WO2003002737A1 - Nouvelle proteine inhibitrice de la topoisomerase humaine 2$g(a) et utilisation associee - Google Patents

Nouvelle proteine inhibitrice de la topoisomerase humaine 2$g(a) et utilisation associee Download PDF

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Publication number
WO2003002737A1
WO2003002737A1 PCT/JP2002/006415 JP0206415W WO03002737A1 WO 2003002737 A1 WO2003002737 A1 WO 2003002737A1 JP 0206415 W JP0206415 W JP 0206415W WO 03002737 A1 WO03002737 A1 WO 03002737A1
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amino acid
acid sequence
seq
cells
human
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PCT/JP2002/006415
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Japanese (ja)
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Akira Nakanishi
Fumio Hanaoka
Shinobu Ohmi
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Riken
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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
    • A61K38/005—Enzyme inhibitors
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a novel human topoisomerase 2 binding factor and use thereof.
  • human topoisomerase 2 ⁇ is referred to as ⁇ 0 ⁇
  • the human topoisomerase 2 ⁇ -binding factor is referred to as ⁇ .
  • the present invention relates to ⁇ capable of interacting with ⁇ to inhibit its activity, an antisense oligonucleotide of the ⁇ , an antibody recognizing the ⁇ la, and a drug using the same.
  • the present invention relates to a method for screening a substance that inhibits the interaction between ⁇ and ⁇ . Background art
  • DNA topoisomerase is present in almost all organisms, from bacteria to humans, and is an essential enzyme for cell growth.
  • type II topoisomerase also referred to as topo II or ⁇ 0 ⁇
  • ⁇ 0 ⁇ is a function that unwinds excessive twisting in DNA due to replication and transcription (relaxation activity), and removes entanglement and entanglement between DNA during chromosome condensation, separation, and distribution (decatenation activity).
  • ⁇ is a basic expression, since its expression kinetics is deeply involved in cell growth and tumorigenesis. He has gained wide interest from the foundation to the clinical field. ⁇ The factors that interact with ⁇ have been energetically investigated, and numerous proteins such as supercoiling factor, p53, casein kinase, c-Jun, sgsl, and Rb protein have been reported. Thus, the discovery of ⁇ function regulators may lead to the discovery of new tumor suppressor gene products related to the cell cycle and the analysis of carcinogenic mechanisms.
  • topoisomerase is a target of anticancer drugs
  • conventional anticancer drugs stop their functions in the form of a clear bubble complex, which is a reaction intermediate with topoisomerase, so that the resulting DNA is cleaved. Occurs. This is considered to be one of the causes of side effects. Finding a regulatory factor of ⁇ 0 ⁇ is useful as a target for new anticancer drugs, and can greatly contribute to the development of anticancer drugs that do not form cleaved complexes and have few side effects.
  • An object of the present invention is to solve the physiological function of ⁇ , which is a protein that binds to ⁇ . Further, the present invention has an object to provide a novel drug based on the physiological function of ⁇ .
  • the present invention utilizes the interaction between Tau0ropai a and Iotataupai alpha, and an object to be achieved by providing a method for screening a new drug.
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and first tried to elucidate the function of ⁇ found from HeLa cells. As a result, ⁇ was found to inhibit ⁇ 0 ⁇ activity, suggesting that ⁇ ⁇ may regulate the function of ⁇ depending on the cell cycle. Was done. Therefore, the present inventors further studied the effects of the physiological function of ⁇ ; particularly the effect of forced expression of ⁇ ; and suppression of the expression by antisense on cells ( ⁇ expression level). The presence or absence of correlation with the expression level of ⁇ ; and the difference in the expression level between normal cells and tumor cells were examined.
  • a human topoisomerase 2 ⁇ inhibitor comprising a human topoisomerase 2 binding factor having any one of the following amino acid sequences: (a) the amino acid sequence of SEQ ID NO: 1; or
  • a human topoisomerase 2 ⁇ inhibitor comprising a protein having a partial amino acid sequence in the amino acid sequence set forth in SEQ ID NO: 1, for example, as set forth in SEQ ID NO: 10.
  • a human topoisomerase 2 ⁇ inhibitor comprising a protein having an amino acid sequence is provided.
  • an apoptosis-inducing agent comprising a human topoisomerase 2 ⁇ -binding factor having any one of the following amino acid sequences:
  • (b) has an amino acid sequence in which one to several amino acids have been deleted, substituted and / or inserted in the amino acid sequence of SEQ ID NO: 1, and interacts with human topoisomerase 2 ⁇ to reduce its activity.
  • a human topoisomerase 2 ⁇ -binding factor having any one of the following amino acid sequences, which is a recombinant protein expressed in insect cells using a baculovirus: Is provided. (a) the amino acid sequence of SEQ ID NO: 1; or
  • amino acid sequence that can be:
  • an antisense oligonucleotide comprising an antisense sequence of any of the following 5 to 100 base sequences in any of the following base sequences.
  • nucleotide sequence in which one to several nucleotides have been deleted, substituted and / or inserted in the nucleotide sequence set forth in SEQ ID NO: 2, and interacting with human topoisomerase 2 ⁇ to inhibit its activity;
  • an inhibitor of human topoisomerase-2 binding factor comprising the antisense oligonucleotide.
  • an expression enhancer for two human topoisomerases comprising the above antisense oligonucleotide.
  • an apoptosis-inducing agent comprising the above antisense oligonucleotide.
  • an anticancer agent comprising the above antisense oligonucleotide.
  • an antibody recognizing a human topoisomerase-2 binding factor having any one of the following amino acid sequences, or a fragment thereof: (a) the amino acid sequence of SEQ ID NO: 1; or
  • a human topoisomerase 2a binding factor having any one of the following amino acid sequences:
  • a screening method for an inhibitor of a binding factor to human topoisomerase 2 comprising measuring an interaction with the enzyme 2 ⁇ and selecting a substance that inhibits the interaction.
  • amino acid sequence of SEQ ID NO: 1 one to several amino acids have a deletion, substitution, Z or insertion amino acid sequence, and interact with human topoisomerase 2 ⁇ to inhibit its activity.
  • Amino acid sequences that can be:
  • the test substance is a small molecule compound, an antibody, an oligonucleotide, or a library thereof.
  • an inhibitor of human Totopoisome hydrolases 2 alpha binding agent is provided.
  • FIG. 1 shows the base sequence and amino acid sequence of ⁇ a.
  • FIG. 2 shows intracellular localization due to overexpression of ⁇ a.
  • FIG. 3 shows detection of apoptosis by overexpression of ⁇ .
  • FIG. 4 shows the construction and purification of a ⁇ a expression system by insect cells.
  • ⁇ antibody column prepared by immobilizing 2.5 rag of purified antibody on CNBr-activated Sepharose 4B (0.5 g)). The eluted fractions (1-8) were dialyzed and subjected to SDS-PAGE and silver staining to confirm protein purification. (B).
  • FIG. 5 shows the inhibitory effect of recombinant ⁇ on ⁇ 0 ⁇ a activity.
  • FIG. 6 shows changes in the expression level of ⁇ in the cell cycle and localization within the cell.
  • Normal human dermal fibroblasts (NB1-RGB) are cultured in a low serum medium (D-MEM containing 0.4% FBS, non-essential amino acids) for 72 hours, and then serum is added to 15% FBS. did.
  • the cells were collected every 4 hours up to 32 hours, taking this time as 0 hour, and the DNA content by laser scanning cytometer was determined by histogram and the G1 / G0, S, G2 / M phase content was calculated as%.
  • A The expression of the ⁇ and T0PI proteins was measured using a simulated knob and their expression levels were measured using NIH Image (B).
  • FIG. 7 shows the examination of the interaction between ⁇ and ⁇ using an anti- ⁇ antibody column.
  • FIG. 8 shows a study of the interaction between ⁇ and ⁇ using plasmon resonance.
  • the measurement by the plasmon resonance method was performed using IAsys plus.
  • ⁇ was immobilized (267 arc seconds) using a CM dextran cuvette as a cuvette for immobilization.
  • T0PII ⁇ was added within the range of 2.9 to 22.2 ⁇ , and this data was analyzed by FASTfit to calculate the association rate constant (kass :), dissociation rate constant (kdiss) and dissociation parallel constant.
  • FIG. 9 shows the expression amount and localization of ⁇ by the antisense oligonucleotide.
  • Sense, sense reverse, random and antisense oligonucleotides were introduced into normal human skin fibroblasts (1 ⁇ 10 6 cells / ml), and 48 hours later, immunoblotting was performed using an anti- ⁇ antibody, and intracellular ⁇ was detected. Was measured. In addition, changes in the expression level of ⁇ were measured by changing the concentration of the antisense oligonucleotide. Furthermore, after introducing the antisense oligonucleotide, the expression levels of ⁇ and ⁇ ⁇ in the cells were measured over time, and changes in the localization of T0PIIa by the antisense oligonucleotide were detected using the indirect tendency antibody method. .
  • Figure 1 0 is, normal cells (human dermal fibroblasts) and cancer cells (Jurcat, HL60, HeLa, renal cancer, rectal cancer, gallbladder cancer, colon cancer) comparisons ⁇ and Tau0roiotaiota alpha expression levels in Show.
  • FIG. 11 shows the effect of the antisense oligonucleotide on normal cells and cancer cells.
  • Liposome 2 / xM antisense against normal cells (NB1-RGB) and cancer cells (HeLa) Introduced by the pectin method. After 48 hours, analysis was performed by LSC (A). At 24, 48, and 69 hours after the introduction of antisense, normal cells and cancer cells were recovered and DNA ladders were detected (B). Furthermore, the presence or absence of PARP cleavage was measured by indirect immunofluorescence on cancer cells 48 hours after transfection with antisense (0).
  • FIG. 12 shows detection of the inhibitory effect of ⁇ ⁇ by the synthetic peptide of ⁇ .
  • the amino acid sequence 132-151 of ⁇ (VTATFPYTTILSIWLATRRV) inhibited ⁇ 0 ⁇ activity in a concentration-dependent manner. Re 1 axation activity was almost completely inhibited at 20 M (A). Decatenation activity was not completely inhibited even at 100 ⁇ M ( ⁇ ), indicating a difference in sensitivity to both activities.
  • the present invention relates to a drug containing ⁇ as an active ingredient, and the drug can be used as a ⁇ 0 ⁇ inhibitor or an apoptosis inducer.
  • ITII c used in the present invention has any of the following amino acids.
  • amino acid sequences (b) one or several amino acids in the amino acid sequence described in SEQ ID NO: 1 have a deleted, substituted and / or inserted amino acid sequence, and may interact with ⁇ 0 ⁇ to inhibit its activity. Possible amino acid sequences:
  • the range of ⁇ 1 to several '' in the ⁇ amino acid sequence in which one to several amino acids have been deleted, substituted and substituted or inserted in the amino acid sequence of SEQ ID NO: 1 '' is not particularly limited.
  • an amino acid sequence capable of interacting with ⁇ 0 ⁇ ⁇ to inhibit its activity refers to a protein having the amino acid sequence described in SEQ ID NO: 1. Means that the protein interacts with ⁇ with the same or higher affinity as a protein having the amino acid sequence of ⁇ to inhibit its activity. Inhibition of ⁇ 0 ⁇ a activity can be assayed by any appropriate method.
  • the inhibition of the activity of ⁇ ⁇ ⁇ ⁇ can be evaluated, for example, by measuring the inhibitory effect of ⁇ on Relaxation activity and Decatenation activity.
  • kinetoplast DNA can be used as the substrate, and for the measurement of the Relaxation activity, the assay can be performed using Supercoiling DNA as the substrate.
  • the inhibitory effect on the activity can be evaluated by, for example, assuming that the amount of DNA after the reaction without adding ⁇ is 100%, and calculating the amount of DNA when adding ⁇ relatively.
  • a protein having a partial amino acid sequence in the amino acid sequence of SEQ ID NO: 1 can be used as a ⁇ inhibitor.
  • the length of the partial amino acid sequence is not particularly limited, and is, for example, from 5 amino acid residues to 100 amino acid residues, preferably from 5 amino acid residues to 50 amino acid residues, and more preferably from 5 amino acid residues to 3 amino acid residues. It is about 0 amino acid residues, particularly preferably about 5 to 20 amino acid residues.
  • a protein having such a partial amino acid sequence for example, a protein having the amino acid sequence of SEQ ID NO: 10 can be mentioned.
  • a protein having such a partial amino acid sequence can be produced by a normal peptide synthesis method using a peptide synthesizer.
  • the method for obtaining the protein having the amino acid sequence of SEQ ID NO: 1 is not particularly limited, and may be a naturally occurring protein, a chemically synthesized protein, or a recombinant protein produced by a genetic recombination technique. Recombinant proteins are preferred because they are relatively easy to operate and can be produced in large quantities.
  • a protein having the amino acid sequence of SEQ ID NO: 1 a protein having the nucleotide sequence encoding the protein (for example, the nucleotide sequence of SEQ ID NO: 2)
  • a target protein can be produced by preparing DNA and introducing it into a suitable expression system.
  • the DNA having the nucleotide sequence of SEQ ID NO: 2 is a suitable primer designed based on the information of the nucleotide sequence of SEQ ID NO: 2 from a human-derived (for example, HeLa cell-derived) cDNA library. It can be obtained by screening using one or a probe. Screening can be performed by plaque hybridization or the like. Alternatively, PCR is performed using a human-derived cDNA library (for example, derived from HeLa cells) as type III and using a suitable primer designed based on the nucleotide sequence information set forth in SEQ ID NO: 2. This allows direct cloning of the target gene.
  • a human-derived cDNA library for example, derived from HeLa cells
  • Expression systems for expressing the recombinant protein are known to those skilled in the art.
  • the DNA is inserted downstream of a promoter in an expression vector, and then the recombinant expression vector is introduced into a host cell suitable for the expression vector.
  • expression vectors for bacteria include pGEMEX-1 (produced by Promega), p QE-9 (produced by QI AG EN), p QE-30 (produced by Q I AG EN), pRSET
  • yeast examples include, for example, YE p13 (ATCC 371 15), YEp 24 (ATCC 37051), Ycp5O (ATCC 374 19), pHS 19, pHS 15 and the like.
  • expression vectors for baculo include pFastBac (manufactured by Gibco BRL), pVL1392 (manufactured by Invitrogen), and the like.
  • PcDNAI pcDM8 (Funakoshi), pcDNAI / AmP (Invitrogen), pREP4 (Invitrogen), and expression vectors for producing recombinant viruses, such as pMFG ( Ta kara And pAd ex (Takara).
  • Promoters which can be used in expression vectors for bacterial e.g., trp promoter (P trp), T7 promoter one, 1 ac promoter (P lac), P L promoter, P R promoter, Ya E. coli, such as P SE promoter
  • Examples include a phage-derived promoter and the like.
  • Examples of promoters that can be used in yeast expression vectors include PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gall promoter, gal10 promoter, heat shock protein promoter, and MF. al promoter, CUP 1 promoter and the like.
  • Examples of a promoter that can be used in an expression vector for baculo include polyhedrin promoter and the like.
  • Examples of a promoter that can be used in an expression vector for animal cells include the promoter of the immediate early (IE) gene of the cytomegalovirus (human CMV), the early promoter of SV40, the promoter of retrowinolas, and the adeno-adenovirus. Winores promoter, metallothionein promoter, heat shock promoter, SRa promoter, actin promoter and the like. Further, the enhancer of IE gene of human CMV may be used together with the promoter.
  • the host cell is not particularly limited as long as it can express the target protein, and includes bacteria, yeast, animal cells, insect cells, and the like. More specifically, bacteria belonging to the genus Etscherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc., Kluyveromyces, Saccharomyces, Yeasts belonging to the genus Schizosaccharomyces, Trichosporon, Genus Schiziomyces, Namalba cells, HeLa cells COS 1 cells, COS 7 cells, CHO cells, animal cells such as 293 cells, Sf9, Sf21, And insect cells such as Hive.
  • Methods for introducing a recombinant vector into a host include, for example, a calcium phosphate method, a protoplast method, an electroporation method, and a suffix :! : Roblast method, lithium acetate method, A lipofection method and the like can be mentioned, and it can be appropriately selected according to the type of the host cell.
  • a recombinant protein expressed in insect cells using a baculovirus.
  • the recombinant gene transfer vector and baculovirus are co-transfected into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is transmitted to the insect cells.
  • examples are described in Baculovirus Expression Vectors, A Laboratory Manual; and Current Protocols in 'Molecular' biology, Bio / Technology, 6, 47 (1988), etc.). ).
  • the baculovirus can be obtained, for example, by the use of Autographs californica nuclear polyhedrosis virus, which is a virus that infects Aspergillus insects, such as Autographi californica, nucleus, polyhedrosis virus, and the like.
  • Autographs californica nuclear polyhedrosis virus which is a virus that infects Aspergillus insects, such as Autographi californica, nucleus, polyhedrosis virus, and the like.
  • Examples of a method of co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include a calcium phosphate method and a Lipofexion method.
  • the transformant having the recombinant expression vector having the target DNA prepared as described above is cultured in a medium, the target protein is produced and accumulated in the culture, and the target protein is collected from the culture. Thereby, the recombinant protein can be isolated.
  • the target recombinant protein is expressed in a lysed state in the cells
  • the cells are collected by centrifugation after cell culture, suspended in an aqueous buffer, and then sonicated, a French press, a Mentongaulin homogenizer, and the like.
  • the cells are disrupted using a dynomill or the like to obtain a cell-free extract.
  • a normal protein isolation and purification method that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resins such as getylaminoethyl (DEAE) Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Kasei), and cations using resins such as S-Sepharose FF (manufactured by Pharmacia) Exchange chromatography, hydrophobic chromatography using resins such as butyl sepharose, phenylsepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing, etc.
  • a purified sample can be obtained using a technique such as electrophoresis alone or in combination.
  • a protein having the amino acid sequence of SEQ ID NO: 1 in which one to several amino acids have been deleted, substituted, Z- or inserted has an amino acid sequence represented by SEQ ID NO: 1 and a base sequence represented by SEQ ID NO: 2.
  • Those skilled in the art can appropriately produce the protein based on the sequence information. For example, it can be produced by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques including PCR, mutagenesis, and the like.
  • a mutant DNA can be obtained by using a DNA having the nucleotide sequence of SEQ ID NO: 2 and introducing a mutation into these DNAs.
  • the method can be carried out using a method in which DNA having the base sequence of SEQ ID NO: 2 is brought into contact with a drug as a mutagen, a method of irradiating ultraviolet rays, a genetic engineering technique, or the like.
  • Site-directed mutagenesis one of the genetic engineering techniques, is useful because it is a technique that can introduce a specific mutation at a specific position, and is useful in Molecular Cloning, 2nd edition, Ryrent's Protocols. 'Molecular' biology, Nucleic Acids Research, 10, 6487, 1982, Nucleic Acids Research, 12, 9441, 1984, Nucleic Acids Research, 13, 4431, 1985, Nucleic Acids Research, 13, 8749, 1985, Proc. Natl. Acad. Sci. USA, 79, 6409, 1982, Proc. Natl. Acad. Sci. USA, 82, 488, 1985. Gene, 34, 315, 1985, Gene, 102, 67, 1991, and the like.
  • the target protein can be produced by obtaining DNA having a nucleotide sequence having a mutation in the nucleotide sequence of SEQ ID NO: 2 by the above-described method, and expressing the DNA in the same manner as described above.
  • the agent of the present invention can be used as a ⁇ inhibitor, and its specific use is not particularly limited. For example, it can be used as an apoptosis inducer.
  • Apoptosis was first discovered and defined as morphologically distinct cell death from the classical cell death, necrosis, and subsequent studies have shown that induction and suppression of apoptosis are governed by genes. It has turned out to be so-called programmed cell death. In apoptosis, a complex biochemical reaction occurs along with the activation of cells, producing various protein and DNA degrading enzymes, which act on their own cells to cause cell death. Apoptosis is a physiological cell death that is essential for normal development and differentiation, and occurs in individual cells during normal biological tissue cell rotation. Therefore, it has been shown that excessive reduction of apoptosis causes many dysfunctions.
  • diseases caused by decreased apoptosis include malignant tumors (cancer), leukemias, autoimmune diseases, viral infectious diseases (such as HIV infection), proliferative skin diseases, rheumatoid arthritis, autoimmune diseases, hepatitis, and kidney diseases. And the like. Therefore, the apoptosis-inducing agent of the present invention can be used as a therapeutic or Z- or prophylactic agent for these diseases caused by a decrease in apoptosis.
  • Morphological features of apoptosis include lack of contact with surrounding cells, cytoplasmic enrichment, chromatin condensation and nucleus condensation associated with endonuclease activity, and nuclear segmentation. Disappearance of microvilli on the cell surface and smoothing of the cell surface (bubble formation on the cell surface: membraneblebbing) are also observed. Also, A phenomenon in which DNA is fragmented due to the nuclease activity is also observed, and the cells themselves form cell fragments called apoptotic bodies, and the formed apoptotic bodies are rapidly mobilized by surrounding cells and macrophages. It is phagocytosed and apoptosis occurs. Therefore, apoptosis can be confirmed by, for example, fragmentation of DNA extracted from cells and morphological observation of the cells.
  • the drug of the present invention When used as a medicine, it is generally provided in the form of a pharmaceutical composition containing ⁇ as an active ingredient and a pharmaceutical additive (carrier, excipient, etc.).
  • a pharmaceutical additive carrier, excipient, etc.
  • the agent of the present invention can be administered as a medicament to mammals including humans.
  • the administration route of the drug of the present invention is not particularly limited, and is orally or parenterally administered (for example, intramuscular administration, intravenous administration, subcutaneous administration, intraperitoneal administration, mucosal administration to nasal cavity, or inhalation administration). Any of
  • the form of the drug of the present invention is not particularly limited, and examples of preparations for oral administration include tablets, capsules, fine granules, powders, granules, liquids, syrups, and the like. Preparations include, for example, injections, drops, suppositories, inhalants, transmucosal absorbers, transdermal absorbers, nasal drops, ear drops, and the like.
  • the dose of the drug of the present invention can be appropriately selected in consideration of the gender, age or weight of the patient, the severity of the symptoms, the administration purpose such as prevention or treatment, and the presence or absence of other complications. .
  • the dosage is generally between 0.000 g / kg body weight / day and 0.1000 g / kg body weight / day, preferably between 0.001 / z g / kg body weight / day and 100 g / kg body weight / day.
  • the agent of the present invention is also useful not only as a pharmaceutical, but also as a ⁇ ⁇ ⁇ inhibitor or an apoptosis inducer as an experimental reagent or the like.
  • ⁇ as an active ingredient is generally provided in a form dissolved in an appropriate solvent or the like.
  • the present invention relates to an antisense oligonucleotide comprising an antisense sequence of 5 to 100 consecutive nucleotide sequences in any of the following nucleotide sequences.
  • the range of ⁇ 1 to several '' in the ⁇ base sequence in which one to several bases are deleted, substituted and / or inserted in the base sequence described in SEQ ID NO: 2 '' is not particularly limited, for example, It means about 1 to 60, preferably about 1 to 30, more preferably about 1 to 20, more preferably about 1 to 10, and particularly preferably about 1 to 5.
  • the antisense oligonucleotide of the present invention is a nucleotide that is complementary or hybridizes to a continuous 5 to 100 nucleotide sequence in a DNA sequence encoding any of the above amino acid sequences, It may be a difference between NA and RNA, and may be modified as long as the function is not hindered.
  • antisense oligonucleotide refers to not only those in which all nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are complementary. As long as stable hybridization is possible, there may be some mismatches.
  • Examples of the antisense oligonucleotide of the present invention include an antisense oligonucleotide having a base sequence (TAGCAGGTCCGACAT) shown in SEQ ID NO: 9 in the sequence listing.
  • the antisense oligonucleotide having such a base sequence was able to suppress the expression of ⁇ very effectively.
  • the antisense oligonucleotide used in the present invention suppresses the expression of ⁇ . It is not limited to the above as long as it is possible.
  • the antisense oligonucleotide may be modified. By performing an appropriate modification, the antisense oligonucleotide is less likely to be degraded in vivo, and ⁇ can be more stably inhibited.
  • modified oligonucleotides include S-oligo type (phosphorothioate type), C-5 thiazole type, D-oligo type (phosphodiester type), and ⁇ ⁇ -oligo type (methylphosphonate type).
  • the antisense oligonucleotide may be one in which at least a part of the oxygen atom constituting the phosphate group is substituted or modified with a zeo atom.
  • Such antisense oligonucleotides are particularly excellent in nuclease resistance, water solubility, and affinity for RNA.
  • Examples of the antisense oligonucleotide in which at least a part of the oxygen atom constituting the phosphate group is substituted and modified with an atom include, for example, S_oligo-type oligonucleotides.
  • the number of bases of the antisense oligonucleotide is preferably 50 or less, more preferably 25 or less. If the number of bases is too large, the labor and cost of synthesis of the oligonucleotide increase, and the yield also decreases. Further, the number of bases of the antisense oligonucleotide is 5 or more, and preferably 9 or more. When the number of bases is 4 or less, the specificity for the target gene is decreased, which is not preferable.
  • the antisense oligonucleotide (or derivative thereof) of the present invention can be synthesized by a conventional method, and can be easily synthesized by, for example, a commercially available DNA synthesizer (for example, Applied Biostems).
  • the synthesis method can be obtained by a solid phase synthesis method using a phosphoramidite, a solid phase synthesis method using a hydrogen phosphonate, or the like.
  • the antisense oligonucleotide of the present invention comprises an inhibitor of ⁇ , It can be used as a current enhancer, an apoptosis inducer, and an anticancer agent. It is considered that the antisense oligonucleotide of the present invention can exert an anticancer action by specifically inducing apoptosis in cancer cells.
  • the antisense oligonucleotide of the present invention is used as a medicament, it is generally provided in the form of a pharmaceutical composition containing the antisense oligonucleotide and a pharmaceutical additive (carrier, excipient, etc.). You.
  • the antisense oligonucleotide of the present invention can be administered as a medicine to mammals including humans.
  • the administration route of the antisense oligonucleotide of the present invention is not particularly limited.
  • oral administration or parenteral administration for example, intramuscular administration, intravenous administration, subcutaneous administration, intraperitoneal administration, mucosal administration to nasal cavity, or inhalation) Administration, etc.).
  • the formulation of the antisense oligonucleotide is not particularly limited, and examples of formulations for oral administration include tablets, capsules, fine granules, powders, granules, solutions, syrups, and the like.
  • Formulations for administration include, for example, injections, drops, suppositories, inhalants, transmucosal absorbents, transdermal absorbents, nasal drops, ear drops and the like.
  • Those skilled in the art can appropriately select the form of the drug containing the antisense oligonucleotide, the additive for the drug to be used, the method for producing the drug, and the like.
  • an antisense encapsulating material that enhances durability and membrane permeability can be used.
  • ribosome, poly-L-lysine, lipid, cholesterol, ribopectyl or derivatives thereof can be mentioned.
  • the dosage of the antisense oligonucleotide can be appropriately selected in consideration of the patient's sex, age or weight, the severity of the symptoms, the administration purpose such as prevention or treatment, and the presence or absence of other complications. .
  • the dose is generally 0.1 ⁇ g / kg body weight / day to 100 mg Z kg body weight / day, preferably 0.1 ⁇ g Z kg body weight Z day to 1 O mg Z kg body weight / day. It is.
  • Antibodies that recognize ⁇ The antibody of the present invention recognizes ⁇ having any of the following amino acid sequences. (a) the amino acid sequence of SEQ ID NO: 1; or
  • amino acids in the amino acid sequence described in SEQ ID NO: 1 have a deleted, substituted and / or inserted amino acid sequence, and interact with ⁇ to inhibit its activity.
  • Amino acid sequences that can:
  • the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and its production can be performed by a conventional method.
  • a polyclonal antibody that recognizes ⁇ is obtained by immunizing a mammal with ⁇ or a partial peptide thereof as an antigen, collecting blood from the mammal, and separating and purifying the antibody from the collected blood.
  • the antigen may be administered, for example, two to three times at an interval of 7 to 30 S.
  • the dose may be, for example, about 0.05 to 2 mg per antigen.
  • the route of administration is not particularly limited, and can be selected as appropriate from subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc., by injecting intravenously, intraperitoneally or subcutaneously. Preferably, it is administered.
  • the antigen can be used by dissolving it in an appropriate buffer, for example, an appropriate buffer containing a commonly used adjuvant such as complete Freund's adjuvant or aluminum hydroxide, depending on the administration route and conditions. In some cases, no adjuvant is used.
  • the serum of the mammal is sampled, and the antibody titer is measured.
  • booster immunization is performed using, for example, 100 ⁇ g to 100 ⁇ g of the antigen.
  • blood is collected from the immunized mammal, and the blood is collected, for example, by centrifugation, precipitation using ammonium sulfate or polyethylene daricol, gel filtration chromatography, ion exchange. Chromatography such as chromatography and affinity chromatography
  • a polyclonal antibody recognizing the protein of the present invention can be obtained as a polyclonal antiserum by separating and purifying by a conventional method such as graphy.
  • the globulin type of the monoclonal antibody that recognizes ⁇ is not particularly limited, and examples include IgG, IgM, IgA, IgE, and IgD.
  • the cell line that produces the monoclonal antibody is not particularly limited. For example, it can be obtained as a hybridoma by cell fusion between the antibody-producing cell and the Myeoma cell line.
  • the hybridoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.
  • spleen cells As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used.
  • the antigen the protein of the present invention or its partial peptide is used. Mice, rats and the like can be used as immunized animals, and administration of the antigen to these animals is performed by a conventional method. For example, a suspension or emulsion of an adjuvant, such as complete Freund's adjuvant or incomplete Freund's adjuvant, and the protein of the present invention, which is an antigen, is administered several times to the vein, subcutaneous, intradermal, intraperitoneal, etc. of an animal. To immunize animals.
  • an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant
  • spleen cells are obtained as antibody-producing cells from the immunized animal, and the spleen cells are fused with myeloid cells by a known method (G. Kohler et al., Nature, 256 495 (1975)) to obtain a hybridoma. Can be produced.
  • myeloma cell lines used for cell fusion include the P3X63 Ag8, P3U1 and Sp2 / 0 strains in mice.
  • Cell fusion is performed using a fusion promoter such as polyethylene glycol or Sendai virus.Hypoxanthine / aminopterin / thymidine (HAT) medium is used for selection of hybridomas after cell fusion in a conventional manner.
  • Hybridomas obtained by cell fusion are cloned by a limiting dilution method or the like. Further, if necessary, screening by enzyme immunoassay using ⁇ ⁇ ; or a partial peptide thereof can obtain a cell line that produces a monoclonal antibody that specifically recognizes ⁇ . it can.
  • the hybridoma may be cultured by a usual cell culture method or ascites formation method, and the monoclonal antibody may be purified from the culture supernatant or ascites. Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used in an appropriate combination.
  • Examples of the method for immunoassay of ⁇ using the antibody of the present invention include enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, and luminescence immunoassay. .
  • Antibody fragments include F (ab ') 2 fragments, Fab, fragments and the like.
  • labeled antibodies of the above-mentioned antibodies are also within the scope of the present invention. That is, the antibody of the present invention prepared as described above can be labeled and used.
  • the types and methods of labeling antibodies are known to those skilled in the art. For example, enzyme labels such as horseradish peroxidase or alkaline phosphatase, fluorescent labels such as FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine B isothiocynate), colloid metal and colored latex labeling with color substances such as roasting two tea labels such Piochin, or 1 2 5 I and isotopic labels, such as can ani gel.
  • enzyme labels such as horseradish peroxidase or alkaline phosphatase
  • fluorescent labels such as FITC (fluorescein isothiocyanate) or TRITC (tetramethylrhodamine B isothiocynate)
  • colloid metal and colored latex labeling with color substances
  • Analysis using the labeled antibody of the present invention can be performed by a method well known to those skilled in the art.
  • the present invention is characterized in that in the presence of a test substance, an interaction between ⁇ having any of the following amino acid sequences and ⁇ is measured, and a substance that inhibits the interaction is selected. , ⁇ ⁇ ⁇ ⁇ ⁇ screening method.
  • select a substance that inhibits the interaction for example, select a substance that inhibits the activity of ⁇ ; can do.
  • a specific screening system for example, a screening system for detecting whether or not the activity of ⁇ is restored by adding a substrate D ⁇ , ⁇ , ⁇ and a test substance can be mentioned.
  • test substance used in the present invention any substance can be used, and its type is not particularly limited.
  • specific examples of the test substance may be a low molecular weight compound, an antibody or an oligonucleotide, a natural product extract, or a compound library, a phage display library or a combinatorial library. Construction of compound libraries is known to those skilled in the art, and commercially available compound libraries can also be used.
  • the test substance is preferably a low molecular compound, an antibody, an oligonucleotide, or a library thereof.
  • an inhibitor of ⁇ obtained by the screening method of the present invention is also within the scope of the present invention.
  • the interaction between ⁇ and ⁇ 0 ⁇ is measured in the presence of a test substance.
  • the method for measuring the interaction is not particularly limited. Further, the above-mentioned interaction itself may be directly measured, or the above-mentioned interaction may be indirectly measured by measuring ⁇ activity.
  • ⁇ activity As a method for measuring ⁇ activity, it can be evaluated by, for example, measuring the inhibitory effect of ⁇ on Relaxation activity and Decatenation activity, and the measuring method is as described above.
  • Example 1 In general, the same Atsushi system is performed in the absence of the test substance, and the above-mentioned interaction in the presence and absence of the test substance is measured. It is preferable to determine whether a substance is inhibiting the above interaction.
  • the present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.
  • HeLa, COS- 1, HL60 distributed from the University of Tokyo Medical Research Institute
  • Jurcat distributed from the University of Tokyo Medical Research Institute
  • the following four strains were purchased from RIKEN Genebank and Cell Development Bank: CW-2 (colorectal cancer cell line) ), TUHR4TKB (kidney cancer cell line), TGBC2TKB (gallbladder cancer cell line), TT1TKB (rectal cancer cell line)
  • the HeLa cell-derived cDNA library was converted to type II and PCR was performed using the following primers (30 seconds at 94 ° C, 30 seconds at 94 ° C, 1 minute at 50 ° C, and 2.5 minutes at 72 ° C). The reaction was carried out for 30 minutes, followed by 72 minutes for 3 minutes.
  • Antiserum was collected from rabbits using the amino acid sequence of ITII c 246- GASSRGWDDGDTRSEHSYSESG-267 (peptide 1) (SEQ ID NO: 5) and 302-LWEPTAPEKGKE-313 (peptide 2) (SEQ ID NO: 6) as antigens.
  • a peptide column was prepared using each peptide, and the antibody was purified from the antiserum. From 4 ral antisera, 1.38 mg / ral (5 ml) of the antibody derived from peptide 1 and 2.4 rag / ml (5 ml) of the antibody derived from peptide 2 were prepared.
  • BAC-T0-BAC Baculovirus Expression Systems (Gibco, BRL) was used as the protein expression system.
  • hgyrl gene design the following primers and perform PCR (2 minutes at 94 ° C, 30 cycles at 94 ° C for 30 seconds and 68 ° C for 30 minutes, and a further 3 minutes reaction at 68 ° C) Amplified by Type ⁇ used 10 ng of pRCl.
  • pRC2 Insert the gene of interest into the Rsr II site of the pFASTBAC Htb vector and A fur vector (pRC2) was constructed. 8. 5 ng of pRC2 was introduced into 100 1 DH10BAC competent cells and cultured at 37 ° C for 24 hours. Remove colonies with 2 ml of LB medium
  • SF9 cells were prepared in 1 ⁇ 10 6 cells (6-well plate) in 2 ml of SF-900 II SFM containing 50 units / ml of penicillin and 50 1 streptomycin. After culturing at 27 ° C. for 1 hour, 0, 5, 101 bacmid DNA was dissolved in 100 1 of SF-900 II SF without antibiotics.
  • a peptide antibody was prepared by binding 12 mg of the antibody derived from peptide 2 to CNBr-activated Sepharose 4B (1 ml of swelling). After collecting High Five cells (1 ⁇ 10 8 cells) expressing the target protein, 2 ml of RX buffer (100 raM KC1, 3 mM NaCl, 3.5 mM MgCl 2 , 1.25 mM EGTA, 100 The suspension was suspended in raM HEPES (pH 7.3, 1 mM PMSF) and sonicated.
  • Decatenation activity was measured using kinetoplast DNA (0.175 ⁇ g / assay) as a substrate, and Relaxation activity was measured using pUC19 (0.3 ⁇ g / assay) as a substrate.
  • ⁇ was used in lU / assay (30 ⁇ 1; 50 mM Tris-HCl (pH 7.5), 120 mM KC1, 10 mM MgCl 2 , 0.5 mM ATP, 0.5 mM DTT, 30 ⁇ g / ml nuclease free BSA) The reaction was performed at 37 ° C for 60 minutes.
  • the measurement by the plasmon resonance method was performed using IAsys plus.
  • a CM dextran cuvette was used as a cuvette for solid phase immobilization of ⁇ .
  • Immobilization methods and measuring methods are carried out according to pro tocol IAsys plus accompanying, 267 arc seconds
  • the corresponding immobilized (200 arc seconds 1 ng / mm2) 0 TOPII c of 2 ⁇ 9 to 22 ⁇ 2 nM
  • This data was analyzed within FASTfit, and the association rate constant (kass), dissociation rate constant (kdiss) and dissociation parallel constant were calculated.
  • Apoptosis can be detected by TUNEL method (In situ Apoptosis Detection Kit; Takara Shuzo Co., Ltd.), indirect fluorescent antibody method using an antibody that recognizes PARP cleaved by apoptosis, detection of DNA ladder, Laser Scanning Cytometry (LSC Olympus Optical Co., Ltd.).
  • S-oligo antisense oligonucleotides were designed for residues 1 to 15 of the ⁇ gene.
  • the synthesis was outsourced to BI0GN0STIK.
  • Antisense was introduced into cells using Lipofectin (Gibco, BRL).
  • Antisense was added to 1 ml of serum-free medium to a volume of ⁇ .
  • 25 ⁇ l of Lipofectin was added to another 1 ml of serum-free medium. Both were mixed and allowed to stand at room temperature for 15 minutes. During this time, 1X10 6
  • the cells cultured in the cells 60 culture dishes) were washed twice with PBS (-), and the mixed solution was added dropwise 15 minutes later. The cells were transferred to a 37 ° C 5% CO 2 incubator and cultured for 4 hours. Thereafter, an equal amount of a medium containing serum was added and cultured, and the state of the cells was checked after 24 48 72 hours.
  • NB1-RBG Normal human dermal fibroblasts
  • low serum medium (0.4% FBS, D-MEM containing non-essential amino acids) for 72 hours, and then serum is added to 15% FBS. did.
  • LSC laser scanning cytometer
  • G0 / G1, S, and G2 / M phase contents were determined. 0/0 shown in (FIG. 6 (A)).
  • the expression of ⁇ and ⁇ at each time was detected with anti- ⁇ and anti- ⁇ antibodies, and the protein mass was quantified using NIH Image. (Fig. 6 (B)).
  • ⁇ was localized in the cytoplasm during the G1 / S phase, and cells localized in the nucleus from the G2 / M phase were observed. Therefore, in order to confirm whether ⁇ is present in the nucleus after the G2 / M phase, a nuclear fraction was prepared from the cells 32 hours after the addition of serum, and anti- ⁇ was expressed in the nucleus. The examination was performed using antibodies. As a result, the presence of ⁇ was confirmed in the prepared nuclear fraction (FIG. 6 (D)), suggesting that ITIIct translocated into the nucleus depending on the cell cycle. At this time, the band disappeared by adding an excess amount of the peptide used as the antigen of the anti- ⁇ antibody, and it was revealed that the band detected in the nuclear fraction was equivalent to ⁇ .
  • Antisense oligonucleotide sequencing is generally 15-20 mer, with 20 mer near the ATG codon for translation initiation, 15-20 mer from ATG, or 20 mer containing the boundary between the first exon and the first intron. There are many. However, this time, since the sequence of genomic DNA was not known, we selected the nucleotide sequence of 15-20 mer from ATG. In determining the number of bases, there is a report that a sequence with a GC content of 50% or more in the sequence and a sequence of three or more Gs has a cell growth inhibitory effect other than the antisense effect. Avoided from.
  • the following 15 mer was selected in consideration of the sequence in which the antisense itself does not form a hairpin. 5'-TAGCAGGTCCGACAT-3 '(SEQ ID NO: 9).
  • the prepared antisense was introduced into cells using Lipofectin. Incorporation of the oligonucleotide into cells by Lipofectin was confirmed using a sense oligonucleotide of FITC labeling (antisense reverse complement).
  • ⁇ antisense oligonucleotides were introduced into normal human skin fibroblasts using Lipofectin. 48 hours after transfection, the amount of intracellular ⁇ protein was detected by immunoblot.As a result, ⁇ ⁇ ⁇ ⁇ of antisense-treated cells was reduced by 66% or more compared to the amount of control oligonucleotide-transfected cells. (Fig. 9 ( ⁇ )). In addition, the amount of ⁇ protein decreased in a concentration-dependent manner of the antisense oligonucleotide (FIG. 9 ( ⁇ )).
  • ⁇ was expressed at lower levels in cancer cells than in normal cells, whereas ⁇ ⁇ was expressed at higher levels in cancer cells than in normal cells. Furthermore, it was confirmed that suppressing the expression of ⁇ in normal cells with antisense increases the expression of T0PIIa.
  • HeLa cells and normal human dermal fibroblasts were transfected with antisense oligonucleotides and introduced. The cells 48 hours after the injection were analyzed by LSC, and as a result, GI / Sarrest occurred in the normal cells, but the number of cells did not decrease. In contrast, the number of HeLa cells decreased, and one-third of the cells measured underwent apoptosis (Fig. 11 (A)).
  • ⁇ A synthetic peptide was prepared every 20 residues (10 residues overlap) from the N-terminal side of ⁇ , and the inhibitory effect on ⁇ activity (Relaxation Decatenation activity) was measured.
  • the ⁇ activity was measured in the same manner as in the method described in (viii) Measurement of ⁇ activity in Example 1.
  • the amino acid sequences 132 to 151 (VTATFPYTTILSIWLATRRV) (SEQ ID NO: 10) showed the strongest inhibitory action. The results are shown in FIG. In addition, other than this sequence, a synthetic peptide that inhibits TOPIIc activity was observed.
  • Industrial applicability The present invention, the physiological function of Iotataupai alpha, a protein that binds to TOPIIc is elucidation. According to the present invention, it is possible to provide a novel drug based on the physiological function of ⁇ , and to provide a method for screening a novel drug by utilizing the interaction between ⁇ and ⁇ . It became possible to do.

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Abstract

L'invention concerne des nouveaux médicaments fondés sur la fonction physiologique de ITIIα, plus précisément, des inhibiteurs de la topoïsomérase humaine 2α renfermant un facteur de liaison à la topoïsomérase humaine 2α comprenant une séquence quelconque parmi les séquences d'acides aminés suivantes : (a) la séquence d'acides aminés représentée par la SEQ ID NO : 1 ; et (b) la séquence d'acides aminés dérivée de la séquence d'acides animés représentée par la SEQ ID NO : 1 par effacement, substitution et/ou introduction d'au moins un acide aminé et étant capable d'interagir avec la topoïsomérase humaine 2α, aux fins d'inhibition de l'activité de celle-ci.
PCT/JP2002/006415 2001-06-27 2002-06-26 Nouvelle proteine inhibitrice de la topoisomerase humaine 2$g(a) et utilisation associee WO2003002737A1 (fr)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998039446A2 (fr) * 1997-03-07 1998-09-11 Human Genome Sciences, Inc. 70 proteines humaines secretees
WO1999016870A1 (fr) * 1997-09-29 1999-04-08 Zymogenetics, Inc. Proteine zsig-11 secretee
WO1999055721A1 (fr) * 1998-04-24 1999-11-04 Alphagene, Inc. Proteines secretees et polynucleotides les codant
WO1999063088A2 (fr) * 1998-06-02 1999-12-09 Genentech, Inc. Proteines membranaires et acides nucleiques codant ces proteines
WO2000037491A2 (fr) * 1998-12-22 2000-06-29 Genset Adn complementaires codant pour des proteines secretees avec des peptides signaux
WO2000073454A1 (fr) * 1999-06-02 2000-12-07 Genentech, Inc. Polypeptides transmembranaires secretes et acides nucleiques codants pour ceux-ci
WO2001000806A2 (fr) * 1999-06-25 2001-01-04 Genset Proteines codant des adn complementaires avec des peptides-signal
WO2001009327A2 (fr) * 1999-07-28 2001-02-08 Genentech, Inc. Procede de prevention de la deterioration ou de la mort des cellules de la retine et de traitement des troubles oculaires
WO2001040466A2 (fr) * 1999-12-01 2001-06-07 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques codant ces polypeptides

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998039446A2 (fr) * 1997-03-07 1998-09-11 Human Genome Sciences, Inc. 70 proteines humaines secretees
WO1999016870A1 (fr) * 1997-09-29 1999-04-08 Zymogenetics, Inc. Proteine zsig-11 secretee
WO1999055721A1 (fr) * 1998-04-24 1999-11-04 Alphagene, Inc. Proteines secretees et polynucleotides les codant
WO1999063088A2 (fr) * 1998-06-02 1999-12-09 Genentech, Inc. Proteines membranaires et acides nucleiques codant ces proteines
WO2000037491A2 (fr) * 1998-12-22 2000-06-29 Genset Adn complementaires codant pour des proteines secretees avec des peptides signaux
WO2000073454A1 (fr) * 1999-06-02 2000-12-07 Genentech, Inc. Polypeptides transmembranaires secretes et acides nucleiques codants pour ceux-ci
WO2001000806A2 (fr) * 1999-06-25 2001-01-04 Genset Proteines codant des adn complementaires avec des peptides-signal
WO2001009327A2 (fr) * 1999-07-28 2001-02-08 Genentech, Inc. Procede de prevention de la deterioration ou de la mort des cellules de la retine et de traitement des troubles oculaires
WO2001040466A2 (fr) * 1999-12-01 2001-06-07 Genentech, Inc. Polypeptides secretes et transmembranaires et acides nucleiques codant ces polypeptides

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BAQUERO M.R. ET AL.: "sbmC, a stationary-phase induced SOS Escherichia coli gene, whose product protects cells from the DNA replication inhibitor microcin B17", MOL. MICROBIOL., vol. 18, no. 2, 1995, pages 301 - 311, XP002169017 *
NAKANISHI A. ET AL.: "Characterization of the interaction between DNA gyrase inhibitor and DNA gyrase of Escherichia coli", J. BIOL. CHEM., vol. 277, no. 11, 15 March 2002 (2002-03-15), pages 8949 - 8964, XP002958940 *
NAKANISHI A. ET AL.: "Identification of DNA gyrase inhibitor (GyrI) in Escherichia coli", J. BIOL. CHEM., vol. 273, no. 4, 1998, pages 1933 - 1938, XP002958938 *

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