JP5517450B2 - 二量体または多量体のマイクロタンパク質 - Google Patents
二量体または多量体のマイクロタンパク質 Download PDFInfo
- Publication number
- JP5517450B2 JP5517450B2 JP2008500131A JP2008500131A JP5517450B2 JP 5517450 B2 JP5517450 B2 JP 5517450B2 JP 2008500131 A JP2008500131 A JP 2008500131A JP 2008500131 A JP2008500131 A JP 2008500131A JP 5517450 B2 JP5517450 B2 JP 5517450B2
- Authority
- JP
- Japan
- Prior art keywords
- polypeptide
- microprotein
- receptor
- amino acid
- protein
- 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
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 233
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 167
- 229920001184 polypeptide Polymers 0.000 claims description 143
- 108090000623 proteins and genes Proteins 0.000 claims description 96
- 108010041111 Thrombopoietin Proteins 0.000 claims description 78
- 102000036693 Thrombopoietin Human genes 0.000 claims description 77
- 102000004169 proteins and genes Human genes 0.000 claims description 76
- 230000027455 binding Effects 0.000 claims description 63
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 45
- 235000018102 proteins Nutrition 0.000 claims description 45
- 230000000694 effects Effects 0.000 claims description 43
- 230000015572 biosynthetic process Effects 0.000 claims description 40
- 108060005989 Tryptase Proteins 0.000 claims description 36
- 102000001400 Tryptase Human genes 0.000 claims description 36
- 239000008194 pharmaceutical composition Substances 0.000 claims description 23
- 102000004190 Enzymes Human genes 0.000 claims description 22
- 108090000790 Enzymes Proteins 0.000 claims description 22
- 235000001014 amino acid Nutrition 0.000 claims description 21
- 239000012634 fragment Substances 0.000 claims description 21
- 150000001413 amino acids Chemical class 0.000 claims description 20
- 229960003067 cystine Drugs 0.000 claims description 19
- 230000002401 inhibitory effect Effects 0.000 claims description 19
- 238000011282 treatment Methods 0.000 claims description 18
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 14
- 230000004936 stimulating effect Effects 0.000 claims description 14
- 206010043554 thrombocytopenia Diseases 0.000 claims description 14
- 201000010099 disease Diseases 0.000 claims description 13
- 239000012528 membrane Substances 0.000 claims description 12
- 125000000539 amino acid group Chemical group 0.000 claims description 10
- 239000003112 inhibitor Substances 0.000 claims description 9
- 239000003937 drug carrier Substances 0.000 claims description 7
- 230000009870 specific binding Effects 0.000 claims description 7
- 230000001588 bifunctional effect Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 230000019491 signal transduction Effects 0.000 claims description 6
- 206010028980 Neoplasm Diseases 0.000 claims description 5
- FARGFPCUTAMIMN-UHFFFAOYSA-N 2,2-bis(2,5-dioxopyrrolidin-1-yl)octanedioic acid Chemical compound O=C1CCC(=O)N1C(C(O)=O)(CCCCCC(=O)O)N1C(=O)CCC1=O FARGFPCUTAMIMN-UHFFFAOYSA-N 0.000 claims description 4
- 206010061218 Inflammation Diseases 0.000 claims description 4
- 201000011510 cancer Diseases 0.000 claims description 4
- 230000004054 inflammatory process Effects 0.000 claims description 4
- 238000005897 peptide coupling reaction Methods 0.000 claims description 4
- 208000032467 Aplastic anaemia Diseases 0.000 claims description 3
- 208000018240 Bone Marrow Failure disease Diseases 0.000 claims description 3
- 206010065553 Bone marrow failure Diseases 0.000 claims description 3
- 201000003793 Myelodysplastic syndrome Diseases 0.000 claims description 3
- 208000019423 liver disease Diseases 0.000 claims description 3
- ALEBYBVYXQTORU-UHFFFAOYSA-N 6-hydrazinyl-6-oxohexanoic acid Chemical compound NNC(=O)CCCCC(O)=O ALEBYBVYXQTORU-UHFFFAOYSA-N 0.000 claims description 2
- 201000001320 Atherosclerosis Diseases 0.000 claims description 2
- 206010020751 Hypersensitivity Diseases 0.000 claims description 2
- 208000029523 Interstitial Lung disease Diseases 0.000 claims description 2
- 201000004681 Psoriasis Diseases 0.000 claims description 2
- 206010039085 Rhinitis allergic Diseases 0.000 claims description 2
- 208000030961 allergic reaction Diseases 0.000 claims description 2
- 201000010105 allergic rhinitis Diseases 0.000 claims description 2
- 208000006673 asthma Diseases 0.000 claims description 2
- 208000007565 gingivitis Diseases 0.000 claims description 2
- 201000006417 multiple sclerosis Diseases 0.000 claims description 2
- 201000008482 osteoarthritis Diseases 0.000 claims description 2
- 201000001245 periodontitis Diseases 0.000 claims description 2
- 208000005069 pulmonary fibrosis Diseases 0.000 claims description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 2
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 claims 2
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 claims 1
- 210000004204 blood vessel Anatomy 0.000 claims 1
- 230000006806 disease prevention Effects 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- 238000000034 method Methods 0.000 description 109
- 102000005962 receptors Human genes 0.000 description 72
- 108020003175 receptors Proteins 0.000 description 72
- 238000009739 binding Methods 0.000 description 59
- 230000014509 gene expression Effects 0.000 description 59
- 108010016529 Bacillus amyloliquefaciens ribonuclease Proteins 0.000 description 39
- 210000004027 cell Anatomy 0.000 description 37
- 102000039446 nucleic acids Human genes 0.000 description 34
- 108020004707 nucleic acids Proteins 0.000 description 34
- 150000007523 nucleic acids Chemical class 0.000 description 34
- 239000000539 dimer Substances 0.000 description 33
- 125000005647 linker group Chemical group 0.000 description 32
- 102000040430 polynucleotide Human genes 0.000 description 30
- 108091033319 polynucleotide Proteins 0.000 description 30
- 239000002157 polynucleotide Substances 0.000 description 30
- 239000013598 vector Substances 0.000 description 27
- 238000003776 cleavage reaction Methods 0.000 description 25
- 230000007017 scission Effects 0.000 description 24
- 108020001507 fusion proteins Proteins 0.000 description 22
- 102000037865 fusion proteins Human genes 0.000 description 22
- 239000000556 agonist Substances 0.000 description 21
- 230000004927 fusion Effects 0.000 description 21
- 210000004899 c-terminal region Anatomy 0.000 description 19
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 19
- 229940088598 enzyme Drugs 0.000 description 19
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 18
- 239000000203 mixture Substances 0.000 description 18
- QJPWUUJVYOJNMH-VKHMYHEASA-N L-homoserine lactone Chemical group N[C@H]1CCOC1=O QJPWUUJVYOJNMH-VKHMYHEASA-N 0.000 description 16
- 238000004519 manufacturing process Methods 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- 239000000126 substance Substances 0.000 description 15
- 239000000758 substrate Substances 0.000 description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- -1 antibody Proteins 0.000 description 14
- 239000003446 ligand Substances 0.000 description 14
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 13
- 230000004913 activation Effects 0.000 description 13
- 125000004122 cyclic group Chemical group 0.000 description 13
- 239000013604 expression vector Substances 0.000 description 13
- 210000003593 megakaryocyte Anatomy 0.000 description 13
- 238000007363 ring formation reaction Methods 0.000 description 13
- 108091026890 Coding region Proteins 0.000 description 12
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 150000007857 hydrazones Chemical class 0.000 description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 11
- 238000003556 assay Methods 0.000 description 11
- 150000001875 compounds Chemical class 0.000 description 11
- 230000006870 function Effects 0.000 description 11
- 230000004048 modification Effects 0.000 description 11
- 238000012986 modification Methods 0.000 description 11
- 125000003729 nucleotide group Chemical group 0.000 description 11
- 238000007254 oxidation reaction Methods 0.000 description 11
- 238000003786 synthesis reaction Methods 0.000 description 11
- 239000002773 nucleotide Substances 0.000 description 10
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- 101000799461 Homo sapiens Thrombopoietin Proteins 0.000 description 9
- 101000694103 Homo sapiens Thyroid peroxidase Proteins 0.000 description 9
- 238000007792 addition Methods 0.000 description 9
- 102000053400 human TPO Human genes 0.000 description 9
- 238000010369 molecular cloning Methods 0.000 description 9
- 239000000178 monomer Substances 0.000 description 9
- 230000003647 oxidation Effects 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 239000002202 Polyethylene glycol Substances 0.000 description 8
- 108090000631 Trypsin Proteins 0.000 description 8
- 102000004142 Trypsin Human genes 0.000 description 8
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 8
- 238000009396 hybridization Methods 0.000 description 8
- 238000001727 in vivo Methods 0.000 description 8
- 229930182817 methionine Natural products 0.000 description 8
- 229920001223 polyethylene glycol Polymers 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- 239000012588 trypsin Substances 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 7
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 239000005557 antagonist Substances 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 230000004071 biological effect Effects 0.000 description 6
- 239000002243 precursor Substances 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 238000002560 therapeutic procedure Methods 0.000 description 6
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 6
- 102000014914 Carrier Proteins Human genes 0.000 description 5
- 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 5
- 102000035195 Peptidases Human genes 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- 238000006471 dimerization reaction Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 239000012636 effector Substances 0.000 description 5
- 210000003630 histaminocyte Anatomy 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 150000002678 macrocyclic compounds Chemical class 0.000 description 5
- 238000003259 recombinant expression Methods 0.000 description 5
- 238000004007 reversed phase HPLC Methods 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 5
- BRQNQZWNPXPCQP-VKHMYHEASA-N (2s)-2-amino-4-hydroxybutanehydrazide Chemical compound NNC(=O)[C@@H](N)CCO BRQNQZWNPXPCQP-VKHMYHEASA-N 0.000 description 4
- 102000054930 Agouti-Related Human genes 0.000 description 4
- 108010078791 Carrier Proteins Proteins 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 101000680845 Luffa aegyptiaca Ribosome-inactivating protein luffin P1 Proteins 0.000 description 4
- 108700012612 McoEeTI Proteins 0.000 description 4
- 101100261153 Mus musculus Mpl gene Proteins 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 229960000583 acetic acid Drugs 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 210000001185 bone marrow Anatomy 0.000 description 4
- 210000002798 bone marrow cell Anatomy 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 4
- 238000010348 incorporation Methods 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 210000001236 prokaryotic cell Anatomy 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 238000012552 review Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000010561 standard procedure Methods 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000002750 tryptase inhibitor Substances 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 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 3
- 102000004378 Melanocortin Receptors Human genes 0.000 description 3
- 108090000950 Melanocortin Receptors Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 108090000190 Thrombin Proteins 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 230000003042 antagnostic effect Effects 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002512 chemotherapy Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical group O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 description 3
- 208000014951 hematologic disease Diseases 0.000 description 3
- 239000000710 homodimer Substances 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 125000000468 ketone group Chemical group 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 238000010647 peptide synthesis reaction Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 230000017854 proteolysis Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 150000007970 thio esters Chemical group 0.000 description 3
- 229960004072 thrombin Drugs 0.000 description 3
- 230000003582 thrombocytopenic effect Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 239000002753 trypsin inhibitor Substances 0.000 description 3
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000001189 Cyclic Peptides Human genes 0.000 description 2
- 108010069514 Cyclic Peptides Proteins 0.000 description 2
- 108060002063 Cyclotide Proteins 0.000 description 2
- 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 2
- 240000006337 Ecballium elaterium Species 0.000 description 2
- 101000609473 Ecballium elaterium Trypsin inhibitor 2 Proteins 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000928179 Homo sapiens Agouti-related protein Proteins 0.000 description 2
- 208000028622 Immune thrombocytopenia Diseases 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical group C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 208000013544 Platelet disease Diseases 0.000 description 2
- 101100084022 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) lapA gene Proteins 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Chemical group CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Chemical group 0.000 description 2
- 241000700618 Vaccinia virus Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000001270 agonistic effect Effects 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000010322 bone marrow transplantation Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000005277 cation exchange chromatography Methods 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 238000010382 chemical cross-linking Methods 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 150000002019 disulfides Chemical class 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 238000007876 drug discovery Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical group NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 102000055839 human AGRP Human genes 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 230000017730 intein-mediated protein splicing Effects 0.000 description 2
- 238000003402 intramolecular cyclocondensation reaction Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 101150109249 lacI gene Proteins 0.000 description 2
- 150000002596 lactones Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 101150009573 phoA gene Proteins 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 239000000902 placebo Substances 0.000 description 2
- 229940068196 placebo Drugs 0.000 description 2
- 230000006337 proteolytic cleavage Effects 0.000 description 2
- 238000001959 radiotherapy Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000003571 reporter gene assay Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- PNGLEYLFMHGIQO-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)-2-hydroxypropane-1-sulfonate;dihydrate Chemical compound O.O.[Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(OC)=C1 PNGLEYLFMHGIQO-UHFFFAOYSA-M 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 230000005030 transcription termination Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 description 2
- 239000008158 vegetable oil Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- MIJDSYMOBYNHOT-UHFFFAOYSA-N 2-(ethylamino)ethanol Chemical compound CCNCCO MIJDSYMOBYNHOT-UHFFFAOYSA-N 0.000 description 1
- 102000012440 Acetylcholinesterase Human genes 0.000 description 1
- 108010022752 Acetylcholinesterase Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 206010002368 Anger Diseases 0.000 description 1
- 244000105975 Antidesma platyphyllum Species 0.000 description 1
- 208000019838 Blood disease Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000701822 Bovine papillomavirus Species 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 229940121981 Carboxypeptidase inhibitor Drugs 0.000 description 1
- 101710127041 Carboxypeptidase inhibitor Proteins 0.000 description 1
- 102000003902 Cathepsin C Human genes 0.000 description 1
- 108090000267 Cathepsin C Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 241000237971 Conus magus Species 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 241000702421 Dependoparvovirus Species 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
- 206010013710 Drug interaction Diseases 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 101710161822 Extracellular ribonuclease Proteins 0.000 description 1
- 108091008794 FGF receptors Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102000044168 Fibroblast Growth Factor Receptor Human genes 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
- 108010010803 Gelatin Proteins 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-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
- 239000007821 HATU Substances 0.000 description 1
- 206010066476 Haematological malignancy Diseases 0.000 description 1
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101001009599 Homo sapiens Granzyme A Proteins 0.000 description 1
- 101001002709 Homo sapiens Interleukin-4 Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- NNJVILVZKWQKPM-UHFFFAOYSA-N Lidocaine Chemical compound CCN(CC)CC(=O)NC1=C(C)C=CC=C1C NNJVILVZKWQKPM-UHFFFAOYSA-N 0.000 description 1
- 206010025323 Lymphomas Diseases 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
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 210000002361 Megakaryocyte Progenitor Cell Anatomy 0.000 description 1
- 101710140999 Metallocarboxypeptidase inhibitor Proteins 0.000 description 1
- 102100039364 Metalloproteinase inhibitor 1 Human genes 0.000 description 1
- 241000872931 Myoporum sandwicense Species 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 108090000189 Neuropeptides Proteins 0.000 description 1
- 102000003797 Neuropeptides Human genes 0.000 description 1
- 101710138657 Neurotoxin Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- 208000020584 Polyploidy Diseases 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- 108010011005 STAT6 Transcription Factor Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 102100023980 Signal transducer and activator of transcription 6 Human genes 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 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
- 229930006000 Sucrose Natural products 0.000 description 1
- 108091005735 TGF-beta receptors 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
- 239000004098 Tetracycline Substances 0.000 description 1
- 208000031981 Thrombocytopenic Idiopathic Purpura Diseases 0.000 description 1
- 108010070774 Thrombopoietin Receptors Proteins 0.000 description 1
- 102100034196 Thrombopoietin receptor Human genes 0.000 description 1
- 102000016715 Transforming Growth Factor beta Receptors Human genes 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 229940022698 acetylcholinesterase Drugs 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001299 aldehydes Chemical group 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 239000000538 analytical sample Substances 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000010775 animal oil Substances 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 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
- 239000003963 antioxidant agent Substances 0.000 description 1
- 229910052586 apatite Inorganic materials 0.000 description 1
- 238000002617 apheresis Methods 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 238000010420 art technique Methods 0.000 description 1
- VSRXQHXAPYXROS-UHFFFAOYSA-N azanide;cyclobutane-1,1-dicarboxylic acid;platinum(2+) Chemical compound [NH2-].[NH2-].[Pt+2].OC(=O)C1(C(O)=O)CCC1 VSRXQHXAPYXROS-UHFFFAOYSA-N 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- BPKIGYQJPYCAOW-FFJTTWKXSA-I calcium;potassium;disodium;(2s)-2-hydroxypropanoate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].C[C@H](O)C([O-])=O BPKIGYQJPYCAOW-FFJTTWKXSA-I 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 229960004562 carboplatin Drugs 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 108010005905 delta-hGHR Proteins 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 238000009585 enzyme analysis Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 229960004887 ferric hydroxide Drugs 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- YQEMORVAKMFKLG-UHFFFAOYSA-N glycerine monostearate Natural products CCCCCCCCCCCCCCCCCC(=O)OC(CO)CO YQEMORVAKMFKLG-UHFFFAOYSA-N 0.000 description 1
- SVUQHVRAGMNPLW-UHFFFAOYSA-N glycerol monostearate Natural products CCCCCCCCCCCCCCCCC(=O)OCC(O)CO SVUQHVRAGMNPLW-UHFFFAOYSA-N 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 229940015043 glyoxal Drugs 0.000 description 1
- 235000009424 haa Nutrition 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 208000018706 hematopoietic system disease Diseases 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229940094991 herring sperm dna Drugs 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 102000055229 human IL4 Human genes 0.000 description 1
- 235000011167 hydrochloric acid Nutrition 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 125000004356 hydroxy functional group Chemical group O* 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- IEECXTSVVFWGSE-UHFFFAOYSA-M iron(3+);oxygen(2-);hydroxide Chemical compound [OH-].[O-2].[Fe+3] IEECXTSVVFWGSE-UHFFFAOYSA-M 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 229960004194 lidocaine Drugs 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000003589 local anesthetic agent Substances 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 238000003468 luciferase reporter gene assay Methods 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000008384 membrane barrier Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 210000000110 microvilli Anatomy 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000004898 n-terminal fragment Anatomy 0.000 description 1
- 239000002581 neurotoxin Substances 0.000 description 1
- 231100000618 neurotoxin Toxicity 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 239000000346 nonvolatile oil Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000002895 organic esters Chemical class 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 230000006320 pegylation Effects 0.000 description 1
- VSIIXMUUUJUKCM-UHFFFAOYSA-D pentacalcium;fluoride;triphosphate Chemical compound [F-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O VSIIXMUUUJUKCM-UHFFFAOYSA-D 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- RGCLLPNLLBQHPF-HJWRWDBZSA-N phosphamidon Chemical compound CCN(CC)C(=O)C(\Cl)=C(/C)OP(=O)(OC)OC RGCLLPNLLBQHPF-HJWRWDBZSA-N 0.000 description 1
- 229940080469 phosphocellulose Drugs 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 108010002543 polyethylene glycol-recombinant human megakaryocyte growth and development factor Proteins 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- BPKIMPVREBSLAJ-UHFFFAOYSA-N prialt Chemical compound N1C(=O)C(CCSC)NC(=O)C(CC(C)C)NC(=O)C(CCCNC(N)=N)NC(=O)C(CO)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(C)NC(=O)CNC(=O)C(CCCCN)NC(=O)CNC(=O)C(CCCCN)NC(=O)C(N)CSSC2)CSSCC(C(NC(CCCNC(N)=N)C(=O)NC(CO)C(=O)NCC(=O)NC(CCCCN)C(=O)NC(CSSC3)C(N)=O)=O)NC(=O)C(CO)NC(=O)CNC(=O)C(C(C)O)NC(=O)C2NC(=O)C3NC(=O)C(CC(O)=O)NC(=O)C1CC1=CC=C(O)C=C1 BPKIMPVREBSLAJ-UHFFFAOYSA-N 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 239000013636 protein dimer Substances 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 239000012268 protein inhibitor Substances 0.000 description 1
- 229940121649 protein inhibitor Drugs 0.000 description 1
- 230000030788 protein refolding Effects 0.000 description 1
- 230000016434 protein splicing Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 1
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000007320 rich medium Substances 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 210000004739 secretory vesicle Anatomy 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- RYYKJJJTJZKILX-UHFFFAOYSA-M sodium octadecanoate Chemical compound [Na+].CCCCCCCCCCCCCCCCCC([O-])=O RYYKJJJTJZKILX-UHFFFAOYSA-M 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 108010004034 stable plasma protein solution Proteins 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- 239000008227 sterile water for injection Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 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
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 101150061166 tetR gene Proteins 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 230000036964 tight binding Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000005891 transamination reaction Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 238000001086 yeast two-hybrid system Methods 0.000 description 1
- 108091058538 ω-conotoxin MVIIA Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/02—Nasal agents, e.g. decongestants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Pulmonology (AREA)
- Gastroenterology & Hepatology (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Rheumatology (AREA)
- Biomedical Technology (AREA)
- Physical Education & Sports Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Pain & Pain Management (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Dermatology (AREA)
- Otolaryngology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
I)このようなペプチドは、立体構造的に高度に柔軟なので、それ自体がタンパク質分解を受けやすく、このため血漿半減期は短い。加えて、胃内および腸内における、ならびに腸管刷子縁膜の表面上におけるプロテアーゼ(ペプシン、トリプシン、エラスターゼ、キモトリプシンなど)によるタンパク質分解性の攻撃に対する感受性があるために、例えば血小板減少症の治療目的の経口投与は行われていない。
II)Cwirla et al. (1997)に記載されているようなペプチドを組換え的に作製することは困難である。小型のペプチドは、細胞プロテアーゼによってしばしば速やかに分解されることから、微生物発現系において高収量で過剰発現させることは極めて困難である。したがって、望ましくない副産物の発生などの、その全ての既知の短所(例えば、合成の成熟前の終結に起因する切断;または立体異性のアミノ酸の取り込み)のある、化学的なペプチド合成が選択される方法である。
(a)SEQ ID NO: 1〜6の任意の1つで表されるアミノ酸配列、
(b)該ポリペプチド内に存在時にTPO受容体を刺激可能な、(a)のアミノ酸配列の断片、ならびに
(c)該ポリペプチド中に存在時にTPO受容体を刺激可能な、(a)もしくは(b)のアミノ酸配列または断片の少なくとも1残基が置換されている、付加されている、および/または欠失されている機能的等価物。
ハイブリダイゼーション用緩衝液:2×SSC;10×デンハート溶液(Fikoll 400 + PEG + BSA;比1:1:1);0.1% SDS;5 mM EDTA;50 mM Na2HPO4;250μg/mlのニシン精子DNA;50μg/mlのtRNA;または0.25 Mリン酸ナトリウム緩衝液、pH 7.2;1 mM EDTA、7% SDS
ハイブリダイゼーション温度T = 60℃
洗浄用緩衝液:2×SSC;0.1% SDS
洗浄温度T = 60℃
(a)N末端に反応性カルボニル基を含み、およびC末端にホモセリンラクトン残基を含むマイクロタンパク質基質を提供する段階;ならびに
(b)前記N末端基およびC末端の残基がヒドラゾン結合に変換するように、マイクロタンパク質基質を反応させる段階。
(i)C末端のホモセリンラクトン残基をホモセリンヒドラジドと反応させる段階;
(ii)ホモセリンヒドラジドとN末端の反応性カルボニル基を反応させてヒドラゾンを生じさせる段階;ならびに
(iii)任意でヒドラゾンを還元させる段階。
(A)グラフトペプチドと置換されるマイクロタンパク質のループアミノ酸の配列を、メチオニンと後続のアミノ酸残基の間の適切なペプチド結合において臭化シアンで切断する段階(ループアミノ酸配列のC末端における後続のアミノ酸残基はセリン残基またはスレオニン残基である);
(B)セリン残基またはスレオニン残基を、グリオキシリル基を形成するように穏やかな酸化によって反応させる段階;
(C)N末端の反応性カルボニル基およびC末端のホモセリンラクトンヒドラジドを含むグラフトペプチド配列を、マイクロタンパク質のグリオキシリル基と反応させてヒドラゾンを生じさせる段階;
(D)段階(A)の切断によって生じたC末端のホモセリンラクトン残基を、ホモセリンヒドラジドと反応させる段階;ならびに
(E)(D)のホモセリンヒドラジドを、グラフトペプチドのN末端の反応性カルボニル基と反応させてヒドラゾンを生じさせる段階。
TPOアゴニストおよびアンタゴニストの産生および測定
材料および方法
分子生物学的手法
実施例において特に言及されない限り、全ての組換えDNA手法は、Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA、またはAusubel et al. (1994), Current Protocols in Molecular Biology, Current Protocolsの第1巻および第2巻に記載されたプロトコルに従って実施される。
バチルス・アミロリクエファシエンス(B. amyloliquefaciens)のRNAseの酵素的に不活性な異型であるバルナーゼ'、および個々のマイクロタンパク質のコード配列からなる融合遺伝子の構築は、基本的に文献に記載された方法で行った。マイクロタンパク質の遺伝子を、標準的なクローニング法(Sambrook et al., 2001)によるポリメラーゼ連鎖反応によって発現ベクターpBar100中に集合させた(図2、EP 04 02 2455.2)。ET-TP-020をコードする遺伝子を、プラスミドpBar100-EETI-II M7I(Schmoldt et al., 2004)をテンプレートとして、ならびにオリゴヌクレオチド対のbarmitte-up
およびETTP21-SOE1lo
、ならびにETTP21-SOE2up-new
、およびcathindmitte-lo
を使用して、Taq DNAポリメラーゼ(NEB)を使用する2段階のSOE PCRで集合させた。結果として得られた産物をゲル電気泳動と、これに続くQIAquick Gel抽出キット(Quiagen)を使用したゲル抽出で精製した。次にこれらを、隣接するオリゴヌクレオチドbarmitte-upおよびcathindmitte-loによる第2のPCR反応のテンプレートとして使用した。得られた生成物をフェノール/クロロフォルム抽出法およびエタノール沈殿法で精製し、NcoIおよびHindIIIで切断し、同様に消化されたpBar100-EETI-II M7I中に連結した。
文献に記載された手順で融合タンパク質を発現させて精製した。個々のマイクロタンパク質を、融合パートナーであるバルナーゼ'から、臭化シアン処理によって放出させた。マイクロタンパク質を逆相HPLCによって、文献(Schmoldt et al., 2004、およびEP 04 02 2455.2)に記載された手順で精製した。
TPOR/4Rαハイブリッド受容体用の発現ベクターであるpcDNATPOR/4Rαを以下の手順で構築した。ヒトTPO受容体のコード配列を含むcDNAクローンを対象に、オリゴヌクレオチドTpoR-Xho-lo
およびTpoR-Xho-up
を使用してPCR反応を行った。結果として得られたPCR産物をXhoIで消化し、同様に消化されたベクターpASKcDNA-NHに連結してpASKcDNA-NH-TPORを得た。pcDNA/4RαのNheI/HindIII断片(Krause et al., 2004)をpASK21TETIsendc1/2 (Christmann et al., 1999)にサブクローニングすることでpASKcDNA-NHを得た。ベクターpASKcDNA-NH-TPORから、TPORのコード配列を含むNheI/HindIII断片を、同様に消化されたpcDNA/4Rαに連結してpcDNATPOR/4Rαを得た。STAT6レポーター遺伝子コンストラクトpIεTATALucを、ヒトIεプロモーター(Ezernieks et al., 1996)のIL-4応答領域に由来するプロモーター/エンハンサー配列の全体を含む合成HindIII/BamHI断片をルシフェラーゼ発現プラスミドpTATALuc+ (Altschmied et al., 1997)に挿入して作製した。
マウスのプレB細胞系列Ba/F3を文献(Lischke et al., 1995)に記載された手順で培養した。細胞を、細胞系列Nucleofector(商標)キットV(Amaxa, Germany)を使用してトランスフェクトした。簡単に説明すると、8×106個の細胞をRPMI 1640/10% FCS中で2時間、飢餓状態におき、遠心分離し、4μgの発現ベクターpcDNATPOR/4Rαおよび1μgのレポーター遺伝子コンストラクトpIεTATALucを添加した100μlのトランスフェクション試薬Vに再懸濁した。トランスフェクションは、プログラムT16を使用するNucleofector(商標)装置で実施した。個々のトランスフェクションバッチを3.5 mlのRPMI 1640/10% FCS中に回収し、1ウェルあたり2×105個の細胞/100μlとなるように96ウェル細胞培養プレートに添加した。細胞を何ら処理せずに1時間、静置した後に、さまざまな濃度のhTPO (Immunotools, Germany)、および個々のマイクロタンパク質候補をそれぞれ総容量が200μlとなるように添加した。TPO競合アッセイ法に関しては、細胞のアリコートを個々のマイクロタンパク質試料と1時間プレインキュベートした後にhTPOで刺激した。37℃、5% CO2で12時間のインキュベーション後に、細胞溶解物を調製し、ルシフェラーゼ活性を文献(Krause et al., 2004)に記載された手順で測定した。
TpoR結合配列(小文字で示す)が移植されたマイクロタンパク質のアミノ酸配列を表1に示す。マイクロタンパク質の足場として、マイクロタンパク質AGRP'、ヒトアグーチ関連タンパク質(McNulty et al., 2001)のメラノコルチン受容体結合ドメイン、およびマイクロタンパク質EETI-II(Christmann et al., 1999)をそれぞれ使用した。下線を付したアミノ酸をスペーサー配列として、またはSE-AG-TP-050の場合にはTPOR結合ペプチドの配列から外れるように導入した。アンタゴニスト活性の決定に際しては、マイクロタンパク質をバルナーゼ'との融合タンパク質として使用した(図2およびEP 04 02 2455.2)。アゴニスト活性の決定に関しては、精製済みのマイクロタンパク質を二量体として使用した。
表1:トロンボポエチン受容体(TPOR)結合配列によるループ置換を有するマイクロタンパク質のアミノ酸配列。タンパク質を、酵素的に不活性なバルナーゼ(バルナーゼ')との融合タンパク質として産生させ、hTPO受容体の活性化の阻害能力または誘導能力(拮抗作用)を検討した。アゴニスト活性の検討対象のマイクロタンパク質は化学的に二量体化されている。適用した試験はTPORの細胞外ドメインの二量体形成を誘導する能力の判定を含んだ。n.d.:決定せず。
マイクロタンパク質の合成後の環化
実験手順
省略形
Boc:tert-ブトキシカルボニル;DTT:ジチオトレイトール;ESI-MS:エレクトロスプレーイオン化質量分析;Fmoc:9-フルオレニルメチルオキシカルボニル;HATU:2-(1H-9-アザベンゾトリアゾール-1-イル)-1,3,3,3-テトラメチルウロニウムヘキサフルオロリン酸;HBTU:2-(1H-ベンゾトリアゾール-1-イル)-1,3,3,3-テトラメチルウロニウムヘキサフルオロリン酸;HOBt:1-ヒドロキシ-1H-ベンゾトリアゾール;HPLC:高圧液体クロマトグラフィー;SPPS:固相ペプチド合成;TFA:トリフルオロ酢酸。
試薬および溶媒は最高品質の市販品とし、さらなる精製を行わずに使用した。シアノホウ化水素ナトリウムおよびm-過ヨウ素酸ナトリウムはSIGMA-Aldrichから購入し、ヒドラジン一水和物および臭化シアンはFluka (Taufkirchen, Germany)から購入した。ESI質量スペクトルは、TSQ 700 Finnegan分光器で測定した。HPLCは、YMC J'sphere ODS H-80、RP C-18カラムを調製実験(250×4.6 mm、4μm、80Å)、および解析用試料(250×4.6μm、80Å)に使用して、Pharmacia Actaをベースとするシステムで実施した。
発現ベクターpBar100-cyclo-McoEeTIの作製では、McoEeTIのコード配列を、Taqポリメラーゼ(Eppendorf)を使用する2段階のSOE-PCRで増幅した。初回のPCRは、プラスミドpBar100-McoEeTI(図2;EP 04 02 2455.2;Schmoldt et al., 2004)をテンプレートとして、ならびにオリゴヌクレオチドBspHI-McoTI-MSDGG-up
およびMCoTI-MSDGGhinten-SOE-lo
、ならびにMCoTI-MSDGGhinten-SOE-up
およびcat-hind-Mitte-lo
をそれぞれ使用して行い、2つの重複する断片を得た。これらの断片を、隣接するオリゴヌクレオチドBspHI-McoTI-MSDGG-upおよびcat-hind-Mitte-loを使用する第2のPCRテンプレートとして使用した。得られた産物をPagIおよびHindIII (MBI Fermentas)で消化し、事前にNcoIおよびHindIIIで切断されたpBar100-EETI-II M7I (Schmoldt et al., 2004)に連結して、発現ベクターpBar100-cyclo-McoEeTIを得た。このプラスミドは、tacプロモーターの制御下に、精製ハンドルとして使用される、バチルス・アミロリクエファシエンス(Bacillus amyloliquefaciens)のRNaseバルナーゼの不活性化突然変異体であるバルナーゼ'、phoA周辺質リーダー配列、およびcyclo-McoEeTI遺伝子の遺伝子融合体を有する。結果として得られるバルナーゼ'-cyclo-McoEeTI融合タンパク質は、2つのメチオニン残基を、バルナーゼ'とcyclo-McoEeTIの境界、およびcyclo-McoEeTIのC末端に有する。これが臭化シアンによって切断されると、融合パートナーであるバルナーゼ'が除去され、ならびにN末端にセリンを、およびC末端にホモセリンラクトンを有するcyclo-McoEeTIペプチドが得られる(例えば図10のa、b参照)。
lacI遺伝子を含むヘルパープラスミドpRep4 (Qiagen)を含む大腸菌株71-18[F' lacIq lacZΔM15 proA+B+ Δlac-proAB supE thi1](入手先はB. Muller-Hill)をエレクトロポレーションによってpBar100-cyclo-McoEeTIで形質転換し、25μg/mlのクロラムフェニコールおよび37.5μg/mlのカナマイシンを含む50 mlのリッチ培地中で37℃で一晩、成長させた。産生は、5 Lの培養槽(Bioengineering)中で、文献(Schmoldt et al., 2004)に記載された手順で実施し、精製は、Schmoldt et al.に記載された手順に若干の変更を施して、バルナーゼ'を精製ハンドルとして使用して実施した。簡単に説明すると、バルナーゼ'-cyclo-McoEeTI融合タンパク質を、細胞培養液1リットルあたり55 mlの氷酢酸による酸性化後に培養液から精製した。この培養液を濾過し、H2Oで1:5の割合で希釈し、600 mlのSP-Sepharose XL (Amersham Biosciences)を含む、直径100 mmのガラス製カラムにアプライした。バルナーゼ'-融合タンパク質を100〜1000 mMのNaClの段階的な勾配で溶出し、および融合タンパク質を含むフラクションを、130 mlのAmberchrom CG-300M(Tosoh Bioscience)を含む26 mmのガラス製カラムに直接アプライした。H2O/0.1%(v/v)酢酸で洗浄後に、融合タンパク質をカラムから、0%(v/v)〜90%イソプロパノール/0.1%(v/v)酢酸の勾配を用いて溶出した。約25〜40%(v/v)のイソプロパノールの範囲の融合タンパク質含有フラクションを混合して凍結乾燥した。8 Mの尿素に再溶解し、および50 mMの酢酸アンモニウムに対して透析した後に、別の陽イオン交換クロマトグラフィーを、Vision BioCadワークステーション(PerSeptive Biosystems)に取り付けられたSP-Sepharose XL (Amersham Biosciences)を含むXK26カラム(2.6 x 20 cm、ベッドボリューム100 ml)を用いて流速8 mL/分で行った。溶出は、0〜0.5 M NaClの勾配を設けて行った。フラクションを含む融合タンパク質を混合し、および1/10容量の37% HClを添加してタンパク質を沈殿させ、ならびにHaereus Omnifuge 3L-R中で4000 rpmで10分間、遠心分離した。
沈殿した融合タンパク質を、タンパク質1 mgあたり20 mlの0.2 M HCl/8 M尿素に可溶化し、1μlの5 M臭化シアン溶液(Fluka)を添加した(Kaiser and Metzka, 1999)。一晩のインキュベーション後に、試料を、Amberchrom CG-300M(Tosoh Bioscience、ベッドボリューム100 ml)を含むXK26カラム(Amersham Biosciences)に直接アプライした。H2O/0.1%(v/v) TFAで洗浄後に、切断されたcyclo-McoEeTIペプチドをバルナーゼ'から、5%(v/v)〜90%アセトニトリル/0.1%(v/v) TFAの勾配を利用して分離した。約20〜30%(v/v)アセトニトリルの範囲のcyclo-McoEeTIを含むフラクションを混合して凍結乾燥した。追加の精製を、Pharmacia Actaをベースとしたシステムで、YMC J'sphere ODS H-80、RP C-18調製用カラムを使用して行い、5 mgの純粋なMcoEeTI-セリンラクトンを得た。
ヒドラジン水和物(7μL、140μmol)を、水に溶解したMcoEeTIホモセリンラクトン(2,4.7 mg、1.4μmol)溶液(2 mL)に添加した。この混合物を室温で1時間、攪拌した。反応は、解析HPLCで制御した。開始時の試料に含まれるラクトンのピークが消失した後に、反応混合物を凍結乾燥して過剰なヒドラジンを除去した。凍結乾燥後の乾燥残渣を水-アセトニトリル混合液に再溶解し、および調製用HPLCで精製した。純粋な収率:2.1 mg(44.3%)。HPLC:tR=ESI MS(メタノール)の結果は以下の通りであった:m/z 856.0 ([M+4H]4+, 100)、1152.6 ([M+3H]3+, 33)、692.2 ([M+5H]5+, 27)、1728.6 ([M+2H]2+, 5)。
McoeETIヒドラジド(1.6 mg、0.46μmol)をリン酸緩衝液(1 mL、0.01 mmol、pH 7)に溶解した。NaIO4(1 mg、4.6μmol)を、リン酸緩衝液を溶媒とする溶液(1 mL)として室温で添加した。5分後にHPLCに注入することで反応を終了させた。モニタリングは、215 nmおよび280 nm(大環状分子の吸光度)で行った。純度は1 mg(63.7%)であった。
Claims (20)
- 非ペプチドカップリングによって連結されている少なくとも2個のマイクロタンパク質(microprotein)を含み、標的タンパク質に特異的に結合するポリペプチド:
ここで、該マイクロタンパク質は、阻害剤シスチンノット(ICK)ポリペプチドファミリーの成員であり、
該少なくとも2個のマイクロタンパク質は、該マイクロタンパク質内に移植された、標的タンパク質に対する特異的な結合活性を有するアミノ酸配列を含み、および
非ペプチドカップリングが、二機能性またはオリゴ機能性(oligofunctional)のリンカー分子を含む。 - リンカー分子が、アジピン酸ヒドラジド、ビス-スクシンイミジル-スベラート(DSS)、およびEDTA-ヒドラジドから選択される、請求項1記載のポリペプチド。
- 個々のマイクロタンパク質が少なくとも6個のシステイン残基を含み、このうち6個のシステイン残基がシスチンノットを形成するように、ジスルフィド結合を介して連結されている、請求項1または2に記載のポリペプチド。
- マイクロタンパク質の少なくとも1個が、アミノ酸モチーフCX3-CX4-CX4-7-CX1-CX4-5-CX5-7(SEQ ID NO: 8)を含み、Xが相互に独立して任意のアミノ酸残基であることを意味する、請求項1〜3のいずれか一項記載のポリペプチド。
- マイクロタンパク質が28〜40アミノ酸の長さを有する、請求項1〜4のいずれか一項記載のポリペプチド。
- 標的タンパク質が膜結合型受容体である、請求項1乃至5のいずれか一項記載のポリペプチド。
- 前記受容体が、該受容体の分子が相互に近接する場合にシグナル伝達カスケードの下流の他のタンパク質を活性化する、請求項6記載のポリペプチド。
- 前記受容体がトロンボポエチン(TPO)受容体である、請求項7記載のポリペプチド。
- TPO受容体に対する結合活性を有するアミノ酸配列が、アミノ酸配列IEGPTLRQWLAARA (SEQ ID NO: 7)を含む、請求項8記載のポリペプチド。
- 少なくとも2個のマイクロタンパク質が、以下からなる群より選択されるアミノ酸配列を含む、請求項8または9記載のポリペプチド:
(a)SEQ ID NO: 2、3、5または6の任意の1つで表されるアミノ酸配列、および
(b)該ポリペプチド内に存在する場合にTPO受容体を刺激可能な、(a)のアミノ酸配列の断片。 - 標的タンパク質が二量体またはオリゴマーの酵素であり、および特異的な結合活性を有するアミノ酸配列が、該酵素の活性部位に結合する、請求項1乃至5のいずれか一項記載のポリペプチド。
- 前記酵素がトリプターゼである、請求項11記載のポリペプチド。
- 請求項1〜12のいずれか一項記載のポリペプチド、および任意で薬学的に許容される担体を含む薬学的組成物。
- TPO受容体を刺激することで処置または予防が可能な疾患もしくは状態の処置用または予防用の薬学的組成物を作製するための、請求項8〜10のいずれか一項記載のポリペプチドの使用。
- 疾患または状態が、血小板減少症、再生不良性貧血、骨髄不全、骨髄異形成症候群、および肝疾患からなる群より選択される、請求項14記載の使用。
- TPO受容体を刺激するための、請求項8〜10のいずれか一項記載のポリペプチド。
- トリプターゼの活性を阻害することで処置または予防が可能な疾患もしくは状態の処置用または予防用の薬学的組成物を作製するための、請求項12記載のポリペプチドの使用。
- 疾患もしくは状態が、喘息、炎症、乾癬、肺線維症、間質性肺疾患、慢性関節リウマチ、歯肉炎、歯周炎、アレルギー反応、アレルギー性鼻炎、骨関節症、アテローム性動脈硬化症、血管形成、多発性硬化症、および癌からなる群より選択される、請求項17記載の使用。
- トリプターゼの活性を阻害するための、請求項12記載のポリペプチド。
- 請求項1〜12、16または19のいずれか一項記載のポリペプチドを含むキット。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP05005292 | 2005-03-10 | ||
EP05005292.7 | 2005-03-10 | ||
PCT/EP2006/002188 WO2006094813A2 (en) | 2005-03-10 | 2006-03-09 | Dimeric or multimeric microproteins |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2008532505A JP2008532505A (ja) | 2008-08-21 |
JP5517450B2 true JP5517450B2 (ja) | 2014-06-11 |
Family
ID=36283202
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2008500131A Active JP5517450B2 (ja) | 2005-03-10 | 2006-03-09 | 二量体または多量体のマイクロタンパク質 |
Country Status (5)
Country | Link |
---|---|
US (1) | US8258258B2 (ja) |
EP (1) | EP1861417B1 (ja) |
JP (1) | JP5517450B2 (ja) |
CA (1) | CA2600749C (ja) |
WO (1) | WO2006094813A2 (ja) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU773891C (en) | 1998-10-23 | 2005-02-17 | Kirin-Amgen Inc. | Dimeric thrombopoietin peptide mimetics binding to MP1 receptor and having thrombopoietic activity |
WO2006032436A2 (en) * | 2004-09-21 | 2006-03-30 | Nascacell Technologies Ag. | Use of microproteins as tryptase inhibitors |
WO2009126290A2 (en) | 2008-04-09 | 2009-10-15 | Cornell University | Coferons and methods of making and using them |
WO2011043817A1 (en) | 2009-10-07 | 2011-04-14 | Cornell University | Coferons and methods of making and using them |
US8765698B2 (en) * | 2010-02-03 | 2014-07-01 | University Of Central Florida Research Foundation, Inc. | Methods and products for reawakening retrocyclins |
CA2957964A1 (en) | 2014-09-03 | 2016-03-10 | Immunogen, Inc. | Conjugates comprising cell-binding agents and cytotoxic agents |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2123062T3 (es) | 1992-08-21 | 1999-01-01 | Biogen Inc | Polipeptidos de transporte derivados de la proteina tat. |
US5869451A (en) | 1995-06-07 | 1999-02-09 | Glaxo Group Limited | Peptides and compounds that bind to a receptor |
EP1961760A3 (en) | 1995-06-07 | 2008-09-03 | Glaxo Group Limited | Peptides and compounds that bind to a thrombopoietin receptor |
WO2000058473A2 (en) * | 1999-03-31 | 2000-10-05 | Curagen Corporation | Nucleic acids including open reading frames encoding polypeptides; 'orfx' |
AUPQ339899A0 (en) | 1999-10-13 | 1999-11-04 | University Of Queensland, The | Novel molecules |
US6380356B1 (en) * | 1999-12-07 | 2002-04-30 | Advanced Medicine, Inc. | Multivalent polymyxin antibiotics |
WO2001053346A1 (en) * | 2000-01-18 | 2001-07-26 | Akzo Nobel N.V. | Human cystine knot polypeptide |
AU2001261363A1 (en) * | 2000-05-09 | 2001-11-20 | The Regents Of The University Of California | Methods and compounds for modulating melanocortin receptor ligand binding and activity |
GB0113657D0 (en) * | 2001-06-05 | 2001-07-25 | Geneprot Inc | Improved native chemical ligation with three or more components |
US7332474B2 (en) | 2001-10-11 | 2008-02-19 | Amgen Inc. | Peptides and related compounds having thrombopoietic activity |
WO2006032436A2 (en) | 2004-09-21 | 2006-03-30 | Nascacell Technologies Ag. | Use of microproteins as tryptase inhibitors |
-
2006
- 2006-03-09 EP EP06707500.2A patent/EP1861417B1/en active Active
- 2006-03-09 JP JP2008500131A patent/JP5517450B2/ja active Active
- 2006-03-09 US US11/886,007 patent/US8258258B2/en active Active
- 2006-03-09 CA CA2600749A patent/CA2600749C/en active Active
- 2006-03-09 WO PCT/EP2006/002188 patent/WO2006094813A2/en active Search and Examination
Also Published As
Publication number | Publication date |
---|---|
WO2006094813A2 (en) | 2006-09-14 |
US8258258B2 (en) | 2012-09-04 |
CA2600749A1 (en) | 2006-09-14 |
EP1861417B1 (en) | 2013-05-15 |
EP1861417A2 (en) | 2007-12-05 |
WO2006094813A3 (en) | 2007-03-08 |
JP2008532505A (ja) | 2008-08-21 |
US20090156476A1 (en) | 2009-06-18 |
CA2600749C (en) | 2014-04-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP4332163B2 (ja) | Mpl受容体に結合し血小板生成活性を有する二量体トロンボポエチンペプチド模倣体 | |
CN106573966B (zh) | 用于治疗代谢异常的组合物和方法 | |
KR101028626B1 (ko) | 혈소판신생 활성을 갖는 펩티드 및 관련 화합물 | |
US6121238A (en) | Peptides and compounds that bind to a receptor | |
JP4949844B2 (ja) | エリスロポエチン受容体に結合する新規ペプチド | |
JP6426103B2 (ja) | c−Metタンパク質アゴニスト | |
JP5517450B2 (ja) | 二量体または多量体のマイクロタンパク質 | |
CZ302303B6 (cs) | Homodimerní fúzní protein vykazující inhibicní aktivitu na angiogenezi, zpusob jeho produkce, molekula DNA a replikovatelný expresní vektor | |
US8956622B2 (en) | Peptidic antagonists of class III semaphorins/neuropilins complexes | |
ZA200602495B (en) | Peptides and compounds that bind to thrombopoietin receptors | |
IL123761A (en) | Truncated neurotrophic factors originating from the glial cell line, and medicinal preparations containing them | |
KR20140101319A (ko) | 인간 페리틴 유래 융합폴리펩티드 | |
JP2003508075A (ja) | Opg融合タンパク質組成物および方法 | |
RU2426745C2 (ru) | Рекомбинантный химерный белок фактора ингибирования нейтрофилов и гиругена и содержащая его фармацевтическая композиция | |
CN109893647A (zh) | 重组的弹性蛋白酶蛋白质及其制备方法和用途 | |
KR20070036057A (ko) | 코브라 베놈 인자-유사 기능을 갖는 사람 보체 c3 유도체 | |
JP2003503426A (ja) | FVIIaアンタゴニスト | |
US20050069987A1 (en) | Modified ciliary neurotrophic factor polypeptides with reduced antigenicity | |
US20210348152A1 (en) | A peptide, a complex and a method for treating cancer | |
US20090088374A1 (en) | Novel use | |
US6833437B2 (en) | Complement receptor type 1 (CR1)-like sequences | |
US7790155B2 (en) | Calbindin-D28K protection against glucocorticoid induced cell death | |
US6280968B1 (en) | Human PEC-60-like protein and DNA encoding the same | |
TWI316961B (en) | Peptides and related compounds having thrombopoietic activity | |
WO2021072056A1 (en) | P53 peptide disrupters of f0x04:p53 protein binding, variants and conjugates thereof. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20090302 |
|
A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20101215 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20101215 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20111121 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20120209 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20120216 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20120518 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20130204 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20130417 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20130424 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20130731 |
|
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: 20140326 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20140401 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 5517450 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 |
|
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 |