WO2013026334A1 - 阻止细胞dna合成、抑制细胞增殖的多肽及其用途 - Google Patents

阻止细胞dna合成、抑制细胞增殖的多肽及其用途 Download PDF

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WO2013026334A1
WO2013026334A1 PCT/CN2012/078378 CN2012078378W WO2013026334A1 WO 2013026334 A1 WO2013026334 A1 WO 2013026334A1 CN 2012078378 W CN2012078378 W CN 2012078378W WO 2013026334 A1 WO2013026334 A1 WO 2013026334A1
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polypeptide
cell
aspartate
cells
ptd
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PCT/CN2012/078378
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French (fr)
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胡俊波
夏献民
王桂华
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常州德健生物科技有限公司
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Priority to EP12825332.5A priority Critical patent/EP2749568B1/en
Priority to ES12825332.5T priority patent/ES2607648T3/es
Publication of WO2013026334A1 publication Critical patent/WO2013026334A1/zh
Priority to US14/187,354 priority patent/US20140179619A1/en
Priority to US15/071,178 priority patent/US9498510B2/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention belongs to the field of medical bioengineering and relates to a polypeptide which prevents cell DNA synthesis from inhibiting cell proliferation and uses thereof. Background technique
  • a tumor is a disease that seriously harms human health.
  • Chemotherapy, radiotherapy and surgical treatment are mainly used in the clinic. These methods have formed a relatively mature treatment system and achieved good results.
  • due to the low selectivity of currently used therapeutic drugs it is difficult to avoid the disadvantages of strong toxic and side effects, small therapeutic window between normal cells and tumors, and there is limited development potential to further improve the therapeutic effect and even achieve the purpose of curing. Therefore, it is urgent to develop new effective treatments. method.
  • BCR-ABL protein is expressed in many leukemia cells.
  • the protein has strong protein kinase activity and plays an important role in the occurrence and development of leukemia.
  • Gleevec is currently the drug of choice for the treatment of a variety of leukemia and other tumors.
  • PCNA proliferating cell nuclear antigen
  • the polypeptide of the present invention has a biological function of blocking PCNA to promote DNA synthesis and replication of cells, thereby inhibiting cell division, and the polypeptide is also effective for inhibiting tumor growth in a growth model of a plurality of tumor animals.
  • a polypeptide that prevents cellular DNA synthesis from inhibiting cell proliferation characterized in that its amino acid sequence is: methionine-valine-tyrosine-serine-threonine-glutamate-leucine-isoleucine- Benzine-tyrosine-isoleucine-glutamate-methionine-aspartate-valine.
  • polypeptide which prevents cell DNA synthesis from inhibiting cell proliferation is useful in the preparation of a medicament for treating diseases associated with tumors and other abnormal cell growth.
  • the corresponding nucleotide coding sequence of the polypeptide which prevents cellular DNA synthesis from inhibiting cell proliferation.
  • nucleotide coding sequence of the polypeptide which prevents cell DNA synthesis from inhibiting cell proliferation is useful in the preparation of a medicament for treating diseases associated with human tumors and other cell growth abnormalities.
  • PTD-P15 fusion polypeptide characterized in that its amino acid sequence is: arginine-aspartate-leucine-tyrosine-aspartate-aspartate-aspartate-aspartate -lysine-aspartate-arginine-methionine-valine-tyrosine-serine-threonine-glutamate-leucine-isoleucine-phenylalanine - tyrosine-isoleucine - glutamate-methionine-aspartate-valine.
  • PTD-P15 fusion polypeptides in the preparation of a medicament for the treatment of diseases associated with human tumors and other abnormal cell growth.
  • the key to the above model is to introduce the P15 polypeptide into the cell and observe the effect of the P15 polypeptide on cell division.
  • the construct encodes a fusion protein consisting of P15 and green fluorescent protein GFP, which is then transferred into cultured human cells, and then the cell cycle changes in cells expressing the P15-GFP fusion protein are observed.
  • fusion polypeptide PTD-P15 consisting of a polypeptide fragment that penetrates the cell membrane and a P15 polypeptide by artificial synthesis; the sequence of PTD is arginine-aspartate-leucine-tyrosine Acid-aspartate-aspartate-aspartate-aspartate-lysine-aspartate-arginine.
  • PTD-P15 was added to the cultured cells, and the effect of the P15 polypeptide entering the cells on the cell cycle was observed. To observe the effect of P15 polypeptide on tumor growth in animal models, we investigated whether PTD-P15 inhibited tumor growth in animal models by injecting PTD-P15 fusion polypeptide into the tail vein of mice.
  • P15 polypeptide was expressed in several different cell lines with growth and division ability or PTD-P15 was added.
  • P15 inhibited the ability of PCNA to bind DNA polymerase, resulting in DNA synthesis of these cells and several other proven cells.
  • the growth indicators were significantly reduced, demonstrating that P15 polypeptide has the ability to inhibit cell proliferation; meanwhile, injection of PTD-P15 polypeptide can inhibit cell proliferation and inhibit tumor growth in animal models.
  • the present invention achieves the following effects:
  • P15 blocks the ability of PCNA to bind to DNA polymerase, thereby inhibiting the role of DNA polymerase in promoting DNA replication, leading to the cessation of the cell cycle, providing a new treatment for the design and screening of new drugs for the treatment of tumor and other abnormal cell growth disorders. Ways and means.
  • PTD-P15 fusion polypeptide can effectively inhibit tumor growth, which proves that P15 polypeptide still has the biological effect of inhibiting cell growth after penetrating cell membrane, and also indicates that polypeptide or molecule with transmembrane function may also be used to help biology.
  • the active P15 penetrates the cell membrane.
  • P15 polypeptide has low toxic side effects and weak antigenicity.
  • the experimental results also found that P15 polypeptide has no obvious effect on apoptosis, so there is no obvious killing effect on normal cells.
  • the PTD-P15 fusion polypeptide was added to the cultured cells and applied to the animals, and no significant toxicity was observed.
  • P15 polypeptide and PTD-P15 fusion polypeptide in cells can effectively inhibit the growth of various human and murine tumor cells in vivo and in vitro culture systems, which proves that P15 is highly efficient and widely used in the treatment of abnormal cell growth diseases such as tumors. Spectrum and other advantages.
  • FIG. 1 PTD-N15 blocks PCNA binding to DNA polymerase.
  • Human rectal cancer cell line HT29 was cultured in culture medium, and control polypeptide or different amount of PTD-P15 polypeptide was added respectively. After the cells were cultured overnight, the cells were collected, and the amount of PCNA protein-binding DNA polymerase in the cells was examined by immunoprecipitation. The results show that the addition of PTD-P15 can reduce the amount of PCNA-bound DNA polymerase. Since the amount of DNA polymerase bound to PCNA is closely related to cellular DNA replication, PTD-P15 can inhibit the DNA replication speed of cells. Blocking the ability of PCNA to bind to DN A polymerase inhibits DNA polymerase from promoting DNA replication and blocks cell division.
  • FIG 2a, Figure 2b, Figure 2c, Figure 2d PTD-P15 inhibits the cell cycle, respectively.
  • the human rectal cancer cell line HT29 was cultured in a culture, and a control polypeptide or a different amount of PTD-P15 polypeptide was separately added. After the cells were cultured for 24 hours, the cells were collected, and the percentage of the cells in different cell cycles was measured. The results showed that PTD-P15 increased the number of cells in Go/d phase and decreased the number of cells in S phase and G 2 /M phase, indicating that PTD-P15 inhibited cell cycle.
  • Detailed ways showed that PTD-P15 increased the number of cells in Go/d phase and decreased the number of cells in S phase and G 2 /M phase, indicating that PTD-P15 inhibited cell cycle.
  • the invention is illustrated by the specific experiments and the results of the data, but it should be noted that these examples do not limit the invention.
  • the P15 polypeptide demonstrated its effectiveness in inhibiting cell proliferation and tumor growth in the following experiments, including but not limited to the following experiments.
  • Example 1 The P15 polypeptide cDNA was introduced into the pEGFP plasmid, and the DNA construct expressed the P15-GFP fusion protein.
  • the pEGFP-N 1 plasmid vector was purchased from Clontech, Inc. (catalog number: 6085-1), and the plasmid contained cDNA encoding green fluorescent protein (GFP) using EcoRI-BamHI (purchased from Promega, USA, catalog number: R6011, R6021)
  • GFP green fluorescent protein
  • EcoRI-BamHI purchased from Promega, USA, catalog number: R6011, R6021
  • the plasmid was digested, and the digested plasmid was separated and purified in agarose gel for subsequent ligation reaction.
  • the kit for recovering DNA fragments from the gel was obtained from Qiagen, Germany (catalog number: 28704).
  • the cDNA encoding the P15 polypeptide is derived from synthetic double-stranded DNA, the sequence of which is:
  • the double-stranded DNA was combined and the product was purified (purification kit from Qiagen, Germany, catalog number: 28704), digested with EcoRI-BamHI, and purified with agarose gel. Then, the vector pEGFP-N1 was subjected to ligation reaction (the connection kit was obtained from Promega, USA, catalog number: M1801).
  • the transfection kit was purchased from Invitrogen (catalog number 11668) in the United States, and the transfection experiments were also performed according to the instructions provided by the manufacturer.
  • COS7 cells were cultured in a concentration of 10% calf serum DMEM nutrient solution. After 48 hours of transfection, COS7 cells were washed twice with normal saline (PBS), lysed by adding cell lysate, disrupted with ultrasonic waves, and then added with appropriate amount of base ethanol and bromophenol blue, and treated in boiling water for 5 minutes. Preserved on water, and then applied to SDS-polyacrylamide gel electrophoresis at a mass concentration of 12%. The isolated protein was transferred to a nylon membrane which was tested for the production of the fusion protein with an anti-GFP antibody (purchased in Invitrogen, USA, catalog number R970), and the expression of P15-GFP in the cells was confirmed.
  • Example 2 Experiments in which P15 polypeptide expression inhibits cell growth and cell cycle.
  • NIH/3T3 mouse fibroblastic cells, purchased from ATCC, USA, catalog number: CRL-1658
  • MCF-7 human breast cancer cell line, purchased from ATCC, USA, catalog number: HTB-22
  • pEGFP-P15 expressing P15-GFP fusion protein
  • pEGFP-N1 as a control plasmid.
  • the plasmid for transfection and the lipofection reagent purchased from Invitrogen, USA, catalog number: 11668
  • the plasmid for transfection and the lipofection reagent were mixed, left at room temperature for 15 minutes, and added to cells cultured in serum-free DMEM nutrient solution (cell density approximately For 50%), culture was carried out for 5 hours at 37 ° C, and 10% fetal bovine serum was added at a mass concentration, and incubation was continued for 48 hours.
  • In fluorescence display Cells expressing fused green fluorescent protein or fluorescent protein alone are easily distinguished from cells that do not express these proteins. In addition, these fluorescent protein-positive cells are also easily isolated and purified by flow cytometry. A marker for cell growth, such as cell cycle, was used to study the effect of P15 polypeptide on cell growth. In the test, only cells expressing fluorescent protein were used as a control group.
  • Example 3 A fusion polypeptide PTD-P15 consisting of a polypeptide having a transmembrane function and a P15 polypeptide was artificially synthesized.
  • P15 polypeptide does not pass through the cell membrane freely.
  • the fusion polypeptide is called PTD-P15, and the amino acid sequence of PTD-P15 is: arginine-aspartate-leucine-tyrosine-aspartate-aspartate-aspartate- Aspartate-lysine-aspartate-arginine-methionine-valine-tyrosine-serine-threonine-glutamate-leucine-isoleucine- Benzene!
  • amino acid sequence of the P15 fragment is: methionine-valine-tyrosine-serine-threonine-glutamate-leucine-isoleucine-phenine-tyrosine- Isoleucine-glutamate-methionine-aspartate-valine;
  • amino acid sequence of a PTD polypeptide fragment having a function of penetrating cell membrane is: arginine-aspartate-leucine-case Aspartate-aspartate-aspartate-aspartate-aspartate-lysine-aspartate-arginine.
  • control peptide whose amino acid sequence is: aspartate-arginine-arginine-aspartate-leucine-tyrosine-aspartate-aspartate - Aspartate-aspartate-lysine-aspartate-arginine-methionine-alanine-glycine-threonine-methionine.
  • Example 4 PTD-N15 blocks PCNA binding to DNA polymerase.
  • the human rectal cancer cell line HT29 was cultured in the culture medium, and the control polypeptide or different amounts of the PTD-P15 fusion polypeptide were separately added. After the cells were cultured overnight, the cells were collected, and the amount of PCNA-binding DNA polymerase in the cells was examined by immunoprecipitation. The results show that PTD-P15 can reduce the amount of DNA polymerase bound by PCNA protein. Since the amount of DNA polymerase bound to PCNA is closely related to cellular DNA replication, PTD-P15 can inhibit the DNA replication speed of cells. Blocking the ability of PCNA to bind to DNA polymerase inhibits DNA polymerase from promoting DNA replication and blocks cell division (Fig. 1).
  • Example 5 PTD-P15 fusion polypeptide affects cell growth and cell cycle experiments.
  • the effect of the PTD-P15 fusion polypeptide on cell growth was examined using a human cervical cancer cell line, Hela cells purchased from ATCC, USA, catalog number: HTB-22. Hela cells were cultured in 10 cm cell culture medium in DMEM medium containing 10% fetal bovine serum at 37 ° C, 5% (by volume) C0 2 /95% (by volume), and the cells were cultured to After the logarithmic growth phase, different concentrations of PTD-P15 polypeptide were added (concentrations: 8, 30 and 50 ⁇ g/ml, respectively), and control peptides (blank control group) were added to the other culture cells for control, culture 24 Cells were collected at an hour, and cell cycle distribution was analyzed by flow cytometry.
  • Example 6 PTD-P15 fusion polypeptide inhibits the growth of human rectal cancer tumors in mice.
  • mice were randomly divided into two groups. One day after the injection of the tumor cells, 1 mg of the PTD-P15 fusion polypeptide was injected into the mice in the treatment group through the tail vein, and the same amount was repeated five times every other day. The number and timing of 1 mg of control polypeptide injections were the same as in the treatment group. Ten days after the tumor was injected, the tumor was sacrificed and the tumor was taken out from the mouse for weight measurement. The results showed that PTD-P15 was injected compared with the control group. Tumor weight in treated mice was reduced by 57%.
  • Example 7 PTD-P15 fusion polypeptide inhibits the growth of human leukemia tumors in mice.
  • mice After subcutaneous injection of 5 ⁇ 10 6 human granulocyte leukemia K562 cells into mice, the mice were randomly divided into five groups. One day after inoculation of tumor cells, mice in each group were injected with 1 mg of blank solution, 1 mg of control peptide, 1 mg of PTD-P15 fusion peptide, Gleevec 1 (H ⁇ and PTD-P151 mg plus Gleevec). 1 (H gram solution, the same injection was repeated every other day, 7 injections, two weeks later, after the animals were sacrificed, the tumors of the mice were taken and weighed. The results showed that compared with the control group, PTD- was injected. P15 can significantly reduce the weight of tumors produced by K562 cells, which is reduced by 34%.
  • Gleevec is currently used clinically as a drug for the treatment of leukemia. In this experiment, Gleevec injection also reduced tumor weight by 48%. If PTD-P15 is combined with Gleevec, the weight of tumors in mice is more reduced than that of the two drugs alone, which is 69% lower than that of the control peptide group, which proves that PTD-P15 is combined. Gleevec has a better therapeutic effect on tumors than two drugs alone, see Table 3.
  • PTD-P15 inhibits the growth of tumors formed by K562 cells in an animal model.

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Abstract

本发明公开了一种阻止细胞DNA合成、抑制细胞增殖的多肽，其氨基酸序列为：甲硫氨酸-脯氨酸-酪氨酸-丝氨酸-苏氨酸-谷氨酸-亮氨酸-异亮氨酸-苯丙氨酸-酪氨酸-异亮氨酸-谷氨酸-甲硫氨酸-天门冬氨酸-脯氨酸，以及所述多肽与穿透肽PTD构成的融合多肽。本发明还公开了所述多肽与所述融合多肽在制备治疗肿瘤及其他细胞生长异常有关疾病的药物中的应用。

Description

阻止细胞 DNA合成、 抑制细胞增殖的多肽及其用途 技术领域
本发明属于医学生物工程领域， 涉及一种阻止细胞 DNA合成抑制细胞增殖的多肽 及用途。 背景技术
肿瘤是一种严重危害人类健康的疾病。 临床中主要应用化疗、 放疗及手术治疗， 这 些方法形成了比较成熟的治疗体系， 取得了较好的效果。 然而， 由于目前常用的治疗肿 瘤药物选择性低， 难以避免毒副作用强、 正常细胞和肿瘤之间治疗窗口小的缺点， 要进 一步提高疗效乃至达到治愈目的发展潜力有限， 因此亟待研发新的有效治疗方法。
近来肿瘤细胞生物学的快速发展，为满足这一社会需求提供了现实的可能性，其中， 随着细胞的信号转导途径 (Signaling transduction pathways)的阐明， 发现了越来越多的可 用作治疗肿瘤的药靶。 有一些已经成功地应用于临床， 取得了非常好的效果。 比如在许 多白血病细胞中表达 BCR-ABL蛋白质， 该蛋白质具有较强的蛋白激酶活性， 在白血病 的发生和发展中起重要的作用， 通过研究 BCR-ABL蛋白质的结构， 人们设计和生产了 多种能特异性抑制该蛋白质生物学活性的化合物， 并成功开发出包括格列卫在内的药物 应用于治疗白血病患者， 格列卫是目前治疗多种白血病和其他肿瘤的首选药物。
细胞生长的信号是由细胞内外促进生长的因子经过一系列蛋白参与传递到细胞核， 引起细胞周期的许多调节蛋白结构和功能变化而导致细胞分裂。 在这些调节细胞生长周 期的蛋白中， 增殖细胞核抗原 (PCNA)担当着一个极其重要的角色， 在细胞 DNA复制过 程中， DNA复制叉是 DNA合成的关键蛋白质复合体， PCNA是 DNA复制叉复合体的 组成成分， PCNA可以结合 DNA和多种蛋白质， 特别是 PCNA结合 DNA复制必需的 DNA聚合酶，并使 DNA聚合酶能催化新生 DNA链的合成，参与 DNA复制过程。 PCNA 存在于所有分裂细胞中， 由于 PCNA在细胞周期中的重要作用， 阻断 PCNA在细胞周期 中的生物功能是一个很有前途的开发治疗肿瘤和其他细胞增殖异常疾病新药的途径。 至 今为止， 由于 PCNA参与细胞周期调节的机制还不清楚， 目前没有有效的方法或化合物 能直接阻断 PCNA的生物功能， 达到抑制细胞生长作用。 发明内容
本发明的目的提供一种阻止细胞 DNA合成抑制细胞增殖的多肽及用途。 本发明所 述的多肽具备阻断 PCNA促进细胞 DNA合成和复制的生物学功能，从而抑制细胞分裂， 该多肽也能有效地抑制多种肿瘤动物体内生长模型中的肿瘤生长。
阻止细胞 DNA合成抑制细胞增殖的多肽， 其特征在于其氨基酸序列为： 甲硫氨酸- 脯氨酸 -酪氨酸-丝氨酸-苏氨酸 -谷氨酸-亮氨酸-异亮氨酸-苯 氨酸-酪氨酸-异亮氨酸-谷 氨酸-甲硫氨酸-天门冬氨酸-脯氨酸。
所述阻止细胞 DNA合成抑制细胞增殖的多肽在制备治疗肿瘤及其他细胞生长异常 有关疾病的药物的应用。
所述阻止细胞 DNA合成抑制细胞增殖的多肽相应的核苷酸编码序列。
所述阻止细胞 DNA合成抑制细胞增殖的多肽相应的核苷酸编码序列在制备治疗人 类肿瘤及其他细胞生长异常有关疾病的药物中的应用。
PTD-P15融合多肽， 其特征在于其氨基酸序列为： 精氨酸-天门冬氨酸-亮氨酸 -酪氨 酸-天门冬氨酸-天门冬氨酸-天门冬氨酸-天门冬氨酸 -赖氨酸-天门冬氨酸-精氨酸-甲硫氨 酸 -脯氨酸-酪氨酸-丝氨酸 -苏氨酸-谷氨酸-亮氨酸 -异亮氨酸-苯丙氨酸-酪氨酸-异亮氨酸- 谷氨酸-甲硫氨酸-天门冬氨酸-脯氨酸。
PTD-P15 融合多肽在制备治疗人类肿瘤及其他细胞生长异常有关疾病的药物中的 应用。
在研究中，我们发现了一个多肽片段和 PCNA的相互作用。该片段的序列为：甲硫氨 酸 -脯氨酸 -酪氨酸 -丝氨酸 -苏氨酸 -谷氨酸 -亮氨酸 -异亮氨酸 -苯丙氨酸 -酪氨 酸 -异亮氨酸 -谷氨酸 -甲硫氨酸-天门冬氨酸-脯氨酸； (见《氨基酸和核苷酸序列表》）， 在以后该多肽称为 P15, 同时，我们还发现 P15和 PCNA结合后， 阻止了 PCNA和 DNA 聚合酶的结合， 而结合到 PCNA蛋白质的 DNA 聚合酶是细胞周期中的一个重要过程 -DNA复制必需的。 根据这一结果， 我们设想， 如果在细胞中表达 P15, 这一多肽能抑 制 PCNA和 DNA聚合酶的结合， 从而抑制 DNA聚合酶介导的 DNA复制过程， 达到阻 止细胞分裂过程的目的。
证明上述模型的关键之处在于把 P15多肽导入细胞中， 再观察 P15多肽对细胞分裂 的影响。 为达到这一目的， 我们釆用分子生物学的手段， 制造一个 DNA构建体， 这一 构建体编码由 P15和绿色荧光蛋白 GFP组成的融合蛋白，再把这种构建体转入培养的人 细胞中， 然后观察表达 P15-GFP融合蛋白的细胞中细胞周期的变化。 另外， 我们还通过 人工合成的方式， 生产一个由具有穿透细胞膜的多肽片段和 P15 多肽构成的融合多肽 PTD-P15; PTD的序列为精氨酸-天门冬氨酸-亮氨酸 -酪氨酸-天门冬氨酸-天门冬氨酸-天 门冬氨酸-天门冬氨酸 -赖氨酸-天门冬氨酸-精氨酸。在培养的细胞中加入 PTD-P15 , 再观 察进入细胞的 P15多肽对细胞周期的影响。 为了观察 P15多肽对动物模型中肿瘤生长的 影响， 我们通过小鼠尾静脉注射 PTD-P15融合多肽， 观察 PTD-P15能否抑制动物模型 中肿瘤的生长。
实验结果表明， 在几种不同的具有生长分裂能力的培养细胞系中表达 P15多肽或加 入 PTD-P15, P15抑制了 PCNA结合 DNA聚合酶的能力， 导致这些细胞的 DNA合成及 其他几个证明细胞生长的观察指标都显著降低， 证明了 P15多肽具有抑制细胞增殖的能 力； 同时， 注射 PTD-P15多肽能够抑制细胞增殖和抑制动物模型中肿瘤的生长。
与现有抑制细胞增殖的方法相比， 釆用本发明达到了以下效果：
① P15阻断 PCNA结合 DNA聚合酶的能力， 从而抑制了 DNA聚合酶促进 DNA复 制的作用， 导致细胞周期的停止， 为治疗肿瘤和其他细胞生长异常疾病新药的设计和筛 选， 提供了一种新的方法和途径。 同时， PTD-P15融合多肽能有效地抑制肿瘤生长， 证 明 P15多肽在穿透细胞膜后仍具有抑制细胞生长的生物学效应， 也说明具有穿膜功能的 多肽或分子也可能用于帮助具有生物学活性 P15穿透细胞膜。
② P15多肽毒副作用低， 抗原性弱， 实验结果也发现 P15多肽对细胞凋亡没有明显 的影响， 所以对正常细胞没有明显的杀伤作用。 由 PTD-P15融合多肽加入培养细胞和应 用于动物体内， 也没有观察到明显的毒性。
③在细胞中表达 P15多肽和 PTD-P15融合多肽能有效抑制多种人类和鼠类肿瘤细 胞在体内和体外培养系统中的生长， 证明 P15在肿瘤等细胞生长异常疾病的治疗方面具 有高效、 广谱等优点。
④ PTD-P15多肽分子量小能化学合成， 便于大规模直接应用于临床， 同时也可用其 他的方法（比如用质粒及病毒载体)把 P15 多肽投人到细胞内， 以达到治疗肿瘤和其他细 胞增殖异常疾病的目的。 附图说明
图 1: PTD-N15阻断 PCNA结合 DNA聚合酶。人直肠癌细胞 HT29培养于培养亚中， 分别加入对照多肽或不同量的 PTD-P15多肽， 细胞培养过夜后， 收集细胞， 用免疫共沉 淀法检查细胞中 PCNA蛋白质结合的 DNA聚合酶的量， 结果显示加入 PTD-P15能减少 PCNA结合的 DNA聚合酶量， 因为结合于 PCNA的 DNA聚合酶的量和细胞 DNA复制 密切相关， 所以， PTD-P15能抑制细胞 DNA复制速度。 阻断了 PCNA结合 DN A聚合酶 的能力， 抑制了 DNA聚合酶促进 DNA复制的作用， 阻断了细胞的分裂。
图 2a、 图 2b、 图 2c、 图 2d:分别为 PTD-P15抑制细胞周期。 人直肠癌细胞 HT29培 养于培养 中， 分别加入对照多肽或不同量的 PTD-P15多肽， 细胞培养 24小时后， 收 集细胞， 测定细胞在不同的细胞周期的百分比。 结果显示 PTD-P15能增加 Go/d期细胞 数量， 同时减少 S期和 G2/M期细胞数量， 表明 PTD-P15抑制了细胞周期。 具体实施方式
下面借助具体实验及其数据结果对本发明进行论证说明， 但应该说明的是， 这些实 施例子并不对本发明加以限制。 P15 多肽在下列实验中证明其抑制细胞增殖和肿瘤生长 的有效性， 包括但不限于下列实验。
例 1:在 pEGFP质粒中引入编码 P15多肽 cDNA, DNA构建体表达 P15-GFP融合蛋 白的检测。
pEGFP-N 1质粒载体购于美国 Clontech公司（目录号为: 6085-1), 质粒包含编码绿色 荧光蛋白（GFP)的 cDNA, 用 EcoRI-BamHI (购于美国 Promega公司， 目录号为: R6011, R6021)酶切质粒， 酶切后的质粒在琼脂糖胶中分离、 纯化， 用于以后的连接反应。 从胶 中回收 DNA片段的试剂盒来自德国 Qiagen公司（产品目录号为 :28704 )。
编码 P15多肽的 cDNA来源于人工合成的双链 DNA, 其序列为：
单链 1:
TGGATCC;
单链 2: TGAATTC。
两个单链混合后， 结合成双链 DNA , 产物经过纯化后（纯化用的试剂盒来自德国 Qiagen公司， 产品目录号为 :28704 ) , 用 EcoRI-BamHI酶切;再用琼脂糖胶纯化， 然后和 酶切纯化后的载体 pEGFP-Nl进行连接反应 (连接试剂盒来自美国 Promega公司， 目录号 为: M1801)。 转化细菌后 (感受态细菌来自美国 Promega公司， 目录号为: L2001) ,选出阳 性克隆， 其 cDNA序列的正确性通过核酸序列测定得到证实后， 大规模纯化制备质粒 (大 规模纯化试剂盒来自美国 Promega公司， 目录号为: A7270 ) , 用于以后的实验。 这种质 粒在真核细胞分别表达一个 P15和 GFP的融合蛋白，表达的 P15连接在 GFP的 N未端， 该融合蛋白称为 P15-GFP。
转染试剂盒购于美国 Invitrogen (目录号为 11668 ) , 转染实验也按照生产厂家提供的 使用说明完成。 COS7细胞培养在质量浓度为 10%小牛血清 DMEM营养液中。 转染 48 小时后， COS7细胞用生理盐水 (PBS)洗两次， 加入细胞裂解液裂解细胞， 用超声波破坏 DNA后， 加入适量的 基乙醇和溴酚蓝， 于沸水中处理 5分钟， 置于水上保存， 随后 上样于质量浓度为 12%的 SDS -聚丙烯酰胺凝胶电泳。 分离后的蛋白转移到尼龙膜上， 此膜用抗 GFP抗体 (购于美国 Invitrogen, 目录号为 R970 )检测融合蛋白的产生， 结果证 实了 P15-GFP在细胞中的表达。
例 2: P15多肽的表达抑制细胞生长及细胞周期的实验。
用 NIH/3T3(小鼠成纤维上皮细胞， 购自美国 ATCC , 目录号为： CRL-1658 )和 MCF-7(人乳腺癌细胞系， 购自美国 ATCC, 目录号为: HTB-22)检测 P15多肽对细胞生长 的影响。 细胞在含质量浓度为 10%胎牛血清 (FBS)的 DMEM培养液， 37°C、 5% (体积） C02/95% (体积） 空气培养条件下培养于 10厘米细胞培养亚。
用于实验质粒: pEGFP-P15(表达 P15-GFP融合蛋白）; pEGFP-Nl作为对照质粒。
用于转染的质粒和脂质体转染试剂（购自购于美国 Invitrogen, 目录号为：11668)混合 后， 室温放置 15分钟， 加入培养于无血清 DMEM营养液的细胞中（细胞密度约为 50%) 在 37°C中培养 5小时， 再加质量浓度为 10%胎牛血清， 继续培养 48小时。 在荧光显 : 镜中， 表达融合绿色荧光蛋白或单独荧光蛋白的细胞很容易和不表达这些蛋白的细胞区 分开来。 另外， 釆用流式细胞仪， 这些荧光蛋白阳性细胞也很容易分离纯化出来。 细胞 周期等检测细胞生长的标志指标被用来研究证实 P15多肽对细胞生长的影响， 在这些实 验中， 仅仅只表达荧光蛋白的细胞作为对照组用。
首先， 我们用上述质粒转染 3Τ3和 MCF7细胞， 两天后， 釆用流式细胞仪把转染了 DNA的 GFP阳性细胞挑选出来， 分析这些细胞的细胞周期分布。
实验结果表明：表达 P15对细胞凋亡没有明显影响， 而 P15 能抑制细胞周期进入 S 期， 诱导细胞进入 G0/G1期， 其作用见下表（表 1 )。
表 1： P15抑制细胞周期， 诱导细胞进入 G0/G1期作用
Figure imgf000007_0001
DNA合成是细胞增殖的标志。 下一个实验中， 我们检测 P15对细胞 DNA合成的影 响。 3T3和 MCF7细胞在转染 pEGFP-N 1及表达 P15-GFP质粒两天后，在细胞培养液加 入能渗入到新合成的 DNA链中的 BrdU标记细胞 15小时， 再用免疫荧光技术检测转染 了质粒的 GFP 阳性细胞， 再确定这些细胞 DNA中 BrdU渗入的阳性率 (这代表了细胞 DNA合成率）。 结果表明， P15也强烈抑制了细胞 DNA的合成。 （见表 2)
表 2: P15抑制细胞 DNA合成
Figure imgf000007_0002
例 3: 人工合成由具有穿膜功能的多肽和 P15多肽组成的融合多肽 PTD-P15。
P15多肽并不能自由通透细胞膜， 为了进一步观察 P15对细胞周期和肿瘤生长的影 响，我们人工合成能穿透细胞膜的 P15融合多肽。该融合多肽被称为 PTD-P15, PTD-P15 的氨基酸序列为： 精氨酸-天门冬氨酸-亮氨酸 -酪氨酸-天门冬氨酸-天门冬氨酸-天门冬氨 酸-天门冬氨酸 -赖氨酸-天门冬氨酸-精氨酸-甲硫氨酸 -脯氨酸-酪氨酸-丝氨酸 -苏氨酸-谷 氨酸 -亮氨酸-异亮氨酸 -苯! ¾氨酸 -酪氨酸-异亮氨酸 -谷氨酸-甲硫氨酸 -天门冬氨酸 -脯氨 酸； 其中 P15片段的氨基酸序列为： 甲硫氨酸-脯氨酸 -酪氨酸-丝氨酸-苏氨酸 -谷氨酸-亮 氨酸-异亮氨酸-苯 氨酸-酪氨酸-异亮氨酸-谷氨酸-甲硫氨酸-天门冬氨酸-脯氨酸； 具有 穿透细胞膜功能的 PTD多肽片段氨基酸序列为： 精氨酸-天门冬氨酸-亮氨酸 -酪氨酸-天 门冬氨酸-天门冬氨酸-天门冬氨酸-天门冬氨酸 -赖氨酸-天门冬氨酸-精氨酸。
我们同时设计和合成了一个对照多肽， 其氨基酸序列为： 天门冬氨酸-精氨酸 -精氨 酸-天门冬氨酸-亮氨酸 -酪氨酸-天门冬氨酸-天门冬氨酸-天门冬氨酸-天门冬氨酸 -赖氨酸- 天门冬氨酸-精氨酸-甲硫氨酸-丙氨酸 -甘氨酸-苏氨酸-甲硫氨酸。
例 4: PTD-N15阻断 PCNA结合 DNA聚合酶。
人直肠癌细胞 HT29培养于培养亚中， 分别加入对照多肽或不同量的 PTD-P15融合 多肽， 细胞培养过夜后， 收集细胞， 用免疫共沉淀法检查细胞中 PCNA结合的 DNA聚 合酶的量， 结果显示 PTD-P15能减少 PCNA蛋白质结合的 DNA聚合酶量， 因为结合于 PCNA的 DNA聚合酶的量和细胞 DNA复制密切相关，所以， PTD-P15能抑制细胞 DNA 复制速度。 阻断了 PCNA结合 DNA聚合酶的能力， 抑制了 DNA聚合酶促进 DNA复制 的作用， 阻断了细胞的分裂（图 1 )。
例 5: PTD-P15融合多肽影响细胞生长及细胞周期的实验。
用人宫颈癌细胞系，购自美国 ATCC, 目录号为： HTB-22的 Hela细胞检测 PTD-P15 融合多肽对细胞生长的影响。 Hela细胞在含质量浓度为 10% 胎牛血清的 DMEM培养液， 37 °C, 5% (体积） C02/95% (体积） 空气培养条件下培养于 10厘米细胞培养亚， 将细胞 培养至对数生长期后， 加入不同浓度的 PTD-P15多肽（浓度分别为： 8,30和 50微克 /毫 升）， 同时在另外的培养细胞中加入对照多肽（空白对照组）用以对照， 培养 24小时收 取细胞， 利用流式细胞仪分析细胞周期分布。 实验结果表明： PTD-P15融合多肽抑制细 胞增殖， 增加 Go/Gi期细胞数,同时， 多肽对细胞凋亡没有明显影响， 见图 2a、 图 2b、 图 2c、 图 2d。
例 6: PTD-P15融合多肽抑制小鼠体内人类直肠癌肿瘤生长的实验。
在 '』、鼠的皮下注射 5x 106个人类直肠癌 HT29细胞后， 将小鼠随机分成两组。 注射 肿瘤细胞一天之后， 1毫克的 PTD-P15融合多肽通过尾静脉向治疗组中的小鼠分别注射， 隔天注射同样的量重复五次。 1 毫克的对照多肽注射次数和时间同治疗组。 注射肿瘤十 天后杀死并从小鼠体内取出肿瘤进行重量测量。结果显示，与对照组相比，注射 PTD-P15 治疗的小鼠体内的肿瘤重量减少了 57%。
例 7: PTD-P15融合多肽抑制小鼠体内人类白血病肿瘤生长的实验。
在小鼠的皮下注射 5xl06个人类粒细胞白血病 K562细胞后， 将小鼠随机分成五组。 接种肿瘤细胞一天以后， 各组小鼠分别通过尾静脉注射空白溶液 1毫克、 对照多肽 1毫 克、 PTD-P15融合多肽 1毫克、格列卫 1(H敫克和 PTD-P151毫克加格列卫 1(H敫克溶液， 同样的注射隔天重复， 注射 7次， 两个星期后， 动物处死后， 取小鼠皮下长的肿瘤， 进 行称重。 结果显示与对照组相比， 注射 PTD-P15能明显减少 K562细胞产生的肿瘤的重 量， 减少达到 34%。 格列卫是目前临床上用作治疗白血病的药物， 在本实验中， 注射格 列卫也减少了肿瘤的重量减少 48% , 如果把 PTD-P15和格列卫结合起来应用， 小鼠体内 的肿瘤的重量比单独用这两个药物有更多的减少， 和对照多肽组相比， 减少 69% , 证明 合用 PTD-P15和格列卫比单独用两个药物具有更好的治疗肿瘤效果， 见表 3。
表 3: PTD-P15抑制动物模型中 K562细胞形成的肿瘤的生长。
Figure imgf000009_0001

Claims

权利要求 书
1、 阻止细胞 DNA合成抑制细胞增殖的多肽， 其特征在于其氨基酸序列为： 甲硫氨 酸 -脯氨酸-酪氨酸-丝氨酸 -苏氨酸-谷氨酸-亮氨酸 -异亮氨酸-苯丙氨酸-酪氨酸-异亮氨酸- 谷氨酸 -甲硫氨酸-天门冬氨酸-脯氨酸。
2、根据权利要求 1所述阻止细胞 DNA合成抑制细胞增殖的多肽在制备治疗肿瘤及 其他细胞生长异常有关疾病的药物的应用。
3、根据权利要求 1所述阻止细胞 DNA合成抑制细胞增殖的多肽相应的核苷酸编码 序列。
4、根据权利要求 3所述阻止细胞 DNA合成抑制细胞增殖的多肽相应的核苷酸编码 序列在制备治疗人类肿瘤及其他细胞生长异常有关疾病的药物中的应用。
5、 PTD-P15融合多肽， 其特征在于其氨基酸序列为： 精氨酸 -天门冬氨酸 -亮氨酸- 酪氨酸-天门冬氨酸-天门冬氨酸-天门冬氨酸-天门冬氨酸 -赖氨酸-天门冬氨酸-精氨酸-甲 硫氨酸-脯氨酸-酪氨酸 -丝氨酸-苏氨酸-谷氨酸 -亮氨酸-异亮氨酸 -苯！ ¾氨酸-酪氨酸 -异亮 氨酸-谷氨酸-甲硫氨酸-天门冬氨酸-脯氨酸。
6、根据权利要求 5所述 PTD-P15融合多肽在制备治疗人类肿瘤及其他细胞生长异 常有关疾病的药物中的应用。
PCT/CN2012/078378 2011-08-23 2012-07-09 阻止细胞dna合成、抑制细胞增殖的多肽及其用途 WO2013026334A1 (zh)

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Families Citing this family (9)

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Publication number Priority date Publication date Assignee Title
CN102321158B (zh) * 2011-08-23 2013-03-27 常州德健生物科技有限公司 阻止细胞dna合成抑制细胞增殖的多肽及用途
CN104043102B (zh) * 2014-03-28 2017-04-19 武汉益承生物科技股份有限公司 一种多肽在制备治疗核因子‑κB异常活化疾病药物中的用途
CN106729609B (zh) * 2016-12-16 2021-04-09 湖北工业大学 N15多肽在制备细菌抑制剂中的用途
CN106729610B (zh) * 2016-12-16 2021-01-29 湖北工业大学 N15多肽在抑制金黄色葡萄球菌抑制剂中的用途
CN107050421A (zh) * 2016-12-16 2017-08-18 湖北工业大学 N15多肽在制备真菌抑制剂中的用途
CN106818746B (zh) * 2016-12-16 2019-08-20 湖北工业大学 N15多肽在制备蓝藻抑制剂中的用途
CN111956781B (zh) * 2019-05-20 2023-11-17 益承康泰(厦门)生物科技有限公司 一种多肽在治疗眼部炎症药物中的应用
CN110882228A (zh) * 2019-11-29 2020-03-17 南京禾瀚医药科技有限公司 一种伊匹乌肽肠溶制剂
CN111803619A (zh) * 2020-07-26 2020-10-23 武汉益承生物科技有限公司 多肽在制备创伤治疗药物中的用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101016340A (zh) * 2007-01-18 2007-08-15 夏献民 融合多肽及其在肿瘤与细胞生长异常相关的疾病治疗中的用途
CN101100679A (zh) * 2007-05-24 2008-01-09 夏献民 一种表达抑制细胞生长多肽的腺病毒的构建及其应用
CN102321158A (zh) * 2011-08-23 2012-01-18 常州德健生物科技有限公司 阻止细胞dna合成抑制细胞增殖的多肽及用途

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5595756A (en) * 1993-12-22 1997-01-21 Inex Pharmaceuticals Corporation Liposomal compositions for enhanced retention of bioactive agents
CN1238052C (zh) * 2003-07-07 2006-01-25 夏献民 抑制细胞生长的多肽的获得及用途
US20080014598A1 (en) * 2006-07-13 2008-01-17 Cell Signaling Technology, Inc. Phospho-specific antibodies to pi3k regulatory subunit and uses thereof
US20080221014A1 (en) * 2006-11-29 2008-09-11 Genentech, Inc. Method of Diagnosing and Treating Glioma
CN101244258B (zh) * 2008-03-21 2010-10-20 夏献民 Tat-n25多肽在制备治疗银屑病药物的用途
CN101759812A (zh) * 2009-12-28 2010-06-30 中国药科大学 一种新型仿穿膜肽结构的壳聚糖衍生物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101016340A (zh) * 2007-01-18 2007-08-15 夏献民 融合多肽及其在肿瘤与细胞生长异常相关的疾病治疗中的用途
CN101100679A (zh) * 2007-05-24 2008-01-09 夏献民 一种表达抑制细胞生长多肽的腺病毒的构建及其应用
CN102321158A (zh) * 2011-08-23 2012-01-18 常州德健生物科技有限公司 阻止细胞dna合成抑制细胞增殖的多肽及用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP2749568A4 *

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