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WO2001036971A1 - Methode de recherche de substance d'inhibition de liaison entre des sous-unites de telomerase - Google Patents

Methode de recherche de substance d'inhibition de liaison entre des sous-unites de telomerase Download PDF

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Publication number
WO2001036971A1
WO2001036971A1 PCT/JP2000/008001 JP0008001W WO0136971A1 WO 2001036971 A1 WO2001036971 A1 WO 2001036971A1 JP 0008001 W JP0008001 W JP 0008001W WO 0136971 A1 WO0136971 A1 WO 0136971A1
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htert
human telomerase
htr
subunit
binding
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PCT/JP2000/008001
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English (en)
Japanese (ja)
Inventor
Yoshinori Yamashita
Akira Asai
Osamu Mikuni
Hideharu Anazawa
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Kyowa Hakko Kogyo Co., Ltd.
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Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Priority to AU13091/01A priority Critical patent/AU1309101A/en
Publication of WO2001036971A1 publication Critical patent/WO2001036971A1/fr

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    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • the present invention relates to a human telomerase catalytic subunit (hTERT) and a human telomerase RNA subunit (hTR), which are considered to be useful as antitumor agents for inhibiting the production of active telomerase in cells. And a method for searching for a binding inhibitor.
  • hTERT human telomerase catalytic subunit
  • hTR human telomerase RNA subunit
  • Telomerase an enzyme that extends telomeres, is expressed in cancer cells [Journal of the NCI, L, 884-894 (1995)]. This enzyme is a reverse transcriptase that extends telomeres using RNA as type II.
  • telomerase inhibits telomere shortening in cancer cells and maintains telomeres stably, enabling infinite growth as immortalized cells
  • telomere shortening a substance that specifically inhibits telomerase can be a new type of anticancer drug that induces telomere shortening and thereby extends the life of cancer cells.
  • Human telomerase is composed of the human telomerase catalytic subunit (hTERT) and the human oral melanase RNA subunit (hTR).
  • hTERT nucleotide sequence, amino acid sequence and production method [Science, 222, 955-959 (199 7); Cell, 785-795 (1997); WO 98/14593], and hTR nucleotide sequence and production method [Science, 1236] -1240 (1995)] are each known.
  • telomere inhibitors Since hTERT and hTR are each synthesized in the cell and then bind to each other to form active telomerase (see Fig. 1), both binding inhibitors become telomerase inhibitors and suppress tumor cell growth. There is a possibility that it can be obtained as an agent.
  • WO 98/14593 includes a method for detecting a change in the activity or expression of hTERT after administration of a test compound or treatment in cells containing hTERT or a polynucleotide thereof.
  • a method for determining whether or not the test compound or modulator of the therapeutic activity ⁇ TRT (synonymous with hTERT) is a modulator of the activity or expression is described, but no specific example is described.
  • FIG. 2 of WO 98 1207 shows that ter-bound hTR in which the polynucleotide ter is bound to hTR has an affinity for ter bound to the solid phase via a conjugate of any protein and its antibody.
  • A a step of contacting the test compound, the above-mentioned r-bound hTR and hTERT,
  • b a step of measuring the binding rate between ter-bound hTR and hTERT
  • the step of determining the difference between the binding rate obtained in the above step and the binding rate between the control and the test compound modulates the binding between ter-bound hTR and hTERT due to the difference in the binding rates between the two.
  • the concept of how to determine whether or not to do so is disclosed, but no specific example is disclosed.
  • an object of the present invention is to provide a method for rapidly and easily searching for a telomerase-subunit binding inhibitor useful as an antitumor agent by inhibiting telomerase in cells.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, by using a solid phase method, it was possible to quickly and easily search for a telomerase-subunit binding inhibitor, and to obtain a large amount at once. And found that it was possible to process the sample of the present invention, thereby completing the present invention.
  • the present invention is characterized in that the Tagi-Danilo human telomerase catalytic subunit is bound to a solid phase via an anti-Tag antibody, and the labeling strength of the labeled human telomerase RNA subunit bound to the parentheses is measured.
  • the present invention also relates to a method for searching for a substance inhibiting the binding of a human telomerase catalytic subunit to a human telomerase RNA subunit, comprising the following steps:
  • a “test compound” a “tagged human telomerase catalytic subunit” (hereinafter, referred to as “tagged hTERT”) and a “labeled human telomerase RNA subunit” (Hereinafter referred to as “labeled hTR”).
  • tagged hTERT a “tagged human telomerase catalytic subunit”
  • labeled hTR a “labeled human telomerase RNA subunit”
  • the reaction solution containing the formed conjugate is reacted with a solid phase to which an anti-Tag antibody is bound, whereby the tagged hTERT in the conjugate is reacted with the anti-Tag on the solid phase.
  • a conjugate of the tagged hTERT and the labeled hTR is formed on the solid phase.
  • the reaction can be carried out by reacting a tagged hTRT with a tagged human telomerase catalytic subunit (hereinafter, referred to as “immobilized hTERT”) bound to a tag antibody on a solid phase in the presence of a test compound.
  • immobilized hTERT a tagged human telomerase catalytic subunit
  • a buffer containing the test compound and the labeled hTR is added to the immobilized hTERT and reacted. As a result of this reaction, the binding of tagged hTERT and Coalescence is formed.
  • test compound is a synthetic or natural chemical substance used for searching for a telomerase inhibitor, and includes a high-molecular or low-molecular compound.
  • Tag hTERT refers to a combination of hTERT and a low-molecular or high-molecular Tag.
  • HTERT used for tagging hTERT may be any product such as a purified product or an unpurified product as long as it has the function of hTERT.
  • a product comprising an amino acid sequence described in WO 98/14593
  • the amino acid sequence preferably has an amino acid in which one or several amino acids have been deleted, substituted or added, and has an hTERT function.
  • the tag may be a low molecular weight or a high molecular weight.
  • the low molecular weight tag include histidine tag, FlagTag. Hemagglutin tag, c-mycTag, and the like. Glutathione S transferase is used as the high molecular weight tag. , Maltose binding protein, protein A, thioredoxin, immunoglobulin G, / 3-lactamase, etc. [His-tag; Eur. J. Biochem., 214.
  • Histidine Tag Histidine Tag, FlagTag, Hemagglutin Tag, c-mycTA
  • amino acid sequence of the peptide G is as follows: Protein Sci., 2.298-2205 (1993), Biotechniques, 11, 754-761 (1994), Gene, il, 285-288 (1992), Gene, iL139- 143 (1993). Also, high-molecular tags such as daltathione S-transferase, maltose-binding protein, protein A, thioredoxin, and immunoglobulin
  • solid phase hTERTJ refers to an anti-tag antibody bound on a solid phase and a tag of the tagged hTERT bound by an antigen-antibody reaction.
  • anti-Tag antibody bound on a solid phase refers to an antibody directly or indirectly bound to the tag portion of Tagged hTERT on the solid phase.
  • anti-Tag antibodies include 6X His Monoclonal Antibody (Clontech), Penta-His Antibody, Tetra-His Antibody (Qiagen) and the like as anti-His-tag antibodies, and 6X His Monoclonal Antibody. It is preferred to use.
  • anti-Flag antibody examples include Ml, M2, and M5 Antibody (manufactured by SIGMA), and the use of M2 Antibody is preferred.
  • solid phase examples include latex, activated agarose, a 96- or 384-well titer plate, and a 96-well titer plate is preferably used.
  • labeling hTR refers to a conjugate of hTR and a labeling substance.
  • the hTR may be any product such as a purified product, an unrefined product, etc., for example, a product comprising an amino acid sequence described in WO 98/1 1207 or the like, or 1 or 1 in the amino acid sequence. It is preferable that the amino acid has several amino acids deleted, substituted or added, and has an hTR function.
  • Labeling substances include radioisotopes such as 3 ⁇ , 13, 1 13, and lH, fluorescent substances such as fluorescein and rhodamine, and antigens such as fluorescein, rhodamine, digoxigenin, and biotin.
  • radioisotopes such as 3 ⁇ , 13, 1 13, and lH
  • fluorescent substances such as fluorescein and rhodamine
  • antigens such as fluorescein, rhodamine, digoxigenin, and biotin.
  • a label other than radioisotope a complex of an antibody against these labeling substances with alfa phosphatase, peroxidase, etc. Immunological detection methods are also possible which detect the signal by using the body and a fluorescent or luminescent substrate for them.
  • the binding between the labeling substance and hTR can be obtained by a conventional method [Nucleic Acids Res., 11, 5843-5851 (1990)].
  • the solid phase is washed after a conjugate of tagged hTERT and labeled hTR is formed on the solid phase by any of the above.
  • the labeling intensity refers to the intensity of the signal emitted by the labeling substance in the conjugate of the tagged hTERT and the labeled hTR bound to the anti-Tag antibody on the solid phase. If the labeling substance is radioisotope, the emission Activity, fluorescence refers to fluorescence intensity, luminescence refers to luminescence intensity, and enzyme refers to enzyme activity. Note that labeling includes the case where a compound having a labeled molecule is bound to hTR.
  • the labeling strength of the conjugate on the solid phase formed in the presence of the test compound as described above and the conjugate on the solid phase similarly formed in the absence of the test compound are determined.
  • the label strength is compared with the measured value.
  • test compound has ⁇ TERT-hTR binding inhibitory activity
  • the measured value of the label strength in the presence of the test compound is lower than the measured value of the label strength in the absence of the test compound. It can be a candidate for an inhibitor.
  • hTERT is a cell in which hTERT is expressed in a cell using the usual enzyme separation and purification method described later, such as Namalwa cell [J. Biol. Chem., 2 ⁇ 14730 (1994)] (ATCC No. CRL-173). ) Force that can be collected by fractionation from cells etc. Generally, DNA encoding hTERT is obtained from the cells and produced by genetic engineering techniques. Tagged hTERT used in the method of the present invention can be produced according to the method for producing hTERT described in WO 98/14593.
  • cDNA encoding Tag is ligated to cDNA encoding hTERT, the cDNA is inserted into an appropriate expression vector, and the expression
  • the vector is introduced into a host cell such as Escherichia coli, yeast, or an animal cell, and is produced by expressing the tagged hTERT in the host cell.
  • it can be produced by attaching a tag to hTERT extracted from blood cells or the like by a protein chemical modification method.
  • the cDNA library was prepared using the conventional method, Molecular Cloning Cloning 2nd edition, Cold Spring Harbor Lab.Press New York (1989) (hereinafter referred to as Molecular Cloning 2nd Edition). Abbreviations) and Current Protocols in Molecular Biology Supplement, 1-38 (hereinafter abbreviated as current protocols).
  • RNA is extracted from a cDNA described in Science, 2L 955 (1997) or a cell expressing hTERT in the cell, and a cDNA is synthesized from the RNA.
  • Methods for preparing total RNA from cells expressing hTERT in cells include the guanidine cesium chloride method and the guanidine thiosinate method [Methods in Enzymo 1., ii ⁇ 3 (1987)] and the like. No. Examples of a method for preparing mRNA from total RNA include a column method or a batch method using oligo dT cellulose or the like. Also, kits such as First Track, mRNA and Isolation 'Kit (Invitrogen), and Quick' Prep 'mRNA and PyuriFicheon Kit (Pharmacia) can be used. mRNA can also be prepared.
  • Methods for synthesizing cDNA from the obtained RNA include the Okamaberg method [Mol. Cell. Biol., Z, 161 (1982)] and the Gubra-Hoffman method [Gene, 21. 263 (1983)]. Are listed. Kits such as Superscript, Plasmid, System for cDNA-Synthesis and Plasmid Cloning (Gibco BRL), Zap-cDNA, Synthesis' Kit (Stratagene), etc. Can be used to synthesize cDNA.
  • a cDNA library is prepared by incorporating the obtained cDNA into a closing vector and introducing the cloning vector into a host cell.
  • the cloning vector When the cloning vector is a plasmid, it is introduced into a host cell by electroporation or calcium chloride. When the cloning vector is a phage, the host cell may be prepared by in vivo packaging. To be introduced.
  • Any phage vector, plasmid vector, or the like may be used as a cloning vector for incorporating the cDNA as long as it can be replicated autonomously in the host cell and can stably maintain the cDNA.
  • ZAP Express [Stratagene, Strat egies, i, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 12, 9494 (1 989)], ⁇ zapll (Stratagene) , ⁇ gtlO, ⁇ gtl1 [DNA Cloning, A Practical Approach, 49 (1985)], ⁇ TriplEx (CLONTECH Laboratolies), ⁇ EXCell (Pharmacia), pT7T3 18U (Pharmacia) , PcD2 [Mol.
  • any microorganism belonging to Escherichia coli can be used. Specifically, Escherichia coli XLI-Blue MRF '[Stratagene, Strategies, ⁇ 81 (1992)], Escherichia QQR C600 [Genetics, 23., 440 (1954)], Escherichia coli YI088 [Science, 222. 778 (1983)], Escherichia oli YIO90 [Science, _222, 778 (1983)], Escherichia QSiR NM522 [J. Mol. Biol., IM 1 (1983)], Escherichia coli K802 [J. Mol. Biol.
  • a transformant containing a cDNA encoding the desired hTERT is selected from one of the prepared libraries.
  • a probe is prepared based on the nucleotide sequence of DNA encoding hT ERT described in Science, T ⁇ 955 (1997), and the probe is labeled with a fluorescent substance, radioisotope, enzyme, etc., and plaque hybridizer By carrying out hybridization, colony hybridization, Southern hybridization, etc., a transformant which hybridizes is selected.
  • the cDNA encoding the Tag can be obtained by chemically synthesizing the same DNA as the Tag.
  • a primer is designed according to the sequence of both ends of the tag cDNA, and the full length of the tag cDNA is obtained by PCR.
  • Any vector can be used as long as it can incorporate the DNA and can be expressed in a host cell.
  • the host cell may be any cell, such as a bacterium, yeast, ff! Il cell, or insect cell, as long as it can express the gene of interest.
  • the bacterium include bacteria of the genus Escherichia such as Escherichia coli and the genus Bacillus such as Bacillus suhtilis.
  • yeast include Saccharomyces cerevisiae and Szocharmyces cerevisiae, and Schi znsaccharomyces es pomhe.
  • animal cells include human cells, such as namaluba cells, monkey cells, COS cells, and Chinese hamster cells, CHO cells.
  • insect cells include Sf9, Sf21 (manufactured by Pharmingen), High Five (manufactured by Invitrogen) and the like.
  • the expression vector may be a promoter, a ribosome binding sequence, a DNA of the present invention, a transcription termination sequence, or, in some cases, a promoter control sequence. It is preferable to use a configured one, and examples thereof include commercially available pGEX (manufactured by Pharmacia) and pET system (manufactured by Novagen).
  • a method for introducing a recombinant vector into bacteria a method for introducing DNA into bacteria, for example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, ⁇ , 21 10 (1972) )], And the protoplast method (JP-A-63-248394).
  • examples of expression vectors include YEpl3 (ATCC37115), YEp24 (ATCC37051), and YCp50 (ATCC37419).
  • Examples of a method for introducing a recombinant vector into yeast include a method for introducing DNA into yeast, such as the electroporation method [Methods. Enzymol., 182 (1990)], the spheroplast method [Pro Natl. Acad. Sci., USA, M, 1929 (1978)], acetic acid Any method such as the lithium method [J. Bacteriol., I5 163 (1983)] can be used.
  • expression vectors include pAGE107 (Japanese Patent Laid-Open No. 3-22979; Cytotechnology, 1, 133 (1990)) and pAGE103 [J. Biochem., 101.
  • any promoter may be used as long as it can be expressed in animal cells.
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • a method for introducing DNA into animal cells includes, for example, the electoral poration method [Cytotechnology, 1, 133 (1990)], Any of these methods can be used, such as the calcium phosphate method (Japanese Patent Application Laid-Open No. 2-227075) and the Lipofection method [Pro Natl. Acad. Sci., USA, M, 7413 (1987)].
  • insect cells When insect cells are used as hosts, for example, the current protocols (supplements 1-34), noculoviruses / exclusions, vectors and / or /
  • the protein can be expressed by the method described in Belle (Baculovirus expression vectors, A laboratory manual). That is, the recombinant gene transfer vector and the defective baculovirus genome described below are co-transfected into insect cells to obtain a recombinant virus in the insect cell culture supernatant, and then the recombinant virus is transferred to the insect cell. To obtain protein-expressing insect cells.
  • pVL1392 As the gene transfer vector, for example, pVL1392, pVL1393, pBlueBacIII (all manufactured by INVITROGEN) and the like are used.
  • AutograDha californica nuclear polyhedrosis virus which is a virus that infects insects of the night roth moth family, such as autographa, californica, nuclei, and the like, is used.
  • the recombinant virus is infected to insect cells such as Sf9, Sf21 or High Five described above, and the protein is produced. Can be produced [Bio / Technology, 47 (1988)].
  • the full-length or partial fragment of hTERT can be produced directly or as a fusion protein in the cells or in the culture supernatant.
  • a secretory production method and a method of expressing the gene as a fusion protein have been developed as a method for expressing the gene, and any method can be used. For example, it can be carried out according to the method described in Molecular Cloning Second Edition.
  • the method for culturing the transformant of the present invention in a medium is performed according to a usual method used for culturing a host.
  • a culture medium for culturing a transformant obtained by using a microorganism such as Escherichia coli or yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like, which can be used by the microorganism, and efficiently cultivates the transformant.
  • a natural medium or a synthetic medium can be used as long as the medium can be used (Molecular Cloning 2nd Edition).
  • Cultivation is usually performed at 15-40 ° C for 16-96 hours under aerobic conditions such as shaking culture or deep aeration stirring culture. During the culture period, the pH is maintained between 3.0 and 9.0.
  • the pH is adjusted by using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • an antibiotic such as ampicillin / tetracycline may be added to the medium as needed.
  • a medium for culturing the transformant obtained using animal cells as a host commonly used RPMI 1640 medium, Eagle's MEM medium, or a medium obtained by adding fetal calf serum or the like to such a medium is used. Cultures, 5% C0 2 presence usually performed 3-7 days at 35 to 37 ° C, the culture if necessary, kanamycin, may be added to the medium antibiotics such as penicillin.
  • TNM-FH medium As a medium for culturing transformants obtained using insect cells as a host, commonly used TNM-FH medium (Pharmingen), Sf900IISFM (Life Technologies) ExCell400, ExCell405 (all manufactured by JRH Biosciences) and the like.
  • the culture is performed at 25 to 30 ° C for 1 to 4 days.
  • an antibiotic such as gentamicin may be added to the medium as needed.
  • the cells are centrifuged, suspended in an aqueous buffer, and subjected to ultrasonic method, French press The cells are disrupted by a method or the like, and the protein is recovered in the supernatant obtained by centrifugation.
  • the insoluble substance when an insoluble substance is formed in the cells, the insoluble substance is solubilized with a protein denaturant, and then the protein denaturant is not contained or the concentration of the protein denaturant is so low that the protein is not denatured. Dilution or dialysis can be used to form the protein conformation.
  • the expressed protein can be recovered in the culture supernatant.
  • the tagged hTERT from the hTERT-expressing host can be extracted by, for example, suspending the expressing cells in a commonly used extraction buffer and using a non-ionic surfactant such as NP-40. After appropriately adding the agent, the reaction can be carried out according to ordinary biochemical methods such as ultrasonication, freeze-thawing, and homogenization.
  • Tagged hTERT For the isolation and purification of Tagged hTERT, solvent extraction, fractional precipitation with organic solvents, salting out, dialysis, centrifugation, ultrafiltration, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity Separation operations such as oral chromatography, reverse phase chromatography, crystallization, and electrophoresis can be performed alone or in combination.
  • HTR used in the present invention according to the method described in WO 98/1 1207, a probe for screening a transformant containing DNA encoding hTR,
  • hTERT and hTR may be prepared by, for example, placing the hTERT or hTR gene downstream of a promoter specific to a certain RNA polymerase and synthesizing in vitro. It may be obtained from a cell extract such as an erythrocyte extract using a TnT coupled Reticulocyte Lysate System of Promega, or may be chemically synthesized in a test tube.
  • Tagged hTERT and hTR can be performed simultaneously or separately.
  • the tagged hTERT gene and the hTR gene can be expressed in the same cell, or hTR prepared in a test tube may be used for the tagged hTERT expressed in a certain cell.
  • a buffer such as Tris' hydrochloric acid, phosphoric acid, or acetic acid.
  • test compound preferably in Tris-HCl buffer, with test compound and either tagged hTERT or labeled hTR at 15-40, preferably 30-37 ° C for 5-180 min Incubate for preferably 30-60 minutes.
  • labeled hTR if tagged hT ERT is present in the mixture
  • add tagged hTERT if labeled hTR is present in the mixture
  • 10-180 minutes preferably 60- Incubate for 120 minutes.
  • the mixed solution after the above reaction is dropped, and the Tag antibody on the solid phase and Tag To the tag of hTERT.
  • the solid phase is left for 1 to 48 hours, preferably 2 to 4 hours.
  • the test compound containing the test compound is added to the immobilized hTERT in which the anti-Tag antibody bound to the solid phase and the tagged hTERT are previously bound by an antigen-antibody reaction using a conventional method.
  • Buffers such as hydrochloric acid, phosphoric acid and acetic acid (pH 5-9, preferably 6-8
  • reducing agents such as salts of NaCl, CL MgCl ⁇ , dithiothreitol, mercaptoethanol, stabilizing agents such as glycerol and sucrose, chelating agents such as EDTA and EGTA, chaperones such as HSP90, HSP70 and p23
  • stabilizing agents such as glycerol and sucrose
  • chelating agents such as EDTA and EGTA
  • chaperones such as HSP90, HSP70 and p23
  • the strength of the label on the solid phase after washing is measured using a liquid scintillation counter or the like when the label is a radioisotope, and using a fluorescence intensity meter when the label is a fluorescent substance.
  • the labeling substance is an antigen
  • a complex of an antibody against the labeling substance and the enzyme is bound to the labeling substance, and a fluorescent or luminescent substrate for the enzyme is added to cause a re-enzyme reaction.
  • a spectrophotometer, a luminometer or the like are examples of the label on the solid phase after washing.
  • FIG. 1 is a diagram showing a mode of hTR-hTERT binding inhibition.
  • FIG. 2 is a diagram showing the activity of reconstituted telomerase enzyme by labeled hTR.
  • the vertical axis shows the enzyme activity determined by Fluorlmager, and the horizontal axis shows the value added during the binding reaction.
  • FIG. 3 shows the results of an experiment of immobilizing reconstituted telomerase on a plate using an anti-Flag antibody.
  • the vertical axis of the graph indicates the enzyme activity (relative value).
  • Lanes 1 to 5 on the horizontal axis show the results when the anti-mouse immunoglobulin antibody was immobilized on the plate (control), and 6 to 10 show the results when the anti-Flag antibody was immobilized.
  • Lane 1 and 6 is telomerase-free control
  • lanes 2 and 7 are 2500-fold after reconstitution
  • lanes 3 and 8 are 500-fold
  • lanes 4 and 9 are 100-fold
  • lanes 5 and 10 are 20
  • the results when the dilution is 1: 2 are shown.
  • FIG. 4 is a diagram showing an operation procedure of a method for searching for an hTR-hTERT binding inhibitor.
  • FIG. 5 is a diagram showing the binding between hTR and hTERT by ELISA. The vertical axis shows the fluorescence intensity by Flu, and the horizontal axis shows the concentration of Flu-hTR during the binding reaction. The black bars indicate the fluorescence in the absence of hTERT, and the hatched bars indicate the fluorescence in the presence of hTERT.
  • FIG. 6 is a diagram showing a comparison between the ELISA method and the enzyme activity detection method. A portion of one binding reaction was used for each assay. A shows the results of the assay by the ELISA method, and B shows the results of the assay by the enzyme activity detection method. The concentration of Flu-hTR during each binding reaction was 0.82 to 200 ng / L. BEST MODE FOR CARRYING OUT THE INVENTION
  • the hTR gene was isolated according to the report of Science, 1236-1240 (1995), and the expression plasmid was constructed by inserting this gene into the EamH Hindlll site of pUC119 by a conventional method.
  • plasmid DNA described above in the form of linearized plasmid DNA with the restriction enzyme E3 ⁇ 4il (Takara Shuzo) as a template
  • a commercially available kit SP6 / T7 Transcription Kit (Boehringer Mannheim) is used.
  • HTR was prepared by in vitro transcription using T7 polymerase.
  • dXTP mixture containing labeled dUTP Fluorescein RNA Labeling Mix (Boehringer Mannheim) and Digoxigenin RNA
  • Example 2 Construction of hTERT-expressing baculovirus vector, expression of hTERT and preparation of nuclear extract
  • the DNA corresponding to the amino acid sequence of Flag-tag was ligated to the C-terminal part of the hTERT gene described in Science, 2ZL 955-959, and this fragment was inserted into the EcoRI site of pVL1392 (Pharmingen), and the transfer was performed.
  • BaculoGold manufactured by Pharmingen
  • Sf21 cells were infected with the virus vector described in the previous section at 3 plaque forming units per cell. After culturing at 27 ° C for 3 days, collect the cells, and add 1 X CHAPS buffer (10 mM Tris / HCl (pH 7.5), ImM MgCl 2 ImM EGTA,
  • TBS solution (10 g / mL) of anti-FLAG M2 antibody (Stratagene) was dispensed into a 96-well micro-tie Yuichi plate (Sumilon black plate) and dispensed at 4: for 12 hours.
  • 100 L of a 3% BSA solution was added and left at room temperature for 2 hours.
  • the same binding reaction as in Example 3 was performed using unlabeled hTR (final concentration: 20 ng / L) and Flag-hTERT. Left at 4 for 4 hours.
  • TBS solution (10 g / mL) of anti-FLAG M2 antibody (Stratagene) into a 96-well microtiter plate (Black plate manufactured by Sumilon) Dispense 100 L into each well, 12:00 at 4 ° C It was left still. After each well was washed three times with 100 L of TBS, 3 L of BSA solution L was added and allowed to stand at room temperature for 2 hours. After removing the BSA solution, 100 L of a solution obtained by diluting the binding reaction solution of labeled hTR and Flag-hTERT obtained in Example 3 with TBS was added, and the mixture was allowed to stand at 4 ° C for 4 hours.
  • telomerase catalytic subunit hTERT
  • hTR human telomerase RNA subunit

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Abstract

L'invention concerne une méthode de recherche d'une substance inhibant la liaison d'une sous-unité catalytique de télomérase humaine à une sous-unité d'ARN de télomérase humaine, ladite méthode étant caractérisée en ce qu'elle consiste à lier une sous-unité catalytique de télomérase humaine marquée à une phase solide par le biais d'un anticorps anti-marqueur et à mesurer l'intensité du marqueur d'une liaison d'une sous-unité d'ARN de télomérase humaine marquée. Cette méthode rend possible la sélection rapide et adéquate d'une substance inhibant la liaison d'une sous-unité catalytique de télomérase humaine (hTERT) à une sous-unité d'ARN de télomérase humaine (hTR), qui est en apparence utile en tant qu'agent antinéoplasique destiné à inhiber la formation de télomérase active dans des cellules, à partir de bibliothèques chimiques de produits chimiques synthétiques, de produits chimiques naturels etc., de milieux de culture microbienne, d'extraits végétaux et similaires.
PCT/JP2000/008001 1999-11-16 2000-11-13 Methode de recherche de substance d'inhibition de liaison entre des sous-unites de telomerase WO2001036971A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU13091/01A AU1309101A (en) 1999-11-16 2000-11-13 Method of searching for substance inhibiting binding between telomerase subunits

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP32552599 1999-11-16
JP11/325525 1999-11-16

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WO2001036971A1 true WO2001036971A1 (fr) 2001-05-25

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PCT/JP2000/008001 WO2001036971A1 (fr) 1999-11-16 2000-11-13 Methode de recherche de substance d'inhibition de liaison entre des sous-unites de telomerase

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AU (1) AU1309101A (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008537109A (ja) * 2005-04-01 2008-09-11 メドベット サイエンス ピーティーワイ． リミティッド 診断法および治療法ならびにそれに有用な薬剤

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10505488A (ja) * 1994-07-07 1998-06-02 ジェロン コーポレイション 哺乳動物のテロメラーゼ
JPH10234384A (ja) * 1996-10-01 1998-09-08 Geron Corp ヒトテロメラーゼ触媒性サブユニット

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10505488A (ja) * 1994-07-07 1998-06-02 ジェロン コーポレイション 哺乳動物のテロメラーゼ
JPH10234384A (ja) * 1996-10-01 1998-09-08 Geron Corp ヒトテロメラーゼ触媒性サブユニット

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008537109A (ja) * 2005-04-01 2008-09-11 メドベット サイエンス ピーティーワイ． リミティッド 診断法および治療法ならびにそれに有用な薬剤

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