WO2020001495A1 - 一类新的Bcl10聚合抑制剂及其应用 - Google Patents

一类新的Bcl10聚合抑制剂及其应用 Download PDF

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WO2020001495A1
WO2020001495A1 PCT/CN2019/093094 CN2019093094W WO2020001495A1 WO 2020001495 A1 WO2020001495 A1 WO 2020001495A1 CN 2019093094 W CN2019093094 W CN 2019093094W WO 2020001495 A1 WO2020001495 A1 WO 2020001495A1
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bcl10
polypeptide
peptide
pharmaceutically acceptable
acceptable salt
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PCT/CN2019/093094
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English (en)
French (fr)
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杨成华
张亮
孙晨霞
鲍伟
于浩澜
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中国科学院上海生命科学研究院
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Priority to CN201980004308.4A priority Critical patent/CN111094357A/zh
Publication of WO2020001495A1 publication Critical patent/WO2020001495A1/zh

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    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • 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 relates to the field of biomedicine, in particular to a new class of Bcl10 polymerization inhibitors and applications thereof.
  • Diffuse large B-cell lymphoma is the most common non-Hodgkin's lymphoma. It can be divided into three subtypes according to its gene expression profile: germinal center B-cell type ( Germinal center, cell-like DLBCL (GCB-DLBCL), activated B-cell type (DLBCL, ABC-DLBCL), and primary mediastinal B-cell type (PMBL).
  • germinal center B-cell type Germinal center, cell-like DLBCL (GCB-DLBCL), activated B-cell type (DLBCL, ABC-DLBCL), and primary mediastinal B-cell type (PMBL).
  • GCB-DLBCL germinal center B-cell type
  • DLBCL activated B-cell type
  • PMBL primary mediastinal B-cell type
  • ABC-DLBCL is the most malignant and has strong resistance to existing immunochemotherapy.
  • the five-year survival rate of GCB-DLBCL can reach 76%, while the five-year survival rate of
  • the object of the present invention is to provide a medicine and a method for effectively treating ABC-DLBCL.
  • Another object of the present invention is to provide a novel drug that inhibits Bcl10 polymerization activity, and also provides the application of Bcl10 inhibitors in the treatment of ABC-DLBCL, and other CBM complex-mediated NF- ⁇ B dependent diseases.
  • Z0 is a N-terminal modification group or a peptide of 1-3 amino acids
  • Z1 is a transmembrane peptide element
  • Z2 is the absence or linking peptide
  • Z3 is a Bcl10 inhibitory peptide element, and the length of said Z3 is 12-25aa, preferably 13-20aa, more preferably 14-18aa;
  • Z4 is a non-C-terminal modification group or a peptide of 1-3 amino acids
  • the length of the polypeptide is 30-50aa, preferably 32-40aa.
  • the N-terminal modification group is selected from the group consisting of acetyl, benzyloxycarbonyl C, aminovaleric acid, palmitic acid, or a combination thereof.
  • the transmembrane peptide element has a sequence as shown in SEQ ID NO.:1.
  • amino acid sequence of the transmembrane peptide element is shown in SEQ ID NO.:1.
  • the length of the connecting peptide is 0-10aa, preferably 0-6aa.
  • the Z3 has a motif (5'-3 ') represented by Formula A1 or a reverse amino acid motif (5'-3') represented by Formula A2:
  • X1 is a peptide of 4-6 amino acids; preferably X1 is HFDHL or TSRRK;
  • X2 is a peptide having no or 1-2 amino acids; preferably X2 is K or G;
  • X3 is a peptide of 1-2 amino acids; preferably, X3 is I or L;
  • X1 ' is a peptide of 4-6 amino acids; preferably X1 is KRSST or LHDFH;
  • X2 ' is a peptide having no or 1-2 amino acids; preferably X2' is K or G.
  • the Z3 has a motif (5'-3 ') represented by Formula A1 or a reverse D-type amino acid motif (5'-3') represented by Formula A2:
  • X1 is a peptide of 4-6 amino acids; preferably X1 is HFDHL or TSRRK;
  • X2 is a peptide having no or 1-2 amino acids; preferably X2 is K or G;
  • X3 is a peptide of 1-2 amino acids; preferably, X3 is I or L;
  • X1 ' is a peptide of 4-6 D amino acids; preferably X1 is KRSST or LHDFH;
  • X2 ' is a peptide having no or 1-2 D-amino acids; preferably X2' is K or G.
  • the C-terminal modification group is selected from the group consisting of amidation, acetaldehyde, or a combination thereof.
  • Z3 has the amino acid sequence shown in SEQ ID No .: 2 or 3 or 15 or 16.
  • amino acid sequence of Z3 is shown as SEQ ID NO .: 2 or 3 or 15 or 16.
  • polypeptide has the sequence shown in SEQ ID No .: 4 or 5 or 8 or 9 or 13 or 14.
  • amino acid sequence of the polypeptide is as shown in SEQ ID NO: 4 or 5 or 8 or 9 or 13 or 14.
  • polypeptide is natural or artificial.
  • polypeptide is selected from the following group:
  • polypeptide is a polypeptide represented by SEQ ID No .: 4 or 5 or 8 or 9 or 13 or 14 after 1-3, preferably 1-2, more preferably 1 Amino acid substitutions, deletions; and / or
  • It is formed by adding 1-5, preferably 1-4, more preferably 1-3, and most preferably 1-2 amino acids.
  • the derived polypeptide retains ⁇ 70% of the activity of the polypeptide shown in SEQ ID NO: 4 or 5 or 8 or 9 or 13 or 14 to inhibit the polymerization of Bcl10.
  • the derivatized polypeptide is identical to SEQ ID NO: 4 or 5 or 8 or 9 or 13 or 14 ⁇ 80%, preferably ⁇ 90%; more preferably ⁇ 95%.
  • the present invention also provides dimer and multimer forms of compounds of Formula I that inhibit the polymerization of Bcl10, and the dimer and multimer forms have the activity of inhibiting the polymerization of Bcl10.
  • a second aspect of the present invention provides an isolated nucleic acid molecule that encodes the polypeptide or the pharmaceutically acceptable salt thereof according to the first aspect of the present invention.
  • a third aspect of the present invention provides a pharmaceutical composition, including:
  • the polypeptide retains ⁇ 70%, 75%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 195%, 195%, or 200
  • the dosage form of the composition includes an oral dosage form, or a parenteral dosage form, such as a topical or topical dosage form.
  • the dosage form of the composition includes tablets, pills, pellets, sustained release agents, emulsions, suspensions, granules, capsules, powders, oral solutions, lyophilizers, syrups, and ointments. , Creams, drops, buccal preparations, intravenous injections, suppositories, sprays, aerosols, lotions, gargles, patches, or eye drops.
  • the pharmaceutical composition also contains other ingredients that can (i) inhibit Bcl10 polymerization; (ii) inhibit the activity of the CBM complex; (iii) selectively inhibit diffuse large B-cell lymphoma (such as ABC-DLBCL); and / or (iv) drugs that prevent and / or treat diseases related to NF- ⁇ B activation induced by the CBM complex.
  • other ingredients that can (i) inhibit Bcl10 polymerization; (ii) inhibit the activity of the CBM complex; (iii) selectively inhibit diffuse large B-cell lymphoma (such as ABC-DLBCL); and / or (iv) drugs that prevent and / or treat diseases related to NF- ⁇ B activation induced by the CBM complex.
  • the other can (i) inhibit Bcl10 polymerization; (ii) inhibit the activity of the CBM complex; (iii) selectively inhibit diffuse large B-cell lymphoma (such as ABC-DLBCL) Growth; and / or (iv) drugs that prevent and / or treat CBM complex-induced NF- ⁇ B activation-related diseases are selected from the group consisting of MI-2, Mepazine, or a combination thereof.
  • the content of the polypeptide or a pharmaceutically acceptable salt thereof is 0.0001-99 wt%, preferably 0.001-90 wt%, more preferably 0.01-50 wt%, Based on the total weight of the composition.
  • the fourth aspect of the present invention provides the use of the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, for preparing a composition or a medicament for (i) inhibiting Bcl10 Polymerization; (ii) inhibits the activity of the CBM complex; (iii) selectively inhibits the growth of diffuse large B-cell lymphoma (such as ABC-DLBCL); and / or (iv) prevents and / or treats the CBM complex Diseases related to induced NF- ⁇ B activation.
  • the disease related to the CBM complex is selected from the group consisting of an autoimmune disease, a long-term inflammatory response, an allergic disease, a NF- ⁇ B dependent cancer, or a combination thereof.
  • the autoimmune disease is selected from the group consisting of psoriasis, lupus erythematosus, rheumatoid arthritis, atherosclerosis, or a combination thereof.
  • composition or medicament is also used to treat one or more diseases selected from the group consisting of:
  • Asthma breast cancer, non-small cell lung cancer, melanoma, colorectal cancer, T / B cell leukemia, or a combination thereof.
  • a fifth aspect of the present invention provides a method for selectively inhibiting the growth of diffuse large B-cell lymphoma, comprising the steps of administering to a subject in need thereof the polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof .
  • the subject is a human or non-human mammal.
  • the concentration of the polypeptide or a pharmaceutically acceptable salt thereof is 10-300 mM, preferably 30-160 mM, and more preferably 130-160 mM.
  • the non-human mammal includes rodents (such as mice, rats, rabbits), and primates (such as monkeys).
  • the method is non-therapeutic and non-diagnostic.
  • the diffuse large B-cell lymphoma cells are selected from the group consisting of ABC-DLBCL, GCB-DLBCL, or a combination thereof.
  • the diffuse large B-cell lymphoma cells include ABC-DLBCL.
  • a sixth aspect of the present invention provides a method for non-therapeutic inhibition of Bcl10 polymerization in vitro, including the steps:
  • tumor cells are cultured, thereby inhibiting the polymerization of Bcl10 in the tumor cells.
  • the tumor cells are selected from the group consisting of diffuse large B-cell lymphoma, breast cancer, non-small cell lung cancer, melanoma, colorectal cancer, T / B cell leukemia, or a combination thereof.
  • a seventh aspect of the present invention provides a method for preventing and / or treating diseases related to NF- ⁇ B activation induced by the CBM complex, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the polypeptide according to the first aspect of the present invention. And / or the pharmaceutical composition according to the third aspect of the present invention.
  • the subject is a human or non-human mammal.
  • the non-human mammal includes rodents (such as mice, rats, rabbits), and primates (such as monkeys).
  • An eighth aspect of the present invention provides a method for screening a candidate compound that selectively inhibits the growth of diffuse large B-cell lymphoma, comprising the steps of:
  • the compound in the test compound library is bound to the Bcl10 protein, it indicates that the compound bound to the Bcl10 protein is a candidate compound.
  • step (b) The candidate compound identified in step (a) is administered to diffuse large B-cell lymphoma cells, and its effect on diffuse large B-cell lymphoma is measured.
  • the method is non-diagnostic and non-therapeutic.
  • the diffuse large B-cell lymphoma cells are selected from the group consisting of ABC-DLBCL, GCB-DLBCL, or a combination thereof.
  • the diffuse large B-cell lymphoma cells include ABC-DLBCL.
  • a ninth aspect of the present invention provides a method for screening a candidate compound that selectively inhibits the growth of diffuse large B-cell lymphoma, comprising the steps of:
  • the method includes step (c): applying the candidate compound determined in step (b) to a mammalian model, and determining its effect on the mammal.
  • the mammal is a mammal with diffuse large B-cell lymphoma.
  • the "significantly lower” means E1 / E2 ⁇ 1 / 2, preferably ⁇ 1 / 3, and more preferably 1/4.
  • the method is non-diagnostic and non-therapeutic.
  • Figure 1 shows the gel filtration chromatographic peak shape of MBP-Bcl10 and the results of the peak product SDS-PAGE gel.
  • Figure 2 shows that one control small molecule peptide and five Bcl10 small molecule peptide inhibitors were designed based on the structural model of the CB complex.
  • Figure 3 shows the effect of Bcl10 small molecule peptide inhibitors on the formation of Bcl10 fiber structure.
  • Figure 4 shows the effect of Bcl10 small molecule peptide inhibitors on the growth of ABC-DLBCL cell lines (HBL-1 and TMD8) and GCB-DLBCL cell lines (OCL-LY1).
  • Figure 5 shows the effect of Bcl10 small molecule peptide inhibitor modified peptides on the growth of ABC-DLBCL cell lines (HBL-1 and TMD8) and GCB-DLBCL cell lines (OCL-LY1).
  • Figure 6 shows the effect of Bcl10 small molecule peptide inhibitors on the growth of ABC-DLBCL tumors in NCG mice.
  • FIG. 7 shows the effect of a Bcl10 small molecule peptide inhibitor on a psoriasis model.
  • FIG. 7A is an H & E staining diagram of mouse epidermal tissue sections of different Bcl10 small molecule inhibitor peptide treatment groups in an imiquimod (IMQ) -induced psoriasis model, showing the effects of different treatment groups on the thickness of mouse epidermis
  • FIG. 7B is a statistical graph of epidermal thickness of mice in different treatment groups.
  • the inventors After extensive and intensive research, the inventors have prepared for the first time a class of small-molecule polypeptides with a molecular weight of less than 5KD (such as only about 2.3-4.4kD) that have the effect of inhibiting the polymerization of Bcl10.
  • the present invention designed several candidate sequences, synthesized them by solid-phase method, isolated and purified small peptides Bcl10-P2, Bcl10-P4 and modified forms thereof, and identified them by HPLC and MS.
  • polypeptide of the present invention and "Bcl10 polymerization inhibitor” are used interchangeably, and both refer to (i) inhibiting the polymerization of Bcl10; (ii) inhibiting the activity of the CBM complex; (iii) selective Inhibits the growth of B-cell lymphoma (such as ABC-DLBCL); and / or (iv) prevents and / or treats CBM complex-induced NF- ⁇ B activation-related disease activity with SEQ ID NO: 4 or 5 or 8 Or a protein or polypeptide having the amino acid sequence shown in 9 or 13 or 14.
  • the term also includes having the ability to inhibit (i) inhibit Bcl10 polymerization; (ii) inhibit the activity of the CBM complex; (iii) selectively inhibit the growth of B-cell lymphomas (such as ABC-DLBCL); and / or (iv) A variant form of the sequence shown in SEQ ID NO: 4 or 5 or 8 or 9 or 13 or 14 for preventing and / or treating disease activity related to NF- ⁇ B activation induced by the CBM complex.
  • variants include (but are not limited to): 1-5 (usually 1-4, preferably 1-3, more preferably 1-2, most preferably 1) amino acid deletions, insertions And / or substitutions, and the addition or deletion of one or several (usually 5 or less, preferably 3 or less, more preferably 2 or less) amino acids at the C-terminus and / or N-terminus.
  • substitution of amino acids with similar or similar properties generally does not change the function of the protein.
  • adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the structure and function of the protein.
  • the term includes monomeric and multimeric forms of the polypeptides of the invention.
  • the term also includes linear and non-linear polypeptides (such as cyclic peptides).
  • the invention also includes active fragments, derivatives and analogs of the polypeptides of the invention.
  • fragment refers to substantially maintaining (i) inhibiting Bcl10 polymerization; (ii) inhibiting the activity of the CBM complex; (iii) selectively inhibiting B cells The growth of lymphomas (such as ABC-DLBCL); and / or (iv) polypeptides that are active in disease related to the prevention and / or treatment of CBM complex-induced NF- ⁇ B activation.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) one or more A polypeptide having a substitution group in three amino acid residues, or (iii) a polypeptide formed by fusion of a polypeptide of the present invention with another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol), or (iv) an additional A polypeptide formed by fusing an amino acid sequence to this polypeptide sequence (a protein formed by fusing with a leader sequence, a secretion sequence, or a tag sequence such as 6His).
  • these fragments, derivatives, and analogs are within the scope of those skilled in the art.
  • a preferred class of active derivatives refers to a maximum of five, preferably up to three, more preferably up to two, and most preferably one amino acid is compared to amino acids of similar or similar nature compared to the amino acid sequence of formula I Substitution to form a polypeptide.
  • These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • substitution Ala Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp
  • the invention also provides analogs of the polypeptides of the invention.
  • the difference between these analogs and the natural polypeptide of the present invention may be a difference in amino acid sequence, a difference in modified form that does not affect the sequence, or both.
  • Analogs also include analogs with residues different from the natural L-amino acid (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modified (usually unchanged primary structure) forms include chemically derived forms of the polypeptide, such as acetylated or carboxylated, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modification in the synthesis and processing of polypeptides or in further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that undergoes glycosylation, such as mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize their solubility.
  • the polypeptides of the invention may also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • These salts include, but are not limited to, salts with hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvate, acetic acid, succinic acid, oxalic acid, fumaric acid, and male Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid or isethionic acid.
  • Other salts include salts with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, and in the form of esters, carbamates or other conventional "prodrugs".
  • the invention also relates to a polynucleotide encoding a polypeptide of the invention.
  • a preferred coding sequence is shown in SEQ ID NO: 6 (Bcl10-P2) or 7 (Bcl10-P4), which encodes the polypeptide shown in SEQ ID NO: 4 (Bcl10-P2) or 5 (Bcl10-P4) Amino acid sequence.
  • nucleotide sequence of Bcl10-P2 is as follows:
  • nucleotide sequence of Bcl10-P4 is as follows:
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 6 or 7 or a degenerate variant.
  • degenerate variant refers to a polypeptide encoding a sequence having the sequence shown in SEQ ID NO: 4 or 5, but the same as SEQ ID NO: 6 Or 7 nucleic acid sequences with different coding region sequences.
  • the full-length nucleotide sequence of a polypeptide of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method, or a synthetic method.
  • a DNA sequence encoding a polypeptide (or a fragment thereof, or a derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into a variety of existing DNA molecules (or such as vectors) and cells known in the art.
  • the invention also relates to a vector comprising a polynucleotide of the invention, and to a host cell genetically engineered using the vector of the invention or a ZY polypeptide coding sequence.
  • the invention also includes polyclonal and monoclonal antibodies, especially monoclonal antibodies, which are specific for the polypeptides of the invention.
  • the term "substantially the same” when comparing and aligning sequences for maximum agreement refers to two or more sequences or subsequences having at least about 80%, such as at least about 85 %, About 90%, about 95%, about 98%, or about 99% of the nucleotide or amino acid residues are identical to a particular reference sequence, as determined using the following sequence comparison methods and / or by visual inspection.
  • the polypeptide of the invention may be a recombinant polypeptide or a synthetic polypeptide.
  • the polypeptides of the invention may be chemically synthesized, or recombinant. Accordingly, the polypeptide of the present invention can be artificially synthesized by conventional methods, and can also be produced by recombinant methods.
  • a preferred method is to use liquid phase synthesis technology or solid phase synthesis technology, such as Boc solid phase method, Fmoc solid phase method or a combination of the two methods. Samples can be obtained quickly by solid-phase synthesis. An appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the peptide of interest.
  • the preferred solid-phase carrier in the Fmoc system is Wang resin with C-terminal amino acid attached to the peptide, the structure of Wang resin is polystyrene, and the arm between amino acid and 4-alkoxybenzyl alcohol; 25% hexahydropyridine / Dimethylformamide was treated at room temperature for 20 minutes to remove the Fmoc protecting group, and extended from the C-terminus to the N-terminus according to the given amino acid sequence.
  • the synthetic proinsulin-related peptide is cleaved from the resin with 4% p-methylphenol trifluoroacetic acid and the protective group is removed.
  • the resin can be filtered to remove the ether to precipitate and isolate the crude peptide.
  • the desired peptide was purified by gel filtration and reversed-phase high-pressure liquid chromatography.
  • the resin is preferably a PAM resin linked to the C-terminal amino acid in the peptide, the structure of the PAM resin is polystyrene, and the arm between the amino acid and 4-hydroxymethylphenylacetamide is synthesized;
  • the protecting group Boc was removed with TFA / dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA / dichloromethane. Peptide chain condensation was completed.
  • DCC dicyclohexylcarbodiimide
  • HOBt hydroxybenzotriazole
  • HBTU 1,1,3,3-tetraurea hexafluorophosphate
  • the polypeptide of the present invention is prepared according to its sequence by a solid-phase synthesis method, and is purified by high-performance liquid chromatography to obtain a lyophilized powder of the peptide of high purity and stored at -20 ° C.
  • polypeptides of the invention Another method is to produce the polypeptides of the invention using recombinant techniques.
  • the polynucleotides of the present invention can be used to express or produce recombinant polypeptides of the present invention by conventional recombinant DNA technology. Generally there are the following steps:
  • Recombinant polypeptides can be expressed or secreted extracellularly within cells or on cell membranes. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation, treatment with a protein precipitant (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment with a protein precipitant (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography
  • polypeptide of the present invention is relatively short, it is conceivable to connect multiple polypeptides in series, obtain an expression product in the form of a multimer after recombinant expression, and then form a desired small peptide by methods such as enzyme digestion.
  • the present invention also provides a pharmaceutical composition containing (a) a safe and effective amount of a polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient .
  • the amount of the polypeptide of the present invention is usually 10 micrograms to 100 mg / dose, preferably 100 to 1000 micrograms / dose.
  • an effective dose is administered to an individual from about 0.01 mg / kg to 50 mg / kg, preferably from 0.05 mg / kg to 10 mg / kg of body weight of a polypeptide of the present invention.
  • the polypeptide of the present invention can be used alone or in combination with other therapeutic agents (such as formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent.
  • pharmaceutical carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the composition and are not excessively toxic after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceuticals Science (Mack Pub. Co., N.J. 1991).
  • Such carriers include, but are not limited to, saline, buffers, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • a pharmaceutically acceptable carrier in a therapeutic composition may contain liquids such as water, saline, glycerol, and ethanol.
  • liquids such as water, saline, glycerol, and ethanol.
  • auxiliary substances in these carriers such as wetting or emulsifying agents, pH buffering substances, and the like.
  • the therapeutic composition can be formulated as an injectable, such as a liquid solution or suspension; it can also be prepared as a solid form suitable as a liquid carrier in a solution or suspension prior to injection.
  • composition of the invention can be administered by conventional routes including, but not limited to: intramuscular, intravenous, subcutaneous, intradermal or topical administration.
  • the subject to be prevented or treated may be an animal; especially a human.
  • the pharmaceutical composition of the present invention When used for actual treatment, various different dosage forms of the pharmaceutical composition can be adopted according to the use situation.
  • tablets, granules, capsules, pills, injections, or oral liquids can be exemplified.
  • compositions can be formulated according to conventional methods by mixing, diluting or dissolving, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity Isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the formulation process can be performed in a conventional manner depending on the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity Isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the formulation process can be performed in a conventional manner depending on the dosage form.
  • the formulation can be carried out by dissolving the polypeptide of the present invention or a pharmaceutically acceptable salt thereof together with the basic substance in sterile water (a surfactant is dissolved in the sterile water), and adjusting the osmotic pressure and pH to a physiological state , And can optionally add suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and tackifiers, and then completely dissolve them.
  • suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and tackifiers, and then completely dissolve them.
  • the pharmaceutical composition of the present invention can also be administered in the form of a sustained release agent.
  • the polypeptide of the present invention or a salt thereof can be incorporated into a pill or microcapsule with a sustained release polymer as a carrier, and then the pill or microcapsule is surgically implanted into a tissue to be treated.
  • the polypeptide of the present invention or a salt thereof can also be applied by inserting an intraocular lens that has been previously coated with a drug.
  • slow-release polymers examples include ethylene-vinyl acetate copolymer, polyhydroxymetaacrylate, polyacrylamide, polyvinylpyrrolidone, methyl cellulose, lactic acid polymer, Lactic-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic-glycolic acid copolymers.
  • the dosage of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient may be based on the weight, age, sex, and degree of symptoms of each patient to be treated And reasonably determined.
  • the transmembrane peptide element of the present invention is a part of the homology box of the antennal foot mutation gene (Antp) of Drosophila, which can enter the cell nucleus and bind to the promoter region of the HOX-1.3 gene to promote neural cell differentiation.
  • the transmembrane peptide element has the amino acid sequence shown in SEQ ID NO.:1.
  • Diffuse large B-cell lymphoma is the most common non-Hodgkin's lymphoma. It can be divided into three subtypes according to its gene expression profile: germinal center B-cell type ( Germinal center, cell-like DLBCL (GCB-DLBCL), activated B-cell type (DLBCL, ABC-DLBCL), and primary mediastinal B-cell type (PMBL).
  • germinal center B-cell type Germinal center, cell-like DLBCL (GCB-DLBCL), activated B-cell type (DLBCL, ABC-DLBCL), and primary mediastinal B-cell type (PMBL).
  • GCB-DLBCL germinal center B-cell type
  • DLBCL activated B-cell type
  • PMBL primary mediastinal B-cell type
  • ABC-DLBCL is the most malignant and has strong resistance to existing immunochemotherapy.
  • the five-year survival rate of GCB-DLBCL can reach 76%, while the five-year survival rate of
  • the CBM complex plays an important role in mediating the activation of NF- ⁇ B.
  • the sustained activity of the CBM complex can lead to the continuous activation of NF- ⁇ B in various cells and the generation of related diseases.
  • CBM-mediated NF- ⁇ B-related diseases include (but are not limited to): autoimmune diseases (such as psoriasis), diffuse large B-cell lymphoma, breast cancer, non-small cell lung cancer, Melanoma, colorectal cancer, or T / B cell leukemia.
  • autoimmune diseases such as psoriasis
  • diffuse large B-cell lymphoma breast cancer, non-small cell lung cancer, Melanoma, colorectal cancer, or T / B cell leukemia.
  • polypeptides of the present invention and their derived polypeptides have a small molecular weight and can penetrate a tissue barrier;
  • the polypeptide of the present invention can effectively inhibit Bcl10 polymerization, thereby inhibiting the growth of diffuse large B-cell lymphoma (especially activated B-cell diffuse large B-cell lymphoma).
  • the polypeptide of the present invention can effectively prevent and / or treat diseases related to the CBM complex (such as autoimmune diseases, diffuse large B-cell lymphoma, breast cancer, non-small cell lung cancer, melanoma, colorectal Cancer, T / B cell leukemia, etc.).
  • diseases related to the CBM complex such as autoimmune diseases, diffuse large B-cell lymphoma, breast cancer, non-small cell lung cancer, melanoma, colorectal Cancer, T / B cell leukemia, etc.
  • polypeptide of the present invention has high safety and has little toxic and side effects on biological tissues.
  • the polypeptide of the present invention can be prepared by a solid-phase synthesis method, and has high purity, large yield, and low cost.
  • polypeptide of the present invention has high specificity and good selectivity.
  • the reagents or materials used in the examples of the present invention are all commercially available products.
  • the expression plasmid of Bcl10 (1-233) was constructed on the pDB-His-MBP vector and transformed into BL21 (DE3) cell line for expression. It was initially purified by Ni-NTA (Qiagen, Valencia, CA) affinity chromatography, and then It was further purified through a gel filtration chromatography column Superdex 200HR 10/300 (GE Healthcare, UK), and the protein of interest was collected, and finally the protein was stored in a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 5 mM DTT.
  • step b Add different amino acids in sequence and make various modifications in the manner of step b.
  • the purified liquid is concentrated in a lyophilizer and lyophilized into a white powder.
  • the purified MBP-Bcl10 is mixed with the Bcl10 small molecule peptide inhibitor at a ratio of 1: 1 or 1: 2. After adding TEV enzyme to react at room temperature for 1 hour, 5ul of the reaction product is dropped on the layer containing carbon and plastic film. On the coated electron microscope copper mesh (Cat: BZ10024a), the droplets were aspirated after one minute, air-dried and then negatively stained with 3% uranyl acetate for one minute, and then the droplets were aspirated and air-dried in Shanghai. Observed under the electron microscope of JEM-1230 in the electron microscope room of the Institute of Neurology.
  • ABC-DLBCL cell lines HBL-1 and TMD8 were cultured in RPMI medium containing 10% FBS, 2 mM glutamine, 10 mM Hepes buffer, and penicillin streptomycin.
  • the GCB-DLBCL cell line OCI-LY1 was cultured in IMEM medium containing 20% fetal bovine serum and penicillin streptomycin. All cell lines were cultured in a 37 ° C constant temperature humidified incubator containing 5% CO 2 .
  • the DLBCL cell line in the logarithmic growth phase was cultured overnight in a 96-well plate containing the corresponding culture solution.
  • the candidate compounds were added to the cells the next day, and each compound was set at a concentration gradient of 3.16 times.
  • Cell proliferation is determined by quantitative detection of intracellular ATP content using fluorescence luminescence (CellTiter-Glo, Promega, Madison, WI).
  • DLBCL cell lines were detected for fluorescence signals at 0 and 72 hours after compound treatment using a multifunctional microplate reader (SpectraMax Paradigm, Molecular Devices, USA).
  • GraphPad Prism 5 software was used to draw a cell growth inhibition curve and calculate the concentration of small molecule compounds that inhibited 50% of cell growth. Three replicates were set up for each experiment.
  • the expression plasmid of Bcl10 (1-233) was constructed on the pDB-His-MBP vector and transformed into BL21 (DE3) cell line for expression. It was initially purified by Ni-NTA (Qiagen, Valencia, CA) affinity chromatography, and then It was further purified by gel filtration chromatography column Superdex 200HR 10/300 (GE Healthcare, UK), and the protein of interest was collected, and finally the protein was stored in a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 5 mM DTT.
  • the horizontal line part is the motif part, and the sequences are SEQ ID NO .: 2 (P2) or 3 (P4), respectively.
  • the purified MBP-Bcl10 protein and Bcl10 small molecule peptide inhibitor were mixed at a molar ratio of 1: 1 or 1: 2 and incubated at room temperature for half an hour, then added The TEV enzyme reacts for one hour at room temperature. The reaction product was dropped on the copper mesh of EM and negatively stained with uranyl acetate. The effect of Bcl10 small molecule peptide inhibitor on the polymerization of Bcl10 was observed under an electron microscope.
  • MBP-Bcl10 can form a fibrous structure, while the small molecule peptide inhibitors Bcl-P2 and Bcl-P4 can effectively inhibit the formation of Bcl10 fibrous structures, while Bcl-P3 cannot inhibit the formation of Bcl10 fibrous structures. , Indicating that Bcl-P2 and Bcl-P4 are effective inhibitors of Bcl10 polymerization.
  • Small molecule peptide inhibitors often have the characteristics of easy degradation and instability in cells, and modification of small molecule peptides is an effective strategy to improve the stability and effectiveness of peptides.
  • Bcl-P2 and BclP4 were modified with N-terminal acetylation (Ac-Bcl10-P2-NH2, Ac-Bcl10-P4-NH2), and reverse D peptide modification (DRI-Bcl10-P2, DRI-Bcl10-P4) (The specific sequence is shown in Table 2), and the effect of these modifications in the cell was tested, and it was found that these modified peptides can also selectively inhibit the growth of ABC-DLBCL cells ( Figure 5).
  • the horizontal line is the reverse amino acid motif and the sequence is SEQ ID NO .: 15 or 16.
  • Example 6 Bcl10 small molecule peptide inhibitor effectively inhibits ABC-DLBCL tumor growth
  • Bcl10 small molecule peptide inhibitor can inhibit the growth of ABC-DLBCL in mice
  • ABC-DLBCL cell line TMD8 cells and GCB-DLBCL cells OCL-LY1 cells were implanted into NCG mice subcutaneously to form tumors.
  • -ctl and Bcl-P4 were administered daily through the tail vein, and the inhibitory effect of Bcl10 small molecule inhibitory peptide on the growth of ABC-DLBCL tumors was observed.
  • the results show that Bcl-P4 can effectively inhibit the growth of ABC-DLBCL tumors in mice without significant side effects.
  • Bcl-P4 has no obvious inhibitory effect on the growth of GCB-DLBCL tumors.
  • BALB / c mice purchased from Nanjing University
  • BALB / c mice were pretreated with Bcl10 inhibitor DRI-Bcl10-P4 (concentration 20mg / kg) or control peptide Bcl10-Ctl (concentration: 10mg / kg) for 2 days, and then imiquimod (Imiquimod, IMQ) (60mg per day) was applied to the skin of mice for 5 days to induce psoriasis, and DRI-Bcl10-P4 (concentration 20mg / kg) was given at the same time.
  • DRI-Bcl10-P4 concentration 20mg / kg
  • FIG. 7A is H & E stained tissue sections of mouse epidermis in different treatment groups, showing the effect of different treatment groups on the thickness of mouse epidermis
  • FIG. 7B is a statistical graph of epidermal thickness of mice in different treatment groups.
  • ABC-DLBCL is currently the most malignant lymphoma and has strong resistance to the immunochemical R-CHOP therapy currently used in clinical practice.
  • One of its characteristics is the continuous activation of the NF- ⁇ B signaling pathway with growth-promoting and anti-apoptotic effects. Therefore, theoretically, looking for targeted drugs to block NF- ⁇ B signaling in ABC-DLBCL is a treatment for ABC-DLBCL Effective method.
  • NF- ⁇ B is a transcription factor with a wide range of important biological activities, it is not suitable as a therapeutic target in itself.
  • tyrosine kinase Syk is activated by binding to phosphorylated ITAM, triggering Signal cascades including Bruton's tyrosine kinase (BTK), phospholipase C ⁇ , and protein kinase C ⁇ (PKC- ⁇ ).
  • BTK Bruton's tyrosine kinase
  • PKC- ⁇ phosphorylates CARM1, prompting it to recruit BCL10 and MALT1 to form a CBM complex, thereby activating I ⁇ B kinase (IKK), and finally triggering the NF- ⁇ B signaling pathway (21).
  • activated MALT1 cleaves and inactivates NF- ⁇ B negative feedback inhibitors such as A20 (22) and CYLD (23,24) through its enzymatic activity, further promoting the activation of NF- ⁇ B signaling pathway.
  • Mutations or translocations of the above signaling pathway molecules are common in tumor cells of ABC-DLBCL, such as CARMA1 mutation (25), CD79A / B mutation (26), BCL10 and MALT1 gene translocation, etc., leading to NF- ⁇ B signaling Continuous activation.
  • MALT1 protein in the CBM complex plays an important role in mediating NF- ⁇ B activation.
  • the only MALT1 protein in the CBM complex with enzymatic activity is a popular molecular target for the development of targeted drugs for the treatment of ABC-DLBCL in recent years.
  • inhibition of MALT1 activity can effectively inhibit the signal of NF- ⁇ B, thereby selectively inhibiting or even
  • the killing of ABC-DLBCL cells demonstrates the effectiveness of the therapy targeting MALT1 enzyme activity.
  • MALT1 knockout mice showed some defects in the activation of T cells and B cells, and were otherwise normal in phenotype, suggesting that the target The side effects of treating ABC-DLBCL by enzymatic activity to MALT1 will be relatively small.
  • MALT1 caspase-like domain of MALT1 is unique in human genes, and inhibition of MALT1 will not produce a wide range of side effects caused by inhibiting other structurally similar proteins.
  • the present inventors have developed MALT1 small molecule inhibitors and demonstrated that MALT1 is an effective target for treating ABC-DLBCL.
  • Bcl10 is the core of the fiber structure of the CBM complex.
  • the fiber structure of Bcl10 provides a central scaffold platform for the activation of the CBM complex and the recruitment and activation of key proteins of the NF- ⁇ B signaling pathway, which is the key to the activation of the NF- ⁇ B signaling pathway.
  • Bcl10 mutants that do not form a fibrous structure have a significant negative inhibitory effect on the activation of the NF- ⁇ B signaling pathway. Therefore, Bcl10 may be another effective target for inhibiting the NF- ⁇ B signaling pathway and treating ABC-DLBCL.
  • the invention designs a small molecule peptide inhibitor that inhibits Bcl10 according to the structural model of Bcl10.
  • Bcl-P2 and Bcl-P4 can effectively inhibit the polymerization of Bcl10 and the formation of fiber structure and the activity of NF- ⁇ B.
  • All mouse xenograft tumor models have shown a very specific killing effect on ABC-DLBCL, proving that Bcl10 is an effective target for inhibiting NF- ⁇ B activity and ABC-DLBCL.

Abstract

本发明提供了一类新的Bcl10聚合抑制剂及其应用。所述Bcl10聚合抑制剂是多肽，且具有(i)抑制Bcl10聚合作用；(ii)抑制CBM复合物的活性；(iii)选择性的抑制B细胞淋巴瘤(如ABC-DLBCL)的生长的活性；和/或(iv)预防和/或治疗CBM复合物介导的NF-kB活化相关疾病的活性。本发明还提供了所述多肽的制备方法和应用以及含所述多肽的药物组合物。

Description

一类新的Bcl10聚合抑制剂及其应用 技术领域
本发明涉及生物医药领域，具体地涉及一类新的Bcl10聚合抑制剂及其应用。
背景技术
弥漫性大B细胞淋巴瘤(Diffuse large B cell lymphoma,DLBCL)是最常见的非霍奇金淋巴瘤，根据其基因表达图谱的不同可将其分为三个亚型：生发中心B细胞型(Germinal center B cell like DLBCL,GCB-DLBCL)、活化B细胞型(Activated B cell like DLBCL,ABC-DLBCL)和原发纵膈B细胞型(Primary mediastinal B cell lymphoma,PMBL)。其中ABC-DLBCL的恶性程度最高，对现有免疫化学疗法有强烈的耐药性，经典的R-CHOP治疗后，GCB-DLBCL的五年存活率能达到76％，而ABC-DLBCL的五年存活率只有30％左右。
因此，寻找能有效治疗ABC-DLBCL的药物和治疗方法是临床上急需解决的问题。
发明内容
本发明的目的是提供一种有效治疗ABC-DLBCL的药物和治疗方法。
本发明的另一目的是提供一种新型的抑制Bcl10聚合活性的药物，同时也提供了Bcl10抑制剂在ABC-DLBCL治疗中的应用，及其他CBM复合体介导的NF-κB依赖性疾病中的应用。
在本发明的第一方面，提供了一种式I所示的多肽，或其药学上可接受的盐，
Z0-Z1-Z2-Z3-Z4   (I)
Z0为无、N端的修饰基团或1-3个氨基酸的肽段；
Z1为穿膜肽元件；
Z2为无或连接肽；
Z3为Bcl10抑制肽元件，所述的Z3的长度为12-25aa，较佳地13-20aa，更佳地14-18aa；
Z4为无、C端的修饰基团或1-3个氨基酸的肽段；
“-”为肽键。
在另一优选例中，所述多肽的长度为30-50aa，较佳地32-40aa。
在另一优选例中，所述N端的修饰基团选自下组：乙酰基、苯甲氧基羰基C、氨基戊酸、棕榈酸、或其组合。
在另一优选例中，所述穿膜肽元件具有如SEQ ID NO.:1所示的序列。
在另一优选例中，所述穿膜肽元件的氨基酸序列如SEQ ID NO.:1所示。
在另一优选例中，所述连接肽的长度为0-10aa，较佳地，0-6aa。
在另一优选例中，所述Z3具有式A1所示的基序(5'-3')或式A2所示的反向氨基酸基序(5'-3')：
R-X1-RA-X2-K-X3-L   (A1)；或
L-X3-K-X2'-AR-X1'-R  (A2)
式中，
X1为4-6个氨基酸的肽段；较佳地X1为HFDHL或TSSRK；
X2为无、或1-2个氨基酸的肽段；较佳地X2为K或G；
X3为1-2个氨基酸的肽段；较佳地，X3为I或L；
X1'为4-6个氨基酸的肽段；较佳地X1为KRSST或LHDFH；
X2'为无、或1-2个氨基酸的肽段；较佳地X2’为K或G。
在另一优选例中，所述Z3具有式A1所示的基序(5'-3')或式A2所示的反向D型氨基酸基序(5'-3')：
R-X1-RA-X2-K-X3-L   (A1)；或
L-X3-K-X2'-AR-X1'-R   (A2)
式中，
X1为4-6个氨基酸的肽段；较佳地X1为HFDHL或TSSRK；
X2为无、或1-2个氨基酸的肽段；较佳地X2为K或G；
X3为1-2个氨基酸的肽段；较佳地，X3为I或L；
X1'为4-6个D型氨基酸的肽段；较佳地X1为KRSST或LHDFH；
X2'为无、或1-2个D型氨基酸的肽段；较佳地X2’为K或G。
在另一优选例中，所述C端的修饰基团选自下组：酰胺化，乙醛化、或其组合。
在另一优选例中，Z3具有SEQ ID No.:2或3或15或16所示的氨基酸序列。
在另一优选例中，Z3的氨基酸序列如SEQ ID NO.:2或3或15或16所示。
在另一优选例中，所述多肽具有SEQ ID NO.:4或5或8或9或13或14所示的序 列。
在另一优选例中，所述多肽的氨基酸序列如SEQ ID NO.:4或5或8或9或13或14所示。
在另一优选例中，所述多肽是天然的或人工合成的。
在另一优选例中，所述多肽选自下组：
(a)具有SEQ ID NO:4或5或8或9或13或14所示氨基酸序列的多肽；
(b)将SEQ ID NO:4或5或8或9或13或14所示氨基酸序列经过1-5个(较佳地1-3，更佳地1-2个)氨基酸残基的取代、缺失或添加而形成的，且具有抑制Bcl10的聚合作用的由(a)衍生的多肽。
在另一优选例中，所述的多肽是由SEQ ID NO.:4或5或8或9或13或14所示的多肽经过1-3个较佳地1-2个，更佳地1个氨基酸取代、缺失；和/或
经过1-5，较佳地1-4个，更佳地1-3个，最佳地1-2个氨基酸的添加形成的。
在另一优选例中，所述的衍生多肽保留了≥70％的SEQ ID NO:4或5或8或9或13或14的所示多肽的抑制Bcl10的聚合作用的活性。
在另一优选例中，所述的衍生多肽与SEQ ID NO:4或5或8或9或13或14的相同性≥80％，较佳地≥90％；更佳地≥95％。
本发明还提供了抑制Bcl10的聚合作用的、式I化合物的二聚体和多聚体形式，且所述二聚体和多聚体形式具有抑制Bcl10的聚合作用的活性。
本发明第二方面提供了一种分离的核酸分子，所述核酸分子编码本发明第一方面所述的多肽或其药学上可接受的盐。
本发明第三方面提供了一种药物组合物，包括：
(a)治疗有效量的权利要求1所述的多肽或其药学上可接受的盐；和
(b)药学上可接受的载体或赋形剂。
在另一优选例中，所述的多肽保留了≥70％、75％、70％、75％、80％、85％、90％、95％、100％、105％、110％、115％、120％、125％、130％、135％、140％、145％、150％、155％、160％、165％、170％、175％、180％、185％、190％、195％、或200％的SEQ ID NO:1所示多肽的抑制Bcl10的聚合作用的活性。
在另一优选例中，所述组合物的剂型包括包括口服剂型、或胃肠外剂型，如外用或局部使用剂型。
在另一优选例中，所述组合物的剂型包括包括片剂、丸剂、微丸剂、缓释剂、乳剂、混悬剂、颗粒剂、胶囊、粉剂、口服液、冻干剂、糖浆、膏剂、霜剂、滴剂、含服剂、静脉注射剂、栓剂、喷雾剂、气雾剂、洗剂、含漱剂、贴剂、或滴眼剂。
在另一优选例中，所述药物组合物还含有其他可(i)抑制Bcl10聚合作用；(ii)抑制CBM复合物的活性；(iii)选择性的抑制弥漫性大B细胞淋巴瘤(如ABC-DLBCL)的生长；和/或(iv)预防和/或治疗CBM复合物诱导的NF-κB活化的相关疾病的药物。
在另一优选例中，所述其他可(i)抑制Bcl10聚合作用；(ii)抑制CBM复合物的活性；(iii)选择性的抑制弥漫性大B细胞淋巴瘤(如ABC-DLBCL)的生长；和/或(iv)预防和/或治疗CBM复合物诱导的NF-κB活化的相关疾病的药物选自下组：MI-2、Mepazine、或其组合。
在另一优选例中，在所述药物组合物中，所述多肽或其药学上可接受的盐含量为0.0001-99wt％，较佳地为0.001-90wt％，更佳地0.01-50wt％，按组合物的总重量计。
本发明第四方面提供了一种本发明第一方面所述的多肽或其药学上可接受的盐的用途，用于制备组合物或药物，所述组合物或药物用于(i)抑制Bcl10聚合作用；(ii)抑制CBM复合物的活性；(iii)选择性的抑制弥漫性大B细胞淋巴瘤(如ABC-DLBCL)的生长；和/或(iv)预防和/或治疗CBM复合物诱导的NF-κB活化的相关疾病。
在另一优选例中，所述CBM复合物的相关疾病选自下组：自身免疫性疾病、长期性炎症反应，过敏性疾病，NF-κB依赖性癌症、或其组合。
在另一优选例中，所述自身免疫性疾病选自下组：银屑病、红斑狼疮、类风湿性关节炎、动脉粥样硬化、或其组合。
在另一优选例中，所述组合物或药物还用于治疗选自下组的一种或多种疾病：
哮喘、乳腺癌、非小细胞肺癌、黑丝素瘤、结直肠癌、T/B细胞性白血病、或其组合。
本发明第五方面提供了一种选择性的抑制弥漫性大B细胞淋巴瘤的生长的方法，包括步骤：给需要的对象施用本发明第一方面所述的多肽或其药学上可接受的盐。
在另一优选例中，所述的对象是人或非人哺乳动物。
在另一优选例中，所述多肽或其药学上可接受的盐的浓度为10-300mM，较佳地，30-160mM，更佳地，130-160mM。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠、兔)、灵长类动物(如猴)。
在另一优选例中，所述方法为非治疗性和非诊断性的。
在另一优选例中，所述弥漫性大B细胞淋巴瘤细胞选自下组：ABC-DLBCL、GCB-DLBCL、或其组合。
在另一优选例中，所述弥漫性大B细胞淋巴瘤细胞包括ABC-DLBCL。
本发明第六方面提供了一种体外非治疗性的抑制Bcl10的聚合作用的方法，包括步骤：
在本发明第一方面所述的多肽或其药学上可接受的盐存在的条件下，培养肿瘤细胞，从而抑制所述肿瘤细胞中Bcl10的聚合作用。
在另一优选例中，所述肿瘤细胞选自下组：弥漫性大B细胞淋巴瘤、乳腺癌、非小细胞肺癌、黑色素瘤、结直肠癌、T/B细胞性白血病、或其组合。
本发明第七方面提供了一种预防和/或治疗CBM复合物诱导的NF-κB活化的相关疾病的方法，包括步骤：向需要的对象施用治疗有效量的本发明第一方面所述的多肽、和/或本发明第三方面所述的药物组合物。
在另一优选例中，所述的对象是人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠、兔)、灵长类动物(如猴)。
本发明第八方面提供了一种筛选选择性抑制弥漫性大B细胞淋巴瘤的生长的候选化合物的方法，包括步骤：
(a)将Bcl10蛋白与化合物库混合，测定化合物库中的化合物与所述Bcl10蛋白的结合情况；
其中，如果所述测试化合物库中的化合物与所述Bcl10蛋白有结合，则表明所述与Bcl10蛋白结合的化合物为候选化合物。
(b)将步骤(a)中所确定的候选化合物施用于弥漫性大B细胞淋巴瘤细胞，测定其对弥漫性大B细胞淋巴瘤的影响。
在另一优选例中，所述的方法是非诊断和非治疗性的。
在另一优选例中，所述弥漫性大B细胞淋巴瘤细胞选自下组：ABC-DLBCL、GCB-DLBCL、或其组合。
在另一优选例中，所述弥漫性大B细胞淋巴瘤细胞包括ABC-DLBCL。
本发明第九方面提供了一种筛选选择性抑制弥漫性大B细胞淋巴瘤的生长的候选化合物的方法，包括步骤：
(a)在测试组中，在培养体系中，在测试化合物的存在下，培养表达Bcl10蛋白的肿瘤细胞一段时间T1，检测测试组所述培养体系中所述Bcl10蛋白的表达情况E1；
并且在不存在所述测试化合物且其他条件相同的对照组中，检测对照组所述培养体系中所述Bcl10蛋白的表达情况E2；
(b)如果所述测试组中的所述Bcl10蛋白的表达情况E1显著低于所述对照组中的所述Bcl10蛋白的表达情况E2，则表示所述测试化合物是候选化合物。
在另一优选例中，所述的方法包括步骤(c)：将步骤(b)中所确定的候选化合物施用于哺乳动物模型，测定其对哺乳动物的影响。
在另一优选例中，所述哺乳动物为患有弥漫性大B细胞淋巴瘤的哺乳动物。
在另一优选例中，所述“显著低于”指E1/E2≤1/2,较佳地，≤1/3，更佳地≤1/4。
在另一优选例中，所述的方法是非诊断和非治疗性的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。
图1显示了MBP-Bcl10的凝胶过滤层析峰形图及峰产物SDS-PAGE胶结果图。
图2显示了根据CB复合体的结构模型设计了1个对照小分子肽和5个Bcl10小分子肽抑制剂。
图3显示了Bcl10小分子肽抑制剂对Bcl10纤维结构形成的影响。
图4显示了Bcl10小分子肽抑制剂对ABC-DLBCL细胞系(HBL-1和TMD8)和GCB-DLBCL细胞系(OCL-LY1)的生长的影响。
图5显示了Bcl10小分子肽抑制剂修饰肽对ABC-DLBCL细胞系(HBL-1和TMD8)和GCB-DLBCL细胞系(OCL-LY1)的生长的影响。
图6显示了Bcl10小分子肽抑制剂对ABC-DLBCL肿瘤在NCG小鼠体内生长的影响。
图7显示了Bcl10小分子肽抑制剂对银屑病模型的作用。其中，图7A为咪喹莫特(IMQ)诱导的银屑病模型中，不同Bcl10小分子抑制肽处理组的小鼠表皮组织切片H&E染色图，显示不同处理组对小鼠表皮厚度的影响，图7B为不同处理组小鼠表皮厚度的统计图。
具体实施方式
本发明人经过广泛而深入的研究，首次制备了一类具有抑制Bcl10聚合作用的，分子量小于5KD(如仅约2.3-4.4kD)的小分子多肽。具体而言，本发明设计了数个候选序列，采用固相法将其合成，分离纯化获得高纯度的小肽Bcl10-P2、Bcl10-P4及其修饰形式，并运用HPLC及MS对之进行鉴定，再经过大量筛选，获得了一类新型的、具有(i)抑制Bcl10聚合作用；(ii)抑制CBM复合物的活性；(iii)选择性的抑制B细胞淋巴瘤(如ABC-DLBCL)的生长；和/或(iv)预防和/或治疗CBM复合物诱导的NF-κB活化的相关疾病活性的小分子多肽。在此基础上，本发明人完成了本发明。
活性多肽
在本发明中，术语“本发明多肽”、“Bcl10聚合抑制剂”可互换使用，都指具有(i)抑制Bcl10聚合作用；(ii)抑制CBM复合物的活性；(iii)选择性的抑制B细胞淋巴瘤(如ABC-DLBCL)的生长；和/或(iv)预防和/或治疗CBM复合物诱导的NF-κB活化的相关疾病活性的具有SEQ ID NO.:4或5或8或9或13或14所示的氨基酸序列的蛋白或多肽。此外，所述术语还包括具有抑制(i)抑制Bcl10聚合作用；(ii)抑制CBM复合物的活性；(iii)选择性的抑制B细胞淋巴瘤(如ABC-DLBCL)的生长；和/或(iv)预防和/或治疗CBM复合物诱导的NF-κB活化的相关疾病活性的、SEQ ID NO：4或5或8或9或13或14 所示序列的变异形式。这些变异形式包括(但并不限于)：1-5个(通常为1-4个，较佳地1-3个，更佳地1-2个，最佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为5个以内，较佳地为3个以内，更佳地为2个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
本发明还包括本发明多肽的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持(i)抑制Bcl10聚合作用；(ii)抑制CBM复合物的活性；(iii)选择性的抑制B细胞淋巴瘤(如ABC-DLBCL)的生长；和/或(iv)与预防和/或治疗CBM复合物诱导的NF-κB活化的相关疾病活性的的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)本发明的多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的然后蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式I的氨基酸序列相比，有至多5个，较佳地至多3个，更佳地至多2个，最佳地1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
发明还提供本发明多肽的类似物。这些类似物与天然本发明多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
本发明多肽还可以以由药学上或生理学可接受的酸或碱衍生的盐形式使用。这些盐包括(但不限于)与如下酸形成的盐：氢氯酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、琥珀酸、草酸、富马酸、马来酸、草酰乙酸、甲磺酸、乙磺酸、苯磺酸或羟乙磺酸。其他盐包括：与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以及以酯、氨基甲酸酯或其他常规的“前 体药物”的形式。
编码序列
本发明还涉及编码本发明多肽的多核苷酸。一种优选的编码序列如SEQ ID NO:6(Bcl10-P2)或7(Bcl10-P4)所示，它编码SEQ ID NO:4(Bcl10-P2)或5(Bcl10-P4)所示的多肽的氨基酸序列。
在一优选实施方式中，Bcl10-P2的核苷酸序列如下所示：
Bcl10-P2:
5’-GACCGCCAGATAAAGATTTGGTTCCAGAATCGGCGCATGAAGTGGAAGAAGAGACATTTTGATCATCTACGTGCAAAAAAAATACTCAGTAGA-3’(SEQ ID No.:6)
在一优选实施方式中，Bcl10-P4的核苷酸序列如下所示：
Bcl10-P4:
5’-GACCGCCAGATAAAGATTTGGTTCCAGAATCGGCGCATGAAGTGGAAGAAGCGAACATCAAGTAGAAAAAGGGCTGGAAAATTGTTAGACTACTTACAGGAA-3’(SEQ ID No.:7)
本发明的多核苷酸可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO:6或7所示的编码区序列相同或者是简并的变异体。如本文所用，以SEQ ID NO:6或7为例，“简并的变异体”在本发明中是指编码具有SEQ ID NO:4或5所示序列的多肽，但与SEQ ID NO:6或7中相应编码区序列有差别的核酸序列。
本发明的多肽的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明多肽(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或ZY多肽编码序列经基因工程产生的宿主细胞。
另一方面，本发明还包括对本发明多肽具有特异性的多克隆抗体和单克隆抗体，尤其是单克隆抗体。
在两个核酸或多肽的背景下，当进行最大相符性序列比较和比对时，术语“基本相同”是指两个或多个序列或亚序列，其具有至少约80％，例如至少约85％、约90％、约95％、约98％或约99％的核苷酸或氨基酸残基与特定的参考序列具有同一性，如使用以下序列比较方法和/或通过肉眼检查所测定。
在本发明中，所有的序列如下表所示。
Figure PCTCN2019093094-appb-000001
Figure PCTCN2019093094-appb-000002
制备方法
本发明多肽可以是重组多肽或合成多肽。本发明的多肽可以是化学合成的，或重组的。相应地，本发明多肽可用常规方法人工合成，也可用重组方法生产。
一种优选的方法是使用液相合成技术或固相合成技术，如Boc固相法、Fmoc固相法或是两种方法联合使用。固相合成可快速获得样品，可根据目的肽的序列特征选用适当的树脂载体及合成系统。例如，Fmoc系统中优选的固相载体如连接有肽中C端氨基酸的Wang树脂，Wang树脂结构为聚苯乙烯，与氨基酸间的手臂是4-烷氧基苄醇；用25％六氢吡啶/二甲基甲酰胺室温处理20分钟，以除去Fmoc保护基团，并按照给定的氨基酸序列由C端逐个向N端延伸。合成完成后，用含4％对甲基苯酚的三氟乙酸将合成的胰岛素原相关肽从树脂上切割下来并除去保护基，可过滤除树脂后乙醚沉淀分离得到粗肽。将所得产物的溶液冻干后，用凝胶过滤和反相高压液相层析法纯化所需的肽。当使用Boc系统进行固相合成时，优选树脂为连接有肽中C端氨基酸的PAM树脂，PAM树脂结构为聚苯乙烯，与氨基酸间的手臂是4-羟甲基苯乙酰胺；在Boc合成系统中，在去保护、中和、偶联的循环中，用TFA/二氯甲烷(DCM)除去保护基团Boc并用二异丙基乙胺(DIEA/二氯甲烷中和。肽链缩合完成后，用含对甲苯酚(5-10％)的氟化氢(HF),在0℃下处理1小时，将肽链从树脂上切下，同时除去保护基团。以50-80％乙酸(含少量巯基乙醇)抽提肽，溶液冻干后进一步用分子筛Sephadex G10或Tsk-40f分离纯化，然后再经高压液相纯化得到所需的肽。可以使用肽化学领域内已知的各种偶联剂和偶联方法偶联各氨基酸残基，例如可使用二环己基碳二亚胺(DCC)，羟基苯骈三氮唑(HOBt)或1,1,3,3-四脲六氟磷酸酯(HBTU)进行直接偶联。对于合成得到的短肽，其纯度与结构可用反相高效液相和质谱分析进行确证。
在一优选例中，本发明的多肽，按其序列，采用固相合成的方法制备，行 高效液相色谱纯化，获得高纯度目的肽冻干粉，-20℃贮存。
另一种方法是用重组技术产生本发明多肽。通过常规的重组DNA技术，可利用本发明的多核苷酸可用来表达或生产重组的本发明的多肽。一般来说有以下步骤：
(1).用本发明的编码本发明多肽的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
重组多肽可在细胞内或在细胞膜上表达或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
由于本发明多肽较短，因此可以考虑将多个多肽串联在一起，重组表达后获得多聚体形式的表达产物，然后通过酶切等方法形成所需的小肽。
药物组合物和施用方法
另一方面，本发明还提供了一种药物组合物，它含有(a)安全有效量的本发明多肽或其药学上可接受的盐；以及(b)药学上可接受的载体或赋形剂。本发明多肽的数量通常为10微克-100毫克/剂，较佳地为100-1000微克/剂。
为了本发明的目的，有效的剂量为给予个体约0.01毫克/千克至50毫克/千克，较佳地0.05毫克/千克至10毫克/千克体重的本发明多肽。此外，本发明的多肽可以单用，也可与其他治疗剂一起使用(如配制在同一药物组合物中)。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在Remington’s Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂及其 组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：肌内、静脉内、皮下、皮内或局部给药。待预防或治疗的对象可以是动物；尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地，可以例举的有片剂、颗粒剂、胶囊、丸剂、注射剂、或口服液。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
例如，配制可这样进行：将本发明的多肽或其药学上可接受的盐与基本物质一起溶解于无菌水(在无菌水中溶解有表面活性剂)中，调节渗透压和酸碱度至生理状态，并可任意地加入合适的药物添加剂如防腐剂、稳定剂、缓冲剂、等渗剂、抗氧化剂和增粘剂，然后使其完全溶解。
本发明的药物组合物还可以缓释剂形式给药。例如，本发明的多肽或其盐可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。此外，本发明的多肽或其盐还可通过插入预先涂有药物的眼内透镜而得以应用。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明的多肽或其药学上可接受的盐的剂量，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定。
穿膜肽元件
本发明的穿膜肽元件是果蝇的触角足突变基因(Antp)同源盒的一部分，可以进入到细胞核内并结合到HOX-1.3基因的启动子区域，促进神经细胞的分化。
在本发明一优选实施方式中，所述穿膜肽元件具有SEQ ID NO.:1所示的氨基酸序列。
弥漫性大B细胞淋巴瘤
弥漫性大B细胞淋巴瘤(Diffuse large B cell lymphoma,DLBCL)是最常见的非霍奇金淋巴瘤，根据其基因表达图谱的不同可将其分为三个亚型：生发中心B细胞型(Germinal center B cell like DLBCL,GCB-DLBCL)、活化B细胞型(Activated B cell like DLBCL,ABC-DLBCL)和原发纵膈B细胞型(Primary mediastinal B cell lymphoma,PMBL)。其中ABC-DLBCL的恶性程度最高，对现有免疫化学疗法有强烈的耐药性，经典的R-CHOP治疗后，GCB-DLBCL的五年存活率能达到76％，而ABC-DLBCL的五年存活率只有30％左右。
CBM介导的NF-κB的相关性疾病
不同的类型的细胞中，CBM复合体在介导NF-κB的活化中有重要作用，CBM复合体持续性活性可以导致多种细胞的NF-κB的持续性活化和相关疾病的产生。
在本发明中，CBM介导的NF-κB的相关性疾病包括(但不限于)：自身免疫性疾病(如银屑病)、弥漫性大B细胞淋巴瘤、乳腺癌、非小细胞肺癌、黑丝素瘤、结直肠癌、或T/B细胞性白血病。
本发明的主要优点包括：
(a)本发明多肽及其衍生多肽的分子量小，可透过组织屏障；
(b)本发明的多肽可有效抑制Bcl10聚合作用，从而抑制弥漫性大B细胞淋巴瘤(尤其是活化B细胞型弥漫性大B细胞淋巴瘤)的生长。
(c)本发明的多肽可有效预防和/或治疗CBM复合物的相关疾病(如自身免疫性疾病、弥漫性大B细胞淋巴瘤、乳腺癌、非小细胞肺癌、黑丝素瘤、结直肠癌、T/B细胞性白血病等)。
(d)本发明的多肽的安全性高，对生物组织毒副作用小。
(e)本发明的多肽可通过固相合成的方法制备，纯度高，产量大，成本低。
(f)本发明多肽的稳定性好。
(g)本发明多肽的特异性高，选择性好。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
如无特别说明，本发明实施例中所用的试剂或材料均为市售产品。
材料和方法
1.1 MBP-Bcl10蛋白表达和纯化
Bcl10(1-233)的表达质粒构建在pDB-His-MBP的载体上，转化到BL21(DE3)细胞株中表达，通过Ni-NTA(Qiagen，Valencia，CA)亲和层析初步纯化，再通过凝胶过滤色谱柱Superdex 200HR 10/300(GE Healthcare，UK)进一步纯化，收集目的蛋白，最后蛋白储存在含20mM Tris(pH 7.5),150mM NaCl,和5mM DTT的缓冲液中。
1.2 Bcl10小分子多肽合成
设计了多条小分子肽，经检验发现其中两条小分子肽能有效抑制Bcl10的聚合及纤维结构的形成。他们的序列分别是：DRQIKIWFQNRRMKWKKRTSSRKRAGKLLDYLQE(SEQ ID No.:5)和DRQIKIWFQNRRMKWKKRHFDHLRAKKILSR(SEQ ID No.:4)。
①小分子肽合成顺序：从序列C端到N端，步骤如下：
a.称取n当量树脂放入反应器，加入DCM(二氯甲烷)溶胀半小时，然后抽掉DCM，加入序列中第一个氨基酸2n当量，加2n当量的DIEA，适量的DMF，DCM(适量是指以可使树脂充分鼓动起来为宜)，DIEA(二异丙基乙胺)、DMF(二甲基甲酰胺)、DCM，氮气鼓泡反应60min。然后加入约5n当量甲醇，反应半小时，抽掉反应液，用DMF、MEOH洗净。
b.往反应器中加入序列中第二个氨基酸(也为2n当量)，2n当量HBTU(1-羟基,苯并,三氯唑四甲基六氟磷酸盐)及DIEA，N2鼓泡反应半小时，洗掉液体，茚三酮 检测，然后用吡啶和乙酸酐封端。最后洗净，加入适量的脱帽液去除Fmoc(9-芴甲氧羰基)保护基，洗净，茚三酮检测。
c.依步骤b的方式依次加入序列中不同的氨基酸并进行各种修饰。
d.将树脂用氮气吹干后从反应柱中取下，倒入烧瓶中，然后往烧瓶中加一定量(切割液和树脂大约以10ml/克的比例)的切割液(组成是95％TFA，2％乙二硫醇，2％三异丙基硅烷，1％水)，震荡，滤掉树脂。
e.得到滤液，然后向滤液中加入大量乙醚，析出粗产物，然后离心，清洗即可得到序列的粗产物。
②多肽纯化：
开发新工艺用高效液相色谱将粗品提纯至要求纯度。
③多肽冻干：
纯化好的液体放入冻干机中进行浓缩，冻干成白色粉末。
1.3透射电镜EM实验
纯化好的MBP-Bcl10与Bcl10小分子肽抑制剂以1:1或1:2的比例混合，加入TEV酶在室温下反应1小时后，将反应产物的5ul滴在含有薄层碳和塑料膜涂层的电镜铜网(中镜科仪cat：BZ10024a)上，一分钟后吸去液滴，风干后再用3％的乙酸铀酰负染一分钟，之后吸去液滴，风干后在上海生科院神经所电镜室的JEM-1230电镜下观察。
1.4细胞培养
ABC-DLBCL细胞株HBL-1和TMD8培养在含10％FBS，2mM谷氨酰胺、10mM Hepes缓冲液和青霉素链霉素的RPMI培养液中。GCB-DLBCL细胞株OCI-LY1培养在含20％胎牛血清和青霉素链霉素的IMEM培养液中。所有的细胞株均培养在含有5％CO 2的37℃恒温湿润培养箱中。
1.5细胞生长抑制实验
对数生长期的DLBCL细胞株在含相应培养液的96孔板中培养过夜，第二天向细胞中加入各候选化合物，每个化合物按3.16倍的稀释比例设置浓度梯度。细胞增殖是通过荧光发光法定量检测细胞内ATP的含量来检测细胞数(CellTiter-Glo,Promega,Madison,WI)。DLBCL细胞株在化合物处理0小时和72小时后，用多功能酶标仪(SpectraMax Paradigm,Molecular Devices,USA)检测荧光信号。小分子肽抑制剂的生长抑制率的计算公式为：如果72小时荧光值(T72)大于0小时荧光值(T0)，生长抑制率＝(T72 cpd-T0)/(T72 DMSO-T0)*100； 如果T72小于T0，生长抑制率＝(T72 cpd-T0)/T0*100。用GraphPad Prism 5软件来绘制细胞生长抑制曲线图，并计算抑制50％细胞生长的小分子化合物浓度。每个实验设置3个复孔。
实施例1 MBP-Bcl10的表达和纯化
Bcl10(1-233)的表达质粒构建在pDB-His-MBP的载体上，转化到BL21(DE3)细胞株中表达，通过Ni-NTA(Qiagen，Valencia，CA)亲和层析初步纯化，再通过凝胶过滤色谱柱Superdex 200HR 10/300(GE Healthcare，UK)进一步纯化，收集目的蛋白，最后蛋白储存在含20mM Tris(pH 7.5),150mM NaCl,and 5mM DTT的缓冲液中。
结果如图1所示，在12-14mL左右的位置开始出现目的蛋白峰，用SDS-PAGE胶验证此峰形下的蛋白确实是MBP-Bcl10蛋白，分子量为75kD，纯度为95％。
实施例2 Bcl10小分子肽抑制剂的设计
根据CB复合体的结构模型设计了1个对照小分子肽和5个Bcl10小分子肽抑制剂。其序列如表1及图2所示。
表1：Bcl10小分子肽抑制剂序列：
肽 序列 SEQ ID No.:
Bcl10-Ctl: DRQIKIWFQNRRMKWKK 1
Bcl10-P1: DRQIKIWFQNRRMKWKK EKIIAERHF 10
Bcl10-P2: DRQIKIWFQNRRMKWKK  RHFDHLRAKKILSR 4
Bcl10-P3: DRQIKIWFQNRRMKWKK REDTEEISCR 11
Bcl10-P4: DRQIKIWFQNRRMKWKK  RTSSRKRAGKLLDYLQE 5
Bcl10-P5: DRQIKIWFQNRRMKWKK KGLDTLVESIRREKTQ 12
其中，横线部分为基序部分，序列分别为SEQ ID NO.:2(P2)或3(P4)。
实施例3 Bcl10小分子抑制剂对Bcl10聚合作用的影响
为了检测Bcl10小分子抑制剂对Bcl10抑制剂作用的影响，将纯化好的MBP-Bcl10蛋白同Bcl10小分子肽抑制剂按照1:1或1:2的摩尔比例混合在室温孵育半小时，然后加入TEV酶在室温反应一小时。反应产物滴在EM的铜网 上经乙酸铀酰负染后在电镜下观察Bcl10小分子肽抑制剂对Bcl10聚合作用的影响。
结果如图3可见，MBP-Bcl10能形成纤维状结构，而小分子肽抑制剂Bcl-P2和Bcl-P4能够有效地抑制Bcl10纤维结构的形成，而Bcl-P3则不能抑制Bcl10纤维结构的形成，说明Bcl-P2和Bcl-P4是有效的Bcl10聚合抑制剂。
实施例4 Bcl10小分子肽抑制剂对ABC-DLBCL细胞系特异性地生长抑制作用
为了检验Bcl10小分子肽抑制剂对ABC-DLBCL和GCB-DLBCL细胞的增殖的影响，采用不同浓度梯度的小分子肽抑制剂，分别作用于两种ABC-DLBCL细胞株即HBL-1、TMD8和一种GCB-DLBCL细胞株OCI-LY1上，0h和72h后测定细胞数量。
结果表明，能够有效抑制Bcl10聚合的小分子肽Bcl-P2和BclP4能够特异性系抑制ABC-DLBCL细胞的生长(GI50：3～5μM)，而对GCB-DLBCL生长没有明显抑制作用(GI50>100μM)；而对Bcl10聚合作用没有影响的小分子肽抑制剂Bcl-P1、Bcl-P3和Bcl-P5则对ABC-DLBCL的生长没有抑制作用，说明Bcl10的聚合作用对ABC-DLBCL的生长有关键作用，而Bcl10也是抑制ABC-DLBCL的生长的有效靶点。
实施例5 Bcl10小分子肽抑制剂修饰肽对ABC-DLBCL细胞系特异性地生长抑制作用
小分子肽抑制剂常常在细胞内有容易降解、不稳定的特点，而将小分子肽进行修饰是提高肽的稳定性及有效性的有效策略。将Bcl-P2和BclP4进行了N端乙酰化修饰(Ac-Bcl10-P2-NH2，Ac-Bcl10-P4-NH2)，以及反向D肽修饰(DRI-Bcl10-P2，DRI-Bcl10-P4)(具体序列见表2)，并测试了这些修饰在细胞内的作用，发现这些修饰肽也能选择性抑制ABC-DLBCL细胞的生长(图5)。
表2：修饰的Bcl10小分子肽抑制剂序列：
Figure PCTCN2019093094-appb-000003
Figure PCTCN2019093094-appb-000004
注释:小写字母代表D型氨基酸
横线部分为反向氨基酸基序，序列分别为SEQ ID NO.:15或16。
实施例6 Bcl10小分子肽抑制剂有效抑制ABC-DLBCL肿瘤的生长
为了检测Bcl10小分子肽抑制剂能不能在小鼠体内抑制ABC-DLBCL的生长，将ABC-DLBCL细胞系TMD8细胞和GCB-DLBCL细胞OCL-LY1细胞植入NCG小鼠皮下成瘤，然后将Bcl-ctl和Bcl-P4经尾静脉每天给药，观察Bcl10小分子抑制肽对ABC-DLBCL肿瘤的生长抑制作用。如图6，结果表明，Bcl-P4能有效抑制ABC-DLBCL肿瘤在小鼠体内的生长，而没有明显的毒副作用。Bcl-P4对GCB-DLBCL肿瘤的生长抑制作用不明显。
实施例7 Bcl10小分子肽抑制剂有效缓解银屑病的发生
为了检测Bcl10抑制剂在IMQ诱导的银屑病小鼠模型中的作用效果，本发明采用BALB/c小鼠(购自南京大学)进行了实验。先用Bcl10抑制剂DRI-Bcl10-P4(浓度20mg/kg)或对照肽Bcl10-Ctl(浓度：10mg/kg)腹腔注射2天预处理BALB/c小鼠，然后用咪喹莫特(Imiquimod，简称IMQ)(每天60mg)涂抹小鼠皮肤5天诱导银屑病，并同时给予DRI-Bcl10-P4(浓度20mg/kg)，结果发现IMQ能有效诱导小鼠表皮增厚等银屑病样特征(图7A、7B)，而DRI-Bcl10-P4处理组小鼠的表皮厚度得到了明显的降低，银屑病样症状得到了明显的缓解(图7A和7B)，表明Bcl10抑制剂能有效抑制ImQ诱导的BALB/c小鼠银屑病样特征的发生发展。图7A为不同处理组小鼠表皮的H&E染色组织切片，显示不同处理组对小鼠表皮厚度的影响，图7B为不同处理组小鼠表皮厚度的统计图。
讨论
ABC-DLBCL是目前恶性程度最高的淋巴瘤，对目前临床上常用的免疫化学R-CHOP疗法有强烈的耐药性。它的特点之一是具有促生长、抗凋亡作用的NF-κB信号通路的持续激活，因此，理论上来说，寻找靶向药物来阻碍ABC-DLBCL中的NF-κB信号是治疗ABC-DLBCL的有效方法。但是，由于NF-κB是具有广泛重要生物活性的转录因子，本身并不适合作为治疗靶点。
在B细胞中，抗原结合BCR后诱导Src家族激酶磷酸化CD79A和CD79B的ITAM结构域的酪氨酸，接着，酪氨酸激酶Syk通过结合到磷酸化的ITAM上被激活，从而引发了包括布鲁顿酪氨酸激酶(Bruton's tyrosine kinase，BTK)、磷酸酶Cγ(phospholipase Cγ)以及蛋白激酶Cβ(protein kinase Cβ，PKC-β)在内的信号级联反应。PKC-β使CARM1发生磷酸化，促使它招募BCL10和MALT1，形成CBM复合物，从而激活IκB激酶(IKK)，最后激发了NF-κB信号通路(21)。另一方面，激活的MALT1通过其酶活性切割灭活A20(22)、CYLD(23,24)等NF-κB负反馈抑制剂，进一步促进NF-κB信号通路的活化。在ABC-DLBCL的肿瘤细胞中常见以上信号通路分子的突变或是转位，例如CARMA1突变(25)、CD79A/B突变(26)、BCL10和MALT1基因的转位等，导致NF-κB信号通路的持续性活化。
由上可见，CBM复合体在介导NF-κB活化中有重要作用。而CBM复合体中唯一具有酶活性的MALT1蛋白是近年来开发治疗ABC-DLBCL的靶向药物的热门分子靶点：首先，抑制MALT1的活性可以有效抑制NF-κB的信号，从而选择性抑制甚至杀伤ABC-DLBCL细胞，说明靶向MALT1酶活的治疗有效性；其次，MALT1敲除的小鼠除了在T细胞和B细胞的激活方面表现出一部分缺陷，在其他方面都表型正常，提示靶向MALT1酶活来治疗ABC-DLBCL的副作用会比较小；第三，MALT1的类caspase结构域在人类基因中是唯一的，抑制MALT1不会产生由于抑制其他结构相似的蛋白等造成的广泛副作用。本发明人已经开发了MALT1小分子抑制剂，并证明了MALT1是治疗ABC-DLBCL的有效靶点。
Bcl10是CBM复合体纤维结构的核心，Bcl10的纤维结构为CBM复合体的活化以及NF-κB信号通路关键蛋白的招募和活化提供了中心支架平台，是NF-κB信号通路激活的关键。不能形成纤维结构的Bcl10的突变体对NF-κB信号通路的活化有显性负作用的抑制作用，因此，Bcl10可能是抑制NF-κB信号通路，治疗ABC-DLBCL的另一个有效靶点。本发明根据Bcl10的结构模型设计了 抑制Bcl10的小分子肽抑制剂，其中Bcl-P2和Bcl-P4能够有效地抑制Bcl10的聚合作用和纤维结构的形成和NF-κB的活性，在细胞水平和小鼠异体移植瘤模型上都显示了对ABC-DLBCL有非常特异的杀伤作用，证明Bcl10是抑制NF-κB活性和ABC-DLBCL的有效靶点。
大量研究表明，异常的NF-κB信号通路的活化在乳腺癌、激素抵抗型前列腺癌、肺癌、甲状腺癌、结肠癌、淋巴瘤等多种癌症及自身免疫性疾病(如银屑病等)中都有着重要作用。近年来的研究显示CBM蛋白复合体及其家族蛋白在这些癌症组织中有广泛的表达，该复合体在除淋巴瘤之外的其它NF-κB相关癌症中也可能有关键作用。因此，Bcl10抑制剂也可能为治疗上述癌症及自身免疫性疾病提供有效的治疗方法。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种式I所示的多肽，或其药学上可接受的盐，
    Z0-Z1-Z2-Z3-Z4  (I)
    Z0为无、N端的修饰基团或1-3个氨基酸的肽段；
    Z1为穿膜肽元件；
    Z2为无或连接肽；
    Z3为Bcl10抑制肽元件，所述的Z3的长度为12-25aa，较佳地13-20aa，更佳地14-18aa；
    Z4为无、C端的修饰基团或1-3个氨基酸的肽段；
    “-”为肽键。
  2. 如权利要求1所述的式I所示的多肽，或其药学上可接受的盐，其特征在于，所述多肽的长度为30-50aa，较佳地32-40aa。
  3. 如权利要求1所述的式I所示的多肽，或其药学上可接受的盐，其特征在于，所述N端的修饰基团选自下组：乙酰基、苯甲氧基羰基C、氨基戊酸、棕榈酸、或其组合。
  4. 如权利要求1所述的式I所示的多肽，或其药学上可接受的盐，其特征在于，所述Z3具有式A1所示的基序(5'-3')或式A2所示的反向氨基酸基序(5'-3')：
    R-X1-RA-X2-K-X3-L  (A1)；或
    L-X3-K-X2'-AR-X1'-R  (A2)
    式中，
    X1为4-6个氨基酸的肽段；较佳地X1为HFDHL或TSSRK；
    X2为无、或1-2个氨基酸的肽段；较佳地X2为K或G；
    X3为1-2个氨基酸的肽段；较佳地，X3为I或L；
    X1'为4-6个氨基酸的肽段；较佳地X1为KRSST或LHDFH；
    X2'为无、或1-2个氨基酸的肽段；较佳地X2’为K或G。
  5. 如权利要求1所述的式I所示的多肽，或其药学上可接受的盐，其特征在于，所述多肽具有SEQ ID NO.:4或5或8或9或13或14所示的序列。
  6. 一种分离的核酸分子，其特征在于，所述核酸分子编码权利要求1所述的多肽或其药学上可接受的盐。
  7. 一种药物组合物，其特征在于，包括：
    (a)治疗有效量的权利要求1所述的多肽或其药学上可接受的盐；和
    (b)药学上可接受的载体或赋形剂。
  8. 一种权利要求1所述的多肽或其药学上可接受的盐的用途，其特征在于，用于制备组合物或药物，所述组合物或药物用于(i)抑制Bcl10聚合作用；(ii)抑制CBM复合物的活性；(iii)选择性的抑制弥漫性大B细胞淋巴瘤的生长；和/或(iv)预防和/或治疗CBM复合物诱导的NF-κB活化的相关疾病。
  9. 一种选择性的抑制弥漫性大B细胞淋巴瘤的生长的方法，其特征在于，包括步骤：给需要的对象施用权利要求1所述的多肽或其药学上可接受的盐。
  10. 一种体外非治疗性的抑制Bcl10的聚合作用的方法，其特征在于，包括步骤：
    在权利要求1所述的多肽或其药学上可接受的盐存在的条件下，培养肿瘤细胞，从而抑制所述肿瘤细胞中Bcl10的聚合作用。
  11. 一种筛选选择性抑制弥漫性大B细胞淋巴瘤的生长的候选化合物的方法，其特征在于，包括步骤：
    (a)将Bcl10蛋白与化合物库混合，测定化合物库中的化合物与所述Bcl10蛋白的结合情况；
    其中，如果所述测试化合物库中的化合物与所述Bcl10蛋白有结合，则表明所述与Bcl10蛋白结合的化合物为候选化合物。
    (b)将步骤(a)中所确定的候选化合物施用于弥漫性大B细胞淋巴瘤细胞，测定其对弥漫性大B细胞淋巴瘤的影响。
  12. 一种筛选选择性抑制弥漫性大B细胞淋巴瘤的生长的候选化合物的方法，其特征在于，包括步骤：
    (a)在测试组中，在培养体系中，在测试化合物的存在下，培养表达Bcl10蛋白的肿瘤细胞一段时间T1，检测测试组所述培养体系中所述Bcl10蛋白的表达情况E1；
    并且在不存在所述测试化合物且其他条件相同的对照组中，检测对照组所述培养体系中所述Bcl10蛋白的表达情况E2；
    (b)如果所述测试组中的所述Bcl10蛋白的表达情况E1显著低于所述对照组中的所述Bcl10蛋白的表达情况E2，则表示所述测试化合物是候选化合物。
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CN105920582A (zh) * 2008-12-10 2016-09-07 普渡研究基金会 基于细胞渗透性肽的激酶抑制剂

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