，随机分为8组：A2、S1、S2、S3、S4、S5、S6和S7组，每组10只。
3.细胞制备：将贴壁培养的肿瘤细胞用胰酶消化，到达胰酶消化时间后(此时细胞状态应为单细胞且刚好贴壁不掉)，吸掉胰酶。用含有1％血清的PBS按2mL/皿终止，将细胞吹下，移至15mL离心管中，1200转离心5min。弃上清，PBS重悬，过100目滤网一次；细胞计数，调整细胞终浓度至2.5×107/mL。该肿瘤细胞为对数生长期的肝癌细胞HepG2、肺癌细胞A549、结肠癌细胞HCT-8和乳腺癌细胞MDA-MB-231。悬浮的白血病细胞K562直接收集至15mL离心管，1200转离心5min。弃上清，PBS重悬，过100目滤网一次；细胞计数，调整细胞终浓度至2.5×107/mL。
4.肿瘤细胞接种：接种5×106个肿瘤细胞(细胞悬液200μl)于裸鼠左上腹部近腋下皮下。
5.肿瘤生长观察：皮下注射肿瘤细胞后一周用多肽进行治疗(5mg/kg体重，每周两次)，游标卡尺纪录肿瘤大小。
其结果如表8-表12所示，各表中多肽S1-S7与对照组A2比较的p值均小于0.001。肿瘤体积越大表明肿瘤生长越快，因此多肽S1、S2、S3、S4、S5、S6和S7可以抑制肿瘤细胞在小鼠体内生长。
表8多肽S1-S7和A2抑制肝癌细胞HepG2在小鼠体内生长
多肽名称 肿瘤体积(mm3)
A2(对照) 2255±208.7
S1 628.2±98.7
S2 608.8±71.7
S3 591.8±69.7
S4 678.1±78.9
S5 696.3±88.3
S6 709.1±94.1
S7 659.8±55.3
表9多肽S1-S7和A2抑制结肠癌细胞HCT-8在小鼠体内生长
多肽名称 肿瘤体积(mm3)
A2(对照) 1754±107.9
S1 583.4±68.2
S2 501.8±81.9
S3 488.8±62.4
S4 496.5±81.7
S5 606.4±74.3
S6 511.1±54.6
S7 619.8±75.5
表10多肽S1-S7和A2抑制乳腺癌细胞MDA-MB-231在小鼠体内生长
多肽名称 肿瘤体积(mm3)
A2(对照) 2379.4±165.4
S1 666.1±74.3
S2 718.7±67.5
S3 570.8±69.4
S4 730.1±84.7
S5 737.3±83.1
S6 629.5±54.1
S7 598.8±75.9
表11多肽S1-S7和A2抑制肺癌细胞A549在小鼠体内生长
多肽名称 肿瘤体积(mm3)
A2(对照) 2065.8±144.5
S1 722.6±86.3
S2 718.7±67.5
S3 692.8±69.7
S4 652.1±57.4
S5 733.3±93.1
S6 649.1±74.1
S7 720.0±83.5
表12多肽S1～S7和A2抑制白血病细胞K562在小鼠体内生长
多肽名称 肿瘤体积(mm3)
A2(对照) 1996.4±1.05
S1 572.6±63.9
S2 638.9±75.1
S3 699.8±69.0
S4 722.1±94.7
S5 703.3±73.6
S6 693.1±54.8
S7 589.9±75.2
上述实施例结果表明，本发明的多肽具有显著的抗肿瘤作用，可作为活性成份用于制备抗肿瘤的药物。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。
Figure PCTCN2016102897-appb-000001
Figure PCTCN2016102897-appb-000002
Figure PCTCN2016102897-appb-000003

Claims (10)

  1. 一种特异性结合TRB3的多肽，其特征在于，所述多肽的氨基酸序列如将序列表SEQ ID No.8所示的氨基酸序列中的两个或两个以上的氨基酸替换为侧链可相连的非天然氨基酸所示。
  2. 如权利要求1所述的特异性结合TRB3的多肽，其特征在于，所述的使其他侧链相连的非天然氨基酸为S-戊烯丙氨酸。
  3. 如权利要求2所述的特异性结合TRB3的多肽，其特征在于，所述替换的氨基酸的数目为两个且所述替换的氨基酸的位置分别为第i位和第i+3位，或者，为第i位和第i+4位，其中1≤i≤7。
  4. 如权利要求3所述的特异性结合TRB3的多肽，其特征在于，所述的多肽的氨基酸序列如序列表SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6和SEQ ID No.7中任一项所示。
  5. 如权利要求1-4中任一项所述的特异性结合TRB3的多肽在制备治疗和/或预防肿瘤的药物中的应用。
  6. 如权利要求5所述的应用，其特征在于，所述的肿瘤为肝癌、肺癌、乳腺癌、肠癌或白血病。
  7. 如权利要求6所述的应用，其特征在于，所述的肝癌为原发性肝癌或继发性肝癌；所述肺癌为小细胞肺癌或非小细胞肺癌；所述乳腺癌为非浸润性乳腺癌、早期浸润性乳腺癌、浸润性特殊类型乳腺癌或浸润性非特殊类型乳腺癌；所述肠癌为结肠癌或直肠癌；所述白血病为淋巴细胞型白血病或非淋巴细胞型白血病。
  8. 一种抗肿瘤的药物组合物，其特征在于，其含有如权利要求1-4中任一项所述的特异性结合TRB3的多肽。
  9. 如权利要求8所述的药物组合物，其特征在于，其还包括一种或多种药用载体。
  10. 如权利要求8所述的药物组合物，其特征在于，其含有如权利要求1-4中任一项所述的特异性结合TRB3的多肽作为活性成分；或者，其含有如权利要求1-4中任一项所述的特异性结合TRB3的多肽和其他具有抗肿瘤活性的化合物一起作为活性成分。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107629114A (zh) * 2017-08-18 2018-01-26 胡卓伟 多肽、其衍生物及其在制备抗肺纤维化的药物中的应用
CN112014564A (zh) * 2020-09-07 2020-12-01 中南大学湘雅医院 p62/SQSTM1在制备PD-L1/PD-1单抗肿瘤免疫治疗药物中的应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110498850B (zh) * 2018-05-17 2021-04-09 胡卓伟 多肽、其衍生物及其在制备防治肿瘤的药物中的应用
CN111333699A (zh) * 2018-12-19 2020-06-26 北京伟峰益民科技有限公司 一种多肽或其衍生物及其在制备防治肿瘤的药物中的应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007146654A1 (en) * 2006-06-08 2007-12-21 The Salk Institute For Biological Studies Methods for identifying candidate fat-mobilizing agents
CN104211765A (zh) * 2013-05-29 2014-12-17 中国医学科学院药物研究所 与trb3蛋白特异性结合的多肽，其筛选方法、鉴定和用途
CN104740604A (zh) * 2013-12-25 2015-07-01 胡卓伟 一种多肽及其衍生物在制备抗肺纤维化药物中的应用
CN104740606A (zh) * 2013-12-25 2015-07-01 胡卓伟 一种多肽在制备治疗或预防糖尿病性心肌病药物中的应用
CN104740605A (zh) * 2013-12-25 2015-07-01 胡卓伟 一种多肽在制备治疗或预防代谢综合征的药物中的应用
CN104740603A (zh) * 2013-12-25 2015-07-01 胡卓伟 一种多肽在制备治疗或预防类风湿性关节炎药物中的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5962224A (en) * 1995-12-19 1999-10-05 Dana-Farber Cancer Institute Isolated DNA encoding p62 polypeptides and uses therefor

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007146654A1 (en) * 2006-06-08 2007-12-21 The Salk Institute For Biological Studies Methods for identifying candidate fat-mobilizing agents
CN104211765A (zh) * 2013-05-29 2014-12-17 中国医学科学院药物研究所 与trb3蛋白特异性结合的多肽，其筛选方法、鉴定和用途
CN104740604A (zh) * 2013-12-25 2015-07-01 胡卓伟 一种多肽及其衍生物在制备抗肺纤维化药物中的应用
CN104740606A (zh) * 2013-12-25 2015-07-01 胡卓伟 一种多肽在制备治疗或预防糖尿病性心肌病药物中的应用
CN104740605A (zh) * 2013-12-25 2015-07-01 胡卓伟 一种多肽在制备治疗或预防代谢综合征的药物中的应用
CN104740603A (zh) * 2013-12-25 2015-07-01 胡卓伟 一种多肽在制备治疗或预防类风湿性关节炎药物中的应用

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107629114A (zh) * 2017-08-18 2018-01-26 胡卓伟 多肽、其衍生物及其在制备抗肺纤维化的药物中的应用
CN112014564A (zh) * 2020-09-07 2020-12-01 中南大学湘雅医院 p62/SQSTM1在制备PD-L1/PD-1单抗肿瘤免疫治疗药物中的应用
CN112014564B (zh) * 2020-09-07 2023-03-21 中南大学湘雅医院 p62/SQSTM1在制备PD-L1/PD-1单抗肿瘤免疫治疗药物中的应用

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