WO2012145890A1 - 钙调磷酸酶抑制因子1在制备治疗与NF-κΒ活性升高相关的疾病的药物中的用途 - Google Patents

钙调磷酸酶抑制因子1在制备治疗与NF-κΒ活性升高相关的疾病的药物中的用途 Download PDF

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WO2012145890A1
WO2012145890A1 PCT/CN2011/073261 CN2011073261W WO2012145890A1 WO 2012145890 A1 WO2012145890 A1 WO 2012145890A1 CN 2011073261 W CN2011073261 W CN 2011073261W WO 2012145890 A1 WO2012145890 A1 WO 2012145890A1
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amino acid
acid sequence
cancer
seq
peptide
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PCT/CN2011/073261
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French (fr)
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李文博
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麦克利科技有限公司
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Priority to PCT/CN2011/073261 priority Critical patent/WO2012145890A1/zh
Priority to CN201180047259.6A priority patent/CN103249428B/zh
Priority to US14/113,993 priority patent/US20140142045A1/en
Priority to JP2014506708A priority patent/JP5898304B2/ja
Priority to EP11864435.0A priority patent/EP2703006A4/en
Publication of WO2012145890A1 publication Critical patent/WO2012145890A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • calcineurin inhibitor 1 in the preparation of a medicament for treating a disease associated with an increase in NF- ⁇ activity
  • the present invention relates to the field of treatment of human tumors and inflammation, in particular to the inhibition of nuclear factor-kappaB (NF- ⁇ ) by calcineurin inhibitor 1 (RCAN1) protein and its use in the treatment of diseases associated with NF-KB, such as tumors.
  • NF- ⁇ nuclear factor-kappaB
  • RCAN1 calcineurin inhibitor 1
  • Cancer is the leading cause of death in developed countries, and the latest data show that one in every four to five deaths in China is due to cancer. And most cancers still lack effective treatments, and cancer poses serious social and economic problems worldwide.
  • NF- ⁇ nuclear factor -kappaB
  • NF- ⁇ acts as a transcriptional regulator under normal physiological conditions and is primarily involved in immune responses, cell proliferation, cell death, and inflammatory responses (Baud and Karin, 2009).
  • NF- ⁇ is the major anti-apoptotic factor in the body.
  • the abnormality of NF-KB signaling pathway is related to the development of many tumors including blood and solid tumors and their tolerance to chemotherapy and radiotherapy. NF- ⁇ has been found.
  • Hematological tumors with persistently abnormal increases include: multiple myeloma, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large cells Lymphoma, Hodgkin's lymphoma, myelodysplastic syndrome, adult T-cell leukemia, and solid tumors with abnormally high NF- ⁇ activity include: breast cancer, ovarian cancer, lung cancer, cervical cancer, prostate cancer, liver cancer , pancreatic cancer, esophageal cancer, gastric cancer, throat cancer, cholangiocarcinoma, thyroid cancer, parathyroid cancer, and squamous cell carcinoma of the skin and head and neck (Basseres and Baldwin, 2006).
  • NF-KB promotes tumor development mainly through cell proliferation, angiogenesis, tumor metastasis, inflammatory response and apoptosis inhibition.
  • the US FDA approved Bortezomib for the treatment of multiple myeloma As a proteasome inhibitor, bortezomib is thought to act primarily as a tumor by inhibiting the NF- ⁇ signaling pathway, but as A broad spectrum of proteasome inhibitors, bortezomib, has widespread and serious side effects that greatly limit its widespread use in cancer therapy (Delforge et al., 2010).
  • Arsenic trioxide also known as arsenic
  • its main role is also to inhibit NF-KB activity, but the toxicity of arsenic trioxide also limits its use in cancer therapy.
  • NF-KB not only has important functions in tumors, but NF-KB is also a very important inflammatory factor, such as interleukin, tumor necrosis factor and other regulatory factors, and therefore has important functions in inflammatory reactions. Also, as an important molecule that affects cell survival, NF-KB also plays an important role in neurodegeneration, including dementia and stroke.
  • the NF- ⁇ signaling pathway is a very important tumor drug target, and it is also a hot spot for drug development at home and abroad. There is an urgent need for drugs that can specifically inhibit NF- ⁇ activity and have fewer side effects for tumor therapy.
  • Calcineurin inhibitor 1 was first discovered to be an endogenous inhibitor of calcineurin, and it has been found that the C-terminus of RCAN1 has calcineurin-inhibiting activity (Arron et al, 2006; Chan Et al, 2005; Fuentes et al, 2000). Due to the difference in the cleavage, RCAN1 has four splicing isoforms, wherein the cleavage isomer 1 is RCAN1.1 (SEQ ID NO: 1) and the cleavage isomer 4 is RCAN1.4 (SEQ ID NO) : 2) The expression in the body is higher.
  • RCAN1.1 and RCAN1.4 differ only in the 28 amino acids at the N-terminus, RCAN1.1 is highly expressed in the nervous tissue, and the expression of RCAN1.4 in the peripheral tissues such as muscle is higher. high. Although the two are expressed differently in each organization, they are functionally similar. Summary of invention
  • the invention provides the use of a peptide or nucleic acid encoding the same for the preparation of a medicament for the treatment of a disease associated with an increase in NF-KB activity, wherein the peptide comprises (a) comprises SEQ ID NO: 3 Or an amino acid sequence comprising (b) the amino acid sequence of SEQ ID NO: 3 modified, for example, by substitution, deletion or addition of one or more amino acids, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway .
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a peptide or a nucleic acid encoding the same, and optionally a pharmaceutically acceptable carrier or excipient, wherein the peptide (a) comprises SEQ ID NO An amino acid sequence of 3; or an amino acid sequence comprising (b) the amino acid sequence of SEQ ID NO: 3 modified, for example, substituted, deleted or added with one or more amino acids Column, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway.
  • the invention provides a method of treating a disease associated with an increase in NF- ⁇ activity, comprising administering a peptide or a nucleic acid encoding the same to a patient having the disease, wherein the peptide (a) comprises SEQ ID NO: An amino acid sequence comprising 3; or (b) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3 modified, for example, by substitution, deletion or addition of one or more amino acids, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway.
  • the peptide (a) consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) the amino acid of SEQ ID NO: Sequence composition, or U) consisting of an amino acid sequence obtained by modifying, for example, a substitution, deletion or addition of one or more amino acids in the amino acid sequence of (a) - (c), wherein the peptide has an inhibitory NF- ⁇ signaling pathway Or (e) consisting of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of (a) - (c), wherein The peptide has the effect of inhibiting the NF- ⁇ signaling pathway.
  • the disease associated with increased NF- ⁇ activity is selected from the group consisting of: multiple myeloma, leukemia, mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large cell lymphoma, Hodgkin Lymphoma, myelodysplastic syndrome, breast cancer, ovarian cancer, lung cancer, cervical cancer, prostate cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, throat cancer, cholangiocarcinoma, thyroid cancer, parathyroid cancer, and skin and neck Squamous cell carcinoma.
  • the disease is preferably leukemia, for example selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, adult T-cell leukemia.
  • leukemia for example selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, adult T-cell leukemia.
  • FIG. 1 RCAN1 reduces nuclear transfer of NF- ⁇ and transcriptional activity of NF- ⁇ .
  • A HEK293 cells were transfected with the RCAN1 expression vector pcDNA3.1 (-) RCANl.l-myc and pcDNA3.1 (-) RCAN 1.4-myc, and the gene knockout vector si-RCAN 1 and cells were harvested 48 hours later. The nuclei were isolated and lysed, subjected to SDS-PAGE, western blot using anti-NF- ⁇ antibody, and TBP (TATA binding protein) detected by anti-TBP antibody as internal reference of nuclear protein.
  • TBP TATA binding protein
  • the vector pNF-KBluc was co-transfected into HEK293 cells, and luciferase activity was measured 24 hours later using a luciferase kit to indicate the transcriptional activity of NF- ⁇ in cells. *: indicates statistical significance, p ⁇ 0.05. Control: empty vector.
  • FIG. 2 Co-immunoprecipitation shows the presence of protein-protein interactions between RCAN1 and 1KB protein.
  • HEK293 cells were transfected with the expression vector pcDNA3.1 (-) RC AN 1.1 -myc of RCAN1, and cells were harvested and lysed 48 hours later, (A) immunoprecipitation using anti-myc antibody (9E10), using anti- ⁇ antibody Western blot; (B) Immunoprecipitation using anti- ⁇ antibody, western blot using anti-myc antibody (9E10). Input for cell division before immunoprecipitation
  • FIG. 3 Low expression of RCAN1 resulted in a significant increase in tyrosine phosphorylation of 1KB at position 42.
  • HEK293 cells were transfected with the gene knockout vector si-RCAN1, cells were harvested 48 h later and lysed,
  • A anti- ⁇ antibody was used Immunoprecipitation was performed using ⁇ 42 tyrosine phosphorylated antibody for western blot;
  • B Immunoprecipitation was performed using IKB42 tyrosine phosphorylated antibody, and western blot was performed using anti- ⁇ .
  • * indicates statistical significance, p ⁇ 0.05. Control: empty vector.
  • FIG. 4 High expression of RCAN1 leads to death of acute leukemia cell lines as well as primary acute leukemia cells.
  • the acute lymphoblastic leukemia cell line (A) and the acute myeloid lymphocytic leukemia cell line (B) were infected with the adenovirus of RCAN1, and the cell viability was measured 48 hours later using the cell viability kit.
  • C Mononuclear cells were isolated from peripheral blood of leukemia patients and remission patients and control normal populations, and primary cultured leukemia cells were infected with adenovirus of RCAN1, and cell viability was measured 48 hours later using the cell viability kit.
  • Av-GFP is an adenovirus expressing green fluorescent protein
  • ⁇ -RCANl.l is an adenovirus expressing RCAN1.1 protein
  • Av-RCANl.4 is expressed Adenovirus of RCAN1.4 protein (Ad-RCAN1.4 vector described in Example 1.2.2).
  • Figure 5 The N-terminal 103 amino acids of RCAN1 bind to 1 KB and inhibit the transcriptional activity of NF-KB and kill acute leukemia cells.
  • A HEK293 cells were transfected with the RCAN1 N-terminal 103 amino acid expression vector pcDNA3.1RCANl-103mychis or
  • B the 103-197 amino acid pcDNA3.1RCAN103-197myc vector. After 48 hours, the cells were harvested and lysed. The -myc antibody (9E10) was immunoprecipitated and subjected to western blot using an anti- ⁇ antibody.
  • the Jurkat leukemia cell line was transfected with the RCAN1 N-terminal 103 amino acid expression vector pcDNA3.1RCANl-103mychis or the 103-197 amino acid pcDNA3.1RCAN103-197myc plasmid, and the cells were assayed using the cell viability kit 48 hours later. Survival rate. * : indicates statistical significance, p ⁇ 0.05. Control: empty vector.
  • FIG. 6 Purified TAT-RCANl-103 polypeptide can cause leukemia cell death.
  • A SDS-PAGE shows that the purified TAT-RCANl-103 polypeptide is a single band.
  • B TAT-RCANl-103 polypeptide was added to Jurkat cells, and cell viability was measured 48 hours later using a cell viability kit. *: indicates statistical significance, p ⁇ 0.05. Control: empty vector.
  • regulation of the NF- ⁇ signaling pathway in vivo is mainly dependent on the 1KB of ⁇ inhibitory factor.
  • Serine phosphorylation of 1KB at positions 32 and 36 promotes degradation of the proteasome by 1KB, and exposure to NF- after 1KB degradation.
  • the nuclear localization sequence of the ⁇ molecule which is subsequently transferred into the nucleus, binds to the ⁇ response element on its target gene to regulate transcription of the target gene (Alkalay et al., 1995).
  • the present inventors have found that the cleavage isomers RCAN1.1 (SEQ ID NO: 1) and RCAN1.4 (SEQ ID NO: 2) of the RCAN1 protein inhibit the NF- ⁇ signaling pathway and reduce the survival of cancer cells; further, The present inventors have found that a peptide having an N-terminal 103 amino acid of RCAN1.1 (SEQ ID NO: 3) can interact with 1 KB and inhibit NF- ⁇ activity, thereby reducing cancer The survival of the cell. Therefore, the inventors have for the first time revealed that the RCAN1 protein is an endogenous regulatory protein of NF- ⁇ , which can affect the NF- ⁇ signaling pathway, which interacts with the NF- ⁇ inhibitor 1KB and affects its tyrosine at position 42. Phosphorylation.
  • the present invention provides the use of a peptide or a nucleic acid encoding the same for the preparation of a medicament for treating a disease associated with an increase in NF- ⁇ activity, wherein the peptide (a) comprises SEQ ID NO: Or an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3 modified, for example, by substitution, deletion or addition of one or more amino acids, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway.
  • the peptide (a) consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) consists of SEQ ID NO An amino acid sequence consisting of 3, or U) consisting of an amino acid sequence obtained by modifying, for example, a substitution, deletion or addition of one or more amino acids in the amino acid sequence of (a) - (c), wherein the peptide has an inhibitory factor of NF
  • a sequence composition in which the peptide has an effect of inhibiting the NF- ⁇ signaling pathway is consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) consists of SEQ ID NO
  • peptide refers to a polymer of amino acid residues comprising two or more amino acids joined by peptide bonds.
  • the polymer may be linear, sub-technical or cyclic, and may comprise naturally occurring and/or amino acid analogs which may be interrupted by non-amino acids.
  • amino acid polymer is long (e.g., more than 50 amino acid residues), it is preferably referred to as a polypeptide or protein, but if it is 50 amino acids long or shorter, it is preferably referred to as a "peptide.”
  • peptides described herein can be prepared by any suitable prior art methods, such as chemical synthesis, recombinant expression, and the like. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001)) and Blackwell Scientific Publications, Nicholson, "Peptide Synthesis", Atherton And Chapter 9 of Sheppard "Peptide Synthesis”.
  • the "modification" is preferably a conservative sequence modification well known in the art, including amino acid substitutions, additions or deletions.
  • Amino acid modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis, molecular cloning, oligonucleotide-directed mutagenesis, and random PCR-mediated mutagenesis in nucleic acids encoding proteins.
  • Conservative amino acid substitutions include those in which the amino acid residues are substituted by amino acid residues having similar structural or chemical properties. A family of amino acid residues having similar side chains has been identified in the art.
  • amino acids with basic side chains eg lysine, arginine, histidine
  • acidic side chain amino acids eg aspartic acid, glutamic acid
  • uncharged polar side chain amino acids eg asparagine, glutamine, serine, threonine, tyrosine, semi-acid, isoleucine, valine, phenylalanine, methionine
  • branched side chain amino acids eg Threonine
  • Guidance on determining which amino acid residues can be substituted, inserted or deleted without eliminating their immunological activity can be found using computer programs well known in the art.
  • Computer algorithms known to those skilled in the art such as Gap or Bestfit, can be used to optimize the comparison of amino acid sequences to compare and define similar or identical.
  • substitution of one or more amino acids herein above can be in SEQ ID NO: 1, 2 or 3.
  • Amino acid residues are substituted with amino acid residues having similar structural or chemical properties, such as mutual substitution between arginine proline, leucine and isoleucine.
  • one or more amino acids deleted herein above may be an amino acid residue lacking position 1 of SEQ ID NO: 1, an amino acid residue at position 1 of SEQ ID NO: 2, or a ninth of SEQ ID NO: Amino acid residue at the position.
  • the above-described addition of one or more amino acids may be the addition of the amino acid residue Ala at position 1 of SEQ ID NO: 1, the addition of amino acid residue Ala at position 1 of SEQ ID NO: 2, or the SEQ ID NO:
  • the amino acid residue Ala is added at the 9th position in 3.
  • inhibiting NF- ⁇ signaling pathway refers to inhibition of NF- ⁇ as a transcription factor. The activity of its downstream target gene.
  • identity refers to the percentage of identical amino acids at the same or similar positions in the two protein chains.
  • the BLAST algorithm was used to calculate the percent homology using the BLAST algorithm, which is available on the NCBI website (http://www.ncbi.nlm.nih.gov/).
  • the peptides having sequence identity are functional homologs, i.e., they exhibit significant identity (e.g., about 50% sequence identity at the amino acid level) and perform the same function as their corresponding proteins.
  • At least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more have the same sequence Sexual functional homologs are preferred functional homologs.
  • nucleic acid As used herein, the terms “nucleic acid”, “nucleic acid molecule”, “polynucleotide” and “nucleotide sequence” are used interchangeably to define a polymer of any length, such as a polydeoxyribonucleotide (DNA X eg eg cDNA, genomic DNA, plasmid, vector, isolated DNA and any mixture thereof) or polyribonucleotide (RNA) molecules (eg mRNA) or mixed polyribose-polydeoxyribonucleotides. They contain single or double stranded, linear or circular, natural or synthetic polynucleotides.
  • DNA X polydeoxyribonucleotide
  • RNA polyribonucleotide
  • mRNA mixed polyribose-polydeoxyribonucleotides
  • encoded refers to information that is included for translation into a given protein.
  • the nucleic acid encoding the protein may comprise a non-translated sequence (e.g., an intron) within the translational region of the nucleic acid, or may lack such an inserted non-translated sequence (e.g., in a cDNA).
  • the information encoding the protein is specified by the use of a codon.
  • the nucleic acid of the invention may also be contained in a vector such as an expression vector, plasmid, virus, phagemid, phage, cosmid or artificial chromosome.
  • a vector such as an expression vector, plasmid, virus, phagemid, phage, cosmid or artificial chromosome.
  • an "expression vector” is a recombinant or synthetically produced nucleic acid construct having a series of designated nucleic acid elements that permit transcription of a particular nucleic acid in a host cell, eg, an expression vector other than the nucleic acid sequence to be expressed.
  • a selection marker derived from a replication and control sequence compatible with the host for expression and a selectable phenotype conferring the transfected cell can also be included.
  • the nucleic acid of the present invention can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment.
  • suitable expression vectors are known in the art and are commercially available, such as prokaryotic expression vectors, yeast expression vectors, mammalian expression vectors, insect cell expression vectors, plant cell expression vectors.
  • the nucleic acid sequence of the invention (SEQ ID NO: 1, 2 or 3) is in an expression vector such as pcDNA3.1 ( - ) mychis ( c ) ( Invitrogen ), pSuper ( Oligoengine ) or Adeno-X (Clontech ) Expressed in.
  • “Host cell” refers to a cell that contains a vector and supports replication and/or expression of the vector.
  • the host cell may be a prokaryotic cell such as an E. coli cell, or a eukaryotic cell such as a yeast cell, an insect cell, an amphibian cell or a mammalian cell.
  • the host cell expressing the protein described herein may be any eukaryotic or prokaryotic cell used in the art, such as HEK293, HEK293T, E. coli cells, and the like.
  • Various cell cultures as well as techniques for expression, collection, purification, and detection of expressed proteins are known to those skilled in the art. See, for example, J.
  • the present invention also provides the use of a peptide or a nucleic acid encoding the same as a medicament for treating a disease associated with an increase in NF- ⁇ activity, wherein the peptide (a) comprises the amino acid sequence of SEQ ID NO: Or (b) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3 modified, for example, by substitution, deletion or addition of one or more amino acids, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway.
  • the present invention provides a pharmaceutical composition for treating a disease associated with an increase in NF- ⁇ activity, comprising a peptide or a nucleic acid encoding the same, and optionally a pharmaceutically acceptable carrier Or an excipient, wherein the peptide (a) comprises the amino acid sequence of SEQ ID NO: 3; or (b) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3 modified, for example, by substitution, deletion or addition of one or more amino acids. Wherein the peptide has the effect of inhibiting the NF- ⁇ signaling pathway.
  • the peptide (a) consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) the amino acid of SEQ ID NO: Sequence composition, or U) consisting of an amino acid sequence obtained by modifying, for example, a substitution, deletion or addition of one or more amino acids in the amino acid sequence of (a) - (c), wherein the peptide has an inhibitory NF- ⁇ signaling pathway
  • the effect, or (e) is at least 95%, at least 96%, at least 97%, at least 98% or from the amino acid sequence in (a) - (c) Amino acid sequence consisting of at least 99% sequence identity, wherein the peptide has an effect of inhibiting the NF- ⁇ signaling pathway.
  • the present invention provides a method of treating a disease associated with an increase in NF- ⁇ activity, comprising administering a peptide or a nucleic acid encoding the same or a pharmaceutical composition comprising the same to a patient having the disease
  • the peptide (a) comprises the amino acid sequence of SEQ ID NO: 3; or (b) an amino acid comprising the amino acid sequence of SEQ ID NO: 3, such as substituted, deleted or added one or more amino acids.
  • a sequence wherein the peptide has an effect of inhibiting the NF-KB signaling pathway.
  • administering means that a predetermined amount of a substance is introduced into a patient by a suitable method.
  • the isolated peptide, isolated nucleic acid molecule, expression vector, recombinant cell, pharmaceutical composition or vaccine of the present invention can be administered by any usual route as long as it can reach the desired tissue.
  • a variety of modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, transdermal, oral, topical, intranasal, intrapulmonary, and rectal, although the invention is not limited to these exemplified modes of administration.
  • the peptide (a) consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) consists of SEQ ID NO An amino acid sequence consisting of 3, or U) consisting of an amino acid sequence obtained by modifying, for example, a substitution, deletion or addition of one or more amino acids in the amino acid sequence of (a) - (c), wherein the peptide has an inhibitory factor of NF
  • a sequence composition in which the peptide has an effect of inhibiting the NF- ⁇ signaling pathway is consists of the amino acid sequence set forth in SEQ ID NO: 1, or (b) consists of the amino acid sequence set forth in SEQ ID NO: 2, or (c) consists of SEQ ID NO
  • the peptides of the invention may also be fused to additional peptides to modify the physiological or pharmaceutical properties of the peptide, such as increasing stability or increasing membrane penetration.
  • the peptide of the invention eg, SEQ ID NO: 3
  • the human immunodeficiency virus HIV Tat protein eg, The 47-57 amino acid RKKRRQRRRG (SEQ ID NO: 14) is fused to facilitate penetration of the polypeptide into the cell membrane.
  • the disease associated with an increase in NF- ⁇ activity refers to any disease associated with the occurrence, development, persistence or recurrence of the disease associated with an increase in NF- ⁇ activity (Basseres and Baldwin, 2006).
  • the disease may be selected from the group consisting of multiple myeloma, leukemia, mantle cell lymphoma, mucosa-associated lymphoid tissue lymphoma, diffuse large cell lymphoma, Hodgkin's lymphoma, myelodysplastic syndrome, breast cancer, ovarian cancer.
  • the disease is preferably leukemia, for example selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, adult T-cell leukemia.
  • treatment refers to: alleviating, alleviating, eliminating, obstructing, preventing the symptoms of the disease and/or delaying the onset and/or progression of the disease. Monitoring and/or measurement of the symptoms of the disease can be performed by the clinician using any means or method.
  • Whether a peptide or a nucleic acid encoding the same can inhibit the activity of NF- ⁇ can be determined by measuring the amount of NF- ⁇ protein in the nucleus.
  • a number of assays for determining NF- ⁇ protein are known in the art, such as immunoblotting of nucleoproteins, electrophoretic mobility experiments using isolated nuclear proteins, and assays for the activity of NF- ⁇ protein as a transcription factor using a reporter gene system (Imbert et al ., 1996).
  • the amount of NF- ⁇ protein in the nucleus is determined by Western blotting to investigate the inhibitory effect of RCAN 1 on NF- ⁇ activity.
  • leukemia is known to be a disease associated with an increase in the activity of NF- ⁇ .
  • Leukemia is a blood-like tumor, and leukemia cell lines such as JURKAT are good leukemia cell models, especially short-term cultured primary leukemia cells, which are directly used for drug testing after isolation from peripheral blood of patients, cell survival based cytology.
  • the method of measurement can reflect the effects and mechanisms of the drug.
  • leukemia cell lines and primary cultured leukemia cells are used as models to study the effect of RCAN1 in the treatment of leukemia, and this model can prove that RCAN1 is a good candidate for treating leukemia.
  • RCAN1 protein of the present invention or the N-terminal 103 amino acid fragment thereof is provided as a novel
  • PCR amplified enzyme and dNTP purchased from Takarra;
  • PCR amplification primers synthesized by Shanghai Shenggong Company;
  • Takara T4 ligase purchased from Dalian Bao Bio Company;
  • Adenovirus expression vector Adeno-X purchased from Clontech;
  • E. coli DH5a was purchased from Invitrogen.
  • HEK293 cell line was purchased from ATCC. HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum at a culture environment of 37. C constant temperature, 5% C0 2 cell culture incubator. Cell culture products were purchased from Invitrogen.
  • Plasmid construction To construct the RCAN1 high expression vector, construct the pcDNA3.1 (-) RCANl.l-myc recombinant plasmid, and use the following primers to PCR-amplify the human cDNA library, and digest it with EcoR 1 and Kpn l and connect it with the same The vector was obtained by digesting the pcDNA3.1 (-) mychis (c) vector.
  • the PCR primers used to clone RCAN1.1 are:
  • Upstream primer ccgCTCGAGgccaccATGGAGGAGG TGGACCTGCA (SEQ ID NO: 4)
  • a human cDNA library purchased from Clontech
  • the above primers were used for conventional PCR (PCR conditions: 94 ° C lOmin, 30 cycles 94 ° C, 30 s, 55 ° C, 30 s, 72 ° C, 30 s, Finally, the gene fragment was amplified at 72 ° C for 5 min, and then the PCR amplified fragment was isolated and recovered by agarose gel electrophoresis, with the EcoR1 restriction site upstream and the Kpn l restriction site downstream.
  • the DNA fragment obtained by PCR was digested with EcoR 1+Kpn I and ligated into pcDNA3.1 (-) mychis (c) vector which was also digested with EcoR 1+Kpn I to obtain pcDNA3.1 (-) RCANl. L-myc recombinant expression plasmid.
  • the PCR primers used to clone RCAN1.4 are:
  • Upstream primer ccgCTCGAGgccaccATGCATTTTA GAAACTTTAA ( SEQ ID NO: 6 )
  • Downstream primer atggtacc CCTCTTCTTCCTCCTTC ( SEQ ID NO: 7 )
  • Ad-RCAN1 .1 and Ad-RCANl .4 vectors The pRCAN1.E-EGFP and pRCANl.4-EGFP vectors were digested with Nhe I and Not I, and a fragment of about 1.4 kb was recovered as an insert and inserted into the same restriction enzyme.
  • pShuttle2 purchased from Clontech
  • pShuttle2-RCAN 1.1 -EGFP and pShuttle2-RCAN 1.4-EGFP was obtained by pShuttle2-RCAN 1.1 -EGFP and pShuttle2-RCAN 1.4-EGFP, and then pShuttle2-RCANl.l-EGFP and pShuttle2-RCAN1.4-EGFP were digested with I-ceuI and Pl-Scel.
  • a fragment of about 1.4 kb was inserted as an insert into the same restriction enzyme Adeno-X adenovirus vector (purchased from Clontech) to obtain Ad-RCAN1.1 and Ad-RCAN 1.4
  • leukemia cell lines cannot be transfected using general transfection methods, we used adenovirus to express RCAN1 protein. About 106 HEK293 cells were seeded one day prior to transfection into 60mm culture dish, cells were transfected using liposome LF2000 (available from invitrogen) and 5 g of Pacl (available from NEB) digestion viral vector or Adeno-X Ad-RCANl.1 and Ad-RCAN 1.4 (see the instructions of Invitrogen's LF2000 Transfection Kit for specific methods). After 7 days, cells were harvested and 3 freeze-thaw lysed cells were used to collect virus-containing lysate.
  • liposome LF2000 available from invitrogen
  • Pacl available from NEB digestion viral vector
  • Adeno-X Ad-RCANl.1 and Ad-RCAN 1.4 see the instructions of Invitrogen's LF2000 Transfection Kit for specific methods. After 7 days, cells were harvested and 3 freeze-thaw lysed cells were used to collect virus-containing lys
  • HEK293 cells inoculated with 60 mm culture dishes were infected with 0.5 ml of cell lysate. After 48 hours, the cells were frozen and thawed to collect virus, and the same method was continued. The virus collected after infection with HEK293 twice was used for the experiment. The virus titer is approximately 4 X 10 8 /ml. In the present invention, the cell line and primary cells use a multiplicity of infection (MOI) of 30 unless otherwise specified.
  • MOI multiplicity of infection
  • HEK293 cells were transfected with RCAN1 expression vector pcDNA3.1 (-) RCANl.l-myc: HEK293 cells were transfected with liposome LF2000 (purchased from invitrogen), and 4 ⁇ l of LF2000 was added to opti-MEM of ⁇ (purchased from Invitrogen) After 5 minutes, it was mixed with 2 g of the RCAN1 expression vector pcDNA3.1 (-) RCANl.l-myc in opt ⁇ of opti-MEM, and then allowed to stand at room temperature for 15 minutes, and added to a 35 mm diameter culture dish containing HEK293. . After 48 hours, the nuclei were separated and lysed: The nuclei were separated and lysed using a nuclear protein extraction kit (purchased from Millipore).
  • the nuclear lysate was separated using 12% glycine SDS-PAGE (purchased from Biorad): The gel and transfer device was Biorad's mini-protean 3 vertical electrophoresis device.
  • NF- ⁇ in the nucleus of cells with high expression of RCAN1 was significantly decreased (see Figure 1), indicating that RCAN1 can significantly inhibit nuclear transfer of NF-KB, and RCAN1 knockdown can significantly increase nuclear transfer of NF-KB (Fig. 1A). .
  • the RCAN1 expression vector was co-transfected with HEK293 cells with pNF-KB-luc (purchased from Clontech;) and pRL-TK (purchased from Promega) (the plasmid usage ratio was 1: 1 : 0.02, see Invitrogen LF2000 transfection for specific methods).
  • the kit instructions lysing the cells 24 hours later, and measuring the fluorescence using a dual fluorescence reporter system (purchased from Promega).
  • HEK293 cells were transfected with RCAN1 expression vector (as described in Example 2 2.1);
  • the supernatant was centrifuged at 2000 g, and the pellet was washed twice with PBS, and the loading buffer was added; 12% glycine SDS-PAGE was used to separate the protein and the protein was transferred to the PVDF membrane; Western blot was used to detect RCAN1 protein using anti-myc antibody (purchased from ABcam) (see ABcam's antibody specification for specific methods).
  • the protein can be precipitated using an anti-myc antibody, and a 1KB antibody can be used for western blot.
  • the method of SDS-PAGE and western blot is specifically described in Example 2 2.1.
  • RCAN1 affects 1KB tyrosine phosphorylation at position 42
  • HEK293 cells were transfected with RCAN1 knockout vector si-RCAN1 (as described in Example 2 2.1, the amount of vector Si-RCAN1 was also 2ug);
  • Leukemia cell lines Jurkat, NALM-6, CEM, MOLT-4, Kasumi, HL-60, THP-1, HEL were purchased from ATCC. Primary leukemia cells and cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum, and cell culture products were purchased from Invitrogen. The culture environment is 37. C constant temperature, cell culture incubator with 5% CO 2 .
  • Cell viability kit Celltiter-Glo Luminescence Kit purchased from Promega.
  • the primary leukemia cells were isolated using Histopaque reagent. 3 ml of Histopaque-1077 reagent was added to a 15 ml centrifuge tube, and the upper layer was 3 ml of fresh peripheral blood of leukemia patients. After centrifugation at 300 g for 30 minutes, the second layer of mononuclear cells was aspirated and washed once with 10 ml of PBS buffer, and inoculated into the culture dish.
  • Leukemia cell lines or primary leukemia cells were infected with adenovirus expressing RCAN1.1 and RCAN1.4 for 48 hours, and cell viability was measured using a cell viability kit.
  • pcDNA3.1RCAN103-197myc plasmid Using primers DSCR1-103F: ccgCTCGAGgccaccatg C AGTTTCTGAT CTCCCCT (SEQ ID NO: 12) and reverse primer BGH (TAGAAGGCACAGTCGAGG (SEQ ID NO: 13)), pcDNA3.1 (-) RCANl.l -myc was used to amplify the 103-197 fragment of the substrate, and the pcDNA3.1RCAN103-197myc recombinant plasmid was obtained by inserting the restriction enzyme sites EcoR 1 and Kpnl into pcDNA3.1 (-) mychis (c).
  • Nuclear transfection kit VCA-1003 purchased from Lonza.
  • Co-immunoprecipitation HEK293 cells were transfected with the vectors pcDNA3.1RCANl-103mychis and pcDNA3.1RCAN103-197myc expressing the N- or C-fragment of RCAN1, respectively. The co-immunoprecipitation experiment was carried out as in the experimental method shown in Example 3.
  • NF- ⁇ activity fluorescent indicator system HEK293 cells were transfected with the vectors pcDNA3.1 RCAN 1 - 103mychis and pcDNA3.1 RCAN 103-197myc expressing the N- or C-fragment of RCAN1, respectively, using the method as in Example 2. NF- ⁇ activity was examined.
  • the TAT sequence is the 47th to 57th amino acids of the human immunodeficiency virus HIV Tat protein (RKKRRQRRRG (SEQ ID NO: 14)), and this TAT sequence is fused to the N-terminal polypeptide of RCAN1 to promote The polypeptide penetrates the cell membrane.
  • the coding oligonucleotide Tatg agg aagaagcggagacagcgacgaaga ggatcc c (SEQ ID NO: 15) containing the TAT fragment was inserted into the prokaryotic expression of pet-28b digested with Nde I and Xho I restriction enzymes (purchased from New England Biolabs).
  • the vector (purchased from Novagen) was used to obtain the pet-28bTAT vector, then the pet-28bTAT vector was digested with Bamh l and Xho I, and the PCR fragment of RCAN1 1-103 was inserted, PCR upstream primer: CGGGATCCATGGAGGAGGTGGACCTG (SEQ ID NO: 16); downstream Primer: CCGCTCGAGCTTGTCTGGATTTGGCGGA (SEQ ID NO: 17).
  • the PCR template was pcDNA3.1 (-) RCANl.l-myc expression vector.
  • RCAN1 N-103 polypeptide 50 ng of pTAT-RCAN1N-103 expression vector was transformed into 21 ⁇ BL21(DE3)pLysS cells (purchased from Promega;) by ice cream for 30 minutes, heat shock for 40 seconds, iced 5 minute.
  • the clone expressing TAT-RCAN1N-103 was inoculated into 200 ml of LB medium containing 100 g/ml of ampicillin, shaken at 225 rpm for 16 hours at 37 ° C, and inoculated again to 1 liter of LB culture containing 100 g/ml of ampicillin. Base, shake at 37 ° C overnight.
  • the cell pellet was collected by centrifugation at 5000 g for 10 minutes, washed once with PBS, and the cell pellet was dissolved in 20 ml of cell suspension (containing 8 M urea, 100 mM sodium chloride, 20 mM HEPES, pH 8.0), and subjected to 3 times of 15 seconds of ultrasound.
  • the cells were pulverized, 16000 g, centrifuged at 4 ° C for 15 minutes, and the supernatant was collected, and the affinity chromatography column HisTrapTM (purchased from amersham pharmacia) was passed. Proteins were resolved from the column using a cell suspension containing 500 mM imidazole, demineralized using a PD-10 column (purchased from GE). Salt was stored at -80 ° C in PBS containing 10% glycerol (purchased from invitrogen) ).
  • Jurkat cells were treated with the purified TAT-RCAN1N-103 polypeptide: Jurkat cells were treated with ⁇ TAT-RCAN1N-103 polypeptide, and cell viability was measured using MTT 24 hours later. The results showed that Jurkat cells were compared in the TAT-RCAN1N-103 treated group. The survival rate of the normal control group was significantly reduced (Fig. 6B).
  • Transcription factor NF-kappaB is constitutively activated in acute lymphoblastic leukemia cells. Leukemia 14, 399-402.
  • Epidermal growth factor activates nuclear factor-kappaB through IkappaBalpha kinase-independent but EGF receptor-kinase dependent tyrosine 42 phosphorylation of IkappaBalpha.
  • Oncogene 26 , 7324-7332.

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Description

钙调磷酸酶抑制因子 1在制备治疗与 NF-κΒ活性升高相关的疾病的药物中的用途
发明领域
本发明涉及人类肿瘤和炎症的治疗领域， 特别涉及钙调磷酸酶抑制因 子 1 ( RCAN1 )蛋白对核因子 -kappaB ( NF-κΒ )的抑制及其在治疗与 NF-KB 相关的疾病例如肿瘤中的应用。 发明背景
在发达国家癌症是死亡的首要原因， 最新数据显示在中国每四到五例 死亡中就有一位死于癌症。 而且大多数癌症仍缺乏有效地治疗方法， 癌症 给全世界都带来了严重的社会和经济问题。
核因子 -kappaB ( NF-κΒ )在正常生理情况下作为转录调节因子主要参 与免疫应答、细胞增殖、细胞死亡及炎症反应 (Baud and Karin, 2009)。 NF-κΒ 是体内主要的细胞抗凋亡因子， NF-KB信号传导通路的异常与很多肿瘤包 括血液以及实体肿瘤的发生发展及其对化疗和放疗的耐受性有关， 已发现 存在 NF-κΒ活性异常持续增高的血液肿瘤包括： 多发性骨髓瘤、 急性淋巴 细胞白血病、 急性髓细胞白血病、 慢性淋巴细胞白血病、 慢性髓细胞白血 病、 套细胞淋巴瘤、 粘膜相关淋巴组织淋巴瘤、 弥漫性大细胞淋巴瘤、 霍 奇金淋巴瘤、 骨髓增生异常综合征、 成人 T-细胞白血病， 而存在 NF-κΒ活 性异常持续增高的实体肿瘤包括： 乳腺癌、 卵巢癌、 肺癌、 宫颈癌、 前列 腺癌、 肝癌、 胰腺癌、 食管癌、 胃癌、 咽喉癌、 胆管癌、 甲状腺癌、 副甲 状腺癌， 以及皮肤和头颈的鳞状细胞癌 (Basseres and Baldwin, 2006)。 对于 人类急性 T-细胞白血病小鼠模型的研究也发现 NF-KB的缺陷可以延缓白血 病的发生 (dos Santos et al., 2008)。研究发现很多类固醇激素， 自然存在或合 成的化合物都是通过对 NF-KB的抑制而起到肿瘤抑制作用的。
NF-KB 主要通过细胞增殖， 血管新生， 肿瘤转移， 炎症反应以及凋亡 抑制等过程促进肿瘤的发生发展。 最近的研究发现大约 1/5 的多发性骨髓 瘤患者存在 NF-KB信号通路的基因突变， 2008年美国 FDA批准硼替佐米 ( Bortezomib )用于治疗多发性骨髓瘤。 作为蛋白酶体抑制剂， 硼替佐米被 认为主要是通过抑制 NF-κΒ信号传导通路而起到肿瘤治疗作用， 但是作为 广谱的蛋白酶体抑制剂， 硼替佐米有很普遍且严重的副作用， 大大限制了 它在肿瘤治疗中的广泛应用 (Delforge et al.， 2010)。 用于急性早幼粒细胞白 血病的三氧化二砷 （又名砒霜）， 它的主要作用也是抑制 NF-KB 活性， 但 三氧化二砷的毒性也限制了它在肿瘤治疗中的应用。
NF-KB不仅在肿瘤中有重要功能， NF-KB也是非常重要的炎症因子， 例如白介素、 肿瘤坏死因子等的调节因子， 因此在炎症反应中也有重要功 能。 还有， 作为影响细胞存活的重要分子， NF-KB 在神经退行性变包括老 年痴呆症和脑中风中也有重要功能。
因此， NF-κΒ信号通路是一个非常重要的肿瘤药物靶点， 也是国内外 药物开发的热点， 现在急需可以特异抑制 NF-κΒ活性， 而且副作用较小的 药物， 以用于肿瘤治疗。
钙调磷酸酶抑制因子 1 ( RCAN1 )最早发现是钙调磷酸酶（ calcineurin ) 的内源性抑制因子， 已发现 RCAN1 的 C-末端具有抑制钙调磷酸酶的活性 (Arron et al, 2006; Chan et al, 2005; Fuentes et al, 2000)。 由于剪切子的不 同， RCAN1有 4个剪切异构体，其中剪切异构体 1即 RCAN1.1( SEQ ID NO: 1 )和剪切异构体 4 即 RCAN1.4 ( SEQ ID NO: 2 )在体内的表达较高， RCAN1.1和 RCAN1.4仅在 N末端 28个氨基酸有区别， RCAN1.1在神经 组织内表达较高， RCAN1.4在外周组织例如肌肉中的表达较高。 虽然二者 在各个组织里的表达不同， 但二者在功能上类似。 发明概述
在一个方面， 本发明提供了一种肽或其编码核酸在制备用于治疗与 NF-KB 活性升高相关的疾病的药物中的应用， 其中所述肽包含（a )包含 SEQ ID NO: 3的氨基酸序列； 或者包含（b ) SEQ ID NO: 3的氨基酸序 列经过修饰例如经过取代、 缺失或添加一或多个氨基酸而获得的氨基酸序 列， 其中所述肽具有抑制 NF-κΒ信号通路的作用。
在一个方面， 本发明提供了一种药物组合物， 其包含一种肽或编码其 的核酸以及任选存在的药物可接受的载体或赋形剂， 其中所述肽（a )包含 SEQ ID NO: 3的氨基酸序列； 或者包含（b ) SEQ ID NO: 3的氨基酸序 列经过修饰例如经过取代、 缺失或添加一或多个氨基酸而获得的氨基酸序 列， 其中所述肽具有抑制 NF-κΒ信号通路的作用。
在一个方面，本发明提供了治疗与 NF-κΒ活性升高相关的疾病的方法， 包括患有所述疾病的患者施用一种肽或编码其的核酸， 其中所述肽（a)包 含 SEQIDNO: 3的氨基酸序列； 或者 （b)包含 SEQIDNO: 3的氨基酸 序列经过修饰例如经过取代、 缺失或添加一或多个氨基酸而获得的氨基酸 序列， 其中所述肽具有抑制 NF-κΒ信号通路的作用。
在一个实施方案中， 所述肽（a) 由 SEQIDNO: 1所示的氨基酸序列 组成， 或者 （b) 由 SEQ ID NO: 2所示的氨基酸序列组成， 或者 （c) 由 SEQIDNO: 3的氨基酸序列组成， 或者 U) 由 （a) - (c) 中的氨基酸序 列经过修饰例如经过取代、 缺失或添加一或多个氨基酸而获得的氨基酸序 列组成， 其中所述肽具有抑制 NF-κΒ信号通路的作用， 或者（e) 由与 （a) - (c) 中的氨基酸序列具有至少 95%、 至少 96%、 至少 97%、 至少 98%或 至少 99%的序列相同性的氨基酸序列组成， 其中所述肽具有抑制 NF-κΒ信 号通路的作用。
在一个实施方案中， 所述与 NF-κΒ活性升高相关的疾病选自： 多发性 骨髓瘤、 白血病、 套细胞淋巴瘤、 粘膜相关淋巴组织淋巴瘤、 弥漫性大细 胞淋巴瘤、 霍奇金淋巴瘤、 骨髓增生异常综合征、 乳腺癌、 卵巢癌、 肺癌、 宫颈癌、 前列腺癌、 肝癌、 胰腺癌、 食管癌、 胃癌、 咽喉癌、 胆管癌、 甲 状腺癌、 副甲状腺癌以及皮肤和头颈的鳞状细胞癌。 在一个实施方案中， 所述疾病优选是白血病， 例如选自急性淋巴细胞白血病、 急性髓细胞白血 病、 慢性淋巴细胞白血病、 慢性髓细胞白血病、 成人 T-细胞白血病。 附图简述
图 1: RCAN1可以降低 NF-κΒ的核转移和 NF-κΒ的转录活性。 （ A ) 使用 RCAN1的表达载体 pcDNA3.1 (-) RCANl.l-myc和 pcDNA3.1 (-) RCAN 1.4-myc , 以及基因敲除载体 si-RCAN 1转染 HEK293细胞， 48小时 后收集细胞， 分离细胞核并裂解， 进行 SDS-PAGE, 使用抗 -NF-κΒ抗体进 行 western blot, 抗 -TBP抗体检测的 TBP ( TATA结合蛋白）作为核蛋白的 内参。（ B )使用 RCAN1的表达载体 pcDNA3.1( - )RCANl.l-myc和 pcDNA3.1 ( - ) RCAN 1.4-myc , 以及基因敲除载体 si-RCAN 1与 NF-κΒ的转录活性指 示载体 pNF-KBluc共同转染 HEK293细胞， 24小时后使用荧光素酶试剂盒 测定荧光素酶活性， 用于指示细胞内 NF-κΒ 的转录活性。 *: 表示统计学 显著性， p<0.05。 对照： 空载体。
图 2: 免疫共沉淀显示 RCAN1与 1KB蛋白存在蛋白-蛋白相互作用。 使用 RCAN1的表达载体 pcDNA3.1 ( - ) RC AN 1.1 -myc转染 HEK293细胞， 48小时后收集细胞并裂解， （A )使用抗 -myc抗体（9E10 )进行免疫沉淀， 使用抗 -ΙΚΒα抗体进行 western blot; ( B )使用抗 -ΙΚΒα抗体进行免疫沉淀， 使用抗 -myc抗体（9E10 )进行 western blot。 输入为免疫沉淀之前的细胞裂
" 图 3: RCAN1低表达使得 1KB在 42位的酪氨酸磷酸化明显增加。 使 用基因敲除载体 si-RCANl转染 HEK293细胞， 48小时后收集细胞并裂解， ( A )使用抗 -ΙΚΒα抗体进行免疫沉淀，使用 ΙΚΒ42位酪氨酸磷酸化抗体进 行 western blot; ( B )使用 IKB42位酪氨酸磷酸化抗体进行免疫沉淀， 使用 抗 -ΙΚΒα进行 western blot。 *: 表示统计学显著性， p<0.05。 对照： 空载体。
图 4: RCAN1高表达导致了急性白血病细胞系以及原代急性白血病细 胞的死亡。 使用 RCAN1的腺病毒感染急性淋巴细胞白血病细胞系 （A )和 急性髓性淋巴细胞白血病细胞系 （B ), 48小时后使用细胞存活率试剂盒检 测细胞存活率。 （C ) 从白血病患者及缓解患者和对照正常人群的外周血中 分离单个核细胞， 并使用 RCAN1 的腺病毒感染原代培养的白血病细胞， 48小时后使用细胞存活率试剂盒检测细胞存活率。 Av-GFP为表达绿色荧 光蛋白的腺病毒，Αν-RCANl.l为表达 RCAN1.1蛋白的腺病毒（实施例 1.2.2 所述 Ad-RCANl.l载体；)， Av-RCANl.4为表达 RCAN1.4蛋白的腺病毒（实 施例 1.2.2所述 Ad-RCAN1.4载体）。
图 5: RCAN1的 N-端 103个氨基酸可以与 1KB结合并抑制 NF-KB 的转录活性， 并且杀伤急性白血病细胞。 （ A )使用 RCANl N-端 103个氨 基酸的表达载体 pcDNA3.1RCANl-103mychis或者（B )表达 103-197氨基 酸的 pcDNA3.1RCAN103-197myc载体转染 HEK293细胞， 48小时后收集 细胞并裂解， 使用抗 -myc抗体（9E10 )进行免疫沉淀， 使用抗 -ΙΚΒα抗体 进行 western blot。 （C ) 使用 RCANl N-端 103 个氨基酸的表达载体 pcDNA3.1 RCAN 1 - 103mychis 或 者 表 达 103-197 氨 基 酸 的 pcDNA3.1 RCAN 103-197myc 载体， 与 NF-κΒ 的转录活性指示载体 pNF-KBluc共同转染 HEK293细胞， 24小时后使用荧光素酶试剂盒测定荧 光素酶活性， 用于指示细胞内 NF-κΒ的转录活性。 （ D )使用 RCAN1 N-端 103个氨基酸的表达载体 pcDNA3.1RCANl-103mychis或者表达 103-197氨 基酸的 pcDNA3.1RCAN103-197myc质粒转染 Jurkat白血病细胞系， 48小 时后使用细胞存活率试剂盒检测细胞存活率。 * : 表示统计学显著性， p<0.05。 对照： 空载体。
图 6: 纯化的 TAT-RCANl-103多肽可以导致白血病细胞死亡。 （A ) SDS-PAGE 显示提纯的 TAT-RCANl-103 多肽为单一条带。 （B )在 Jurkat 细胞中加入 TAT-RCANl-103多肽， 48小时后使用使用细胞存活率试剂盒 检测细胞存活率。 *: 表示统计学显著性， p<0.05。 对照： 空载体。 发明详细描述
除非特别指出， 本文使用的科学和技术术语应具有本领域技术人员通 常已知的含义。 另外除非特别需要， 则单数术语应包括复数， 复数术语应 包括单数。 前述技术和方法通常根据本领域熟知的及在本说明书引用的参 考文献所述的常规方法进行。 见例如并入作参考的 Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001》所述。
不希望受任何理论所局限， 在体内 NF-κΒ信号通路的调节主要依赖于 κΒ抑制因子 1KB, 1KB在 32和 36位的丝氨酸磷酸化促进了 1KB在蛋白酶 体的降解， 1KB降解后暴露 NF-κΒ分子的核定位序列， NF-κΒ分子随后转 入细胞核内， 与其靶基因上的 κΒ反应元件结合调节靶基因的转录 (Alkalay et al., 1995)。 此外， 研究证实 1KB在 42位酪氨酸的磷酸化也可以影响 1KB 的稳定性， 从而影响 NF-κΒ信号通路的活性 (Imbert et al., 1996; Sethi et al., 2007; Waris et al" 2003)。
本发明发现了 RCAN1蛋白的剪切异构体 RCAN1.1 ( SEQ ID NO: 1 ) 和 RCAN1.4 ( SEQ ID NO: 2 )抑制 NF-κΒ信号通路， 降低了癌症细胞的 存活； 进一步地， 本发明人发现具有 RCAN1.1 的 N-端 103个氨基酸的肽 ( SEQ ID NO: 3 ) 可以与 1KB相互作用并抑制 NF-κΒ活性， 降低癌症细 胞的存活。 因此， 本发明人首次揭示了 RCAN1蛋白是 NF-κΒ的内源性调 节蛋白，其可以影响 NF-κΒ信号通路，其与 NF-κΒ抑制因子 1KB相互作用 并影响它在第 42位酪氨酸的磷酸化。
在一个方面， 本发明提供了一种肽或其编码核酸在制备用于治疗与 NF-κΒ活性升高相关的疾病的药物中的应用，其中所述肽（a )包含 SEQ ID NO: 3的氨基酸序列； 或者（b )包含 SEQ ID NO: 3的氨基酸序列经过修 饰例如经过取代、 缺失或添加一或多个氨基酸而获得的氨基酸序列， 其中 所述肽具有抑制 NF-κΒ信号通路的作用。
在一个实施方案中， 所述肽（a ) 由 SEQ ID NO: 1所示的氨基酸序列 组成， 或者 （b ) 由 SEQ ID NO: 2所示的氨基酸序列组成， 或者 （c ) 由 SEQ ID NO: 3的氨基酸序列组成， 或者 U ) 由 （a ) - ( c ) 中的氨基酸序 列经过修饰例如经过取代、 缺失或添加一或多个氨基酸而获得的氨基酸序 列组成， 其中所述肽具有抑制 NF-κΒ信号通路的作用， 或者（e ) 由与 （a ) - ( c ) 中的氨基酸序列具有至少 95%、 至少 96%、 至少 97%、 至少 98%或 至少 99%的序列相同性的氨基酸序列组成， 其中所述肽具有抑制 NF-κΒ信 号通路的作用。
当术语"包含"被用于本发明说明书和权利要求书中时， 其不排除其它 元件或步骤。 为本发明目的， 术语 "由…组成"被认为是术语"包含"的优选 实施方案。
本文所用术语"肽"， "多肽 "和"蛋白质"可互换使用，指氨基酸残基的聚 合物， 其包含经肽键结合的两个或更多个氨基酸。 聚合物可以是线性的， 分技的或环状的， 可包含天然存在的和 /或氨基酸类似物， 它可被非氨基酸 间断。 通常， 若氨基酸聚合物是长的 （例如超过 50个氨基酸残基）， 其优 选称为多肽或蛋白质， 但是若其是 50个氨基酸长或更短， 优选称为"肽"。
本文中所述肽可以由任何合适的现有技术方法制备， 例如化学合成、 重组表达等等。 对此可以参见例如 Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001))以及 Blackwell Scientific Publications出版、 Nicholson 编辑的 "Peptide Synthesis"中 Atherton 和 Sheppard 的第 9 章" Peptide Synthesis"所述。
在本文中， 所述"修饰"优选是本领域所熟知的保守序列修饰， 包括氨 基酸取代、 添加或缺失。 氨基酸修饰可以通过本领域已知的标准技术导入， 例如在编码蛋白的核酸中进行定点诱变、 分子克隆、 寡核苷酸指导的诱变 和随机 PCR介导的诱变。 保守氨基酸取代包括其中氨基酸残基由具有相似 结构或化学性质的氨基酸残基取代的那些。 本领域已经确定了具有相似侧 链的氨基酸残基家族。 这些家族包括具有碱性侧链的氨基酸 (例如赖氨酸、 精氨酸、 组氨酸)， 酸性侧链的氨基酸 (例如天冬氨酸、 谷氨酸)， 无电荷的 极性侧链氨基酸 (例如天冬酰胺、 谷氨酰胺、 丝氨酸、 苏氨酸、 酪氨酸、 半 酸、 异亮氨酸、 脯氨酸、 苯丙氨酸、 甲硫氨酸)， 分支的侧链氨基酸 (例如苏 氨酸)。 本领域技术人员已知也可以使用除 上述之外的其、他类别氨基酸家 族。 相似的较小变化也包括氨基酸缺失或插入， 或者这二者。 使用本领域 熟知的计算机程序可以找到关于确定哪些氨基酸残基可以被取代、 插入或 缺失而不消除其免疫学活性的指导。 本领域技术人员已知的计算机算法例 如 Gap或者 Bestfit可用于优化对比氨基酸序列以对比及限定相似或相同的 例如， 本文上述取代一或多个氨基酸可以是将 SEQ ID NO: 1、 2或 3 中氨基酸残基取代为具有相似结构或化学性质的氨基酸残基， 例如精氨酸 缬氨酸、 亮氨酸和异亮氨酸之间的相互取代。
例如，本文上述缺失一或多个氨基酸可以是缺失 SEQ ID NO: 1中第 1 位的氨基酸残基， SEQ ID NO: 2中第 1位的氨基酸残基，或者 SEQ ID NO: 3中第 9位的氨基酸残基。
例如， 本文上述添加一或多个氨基酸可以是在 SEQ ID NO: 1中第 1 位添加氨基酸残基 Ala, 在 SEQ ID NO: 2中第 1位添加氨基酸残基 Ala, 或者在 SEQ ID NO: 3中第 9位添加氨基酸残基 Ala。
本文所用术语"抑制 NF-κΒ信号通路"是指抑制 NF-κΒ作为转录因子调 节其下游靶基因的活性。
本文所用术语 "相同性"是指对应于两个蛋白质链中位于相同或类似位 置的相同氨基酸的百分比。用 BLAST算法利用 BLOSUM 62阵列计算同源 性百分比， 该算法可在 NCBI网站 (http://www.ncbi.nlm.nih. gov/)获得。 优选 所述具有序列相同性的肽是功能性同源物， 即其显示显著的相同性 （例如 在氨基酸水平大约 50%序列相同性） 并且如其相应蛋白质一样执行相同功 能。 至少大约 60%, 至少大约 70%, 至少大约 80%、 至少大约 90%、 至少 大约 95%、 至少大约 96%、 至少大约 97%、 至少大约 98%、 至少大约 99% 或更高的序列相同性的功能性同源物是优选的功能性同源物。
在本文中， 术语"核酸"， "核酸分子"， "多核苷酸"和"核苷酸序列"可互 换使用，定义任何长度的多聚物，如聚脱氧核糖核苷酸（ DNA X例如 cDNA, 基因组 DNA, 质粒， 载体， 分离的 DNA和其任何混合物）或聚核糖核苷 酸（RNA )分子（例如 mRNA )或混合的聚核糖-聚脱氧核糖核苷酸。 它们 包含单链或双链， 线性或圆形， 天然或合成的多核苷酸。
对于指定的核酸而言， "编码 "或者 "被编码的"是指包含用于翻译为指 定蛋白的信息。 编码蛋白质的核酸在核酸的翻译区内可包含非翻译序列 (例 如内含子)， 或者可以缺少这种插入的非翻译序列 (例如在 cDNA中）。 编码 蛋白的信息由使用密码子而指定。
在一个实施方案中， 本发明的核酸还可以含在载体例如表达载体、 质 粒、 病毒， 噬菌粒、 噬菌体、 粘粒或人工染色体中。 如本文所用， "表达载 体"是重组或者合成产生的核酸构建体， 其具有一系列指定核酸元件， 所述 核酸元件允许特定核酸在宿主细胞中转录， 例如表达载体除了编码要表达 的核酸序列以外还可包括衍生自与用于表达的宿主相容的复制和控制序列 以及赋予转染的细胞可选择表型的选择标记。 本发明的核酸可以被掺入质 粒、 染色体、 线粒体 DNA、 质体 DNA、 病毒或者核酸片段中。 本领域已 知且可商购许多合适的表达载体， 例如原核表达载体、 酵母表达载体、 哺 乳动物表达载体、 昆虫细胞表达载体、 植物细胞表达载体。 在一个实施方 案中，本发明的核酸序列（SEQ ID NO: 1、 2或 3 )在表达载体如 pcDNA3.1 ( - )mychis( c ) ( Invitrogen )、pSuper ( Oligoengine )或者 Adeno-X (Clontech) 中表达。
"宿主细胞"是指含有载体且支持该载体复制和 /或表达的细胞。 宿主细 胞可以是原核细胞如大肠杆菌细胞， 或者真核细胞如酵母细胞、 昆虫细胞、 两栖动物细胞或者哺乳动物细胞。 表达本文所述蛋白的宿主细胞可以是本 领域使用的任何真核细胞或原核细胞， 例如 HEK293, HEK293T, 大肠杆 菌细胞等。 本领域技术人员已知各种细胞培养以及表达、 收集、 纯化以及 检测被表达的蛋白的技术。 对此可参见例如 J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989) 禾口 KM. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988), Ed Harlow and David Lane (editors)。
在另一个方面， 本发明还提供了一种肽或其编码核酸作为治疗与 NF-κΒ活性升高相关的疾病的药物的应用， 其中所述肽（a)包含 SEQ ID NO: 3的氨基酸序列； 或者（b)包含 SEQIDNO: 3的氨基酸序列经过修 饰例如经过取代、 缺失或添加一或多个氨基酸而获得的氨基酸序列， 其中 所述肽具有抑制 NF-κΒ信号通路的作用。
在另一个方面， 本发明还提供了一种用于治疗与 NF-κΒ活性升高相关 的疾病的药物组合物， 其包含一种肽或编码其的核酸以及任选存在的药物 可接受的载体或赋形剂， 其中所述肽（a)包含 SEQIDNO: 3的氨基酸序 列； 或者 （b)包含 SEQIDNO: 3的氨基酸序列经过修饰例如经过取代、 缺失或添加一或多个氨基酸而获得的氨基酸序列， 其中所述肽具有抑制 NF-κΒ信号通路的作用。
在一个实施方案中， 所述肽（a) 由 SEQIDNO: 1所示的氨基酸序列 组成， 或者 （b) 由 SEQ ID NO: 2所示的氨基酸序列组成， 或者 （c) 由 SEQIDNO: 3的氨基酸序列组成， 或者 U) 由 （a) - (c) 中的氨基酸序 列经过修饰例如经过取代、 缺失或添加一或多个氨基酸而获得的氨基酸序 列组成， 其中所述肽具有抑制 NF-κΒ信号通路的作用， 或者（e) 由与 （a) - (c) 中的氨基酸序列具有至少 95%、 至少 96%、 至少 97%、 至少 98%或 至少 99%的序列相同性的氨基酸序列组成， 其中所述肽具有抑制 NF-κΒ信 号通路的作用。将肽或核酸分子配制为药物组合物可参见 Gennaro, A丄. and Gennaro, A.R. (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA; Crowder, T.M. et al. (2003 ) A Guide to Pharmaceutical Particulate Science. Interpharm/CRC, Boca Raton, FL;禾口 Niazi, S.K. (2004) Handbook of Pharmaceutical Manufacturing Formulations, CRC Press, Boca Raton, FL。
在另一个方面， 本发明还提供了一种治疗与 NF-κΒ活性升高相关的疾 病的方法， 包括给患有所述疾病的患者施用一种肽或编码其的核酸或者包 含其的药物组合物， 其中所述肽（a )包含 SEQ ID NO: 3的氨基酸序列； 或者（b )包含 SEQ ID NO: 3的氨基酸序列经过修饰例如经过取代、 缺失 或添加一或多个氨基酸而获得的氨基酸序列， 其中所述肽具有抑制 NF-KB 信号通路的作用。
本发明使用的术语"施用"表示通过合适的方法将预定量的物质导入病 人。 本发明的分离的肽、 分离的核酸分子、 表达载体、 重组细胞、 药物组 合物或疫苗可以通过任何通常途径施用， 只要其能到达所希望的组织。 可 以考虑多种施用方式， 包括腹膜内、 静脉内、 肌内、 皮下、 透皮、 口服、 局部、 鼻内、 肺内和直肠内， 但是本发明不限于这些举例说明的施用方式。
在一个实施方案中， 所述肽（a ) 由 SEQ ID NO: 1所示的氨基酸序列 组成， 或者 （b ) 由 SEQ ID NO: 2所示的氨基酸序列组成， 或者 （c ) 由 SEQ ID NO: 3的氨基酸序列组成， 或者 U ) 由 （a ) - ( c ) 中的氨基酸序 列经过修饰例如经过取代、 缺失或添加一或多个氨基酸而获得的氨基酸序 列组成， 其中所述肽具有抑制 NF-κΒ信号通路的作用， 或者（e ) 由与 （a ) - ( c ) 中的氨基酸序列具有至少 95%、 至少 96%、 至少 97%、 至少 98%或 至少 99%的序列相同性的氨基酸序列组成， 其中所述肽具有抑制 NF-κΒ信 号通路的作用。
本文中， 本发明所述肽还可以与另外的肽融合， 从而修饰肽的生理学 或药学性质， 例如增加稳定性或增加膜穿透性。 在一个实施方案中， 本发 明的肽（例如 SEQ ID NO: 3 )可以与人类免疫缺陷病毒 HIV的 Tat蛋白 （例 如第 47-57个氨基酸 RKKRRQRRRG ( SEQ ID NO: 14 ) )融合以促进多肽穿 透细胞膜。
本文中， 所述与 NF-κΒ活性升高相关的疾病是指疾病的发生、 发展、 持续或者复发与 NF-κΒ活性升高相关的任何疾病（Basseres and Baldwin, 2006)。 例如所述疾病可以选自多发性骨髓瘤、 白血病、 套细胞淋巴瘤、 粘 膜相关淋巴组织淋巴瘤、 弥漫性大细胞淋巴瘤、 霍奇金淋巴瘤、 骨髓增生 异常综合征、 乳腺癌、 卵巢癌、 肺癌、 宫颈癌、 前列腺癌、 肝癌、 胰腺癌、 食管癌、 胃癌、 咽喉癌、 胆管癌、 甲状腺癌、 副甲状腺癌以及皮肤和头颈 的鳞状细胞癌。 在一个实施方案中， 所述疾病优选是白血病， 例如选自急 性淋巴细胞白血病、 急性髓细胞白血病、 慢性淋巴细胞白血病、 慢性髓细 胞白血病、 成人 T-细胞白血病。
在本文中， 治疗是指： 减轻、 缓解、 消除、 阻碍、 阻止所述疾病的症 状和 /或者延缓所述疾病的发生和 /或发展。 对于疾病症状的监测和 /或测量 可以通过临床医师可以使用任何手段或方法进行。
一种肽或编码其的核酸是否能够抑制 NF-κΒ的活性可以通过测定细胞 核内 NF-κΒ蛋白的量来确定。 本领域已知许多测定 NF-κΒ蛋白的测定方法， 例如细胞核蛋白免疫印迹， 使用分离的核蛋白的电泳迁移率实验， 以及使 用报告基因系统测定 NF-κΒ蛋白作为转录因子的活性 (Imbert et al., 1996)。 在本发明的一个实施方案中， 通过 Western印迹测定细胞核内 NF-κΒ蛋白的 量以研究 RCAN 1对于 NF-κΒ活性的抑制作用。
本领域中， 已知很多体内例如动物实验或者体外实验例如细胞学测定 可以用来研究一种肽或编码其的核酸是否能够用于治疗与 NF-κΒ活性升高 相关的疾病 (Kordes et al., 2000)。 例如， 白血病已知是一种与 NF-κΒ活性升 高相关的疾病。 白血病为血液类肿瘤， 白血病细胞系例如 JURKAT等是良好 的白血病细胞模型， 尤其是短期培养的原代白血病细胞， 直接从患者外周 血中分离后立即用于药物测试， 基于细胞存活率的细胞学测定方法能够反 映药物的效果及机制。 在本发明中， 使用了白血病细胞系和原代培养的白 血病细胞作为模型研究了 RCAN1治疗白血病的效果， 该模型能够证明 RCAN1是治疗白血病的良好候选药物。
本发明所述 RCAN1蛋白或者其 N端 103个氨基酸片段具备作为新型 NF-κΒ抑制剂的如下优势： RCAN1是人体自然存在的内源性蛋白， 因此在 人体中的免疫原性很低， 不会被人体的免疫系统快速清除； 多肽类药物比 之于小分子化合物还具有生物活性高， 作用特异性强等特点， 因此多肽类 药物的副作用较小而且治疗效果明显。 实施例
本发明通过下述实施例进一步阐明 , 但任何实施例或其组合不应当理 解为对本发明的范围或实施方式的限制。 本发明的范围由所附权利要求书 限定， 结合本说明书和本领域一般常识 , 本领域普通技术人员可以清楚地 明白权利要求书所限定的范围。 在不偏离本发明的精神和范围的前提下， 本领域技术人员可以对本发明的技术方案进行任何修改或改变， 这种修改 和改变也包含在本发明的范围内。 实施例 1、 RCAN1表达载体的制备
1.1 材料
PCR扩增的酶及 dNTP： 购自 Takarra公司；
PCR扩增引物： 由上海生工公司合成；
Takara限制性内切酶（ EcoR I、 Kpn I、 Hind III、 Bgl II ):购自 New England Biolab公司；
Takara T4连接酶： 购自大连宝生物公司；
真核表达载体 pcDNA3.1 ( - ) mychis ( c ): 购自 Invitrogen公司； 基因敲除载体 pSuper: 购自 Oligoengine;
腺病毒表达载体 Adeno-X: 购自 Clontech;
大肠杆菌 DH5a购自 Invitrogen。
其余常规化学试剂均为国产分析纯， 除非另外指明。
细胞系： HEK293细胞系购自 ATCC。 HEK293细胞培养于含 10%胎牛 血清的 DMEM培养基中，培养环境为 37。C恒温，含 5%C02的细胞培养箱。 细胞培养产品均购自 Invitrogen公司。
1.2 实验步骤
1.2.1 质粒构建 (1)为构建 RCAN1 高表达载体， 构建了 pcDNA3.1 ( - ) RCANl.l-myc 重组质粒，使用如下引物 PCR扩增人类 cDNA文库，并经 EcoR 1 和 Kpn l 酶切后连接入用相同酶切的 pcDNA3.1 ( - ) mychis ( c )载体后获得。
用于克隆 RCAN1.1的 PCR引物为：
上游引物： ccgCTCGAGgccaccATGGAGGAGG TGGACCTGCA ( SEQ ID NO: 4 )
下游引物： atggtacc CCTCTTCTTCCTCCTTC ( SEQ ID NO: 5 )
使用人类 cDNA文库 （购自 Clontech公司） 为模板， 分别以上述引物 通过常规 PCR ( PCR条件： 94°C lOmin, 30个循环 94°C、 30s, 55°C、 30s, 72°C、 30s, 最后 72°C 5min ) 扩增基因片段， 然后通过琼脂糖凝胶电泳分 离并回收 PCR扩增片段， 所述片段的上游带有 EcoRl酶切位点， 下游带有 Kpn l酶切位点。 PCR得到的 DNA片段通过 EcoR 1+Kpn I双酶切， 然后连 接到同样利用 EcoR 1+Kpn I双酶切的 pcDNA3.1 ( - ) mychis ( c )载体， 获 得 pcDNA3.1 ( - ) RCANl.l-myc重组表达质粒。
(2)使用与 (1)同样的方法， 构建了 RCAN1 同源异构体 4的真核表达载 体 pcDNA3.1 ( - ) RCAN 1.4-myc。
用于克隆 RCAN1.4的 PCR引物为：
上游引物： ccgCTCGAGgccaccATGCATTTTA GAAACTTTAA ( SEQ ID NO: 6 )
下游引物： atggtacc CCTCTTCTTCCTCCTTC ( SEQ ID NO: 7 )
( 为获得 RCAN1 基因敲除载体， 两条核苷酸经过退火， 即 95°C、 5 分钟， 然后自然冷却， 变为双链后插入被 Hind III和 Bgl II酶切的 pSuper 载体， 获得 Si-RCAN1。 两条核苷酸的序列为：
DS-511 super sense：
GatccccGAGGAAATGGAAAGAATGAGGttcaagagaCCTCATTCTTTCCATT TCCTCttttta ( SEQ ID NO: 8 )
DS-511 super AS:
CCATTTCCTC ggg ( SEQ ID NO: 9 )
1.2.2腺病毒载体的构建 首先构建 pRCAN 1.1 -EGFP和 pRCAN 1.4-EGFP载体： 使用 EcoRI和 Kpnl 酶切 pcDNA3.1 ( - ) RCANl.l-myc或者 pcDNA3.1 ( - ) RCAN1.4-myc质粒， 回收 0.7kb的 DNA片段作为插入物， 插入到使用相同酶切的 pEGFP-N3 (购 自 clonetech公司）载体， 获得 pRCANl.l-EGFP和 pRCAN1.4-EGFP载体。
构建 Ad-RCANl .1和 Ad-RCANl .4载体： 使用 Nhe I和 Not I酶切 pRCANl.1 -EGFP和 pRCANl .4-EGFP载体， 回收约 1.4kb的片段作为插入物， 插 入 到 相 同 酶 切 的 pShuttle2 ( 购 自 Clontech公 司 ） 得 到 pShuttle2-RCAN 1.1 -EGFP和 pShuttle2-RCAN 1.4-EGFP , 然后使用 I-ceuI和 Pl-Scel酶切 pShuttle2-RCANl.l-EGFP和 pShuttle2-RCAN1.4-EGFP回收约 1.4kb的片段作为插入物， 插入到相同酶切的 Adeno-X腺病毒载体（购自 Clontech公司 )得到 Ad-RCANl.1和 Ad-RCAN 1.4载体。
1.2.3腺病毒的生产
由于白血病细胞系使用一般的转染方法都无法转染，在此我们使用了腺 病毒来表达 RCAN1 蛋白。 约 106个 HEK293细胞在转染之前一天接种到 60mm培养皿， 细胞转染使用脂质体 LF2000 (购自 invitrogen )和 5 g的 Pacl (购自 NEB 公司 ) 酶切的病毒载体 Adeno-X或者 Ad-RCANl.1 和 Ad-RCAN 1.4 (具体方法参见 Invitrogen的 LF2000转染试剂盒的说明书）。 7天后， 收取细胞并使用 3次冻融裂解细胞收取含病毒的裂解液，使用细胞 裂解液 0.5ml感染 60mm培养皿接种的 HEK293细胞， 48小时后冻融裂解 细胞收取病毒， 同样的方法再连续感染两次 HEK293后收取的病毒用于实 验。 病毒的滴度大约为 4 X 108 /ml。 在本发明中， 除非特别指明， 细胞系 及原代细胞使用的感染复数（MOI )为 30。 实施例 2: RCAN1高表达抑制 NF-κΒ活性，低表达可以增高 NF-κΒ活性 2.1 NF-κΒ核转移实验步骤
HEK293细胞转染 RCAN1表达载体 pcDNA3.1 ( - ) RCANl.l-myc: 使 用脂质体 LF2000 (购自 invitrogen ) 转染 HEK293细胞， 4μ1的 LF2000加 入至 ΙΟΟμΙ的 opti-MEM (购自 Invitrogen公司）， 5分钟后与加入 2 g的 RCAN1表达载体 pcDNA3.1 ( - ) RCANl.l-myc的 ΙΟΟμΙ的 opti-MEM混合 后， 室温静置 15分钟， 加入至养有 HEK293的 35mm直径的培养皿中。 48小时后分离细胞核并裂解： 细胞核使用细胞核蛋白提取试剂盒（购 自 Millipore公司 )分离并裂解。
细胞核裂解液使用 12%甘氨酸 SDS-PAGE 分离蛋白 （购自 Biorad公 司 ): 跑胶及转膜装置为 Biorad公司的 mini-protean 3垂直电泳装置。
Western Blot方法： 使用 anti-NF-κΒ 抗体（购自 Sigma公司 )检测细 胞核内的 NF-KB水平（具体方法参见 Sigma公司的使用说明书）。 Anti-TBP 抗体（购自 Sigma公司）检测的 TBP蛋白作为内参。 SDS-PAGE胶及 western blot的具体方法参见 Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989)
2.2 NF-κΒ核转移实验结果
显示 RCAN1高表达的细胞的细胞核内的 NF-κΒ明显降低 （见图 1 ), 说明 RCAN1 可以明显抑制 NF-KB的核转移， RCAN1 敲除可以明显升高 NF-KB的核转移 （图 1A )。
2.3 NF-KB活性荧光指示系统的实验步骤
RCAN1表达载体与 pNF-KB-luc (购自 Clontech公司；)， pRL-TK (购 自 Promega公司 )共同转染 HEK293细胞（质粒使用比例为 1 : 1 :0.02 , 具体 方法参见 Invitrogen的 LF2000转染试剂盒的说明书 ) , 24小时后裂解细胞 釆用双荧光报告系统 （购自 Promega公司 ) 测试荧光。
2.4 NF-KB活性荧光指示系统的实验结果
结果显示 RCAN1高表达可以显著抑制 NF-KB活性， RCAN1敲除可以 明显升高 NF-κΒ活性 （图 1B )。 实施例 3: RCAN1与 1KB结合
3.1 方法步驟
HEK293细胞转染 RCAN1表达载体（如实施例 2 2.1所述）；
48小时后收取细胞并裂解；
力口入 20μ1 免疫共沉淀试剂 Protein A/G agarose (购自 Santa Cruz公司） 和 2μ1抗 -ΙΚΒα抗体（购自 Cell Signaling公司；)， 4°C摇动过夜。
2000g离心去上清， 并使用 PBS洗沉淀两次， 加入上样缓冲液； 12%甘氨酸 SDS-PAGE分离蛋白并将蛋白转至 PVDF膜； Western blot使用 anti-myc抗体（购自 ABcam公司）检测 RCAN1蛋白 (具体方法参见 ABcam的抗体说明书）。
反之， 可以使用 anti -myc抗体沉淀蛋白, 再使用 1KB抗体做 western blot。 SDS-PAGE和 western blot的方法具体参见实施例 2 2.1。
3.2 结果
如图所示， RCAN1蛋白与 1KB蛋白存在相互作用 （图 2 )。 实施例 4: RCAN1影响 1KB在 42位的酪氨酸磷酸化
4.1 实验步驟
HEK293细胞转染 RCAN1敲除载体 si-RCANl (如实施例 2 2.1所述， 载体 Si-RCANl用量也是 2ug );
48小时后收取细胞并裂解， 使用 1KB抗体（购自 Cell Signaling公司） 做免疫沉淀（具体方法参见 Cell signaling的说明书；)；
免疫沉淀用 12% SDS-PAGE胶分离并转至 PVDF膜；
Western blot使用抗 IKB-Y42磷酸化抗体 (购自 ABcam公司 )检测。 SDS-PAGE和 western blot的方法具体参见实施例 2 2.1。
4.2 结果
结果显示 RCAN1的敲除可以明显增加 1KB在 Y42的磷酸化 （图 3 )。 实施例 5: RCAN1腺病毒感染导致白血病细胞死亡
5.1 材料
白血病细胞株 Jurkat, NALM-6, CEM, MOLT-4, Kasumi, HL-60, THP-1 , HEL均购自 ATCC。 白血病原代细胞以及细胞株均培养在含 10% 胎牛血清的 RPMI 1640培养基中， 细胞培养产品均购自 Invitrogen公司。 培养环境为 37。C恒温， 含 5%C02的细胞培养箱。
单个核细胞分离液 Histopaque- 1077： 购自 Sigma公司。
细胞存活率试剂盒 Celltiter-Glo发光试剂盒： 购自 Promega公司。
5.2 实验步驟
白血病原代细胞的分离釆用的是 Histopaque试剂，在 15ml的离心管内 加入 3ml的 Histopaque-1077试剂， 上层为 3ml白血病患者的新鲜外周血， 在 300g离心 30分钟， 吸取第二层的单个核细胞并使用 10ml的 PBS缓冲 液洗涤一次， 接种至培养皿。
白血病细胞株或者原代白血病细胞使用表达 RCAN1.1和 RCAN1.4的 腺病毒感染 48小时后， 使用细胞存活率试剂盒检测细胞存活率。
5.3 结果
结果显示 RCAN1.1 或者 RCAN1.4的高表达可以引起急性淋巴白血病 细胞系 Jurkat, NALM-6, CEM, MOLT-4死亡， 存活率大约为 30-60% (图 4A )。 RCANl.l 或者 RCAN1.4 高表达可以引起急性髓细胞白血病细胞系 Kasumi, HL-60, THP-1 , HEL的死亡， 存活率大约为 40-80% (图 4B )。
RCANl.l在急性白血病原代细胞中的高表达可以引起明显的细胞死亡， 存 活率大约为 55%, 而对于正常的血细胞及缓解白血病患者的血细胞没有明 显的杀伤作用， 说明 RCAN1可以特异的引起白血病细胞的死亡 （图 4C )。 实施例 6: 确定抑制 NF-κΒ的 RCAN1结构域
6.1 材料
构建 pcDNA3.1RCANl-103mychis 质粒： 使用引物 T7 ( TAATACGA CTCACTATAGGG ( SEQ ID NO: 10 ) )和逆向引物 DS103KpnR: AtggtaccG CTTGTCTGGATTTGGCGGA ( SEQ ID NO : 11 ) , 以 pcDNA3.1 ( - ) RCANl.l-myc为底物扩增 1-103片段， 使用酶切位点 EcoR 1和 Kpnl插入 到 pcDNA3.1 ( - ) mychis ( c ) 获得 pcDNA3.1 RCAN 1 - 103mychis重组质粒。
构建 pcDNA3.1RCAN103-197myc质粒： 使用引物 DSCR1-103F： ccgCTCGAGgccaccatg C AGTTTCTGAT CTCCCCT ( SEQ ID NO: 12 )和逆 向引物 BGH( TAGAAGGCACAGTCGAGG ( SEQ ID NO: 13 ) ),以 pcDNA3.1 ( - ) RCANl.l-myc为底物扩增 103-197片段， 使用酶切位点 EcoR 1和 Kpnl 插入到 pcDNA3.1 ( - ) mychis ( c ) 获得 pcDNA3.1RCAN103-197myc重组质 粒。
核转染试剂盒 VCA-1003: 购自 Lonza公司。
6.2 实验步骤
(1) 免疫共沉淀： HEK293细胞分别转染表达 RCAN1的 N-或者 C-片段 的载体 pcDNA3.1RCANl-103mychis和 pcDNA3.1RCAN103-197myc, 使用 如实施例 3所示实验方法进行免疫共沉淀实验。
(2) NF-κΒ活性荧光指示系统： HEK293细胞分别转染表达 RCAN1的 N- 或 者 C- 片 段 的 载 体 pcDNA3.1 RCAN 1 - 103mychis 和 pcDNA3.1 RCAN 103-197myc , 使用如实施例 2的方法检测 NF-κΒ活性。
(3)白血病细胞株 Jurkat的存活率检测： 表达 RCAN1的 N端 103个氨 基酸的质粒 pcDNA3.1RCANl-103mychis使用核转染法 （方法参见 Amaxa VCA-1003试剂盒说明书 ) 转染 Jurkat细胞， 细胞存活率使用 Celltiter-Glo 发光试剂盒 ( Promega )检测。
6.3 结果
结果显示 RCAN1 的 N-端 103个氨基酸可以与 1KB相互作用并抑制 NF-κΒ活性， RCAN1的 C-端与 NF-κΒ活性无关 （图 5A、 B )。 RCAN1的 N-端 103个氨基酸可以降低白血病细胞株 Jurkat的存活率 （图 5C )。 实施例 7: 抑制 NF-κΒ的 RCAN1多肽的纯化及活性测试
7.1 实验步骤
构建 pTAT-RCANlN-103质粒： TAT序列为人类免疫缺陷病毒 HIV的 Tat蛋白的第 47-57个氨基酸（ RKKRRQRRRG ( SEQ ID NO: 14 ) ), 此 TAT 序列与 RCAN1的 N端多肽融合， 可以促进多肽穿透细胞膜。 将含有 TAT 片段的编码寡核苷酸 Tatg agg aagaagcggagacagcgacgaaga ggatcc c ( SEQ ID NO: 15 )插入到使用 Nde I和 Xho I限制性内切酶（购自 New England Biolabs )酶切的 pet-28b原核表达载体（购自 Novagen公司）获得 pet-28bTAT 载体， 然后使用 Bamh l和 Xho I酶切 pet-28bTAT载体，插入 RCAN1 1-103 的 PCR片段， PCR上游引物： CGGGATCCATGGAGGAGGTGGACCTG ( SEQ ID NO: 16 )；下游引物： CCGCTCGAGCTTGTCTGGATTTGGCGGA ( SEQ ID NO: 17 )。 PCR模板为 pcDNA3.1 ( - ) RCANl.l-myc表达载体。
RCAN1 N-103多肽的纯化： 50ng的 pTAT-RCANlN-103表达载体转化 至 ΙΟΟμΙ的 BL21(DE3)pLysS细胞 （购自 Promega公司；)， 方法为冰洛 30 分钟， 热休克 40秒， 冰洛 5分钟。 表达 TAT-RCAN1N-103的克隆接种至 200ml含 lOO g/ml的氨苄西林的 LB培养基， 37°C摇床 225rpm摇 16小时， 再次接种至 1升含 lOO g/ml的氨苄西林的 LB培养基中， 37°C摇床过夜。 5000g离心 10分钟收集细胞沉淀，使用 PBS洗一次，将细胞沉淀溶于 20ml 细胞混悬液（含 8M尿素， l OOmM氯化纳， 20mM HEPES , PH8.0 ), 釆 用 3次 15秒的超声粉碎细胞， 16000g, 4°C离心 15分钟， 收集上清， 过亲 和层析柱子 HisTrapTM (购自 amersham pharmacia公司）。 使用含 500mM 咪唑的细胞混悬液从柱子上解析蛋白， 使用 PD-10去盐化柱子 （购自 GE 公司 ) 去盐后在 -80°C保存于含 10%甘油的 PBS (购自 invitrogen公司 )。
SDS-PAGE 凝胶电泳后使用考马斯亮蓝染色后显示分离纯化的蛋白条带 (图 6A )。
使用该纯化的 TAT-RCAN1N-103多肽处理 Jurkat细胞： 使用 ΙΟμΜ的 TAT-RCAN1N- 103多肽处理 Jurkat细胞， 24小时后使用 MTT检测细胞存 活率， 实验结果显示 TAT-RCAN1N-103处理组 Jurkat细胞比正常对照组存 活率明显减少 （图 6B )。
参考文献：
Alkalay, I., Yaron, A., Hatzubai, A., Orian, A., Ciechanover, A., and Ben-Neriah, Y. (1995). Stimulation-dependent I kappa B alpha phosphorylation marks the NF-kappa B inhibitor for degradation via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A 92, 10599-10603.
Arron, J.R., Winslow, M.M., Polleri, A" Chang, CP., Wu, H., Gao, X" Neilson, J.R., Chen, L., Heit, J.J., Kim, S.K., et al. (2006). NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21. Nature 441, 595-600.
Basseres, D.S., and Baldwin, A.S. (2006). Nuclear factor-kappaB and inhibitor of kappaB kinase pathways in oncogenic initiation and progression. Oncogene 25, 6817-6830.
Baud, V., and Karin, M. (2009). Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat Rev Drug Discov 8, 33-40.
Chan, B., Greenan, G" McKeon, F" and Ellenberger, T. (2005). Identification of a peptide fragment of DSCR1 that competitively inhibits calcineurin activity in vitro and in vivo. Proc Natl Acad Sci U S A 102, 13075- 13080.
Delforge, M" Blade, J" Dimopoulos, M.A., Facon, T" Kropff, M" Ludwig, Η·, Palumbo, A., Van Damme, P., San-Miguel, J.F., and Sonneveld, P. (2010). Treatment-related peripheral neuropathy in multiple myeloma: the challenge continues. Lancet Oncol 11, 1086-1095.
dos Santos, N.R., Williame, M., Gachet, S., Cormier, R, Janin, A., Weih, D., Weih, R, and Ghysdael, J. (2008). RelB-dependent stromal cells promote T-cell leukemogenesis. PLoS One 3, e2555.
Fuentes, J.J., Genesca, L., Kingsbury, T.J., Cunningham, K.W., Perez-Riba, M., Estivill, X., and de la Luna, S. (2000). DSCR1, overexpressed in Down syndrome, is an inhibitor of calcineurin-mediated signaling pathways. Hum Mol Genet 9, 1681-1690.
Imbert, V" Rupee, R.A., Livolsi, A., Pahl, H.L., Traenckner, E.B., Mueller-Dieckmann, C, Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P.A., et al. (1996). Tyrosine phosphorylation of I kappa B-alpha activates NF-kappa B without proteolytic degradation of I kappa B-alpha. Cell 86, 787-798.
Kordes, U., Krappmann, D., Heissmeyer, V" Ludwig, W.D., and Scheidereit, C. (2000). Transcription factor NF-kappaB is constitutively activated in acute lymphoblastic leukemia cells. Leukemia 14, 399-402.
Sethi, G" Ahn, K.S., Chaturvedi, M.M., and Aggarwal, B.B. (2007). Epidermal growth factor (EGF) activates nuclear factor-kappaB through IkappaBalpha kinase-independent but EGF receptor-kinase dependent tyrosine 42 phosphorylation of IkappaBalpha. Oncogene 26, 7324-7332.
Waris, G" Livolsi, A., Imbert, V., Peyron, J.F., and Siddiqui, A. (2003). Hepatitis C virus NS5A and subgenomic replicon activate NF-kappaB via tyrosine phosphorylation of IkappaBalpha and its degradation by calpain protease. J Biol Chem 278, 40778-40787.

Claims

权 利 要 求 书
1. 一种肽或其编码核酸在制备用于治疗与 NF-KB 活性升高相关的疾 病的药物中的应用， 其中所述肽
(a)包含 SEQIDNO: 3的氨基酸序列； 或者
(b)包含 SEQIDNO: 3的氨基酸序列经过修饰例如经过取代、 缺失 或添加一或多个氨基酸而获得的氨基酸序列， 并且所述肽具有抑制 NF-KB 信号通路的作用。
2. 权利要求 1的应用， 其中所述肽
(a) 由 SEQIDNO: 1所示的氨基酸序列组成， 或者
(b) 由 SEQIDNO: 2所示的氨基酸序列组成， 或者
(c) 由 SEQIDNO: 3的氨基酸序列组成， 或者
(d) 由 （a) - (c)中的氨基酸序列经过修饰例如经过取代、 缺失或添 加一或多个氨基酸而获得的氨基酸序列组成， 并且所述肽具有抑制 NF-KB 信号通路的作用， 或者
(e) 由与 （a) - (c) 中的氨基酸序列具有至少 95%、 至少 96%、 至 少 97%、 至少 98%或至少 99%的序列相同性的氨基酸序列组成， 且所述肽 具有抑制 NF-KB信号通路的作用。
3. 权利要求 1或 2的应用，其中所述与 NF-KB活性升高相关的疾病选 自： 多发性骨髓瘤、 白血病、 套细胞淋巴瘤、 粘膜相关淋巴组织淋巴瘤、 弥漫性大细胞淋巴瘤、 霍奇金淋巴瘤、 骨髓增生异常综合征、 乳腺癌、 卵 巢癌、 肺癌、 宫颈癌、 前列腺癌、 肝癌、 胰腺癌、 食管癌、 胃癌、 咽喉癌、 胆管癌、 甲状腺癌、 副甲状腺癌以及皮肤和头颈的鳞状细胞癌。
4. 权利要求 3的应用， 其中所述疾病是白血病， 例如选自急性淋巴细 胞白血病、 急性髓细胞白血病、 慢性淋巴细胞白血病、 慢性髓细胞白血病、 成人 T-细胞白血病。
5. 一种药物组合物， 其包含一种肽或编码其的核酸以及任选存在的药 物可接受的载体或赋形剂， 其中所述肽
(a)包含 SEQIDNO: 3的氨基酸序列； 或者
(b)包含 SEQIDNO: 3的氨基酸序列经过修饰例如经过取代、 缺失 或添加一或多个氨基酸而获得的氨基酸序列， 并且所述肽具有抑制 NF-KB 信号通路的作用。
6. 权利要求 5的药物组合物， 其中所述肽
(a) 由 SEQIDNO: 1所示的氨基酸序列组成， 或者
(b) 由 SEQIDNO: 2所示的氨基酸序列组成， 或者
(c) 由 SEQIDNO: 3的氨基酸序列组成， 或者
(d) 由 （a) - (c)中的氨基酸序列经过修饰例如经过取代、 缺失或添 加一或多个氨基酸的氨基酸序列组成， 且所述肽具有抑制 NF-κΒ信号通路 的作用， 或者
(e) 由与 （a) - (c) 中的氨基酸序列具有至少 95%、 至少 96%、 至 少 97%、 至少 98%或至少 99%的序列相同性的氨基酸序列组成， 且所述肽 具有抑制 NF-KB信号通路的作用。
7. —种治疗与 NF-KB活性升高相关的疾病的方法，包括患有所述疾病 的患者施用一种肽或编码其的核酸， 其中所述肽包含
(a)包含 SEQIDNO: 3的氨基酸序列； 或者
(b)包含 SEQIDNO: 3的氨基酸序列经过修饰例如经过取代、 缺失 或添加一或多个氨基酸而获得的氨基酸序列， 且所述肽具有抑制 NF-κΒ信 号通路的作用。
8. 权利要求 7的方法， 其中所述肽
(a) 由 SEQIDNO: 1所示的氨基酸序列组成， 或者
(b) 由 SEQIDNO: 2所示的氨基酸序列组成， 或者 (c) 由 SEQIDNO: 3的氨基酸序列组成， 或者
(d) 由（a) - (c)中的氨基酸序列经过修饰例如经过取代、 缺失或添 加一或多个氨基酸的氨基酸序列组成， 且所述肽具有抑制 NF-κΒ信号通路 的作用， 或者
(e) 由与 （a) - (c) 中的氨基酸序列具有至少 95%、 至少 96%、 至 少 97%、 至少 98%或至少 99%的序列相同性的氨基酸序列组成， 且所述肽 具有抑制 NF-KB信号通路的作用。
9. 权利要求 7或 8的方法，其中所述与 NF-κΒ活性升高相关的疾病选 自： 多发性骨髓瘤、 白血病、 套细胞淋巴瘤、 粘膜相关淋巴组织淋巴瘤、 弥漫性大细胞淋巴瘤、 霍奇金淋巴瘤、 骨髓增生异常综合征、 乳腺癌、 卵 巢癌、 肺癌、 宫颈癌、 前列腺癌、 肝癌、 胰腺癌、 食管癌、 胃癌、 咽喉癌、 胆管癌、 甲状腺癌、 副甲状腺癌以及皮肤和头颈的鳞状细胞癌。
10. 权利要求 9的方法，其中所述疾病是白血病，例如选自急性淋巴细 胞白血病、 急性髓细胞白血病、 慢性淋巴细胞白血病、 慢性髓细胞白血病、 成人 T-细胞白血病。
PCT/CN2011/073261 2011-04-25 2011-04-25 钙调磷酸酶抑制因子1在制备治疗与NF-κΒ活性升高相关的疾病的药物中的用途 WO2012145890A1 (zh)

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US14/113,993 US20140142045A1 (en) 2011-04-25 2011-04-25 Use of regulator of calcineurin 1 for manufacturing medicament for treatment of diseases associated with increased nf-kb activity
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EP11864435.0A EP2703006A4 (en) 2011-04-25 2011-04-25 USE OF A CALCINEURIN 1 REGULATOR FOR THE MANUFACTURE OF A MEDICAMENT FOR THE TREATMENT OF DISEASES ASSOCIATED WITH ENHANCED NF-KB ACTIVITY

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