CN111094357A - Novel Bcl10 polymerization inhibitor and application thereof - Google Patents

Novel Bcl10 polymerization inhibitor and application thereof Download PDF

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Publication number
CN111094357A
CN111094357A CN201980004308.4A CN201980004308A CN111094357A CN 111094357 A CN111094357 A CN 111094357A CN 201980004308 A CN201980004308 A CN 201980004308A CN 111094357 A CN111094357 A CN 111094357A
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bcl10
polypeptide
peptide
pharmaceutically acceptable
acceptable salt
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杨成华
张亮
孙晨霞
鲍伟
于浩澜
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Shanghai Institutes for Biological Sciences SIBS of CAS
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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
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    • 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
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    • 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

Abstract

The invention provides a novel Bcl10 polymerization inhibitor and application thereof. The Bcl10 polymerization inhibitor is a polypeptide and has (i) inhibiting Bcl10 polymerization; (ii) inhibiting the activity of the CBM complex; (iii) activity to selectively inhibit the growth of B cell lymphoma (e.g., ABC-DLBCL); and/or (iv) preventing and/or treating disease activity associated with CBM complex mediated NF-kB activation. The invention also provides a preparation method and application of the polypeptide and a pharmaceutical composition containing the polypeptide.

Description

Novel Bcl10 polymerization inhibitor and application thereof Technical Field
The invention relates to the field of biological medicines, in particular to a novel Bcl10 polymerization inhibitor and application thereof.
Background
Diffuse large B-cell lymphoma (DLBCL) is the most common non-hodgkin lymphoma, and can be divided into three subtypes according to the difference in gene expression profiles: germinal center B cell like DLBCL (GCB-DLBCL), Activated B cell type (ABC-DLBCL) and Primary mediastinal B cell type (PMBL). The ABC-DLBCL has the highest malignancy degree and has strong drug resistance to the existing immunochemistry therapy, after the classical R-CHOP treatment, the five-year survival rate of the GCB-DLBCL can reach 76 percent, and the five-year survival rate of the ABC-DLBCL is only about 30 percent.
Therefore, the search for drugs and therapeutic methods that can effectively treat ABC-DLBCL is a problem that needs to be solved clinically.
Disclosure of Invention
The invention aims to provide a medicament and a treatment method for effectively treating ABC-DLBCL.
Another objective of the invention is to provide a novel drug for inhibiting the polymerization activity of Bcl10, and also to provide application of Bcl10 inhibitors in the treatment of ABC-DLBCL and other CBM complex-mediated NF-kB dependent diseases.
In a first aspect of the invention, there is provided a polypeptide of formula I, or a pharmaceutically acceptable salt thereof,
Z0-Z1-Z2-Z3-Z4 (I)
z0 is nothing, a modification group at the N end or a peptide segment of 1-3 amino acids;
z1 is a cell penetrating peptide element;
z2 is nothing or a linker peptide;
z3 is a Bcl10 inhibitory peptide element, and the length of Z3 is 12-25aa, preferably 13-20aa, more preferably 14-18 aa;
z4 is nothing, a modification group at the C terminal or a peptide segment of 1-3 amino acids;
"-" is a peptide bond.
In another preferred embodiment, the length of the polypeptide is 30-50aa, preferably 32-40 aa.
In another preferred embodiment, the N-terminal modifying group is selected from the group consisting of: acetyl, benzyloxycarbonyl C, aminopentanoic acid, palmitic acid, or combinations thereof.
In another preferred embodiment, the cell-penetrating peptide element has a sequence as shown in SEQ ID No. 1.
In another preferred embodiment, the amino acid sequence of the cell-penetrating peptide element is shown in SEQ ID No. 1.
In another preferred embodiment, the length of the linking peptide is 0-10aa, preferably 0-6 aa.
In another preferred embodiment, said Z3 has the motif (5'-3') represented by formula a1 or the inverted amino acid motif (5'-3') represented by formula a 2:
R-X1-RA-X2-K-X3-L (A1); or
L-X3-K-X2'-AR-X1'-R (A2)
In the formula (I), the compound is shown in the specification,
x1 is a peptide segment of 4-6 amino acids; preferably X1 is HFDHL or TSSRK;
x2 is nothing, or a peptide segment of 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 segment of 4-6 amino acids; preferably X1 is KRSST or LHDFH;
x2' is nothing, or a peptide fragment of 1-2 amino acids; preferably X2' is K or G.
In another preferred embodiment, said Z3 has the motif (5'-3') represented by formula a1 or the inverted D-form amino acid motif (5'-3') represented by formula a 2:
R-X1-RA-X2-K-X3-L (A1); or
L-X3-K-X2'-AR-X1'-R (A2)
In the formula (I), the compound is shown in the specification,
x1 is a peptide segment of 4-6 amino acids; preferably X1 is HFDHL or TSSRK;
x2 is nothing, or a peptide segment of 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 segment of 4-6D-amino acids; preferably X1 is KRSST or LHDFH;
x2' is peptide segment without or 1-2D amino acids; preferably X2' is K or G.
In another preferred embodiment, the C-terminal modifying group is selected from the group consisting of: amidation, hydroformylation, or a combination thereof.
In another preferred embodiment, Z3 has the amino acid sequence shown in SEQ ID No. 2 or 3 or 15 or 16.
In another preferred embodiment, the amino acid sequence of Z3 is as shown in SEQ ID NO. 2 or 3 or 15 or 16.
In another preferred embodiment, the polypeptide has the sequence shown in SEQ ID No. 4 or 5 or 8 or 9 or 13 or 14.
In another preferred embodiment, the amino acid sequence of the polypeptide is as shown in SEQ ID No. 4 or 5 or 8 or 9 or 13 or 14.
In another preferred embodiment, the polypeptide is natural or synthetic.
In another preferred embodiment, the polypeptide is selected from the group consisting of:
(a) a polypeptide having an amino acid sequence shown as SEQ ID NO 4 or 5 or 8 or 9 or 13 or 14;
(b) a polypeptide derived from (a) which is formed by substituting, deleting or adding 1-5 (preferably 1-3, more preferably 1-2) amino acid residues in an amino acid sequence shown in SEQ ID NO. 4, 5, 8, 9, 13 or 14, and has the function of inhibiting the polymerization of Bcl 10.
In another preferred embodiment, the polypeptide is a polypeptide represented by SEQ ID No. 4 or 5 or 8 or 9 or 13 or 14, which is substituted or deleted by 1-3, preferably 1-2, more preferably 1 amino acid; and/or
Formed by the addition of 1 to 5, preferably 1 to 4, more preferably 1 to 3, most preferably 1 to 2 amino acids.
In another preferred embodiment, the derivative 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 in inhibiting Bcl10 polymerization.
In another preferred embodiment, the derived polypeptide has an identity of 80% or more, preferably 90% or more, to SEQ ID NO 4 or 5 or 8 or 9 or 13 or 14; more preferably not less than 95%.
The invention also provides dimeric and multimeric forms of the compounds of formula I that inhibit polymerization of Bcl10, and which have activity in inhibiting polymerization of Bcl 10.
In a second aspect, the present invention provides an isolated nucleic acid molecule encoding a polypeptide according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof.
In a third aspect, the present invention provides a pharmaceutical composition comprising:
(a) a therapeutically effective amount of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof; and
(b) a pharmaceutically acceptable carrier or excipient.
In another preferred embodiment, the polypeptide retains ≥ 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%, or 200% of the activity of the polypeptide set forth in SEQ ID NO:1 in inhibiting polymerization of Bcl 10.
In another preferred embodiment, the dosage form of the composition comprises an oral dosage form, or a parenteral dosage form, such as a topical or topical dosage form.
In another preferred embodiment, the dosage form of the composition comprises a tablet, a pill, a pellet, a sustained release formulation, an emulsion, a suspension, a granule, a capsule, a powder, an oral liquid, a lyophilized formulation, a syrup, an ointment, a cream, a drop, an buccal formulation, an intravenous injection, a suppository, a spray, an aerosol, a lotion, a gargle, a patch, or an eye drop.
In another preferred embodiment, the pharmaceutical composition further comprises an additional agent that (i) inhibits polymerization of Bcl 10; (ii) inhibiting the activity of the CBM complex; (iii) selectively inhibiting the growth of diffuse large B-cell lymphoma (such as ABC-DLBCL); and/or (iv) an agent for preventing and/or treating a disease associated with NF-. kappa.B activation induced by CBM complex.
In another preferred embodiment, the other(s) inhibit Bcl10 polymerization; (ii) inhibiting the activity of the CBM complex; (iii) selectively inhibiting the growth of diffuse large B-cell lymphoma (such as ABC-DLBCL); and/or (iv) an agent for preventing and/or treating a disease associated with CBM complex-induced NF- κ B activation selected from the group consisting of: MI-2, Mepazine, or a combination thereof.
In another preferred embodiment, the polypeptide or its pharmaceutically acceptable salt is contained in the pharmaceutical composition in an amount of 0.0001-99 wt%, preferably 0.001-90 wt%, more preferably 0.01-50 wt%, based on the total weight of the composition.
In a fourth aspect, the invention provides a use of a polypeptide according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, for the manufacture of a composition or medicament for (i) inhibiting Bcl10 polymerisation; (ii) inhibiting the activity of the CBM complex; (iii) selectively inhibiting the growth of diffuse large B-cell lymphoma (such as ABC-DLBCL); and/or (iv) preventing and/or treating diseases associated with CBM complex-induced NF- κ B activation.
In another preferred embodiment, the disease associated with the CBM complex is selected from the group consisting of: autoimmune diseases, chronic inflammatory reactions, allergic diseases, NF- κ B dependent cancers, or a combination thereof.
In another preferred embodiment, the autoimmune disease is selected from the group consisting of: psoriasis, lupus erythematosus, rheumatoid arthritis, atherosclerosis, or a combination thereof.
In another preferred embodiment, the composition or medicament is also for the treatment of 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.
In a fifth aspect, 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 a polypeptide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof.
In another preferred embodiment, the subject is a human or non-human mammal.
In another preferred embodiment, the concentration of the polypeptide or the pharmaceutically acceptable salt thereof is 10-300mM, preferably 30-160mM, more preferably 130-160 mM.
In another preferred embodiment, the non-human mammal includes a rodent (e.g., mouse, rat, rabbit), primate (e.g., monkey).
In another preferred embodiment, the method is non-therapeutic and non-diagnostic.
In another preferred embodiment, the diffuse large B-cell lymphoma cells are selected from the group consisting of: ABC-DLBCL, GCB-DLBCL, or a combination thereof.
In another preferred embodiment, the diffuse large B-cell lymphoma cells comprise ABC-DLBCL.
In a sixth aspect, the present invention provides an in vitro non-therapeutic method of inhibiting polymerization of Bcl10, comprising the steps of:
culturing a tumor cell in the presence of a polypeptide of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, thereby inhibiting polymerization of Bcl10 in said tumor cell.
In another preferred embodiment, the tumor cell is 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.
In a seventh aspect, the present invention provides a method for preventing and/or treating a disease associated with NF- κ B activation induced by CBM complex, comprising the steps of: administering to a subject in need thereof a therapeutically effective amount of a polypeptide according to the first aspect of the invention, and/or a pharmaceutical composition according to the third aspect of the invention.
In another preferred embodiment, the subject is a human or non-human mammal.
In another preferred embodiment, the non-human mammal includes a rodent (e.g., mouse, rat, rabbit), primate (e.g., monkey).
An eighth aspect of the present invention provides a method of screening a candidate compound for selective inhibition of the growth of diffuse large B-cell lymphoma, comprising the steps of:
(a) mixing the Bcl10 protein with a library of compounds, and determining binding of compounds in the library of compounds to the Bcl10 protein;
wherein, if a compound in the test compound library binds to the Bcl10 protein, it indicates that the compound that binds to the Bcl10 protein is a candidate compound.
(b) Administering the candidate compound identified in step (a) to diffuse large B-cell lymphoma cells and determining its effect on diffuse large B-cell lymphoma.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
In another preferred embodiment, the diffuse large B-cell lymphoma cells are selected from the group consisting of: ABC-DLBCL, GCB-DLBCL, or a combination thereof.
In another preferred embodiment, the diffuse large B-cell lymphoma cells comprise ABC-DLBCL.
In a ninth aspect, the present invention provides a method of screening a candidate compound for selective inhibition of the growth of diffuse large B-cell lymphoma comprising the steps of:
(a) culturing tumor cells expressing Bcl10 protein in a test group in a culture system in the presence of a test compound for a period of time T1, and detecting the expression of the Bcl10 protein in the culture system of the test group E1;
and detecting expression of said Bcl10 protein in said culture system of a control group in a control group otherwise identical in the absence of said test compound E2;
(b) if the expression profile of the Bcl10 protein in the test group, E1, is significantly lower than the expression profile of the Bcl10 protein in the control group, E2, then it is an indication that the test compound is a candidate compound.
In another preferred embodiment, the method comprises the step (c): administering the candidate compound identified in step (b) to a mammalian model and determining its effect on the mammal.
In another preferred example, the mammal is a mammal suffering from diffuse large B-cell lymphoma.
In another preferred embodiment, the phrase "substantially less than" means E1/E2 ≦ 1/2, preferably ≦ 1/3, more preferably ≦ 1/4.
In another preferred embodiment, the method is non-diagnostic and non-therapeutic.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.
FIG. 1 shows the peak shape of MBP-Bcl10 by gel filtration chromatography and the result of SDS-PAGE gel.
Figure 2 shows that 1 control small peptide and 5 Bcl10 small peptide inhibitors were designed based on a structural model of the CB complex.
FIG. 3 shows the effect of Bcl10 small molecule peptide inhibitors on the formation of Bcl10 fibrous structures.
FIG. 4 shows the effect of a Bcl10 small molecule peptide inhibitor on the growth of ABC-DLBCL cell line (HBL-1 and TMD8) and GCB-DLBCL cell line (OCL-LY 1).
FIG. 5 shows the effect of a Bcl10 small molecule peptide inhibitor modified peptide on the growth of ABC-DLBCL cell line (HBL-1 and TMD8) and GCB-DLBCL cell line (OCL-LY 1).
FIG. 6 shows the effect of Bcl10 small peptide inhibitors on the growth of ABC-DLBCL tumors in NCG mice.
Figure 7 shows the effect of Bcl10 small molecule peptide inhibitors on psoriasis models. Wherein, fig. 7A is H & E staining graph of mouse epidermal tissue sections of different Bcl10 small molecule inhibitory peptide treatment groups in an Imiquimod (IMQ) -induced psoriasis model, showing the effect of different treatment groups on mouse epidermal thickness, and fig. 7B is a statistical graph of mouse epidermal thickness of different treatment groups.
Detailed Description
The present inventors have conducted extensive and intensive studies and have for the first time prepared a class of small polypeptides having a molecular weight of less than 5KD (e.g., only about 2.3-4.4KD) that inhibit the polymerization of Bcl 10. Specifically, the invention designs a plurality of candidate sequences, and the candidate sequences are synthesized by a solid phase method, separated and purified to obtain high-purity small peptides Bcl10-P2, Bcl10-P4 and modified forms thereof, and are identified by HPLC and MS, and then are screened in a large amount to obtain a novel peptide which has (i) the function of inhibiting Bcl10 polymerization; (ii) inhibiting the activity of the CBM complex; (iii) selectively inhibiting the growth of B cell lymphoma (e.g., ABC-DLBCL); and/or (iv) small molecule polypeptides that prevent and/or treat disease activities associated with CBM complex-induced NF- κ B activation. On this basis, the present inventors have completed the present invention.
Active polypeptide
In the present invention, the terms "polypeptide of the present invention", "Bcl 10 polymerization inhibitor" are used interchangeably and all refer to a polypeptide having (i) inhibitory activity against Bcl10 polymerization; (ii) inhibiting the activity of the CBM complex; (iii) selectively inhibiting the growth of B cell lymphoma (e.g., ABC-DLBCL); and/or (iv) a protein or polypeptide having an amino acid sequence as set forth in SEQ ID No. 4 or 5 or 8 or 9 or 13 or 14 for preventing and/or treating disease activity associated with NF- κ B activation induced by CBM complex. In addition, the term also includes compositions having an inhibitory activity (i) against Bcl10 polymerization; (ii) inhibiting the activity of the CBM complex; (iii) selectively inhibiting the growth of B cell lymphoma (e.g., ABC-DLBCL); and/or (iv) the activity of SEQ ID NO:4 or 5 or 8 or 9 or 13 or 14. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 5 (usually 1 to 4, preferably 1 to 3, more preferably 1 to 2, most preferably 1) amino acids, and addition or deletion of one or several (usually within 5, preferably within 3, more preferably within 2) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).
The invention also includes active fragments, derivatives and analogs of the polypeptides of the invention. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains (i) inhibition of Bcl10 polymerization; (ii) inhibiting the activity of the CBM complex; (iii) selectively inhibiting the growth of B cell lymphoma (e.g., ABC-DLBCL); and/or (iv) polypeptides associated with disease activity for the prevention and/or treatment of CBM complex-induced NF- κ B activation. The fragment, derivative or analogue of the polypeptide of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the polypeptide of the present invention is fused to another compound (such as a compound for increasing the half-life of the polypeptide, for example, polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (a protein which is then fused to a leader sequence, a secretory sequence or a tag sequence such as 6 His). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.
A preferred class of reactive derivatives refers to polypeptides formed by the replacement of up to 5, preferably up to 3, more preferably up to 2, most preferably 1 amino acid with an amino acid of similar or analogous nature compared to the amino acid sequence of formula I. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.
TABLE 1
Initial residue(s) Representative substitutions Preferred substitutions
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
Analogs also include analogs having residues other than the natural L-amino acid (e.g., the D-amino acid), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β, gamma-amino acids).
Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.
The polypeptides of the invention can also be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases. These salts include, but are not limited to, salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulphuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulphonic acid, ethanesulphonic acid, benzenesulphonic 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 "prodrug" forms.
Coding sequence
The present invention also relates to polynucleotides encoding the polypeptides of the invention. A preferred coding sequence is shown in SEQ ID NO 6(Bcl10-P2) or 7(Bcl10-P4) which encodes the amino acid sequence of the polypeptide shown in SEQ ID NO 4(Bcl10-P2) or 5(Bcl 10-P4).
In a preferred embodiment, the nucleotide sequence of Bcl10-P2 is as follows:
Bcl10-P2:
5’-GACCGCCAGATAAAGATTTGGTTCCAGAATCGGCGCATGAAGTGGAAGAAGAGACATTTTGATCATCTACGTGCAAAAAAAATACTCAGTAGA-3’(SEQ ID No.:6)
in a preferred embodiment, the nucleotide sequence of Bcl10-P4 is as follows:
Bcl10-P4:
5’-GACCGCCAGATAAAGATTTGGTTCCAGAATCGGCGCATGAAGTGGAAGAAGCGAACATCAAGTAGAAAAAGGGCTGGAAAATTGTTAGACTACTTACAGGAA-3’(SEQ ID No.:7)
the polynucleotide of the present invention may be in the form of DNA or RNA. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region as shown in SEQ ID NO 6 or 7 or may be a degenerate variant. As used herein, in the case of SEQ ID NO 6 or 7, a "degenerate variant" refers herein to a nucleic acid sequence which encodes a polypeptide having the sequence shown in SEQ ID NO 4 or 5, but differs from the corresponding coding region sequence in SEQ ID NO 6 or 7.
The full-length nucleotide sequence of the polypeptide of the present invention or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the polypeptides of the present invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.
The invention also relates to vectors comprising the polynucleotides of the invention, and to genetically engineered host cells produced with the vectors or ZY polypeptide coding sequences of the invention.
In another aspect, the invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the polypeptides of the invention.
The term "substantially identical" in the context of two nucleic acids or polypeptides, when compared and aligned for maximum correspondence, refers to two or more sequences or subsequences that have at least about 80%, e.g., at least about 85%, about 90%, about 95%, about 98%, or about 99% nucleotide or amino acid residue identity to a particular reference sequence, as determined using the following sequence comparison method and/or by visual inspection.
In the present invention, all sequences are shown in the following table.
Figure PCTCN2019093094-APPB-000001
Figure PCTCN2019093094-APPB-000002
Preparation method
The polypeptides of the invention may be recombinant polypeptides or synthetic polypeptides. The polypeptides of the invention may be chemically synthesized, or recombinant. Accordingly, the polypeptides of the present invention can be artificially synthesized by a conventional method or can be produced by a recombinant method.
A preferred method is to use liquid phase synthesis techniques or solid phase synthesis techniques, such as Boc solid phase method, Fmoc solid phase method or a combination of both. The solid phase synthesis can quickly obtain samples, and can select proper resin carriers and synthesis systems according to the sequence characteristics of target peptides. For example, the preferred solid support in the Fmoc system is Wang resin with C-terminal amino acid attached to the peptide, Wang resin is polystyrene in structure, and the arm between the Wang resin and the amino acid is 4-alkoxybenzyl alcohol; the Fmoc protecting group was removed by treatment with 25% piperidine/dimethylformamide for 20 minutes at room temperature and extended from the C-terminus to the N-terminus one by one according to the given amino acid sequence. After completion of the synthesis, the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol and the protecting groups are removed, optionally by filtration and isolated as a crude peptide by ether precipitation. After lyophilization of the resulting solution of the product, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography. When the solid phase synthesis is performed using the Boc system, it is preferable that the resin is a PAM resin to which a C-terminal amino acid in a peptide is attached, the PAM resin has a structure of polystyrene, and an arm between the PAM resin and the amino acid is 4-hydroxymethylphenylacetamide; in the Boc synthesis system, after the cycle of deprotection, neutralization and coupling, Boc of the protecting group is removed with TFA/Dichloromethane (DCM) and diisopropylethylamine (DIEA/dichloromethane neutralization. peptide chain condensation is completed, the peptide chain is cleaved from the resin by treatment with Hydrogen Fluoride (HF) containing p-cresol (5-10%) at 0 ℃ for 1 hour while removing the protecting group, the peptide is extracted with 50-80% acetic acid (containing a small amount of mercaptoethanol), the solution is lyophilized and then further separated and purified with molecular sieves Sephadex G10 or Tsk-40f, followed by high pressure liquid phase purification to obtain the desired peptide, various coupling agents and coupling methods known in the field of peptide chemistry can be used to couple each amino acid residue, for example, Dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3, 3-tetraurea Hexafluorophosphate (HBTU) can be used for direct coupling of the synthesized short peptide, the purity and structure of the product can be confirmed by reversed-phase high performance liquid chromatography and mass spectrometry.
In a preferred embodiment, the polypeptide of the present invention is prepared by solid phase synthesis according to its sequence, and purified by high performance liquid chromatography to obtain high purity target peptide lyophilized powder, which is stored at-20 ℃.
Another method is to produce the polypeptide of the invention by recombinant techniques. The polynucleotides of the present invention may be used to express or produce recombinant polypeptides of the present invention by conventional recombinant DNA techniques. Generally, the following steps are performed:
(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a polypeptide of the invention, or with a recombinant expression vector comprising the polynucleotide;
(2) a host cell cultured in a suitable medium;
(3) isolating and purifying the protein from the culture medium or the cells.
The recombinant polypeptide may be expressed or secreted intracellularly or on the cell membrane outside the cell. 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 such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
Because the polypeptide of the invention is short, a plurality of polypeptides can be considered to be connected in series, a multimeric expression product is obtained after recombinant expression, and then the required small peptide is formed by enzyme digestion and other methods.
Pharmaceutical compositions and methods of administration
In another aspect, the present invention provides a pharmaceutical composition comprising (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. mu.g to 100 mg/dose, preferably 100. mu.g to 1000. mu.g/dose.
For the purposes of the present invention, an effective dose is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10mg/kg, of the polypeptide of the invention to a subject. In addition, the polypeptides of the invention may be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. co., n.j.1991). Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.
Pharmaceutically acceptable carriers in therapeutic compositions can comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
Generally, the therapeutic compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; solid forms suitable for constitution with a solution or suspension, or liquid carrier, before injection, may also be prepared.
Once formulated, the compositions of the present invention may 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.
When the pharmaceutical composition of the present invention is used for practical treatment, various dosage forms of the pharmaceutical composition may be used depending on the use case. Preferably, tablets, granules, capsules, pills, injections, or oral liquids are exemplified.
These pharmaceutical compositions may be formulated by mixing, dilution or dissolution according to a conventional method, and occasionally, suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents are added, and the formulation process may be carried out in a conventional manner according to the dosage form.
For example, the formulation may be carried out as follows: the polypeptide of the present invention or a pharmaceutically acceptable salt thereof is dissolved in sterile water (in which a surfactant is dissolved) together with a basic substance, the osmotic pressure and the pH value are adjusted to physiological conditions, and suitable pharmaceutical additives such as a preservative, a stabilizer, a buffer, an isotonizing agent, an antioxidant and a tackifier may be optionally added and then completely dissolved.
The pharmaceutical compositions of the present invention may also be administered in the form of sustained release formulations. For example, the polypeptide of the invention or a salt thereof can be incorporated into a pellet or microcapsule carried by a slow release polymer and then surgically implanted into the tissue to be treated. In addition, the polypeptide of the present invention or a salt thereof can be used by inserting an intraocular lens previously coated with a drug. As examples of the sustained-release polymer, ethylene-vinyl acetate copolymer, polyhydroxymethacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer and the like can be exemplified, and biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be preferably exemplified.
When the pharmaceutical composition of the present invention is used for practical treatment, the dosage of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient can be determined reasonably according to the body weight, age, sex, and degree of symptoms of each patient to be treated.
Cell-penetrating peptide element
The cell-penetrating peptide element is a part of an antennapedia mutation gene (Antp) homology box of drosophila, can enter into cell nucleus and is combined with a promoter region of HOX-1.3 gene to promote differentiation of nerve cells.
In a preferred embodiment of the invention, the cell-penetrating peptide element has the amino acid sequence shown in SEQ ID No. 1.
Diffuse large B cell lymphoma
Diffuse large B-cell lymphoma (DLBCL) is the most common non-hodgkin lymphoma, and can be divided into three subtypes according to the difference in gene expression profiles: germinal center B cell like DLBCL (GCB-DLBCL), Activated B cell type (ABC-DLBCL) and Primary mediastinal B cell type (PMBL). The ABC-DLBCL has the highest malignancy degree and has strong drug resistance to the existing immunochemistry therapy, after the classical R-CHOP treatment, the five-year survival rate of the GCB-DLBCL can reach 76 percent, and the five-year survival rate of the ABC-DLBCL is only about 30 percent.
CBM-mediated NF-kB related diseases
In different cell types, the CBM complex plays an important role in mediating NF-. kappa.B activation, and the persistent activity of the CBM complex can lead to the persistent activation of NF-. kappa.B in various cells and the production of related diseases.
In the present invention, CBM-mediated NF-. kappa.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.
The main advantages of the invention include:
(a) the polypeptide and the derived polypeptide thereof have small molecular weight and can permeate a tissue barrier;
(b) the polypeptide of the invention can effectively inhibit Bcl10 polymerization, thereby inhibiting the growth of diffuse large B-cell lymphoma (especially activated B-cell type diffuse large B-cell lymphoma).
(c) The polypeptide of the 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 and the like).
(d) The polypeptide of the invention has high safety and small toxic and side effects on biological tissues.
(e) The polypeptide of the invention can be prepared by a solid phase synthesis method, and has high purity, large yield and low cost.
(f) The polypeptide of the invention has good stability.
(g) The polypeptide of the invention has high specificity and good selectivity.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.
Unless otherwise specified, all reagents and materials used in examples of the present invention are commercially available products.
Materials and methods
1.1 MBP-Bcl10 protein expression and purification
Expression plasmids of Bcl10(1-233) were constructed on a vector of pDB-His-MBP, transformed into BL21(DE3) cell line for expression, preliminarily purified by Ni-NTA (Qiagen, Valencia, CA) affinity chromatography, further purified by gel filtration chromatography Superdex 200HR 10/300(GE Healthcare, UK), collected for the protein of interest, and finally stored in a buffer containing 20mM Tris (pH 7.5),150mM NaCl, and 5mM DTT.
1.2 Bcl10 Small molecule polypeptide Synthesis
A plurality of small-molecule peptides are designed, and two of the small-molecule peptides are proved to be capable of effectively inhibiting the polymerization of Bcl10 and the formation of a fiber structure through tests. Their sequences were respectively: DRQIKIWFQNRRMKWKKRTSSRKRAGKLLDYLQE (SEQ ID No.:5) and DRQIKIWFQNRRMKWKKRHFDHLRAKKILSR (SEQ ID No.: 4).
① Synthesis sequence of small molecule peptide, from C end to N end of the sequence, the steps are as follows:
a. weighing n equivalents of resin, putting the resin into a reactor, adding DCM (dichloromethane) to swell for half an hour, then pumping out DCM, adding 2n equivalents of the first amino acid in the sequence, adding 2n equivalents of DIEA, an appropriate amount of DMF (dimethyl formamide), DCM (the appropriate amount is that the resin can be fully stirred), DIEA (diisopropylethylamine), DMF (dimethyl formamide), DCM, and nitrogen for bubbling reaction for 60 min. Then, about 5n equivalent of methanol was added thereto, and the reaction mixture was reacted for half an hour, and then the reaction mixture was taken out and washed with DMF and MEOH.
b. The second amino acid in the sequence (also 2N equivalents), 2N equivalents HBTU (1-hydroxy, benzo, trichloroazol tetramethyl hexafluorophosphate) and DIEA, N2 were bubbled through the reactor for half an hour, washed off the liquid, assayed for ninhydrin, and then capped with pyridine and acetic anhydride. And finally, washing, adding a proper amount of decapping liquid to remove the Fmoc (9-fluorenylmethyloxycarbonyl) protecting group, washing, and detecting ninhydrin.
c. And (c) adding different amino acids in the sequence in sequence according to the mode of the step b and carrying out various modifications.
d. The resin was blown dry with nitrogen, taken out of the reaction column, poured into a flask, and then a certain amount (about 10 ml/g ratio of cleavage solution to resin) of a cleavage solution (composition 95% TFA, 2% ethanedithiol, 2% triisopropylsilane, 1% water) was added to the flask, shaken, and the resin was filtered off.
e. Obtaining filtrate, then adding a large amount of ether into the filtrate to separate out a crude product, then centrifuging and cleaning to obtain the crude product of the sequence.
② polypeptide purification:
the crude product is purified to the required purity by high performance liquid chromatography.
③ lyophilization of the polypeptide:
and (4) putting the purified liquid into a freeze dryer for concentration, and freeze-drying to obtain white powder.
1.3 Transmission Electron microscopy EM experiment
Mixing the purified MBP-Bcl10 and a Bcl10 small-molecule peptide inhibitor in a ratio of 1:1 or 1:2, adding TEV enzyme, reacting at room temperature for 1 hour, dripping 5ul of a reaction product on an electron microscope copper net (BZ 10024a) containing thin-layer carbon and a plastic film coating, sucking the liquid drops after one minute, air-drying, carrying out negative dyeing by 3% uranyl acetate for one minute, sucking the liquid drops, air-drying, and observing under a JEM-1230 electron microscope in a nerve endoscope room of Shanghai institute of sciences.
1.4 cell culture
ABC-DLBCL cell lines HBL-1 and TMD8 were cultured in RPMI medium containing 10% FBS, 2mM glutamine, 10mM Hepes buffer and penicillin streptomycin. 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 the presence of 5% CO237 ℃ in a constant temperature humid incubator.
1.5 cell growth inhibition assay
DLBCL cell lines in the logarithmic growth phase were cultured overnight in 96-well plates containing the respective culture liquids, and each candidate compound was added to the cells the next day, and a concentration gradient was set at a dilution ratio of 3.16 times for each compound. Cell proliferation is the number of cells measured by quantitative measurement of intracellular ATP content by fluorescence spectrometry (CellTiter-Glo, Promega, Madison, Wis.). The DLBCL cell line was treated with the compound for 0 hours and 72 hours, and then the fluorescence signal was detected using a multifunctional microplate reader (SpectraMax Paradigm, Molecular Devices, USA). The calculation formula of the growth inhibition rate of the small molecule peptide inhibitor is as follows: if the fluorescence value at 72 hours (T72) is greater than the fluorescence value at 0 hours (T0), the growth inhibition rate is (T72)cpd-T0)/(T72DMSO-T0) × 100; if T72 is less than T0, the growth inhibition rate is (T72)cpd-T0)/T0 x 100. Cell growth inhibition was plotted using GraphPad Prism 5 software and the concentration of small molecule compound that inhibited 50% of cell growth was calculated. 3 duplicate wells were set for each experiment.
Example 1 expression and purification of MBP-Bcl10
Expression plasmids of Bcl10(1-233) were constructed on a vector of pDB-His-MBP, transformed into BL21(DE3) cell line for expression, preliminarily purified by Ni-NTA (Qiagen, Valencia, CA) affinity chromatography, further purified by gel filtration chromatography Superdex 200HR 10/300(GE Healthcare, UK), collected for the protein of interest, and finally stored in a buffer containing 20mM Tris (pH 7.5),150mM NaCl, and 5mM DTT.
As a result, as shown in FIG. 1, the peak of the desired protein began to appear at a position of about 12-14mL, and it was confirmed by SDS-PAGE that the protein in this peak form was indeed MBP-Bcl10 protein, and had a molecular weight of 75kD and a purity of 95%.
Example 2 design of Bcl10 Small molecule peptide inhibitors
1 control small peptide and 5 Bcl10 small peptide inhibitors were designed based on a structural model of the CB complex. The sequences are shown in Table 1 and FIG. 2.
Table 1: bcl10 small molecule peptide inhibitor sequence:
peptides Sequence of 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
Wherein, the horizontal line part is a base sequence part, and the sequence is SEQ ID NO. 2(P2) or 3 (P4).
Example 3 Effect of Small molecule inhibitors of Bcl10 on Bcl10 polymerization
In order to test the influence of the Bcl10 small-molecule inhibitor on the action of the Bcl10 inhibitor, the purified MBP-Bcl10 protein and the Bcl10 small-molecule peptide inhibitor are mixed according to the molar ratio of 1:1 or 1:2, and are incubated at room temperature for half an hour, and then TEV enzyme is added to react at room temperature for one hour. The reaction product is dripped on a copper net of EM and is negatively stained with uranyl acetate, and the influence of the Bcl10 small-molecule peptide inhibitor on the polymerization of Bcl10 is observed under an electron microscope.
As shown in FIG. 3, MBP-Bcl10 can form fibrous structures, while the small-molecule peptide inhibitors Bcl-P2 and Bcl-P4 can effectively inhibit the formation of the fibrous structures of Bcl10, while Bcl-P3 cannot inhibit the formation of the fibrous structures of Bcl10, which indicates that the Bcl-P2 and Bcl-P4 are effective Bcl10 polymerization inhibitors.
Example 4 growth inhibition of ABC-DLBCL cell lines with Small molecule peptide inhibitors of Bcl10
In order to test the influence of the Bcl10 small-molecule peptide inhibitor on the proliferation of ABC-DLBCL and GCB-DLBCL cells, the small-molecule peptide inhibitor with different concentration gradients is adopted to act on two ABC-DLBCL cell strains, namely HBL-1 and TMD8 and one GCB-DLBCL cell strain OCI-LY1 respectively, and the cell number is measured after 0h and 72 h.
The result shows that the small molecule peptides Bcl-P2 and BclP4 capable of effectively inhibiting the polymerization of Bcl10 can specifically inhibit the growth of ABC-DLBCL cells (GI 50: 3-5 mu M), and have no obvious inhibition effect on the growth of GCB-DLBCL (GI50>100 mu M); and small molecule peptide inhibitors Bcl-P1, Bcl-P3 and Bcl-P5 which have no influence on the polymerization of Bcl10 have no inhibition effect on the growth of ABC-DLBCL, which shows that the polymerization of Bcl10 has a key effect on the growth of ABC-DLBCL, and Bcl10 is an effective target for inhibiting the growth of ABC-DLBCL.
Example 5 Bcl10 Small molecule peptide inhibitor modified peptides specifically inhibit the growth of ABC-DLBCL cell line
Small-molecule peptide inhibitors are often easy to degrade and unstable in cells, and modification of small-molecule peptides is an effective strategy for improving stability and effectiveness of the peptides. Bcl-P2 and BclP4 were subjected to N-terminal acetylation (Ac-Bcl10-P2-NH2, Ac-Bcl10-P4-NH2) and reverse D-peptide modification (DRI-Bcl10-P2, DRI-Bcl10-P4) (see Table 2 for specific sequences), and the effect of the modifications in cells was tested, and it was found that these modified peptides can also selectively inhibit the growth of ABC-DLBCL cells (FIG. 5).
Table 2: modified Bcl10 small molecule peptide inhibitor sequence:
Figure PCTCN2019093094-APPB-000003
Figure PCTCN2019093094-APPB-000004
note that lower case letters represent the D form of amino acids
The horizontal line part is an inverted amino acid motif, and the sequence is SEQ ID No. 15 or 16 respectively.
Example 6 Bcl10 Small molecule peptide inhibitors effectively inhibit the growth of ABC-DLBCL tumors
To test the inability of Bcl10 small-molecule peptide inhibitors to inhibit the growth of ABC-DLBCL in mice, the ABC-DLBCL cell line TMD8 cells and GCB-DLBCL cells OCL-LY1 cells were implanted into NCG mice for subcutaneous neoplasia, then Bcl-ctl and Bcl-P4 were administered daily via the caudal vein and the growth inhibitory effect of Bcl10 small-molecule inhibitory peptides on ABC-DLBCL tumors was observed. As shown in FIG. 6, the result shows that Bcl-P4 can effectively inhibit the growth of ABC-DLBCL tumor in mice without obvious toxic and side effects. The growth inhibition effect of the Bcl-P4 on GCB-DLBCL tumor is not obvious.
Example 7 Bcl10 Small molecule peptide inhibitors are effective in reducing the incidence of psoriasis
To test the effect of Bcl10 inhibitors in an IMQ-induced psoriasis mouse model, experiments were performed using BALB/c mice (purchased from the university of tokyo). BALB/c mice were pretreated by intraperitoneal injection of a Bcl10 inhibitor DRI-Bcl10-P4 (concentration 20mg/kg) or a control peptide Bcl10-Ctl (concentration: 10mg/kg) for 2 days, and then the skin of the mice was smeared with Imiquimod (IMQ) (60 mg per day) for 5 days to induce psoriasis, and at the same time DRI-Bcl10-P4 (concentration 20mg/kg) was administered, so that IMQ was found to be effective in inducing psoriasis-like features such as thickening of the epidermis in the mice (FIGS. 7A and 7B), whereas mice treated with DRI-Bcl10-P4 had a marked reduction in the thickness of the epidermis and marked alleviation of the psoriasis-like symptoms (FIGS. 7A and 7B), indicating that the Bcl10 inhibitor was effective in inhibiting the development of ImQ-induced psoriasis-like features in BALB/c mice. Fig. 7A is a H & E stained histological section of the epidermis of mice of different treatment groups showing the effect of different treatment groups on the thickness of the epidermis of the mice, and fig. 7B is a statistical graph of the thickness of the epidermis of mice of different treatment groups.
Discussion of the related Art
ABC-DLBCL is the lymphoma with the highest degree of malignancy at present and has strong resistance to the immunochemical R-CHOP therapy which is commonly used clinically at present. One of the characteristics of the method is that the NF-kB signal path with the functions of promoting growth and resisting apoptosis is continuously activated, so that theoretically, finding a targeted drug for blocking NF-kB signals in ABC-DLBCL is an effective method for treating ABC-DLBCL. However, NF-. kappa.B is a transcription factor with a wide range of important biological activities and is not suitable as a therapeutic target per se.
In B cells, Src family kinases are induced to phosphorylate tyrosine in the ITAM domain of CD79A and CD79B upon antigen binding to BCR, followed by activation of tyrosine kinase Syk by binding to phosphorylated ITAM, which triggers a signaling cascade including Bruton's Tyrosine Kinase (BTK), phosphatase C γ (phosphoipase C γ), and protein kinase C β (protein kinase C β - β). PKC- β phosphorylates CARM1, causing it to recruit BCL10 and MALT1, forming a CBM complex, which activates ikb kinase (IKK), which finally stimulates NF- κ B signaling pathway (21). on the other hand, activated mam 1 cleaves through its enzymatic activity an NF- κ B inhibitor such as a20(22), CYLD (23,24), further promotes activation of the NF- κ B signaling pathway, mutations in tumor cells such as CD 3825, CD 7342, CD 1/CD 7375, or the above mutation of the gene translocation of BCL-B19, which results in negative feedback mutations of tumor cells such as CD 1, CD 7342, and/isb 7374.
As can be seen from the above, the CBM complex plays an important role in mediating NF- κ B activation. While the only enzymatically active MALT1 protein in the CBM complex is the hot molecular target for the recent development of targeted drugs for the treatment of ABC-DLBCL: firstly, the inhibition of the activity of MALT1 can effectively inhibit the signal of NF-kB, thereby selectively inhibiting or even killing ABC-DLBCL cells, and the therapeutic effectiveness of targeting MALT1 enzyme activity is demonstrated; secondly, the MALT1 knockout mouse shows a part of defects in the activation of T cells and B cells and has normal phenotype in other aspects, which suggests that the side effect of targeting MALT1 enzyme activity to treat ABC-DLBCL is smaller; third, the caspase-like domain of MALT1 is unique among human genes, and inhibition of MALT1 does not produce a wide range of side effects due to inhibition of other structurally similar proteins, etc. The inventors have developed small molecule inhibitors of MALT1 and demonstrated that MALT1 is an effective target for the treatment of ABC-DLBCL.
The Bcl10 is the core of the fiber structure of the CBM complex, and the fiber structure of the Bcl10 provides a central scaffold platform for the activation of the CBM complex and the recruitment and activation of key proteins of the NF-kB signaling pathway, and is the key to the activation of the NF-kB signaling pathway. The mutant of Bcl10 which can not form a fiber structure has the inhibiting effect of dominant negative effect on the activation of NF-kB signal path, therefore, the Bcl10 can be another effective target point for inhibiting the NF-kB signal path and treating ABC-DLBCL. According to the invention, a small-molecule peptide inhibitor for inhibiting Bcl10 is designed according to a structure model of Bcl10, wherein Bcl-P2 and Bcl-P4 can effectively inhibit the polymerization of Bcl10, the formation of a fibrous structure and the activity of NF-kB, and show very specific killing effect on ABC-DLBCL on a cell level and a mouse allograft tumor model, so that Bcl10 is an effective target for inhibiting the activity of NF-kB and ABC-DLBCL.
A large number of researches show that the activation of abnormal NF-kB signal channels plays an important role in various cancers such as breast cancer, hormone-resistant prostate cancer, lung cancer, thyroid cancer, colon cancer, lymphoma and the like and autoimmune diseases (such as psoriasis and the like). Recent studies have shown that the CBM protein complex and its family proteins are widely expressed in these cancer tissues, and that this complex may also have a key role in NF-. kappa.B-related cancers other than lymphoma. Therefore, Bcl10 inhibitors may also provide effective therapeutic approaches for the treatment of the above-mentioned cancers and autoimmune diseases.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (12)

  1. A polypeptide of formula I, or a pharmaceutically acceptable salt thereof,
    Z0-Z1-Z2-Z3-Z4 (I)
    z0 is nothing, a modification group at the N end or a peptide segment of 1-3 amino acids;
    z1 is a cell penetrating peptide element;
    z2 is nothing or a linker peptide;
    z3 is a Bcl10 inhibitory peptide element, and the length of Z3 is 12-25aa, preferably 13-20aa, more preferably 14-18 aa;
    z4 is nothing, a modification group at the C terminal or a peptide segment of 1-3 amino acids;
    "-" is a peptide bond.
  2. The polypeptide of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, wherein said polypeptide has a length of 30-50aa, preferably 32-40 aa.
  3. The polypeptide of formula I of claim 1, or a pharmaceutically acceptable salt thereof, wherein the N-terminal modification group is selected from the group consisting of: acetyl, benzyloxycarbonyl C, aminopentanoic acid, palmitic acid, or combinations thereof.
  4. The polypeptide of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Z3 has the motif (5'-3') of formula a1 or the inverted amino acid motif (5'-3') of formula a 2:
    R-X1-RA-X2-K-X3-L (A1); or
    L-X3-K-X2'-AR-X1'-R (A2)
    In the formula (I), the compound is shown in the specification,
    x1 is a peptide segment of 4-6 amino acids; preferably X1 is HFDHL or TSSRK;
    x2 is nothing, or a peptide segment of 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 segment of 4-6 amino acids; preferably X1 is KRSST or LHDFH;
    x2' is nothing, or a peptide fragment of 1-2 amino acids; preferably X2' is K or G.
  5. The polypeptide of formula I of claim 1, or a pharmaceutically acceptable salt thereof, wherein the polypeptide has the sequence of SEQ ID No. 4 or 5 or 8 or 9 or 13 or 14.
  6. An isolated nucleic acid molecule encoding the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof.
  7. A pharmaceutical composition, comprising:
    (a) a therapeutically effective amount of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof; and
    (b) a pharmaceutically acceptable carrier or excipient.
  8. Use of the polypeptide of claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of a composition or medicament for (i) inhibiting Bcl10 polymerization; (ii) inhibiting the activity of the CBM complex; (iii) selectively inhibiting the growth of diffuse large B-cell lymphoma; and/or (iv) preventing and/or treating diseases associated with CBM complex-induced NF- κ B activation.
  9. A method of selectively inhibiting the growth of diffuse large B-cell lymphoma comprising the steps of: administering to a subject in need thereof a polypeptide of claim 1 or a pharmaceutically acceptable salt thereof.
  10. An in vitro non-therapeutic method of inhibiting polymerization of Bcl10, comprising the steps of:
    culturing a tumor cell in the presence of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, thereby inhibiting polymerization of Bcl10 in the tumor cell.
  11. A method of screening a candidate compound for selective inhibition of the growth of diffuse large B-cell lymphoma comprising the steps of:
    (a) mixing the Bcl10 protein with a library of compounds, and determining binding of compounds in the library of compounds to the Bcl10 protein;
    wherein, if a compound in the test compound library binds to the Bcl10 protein, it indicates that the compound that binds to the Bcl10 protein is a candidate compound.
    (b) Administering the candidate compound identified in step (a) to diffuse large B-cell lymphoma cells and determining its effect on diffuse large B-cell lymphoma.
  12. A method of screening a candidate compound for selective inhibition of the growth of diffuse large B-cell lymphoma comprising the steps of:
    (a) culturing tumor cells expressing Bcl10 protein in a test group in a culture system in the presence of a test compound for a period of time T1, and detecting the expression of the Bcl10 protein in the culture system of the test group E1;
    and detecting expression of said Bcl10 protein in said culture system of a control group in a control group otherwise identical in the absence of said test compound E2;
    (b) if the expression profile of the Bcl10 protein in the test group, E1, is significantly lower than the expression profile of the Bcl10 protein in the control group, E2, then it is an indication that the test compound is a candidate compound.
CN201980004308.4A 2018-06-29 2019-06-26 Novel Bcl10 polymerization inhibitor and application thereof Pending CN111094357A (en)

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CN2018106945845 2018-06-29
PCT/CN2019/093094 WO2020001495A1 (en) 2018-06-29 2019-06-26 Novel bcl10 polymerization inhibitor and application thereof

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105920582A (en) * 2008-12-10 2016-09-07 普渡研究基金会 Cell-permeant Peptide-based Inhibitor Of Kinases

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105920582A (en) * 2008-12-10 2016-09-07 普渡研究基金会 Cell-permeant Peptide-based Inhibitor Of Kinases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DANIELA M.等: "Generation and functional characterization of a BCL10-inhibitory peptide that represses NF-κB activation", 《BIOCHEM. J.》 *

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