JP2016065869A - Tacc3 inhibitor screening method - Google Patents
Tacc3 inhibitor screening method Download PDFInfo
- Publication number
- JP2016065869A JP2016065869A JP2015186097A JP2015186097A JP2016065869A JP 2016065869 A JP2016065869 A JP 2016065869A JP 2015186097 A JP2015186097 A JP 2015186097A JP 2015186097 A JP2015186097 A JP 2015186097A JP 2016065869 A JP2016065869 A JP 2016065869A
- Authority
- JP
- Japan
- Prior art keywords
- tacc3
- togp
- amino acid
- compound
- fragment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000012216 screening Methods 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 38
- 229940086614 TACC3 inhibitor Drugs 0.000 title 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 54
- 150000001875 compounds Chemical class 0.000 claims abstract description 53
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 16
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 16
- 230000000694 effects Effects 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 101000836150 Homo sapiens Transforming acidic coiled-coil-containing protein 3 Proteins 0.000 claims abstract 9
- 102100027048 Transforming acidic coiled-coil-containing protein 3 Human genes 0.000 claims abstract 9
- 102100028624 Cytoskeleton-associated protein 5 Human genes 0.000 claims abstract 6
- 101000766864 Homo sapiens Cytoskeleton-associated protein 5 Proteins 0.000 claims abstract 6
- 239000012634 fragment Substances 0.000 description 41
- 210000004027 cell Anatomy 0.000 description 19
- 206010028980 Neoplasm Diseases 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 13
- 230000032823 cell division Effects 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 210000004881 tumor cell Anatomy 0.000 description 7
- 102000004243 Tubulin Human genes 0.000 description 6
- 108090000704 Tubulin Proteins 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 6
- 239000000546 pharmaceutical excipient Substances 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 5
- 239000002246 antineoplastic agent Substances 0.000 description 5
- 239000008194 pharmaceutical composition Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 238000001114 immunoprecipitation Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 229920005862 polyol Polymers 0.000 description 4
- 150000003077 polyols Chemical class 0.000 description 4
- 239000012453 solvate Chemical class 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 206010009944 Colon cancer Diseases 0.000 description 3
- 108010024636 Glutathione Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102000029749 Microtubule Human genes 0.000 description 3
- 108091022875 Microtubule Proteins 0.000 description 3
- 108700020796 Oncogene Proteins 0.000 description 3
- 206010033128 Ovarian cancer Diseases 0.000 description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 210000004748 cultured cell Anatomy 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 239000007903 gelatin capsule Substances 0.000 description 3
- 229960003180 glutathione Drugs 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 210000004688 microtubule Anatomy 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 2
- 208000032612 Glial tumor Diseases 0.000 description 2
- 206010018338 Glioma Diseases 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- 206010025323 Lymphomas Diseases 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 208000002495 Uterine Neoplasms Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000000556 agonist Substances 0.000 description 2
- -1 amine salts Chemical class 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 210000003793 centrosome Anatomy 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 230000009918 complex formation Effects 0.000 description 2
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 239000008298 dragée Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 201000004101 esophageal cancer Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 210000000479 mitotic spindle apparatus Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000002331 protein detection Methods 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 206010046766 uterine cancer Diseases 0.000 description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 description 2
- 239000008158 vegetable oil Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000001993 wax Substances 0.000 description 2
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 125000004172 4-methoxyphenyl group Chemical group [H]C1=C([H])C(OC([H])([H])[H])=C([H])C([H])=C1* 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 206010023825 Laryngeal cancer Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 102000041330 TACC family Human genes 0.000 description 1
- 108091075724 TACC family Proteins 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 206010062129 Tongue neoplasm Diseases 0.000 description 1
- 206010044002 Tonsil cancer Diseases 0.000 description 1
- 208000006842 Tonsillar Neoplasms Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 208000036815 beta tubulin Diseases 0.000 description 1
- 201000009036 biliary tract cancer Diseases 0.000 description 1
- 208000020790 biliary tract neoplasm Diseases 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 210000004900 c-terminal fragment Anatomy 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000008011 inorganic excipient Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229960004903 invert sugar Drugs 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 206010023841 laryngeal neoplasm Diseases 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 208000026037 malignant tumor of neck Diseases 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 102000021160 microtubule binding proteins Human genes 0.000 description 1
- 108091011150 microtubule binding proteins Proteins 0.000 description 1
- 230000029115 microtubule polymerization Effects 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000008012 organic excipient Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 238000010379 pull-down assay Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000020347 spindle assembly Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 201000002341 thymus lymphoma Diseases 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 201000006134 tongue cancer Diseases 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 125000005270 trialkylamine group Chemical group 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Abstract
Description
本発明は、TACC3に結合し、その活性を阻害する化合物のスクリーニング方法に関する。特に、TACC3活性を阻害する小化合物をスクリーニングする方法に関する。 The present invention relates to a method for screening a compound that binds to TACC3 and inhibits its activity. In particular, the present invention relates to a method for screening small compounds that inhibit TACC3 activity.
TACC3(Transforming acidic coiled-coil 3)は、多発性骨髄腫の染色体の切断点から単離された遺伝子であるが、その後、多くの癌で発現異常が示されている癌関連遺伝子である(例えば、非特許文献1〜5、特許文献1)。 TACC3 (Transforming acidic coiled-coil 3) is a gene isolated from the chromosomal breakpoint of multiple myeloma, but is a cancer-related gene that has been shown to be abnormally expressed in many cancers (for example, Non-patent documents 1 to 5, Patent document 1).
TACC3は細胞***期にのみ発現し、中心体及び紡錘体に局在することが知られている。また、XenopusやDrosophilaで得られた知見をもとに、TACC3がTOGpと結合し、有糸***の際に中心体で微小管の重合を安定化し、細胞***を制御するというモデルが提示されている(非特許文献6)。 It is known that TACC3 is expressed only in the cell division phase and is localized in the centrosome and spindle. In addition, based on the knowledge obtained in Xenopus and Drosophila, a model has been proposed in which TACC3 binds to TOGp, stabilizes microtubule polymerization in the centrosome during mitosis, and controls cell division. (Non-patent document 6).
腫瘍細胞は細胞***が盛んであることから、一般的に、細胞***を阻害する薬剤は抗癌剤として有効であると考えられている。実際に従来から***装置の主要構造である微小管や、構成タンパク質であるチューブリンを標的とするチューブリン作用薬は抗癌剤として用いられている。 Since tumor cells are actively divided, it is generally considered that drugs that inhibit cell division are effective as anticancer agents. Actually, tubulin agonists that target tubulin, which is the main structure of the mitotic apparatus, and tubulin, which is a constituent protein, have been used as anticancer agents.
しかしながら、ビンクリスチンやパクリタキセルのような従来から用いられているチューブリン作用薬は***装置の微小管だけではなく、正常細胞の微小管を同時に標的と
することから、末梢性ニューロパシーのような重篤な副作用を生じることが知られている(例えば、特許文献2)。
However, traditionally used tubulin agonists such as vincristine and paclitaxel target not only the microtubules of the mitotic apparatus but also the microtubules of normal cells at the same time, which can cause serious problems such as peripheral neuropathy. It is known to cause side effects (for example, Patent Document 2).
そこで、腫瘍細胞の***装置に特異的に作用する治療薬の開発が望まれている。TACC3は、上述のように多くの癌で発現に異常が見られることから、発癌との関連が指摘されてきた。実際に、本発明者はTACC3のコンディショナルノックアウトマウスを用い、TACC3発現の枯渇が胸腺リンパ腫をアポトーシスにより退縮させるものの、正常細胞においてはTACC3が高発現であっても、何ら異常を示さないことを明らかにした(非特許文献7)。 Therefore, development of therapeutic agents that specifically act on tumor cell division apparatus is desired. Since TACC3 is abnormally expressed in many cancers as described above, its association with carcinogenesis has been pointed out. In fact, the present inventor used a TACC3 conditional knockout mouse, and although depletion of TACC3 expression caused thymic lymphoma to regress by apoptosis, normal cells did not show any abnormality even if TACC3 was highly expressed. Clarified (Non-Patent Document 7).
以上のようなTACC3の発癌との関連性や、細胞***を制御するという知見から、TACC3を標的とする化合物は、腫瘍細胞に選択的に作用する優れた抗癌剤となることが期待できる。 From the above-described relevance of TACC3 to carcinogenesis and the knowledge that it controls cell division, a compound targeting TACC3 can be expected to be an excellent anticancer agent that selectively acts on tumor cells.
すでに、本発明者は、TACC3を標的とするTACC3活性阻害剤のスクリーニング方法を開発し、該スクリーニング方法により、TACC3を標的とし、その活性を抑制する化合物について開示している(非特許文献8、特許文献3、特許文献4)。 The present inventor has already developed a method for screening a TACC3 activity inhibitor targeting TACC3, and disclosed a compound that targets TACC3 and suppresses its activity by the screening method (Non-patent Document 8, Patent Document 3 and Patent Document 4).
本発明の課題は、TACC3やTACC3-TOGp複合体と相互作用し、TACC3活性を阻害する物質の新たなスクリーニング方法を提供することにある。従来のスクリーニング方法とは異なるスクリーニング方法を用いることによって、今までに得られている化合物とは作用機序の異なる化合物をスクリーニングできる可能性がある。 An object of the present invention is to provide a new screening method for a substance that interacts with TACC3 or TACC3-TOGp complex and inhibits TACC3 activity. By using a screening method different from the conventional screening method, there is a possibility that a compound having a different mechanism of action from a compound obtained so far can be screened.
TACC3と相互作用することが知られているTOGp(Tumor over-expressed gene)も、種々の癌で過剰発現しており、いわゆる癌遺伝子であると考えられている(非特許文献9)。また、タンパク質の構造解析からα/βチューブリン二量体に結合することが示されている。また、TACC3はTOGpの微小管ポリメラーゼ活性を制御することにより癌細胞の紡錘体形成に関わると考えられている。 TOGp (Tumor over-expressed gene), which is known to interact with TACC3, is also overexpressed in various cancers and is considered to be a so-called oncogene (Non-patent Document 9). Protein structure analysis indicates that it binds to α / β tubulin dimers. TACC3 is also thought to be involved in the spindle formation of cancer cells by controlling the microtubule polymerase activity of TOGp.
本発明は、TACC3とTOGpとの複合体形成を阻害する化合物を得ることのできるスクリーニング方法を提供することを課題とする。本発明のスクリーニング方法により、腫瘍細胞に対して、より選択性が高く、優れた効果を備えた化合物を得ることができる。 An object of the present invention is to provide a screening method capable of obtaining a compound that inhibits complex formation between TACC3 and TOGp. By the screening method of the present invention, it is possible to obtain a compound having higher selectivity and superior effect on tumor cells.
本発明のスクリーニング方法は、TACC3のアミノ酸配列番号593〜838を含むアミノ酸断片とTOGpのアミノ酸配列番号1890〜2032を含むアミノ酸断片との結合を指標とすることを特徴とする。 The screening method of the present invention is characterized by using as an index the binding between an amino acid fragment containing amino acid sequence numbers 593 to 838 of TACC3 and an amino acid fragment containing amino acid sequence numbers 1890 to 2032 of TOGp.
本発明者は、TACC3のアミノ酸配列番号593〜838の領域とTOGpのアミノ酸配列番号1890〜2032の領域が相互作用することを見出した。したがって、上記アミノ酸領域を用いて化合物をスクリーニングすることによって、TACC3とTOGpとの結合を阻害する化合物を得ることができる。 The present inventor has found that the region of amino acid sequence numbers 593 to 838 of TACC3 and the region of amino acid sequence numbers 1890 to 2032 of TOGp interact. Therefore, a compound that inhibits the binding between TACC3 and TOGp can be obtained by screening a compound using the amino acid region.
TOGpはTACC3とともにいわゆる癌関連遺伝子であると考えられており、両者の結合を阻害する化合物は腫瘍細胞に特異的に作用することが期待される。 TOGp is considered to be a so-called cancer-related gene together with TACC3, and a compound that inhibits the binding of both is expected to act specifically on tumor cells.
本発明のスクリーニング方法を用いることによって、TACC3-TOGp複合体の形成を阻害する新たな化合物を同定することができた。また、これら化合物のTACC3活性阻害や細胞***阻害を確認することができた。 By using the screening method of the present invention, a new compound that inhibits the formation of the TACC3-TOGp complex could be identified. Moreover, TACC3 activity inhibition and cell division inhibition of these compounds could be confirmed.
本発明のスクリーニング方法は、前記TACC3のアミノ酸配列番号593〜838を含むアミノ酸断片が、TACC3のアミノ酸配列番号593〜838からなるアミノ酸断片を含む組換えタンパク質であり、TOGpのアミノ酸配列番号1890〜2032を含むアミノ酸断片が、TOGpのアミノ酸配列番号1890〜2032からなるアミノ酸断片を含む組換えタンパク質であり、少なくともいずれか一方の前記組換えタンパク質が検出に必要な配列を含むことを特徴とする。 In the screening method of the present invention, the amino acid fragment containing the amino acid sequence number 593-838 of TACC3 is a recombinant protein containing the amino acid fragment consisting of amino acid sequence numbers 593-838 of TACC3, and the amino acid sequence number 1890-2032 of TOGp. Is a recombinant protein containing an amino acid fragment consisting of amino acid sequence numbers 1890-2032 of TOGp, and at least one of the recombinant proteins contains a sequence necessary for detection.
TACC3、TOGpの結合に必要な領域を含む組換えタンパク質を用いることにより、精製したアミノ酸断片を容易に準備することが可能である。また、これら組換えタンパク質のどちらか一方に検出に必要なエピトープが含まれるようにデザインした組換えタンパク質を用いることによって、ELISA法等、免疫アッセイによって、簡便に精度良くスクリーニングを行うことができる。 By using a recombinant protein containing a region necessary for binding of TACC3 and TOGp, a purified amino acid fragment can be easily prepared. Further, by using a recombinant protein designed so that an epitope necessary for detection is contained in either one of these recombinant proteins, screening can be performed easily and accurately by immunoassay such as ELISA.
本発明は、TACC3を標的とする化合物をスクリーニングする方法を提供することを課題とする。TACC3を標的とする化合物とは、化合物がTACC3タンパク質、又はTACC3-TOGp複合体と結合することによって、その作用を阻害する化合物を意味する。 An object of the present invention is to provide a method for screening a compound targeting TACC3. The compound that targets TACC3 means a compound that inhibits its action by binding to the TACC3 protein or the TACC3-TOGp complex.
TACC3は、TACCファミリーに属するタンパク質の1種である。TACC3をコードする遺伝子のヌクレオチド配列及びアミノ酸配列は、例えば、GenBankアクセッション番号NM_006342.1、UniProtアクセッション番号Q9Y6A5等、データベース上に開示されている。 TACC3 is one type of protein belonging to the TACC family. The nucleotide sequence and amino acid sequence of the gene encoding TACC3 are disclosed on databases such as GenBank accession number NM_006342.1, UniProt accession number Q9Y6A5, and the like.
TOGpは、微小管結合タンパク質として知られている。TOGpをコードする遺伝子のヌクレオチド配列及びアミノ酸配列は、例えば、GenBankアクセッション番号NM_001008938.3、 NM_014756.3、UniProtアクセッション番号Q14008等、データベース上に開示されている。 TOGp is known as a microtubule binding protein. The nucleotide sequence and amino acid sequence of the gene encoding TOGp are disclosed on databases such as GenBank accession numbers NM_001008938.3, NM_014756.3, UniProt accession number Q14008, and the like.
本発明のスクリーニング方法では、化合物として、化学合成により得られる化合物だけではなく、抗体、抗体断片、ペプチド等、生物由来の物質天然物等、どのような物質を用いてスクリーニングしてもよい。 In the screening method of the present invention, not only compounds obtained by chemical synthesis but also substances such as antibodies, antibody fragments, peptides, etc. may be used for screening.
本発明のスクリーニング方法で得られた化合物は、医薬組成物の薬学的に許容されうるその塩、溶媒和物、又はエステル誘導体を有効成分とし、薬学的に許容し得る賦形剤を含有する医薬組成物とすることができる。 The compound obtained by the screening method of the present invention is a pharmaceutical comprising a pharmaceutically acceptable salt, solvate or ester derivative of a pharmaceutical composition as an active ingredient and a pharmaceutically acceptable excipient. It can be a composition.
医薬組成物の薬学的に許容されうる塩とは、塩酸、臭化水素酸、硝酸、硫酸、リン酸、クエン酸、ギ酸、マレイン酸、酢酸、コハク酸、酒石酸、メタンスルホン酸、パラトルエンスルホン酸等の無機酸及び有機酸との塩が挙げられる。また、水酸化ナトリウム、水酸化カリウム、炭酸カリウム、重炭酸ナトリウム、アンモニア、アミン塩、トリアルキルアミン塩等の塩基塩も挙げられる。 Pharmaceutically acceptable salts of pharmaceutical compositions include hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, citric acid, formic acid, maleic acid, acetic acid, succinic acid, tartaric acid, methanesulfonic acid, paratoluenesulfone Examples thereof include salts with inorganic acids such as acids and organic acids. In addition, base salts such as sodium hydroxide, potassium hydroxide, potassium carbonate, sodium bicarbonate, ammonia, amine salts, trialkylamine salts and the like are also included.
また、薬学的に許容されうるエステル誘導体には、炭素数1〜10個のアルコールまたはカルボン酸など、好ましくは、メチルアルコール、エチルアルコール、酢酸、又はプロピオン酸などとのエステル化合物が挙げられる。 Examples of the pharmaceutically acceptable ester derivative include an ester compound with an alcohol or carboxylic acid having 1 to 10 carbon atoms, preferably methyl alcohol, ethyl alcohol, acetic acid, or propionic acid.
薬学的に許容されうる溶媒和物には、好ましくは、水との溶媒和物が挙げられる。このような塩、エステル誘導体、溶媒和物は、標準的な技術を使用して当業者により形成することができる。 The pharmaceutically acceptable solvate preferably includes a solvate with water. Such salts, ester derivatives, solvates can be formed by those skilled in the art using standard techniques.
本発明のスクリーニング方法により得られた化合物を有効成分とする医薬組成物は、TACC3及びTOGpを発現し、細胞***を盛んに行っている細胞、腫瘍を処置するための組成物として用いることができる。処置とは、TACC3及びTOGp発現細胞において異常な細胞増殖を抑制する、または、TACC3高発現細胞、又はTOGp高発現細胞、特に腫瘍細胞にアポトーシスを誘導することにより、対象における疾患もしくは障害、特に腫瘍を退縮させる、または、腫瘍の増殖を遅延もしくは抑制することを意味する。 The pharmaceutical composition comprising the compound obtained by the screening method of the present invention as an active ingredient can be used as a composition for treating cells and tumors that actively express cell division and express TACC3 and TOGp. . Treatment refers to a disease or disorder in a subject, particularly a tumor, by suppressing abnormal cell growth in TACC3 and TOGp-expressing cells or inducing apoptosis in TACC3 high-expressing cells or TOGp-highly expressing cells, particularly tumor cells. Or regression or suppression of tumor growth.
本発明のスクリーニング方法によって得られた化合物を有効成分とする医薬組成物は、例えば錠剤、コーティング錠、糖衣錠、硬若しくは軟ゼラチンカプセル剤、液剤、乳剤又は懸濁剤の剤形で経口投与してよい。また、例えば坐剤を使用して直腸内に投与してよい。また、例えば軟膏剤、クリーム剤、ゲル剤又は液剤を使用して局所的又は経皮的に投与してよい。また、例えば、注射剤を使用して非経口的、例えば静脈内、筋肉内、皮下、脊髄内又は皮内的に投与してよい。好ましくは、静脈内、筋肉内又は経口投与であり、最も好ましくは経口投与である。限定されないが、1日に1回または数回投与できる。 The pharmaceutical composition containing the compound obtained by the screening method of the present invention as an active ingredient is orally administered in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions, for example. Good. It may also be administered rectally, for example using suppositories. Alternatively, it may be administered topically or transdermally using, for example, an ointment, cream, gel or solution. It may also be administered parenterally, for example intravenously, intramuscularly, subcutaneously, intraspinally or intradermally using injections. Intravenous, intramuscular or oral administration is preferred, and oral administration is most preferred. Without limitation, it can be administered once or several times a day.
本発明のスクリーニング方法によって得られた化合物を有効成分とする医薬組成物は、薬学的に不活性な無機又は有機の賦形剤と混合してもよい。錠剤、糖衣錠又は硬ゼラチンカプセル剤の適切な賦形剤の例としては、乳糖、トウモロコシデンプン若しくはその誘導体、タルク又はステアリン酸若しくはその塩などが挙げられる。軟ゼラチンカプセル剤に使用される適切な賦形剤の例には、植物油、ロウ、脂肪、半固体又は液体ポリオール等が挙げられる。液剤及びシロップ剤の調製のための賦形剤の例には、水、ポリオール、サッカロース、転化糖及びグルコースなどが挙げられる。注射剤のための賦形剤の例には、水、アルコール、ポリオール、グリセリン及び植物油などが挙げられる。坐剤及び局所又は経皮適用のための賦形剤の例には、天然又は硬化油、ロウ、脂肪及び半固体又は液体ポリオールなどが挙げられる。また、防腐剤、可溶化剤、安定剤、湿潤剤、乳化剤、甘味料、着色剤、着香剤、浸透圧を変える塩、緩衝剤、被膜剤又は酸化防止剤などを含んでよい。さらに、他の治療上有用な薬剤を含んでよい。 The pharmaceutical composition containing the compound obtained by the screening method of the present invention as an active ingredient may be mixed with a pharmaceutically inert inorganic or organic excipient. Examples of suitable excipients for tablets, dragees or hard gelatin capsules include lactose, corn starch or derivatives thereof, talc or stearic acid or salts thereof. Examples of suitable excipients used in soft gelatin capsules include vegetable oils, waxes, fats, semi-solid or liquid polyols and the like. Examples of excipients for the preparation of solutions and syrups include water, polyols, saccharose, invert sugar and glucose. Examples of excipients for injection include water, alcohol, polyol, glycerin and vegetable oil. Examples of suppositories and excipients for topical or transdermal application include natural or hardened oils, waxes, fats and semi-solid or liquid polyols. Further, it may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts that change osmotic pressure, buffers, coating agents or antioxidants. In addition, other therapeutically useful agents may be included.
本発明のスクリーニング方法によって得られた化合物の有効量または投与量は、特に制限されないが、投与形態、年齢、体重、症状に応じて適宜選択すればよい。 The effective amount or dose of the compound obtained by the screening method of the present invention is not particularly limited, and may be appropriately selected depending on the administration form, age, weight, and symptoms.
TACC3やTOGpの遺伝子またはタンパク質の働きを阻害することによって利益が得られる疾患は、限定されないが、好ましくは腫瘍である。本発明において、癌と腫瘍は交換可能に用いられる。 The disease that can benefit from inhibiting the action of TACC3 or TOGp gene or protein is not limited, but is preferably a tumor. In the present invention, cancer and tumor are used interchangeably.
腫瘍は、限定されないが、肉腫、白血病、胆道癌、乳癌、子宮癌、結腸直腸癌、喉頭癌、食道癌、胃癌、大腸癌、扁桃癌、舌癌、首の癌、リンパ腫、肺癌、甲状腺癌、卵巣癌、腎臓癌、すい臓癌、脳腫瘍、骨髄腫、神経膠腫、メラノーマ、肝癌、前立腺癌及び膀胱癌、好ましくは、大腸癌、卵巣癌、子宮癌、乳癌、食道癌、リンパ腫、神経膠腫、前立腺癌、腎臓癌、メラノーマからなる群から選択される。 Tumors include, but are not limited to, sarcoma, leukemia, biliary tract cancer, breast cancer, uterine cancer, colorectal cancer, laryngeal cancer, esophageal cancer, stomach cancer, colon cancer, tonsil cancer, tongue cancer, neck cancer, lymphoma, lung cancer, thyroid cancer Ovarian cancer, kidney cancer, pancreatic cancer, brain tumor, myeloma, glioma, melanoma, liver cancer, prostate cancer and bladder cancer, preferably colon cancer, ovarian cancer, uterine cancer, breast cancer, esophageal cancer, lymphoma, glioma Selected from the group consisting of tumor, prostate cancer, kidney cancer, melanoma.
以下、本発明のスクリーニング方法について、詳細に説明する。本発明のスクリーニング方法は、検出方法としてELISAを用いているが、TACC3とTOGpの複合体形成が検出できれば、以下に示す方法に限らずどのような方法を用いてもよい。 Hereinafter, the screening method of the present invention will be described in detail. The screening method of the present invention uses ELISA as a detection method, but any method may be used as long as the complex formation between TACC3 and TOGp can be detected.
[TACC3とTOGpとの結合を指標とするスクリーニング方法]
1.スクリーニング方法の概略
図1にTACC3とTOGpとの結合を指標とするスクリーニング方法の概略を示す。
[Screening method using binding of TACC3 and TOGp as an index]
1. Outline of Screening Method FIG. 1 shows an outline of a screening method using the binding between TACC3 and TOGp as an index.
TOGpをELISAプレート(Nunc Maxisorp)に吸着させ、洗浄した後、5% skim milkを含むPBSでブロッキングする。TACC3と化合物は混合し、室温で一定時間放置した後、TOGpが固定化されたプレートに添加する。その後所定の時間静置し、洗浄後、結合したTACC3をHRP標識抗GST抗体を用いて検出する。 TOGp is adsorbed on an ELISA plate (Nunc Maxisorp), washed, and then blocked with PBS containing 5% skim milk. TACC3 and the compound are mixed, allowed to stand at room temperature for a certain period of time, and then added to the plate on which TOGp is immobilized. Then, it is allowed to stand for a predetermined time, and after washing, bound TACC3 is detected using an HRP-labeled anti-GST antibody.
ここでは、タンパク質の精製、検出のために、夫々HISタグ、GSTを融合させたTOGp、TACC3を用いているが、精製、検出が可能であれば、どのように構築した融合タンパク質を用いてもよい。 Here, TOGp and TACC3 fused with HIS tag and GST are used for protein purification and detection, respectively, but any purified fusion protein can be used as long as purification and detection are possible. Good.
また、ここでは、GST-TACC3融合タンパク質を用いていることから、TOGpとTACC3の結合を検出するために、HRP標識をした抗GST抗体を用いているが、標識、抗体等、TACC3-TOGp複合体を検出することができればどのようなものを用いてもよい。 Here, since the GST-TACC3 fusion protein is used, an HRP-labeled anti-GST antibody is used to detect the binding between TOGp and TACC3. However, the TACC3-TOGp complex such as a label, an antibody, etc. Any object can be used as long as the body can be detected.
さらに、TOGpを固相に吸着させるのではなく、TACC3を固相に吸着させ、その後TOGpと化合物を添加して、結合したTOGpを検出する構成としてもよい。 Furthermore, it is good also as a structure which does not adsorb | suck TOGp to a solid phase, but adsorb | sucks TACC3 to a solid phase, adds TOGp and a compound after that, and detects the bound TOGp.
また、本発明のスクリーニング方法は、TACC3-TOGp複合体の形成を指標としていることから、TACC3-TOGp複合体形成を検出することができれば、ELISAを応用した系に限らず、公知のどのような系を用いてもよい。 In addition, since the screening method of the present invention uses the formation of the TACC3-TOGp complex as an index, as long as it can detect the formation of the TACC3-TOGp complex, it is not limited to the system to which the ELISA is applied, and any known type A system may be used.
2.TACC3、TOGpの結合領域の特定
TACC3、TOGpともに、全長が含まれるタンパク質を用いてもよいが、タンパク質精製、検出感度の点から、結合領域が含まれるなるべく狭い領域をスクリーニングに用いた方がよい。そこで、両者の結合領域の特定を行った。
2. Identification of the binding region of TACC3 and TOGp For both TACC3 and TOGp, a protein containing the full length may be used, but from the viewpoint of protein purification and detection sensitivity, it is better to use a region as narrow as possible containing the binding region for screening. . Therefore, the coupling region between the two was specified.
以下、培養細胞での組換えタンパク質の発現、免疫沈降等は、特に断りのない限り周知の分子遺伝学的手法により行った。 Hereinafter, expression of the recombinant protein in the cultured cells, immunoprecipitation, and the like were performed by well-known molecular genetic techniques unless otherwise specified.
2.1.TOGpに対するTACC3の結合領域の検討
TACC3を3分割したフラグメントd1(アミノ酸配列番号1〜332)、d3(アミノ酸配列番号327〜600)、d4(アミノ酸配列番号593〜838)をpcDNA3にクローニングし、HA-タグを付与し(図2A)、293細胞で発現させ、内因性のTOGpとの相互作用を解析した。
2.1. Examination of the binding region of TACC3 to TOGp Fragments d1 (amino acid sequence numbers 1 to 332), d3 (amino acid sequence numbers 327 to 600) and d4 (amino acid sequence numbers 593 to 838) obtained by dividing TACC3 into three were cloned into pcDNA3, and HA A tag was added (FIG. 2A) and expressed in 293 cells, and the interaction with endogenous TOGp was analyzed.
抗HA抗体(シグマ社製 E6779)で免疫沈降し、上記各TACC3フラグメントとTOGpが共沈するかをウェスタンブロットにより解析した(図2B右)。その結果、TACC3ドメインを含むd4フラグメントとTOGpが相互作用することが明らかとなった。 Immunoprecipitation was performed with an anti-HA antibody (E6779 manufactured by Sigma), and whether or not each TACC3 fragment and TOGp co-precipitated was analyzed by Western blotting (right of FIG. 2B). As a result, it became clear that d4 fragment containing TACC3 domain interacts with TOGp.
なお、ウェスタンブロットは各パネルの下に記載した抗体を用いて検出を行っている(以下に示す免疫沈降の解析結果も同様に表示している。)。図2B左は293細胞内で発現している内因性のTOGp、図2中央は293細胞内で発現させたTACC3フラグメントの検出結果を示す。 Western blotting was detected using the antibodies described under each panel (the immunoprecipitation analysis results shown below are also shown). FIG. 2B left shows the detection result of endogenous TOGp expressed in 293 cells, and FIG. 2 center shows the detection result of TACC3 fragment expressed in 293 cells.
2.2. TACC3に対するTOGpの結合領域の検討
TOGpを4分割したフラグメントF1(アミノ酸配列番号1〜619)、F2(アミノ酸配列番号620〜1094)、F3(アミノ酸配列番号1095〜1509)、F4(アミノ酸配列番号1502〜2032)をpcDNAにクローニングし、FLAG-タグを付与し(図3A)、293細胞で発現させ、内因性のTACC3との相互作用を解析した。
2.2. Examination of the binding region of TOGp to TACC3 Fragments F1 (amino acid sequence numbers 1 to 619), F2 (amino acid sequence numbers 620 to 1094), F3 (amino acid sequence numbers 1095 to 1509), F4 ( Amino acid sequence numbers 1502 to 2032) were cloned into pcDNA, added with a FLAG-tag (FIG. 3A), expressed in 293 cells, and analyzed for interaction with endogenous TACC3.
抗FLAG抗体(シグマ社製 F2426)で免疫沈降し、上記TOGpフラグメントとTACC3が共沈するかをウェスタンブロットにより解析した(図3B右)。その結果、F4フラグメントとTACC3が結合することが明らかとなった。 It was immunoprecipitated with an anti-FLAG antibody (F2426 manufactured by Sigma) and analyzed by Western blotting whether the TOGp fragment and TACC3 co-precipitated (FIG. 3B right). As a result, it was revealed that the F4 fragment and TACC3 were bound.
さらに、F4フラグメントを、F4−6(アミノ酸配列番号1502〜1889)、F4−3(アミノ酸配列番号1890〜2032)に分割し、上記と同様に、293細胞で発現させ、免疫沈降実験を行った。その結果、C末のフラグメントF4−3とTACC3との結合が認められた(図3C右)。 Further, the F4 fragment was divided into F4-6 (amino acid sequence numbers 1502 to 1889) and F4-3 (amino acid sequence numbers 1890 to 2032) and expressed in 293 cells in the same manner as described above, and immunoprecipitation experiments were performed. . As a result, binding between the C-terminal fragment F4-3 and TACC3 was observed (FIG. 3C right).
2.3.TACC3、TOGp各フラグメントのin vivoでの相互作用
内因性のTOGp、又はTACC3と、各フラグメントの結合が観察されたことから、in vivoでこれらフラグメント同士が結合するか解析した。
2.3. Interaction of TACC3 and TOGp fragments in vivo Since binding of each fragment to endogenous TOGp or TACC3 was observed, it was analyzed whether these fragments bind to each other in vivo.
まず、TACC3-d4フラグメントに、TOGpのF1からF4までのフラグメントが結合するか解析した。 First, it was analyzed whether a fragment from F1 to F4 of TOGp was bound to the TACC3-d4 fragment.
HAタグを付与されているTACC3-d4フラグメントと、FLAGタグが付与されているTOGpのF1〜F4までの各フラグメントを293細胞で発現させる。その後、抗HA抗体でTACC3-d4フラグメントを免疫沈降し、TACC3-d4と共沈するTOGpのフラグメントの解析を行った(図4A)。その結果、TACC3-d4(アミノ酸配列番号593〜838)フラグメントは、TOGpのF4(アミノ酸配列番号1502〜2032)フラグメントと結合することが示された(図4A右)。 The TACC3-d4 fragment to which the HA tag is attached and the fragments from F1 to F4 of TOGp to which the FLAG tag is attached are expressed in 293 cells. Thereafter, the TACC3-d4 fragment was immunoprecipitated with an anti-HA antibody, and the TOGp fragment co-precipitated with TACC3-d4 was analyzed (FIG. 4A). As a result, it was shown that the TACC3-d4 (amino acid sequence number 593-838) fragment bound to the F4 (amino acid sequence number 1502-2032) fragment of TOGp (FIG. 4A right).
次に、TOGp-F4−3(アミノ酸配列番号1890〜2032)フラグメントに結合するTACC3フラグメントを解析した。FLAGタグが付与されているTOGp-F4−3と、HAタグが付与されているTACC3のd1、d3、d4の各フラグメントを293細胞で発現させ、抗FLAG抗体で免疫沈降し、TOGp-F4−3フラグメントと共沈するTACC3のフラグメントの解析を行った。その結果、TOGp-F4−3フラグメントは、TACC3のd4(アミノ酸配列番号593〜838)と結合することが示された(図4B右)。 Next, the TACC3 fragment that binds to the TOGp-F4-3 (amino acid sequence number 1890-2032) fragment was analyzed. The FLAG tag-tagged TOGp-F4-3 and the HAC-tagged tacc3 d1, d3, and d4 fragments were expressed in 293 cells, immunoprecipitated with anti-FLAG antibody, and TOGp-F4- TACC3 fragment co-precipitated with 3 fragments was analyzed. As a result, it was shown that the TOGp-F4-3 fragment binds to d4 (amino acid sequence numbers 593 to 838) of TACC3 (FIG. 4B right).
以上の結果は、前述の内因性のTACC3、TOGpと、各フラグメントとの結合実験の結果と一致している。 The above results are consistent with the results of the above-described binding experiment between endogenous TACC3 and TOGp and each fragment.
2.4.TACC3、TOGp各フラグメントのin vitroでの相互作用
上述のin vivoアッセイで相互作用が確認されたTOGp-F4−3(アミノ酸配列番号1890〜2032)をさらに分割したF4−4(アミノ酸配列番号1890〜1963)、F4−5(アミノ酸配列番号1956〜2032)(図5A)とTACC3-d4フラグメントを用いて、in vitro pull-downアッセイにより結合解析を行った(図5B)。
2.4. Interaction of TACC3 and TOGp fragments in vitro TOGp-F4-3 (amino acid SEQ ID NO: 1890-2032) whose interaction was confirmed by the above in vivo assay was further divided into F4-4 (amino acid SEQ ID NO: 1890- 1963), F4-5 (amino acid sequence numbers 1956-2032) (FIG. 5A) and the TACC3-d4 fragment were analyzed for binding by an in vitro pull-down assay (FIG. 5B).
GST融合TACC3-d4フラグメント、HIS-V5タグ融合TOGpの各フラグメントを夫々大腸菌BL21DE3中で発現させ、GST融合タンパク質はGlutathione Sepharose 4B(GE healthcare社製)、Hisタグ融合タンパク質はTALONレジン(clontech社製)を用いて精製した。 GST fusion TACC3-d4 fragment and HIS-V5 tag fusion TOGp fragment are expressed in E. coli BL21DE3, GST fusion protein is Glutathione Sepharose 4B (GE healthcare), His tag fusion protein is TALON resin (clontech) ).
Glutathione Sepharos 4BにGST融合TACC3-d4フラグメントを結合させ、Glutathione Sepharose-GST-TACC3-d4カラムを作製し、精製した各TOGpフラグメントをアプライした。洗浄後、SDSbufferで溶出し、結合したTOGpフラグメントを抗V5抗体(invitrogen社製)を用い、ウエスタンブロットにより解析した(図5B上)。 Glutathione Sepharos 4B was combined with the GST-fused TACC3-d4 fragment to prepare a Glutathione Sepharose-GST-TACC3-d4 column, and each purified TOGp fragment was applied. After washing, elution was performed with SDS buffer, and the bound TOGp fragment was analyzed by Western blotting using an anti-V5 antibody (manufactured by Invitrogen) (FIG. 5B top).
その結果、TACC3-d4と結合するのはTOGp−F4−3フラグメントであり、さらに短くしたF4−4、F4−5とは結合が確認できなかった。 As a result, it was TOGp-F4-3 fragment that bound to TACC3-d4, and binding to F4-4 and F4-5 which were further shortened could not be confirmed.
2.5.TACC3-TOGpの結合を用いた化合物のスクリーニング
上記アッセイより、in vivo、in vitroで結合が確認できたTACC3-d4、TOGp-F4-3を用いて理研NPDepo化合物ライブラリーを用いて化合物のスクリーニングを行った。ここでは、化合物のスクリーニングを例に挙げて説明するが、抗体、ペプチドなど医薬候補となるものであればどのようなものを用いてもよい。
2.5. Screening of compounds using TACC3-TOGp binding Screening of compounds using the RIKEN NPDepo compound library using TACC3-d4 and TOGp-F4-3 confirmed binding in vivo and in vitro from the above assay. went. Here, the screening of compounds will be described as an example, but any type of drug candidate such as an antibody or peptide may be used.
候補化合物は、100μMの濃度で、図1に示したようにTOGpフラグメントを吸着させた固相に、TACC3-d4フラグメントとともに添加して、結合をELISAにより測定した。図6にELISAの結果を示す。図中矢印で示したTOGpとTACC3との結合を阻害し、50%以下の結合しか示さない化合物をTACC3-TOGpスクリーニングにおいて、陽性を示す化合物とした。 Candidate compounds were added at a concentration of 100 μM to the solid phase on which the TOGp fragment was adsorbed as shown in FIG. 1 together with the TACC3-d4 fragment, and binding was measured by ELISA. FIG. 6 shows the result of ELISA. A compound that inhibits the binding between TOGp and TACC3 indicated by an arrow in the figure and shows only 50% or less of the binding was determined as a positive compound in the TACC3-TOGp screening.
さらに、TACC3とTOGpとの結合を阻害する化合物について、培養細胞を用いて細胞アッセイを行った。細胞アッセイは、上記スクリーニングにより得られた化合物を培養細胞(卵巣癌細胞、SKOV−3)の培地に40μg/mlの濃度で添加して、細胞数低下、mitotic index、紡錘体サイズなど、有糸***の停止を示す表現型を誘導するか解析を行った。 Furthermore, a cell assay was performed using cultured cells for compounds that inhibit the binding between TACC3 and TOGp. In the cell assay, the compound obtained by the above screening was added to the culture medium of cultured cells (ovarian cancer cells, SKOV-3) at a concentration of 40 μg / ml, and the number of cells, mitotic index, spindle size, etc. We analyzed whether to induce a phenotype indicating arrest of division.
TACC3-TOGpスクリーニングでは、69の候補化合物のうち、17の化合物がTACC3とTOGpの結合を阻害し、さらに、細胞***を停止する表現型を備えた8つの化合物得ることができた。 In the TACC3-TOGp screening, out of 69 candidate compounds, 17 compounds inhibited the binding between TACC3 and TOGp, and furthermore, 8 compounds with a phenotype that stopped cell division could be obtained.
本発明のスクリーニングによって得られた化合物の例を示す。下記構造式で示した化合物(NPD13486、2H-1-Benzopyran-2-one,7,8-dihydroxy-4-[[4-(4-methoxyphenyl)-1-piperazinyl]methyl]-)は、コントロール(候補化合物無添加)の22.5%しか結合せず、ELISAのスクリーニング系で最も強い結合阻害を示した化合物である(図6において、黒い矢印で示している。)。本化合物は、従来のスクリーニング方法では検出することのできなかった化合物であり、類似の化合物も抗癌剤として報告された例はない。 The example of the compound obtained by the screening of this invention is shown. The compound represented by the following structural formula (NPD13486, 2H-1-benzopyran-2-one, 7,8-dihydroxy-4-[[4- (4-methoxyphenyl) -1-piperazinyl] methyl]-) was used as a control ( This is a compound that binds only 22.5% of the candidate compound (without addition of a candidate compound) and shows the strongest binding inhibition in the ELISA screening system (indicated by a black arrow in FIG. 6). This compound is a compound that could not be detected by a conventional screening method, and no similar compound has been reported as an anticancer agent.
以上示してきたように、本発明のスクリーニング方法を用い、TACC3-TOGp複合体の形成を指標とすることによって、TACC3活性を特異的に阻害し、細胞***の進行を阻害する化合物を得ることができた。 As described above, by using the screening method of the present invention and using the formation of a TACC3-TOGp complex as an index, a compound that specifically inhibits TACC3 activity and inhibits the progression of cell division can be obtained. did it.
さらにこれら化合物をリード化合物としてより強くTACC3活性を阻害する化合物を得ることも可能である。本発明のスクリーニング方法によって、腫瘍細胞に選択性の高い化合物のスクリーニングし、新しい抗癌剤を開発することが期待できる。 Furthermore, it is also possible to obtain compounds that strongly inhibit TACC3 activity using these compounds as lead compounds. With the screening method of the present invention, it is expected that a compound having high selectivity for tumor cells will be screened to develop a new anticancer agent.
Claims (2)
TACC3のアミノ酸配列番号593〜838を含むアミノ酸断片と
TOGpのアミノ酸配列番号1890〜2032を含むアミノ酸断片との結合を指標とすることを特徴とする化合物のスクリーニング方法。 A method for screening a compound that inhibits TACC3 activity, comprising:
A method for screening a compound, characterized by using, as an index, the binding between an amino acid fragment containing amino acid sequences 593 to 838 of TACC3 and an amino acid fragment containing amino acid sequences 1890 to 2032 of TOGp.
TACC3のアミノ酸配列番号593〜838からなるアミノ酸断片を含む組換えタンパク質であり、
前記TOGpのアミノ酸配列番号1890〜2032を含むアミノ酸断片が、
TOGpのアミノ酸配列番号1890〜2032からなるアミノ酸断片を含む組換えタンパク質であり、
少なくともいずれか一方の前記組換えタンパク質が検出に必要な配列を含むことを特徴とする請求項1記載の化合物のスクリーニング方法。 The amino acid fragment comprising the amino acid sequence number 593-838 of TACC3 is a recombinant protein comprising an amino acid fragment consisting of amino acid sequence numbers 593-838 of TACC3,
An amino acid fragment comprising the amino acid sequence number 1890-2032 of the TOGp,
A recombinant protein comprising an amino acid fragment consisting of amino acid sequences of 1890-2032 of TOGp,
2. The method for screening a compound according to claim 1, wherein at least one of the recombinant proteins contains a sequence necessary for detection.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014194353 | 2014-09-24 | ||
JP2014194353 | 2014-09-24 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2016065869A true JP2016065869A (en) | 2016-04-28 |
JP6682221B2 JP6682221B2 (en) | 2020-04-15 |
Family
ID=55804127
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2015186097A Active JP6682221B2 (en) | 2014-09-24 | 2015-09-18 | Method for screening TACC3 inhibitor |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP6682221B2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3801529A4 (en) * | 2018-05-25 | 2022-04-06 | OncoCube Therapeutics LLC | Highly potent tacc3 inhibitor as a novel anticancer drug candidate |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013165008A1 (en) * | 2012-05-02 | 2013-11-07 | 公益財団法人がん研究会 | Small compound targeting at tacc3 |
-
2015
- 2015-09-18 JP JP2015186097A patent/JP6682221B2/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013165008A1 (en) * | 2012-05-02 | 2013-11-07 | 公益財団法人がん研究会 | Small compound targeting at tacc3 |
Non-Patent Citations (2)
Title |
---|
FIONA E. HOOD ET AL., J. CELL.BIOL., vol. 202, no. 3, JPN6019020512, 2013, pages 463 - 478, ISSN: 0004181955 * |
STEPHEN J. ROYLE, J CELL SCI., vol. 125 pt1, JPN6019020511, 2012, pages 1 - 19, ISSN: 0004181954 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3801529A4 (en) * | 2018-05-25 | 2022-04-06 | OncoCube Therapeutics LLC | Highly potent tacc3 inhibitor as a novel anticancer drug candidate |
US11622966B2 (en) | 2018-05-25 | 2023-04-11 | A2A Pharmaceuticals, Inc. | Highly potent TACC3 inhibitor as a novel anticancer drug candidate |
Also Published As
Publication number | Publication date |
---|---|
JP6682221B2 (en) | 2020-04-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Tang et al. | Selective inhibition of STRN3-containing PP2A phosphatase restores hippo tumor-suppressor activity in gastric cancer | |
US20210003557A1 (en) | Compositions and methods for inducing conformational changes in cereblon and other e3 ubiquitin ligases | |
Sun et al. | Rab34 regulates adhesion, migration, and invasion of breast cancer cells | |
De Palma et al. | The NMR-based characterization of the FTY720-SET complex reveals an alternative mechanism for the attenuation of the inhibitory SET-PP2A interaction | |
KR20160074000A (en) | Cancer models and associated methods | |
US20190271705A1 (en) | Sh2 domain variants | |
US20230053231A1 (en) | Compositions targeting the interaction domain between p27kip1 and brk and methods of use thereof to inhibit p27 y phosphorylation and cdk4 activity | |
JP6302674B2 (en) | Use of stathmin as a biomarker of drug response to flazanobenzimidazole | |
Qiu et al. | A small peptide derived from p53 linker region can resume the apoptotic activity of p53 by sequestering iASPP with p53 | |
US20160052918A1 (en) | Small compounds targeting tacc3 | |
US20190369104A1 (en) | P27 tyrosine phosphorylation as a marker of cdk4 activity and methods of use thereof | |
JP6682221B2 (en) | Method for screening TACC3 inhibitor | |
JP6914269B2 (en) | Anti-cancer drug screening method that inhibits the binding between AIMP2-DX2 and HSP70 | |
EP2845588A1 (en) | PIN1 inhibitors for use in the prevention and/or treatment of theileriosis, and related applications | |
WO2012090939A1 (en) | Method for suppressing receptor tyrosine kinase-mediated prosurvival signaling in cancer cells | |
US20170291958A1 (en) | Compositions and Methods For the Modulation of Ras Proteins | |
Takai et al. | Casein kinase 2 phosphorylates and stabilizes C/EBPβ in pancreatic β cells | |
JP6057408B2 (en) | Cancer preventive and / or therapeutic agent | |
US20240139125A1 (en) | Bi-1 antagonists and their uses | |
US11351144B2 (en) | Compounds for inhibiting secretory leukocyte protease inhibitor (SLPI) | |
US11672794B2 (en) | Therapeutic targeting of the BAP1 complex in cancer | |
GB2546773A (en) | Cancer | |
US20130345145A1 (en) | Atip3 and biologically active fragments thereof for use in the treatment of cancer | |
WO2023161443A1 (en) | PEPTIDES TARGETING THE INTERACTION BETWEEN KINDLIN-1 AND ß-INTEGRIN | |
Eguchi et al. | Stress-inducible SCAN factors suppress the stress response and are biomarkers for enhanced prognosis in cancers |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20180824 |
|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20190522 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20190611 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20190729 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20200107 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200210 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20200303 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20200325 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6682221 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |