WO2018166119A1 - 一种多肽或其衍生物及其在制备治疗肿瘤的药物中的应用 - Google Patents

一种多肽或其衍生物及其在制备治疗肿瘤的药物中的应用 Download PDF

Info

Publication number
WO2018166119A1
WO2018166119A1 PCT/CN2017/091644 CN2017091644W WO2018166119A1 WO 2018166119 A1 WO2018166119 A1 WO 2018166119A1 CN 2017091644 W CN2017091644 W CN 2017091644W WO 2018166119 A1 WO2018166119 A1 WO 2018166119A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
amino acid
derivative
cancer
Prior art date
Application number
PCT/CN2017/091644
Other languages
English (en)
French (fr)
Inventor
胡卓伟
花芳
Original Assignee
北京伟峰益民科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京伟峰益民科技有限公司 filed Critical 北京伟峰益民科技有限公司
Publication of WO2018166119A1 publication Critical patent/WO2018166119A1/zh

Links

Classifications

    • 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
    • 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
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a polypeptide or a derivative thereof and the use thereof in preparing a medicament for treating tumor.
  • the epidermal growth factor receptor is a member of the ErbB transmembrane receptor tyrosine kinase family and is also named ErbBl or HER-1.
  • EGFR is highly expressed or over-activated in a variety of human tumors. Activated EGFR activates MAPK/ERK, PI3K/Akt and other signaling pathways, and plays a role in tumor proliferation, angiogenesis, tumor metastasis, tumor immune escape, tumor resistance, and tumor metabolic reprogramming.
  • EGFR can also be translocated to the nucleus as a novel transcription factor that acts independently in the nucleus or synergizes with other transcription factors for target genes closely related to cell cycle progression or cell proliferation. To promote the development of tumors.
  • tumor molecular targeted drugs against EGFR are mainly divided into two categories according to their nature: one is a monoclonal antibody, which blocks the activation of EGFR signal mainly by blocking the binding of ligand to EGFR.
  • the other is a small molecule inhibitor that blocks its interaction with ATP by competitively binding to the phosphorylation site of EGFR intracellular tyrosine kinase, which in turn inhibits tyrosine phosphorylation of EGFR and downstream Signaling.
  • targeting EGFR has successfully entered the clinical application stage in the treatment of non-small cell lung cancer, glioblastoma, colorectal cancer, pancreatic cancer and head and neck cancer, its long-term efficacy has not achieved satisfactory results.
  • the amount of EGFR expression is not directly related to the gene copy number of efgr, suggesting that EGFR overexpression may be due to abnormal regulation of EGFR degradation.
  • Overexpression of EGFR not only leads to tolerance to targeted EGFR drugs, but also to tumors that are tolerant to multiple chemotherapeutic drugs. It is precisely because of the important regulatory role of EGFR in tumors that it is not completely dependent on its kinase activity, and that existing EGFR is targeted. The current status of high drug resistance rate, substances that directly regulate EGFR expression levels or protein stability have a good prospect of inhibiting tumorigenesis and development.
  • the technical problem to be solved by the present invention is to provide a polypeptide or a derivative thereof which promotes degradation of EGFR protein and a preparation thereof for treating tumors, aiming at the high resistance rate of EGFR molecular targeted drugs and the lack of direct targeting of EGFR protein stability drugs.
  • the application of the drug is to provide a polypeptide or a derivative thereof which promotes degradation of EGFR protein and a preparation thereof for treating tumors, aiming at the high resistance rate of EGFR molecular targeted drugs and the lack of direct targeting of EGFR protein stability drugs.
  • the inventors of the present invention have found through intensive research and repeated experiments that a polypeptide EJ4 capable of targeting EGFR degradation (the amino acid sequence thereof is shown in SEQ ID No. 1 of the Sequence Listing) is obtained, but the biostability of the polypeptide EJ4 is compared. low. This low biostable defect is directly related to the alpha helical conformation required for the polypeptide EJ4 to be stable in solution.
  • the inventors conducted targeted studies and experiments and found that if an amino acid residue at a specific position in the polypeptide EJ4 is replaced with an unnatural amino acid to which a 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, thereby having extremely high ⁇ -helical stability and metabolic stability. It can inhibit the proliferation and metastasis of a variety of tumor cells, and thus can be applied to the preparation of drugs for treating tumors.
  • the present invention provides the following technical solutions.
  • a polypeptide targeting the degradation of EGFR protein or a derivative of the polypeptide the amino acid sequence of the polypeptide is shown in SEQ ID No. 1 of the sequence listing, or, as in the order The substitution of two or more amino acids in the amino acid sequence shown in SEQ ID No. 1 is shown by the unnatural amino acid to which the side chain can be linked.
  • polypeptide EJ4 the polypeptide having the amino acid sequence shown in SEQ ID No. 1 of the Sequence Listing is referred to as polypeptide EJ4.
  • amino acid sequence of the polypeptide which is targeted to promote EGFR protein degradation may also be replaced by replacing two or more amino acids in the amino acid sequence shown in SEQ ID No. 1 of the sequence listing into side chains.
  • the unnatural amino acids are shown.
  • the unnatural amino acid to which the side chain can be linked is conventional in the art, preferably S-pentene alanine (S5), R-pentene alanine (R5) or R-octene Amino acid (R8).
  • the side chain of the unbonded unnatural amino acid of the side chain is reacted, and the cyclization forms a structure in which the side chain is linked. More preferably, the side chain of the adjacent unnatural amino acid is cyclized by olefin metathesis (RCM) under the catalysis of hydrazine.
  • RCM olefin metathesis
  • the number of the substituted amino acids is two.
  • the substituted amino acids are
  • i is an integer and 1 ⁇ i ⁇ 11.
  • the unnatural amino acid substituted with the ith position is R-pentene alanine, S-pentene alanine or R-octene alanine, the i+3, i+4 Or the unnatural amino acid substituted with i+7 is S-pentene alanine.
  • the amino acid sequence of the polypeptide substituted with a non-natural amino acid to which a side chain can be linked is SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. .5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, or SEQ ID No. 12, SEQ ID No. 13. SEQ ID No. 14 and SEQ ID No. 15.
  • the derivative is a chimeric peptide formed by the polypeptide and the cell penetrating peptide.
  • the cell penetrating peptide of the present invention is a cell penetrating peptide which is conventional in the art as long as it can assist in bringing the polypeptide into a cell to function.
  • the cell penetrating peptide is a short peptide molecule composed of 10 to 30 amino acids.
  • the cell penetrating peptide is linked to the N-terminus or C-terminus of the polypeptide, more preferably to the N-terminus of the polypeptide; further preferably the cell penetrating peptide and the polypeptide of the invention EJ4 It is linked by two glycines (Gly-Gly).
  • the cell penetrating peptide is a TAT peptide as shown in SEQ ID No. 19 of the Sequence Listing.
  • the amino acid sequence of the chimeric polypeptide formed, that is, the polypeptide derivative of the present invention is shown in SEQ ID No. 18.
  • the amino acid sequence shown in the above SEQ ID No. 1 to SEQ ID No. 15 can be appropriately substituted, deleted or added as long as the modified amino acid sequence can still promote EGFR protein degradation and maintain the transformation.
  • the activity can be.
  • the amino acid at position 8 and/or 9 may be mutated to arginine (Arg); preferably, the amino acid sequence thereof is shown in SEQ ID No. 16 or SEQ ID No. 17 of the Sequence Listing.
  • the second technical solution provided by the present invention is: a polypeptide targeted to promote degradation of EGFR protein or a derivative of the polypeptide for use in preparing a medicament for treating tumor.
  • the tumor is conventional in the art, preferably lung cancer, intestinal cancer, pancreatic cancer, breast cancer or liver cancer.
  • the lung cancer is conventional in the art, preferably non-small cell lung cancer or small cell lung cancer.
  • the intestinal cancer is conventional in the art, preferably colon cancer or rectal cancer.
  • the pancreatic cancer is conventional in the art, preferably pancreatic ductal adenocarcinoma or pancreatic acinar cell carcinoma.
  • the breast cancer is conventional in the art, preferably non-invasive breast cancer, early invasive breast cancer, invasive special type breast cancer or invasive non-special type breast cancer.
  • the liver cancer is conventional in the art, preferably primary liver cancer or secondary liver cancer.
  • the anti-tumor is conventional in the art, preferably refers to preventing or reducing the occurrence of tumors in the presence of possible tumor factors, and also refers to reducing the degree of tumors in the presence of tumor lesions, or curing tumors. Normalize it, either slow or delay the progression of the tumor, or alleviate the symptoms caused by the tumor.
  • the third technical solution provided by the present invention is: an antitumor pharmaceutical composition comprising the above-mentioned polypeptide which promotes degradation of EGFR protein or a derivative of the polypeptide.
  • the active ingredient refers to a component having an antitumor function.
  • the above-mentioned polypeptide which promotes degradation of EGFR protein or a derivative of the polypeptide may be used alone as an active ingredient or as an active ingredient together with other components having antitumor activity.
  • the pharmaceutical composition further comprises 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, preferably including pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the above-mentioned polypeptide which promotes degradation of EGFR protein or a derivative of said polypeptide, and 0.01 to 99.99% of a pharmaceutically acceptable carrier, said percentage being Percentage by mass of the pharmaceutical composition.
  • the pharmaceutical composition of the present invention is administered by a conventional polypeptide drug administration route, preferably by injection or oral administration.
  • the administration by injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection.
  • the pharmaceutical composition is in various 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 composition is administered in an amount effective to reduce or delay the progression of the disease 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 progressive effect of the present invention is that the polypeptide or polypeptide derivative of the present invention can be targeted to promote EGFR protein degradation and inhibit EGFR signaling pathway activity, thereby being applied to the preparation of antitumor drugs.
  • the prepared drug has the advantages of remarkable curative effect, less toxic side effects and safe use in anti-tumor.
  • the PBS described 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 described in the examples is room temperature conventional in the art, preferably 15 to 30 °C.
  • the amino acid sequence of the polypeptide EJ4 is shown in SEQ ID No. 1 of the Sequence Listing.
  • the peptide EJ4 was synthesized and purified by Beijing Saibaisheng Gene Technology Co., Ltd.
  • EJ4 Asn-Gln-Ala-Leu-Leu-Arg-Ile-Leu-Lys-Glu-Thr-Glu-Phe-Lys-Lys;
  • EJ4-S1 S5- Gln-Ala-Leu- S5- Arg-Ile-Leu-Lys-Glu-Thr-Glu-Phe-Lys-Lys;
  • EJ4-S2 Asn- S5 -Ala- Leu-Leu- S5 -Ile-Leu-Lys-Glu-Thr-Glu-Phe-Lys-Lys;
  • EJ4-S3 Asn-Gln- S5 -Leu-Leu-Arg- S5- Leu-Lys-Glu-Thr-Glu-Phe-Lys-Lys;
  • EJ4-S4 Asn-Gln- Ala- S5 -Leu-Arg-Ile- S5 -Lys-Glu-Thr-Glu-Phe-Lys-Lys;
  • EJ4-S5 Asn-Gln-Ala-Leu- S5- Arg-Ile-Leu- S5 -Glu-Thr-Glu-Phe-Lys-Lys;
  • EJ4-S6 Asn-Gln- Ala-Leu-Leu- S5 -Ile-Leu-Lys- S5 -Thr-Glu-Phe-Lys-Lys;
  • EJ4-S7 Asn-Gln- Ala-Leu-Leu-Arg- S5 -Leu-Lys-Glu- S5 -Glu-Phe-Lys-Lys;
  • EJ4-S8 Asn-Gln-Ala-Leu-Leu-Arg-Ile- S5- Lys-Glu-Thr- S5- Phe-Lys-Lys;
  • EJ4-S9 Asn-Gln- Ala-Leu-Leu-Arg-Ile-Leu- S5 -Glu-Thr-Glu- S5 -Lys-Lys;
  • EJ4-S10 Asn-Gln-Ala-Leu-Leu-Arg-Ile-Leu-Lys- S5- Thr-Glu-Phe- S5- Lys;
  • EJ4-S11 Asn-Gln-Ala-Leu-Leu-Arg-Ile-Leu-Lys-Glu- S5 -Glu-Phe-Lys- S5 ;
  • EJ4-S12 Asn-Gln-Ala-Leu-Leu- R5 -Ile-Leu- S5 -Glu-Thr-Glu-Phe-Lys-Lys;
  • EJ4-S13 Asn-Gln-Ala-Leu-Leu- R8- Ile-Leu-Lys-Glu-Thr-Glu- S5- Lys-Lys;
  • EJ4-S14 Asn-Gln- R8 -Leu-Leu-Arg-Ile-Leu-Lys- S5 -Thr-Glu-Phe-Lys-Lys;
  • EJ4-S15 Asn-Gln-Ala-Leu-Leu- S5- Ile- Arg-Arg - S5- Thr-Glu-Phe-Lys-Lys;
  • EJ4-S16 Asn-Gln-Ala-Leu-Leu- S5 -Ile-Leu- Arg - S5- Thr-Glu-Phe-Lys-Lys;
  • TAT-EJ4 TAT peptide-Gly-Gly-Asn-Gln-Ala-Leu-Leu-Arg-Ile-Leu-Lys-Glu-Thr-Glu-Phe-Lys-Lys.
  • the polypeptide after amino acid substitution of the polypeptide EJ4 is shown as EJ4-S15 and EJ4-S16 (the amino acid sequence is shown in SEQ ID No. 16, SEQ ID No. 17 of the Sequence Listing).
  • the chimeric peptide formed by the cell membrane-penetrating peptide TAT-peptide is shown as TAT-EJ4 (amino acid sequence is shown in SEQ ID No. 18 of the Sequence Listing).
  • the alpha helix ratio of the polypeptide was measured by a circular dichroism (purchased from Jasco, Japan).
  • the alpha helix ratio refers to the percentage of the number of peptides of the polypeptide which maintains the alpha helix of the secondary structure as a percentage of the total polypeptide.
  • Table 1 illustrates that the polypeptides EJ4-S1, EJ4-S2, EJ4-S3, EJ4-S4, EJ4-S5, EJ4-S6, EJ4-S7, EJ4-S8, EJ4-S9, EJ4-S10, EJ4-S11, EJ4
  • the ⁇ helix ratio of -S12, EJ4-S13, EJ4-S14, EJ4-S15, EJ4-S16 and TAT-EJ4 is significantly higher than that of polypeptide EJ4, and the maintenance of the alpha helix rate of the polypeptide is an important indicator for increasing the stability of the polypeptide. Therefore, an increase in the alpha helix ratio of the polypeptides EJ4-S1 to EJ4-S11 enhances its stability.
  • Example 3 Flow cytometry to detect peptide transmembrane ability
  • Flow cytometry detects the ability of a polypeptide to cross a cell membrane. The specific steps are as follows:
  • A549 lung cancer cells A549 (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were collected, and the cell concentration was adjusted with 1640 medium (purchased from Invitrogen, USA) to prepare a cell suspension of 200,000/mL.
  • Table 2 illustrates the administration of polypeptides EJ4-S1, EJ4-S2, EJ4-S3, EJ4-S4, EJ4-S5, EJ4-S6, EJ4-S7, EJ4-S8, EJ4-S9, EJ4-S10, EJ4-S11, After treatment with EJ4-S12, EJ4-S13, EJ4-S14, EJ4-S15, EJ4-S16 and TAT-EJ4, the proportion of cells containing fluorescence was significantly higher than that of EJ4, so the transmembrane ability of peptides EJ4-S1 ⁇ TAT-EJ4 was obvious. Better than EJ4.
  • Lung cancer cells A549 in logarithmic growth phase were collected, and the cell concentration was adjusted with 1640 medium to prepare a cell suspension of 200,000/mL.
  • the protein synthesis inhibitor cycloheximide (CHX) was added at time, and the action time was 24h, 12h, 8h, 4h, 2h, 0h. 1 ⁇ g/mL of the polypeptides prepared in Example 1 were prepared for each 12 h, EJ4, EJ4-S1, EJ4-S2, EJ4-S3, EJ4-S4, EJ4-S5, EJ4-S6, EJ4-S7, EJ4-S8. , EJ4-S9, EJ4-S10, EJ4-S11, EJ4-S12, EJ4-S13, EJ4-S14, EJ4-S15, EJ4-S16 and TAT-EJ4.
  • the control group was added an equal volume of solvent.
  • Table 3 shows that compared to the polypeptide EJ4, the polypeptides EJ4-S1, EJ4-S2, EJ4-S4, EJ4-S6, EJ4-S7, EJ4-S8, EJ4-S9, EJ4-S10, EJ4-S11, EJ4-S12 EJ4-S13, EJ4-S14, EJ4-S15, EJ4-S16 and TAT-EJ4 can significantly reduce the half-life of EGFR protein.
  • EJ4-S8 8.4 EJ4-S9 6.7 EJ4-S10 5.9 EJ4-S11 6.2 EJ4-S12 6.3 EJ4-S13 5.3 EJ4-S14 7.4 EJ4-S15 5.7 EJ4-S16 5.1 TAT-EJ4 9.6
  • the medium used for HepG2, HCT-8 and MDA-MB-231 cells is DMEM medium, and the culture medium for A549 and SW1990 cells
  • the base was RPMI1640 medium, which 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 polypeptide EJ4 prepared in Example 1 was separately added.
  • the control group was added an equal volume of solvent. Pass once every other day and count. As the number of cells increases, the cells are replaced by changing to the corresponding bottom area culture dish. After 12 days of culture, all cells were collected into 1 ml of medium for cell counting, and the total number of cells was counted.
  • Tables 4-8 illustrate polypeptides EJ4-S1, EJ4-S2, EJ4-S3, EJ4-S4, EJ4-S5, EJ4-S6, EJ4-S7, EJ4-S8, EJ4-S9, EJ4-S10, EJ4-S11, EJ4-S12, EJ4-S13, EJ4-S14, EJ4-S15, EJ4-S16 and TAT-EJ4 are more capable of inhibiting the growth of tumor cells than EJ4.
  • EJ4-S2 217.462 ⁇ 26.359 0.0004 EJ4-S3 488.323 ⁇ 40.747 0.0389 EJ4-S4 196.785 ⁇ 23.865 0.0003 EJ4-S5 464.872 ⁇ 48.388 0.0277 EJ4-S6 167.786 ⁇ 23.487 0.0002 EJ4-S7 179.487 ⁇ 24.486 0.0003 EJ4-S8 222.376 ⁇ 28.476 0.0004 EJ4-S9 219.775 ⁇ 24.460 0.0004 EJ4-S10 198.438 ⁇ 23.487 0.0003 EJ4-S11 204.476 ⁇ 34.186 0.0004 EJ4-S12 284.391 ⁇ 31.331 0.0009 EJ4-S13 278.482 ⁇ 40.132 0.0011 EJ4-S14 256.743 ⁇ 30.486 0.0006 EJ4-S15 213.592 ⁇ 26.444 0.0004 EJ4-S16 284.335 ⁇ 38.482
  • 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 lung cancer cells A549, colon cancer cells HCT-8, pancreatic cancer cells SW1990, breast cancer cells MDA-MB-231, and liver cancer cells HepG2 in logarithmic growth phase.
  • Example 1 EJ4, EJ4-S1, EJ4-S2, EJ4-S3, EJ4-, respectively.
  • the control group was given an equal volume of solvent. Take a sample and take a picture and calculate the scratch area, which is the 0h scratch area.
  • EJ4-S2 25.3 ⁇ 3.06 ⁇ 0.0001 EJ4-S3 72.3 ⁇ 6.50 0.0184 EJ4-S4 28.4 ⁇ 3.22 ⁇ 0.0001 EJ4-S5 69.4 ⁇ 7.92 0.0187 EJ4-S6 28.0 ⁇ 3.24 ⁇ 0.0001 EJ4-S7 33.9 ⁇ 2.64 ⁇ 0.0001 EJ4-S8 20.33 ⁇ 2.38 ⁇ 0.0001 EJ4-S9 28.5 ⁇ 4.16 ⁇ 0.0001 EJ4-S10 30.0 ⁇ 1.99 ⁇ 0.0001 EJ4-S11 30.4 ⁇ 5.01 ⁇ 0.0001 EJ4-S12 29.4 ⁇ 3.21 ⁇ 0.0001 EJ4-S13 31.5 ⁇ 4.13 ⁇ 0.0001 EJ4-S14 27.7 ⁇ 1.98 ⁇ 0.0001 EJ4-S15 26.1 ⁇ 2.77 ⁇ 0.0001 EJ4-S16 22.8 ⁇ 3.10 ⁇ 0.0001 TAT-EJ4 37.2 ⁇ 5.16 0.0001
  • EJ4-S10 19.8 ⁇ 2.24 ⁇ 0.0001 EJ4-S11 29.3 ⁇ 4.19 0.0002 EJ4-S12 28.4 ⁇ 4.14 0.0002 EJ4-S13 29.3 ⁇ 3.10 0.0001 EJ4-S14 41.2 ⁇ 4.17 0.0004 EJ4-S15 34.7 ⁇ 5.13 0.0003 EJ4-S16 28.3 ⁇ 3.19 0.0001 TAT-EJ4 31.6 ⁇ 4.18 0.0002
  • Example 7 Tumor subcutaneous growth assay to verify that polypeptide inhibits 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 110 male nude mice aged 4-6 weeks (purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.), randomly divided into 1 group: EJ4, EJ4-S3, EJ4-S4, EJ4-S5 , EJ4-S6, EJ4-S8, EJ4-S12, EJ4-S13, EJ4-S15, TAT-EJ4 group and solvent control group, 10 in each group.
  • Cell preparation The adherent cultured tumor cells were trypsinized and digested until the trypsin digestion time (the cell state should be single cells and just stuck to the wall), and the trypsin was aspirated. The cells were stopped with 2%/dish with PBS containing 1% serum, and the cells were blown off, transferred to 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.
  • the tumor cells were collected in a logarithmic growth phase of lung cancer cells A549, colon cancer cells HCT-8, pancreatic cancer cells SW1990, breast cancer cells MDA-MB-231, and liver cancer cells HepG2 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.
  • Tumor growth observation 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.
  • Tumor volume (length ⁇ width ⁇ width) / 2;
  • the subcutaneous tumor volume of each group of mice was as shown in Table 14 to Table 18.
  • EJ4-S4 394.2 ⁇ 44.1 ⁇ 0.0001 EJ4-S5 1330.4 ⁇ 135.5 0.0223
  • EJ4-S6 298.3 ⁇ 30.5 ⁇ 0.0001
  • EJ4-S8 324.5 ⁇ 27.8 ⁇ 0.0001
  • EJ4-S12 394.3 ⁇ 40.8 ⁇ 0.0001
  • EJ4-S13 417.4 ⁇ 44.2
  • EJ4-S15 631.4 ⁇ 48.3 ⁇ 0.0001 TAT-EJ4 430.0 ⁇ 41.5 ⁇ 0.0001

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

一种靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物及其在制备治疗肿瘤的药物中的应用。所述多肽的氨基酸序列如序列表SEQ ID No.1所示，或者，如将序列表SEQ ID No.1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；所述的衍生物包括所述多肽与细胞穿膜肽接连所形成的嵌合肽。所述多肽或多肽衍生物能够促进EGFR蛋白降解，抑制EGFR信号通路活性，因此应用于治疗肿瘤的药物的制备中。所制备的药物可用于治疗肿瘤，例如肺癌、肠癌、胰腺癌、乳腺癌以及肝癌等。

Description

一种多肽或其衍生物及其在制备治疗肿瘤的药物中的应用
本申请要求申请日为2017年3月14日的中国专利申请CN201710150282.7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，具体地涉及一种多肽或其衍生物及其在制备治疗肿瘤的药物中的应用。
背景技术
表皮生长因子受体(epidermal growth factor receptor,EGFR)是ErbB跨膜受体酪氨酸激酶家族成员，也被命名为ErbBl或HER-1。EGFR在多种人类肿瘤中高表达或信号过度活化。活化的EGFR激活MAPK/ERK、PI3K/Akt等信号途径，在肿瘤增殖、血管新生、肿瘤转移，肿瘤免疫逃逸、肿瘤耐药以及肿瘤代谢重编程等方面发挥促进作用。EGFR除了作为膜受体激活信号途径参与肿瘤调节外，还可转位至细胞核作为一种新型转录因子在核内独立或与其他转录因子协同作用于与细胞周期行进或细胞增殖密切相关的靶基因，促进肿瘤发生发展。
目前，针对EGFR的肿瘤分子靶向药物，按其性质主要分为两大类：一类是单克隆抗体，主要通过阻碍配体与EGFR结合，阻断EGFR信号活化。另一类是小分子抑制剂，主要通过竞争性结合EGFR胞内区酪氨酸激酶的磷酸化位点，阻断其与ATP的相互作用，继而抑制EGFR的酪氨酸磷酸化及下游一系列的信号传导。虽然靶向EGFR在非小细胞肺癌、恶性胶质瘤、结直肠癌、胰腺癌以及头颈部肿瘤的治疗中已成功进入临床应用阶段，但其长期疗效并没有取得令人满意的效果。主要原因在于有的肿瘤患者对EGFR靶向治疗不敏感，而那些最初对EGFR靶向治疗敏感的患者往往在一年内出现耐药现象。有多种因素导致EGFR靶向治疗耐受，EGFR突变(T790M)导致激酶与ATP的亲和力提高，或阻碍药物与靶点结合是导致耐受的最常见原因。近年来，有研究表明靶向EGFR治疗导致的继发性耐药即使在没有EGFR突变的情况下也会发生。事实上，在肿瘤中EGFR过表达与EGFR突变相比是更为常见的现象。在很多情况下，EGFR表达量与efgr的基因拷贝数并不直接相关，表明EGFR过表达可能是由于EGFR降解调节异常所致。EGFR过表达不仅导致对靶向EGFR药物的耐受，也导致肿瘤对多种化疗药物耐受。正是由于EGFR在肿瘤中的重要调节作用并不完全依赖其激酶活性，以及现有靶向EGFR 药物高耐药率的现状，直接调节EGFR表达水平或蛋白稳定性的物质具有良好的抑制肿瘤发生和发展的成药前景。
发明内容
本发明所要解决的技术问题是针对EGFR分子靶向药物高耐药率以及缺乏直接靶向EGFR蛋白稳定性药物的现状，提供一种促进EGFR蛋白降解的多肽或其衍生物及其在制备治疗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，获得了能够靶向促进EGFR降解的多肽EJ4(其氨基酸序列如序列表SEQ ID No.1所示)，然而多肽EJ4的生物稳定性比较低。该生物稳定性低的缺陷与多肽EJ4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EJ4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，从而具有极高的α螺旋稳定性、代谢稳定性，能够抑制多种肿瘤细胞增殖和转移，从而应用于制备治疗肿瘤的药物中。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：一种靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物，所述多肽的氨基酸序列如序列表SEQ ID No.1所示，或者，如将序列表SEQ ID No.1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。
本发明中，所述的氨基酸序列如序列表SEQ ID No.1所示的多肽称为多肽EJ4。
本发明中，所述的靶向促进EGFR蛋白降解的多肽的氨基酸序列也可以是将序列表SEQ ID No.1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。
其中，所述的侧链可相连的非天然氨基酸是本领域常规的，较佳的为S-戊烯丙氨酸(S5)、R-戊烯丙氨酸(R5)或者R-辛烯丙氨酸(R8)。
较佳的，所述的侧链可相连的非天然氨基酸的侧链进行反应，环化形成侧链相连的结构。更佳的，相邻非天然氨基酸的侧链在钌的催化作用下进行烯烃复分解反应(RCM)环化即得。
其中，所述的多肽中，所述替换的氨基酸的数目为两个。所述替换的氨基酸分别为
Figure PCTCN2017091644-appb-000001
序列表SEQ ID No.1所示的氨基酸序列的第i位和第i+3位的氨基酸、第i位和第i+4位的氨基酸，或者，第i位和第i+7位的氨基酸，其中i为整数，且1≤i≤11。较佳地，所述第i位替换成的非天然氨基酸为R-戊烯丙氨酸、S-戊烯丙氨酸或者R-辛烯丙氨酸，所述第i+3、i+4或i+7位替换成的非天然氨基酸为S-戊烯丙氨酸。
本发明中，更佳地，替换有侧链可相连的非天然氨基酸的所述的多肽的氨基酸序列如序列表SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8、SEQ ID No.9、SEQ ID No.10、SEQ ID No.11、或SEQ ID No.12、SEQ ID No.13、SEQ ID No.14和SEQ ID No.15所示。
本发明中，所述的衍生物为所述多肽与细胞穿膜肽接连所形成的嵌合肽。
其中，本发明所述的细胞穿膜肽为本领域常规的细胞穿膜肽，只要其能辅助将所述多肽送入细胞以发挥作用即可。一般，所述的细胞穿膜肽为由10～30个氨基酸组成的短肽分子。较佳地，所述细胞穿膜肽连接在所述多肽的N端或者C端，更佳地连接在所述多肽的N端；进一步更佳地所述细胞穿膜肽和本发明多肽EJ4之间以两个甘氨酸(Gly-Gly)连接。
进一步更佳地，所述细胞穿膜肽为如序列表SEQ ID No.19所示的TAT肽(peptide)， 所形成的嵌合多肽即本发明的多肽衍生物的氨基酸序列如SEQ ID No.18所示。本发明中，上述SEQ ID No.1～SEQ ID No.15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促进EGFR蛋白降解并且保持改造前的活性即可。例如，第8和/或第9位的氨基酸可突变为精氨酸(Arg)；较佳地，其氨基酸序列如序列表SEQ ID No.16或SEQ ID No.17所示。
本发明提供的技术方案之二是：一种靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物在制备治疗肿瘤的药物中的应用。
本发明中，所述的肿瘤为本领域常规的，较佳地为肺癌、肠癌、胰腺癌、乳腺癌或者肝癌。其中，所述肺癌为本领域常规，较佳的为非小细胞肺癌或小细胞肺癌。所述肠癌为本领域常规，较佳的为结肠癌或直肠癌。所述胰腺癌为本领域常规，较佳的为胰腺导管腺癌或胰腺腺泡细胞癌。所述乳腺癌为本领域常规，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。所述肝癌为本领域常规，较佳的为原发性肝癌或继发性肝癌。
本发明中，所述抗肿瘤是本领域常规的，较佳地指在存在可能的肿瘤因素时，使用后防止或降低肿瘤的产生，也指存在肿瘤病灶时，减轻肿瘤的程度，或者治愈肿瘤使之正常化，或者减缓或延迟肿瘤的进程，或者减轻肿瘤引起的症状。
本发明提供的技术方案之三是：一种抗肿瘤的药物组合物，其含有上述靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物。
本发明中，所述的活性成分是指具有抗肿瘤功能的成分。在所述药物组合物中，上述靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物可以单独作为活性成分或和其他具有抗肿瘤活性的成分一起作为活性成分。
本发明中，较佳地，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，较佳地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％的上述靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物，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。
本发明所述的药物组合物的给药途径是常规的多肽药物给药途径，较佳地为注射给药或口服给药。所述注射给药较佳地包括静脉注射、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，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂 或输注剂等。
本发明中，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：本发明的多肽或多肽衍生物能够靶向促进EGFR蛋白降解，抑制EGFR信号通路活性，从而应用于抗肿瘤的药物的制备中。所制备的药物在抗肿瘤中，具有疗效显著，毒副作用少，使用安全的优点。
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。
实施例中所述的PBS，指浓度为0.1M，pH值为7.2的磷酸盐缓冲液。
实施例中所述的室温为本领域常规的室温，较佳地为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被认为有显著性差异，p<0.01被认为有极其显著性差异。
实施例1多肽的合成
多肽EJ4的氨基酸序列参见序列表SEQ ID No.1。多肽EJ4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(S5)插入在多肽EJ4氨基酸序列中的第i、i+4位，R-戊烯丙氨酸(R5)和S5分别插入在多肽EJ4的第i、i+3位；R-辛烯丙氨酸(R8)和S5分别插入在多肽EJ4的第i、i+7位。由此得到不同的序列改造后的多肽(氨基酸序列参见序列表SEQ ID No.2～SEQ ID No.15)，其具体插入位点如下所示：
EJ4：Asn-Gln-Ala-Leu-Leu-Arg-Ile-Leu-Lys-Glu-Thr-Glu-Phe-Lys-Lys；
EJ4-S1：S5-Gln-Ala-Leu-S5-Arg-Ile-Leu-Lys-Glu-Thr-Glu-Phe-Lys-Lys；
EJ4-S2：Asn-S5-Ala-Leu-Leu-S5-Ile-Leu-Lys-Glu-Thr-Glu-Phe-Lys-Lys；
EJ4-S3：Asn-Gln-S5-Leu-Leu-Arg-S5-Leu-Lys-Glu-Thr-Glu-Phe-Lys-Lys；
EJ4-S4：Asn-Gln-Ala-S5-Leu-Arg-Ile-S5-Lys-Glu-Thr-Glu-Phe-Lys-Lys；
EJ4-S5：Asn-Gln-Ala-Leu-S5-Arg-Ile-Leu-S5-Glu-Thr-Glu-Phe-Lys-Lys；
EJ4-S6：Asn-Gln-Ala-Leu-Leu-S5-Ile-Leu-Lys-S5-Thr-Glu-Phe-Lys-Lys；
EJ4-S7：Asn-Gln-Ala-Leu-Leu-Arg-S5-Leu-Lys-Glu-S5-Glu-Phe-Lys-Lys；
EJ4-S8：Asn-Gln-Ala-Leu-Leu-Arg-Ile-S5-Lys-Glu-Thr-S5-Phe-Lys-Lys；
EJ4-S9：Asn-Gln-Ala-Leu-Leu-Arg-Ile-Leu-S5-Glu-Thr-Glu-S5-Lys-Lys；
EJ4-S10：Asn-Gln-Ala-Leu-Leu-Arg-Ile-Leu-Lys-S5-Thr-Glu-Phe-S5-Lys；
EJ4-S11：Asn-Gln-Ala-Leu-Leu-Arg-Ile-Leu-Lys-Glu-S5-Glu-Phe-Lys-S5；
EJ4-S12：Asn-Gln-Ala-Leu-Leu-R5-Ile-Leu-S5-Glu-Thr-Glu-Phe-Lys-Lys；
EJ4-S13：Asn-Gln-Ala-Leu-Leu-R8-Ile-Leu-Lys-Glu-Thr-Glu-S5-Lys-Lys；
EJ4-S14：Asn-Gln-R8-Leu-Leu-Arg-Ile-Leu-Lys-S5-Thr-Glu-Phe-Lys-Lys；
EJ4-S15：Asn-Gln-Ala-Leu-Leu-S5-Ile-Arg-Arg-S5-Thr-Glu-Phe-Lys-Lys；
EJ4-S16：Asn-Gln-Ala-Leu-Leu-S5-Ile-Leu-Arg-S5-Thr-Glu-Phe-Lys-Lys；
TAT-EJ4：TAT peptide-Gly-Gly-Asn-Gln-Ala-Leu-Leu-Arg-Ile-Leu-Lys-Glu-Thr-Glu-Phe-Lys-Lys。
多肽EJ4进行氨基酸替换后的多肽如EJ4-S15、EJ4-S16所示(氨基酸序列参见序列表SEQ ID No.16、SEQ ID No.17)。与细胞穿膜肽TAT-肽(peptide)(氨基酸序列参见序列表SEQ ID No.19)接连所形成的嵌合肽如TAT-EJ4所示(氨基酸序列参见序列表SEQ ID No.18)。
上述多肽均由北京赛百盛基因技术有限公司合成并纯化。
实施例2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EJ4、EJ4-S1、EJ4-S2、EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S7、EJ4-S8、EJ4-S9、EJ4-S10、EJ4-S11和EJ4-S12、EJ4-S13、EJ4-S14、EJ4-S15、EJ4-S16和TAT-EJ4溶解到水溶液中，将圆二色谱仪的上机浓度调整为1mg/mL，结果如表1所示。其中，α螺旋率指保持二级结构α螺旋的多肽的肽段数量占总多肽的肽段数量的百分比。
表1说明，多肽EJ4-S1、EJ4-S2、EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S7、EJ4-S8、EJ4-S9、EJ4-S10、EJ4-S11、EJ4-S12、EJ4-S13、EJ4-S14、EJ4-S15、EJ4-S16和TAT-EJ4的α螺旋率明显高于多肽EJ4，多肽的α螺旋率的维持是增加多肽稳定性的重要指标， 因此，多肽EJ4-S1～EJ4-S11的α螺旋率的提高会增强其稳定性。
表1.圆二色谱法测定多肽α螺旋率
多肽名称 α螺旋率(％)
EJ4 18
EJ4-S1 56
EJ4-S2 45
EJ4-S3 37
EJ4-S4 39
EJ4-S5 32
EJ4-S6 65
EJ4-S7 54
EJ4-S8 66
EJ4-S9 74
EJ4-S10 66
EJ4-S11 48
EJ4-S12 52
EJ4-S13 45
EJ4-S14 39
EJ4-S15 62
EJ4-S16 49
TAT-EJ4 25
实施例3流式细胞术检测多肽穿膜能力
流式细胞术检测多肽穿过细胞膜的能力。具体操作步骤如下：
1.收集对数生长期的肺癌细胞A549(购自中国医学科学院基础医学研究所)，用1640培养基(购自美国Invitrogen公司)调整细胞浓度，制成20万个/mL的细胞悬液。
2.将1mL步骤1所制得的细胞悬液加入6孔板进行培养，12小时后换成新的培养基，并且分别加入1μg/mL实施例1所制得的FAM荧光基团标记的多肽EJ4、EJ4-S1、EJ4-S2、EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S7、EJ4-S8、EJ4-S9、EJ4-S10、EJ4-S11、EJ4-S12、EJ4-S13、EJ4-S14、EJ4-S15、EJ4-S16和TAT-EJ4。
3.6小时后，用胰酶消化制备成单细胞悬液，冷PBS重悬细胞。
4.运用流式细胞计数仪，激发波长为465nm，发射波长为520nm，测定细胞内荧光 的强弱，计算含荧光细胞占总细胞的百分比。结果如表2所示，含荧光细胞占总细胞百分比越高，说明多肽能穿过的细胞数越多，即多肽的穿膜能力越好。
表2说明，给予多肽EJ4-S1、EJ4-S2、EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S7、EJ4-S8、EJ4-S9、EJ4-S10、EJ4-S11、EJ4-S12、EJ4-S13、EJ4-S14、EJ4-S15、EJ4-S16和TAT-EJ4处理后，含荧光的细胞比例明显多于EJ4，因此多肽EJ4-S1～TAT-EJ4的穿膜能力明显优于EJ4。
表2.流式细胞术检测多肽穿膜能力
多肽名称 含荧光细胞比例％
EJ4 1.2
EJ4-S1 46
EJ4-S2 34
EJ4-S3 29
EJ4-S4 59
EJ4-S5 31
EJ4-S6 51
EJ4-S7 48
EJ4-S8 44
EJ4-S9 51
EJ4-S10 62
EJ4-S11 47
EJ4-S12 64
EJ4-S13 46
EJ4-S14 47
EJ4-S15 43
EJ4-S16 63
TAT-EJ4 39
实施例4免疫荧光染色验证多肽对EGFR蛋白的半衰期的影响
1.收集对数生长期的肺癌细胞A549，用1640培养基调整细胞浓度，制成20万个/mL的细胞悬液。
2.将2mL步骤1所制得的细胞悬液加入6孔板进行培养，12小时后换成新的培养 基，并且分别加入1μg/mL实施例1所制得的多肽EJ4、EJ4-S1、EJ4-S2、EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S7、EJ4-S8、EJ4-S9、EJ4-S10、EJ4-S11、EJ4-S12、EJ4-S13、EJ4-S14、EJ4-S15、EJ4-S16和TAT-EJ4。对照组加入等体积的溶剂PBS。
3.12小时后，按时间点加入蛋白合成抑制剂放线菌酮(Cycloheximide，CHX)，使其作用时间分别为24h，12h，8h，4h，2h，0h。每过12h分别补加1μg/mL实施例1所制得的多肽EJ4、EJ4-S1、EJ4-S2、EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S7、EJ4-S8、EJ4-S9、EJ4-S10、EJ4-S11、EJ4-S12、EJ4-S13、EJ4-S14、EJ4-S15、EJ4-S16和TAT-EJ4。对照组加入等体积的溶剂。
4.收集细胞，加入RIPA裂解液(购自上海碧云天生物技术有限公司)(按照使用说明书，用前加入蛋白酶抑制剂PMSF和leupeptin，aprotinin等其它抑制剂)，冰上裂解30min；12000rpm，4℃离心30min；吸取上清，用BCA法对蛋白进行定量，按照定量结果将蛋白调整成统一浓度，加入5×上样缓冲液，98℃变性10min。
5.取部分样品按照《分子克隆》所述的方法进行SDS-聚丙烯酰胺凝胶电泳。电泳结束后，进行免疫印迹检测。
6.免疫印迹结果利用Gel-Pro Analyzer32Analyzer4.0进行定量分析，并绘制时间相关EGFR含量变化曲线，确定EGFR蛋白含量下降至CHX作用0h时的50％所需时间，即为EGFR蛋白半衰期。结果如表3所示。
表3说明，与多肽EJ4相比，多肽EJ4-S1、EJ4-S2、EJ4-S4、EJ4-S6、EJ4-S7、EJ4-S8、EJ4-S9、EJ4-S10、EJ4-S11、EJ4-S12、EJ4-S13、EJ4-S14、EJ4-S15、EJ4-S16和TAT-EJ4能明显降低EGFR蛋白半衰期。
表3.多肽对细胞EGFR蛋白半衰期的影响
多肽名称 EGFR蛋白半衰期(小时)
对照 17.4
EJ4 14.3
EJ4-S1 5.1
EJ4-S2 4.4
EJ4-S3 11.6
EJ4-S4 3.9
EJ4-S5 12.1
EJ4-S6 7.1
EJ4-S7 4.9
EJ4-S8 8.4
EJ4-S9 6.7
EJ4-S10 5.9
EJ4-S11 6.2
EJ4-S12 6.3
EJ4-S13 5.3
EJ4-S14 7.4
EJ4-S15 5.7
EJ4-S16 5.1
TAT-EJ4 9.6
实施例5细胞计数实验验证多肽抑制肿瘤细胞的生长
1.收集对数生长期的肺癌细胞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)、胰腺癌细胞SW1990(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)和肝癌细胞HepG2(购自中国医学科学院基础医学研究所)，将其制备为1.5×105/ml的细胞悬液。
2.取步骤1所制得的细胞悬液1ml，将其加入12孔板进行培养(其中HepG2、HCT-8和MDA-MB-231细胞所用培养基为DMEM培养基，A549、SW1990细胞所用培养基为RPMI1640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，并且分别加入1μg/mL实施例1所制得的多肽EJ4、EJ4-S1、EJ4-S2、EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S7、EJ4-S8、EJ4-S9、EJ4-S10、EJ4-S11、EJ4-S12、EJ4-S13、EJ4-S14、EJ4-S15、EJ4-S16和TAT-EJ4。对照组加入等体积溶剂。每隔一天传代一次，并进行计数。随着细胞数量增多，更换至相应底面积培养皿中进行培养。培养12天后，收集所有细胞至1ml培养基中进行细胞计数，并计算总的细胞数量。实验结果以mean±SD表示，并采用t test检验各组与EJ4组间的差异。实验结果见表4～8。表4～8说明多肽EJ4-S1、EJ4-S2、EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S7、EJ4-S8、EJ4-S9、EJ4-S10、EJ4-S11、EJ4-S12、EJ4-S13、EJ4-S14、EJ4-S15、EJ4-S16和TAT-EJ4相比于EJ4更能够抑制肿瘤细胞的生长。
表4.多肽抑制肺癌细胞A549的生长
多肽名称 细胞数量(104) P值
对照 647.521±45.348  
EJ4 621.342±44.201  
EJ4-S1 278.432±30.561 0.0004
EJ4-S2 199.893±29.346 0.0002
EJ4-S3 478.261±56.724 0.0261
EJ4-S4 256.833±41.281 0.0005
EJ4-S5 500.384±48.391 0.0330
EJ4-S6 283.347±29.456 0.0004
EJ4-S7 246.461±18.392 0.0002
EJ4-S8 236.468±29.573 0.0002
EJ4-S9 267.351±24.572 0.0003
EJ4-S10 244.672±31.435 0.0003
EJ4-S11 301.457±35.941 0.0006
EJ4-S12 333.253±41.450 0.0012
EJ4-S13 295.594±31.456 0.0005
EJ4-S14 292.482±30.491 0.0004
EJ4-S15 284.595±43.135 0.0007
EJ4-S16 374.591±41.459 0.0021
TAT-EJ4 314.597±34.472 0.0007
表5.多肽抑制肠癌细胞HCT-8的生长
多肽名称 细胞数量(104) P值
对照 633.248±36.356  
EJ4 602.402±43.298  
EJ4-S1 212.124±39.271 0.0003
EJ4-S2 254.345±31.346 0.0004
EJ4-S3 492.294±40.201 0.0319
EJ4-S4 234.442±19.895 0.0002
EJ4-S5 504.730±39.485 0.0445
EJ4-S6 192.358±24.579 0.0001
EJ4-S7 178.314±33.137 0.0002
EJ4-S8 214.536±35.365 0.0003
EJ4-S9 222.585±35.258 0.0003
EJ4-S10 257.573±29.467 0.0003
EJ4-S11 249.468±32.146 0.0003
EJ4-S12 312.383±28.301 0.0003
EJ4-S13 284.482±30.193 0.0002
EJ4-S14 233.482±29.492 0.0001
EJ4-S15 312.490±29.443 0.0003
EJ4-S16 248.477±30.482 0.0001
TAT-EJ4 294.418±28.747 0.0002
表6.多肽抑制胰腺癌细胞SW1990的生长
多肽名称 细胞数量(104) P值
对照 629.247±55.146  
EJ4 628.591±48.249  
EJ4-S1 278.357±36.148 0.0007
EJ4-S2 189.468±24.520 0.0002
EJ4-S3 523.436±38.579 0.0420
EJ4-S4 257.486±33.567 0.0004
EJ4-S5 527.498±40.482 0.0498
EJ4-S6 303.465±23.935 0.0005
EJ4-S7 213.460±31.351 0.0002
EJ4-S8 218.462±29.462 0.0002
EJ4-S9 222.456±35.456 0.0003
EJ4-S10 188.375±30.948 0.0002
EJ4-S11 283.462±29.462 0.0005
EJ4-S12 294.498±31.341 0.0005
EJ4-S13 248.592±29.493 0.0003
EJ4-S14 312.421±35.291 0.0008
EJ4-S15 193.442±22.236 0.0001
EJ4-S16 341.244±31.453 0.0010
TAT-EJ4 334.592±21.492 0.0006
表7.多肽抑制乳腺癌细胞MDA-MB-231的生长
多肽名称 细胞数量(104) P值
对照 614.564±56.876  
EJ4 585.392±39.299  
EJ4-S1 256.852±41.468 0.0006
EJ4-S2 193.467±20.462 0.0001
EJ4-S3 489.382±40.234 0.0417
EJ4-S4 245.945±42.468 0.0005
EJ4-S5 488.491±40.923 0.0416
EJ4-S6 187.487±24.485 0.0001
EJ4-S7 199.376±25.687 0.0001
EJ4-S8 284.475±36.381 0.0006
EJ4-S9 193.486±34.143 0.0002
EJ4-S10 276.365±22.486 0.0003
EJ4-S11 197.476±24.476 0.0001
EJ4-S12 288.218±30.332 0.0005
EJ4-S13 259.391±31.436 0.0004
EJ4-S14 247.326±32.492 0.0003
EJ4-S15 216.391±33.445 0.0002
EJ4-S16 294.313±31.291 0.0006
TAT-EJ4 312.492±29.325 0.0006
表8.多肽抑制肝癌细胞HepG2的生长
多肽名称 细胞数量(104) P值
对照 648.364±34.572  
EJ4 608.475±55.342  
EJ4-S1 233.458±34.135 0.0006
EJ4-S2 217.462±26.359 0.0004
EJ4-S3 488.323±40.747 0.0389
EJ4-S4 196.785±23.865 0.0003
EJ4-S5 464.872±48.388 0.0277
EJ4-S6 167.786±23.487 0.0002
EJ4-S7 179.487±24.486 0.0003
EJ4-S8 222.376±28.476 0.0004
EJ4-S9 219.775±24.460 0.0004
EJ4-S10 198.438±23.487 0.0003
EJ4-S11 204.476±34.186 0.0004
EJ4-S12 284.391±31.331 0.0009
EJ4-S13 278.482±40.132 0.0011
EJ4-S14 256.743±30.486 0.0006
EJ4-S15 213.592±26.444 0.0004
EJ4-S16 284.335±38.482 0.0011
TAT-EJ4 313.491±38.206 0.0016
实施例6细胞划痕实验验证多肽抑制肿瘤细胞划痕后的愈合
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肺癌细胞A549、结肠癌细胞HCT-8、胰腺癌细胞SW1990、乳腺癌细胞MDA-MB-231和肝癌细胞HepG2。
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，同时分别加入1μg/mL实施例1所制备的多肽EJ4、EJ4-S1、EJ4-S2、EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S7、EJ4-S8、EJ4-S9、EJ4-S10、EJ4-S11、EJ4-S12、EJ4-S13、EJ4-S14、EJ4-S15、EJ4-S16和TAT-EJ4。对照组给予等体积的溶剂。取样拍照，计算划痕面积，即为0h划痕面积。
5.然后放入37℃、5％(v/v)CO2培养箱培养，24小时后取样拍照，并计算此时未修复的剩余面积，即24h剩余面积。按损伤修复比＝(0h划痕面积-24h剩余面积)/0h划痕面积*100％计算损伤修复比。
实验结果以mean±SD表示，并采用t test检验各组与EJ4组间的差异。实验结果见表9～13。
表9～13的结果表明，损伤修复面积比越大，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EJ4-S1、EJ4-S2、EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S7、EJ4-S8、EJ4-S9、EJ4-S10、EJ4-S11、EJ4-S12、EJ4-S13、EJ4-S14、EJ4-S15、EJ4-S16和TAT-EJ4可以降低肿瘤细胞划痕后的愈合能力。
表9.多肽抑制肺癌细胞A549迁移
多肽名称 损伤修复面积比 P值
对照 91.7±4.26  
EJ4 88.3±7.34  
EJ4-S1 23.2±2.87 0.0001
EJ4-S2 33.7±1.65 0.0002
EJ4-S3 60.3±6.38 0.0076
EJ4-S4 35.2±2.79 0.0003
EJ4-S5 70.4±5.49 0.0277
EJ4-S6 27.0±1.30 0.0001
EJ4-S7 25.8±2.55 0.0002
EJ4-S8 27.7±3.94 0.0002
EJ4-S9 29.4±5.19 0.0003
EJ4-S10 22.0±1.80 0.0001
EJ4-S11 31.3±3.86 0.0003
EJ4-S12 30.4±4.02 0.0003
EJ4-S13 28.6±3.13 0.0002
EJ4-S14 33.4±3.78 0.0003
EJ4-S15 30.7±4.10 0.0003
EJ4-S16 38.5±4.04 0.0005
TAT-EJ4 41.5±5.14 0.0008
表10.多肽抑制肠癌细胞HCT-8迁移
多肽名称 损伤修复面积比 P值
对照 89.9±3.50  
EJ4 87.8±2.57  
EJ4-S1 32.4±1.54 <0.0001
EJ4-S2 25.3±3.06 <0.0001
EJ4-S3 72.3±6.50 0.0184
EJ4-S4 28.4±3.22 <0.0001
EJ4-S5 69.4±7.92 0.0187
EJ4-S6 28.0±3.24 <0.0001
EJ4-S7 33.9±2.64 <0.0001
EJ4-S8 20.33±2.38 <0.0001
EJ4-S9 28.5±4.16 <0.0001
EJ4-S10 30.0±1.99 <0.0001
EJ4-S11 30.4±5.01 <0.0001
EJ4-S12 29.4±3.21 <0.0001
EJ4-S13 31.5±4.13 <0.0001
EJ4-S14 27.7±1.98 <0.0001
EJ4-S15 26.1±2.77 <0.0001
EJ4-S16 22.8±3.10 <0.0001
TAT-EJ4 37.2±5.16 0.0001
表11.多肽抑制胰腺癌细胞SW1990迁移
多肽名称 损伤修复面积比 P值
对照 91.2±5.18  
EJ4 88.4±6.31  
EJ4-S1 28.3±3.39 0.0001
EJ4-S2 30.1±1.98 0.0001
EJ4-S3 73.4±4.40 0.0279
EJ4-S4 22.8±2.06 <0.0001
EJ4-S5 71.3±5.39 0.0234
EJ4-S6 33.5±3.47 0.0002
EJ4-S7 28.3±4.12 0.0002
EJ4-S8 27.7±3.12 0.0001
EJ4-S9 30.1±4.91 0.0002
EJ4-S10 19.8±2.24 <0.0001
EJ4-S11 29.3±4.19 0.0002
EJ4-S12 28.4±4.14 0.0002
EJ4-S13 29.3±3.10 0.0001
EJ4-S14 41.2±4.17 0.0004
EJ4-S15 34.7±5.13 0.0003
EJ4-S16 28.3±3.19 0.0001
TAT-EJ4 31.6±4.18 0.0002
表12.多肽抑制乳腺癌细胞MDA-MB-231迁移
多肽名称 损伤修复面积比 P值
对照 85.0±3.85  
EJ4 76.3±5.73  
EJ4-S1 32.0±4.73 0.0005
EJ4-S2 23.0±2.07 0.0001
EJ4-S3 62.3±4.29 0.0276
EJ4-S4 27.2±2.58 0.0002
EJ4-S5 60.4±4.20 0.0179
EJ4-S6 25.1±3.54 0.0002
EJ4-S7 23.2±2.19 0.0001
EJ4-S8 26.7±3.56 0.0002
EJ4-S9 25.7±4.52 0.0003
EJ4-S10 32.8±2.44 0.0003
EJ4-S11 29.0±2.55 0.0002
EJ4-S12 22.5±3.11 0.0001
EJ4-S13 28.4±3.05 0.0002
EJ4-S14 30.1±3.45 0.0003
EJ4-S15 42.6±5.19 0.0016
EJ4-S16 22.6±4.15 0.0002
TAT-EJ4 28.5±5.82 0.0005
表13.多肽抑制肝癌细胞HepG2迁移
多肽名称 损伤修复面积比 P值
对照 89.1±3.45  
EJ4 87.5±5.32  
EJ4-S1 25.6±4.13 <0.0001
EJ4-S2 19.7±2.56 <0.0001
EJ4-S3 59.4±4.59 0.0023
EJ4-S4 30.1±4.39 0.0001
EJ4-S5 63.3±5.32 0.0051
EJ4-S6 27.8±4.11 0.0001
EJ4-S7 23.4±1.35 <0.0001
EJ4-S8 29.3±1.88 <0.0001
EJ4-S9 31.4±3.31 0.0001
EJ4-S10 27.7±3.45 <0.0001
EJ4-S11 34.1±4.13 0.0002
EJ4-S12 22.6±2.58 <0.0001
EJ4-S13 25.7±3.19 <0.0001
EJ4-S14 28.9±3.17 <0.0001
EJ4-S15 26.3±3.51 <0.0001
EJ4-S16 23.4±3.20 <0.0001
TAT-EJ4 41.3±5.15 0.0004
实施例7肿瘤皮下生长实验验证多肽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110只(购自北京维通利华实验动物有限公司)，随机分为1组：EJ4、EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S8、EJ4-S12、EJ4-S13、EJ4-S15、TAT-EJ4组及溶剂对照组，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肺癌细胞A549、结肠癌细胞HCT-8、胰腺癌细胞SW1990、乳腺癌细胞MDA-MB-231和肝癌细胞HepG2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。肿瘤体积＝(长×宽×宽)/2；
实验结果以mean±SEM表示，并采用t test检验各组与EJ4组间的差异。
接种肿瘤后4周，各组小鼠皮下肿瘤体积如表14～表18所示，肿瘤体积越大表明肿瘤生长越快，因此多肽EJ4-S3、EJ4-S4、EJ4-S5、EJ4-S6、EJ4-S8、EJ4-S12、EJ4-S13、EJ4-S15、TAT-EJ4均能抑制肿瘤细胞在小鼠体内生长。
表14.多肽抑制肺癌细胞A549在小鼠体内生长
多肽名称 肿瘤体积(mm3) P值
对照 2445.2±180.8  
EJ4 2381.4±201.4  
EJ4-S3 1789.3±100.3 0.0169
EJ4-S4 664.3±56.8 <0.0001
EJ4-S5 1738.4±201.8 0.0368
EJ4-S6 498.5±46.4 <0.0001
EJ4-S8 511.8±57.4 <0.0001
EJ4-S12 409.7±44.3 <0.0001
EJ4-S13 428.4±39.6 <0.0001
EJ4-S15 399.2±44.0 <0.0001
TAT-EJ4 441.4±53.9 <0.0001
表15.多肽抑制肠癌细胞HCT-8在小鼠体内生长
多肽名称 肿瘤体积(mm3) P值
对照 2231.5±240.8  
EJ4 1984.5±183.8  
EJ4-S3 1439.4±123.4 0.0241
EJ4-S4 410.6±33.9 <0.0001
EJ4-S5 1305.3±100.4 0.0045
EJ4-S6 477.3±45.3 <0.0001
EJ4-S8 444.3±56.3 <0.0001
EJ4-S12 384.5±29.5 <0.0001
EJ4-S13 400.6±38.7 <0.0001
EJ4-S15 304.7±33.5 <0.0001
TAT-EJ4 408.5±44.6 <0.0001
表16.多肽抑制胰腺癌细胞SW1990在小鼠体内生长
多肽名称 肿瘤体积(mm3) P值
对照 2089.4±189.3  
EJ4 1893.4±135.6  
EJ4-S3 1338.6±100.4 0.0041
EJ4-S4 403.5±44.6 <0.0001
EJ4-S5 1250.5±123.4 0.0025
EJ4-S6 422.8±44.7 <0.0001
EJ4-S8 403.8±44.2 <0.0001
EJ4-S12 513.8±60.3 <0.0001
EJ4-S13 502.5±41.9 <0.0001
EJ4-S15 403.8±51.2 <0.0001
TAT-EJ4 664.5±56.8 <0.0001
表17.多肽抑制乳腺癌细胞MDA-MB-231在小鼠体内生长
多肽名称 肿瘤体积(mm3) P值
对照 2234.2±189.3  
EJ4 1938.3±201.8  
EJ4-S3 1439.4±104.5 0.0415
EJ4-S4 394.2±44.1 <0.0001
EJ4-S5 1330.4±135.5 0.0223
EJ4-S6 298.3±30.5 <0.0001
EJ4-S8 324.5±27.8 <0.0001
EJ4-S12 394.3±40.8 <0.0001
EJ4-S13 417.4±44.2 <0.0001
EJ4-S15 631.4±48.3 <0.0001
TAT-EJ4 430.0±41.5 <0.0001
表18.多肽抑制肝癌细胞HepG2在小鼠体内生长
多肽名称 肿瘤体积(mm3) P值
对照 2038.4±164.2  
EJ4 1739.2±157.1  
EJ4-S3 1231.4±114.5 0.0176
EJ4-S4 427.4±44.2 <0.0001
EJ4-S5 1279.5±104.6 0.0255
EJ4-S6 488.1±50.3 <0.0001
EJ4-S8 503.3±48.2 <0.0001
EJ4-S12 482.4±40.5 <0.0001
EJ4-S13 601.2±49.8 <0.0001
EJ4-S15 438.4±44.5 <0.0001
TAT-EJ4 471.0±45.6 <0.0001
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。

Claims (10)

  1. 一种靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物，其特征在于，所述多肽的氨基酸序列如序列表SEQ ID No.1所示，或者，如将序列表SEQ ID No.1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，所述的衍生物为所述多肽与细胞穿膜肽接连所形成的嵌合肽。
  2. 如权利要求1所述的靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物，其特征在于，所述侧链可相连的非天然氨基酸为S-戊烯丙氨酸、R-戊烯丙氨酸或者R-辛烯丙氨酸。
  3. 如权利要求2所述的靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物，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分别为序列表SEQ ID No.1所示的氨基酸序列的第i位和第i+3位的氨基酸、第i位和第i+4位的氨基酸，或者，第i位和第i+7位的氨基酸；其中，i为整数，且1≤i≤11。
  4. 如权利要求3所述的靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物，其特征在于，所述第i位替换成的非天然氨基酸为R-戊烯丙氨酸、S-戊烯丙氨酸或者R-辛烯丙氨酸，所述第i+3、i+4或i+7位替换成的非天然氨基酸为S-戊烯丙氨酸。
  5. 如权利要求4所述的靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物，其特征在于，所述的多肽为a.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、SEQ ID No.7、SEQ ID No.8、SEQ ID No.9、SEQ ID No.10、SEQ ID No.11和SEQ ID No.12、SEQ ID No.13、SEQ ID No.14和SEQ ID No.15中任一项所示；或者b.在a.中的氨基酸序列经过替换、缺失或添加一个或几个氨基酸后形成的仍然具有改造前促进EGFR蛋白降解的活性的多肽，例如第8和/或第9位的氨基酸突变为精氨酸，较佳地其氨基酸序列如序列表SEQ ID No.16或SEQ ID No.17所示。
  6. 如权利要求1～5任一项所述的靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物，其特征在于，所述的细胞穿膜肽连接在所述多肽的N端或者C端；较佳地，所述细胞穿膜肽和多肽之间以两个甘氨酸连接；更佳地，当细胞穿膜肽为TAT肽时，所述多肽的衍生物的氨基酸序列如序列表SEQ ID No.18所示。
  7. 如权利要求1～6任一项所述的靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物在制备治疗肿瘤的药物中的应用；较佳地，所述的肿瘤为肺癌、肠癌、胰腺癌、乳腺癌或者肝癌；更佳地，所述肺癌为非小细胞肺癌或小细胞肺癌；所述肠癌为结肠癌或直肠 癌；所述胰腺癌为胰腺导管腺癌、胰腺腺泡细胞癌；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；所述的肝癌为原发性肝癌或继发性肝癌。
  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1～6任一项所述的靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物。
  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或9所述的药物组合物，其特征在于，其含有如权利要求1～6任一项所述的靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物作为单一活性成分；或者，其含有如权利要求1～6任一项所述的靶向促进EGFR蛋白降解的多肽或所述多肽的衍生物和其他具有抗肿瘤活性的化合物一起作为活性成分。
PCT/CN2017/091644 2017-03-14 2017-07-04 一种多肽或其衍生物及其在制备治疗肿瘤的药物中的应用 WO2018166119A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710150282 2017-03-14
CN201710150282.7 2017-03-14

Publications (1)

Publication Number Publication Date
WO2018166119A1 true WO2018166119A1 (zh) 2018-09-20

Family

ID=63522694

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/091644 WO2018166119A1 (zh) 2017-03-14 2017-07-04 一种多肽或其衍生物及其在制备治疗肿瘤的药物中的应用

Country Status (2)

Country Link
CN (1) CN108570096B (zh)
WO (1) WO2018166119A1 (zh)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN111732631B (zh) * 2019-03-24 2023-07-28 中国医学科学院药物研究所 一种多肽及其在制备治疗和预防肿瘤的药物中的应用
CN114457052B (zh) * 2021-09-17 2024-02-27 厦门大学 一种抗肿瘤的多肽组合物及其应用
CN114133429B (zh) * 2021-11-22 2022-06-24 哈尔滨吉象隆生物技术有限公司 一种杀灭肿瘤细胞的多肽及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1958572A (zh) * 2005-11-04 2007-05-09 中国科学院上海药物研究所 二氢喹啉类化合物、其制备方法及药物组合物
CN101675075A (zh) * 2007-03-01 2010-03-17 西福根有限公司 重组抗表皮生长因子受体抗体组合物
CN103992382A (zh) * 2014-05-14 2014-08-20 南方医科大学 一种靶向eps8与egfr结合的短肽及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993014781A1 (en) * 1992-01-24 1993-08-05 The Regents Of The University Of California Novel peptides and method for altering the activity of allosteric proteins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1958572A (zh) * 2005-11-04 2007-05-09 中国科学院上海药物研究所 二氢喹啉类化合物、其制备方法及药物组合物
CN101675075A (zh) * 2007-03-01 2010-03-17 西福根有限公司 重组抗表皮生长因子受体抗体组合物
CN103992382A (zh) * 2014-05-14 2014-08-20 南方医科大学 一种靶向eps8与egfr结合的短肽及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank [O] 4 September 2014 (2014-09-04), GLADYSHEV, V.N. ET AL.: "Epidermal Growth Factor Receptor [Fukomys Damarensis]", Database accession no. KF033012.1 *
HART, M.R. ET AL.: "Inactive ERBB Receptors Cooperate with Reactive Oxygen Species to Suppress Cancer Progression", MOLECULAR THERAPY, vol. 21, no. 11, November 2013 (2013-11-01), pages 1996 - 2007, XP055540256 *
KIRISITS, A. ET AL.: "Epidermal Growth Factor Receptor Degradation: An Alternative View of Oncogenic Pathways", THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY, vol. 39, no. 12, 1 August 2007 (2007-08-01), pages 2173 - 2182, XP022277792 *
ZHENG, B. ET AL.: "Regulation of Epidermal Growth Factor Receptor Degradation by Heterotrimeric Gas Protein", MOLECULAR BIOLOGY OF THE CELL, vol. 15, no. 12, 1 December 2004 (2004-12-01), pages 5538 - 5550, XP055540118 *

Also Published As

Publication number Publication date
CN108570096A (zh) 2018-09-25
CN108570096B (zh) 2021-07-02

Similar Documents

Publication Publication Date Title
CN110194787B (zh) 靶向抑制Wnt/β-catenin信号活性的多肽及其用途
WO2018166119A1 (zh) 一种多肽或其衍生物及其在制备治疗肿瘤的药物中的应用
JP6298960B2 (ja) 抗腫瘍活性のあるペプチド及びその用途
US20220267770A1 (en) Compositions and methods of using c/ebp alpha sarna
CN112543809A (zh) 包含C/EBPα saRNA的组合疗法
CN107474115B (zh) 一种多肽及其在制备治疗和/或预防肿瘤的药物中的应用
CN107056887B (zh) 一种多肽及其在制备治疗和预防肿瘤的药物中的应用
US11576873B2 (en) Compositions for the treatment of drug-resistant tumors and methods of use thereof
CN110498850B (zh) 多肽、其衍生物及其在制备防治肿瘤的药物中的应用
WO2021243797A1 (zh) 一种果蝇Hsp22蛋白在制备用于抗肿瘤的药物中的应用
CN113214357A (zh) 一种多肽或其衍生物及其在制备治疗肿瘤的药物中的应用
US20210024928A1 (en) C/ebp alpha sarna compositions and methods of use
JP2011512364A (ja) 治療用ペプチド
KR102658844B1 (ko) 펩티드 유도체 및 약학적 조성물
TW201725043A (zh) 化合物在製備用於治療腦膠質瘤的藥物中的用途
CN111732631B (zh) 一种多肽及其在制备治疗和预防肿瘤的药物中的应用
EP2344528B1 (en) Pharmaceutical being used for treating cancer and fibrosis disease and the composition and uses thereof
CN111333699A (zh) 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
US20220096594A1 (en) Macrocyclic peptides for targeted inhibition of autophagy
ES2742856T3 (es) Composiciones farmacéuticas y procedimientos para el tratamiento y prevención del cáncer metastático
TWI842966B (zh) 一種具緩解抗癌藥物抗藥性及增加抗癌藥物敏感性之醫藥組合物及其用途
Ruan et al. Growth Arrest Specific Gene 6 Promotes Proliferation and Invasion of Laryngeal Cancer Cells Through the Vascular Endothelial Growth Factor (VEGF)/Protein Kinase B (AKT) Pathway
US20240207304A1 (en) Combination Therapies Comprising C/EBP Alpha saRNA
CN110520144B (zh) 肽激酶抑制剂及其使用方法
US20240207301A1 (en) Pharmaceutical composition and anti-tumor agent for tumor having decreased function of at least one of bap1 or pbrm1

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17900455

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17900455

Country of ref document: EP

Kind code of ref document: A1