JP5696830B2 - Par−2活性化阻害物質 - Google Patents
Par−2活性化阻害物質 Download PDFInfo
- Publication number
- JP5696830B2 JP5696830B2 JP2010102444A JP2010102444A JP5696830B2 JP 5696830 B2 JP5696830 B2 JP 5696830B2 JP 2010102444 A JP2010102444 A JP 2010102444A JP 2010102444 A JP2010102444 A JP 2010102444A JP 5696830 B2 JP5696830 B2 JP 5696830B2
- Authority
- JP
- Japan
- Prior art keywords
- par
- aptamer
- peptide
- trypsin
- activation
- 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.)
- Active
Links
- 230000004913 activation Effects 0.000 title claims description 47
- NTQVODZUQIATFS-WAUHAFJUSA-N (2s)-2-[[(2s)-6-amino-2-[[2-[[(2s,3s)-2-[[(2s)-2-[[(2s)-2-amino-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-methylpentanoyl]amino]acetyl]amino]hexanoyl]amino]-3-methylbutanoic acid Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O NTQVODZUQIATFS-WAUHAFJUSA-N 0.000 title claims 8
- 102100037132 Proteinase-activated receptor 2 Human genes 0.000 title claims 8
- 101710121435 Proteinase-activated receptor 2 Proteins 0.000 title claims 8
- 239000003112 inhibitor Substances 0.000 title description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 51
- 108091023037 Aptamer Proteins 0.000 claims description 48
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 12
- 201000010099 disease Diseases 0.000 claims description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- 229940121363 anti-inflammatory agent Drugs 0.000 claims description 4
- 239000002260 anti-inflammatory agent Substances 0.000 claims description 4
- 230000000069 prophylactic effect Effects 0.000 claims description 4
- 229940124597 therapeutic agent Drugs 0.000 claims description 4
- 238000009007 Diagnostic Kit Methods 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 108050009310 Protease-activated receptors Proteins 0.000 description 96
- 102000002020 Protease-activated receptors Human genes 0.000 description 96
- 239000012588 trypsin Substances 0.000 description 49
- 108090000631 Trypsin Proteins 0.000 description 48
- 102000004142 Trypsin Human genes 0.000 description 48
- 210000004027 cell Anatomy 0.000 description 47
- 101001098560 Homo sapiens Proteinase-activated receptor 2 Proteins 0.000 description 42
- 108020004414 DNA Proteins 0.000 description 38
- 108010079855 Peptide Aptamers Proteins 0.000 description 27
- 150000001413 amino acids Chemical group 0.000 description 20
- 238000000034 method Methods 0.000 description 19
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 15
- 108091008102 DNA aptamers Proteins 0.000 description 15
- 229910001424 calcium ion Inorganic materials 0.000 description 15
- 230000003834 intracellular effect Effects 0.000 description 15
- 230000005764 inhibitory process Effects 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 239000000872 buffer Substances 0.000 description 12
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 12
- 239000002953 phosphate buffered saline Substances 0.000 description 12
- 206010061218 Inflammation Diseases 0.000 description 10
- 239000011324 bead Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 230000008878 coupling Effects 0.000 description 10
- 238000010168 coupling process Methods 0.000 description 10
- 238000005859 coupling reaction Methods 0.000 description 10
- 230000004054 inflammatory process Effects 0.000 description 10
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 102000053602 DNA Human genes 0.000 description 8
- 108020004682 Single-Stranded DNA Proteins 0.000 description 8
- 239000013604 expression vector Substances 0.000 description 8
- 210000004408 hybridoma Anatomy 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- -1 9-fluorenylmethoxycarbonyl (Fmoc) Chemical class 0.000 description 6
- 108060001084 Luciferase Proteins 0.000 description 6
- 102000003945 NF-kappa B Human genes 0.000 description 6
- 108010057466 NF-kappa B Proteins 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 230000004941 influx Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- 210000004989 spleen cell Anatomy 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 239000013076 target substance Substances 0.000 description 5
- 239000005089 Luciferase Substances 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 210000000988 bone and bone Anatomy 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 210000002249 digestive system Anatomy 0.000 description 4
- 210000003499 exocrine gland Anatomy 0.000 description 4
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 4
- 210000000653 nervous system Anatomy 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 210000002345 respiratory system Anatomy 0.000 description 4
- 210000003491 skin Anatomy 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 230000002485 urinary effect Effects 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 3
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 239000003114 blood coagulation factor Substances 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- 239000012521 purified sample Substances 0.000 description 3
- 230000000241 respiratory effect Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 108090000932 Calcitonin Gene-Related Peptide Proteins 0.000 description 2
- 102000004414 Calcitonin Gene-Related Peptide Human genes 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 102100037136 Proteinase-activated receptor 1 Human genes 0.000 description 2
- 101710121440 Proteinase-activated receptor 1 Proteins 0.000 description 2
- 102100023710 Proteinase-activated receptor 4 Human genes 0.000 description 2
- 101710121439 Proteinase-activated receptor 4 Proteins 0.000 description 2
- 108091008103 RNA aptamers Proteins 0.000 description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 2
- 229960003896 aminopterin Drugs 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 230000009460 calcium influx Effects 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 239000005289 controlled pore glass Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 238000003359 percent control normalization Methods 0.000 description 2
- 210000003200 peritoneal cavity Anatomy 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000005167 vascular cell Anatomy 0.000 description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- KZDCMKVLEYCGQX-UDPGNSCCSA-N 2-(diethylamino)ethyl 4-aminobenzoate;(2s,5r,6r)-3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid;hydrate Chemical compound O.CCN(CC)CCOC(=O)C1=CC=C(N)C=C1.N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 KZDCMKVLEYCGQX-UDPGNSCCSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 238000003718 Dual-Luciferase Reporter Assay System Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 108091006027 G proteins Proteins 0.000 description 1
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 1
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 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
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 101001028689 Homo sapiens Protein JTB Proteins 0.000 description 1
- 101900297506 Human immunodeficiency virus type 1 group M subtype B Reverse transcriptase/ribonuclease H Proteins 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108010070503 PAR-2 Receptor Proteins 0.000 description 1
- 102000032628 PAR-2 Receptor Human genes 0.000 description 1
- 101710186509 Partitioning defective 3 homolog Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940118430 Protease-activated receptor-2 antagonist Drugs 0.000 description 1
- 102100037171 Protein JTB Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 102100037133 Proteinase-activated receptor 3 Human genes 0.000 description 1
- 101710121425 Proteinase-activated receptor 3 Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 108060005989 Tryptase Proteins 0.000 description 1
- 102000001400 Tryptase Human genes 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- QOMNQGZXFYNBNG-UHFFFAOYSA-N acetyloxymethyl 2-[2-[2-[5-[3-(acetyloxymethoxy)-2,7-difluoro-6-oxoxanthen-9-yl]-2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]-n-[2-(acetyloxymethoxy)-2-oxoethyl]-4-methylanilino]acetate Chemical compound CC(=O)OCOC(=O)CN(CC(=O)OCOC(C)=O)C1=CC=C(C)C=C1OCCOC1=CC(C2=C3C=C(F)C(=O)C=C3OC3=CC(OCOC(C)=O)=C(F)C=C32)=CC=C1N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O QOMNQGZXFYNBNG-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 206010064930 age-related macular degeneration Diseases 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 238000007846 asymmetric PCR Methods 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000006642 detritylation reaction Methods 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000000132 electrospray ionisation Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 208000030603 inherited susceptibility to asthma Diseases 0.000 description 1
- 125000002346 iodo group Chemical group I* 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000003468 luciferase reporter gene assay Methods 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001992 poloxamer 407 Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000034190 positive regulation of NF-kappaB transcription factor activity Effects 0.000 description 1
- DBABZHXKTCFAPX-UHFFFAOYSA-N probenecid Chemical compound CCCN(CCC)S(=O)(=O)C1=CC=C(C(O)=O)C=C1 DBABZHXKTCFAPX-UHFFFAOYSA-N 0.000 description 1
- 229960003081 probenecid Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 229940127293 prostanoid Drugs 0.000 description 1
- 150000003814 prostanoids Chemical class 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
Images
Description
(1−1:一本鎖ランダムオリゴDNAの作製)
50塩基のランダム領域およびその両側にプライマー部位(配列番号4および5)を含む種々の一本鎖ランダムオリゴDNAを調製した。これらの一本鎖ランダムオリゴDNAの合成は、オペロンバイオテクノロジー株式会社に委託し、精製標品を得た。精製標品をSELEXライブラリーのテンプレートDNAとした。
ヒトPAR−2のアミノ酸配列(配列番号2)の212位〜226位に相当するアミノ酸配列(配列番号3:VKQTIFIPALNITTC)のペプチドを調製した。このペプチドの合成は、オペロンバイオテクノロジー株式会社に委託し、精製標品を得た。
上記1−2で得たヒトPAR−2ペプチドを、PIERCE社製のSulfoLink(登録商標)カップリングゲル(カタログ番号20401)を用いて製品の指示書に従って、次のようにビーズに固定化した。
上記1−1で合成したランダムオリゴDNAを鋳型にして、forwardプライマー(配列番号4)とreverseプライマー(配列番号5)とによるPCR(1サイクル:94℃,15秒;55℃,15秒;72℃,15秒×12サイクル)を行った。増幅後、forwardプライマーのみを用いた非対称PCRによりプラス鎖を増幅した(1サイクル:94℃,15秒;55℃,15秒;72℃,15秒×45サイクル)。増幅したプラス鎖をアガロースゲル電気泳動によって精製し、SELEX用DNAライブラリーとした。このSELEX用DNAライブラリーをPBSに溶解し、95℃で5分間加熱し、室温に戻した。次いで、SELEX用DNAライブラリーと上記1−3で調製したヒトPAR−2ペプチドを固定化させたビーズとを混合し、室温で30分間インキュベートした。インキュベーション後、PBSでビーズを数回洗浄した。適量の水をビーズに加えて混合し、100℃で5分間加熱し、PAR−2ペプチドビーズに結合したDNAを解離させて回収した。回収したDNAと上記1−3で調製したヒトPAR−2ペプチドを固定化していないビーズとを混合し、室温で10分間インキュベートした。素通りしたDNA(ヒトPAR−2ペプチドに結合するDNA)を回収して、エタノール沈殿法によって濃縮した。濃縮したDNAを鋳型にして、上記の操作を5〜15ラウンド繰り返した。この時、5〜8mMのMg2+存在下でPCRを行って、変異を導入した。
第15ラウンドで得られたDNAを、forwardプライマー(配列番号4)およびreverseプライマー(配列番号5)を用いるPCRによって増幅し、アガロースゲル電気泳動によって精製し、ヒトPAR−2ペプチド特異的DNAを得た。このDNAを平滑末端クローニングベクター(Invitrogen社製Zero Blunt(登録商標)TOPO(登録商標)PCR Cloning Kit for Sequencing(カタログ番号K2875J10))に導入し、次のようにして配列を決定した。
上記1−5で得られたプラスミドDNA中のヒトPAR−2ペプチド特異的DNAの配列を、Applied Biosystem社製のABI377を用いてBigDye Terminator Cycle sequence法によって決定した。
配列番号7:5’−GCCACGCTTTGGCATGTGCGGGGCTGAGCGGTGGGCCAATGTGGGGTGTA−3’(EEL2)
配列番号8:5’−CCCGCAAGGCGAGGTGAGCGCATGGGCACACAAAAGGCACAGAACGGGCT−3’(EEL3)
配列番号9:5’−CCGGCGATACGCGCCGGGGAGCGGGAGGATTAAAGTAACTTTTAGCAAGC−3’(EEL4)
(2−1:免疫原の調製)
上記1−2で合成したペプチド(配列番号3:配列番号2のヒトPAR−2のアミノ酸配列の212位〜226位に相当するペプチド)のシステイン残基に、ペプチドの免疫活性を高めるために、KLH(Keyhole limpet hemocyanin)を結合させて免疫原とした(これをKLH−ペプチドと呼ぶ)。
PBSに溶解した25μgのKLH−ペプチドを、フロイント完全アジュバントと共に懸濁して、Balb/cマウスの腹腔に注射した。その2週間後に、同量の抗原をフロイント不完全アジュバントに懸濁して、同じマウスの腹腔に注射した。これをさらに2〜4回繰り返した。
最終免疫から3日後に脾臓を摘出し、脾臓細胞を分散させた。ポリエチレングリコールを用いて、脾臓細胞をミエローマSP2/0−Ag14細胞と融合させた(融合により脾臓細胞の増殖が半永久化される)。融合した細胞(ハイブリドーマ)のみが生育するHAT培地(ヒポキサンチン(H)、アミノプテリン(A)およびチミジン(T)を含む培地)(Sigma社製)で培養し、増殖したハイブリドーマをクローン化した。
クローン化したハイブリドーマの培養上清について、ELISA(enzyme-linked immunosorbent assay)法を利用してヒトPAR−2ペプチドに結合する抗体の有無を検定した。その結果、16A、16B、22A、22Cf、27A、27Dの6クローンが抗ヒトPAR−2ペプチド抗体を生産するハイブリドーマとして得られた。
単一クローン化されたハイブリドーマを拡大培養した後、培養上清からプロテインGカラム(ファルマシア社製MAbTrap GII)を用いてマニュアルに従って抗体を精製した。それぞれの抗体はIsoStrip(登録商標)mouse monoclonal antibody isotyping kit(ロシュ・ダイアグノスティクス社製)を用いてアイソタイプを解析した。その結果、22CfはIgG2a/κで、それ以外はIgG1/κであった。
トリプシン刺激によるPAR−2活性化は、転写因子NF−κBの活性化を誘導する。実施例1で得られたアプタマーあるいは実施例2で得られたモノクローナル抗体がPAR−2活性化の阻害能を示すかをNF−κBルシフェラーゼレポーターアッセイにより評価した。以下にその手順を示す。
PAR−2の活性化により誘導される細胞内カルシウムイオン濃度の上昇が、抗ヒトPAR−2ペプチドアプタマーあるいは抗ヒトPAR−2ペプチドモノクローナル抗体によって阻害されるかどうかをカルシウム蛍光指示薬(Dojindo社製Fluo 4−AM)により評価した。以下にその手順を示した。使用した試薬は特に記載がある場合を除いてナカライテスク社製である。
実施例3および4においてトリプシン誘導性のPAR−2活性化のより高い抑制能を示したアプタマーEEL4ならびにモノクローナル抗体22Cfおよび27Dについて、ヒトPAR−2の認識能を調べた。
Claims (7)
- 配列表の配列番号2の212位から226位までのアミノ酸配列で示されるPAR−2ペプチド領域に結合し、PAR−2の活性化を阻害し、配列表の配列番号9の1位から50位までの塩基配列で示されるDNAを含む、アプタマー。
- 請求項1に記載のアプタマーを含む、抗炎症剤。
- 請求項1に記載のアプタマーを含む、PAR−2が関与する疾患の診断試薬。
- 請求項1に記載のアプタマーを含む、PAR−2が関与する疾患の診断用キット。
- 請求項1に記載のアプタマーを含む、PAR−2が関与する疾患の予防剤。
- 請求項1に記載のアプタマーを含む、PAR−2が関与する疾患の治療剤。
- 請求項1に記載のアプタマーを含む、PAR−2の検出試薬。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010102444A JP5696830B2 (ja) | 2010-04-27 | 2010-04-27 | Par−2活性化阻害物質 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010102444A JP5696830B2 (ja) | 2010-04-27 | 2010-04-27 | Par−2活性化阻害物質 |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2011229452A JP2011229452A (ja) | 2011-11-17 |
JP5696830B2 true JP5696830B2 (ja) | 2015-04-08 |
Family
ID=45319466
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2010102444A Active JP5696830B2 (ja) | 2010-04-27 | 2010-04-27 | Par−2活性化阻害物質 |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP5696830B2 (ja) |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2009273052B2 (en) * | 2008-07-14 | 2015-09-17 | The University Of Tokyo | Aptamer against IL-17 and use thereof |
ES2543222T3 (es) * | 2008-09-24 | 2015-08-17 | Ribomic Inc. | Aptámero para NGF y uso del mismo |
-
2010
- 2010-04-27 JP JP2010102444A patent/JP5696830B2/ja active Active
Also Published As
Publication number | Publication date |
---|---|
JP2011229452A (ja) | 2011-11-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220098294A1 (en) | Activatable Binding Polypeptides and Methods of Identification and Use Thereof | |
ES2567437T3 (es) | Oligómeros beta-(1,42) amiloides, derivados de los mismos y anticuerpos para estos, métodos para la preparación de los mismos y uso de los mismos | |
JP5861223B2 (ja) | プロタンパク質およびその使用方法 | |
JP5851842B2 (ja) | 改変した抗体組成物、それを作製および使用する方法 | |
ES2647823T3 (es) | Anticuerpos anti-CD100 y métodos de uso de los mismos | |
AU2008202992B2 (en) | Antibody against enzyme specifically cleaving von villebrand factor and assay system using the same | |
CN114106099A (zh) | 蛋白裂解酶和u型纤溶酶原激活物的底物和其它可裂解部分及其使用方法 | |
JP2001510987A (ja) | 哺乳動物のCD97αサブユニットを含む炎症および脈管形成を阻害するための方法および組成物 | |
WO2002010217A9 (en) | Endothelial cell expression patterns | |
US20100093624A1 (en) | Peptide therapeutics that bind vegf and methods of use thereof | |
KR102150419B1 (ko) | Pd-l1에 결합하는 펩타이드 및 이의 용도 | |
JPWO2002061076A1 (ja) | アディポネクチン関連蛋白質 | |
AU2002258543A1 (en) | Endothelial cell expression patterns | |
EP1572867A2 (en) | Endothelial cell expression patterns | |
AU2013202755A1 (en) | Activatable binding polypeptides and methods of identification and use thereof | |
JP5696830B2 (ja) | Par−2活性化阻害物質 | |
AU2016213702B2 (en) | Activatable binding polypeptides and methods of identification and use thereof | |
Skrzypek et al. | Interaction between Yersinia pestis YopM protein and human α-thrombin | |
JP2003503040A (ja) | 合成ペプチド免疫原およびそれに対する抗体 | |
JP2007527201A (ja) | 抗インテグリン免疫グロブリン | |
WO2003084998A1 (fr) | Anticorps monoclonal neutralisant la megsine | |
JPWO2005014809A1 (ja) | 肝臓ガン特異的ポリペプチド、該ポリペプチドをコードするポリヌクレオチド、及び該ポリペプチドの発現を抑制するrna分子 | |
JP2003038186A (ja) | 新規セリンプロテアーゼモジュレーター |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20130123 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20140617 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20140808 |
|
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: 20150106 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20150127 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 5696830 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: R3D02 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |