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WO2009070957A1 - Inhibiteur de l'interaction entre blys et ngr et son utilisation - Google Patents

Inhibiteur de l'interaction entre blys et ngr et son utilisation Download PDF

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Publication number
WO2009070957A1
WO2009070957A1 PCT/CN2008/001410 CN2008001410W WO2009070957A1 WO 2009070957 A1 WO2009070957 A1 WO 2009070957A1 CN 2008001410 W CN2008001410 W CN 2008001410W WO 2009070957 A1 WO2009070957 A1 WO 2009070957A1
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Prior art keywords
blys
substance
ngr
neurite outgrowth
blocking
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Chinese (zh)
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Lei Zhang
Jian Zhang
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Institute of Genetics and Developmental Biology of CAS
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Institute of Genetics and Developmental Biology of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders

Definitions

  • the present invention relates to a substance which blocks BLyS inhibition of neuronal neurite outgrowth information transmission and its use in nerve regeneration. Background technique
  • the neuronal cells of the nervous system are damaged, they are usually divided into two types according to the injury site: one is cell body damage, in which case the cells die quickly; the other is axonal damage, and the damaged neuron cells can It lasts for a long time and can last for several years in the human body.
  • Neuronal axons of the peripheral nervous system are relatively easy to regenerate after injury, but adult mammalian central neuron axons are difficult to regenerate after being damaged, mainly due to the central nervous system's own inhibitory environment.
  • oligodendrocytes which constitute the myelin of the neuron, express a factor that inhibits axonal growth and regulate axonal growth of damaged neurons, among which NogoA, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin Glycoprotein (Omgp) is a molecule with three significant inhibitory effects (He and Koprivica, 2004).
  • NogoA, MAG and Omgp have no similarities in protein structure, but are capable of interacting with the same glycosylphosphatidylinesitol (GPI) domain.
  • Nogo-66 receptor (NgR) specifically binds (McGee and Strittmatter, 2003).
  • central nervous system injury diseases not only the damage of neuronal cells, but also the participation of inflammatory reactions.
  • the inflammatory response is mainly mediated by lymphocytes that enter the blood-brain barrier and glial cells of the nervous system itself.
  • Most of the current clinical drugs are used to suppress the immune response caused by T cells and fail to achieve the desired therapeutic effect.
  • MS multiple sclerosis
  • IFN- ⁇ can only relieve the symptoms of some patients. The disease cannot be cured, and the drug has no clinical effect on some patients.
  • BLyS B lymphocyte stimulator
  • ⁇ Tumor Necrosis Factor
  • BAFF Tumor Necrosis Factor
  • TALL-K zTNF THANK
  • TNFSF13B Tumor Necrosis Factor
  • BLyS plays an important role in B cell development and antibody production. The effect can also promote the inflammatory response of lymphocyte Th1 (Sutherland et al., 2005). It is generally accepted that BLyS, like most TNF ligands, is active in soluble trimers. It has been found that BLyS has three receptors.
  • B-cell maturation antigen BCMA
  • BR3 BLyS receptor 3
  • TACI transmembrane activator And CAML-interactor
  • the present invention clarifies the role of BLyS in inhibiting the growth of neuronal processes in the nervous system, and finds that the new receptors for BLyS, NgR, BLyS, can inhibit the growth of neurites through the receptor NgR, thereby providing a therapeutic effect by blocking the inhibition of BLyS.
  • the theoretical basis of systemic disease The theoretical basis of systemic disease.
  • BLyS B lymphocyte stimulator cDNA complementary DNA complementary deoxyribonucleotides
  • COS-7 cells are derived from the cell line of African green monkey kidney cells and are capable of expressing large ⁇ antigens
  • the Fc Fc fragment of IgG when fused to a protein of interest as a protein tag, can be used for detecting the expression of a protein of interest as well as improving the stability of expression of a protein of interest;
  • FLAG refers specifically to DYKDDD polypeptide and can be used as a protein tag
  • Laminin laminin which is coated with a cell culture dish to enhance cell adhesion
  • a fragment of Nogo-66 NogoA protein outside the cell membrane also has the effect of inhibiting neurite outgrowth
  • PCR Polymerase Chain Reaction a molecular biology technique used to amplify specific DNA fragments and can be considered as special DNA copies in vitro;
  • Phalloidin which specifically binds to the cytoskeletal actin Poly-L-lysine polylysine, which is coated with a cell culture dish to enhance cell adhesion;
  • PI-PLC phosphatidylinositol-specific phospholipase C, which specifically releases GPI-anchored proteins from the cell membrane;
  • RhoA A small molecule of GTPase that regulates cytoskeletal actin
  • the present invention discloses that BLyS inhibits the growth and regeneration of neurites by binding to NgR, and also regulates the signaling pathway of neurite outgrowth by RhoA.
  • BLyS By inhibiting the binding of BLyS to NgR, BLyS inhibits the transmission of growth information of neuronal neurite outgrowth by blocking the binding of BLyS to NgR.
  • the present invention also provides the use of the above-mentioned substance which inhibits the binding of BLyS and NgR in the preparation of a medicament for promoting neurite outgrowth.
  • the substance which inhibits binding of BLyS to NgR may be a polypeptide, a protein, a small molecule of a compound, a double-stranded RNA, a small RA or an RNA aptamer, and preferably has an amino terminal position 1-47 amino acid of GenBank Accession No. NP-075380.
  • the polypeptide of the sequence shown by the residue is designated as sNgR, and the amino acid sequence is shown in SEQ ID No: 1.
  • sequence-derived polypeptide can also achieve the object of the present invention. ⁇ , by blocking the binding of BLyS to NgR, BLyS can inhibit the growth of neuronal neurite outgrowth. Passing the block.
  • the present invention still further provides a composition
  • a composition comprising the above-mentioned substance which inhibits the binding of BLyS to NgR.
  • the above compositions may also comprise a pharmaceutically acceptable carrier such as water, an aqueous buffer, a pharmaceutically acceptable salt and/or hyaluronic acid.
  • compositions may also contain suitable excipients and/or suitable additives which are pharmaceutically acceptable.
  • suitable excipients include stabilizers, antioxidants, permeability modifiers, buffers and/or pH modifiers.
  • Suitable additives include suitable buffers, additional chelating masking agents (such as DTPA or DTPA bisamide) or calcium chelating complexes (such as DTPA, CaNaDTPA bisamide) or calcium or sodium salts (calcium chloride, calcium ascorbate, glucose). Calcium acid or calcium lactate).
  • the compositions of the invention may be packaged in liquid form or lyophilized.
  • compositions of the present invention may be used in conventional pharmaceutically acceptable non-toxic carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate.
  • the above composition may be introduced into the body by injection, spray, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue; or may be mixed or wrapped by other substances. After importing into the body.
  • the effective dosage is based on the purpose of promoting the growth of injured neurites.
  • NgRl-447 is an effective medicinal ingredient
  • the specific dosage of 4 mg NgRl-447/kg body weight can be referred to.
  • the present invention also provides a substance which blocks a BLyS-related nerve cell growth signaling pathway by inhibiting the binding of BLyS to a nerve cell growth signaling pathway; and blocking a BLyS-related neuronal cell growth signaling pathway by binding to BLyS A substance that blocks the growth signaling pathways involved in BLyS and RhoA by inhibiting the binding of BLyS to the neuronal growth signaling pathway.
  • the present invention also provides a method for screening a human fetal brain cDNA library using a fusion protein of AP and BLyS (AP-BLyS) as a probe to obtain NgR.
  • AP-BLyS fusion protein of AP and BLyS
  • the above method for screening human fetal brain cDNA library with AP-BLyS to obtain NgR Using alkaline phosphatase-labeled BLyS fusion protein (AP-BLyS) as a soluble probe, the COS-7 cells transfected with the human brain cDNA library were subjected to the above method. Screening, alkaline phosphatase staining was used to detect whether AP-BLyS binds to COS-7 cells expressing a protein in a human fetal brain cDNA library.
  • AP-BLyS alkaline phosphatase-labeled BLyS fusion protein
  • BLyS can inhibit the growth and regeneration of central neurites by transmitting information in combination with NgR and play a role in the signaling pathway by which RhoA regulates nerve cell growth. Based on this fact, it will be apparent to those skilled in the art that substances which inhibit the binding of BLyS to NgR and substances which block the signaling pathway of RhoA regulation of nerve cell growth by inhibiting the binding of BLyS to downstream components can be used to promote damage to neurites. Growing. To inhibit the binding of BLyS to NgR and/or to inhibit Drugs that bind BLyS to downstream components and block RhoA signaling pathways that regulate neural cell growth are also active ingredients that can be used to promote the growth and regeneration of damaged neurites. DRAWINGS
  • Figure 1 shows the interaction between AP-BLyS and NgR:
  • a is the binding of AP-BLyS to COS-7 cells expressing NgR;
  • b is Flag-BLyS specifically blocking the binding of AP-BLyS to NgR-expressing COS-7 cells;
  • c is the binding of AP to COS-7 cells expressing NgR
  • d is the binding of AP-BLyS to COS-7 cells expressing TACI
  • e is the binding of AP-BLyS to COS-7 cells expressing DAF
  • Figure 2 shows the results of in vitro co-immunoprecipitation of soluble protein NgR and BLyS.
  • Figure 3 shows the effect of BLyS on the growth cone structure of chick embryo dorsal root neurons.
  • Figure 4 shows the growth of neurites in chick embryo dorsal root neurons of BLyS inhibiting embryos for 12 days.
  • Figure 5 shows that sNgR-Fc blocks the binding of AP-BLyS to COS-7 cells expressing NgR.
  • Figure 6 shows that sNgR-Fc blocks BLyS inhibition of neurite outgrowth in chick embryo dorsal root neurons.
  • Figure 7 shows the inhibition of BLyS on the growth of neurites in chick embryo dorsal root neurons by PI-PLC.
  • Figure 8 shows that BLyS affects the growth of neurite outgrowth in chick embryo dorsal root neurons by activating RhoA.
  • Example 1 AP-BLyS inhibits the growth of damaged neurites
  • AP-BLyS and NgR bind to each other and cannot directly interact with other members of the NgR receptor complex.
  • the human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 2 and SEQ ID NO: 3 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Xba1.
  • Ligation with pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba1, and ligated the product into E. coli DH5a competent cells.
  • the amplified fragment (the coding gene of NgR) is identical to the sequence of the deoxyribonucleotide of the gene coding region of GenBank Accession No. NM-023004, encoding GenBank. Accession No. NP- 075380 amino acid at position 1-473 from the amino terminus.
  • the recombinant vector containing the NgR-encoding gene was named pcDNA-NgR.
  • the human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 4 and SEQ ID NO: 5 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Xba I. Ligation was carried out with pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I, and the ligation product was transformed into E. coli DH5a competent cells. According to sequencing, the amplified gene of the fragment iNg i is identical to the sequence of the deoxyribonucleotide of the gene coding region of GenBank Accession No. NM-178570, and encodes the amino terminal of GenBank Accession No. NP-848665. - 420 amino acids.
  • the heavy expression vector containing the N g R /7 coding gene was named pcDNA-NgRH1.
  • the human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 6 and SEQ ID NO: 7 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Xba I. Ligation was carried out with pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I, and the ligation product was transformed into E. coli DH5a competent cells. According to sequencing, the amplified gene of the fragment NgRH2 is identical to the sequence of the deoxyribonucleotide of the gene coding region of GenBank Accession No. NM-178568, encoding the amino terminal of the GenBank Accession No. NP-848663. 442 amino acids.
  • the re-expression vector containing the gene encoding the ⁇ 2 was named pcDNA-NgRH2.
  • the human spleen cDNA library (Invitrogen, Cat. 10425-015) was used as a template, and SEQ ID NO: 8 and SEQ ID NO: 9 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Xba I.
  • the pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I was ligated, and the ligated product was transformed into E. coli DH5 (x competent cells. According to sequencing, the amplified fragment was The coding gene of 73 ⁇ 4C is identical to the sequence of the degenerate ribonucleotide of the gene coding region of GenBank Accession No. NM-012452, and encodes the amino acid residue at position 1-194 from the amino terminus of GenBank Accession No. NP-036584.
  • the volume group expression vector of the 73 ⁇ 4 C/encoding gene was named pcDNA
  • the human spleen cDNA library (Invitrogen, Cat. 10425-015) was used as a template, and SEQ ID NO: 10 and SEQ ID NO: 11 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Xba I.
  • the pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I was ligated, and the ligation product was transformed into E. coli DH5a competent cells.
  • the amplified fragment (the coding gene of DAF) is identical to the sequence of the deoxyribonucleotide of the gene coding region of GenBank Accession No. NM_000574.3, and encodes the amino terminal of GenBank Accession No. NP-5000565. -382 amino acid residues.
  • One set of expression vectors containing the coding gene was named pcDNA-DAF.
  • the amplified fragment (the coding gene of ' «go7) is identical to the sequence of the 3 ⁇ 4 coding region deoxyribonucleotide of GenBank Accession No. NM-032808.5, encoding the amino terminal of GenBank Accession No. NP_116197. Amino acid residue at position 1-620.
  • the recombinant expression vector containing the Lingo 1 encoding gene was named pcDNA-Lingol.
  • the human placenta cDNA library (Invitrogen, Cat. SL. NB2HP) was used as a template, SEQ ID NO: 16 and SEQ ID NO: 17 were used as primers for PCR amplification, and the PCR amplification products were digested with Not I and used with Not I.
  • the digested pFLAG-CMV1 (Sigma, Cat. E7273) was ligated and the ligation product was transformed into E. coli DH5a competent cells.
  • the gene encoding the amplified fragment has the same sequence as the deoxyribonucleotide of the gene coding region of GenBank Accession No. X55958, and encodes the amino acid residue of GenBank Accession No. CAA39425.
  • the recombinant expression vector containing the /4 cadaver-encoding gene was named pFLAG-AP-CMV1.
  • the human spleen cDNA library (Invitrogen, Cat. 10425-015) was used as a template, and SEQ ID NO: 18 and SEQ ID NO: 19 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Sal I.
  • the amplified coding gene is identical to the sequence of the deoxyribonucleotide of the gene coding region of GenBank Accession No.
  • NM_006573 encodes the amino acid residue at positions 137-285 from the amino terminus of GenBank Accession No. NP-006564. base.
  • the recombinant expression vector containing the coding gene was designated as pFLAG-AP-BLyS-CMV1.
  • the human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 20 and SEQ ID NO: 21 were used as primers for PCR amplification, and the PCR amplification products were digested with EcoR I and Sal I.
  • the recombinant expression vector pFLAG-AP-CMV1 of the above step 8) digested with EcoR I and Sal I was ligated, and the ligation product was transformed into E. coli DH5a competent cells. Root According to sequencing, the amplified fragment (N O-encoding gene) is identical to the sequence of GenBank Accession No.
  • the recombinant expression vector containing the Nogo-66 encoding gene was named pFLAG-AP-Nogo66-CMV 1.
  • the recombinant expression vector pFLAG-AP-BLyS-CMV1 constructed in the above step 9) was digested with the restriction enzymes EcoR I and Sal I, and the BLyS fragment recovered by the gel was pFLAG-digested with EcoR I and Sal I. CMV1 was ligated and the resulting recombinant expression vector was named pFLAG-BLyS-CMVl o
  • the human spleen cDNA library (Invitrogen, Cat. 10425-015) was used as a template, and SEQ ID NO: 22 and SEQ ID NO: 23 were used as primers for PCR amplification, and the PCR amplification products were digested with Xba I and Xba l
  • the digested pcDNA3.1 (+) (Invitrogen, Cat. V790-20) was ligated and the ligation product was transformed into E. coli DH 5 ⁇ competent cells.
  • the amplified fragment (the coding gene of Fc) has the same sequence as the deoxyribonucleotide of the gene coding region of GenBank Accession No. BC073782, and encodes the amino acid position 254-475 of GenBank Accession No. AAH73782. Amino acid residues.
  • the recombinant expression vector containing the Fc-encoding gene was named pcDNA-Fc.
  • a human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 24 and SEQ ID NO: 25 were used as primers for PCR amplification, and a fragment of the NgR-encoding gene which does not contain a GPI domain portion was amplified. (designated as sNgK), the PCR amplification product was digested with EcoR I and Xba I and ligated with pcDNA3.1 (+) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I to ligate the product. Transformation of E. coli DH5 a competent cells.
  • the amplified gene encoding NgR is identical to the sequence of GenBank Accession No. NM-023004 from the 5' end of 202-1542 deoxyribonucleotides, encoding the amino group of GenBailk Accession No. NP_075380. Amino acid residues at positions 1-447.
  • the expression vector containing the sNgR-encoding gene was named pcDNA-sNgR.
  • the recombinant expression vector pcDNA-Fc constructed in the above step 12) was digested with Xba I, and the Fc fragment recovered from the gel was ligated with the above pcDNA-sNgR digested with Xba1, and the resulting recombinant expression vector was named as pcDNA-sNgR-Fc.
  • a human brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was used as a template, and SEQ ID NO: 26 and SEQ ID NO: 27 were used as primers for PCR amplification, and a fragment of the NgRH2 encoding gene which does not contain a domain portion was amplified ( Named sNgR /2), the PCR amplification products were digested with EcoR I and Xba I and ligated with pcDNA3.1 ( +) (Invitrogen, Cat. V790-20) digested with EcoR I and Xba I. The product was transformed into E. coli DH5a competent cells.
  • the amplified gene encoding the fragment Ng 2 is identical to the sequence of the 1st to 1242 deoxyribonucleotides from the 5' end of the gene coding region of GenBank Accession No. NM-012452.
  • the recombinant expression vector containing the sNgRH2 encoding gene I was named pcDNA-sNgRH2.
  • the recombinant expression vector pcDNA-Fc constructed in the above step 12) was digested with Xba I, and the Fc fragment recovered in the gel was ligated with the above-mentioned pcDNA-sNgRH2 digested with Xba I, and the resulting recombinant expression vector was named as pcDNA-sNgRH2-Fc.
  • HEK 293T cells were cultured in a 010 cm cell culture plate at a cell density of 30-50%.
  • the recombinant expression vectors pFLAG-AP-CMV1, pFLAG-AP-BLyS-CMV1, pFLAG-AP-Nogo-66-CMV1, pFLAG-BLyS-CMV pcDNA-sNgRH2-Fc and pcDNA constructed in the above step 1 were respectively prepared as follows. -sNgR-Fc was transfected into HEK 293T cells for transient expression of the protein.
  • 4 ⁇ g of the above recombinant expression vector was diluted into 170 ⁇ 2 ⁇ HBS (50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HP0 4 , 11 7.10), and 170 ⁇ 250 mM CaCl 2 was added dropwise to the above diluted recombinant expression vector. , fully mixed.
  • the diluted recombinant expression vector and the mixture of CaCl 2 were added to the cell supernatant, and after 4 hours, the culture solution was aspirated, and the cells were treated with 10% DMSO (diluted in PBS) for 2 min, and replaced with fresh normal cell culture. The solution is placed in a cell culture incubator.
  • the cell supernatant was collected and protease inhibitor (final concentration 1 g/ml aprotinin, 1 Mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride), 4 ° 4,000 was added.
  • protease inhibitor final concentration 1 g/ml aprotinin, 1 Mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride
  • 4 ° 4,000 was added.
  • 111 centrifugation 10 1 ⁇ 11 the collected cell supernatant was poured into a 19 ml ultrafiltration tube (Centricon tubes Plus-20, Millipore, Beijing, China), and the horizontal rotor was centrifuged at 4 ° C, 3,800 rpm. Protein concentration. The concentrated protein was subjected to immunoblot detection.
  • the size of AP-BLyS expressed by cells of pFLAG-AP-BLyS-CMV1 was 75 kDa; the size of AP-Nogo66 expressed by cells transfected into recombinant expression vector pFLAG-AP-Nogo-66-CMV1 was 65.7 kDa, neurite outgrowth
  • the Nogo66 used in the inhibition assay was AP-Nogo66 prepared by this method; the FLAG-BLyS expressed by the cells transfected into the recombinant expression vector pFLAG-BLyS-CMV1 was 16.7 kDa.
  • the backbone of the vector is pRK5 (BD Biosciences Pharmingen, Cat. 556104), and a linker is inserted through EcoR I/Xba I to introduce MIu I and Not I sites for easy ligation to the library.
  • the sequence of Linker is shown in SEQ ID NO: 28 and SEQ ID NO: 29, and the recombinant vector ligated into Linker was named pRK5L.
  • pRK5L was linearized with Not I /Mlu I, then dephosphorylated and purified using a DNA purification kit (Promega, Cat. A9281) to obtain a carrier concentration of approximately 100 ng ⁇ l.
  • the human fetal brain cDNA library (Invitrogen, Cat. SL.B4HB3MA) was digested with Not I /Mlu I, and the digested library fragments were separately recovered using a gel recovery kit (QIANGEN).
  • the recombinant human brain cDNA library was divided into different pools and transfected into COS-7 cells by transfection reagent Polyfect (QIAGEN, Cat. 301105).
  • the recombinant expression vector pFLAG-AP-BLyS-CMV1 was used in HEK 293T.
  • the protein product AP-BLyS expressed in the cell was used as a recombinant probe, incubated with COS-7 cells expressing the cDNA pool, and stained by alkaline phosphatase.
  • COS-7 cells in good growth state were passaged to poly-L-lysine (Sigma, Cat. P7890) coated cell culture plates one day prior to transfection with a cell density of 30-40%.
  • poly-L-lysine Sigma, Cat. P7890
  • the recombinant expression vector pcDNA-NgR was transfected into the above COS-7 cells using polyfect (QIAGEN, Cat. 301105) according to the instructions for transfection reagent.
  • polyfect QIAGEN, Cat. 301105
  • step b) Wash the cells of step b) above for 6 times with pre-cooled HBHA solution for 7 min each time.
  • the cells were heated in a 65 °C electrothermal incubator for 2 h to inactivate the endogenous alkaline phosphatase.
  • the recombinant expression vector pcDNA-NgRH2 constructed in the above step 1 was transfected into COS-7 cells by lipofection, transfected for 36 h, and then treated with AP-BLyS according to the above ( 3) The method is performed by alkaline phosphatase staining.
  • FIG. 1a AP-BLyS binds to COS-7 cells expressing NgR; in Figure 1 b, FLAG-BLyS blocks AP-BLyS binding to NgR; Figure 1 c, when AP acts as a probe , can not bind to NgR; Figure I d, TACI as a known receptor for BLyS, can bind to AP-BLyS, is the experimental positive control; Figure 1 e, DAF as another protein of the GPI protein family, Can not bind to AP-BLyS, is the negative control of the experiment; Figure I f, Figure 1 g, the other two homologous molecules NgR NgRHl and NgRH2 are transiently expressed in COS-7 cells, AP-BLyS can not Direct binding; In Figure lh, Figure 1 i, the other two components of the NgR receptor complex, p75 and Lingol, were expressed on the surface of COS-7 cells, respectively
  • the cells were treated with a 300 ⁇ M cell lysate (10 mM Tris, pH 8.0, 1% Triton) at 4 °C for 60 min on a shaker to maximize the cell lysate on the cell surface.
  • the broken cells were blown down with a pipette tip, centrifuged at 10,000 rpm, and the precipitate was discarded. The supernatant was incubated in a 65 °C water bath for 1 h.
  • the specific experimental procedures are as follows: 5 ⁇ ⁇ sNgR-Fc and 3 ⁇ ⁇ FLAG-BLyS are co-dissolved in 1.5 ml TBS (10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5), and the protease inhibitor cocktail ( Roche Applied Science, Mannheim, Germany), 1 h at room temperature. Add Protein A beads or Anti-FLAG M2 beads to the reaction solution, 4. C acts for 1 h.
  • the agar gel of Protein A combined with sNgR-Fc was washed 3 times with 100 mM Tris (pH 8.0) and 3 times with 10 mM Tris (pH 8.0); Anti-FLAG M2 combined with FLAG-BLyS
  • the agarose gel was washed 5 times with TBS and centrifuged at 800 °g for 5 min each time at 4 °C.
  • the above-prepared sample was added to 5x loading buffer (0.1 M Tris-cl; 4% SDS; 0.2% bromophenol blue, 20% glycerol, 0.1 M DTT), and the sample treated with Anti-FLAG M2 agar gel
  • the sample buffer does not contain DTT to prevent antibodies in the agarose gel from eluting.
  • Samples treated with Protein A agarose and samples treated with Anti-FLAG M2 agarose were incubated for 3 min at 100 °C on a heater and used for immunoblotting.
  • sNgR-Fc was detected with horseradish peroxidase-labeled anti-human Fc (Beijing Dingguo Biotechnology Co., Ltd.), and FLAG-BLyS was detected with anti-FLAG (Sigma, Cat. F 1804).
  • lane 1 indicates that no target protein is added to the reaction system, and only Protein A agar is added.
  • the lane 2 is a protein of sNgR-Fc that binds to Protein A agarose and reacts with anti-human Fc.
  • Lane 3 the lane 3 indicates that the FLAG-BLyS protein cannot bind to the Protein A agar gel, and only the FLAG-BLyS protein and the Protein A agar gel cannot be immunoprecipitated to any protein; in lane 4, from the top, the first band is a protein band of sNgR-Fc that binds to Protein A agarose and reacts with anti-human Fc. The second band is FLAG that is simultaneously immunoprecipitated by Protein A agar gel and recognized by anti-FLAG by binding to sNgR-Fc.
  • Lane 5 indicates that no protein of interest is added to the reaction system, and anti-human Fc and anti-FLAG do not recognize any protein when only Anti-FLAG M2 beads are added. Can be used in this experiment; Lane 6 indicates that sNgR-Fc protein cannot bind to Anti-FLAG M2 beads, only sNgR-Fc protein and Anti-FLAG M2 beads can not be immunoprecipitated to any protein; Lane 7 is capable of Anti-FLAG M2 beads bind to the FLAG-BLyS protein band recognized by anti-FLAG; in lane 8, from the top, the first band is immunoprecipitated by Anti-FLAG M2 beads by binding to FLAG-BLyS The sNgR-Fc protein band, which is recognized by anti-human Fc, and the second band, is a FLAG-BLyS protein band that is immunoprecipitated by Anti-FLAG M2 beads and can be recognized by anti-FLAG.
  • Figure 3-a The growth of the stained DRG neurons in the growth cone can be seen in Figure 3-a.
  • Figure 3-b is the statistical result of three replicate experiments. The number of damaged growth cones is counted according to the double-blind standard. Statistics were performed using the student's t test method. */? ⁇ 0.04, compared to the PBS treated group; **/? ⁇ 0.009, compared to the PBS treated group. The experimental results show that the degree of damage in the growth cone of DRG explants treated with BLyS is significantly increased.
  • the cells were fixed with the above paraformaldehyde fixative for 1 h at room temperature, and then blocked with a blocking solution (0.1 g Gelatin, 1 g BSA and 0.02 g NaN 3 , pH 7.4 per 100 ml PBS buffer) for 1 h at room temperature; Add anti- ⁇ III tubulin (Millipore, Cat. MAB5544) specific for neurons.
  • Figure 4-a is the statistical result of three replicates of the experiment in Figure 4-a. Each experimental group randomly takes 100 DRG neurites. The length of the double-blind method was statistically analyzed using the Student's t test method.
  • Example 2 BLyS inhibits neurite outgrowth by binding to NgR, and sNgR inhibits BLyS inhibition of neurite outgrowth by blocking the binding of BLyS and NgR.
  • the pcDNA-NgR recombinant vector was transfected into COS-7 cells, and the same amount of AP, AP-Nogo66 and AP-BLyS probes were combined with NgR-expressing COS-7 cells to perform alkaline phosphatase staining quantitative experiments.
  • the NgR-expressing COS-7 cells incubated with the recombinant alkaline phosphatase-labeled probe were lysed, and the alkaline phosphatase bound to NgR was measured at OD 405 nm.
  • the experimental results are shown in Figure 5.
  • BLyS affects the growth of chicken embryo neurites through NgR
  • the embryonic dorsal root neurons of 12.5-day-old embryos were isolated and cultured in a cover glass coated with polylysine and the following proteins expressed in the above Example 1 after trypsinization: protein-free PBS solution, dissolved 100 ng sNgR-Fc in PBS, 1 Nogo-66 in PBS, 1 Nogo-66 in PBS, 200 ng BLyS in PBS, 200 ng BLyS in PBS, and 100 ng sNgR-Fc or 100 a mixture of ng sNgRH2-Fc.
  • the cells were fixed with the above 4% paraformaldehyde fixative for 1 h at room temperature; then blocked with the above blocking solution for 1 h at room temperature; add anti- ⁇ III tubulin specific for nerve cells and incubate for 1 h at room temperature, wash with PBS 5 times, 5 min each time; add anti-mouse conjugated Rhodamine for 1 h at room temperature, wash 5 times with PBS for 5 min each time; Finally, place the coverslip on absorbent paper, dry in air, seal sheet. Different fields of view were randomly selected under a fluorescence microscope (the intersections could not be repeated between the fields of view), and the Nikon ECLIPSE TE2000-U fluorescence microscope was photographed under the objective lens.
  • the length of the radii of the dorsal root neurons was measured by RS image software, and the results are shown in Fig. 6.
  • Statistical analysis was performed using Student's t test method, **/? ⁇ 0.0001, compared to the experimental group treated with PBS.
  • the results showed that sNgR-Fc blocked the inhibition of BLyS induction, while sNgRH2-Fc, which also carries the Fc protein fragment, could not block the inhibition induced by BLyS, suggesting that sNgR-Fc specifically blocks BLyS-induced inhibition of neurite outgrowth. effect.
  • the substance capable of blocking the BLyS-induced inhibition of neurite outgrowth is sNgR, a polypeptide of the sequence represented by amino acid residues 1-447 of the NgR full-length amino terminus.
  • PI-PLC phosphatidylinositol-specific phospholipase C
  • Chicken embryo DRG isolated from 12-day embryos was trypsinized and cultured on coverslips coated with the following proteins expressed in Example 1 above: protein-free PBS solution, 1 ⁇ ⁇ Nogo-66 in PBS And 200 ng BLyS in PBS.
  • a neuron medium was added while adding 1 U/ml PI-PLC (Sigma, Cat. 5542).
  • the cells were fixed with the above-mentioned paraformaldehyde fixative for 1 h at room temperature; then blocked with the above blocking solution for 1 h at room temperature; the anti- ⁇ III tubulin specific for the nerve cell was added and incubated for 1 h at room temperature, and washed with PBS for 5 times.
  • BLyS affects the growth of chicken embryo neuron by activating RoA
  • BLyS can affect the growth cone of neurons, and the shape of the growth cone is mainly controlled by cytoskeletal proteins.
  • RhoA activation has a great influence on the growth cone of neurons, and studies have used RhoA inhibitors to promote neuronal regeneration.
  • RhoA inhibitors to promote neuronal regeneration.
  • Chicken embryo DRG isolated from 12-day embryos was trypsinized and cultured on coverslips coated with the following proteins of different protein expression in Example 1 above: protein-free PBS solution, 1 ⁇ g Nogo- dissolved in PBS 66 and 200 ng BLyS in PBS, 15 ⁇ ⁇ 27632 was added to the culture solution. After 18-19 h, the above paraformaldehyde fixative was fixed at room temperature for 1 h; then blocked with the above blocking solution at room temperature for 1 h; add anti- ⁇ III tubulin specific for nerve cells for 1 h at room temperature, and wash 5 times with PBS.
  • the photos of the different experimental groups of each batch are regrouped, and the names of the original encoded files are retained.
  • the scrambled pictures were opened in Adobe photoshop software to see the damage and intact number of growth cones.
  • the counting of ganglion lengths was performed in the RS image software (Roper Scientific, Arlington, AZ), and the method was referenced (Lagenaur and Lemmon, 1987). Pick up neurites that are clearly not intersecting the surroundings, measured from the middle of the cell body to the tip of the neurite. The number of each photo is counted, corresponding to the original encoded file name, and the number of each experimental group is counted.
  • the mean value of the lesion growth cone or neurite length was calculated using Graph Prism 5.0 software and plotted, and the error bars represent SEM.

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Abstract

L'invention porte sur un inhibiteur de l'interaction entre BLyS et NgR et sur l'utilisation de l'inhibiteur pour la fabrication d'un médicament permettant de favoriser l'excroissance de neurites. L'inhibiteur peut être sélectionné dans le groupe consistant en polypeptide, protéine, petit composé moléculaire, ARN double brin, petit ARN et aptamère d'ARN ; de préférence, l'inhibiteur est le polypeptide ayant la séquence d'acides aminés représentée dans SEQ ID NO : 1. L'invention porte également sur un procédé de criblage de l'inhibiteur de l'interaction entre BLyS et NgR par l'utilisation du système de phosphatase alcaline (système AP).
PCT/CN2008/001410 2007-12-05 2008-08-01 Inhibiteur de l'interaction entre blys et ngr et son utilisation Ceased WO2009070957A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050182008A1 (en) * 2000-02-11 2005-08-18 Sirna Therapeutics, Inc. RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering nucleic acid (siNA)
CN1681838A (zh) * 2002-08-10 2005-10-12 拜奥根Idec马萨诸塞公司 Nogo受体拮抗剂
CN1781936A (zh) * 2004-12-03 2006-06-07 中国人民解放军第三军医大学 B淋巴细胞刺激因子抑制肽及其筛选、制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050182008A1 (en) * 2000-02-11 2005-08-18 Sirna Therapeutics, Inc. RNA interference mediated inhibition of NOGO and NOGO receptor gene expression using short interfering nucleic acid (siNA)
CN1681838A (zh) * 2002-08-10 2005-10-12 拜奥根Idec马萨诸塞公司 Nogo受体拮抗剂
CN1781936A (zh) * 2004-12-03 2006-06-07 中国人民解放军第三军医大学 B淋巴细胞刺激因子抑制肽及其筛选、制备方法

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Title
DATABASE EMBL DATABASE 4 December 2007 (2007-12-04), Database accession no. Q9BZR6 *
MACDERMID, V E. ET AL.: "A soluble Nogo receptor differentially affects plasticity of spinally projecting axons.", EUROPEAN JOURNAL OF NEUROSCIENCE., vol. 20, no. 10, November 2004 (2004-11-01), pages 2567 - 2579 *
WANG, Y. T ET AL.: "Nogo and axonal regeneration.", JOURNAL OF BRAIN AND NERVOUS DISEASES., vol. 13, no. 5, 2005, pages 400 - 402 *
WANQ Y. T. ET AL.: "Research progress of repairing the injured spinal cord on the target of NgR.", CHIN J NEUROMED., vol. 4, no. 12, December 2005 (2005-12-01), pages 1281 - 1283 *

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