JPH032195A - Peptide and protease inhibitor - Google Patents
Peptide and protease inhibitorInfo
- Publication number
- JPH032195A JPH032195A JP1135678A JP13567889A JPH032195A JP H032195 A JPH032195 A JP H032195A JP 1135678 A JP1135678 A JP 1135678A JP 13567889 A JP13567889 A JP 13567889A JP H032195 A JPH032195 A JP H032195A
- Authority
- JP
- Japan
- Prior art keywords
- peptide
- amino acid
- arg
- gly
- acid sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 120
- 239000000137 peptide hydrolase inhibitor Substances 0.000 title claims description 14
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 title claims description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 22
- 150000003839 salts Chemical class 0.000 claims abstract description 16
- 239000004480 active ingredient Substances 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 abstract description 20
- 150000001413 amino acids Chemical class 0.000 abstract description 12
- 108091005804 Peptidases Proteins 0.000 abstract description 7
- 239000004365 Protease Substances 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 4
- 125000003277 amino group Chemical group 0.000 abstract description 3
- 241000124008 Mammalia Species 0.000 abstract description 2
- 239000002260 anti-inflammatory agent Substances 0.000 abstract description 2
- 239000002246 antineoplastic agent Substances 0.000 abstract description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 238000005227 gel permeation chromatography Methods 0.000 abstract description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 229940124599 anti-inflammatory drug Drugs 0.000 abstract 1
- 229940041181 antineoplastic drug Drugs 0.000 abstract 1
- 210000004899 c-terminal region Anatomy 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 22
- 102000004196 processed proteins & peptides Human genes 0.000 description 19
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 18
- 108010027597 alpha-chymotrypsin Proteins 0.000 description 14
- 230000015572 biosynthetic process Effects 0.000 description 14
- 238000003786 synthesis reaction Methods 0.000 description 13
- 108090000631 Trypsin Proteins 0.000 description 12
- 102000004142 Trypsin Human genes 0.000 description 12
- 239000012588 trypsin Substances 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 10
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 10
- 229940024606 amino acid Drugs 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 9
- -1 Organic acid salts Chemical class 0.000 description 9
- 238000009472 formulation Methods 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 229940088598 enzyme Drugs 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- 108090000317 Chymotrypsin Proteins 0.000 description 6
- MQUQNUAYKLCRME-INIZCTEOSA-N N-tosyl-L-phenylalanyl chloromethyl ketone Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N[C@H](C(=O)CCl)CC1=CC=CC=C1 MQUQNUAYKLCRME-INIZCTEOSA-N 0.000 description 6
- 102000035195 Peptidases Human genes 0.000 description 6
- 229960002376 chymotrypsin Drugs 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 125000006239 protecting group Chemical group 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 5
- 239000004809 Teflon Substances 0.000 description 5
- 229920006362 Teflon® Polymers 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000002674 ointment Substances 0.000 description 5
- 238000010647 peptide synthesis reaction Methods 0.000 description 5
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 4
- 101710116435 Outer membrane protein Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 4
- 238000009833 condensation Methods 0.000 description 4
- 230000005494 condensation Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 3
- QMMFVYPAHWMCMS-UHFFFAOYSA-N Dimethyl sulfide Chemical compound CSC QMMFVYPAHWMCMS-UHFFFAOYSA-N 0.000 description 3
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 description 3
- 101710132601 Capsid protein Proteins 0.000 description 2
- 101710094648 Coat protein Proteins 0.000 description 2
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 101710125418 Major capsid protein Proteins 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- 101710141454 Nucleoprotein Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 101710083689 Probable capsid protein Proteins 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- DANUORFCFTYTSZ-UHFFFAOYSA-N epinigericin Natural products O1C2(C(CC(C)(O2)C2OC(C)(CC2)C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)C)C(C)C(OC)CC1CC1CCC(C)C(C(C)C(O)=O)O1 DANUORFCFTYTSZ-UHFFFAOYSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 238000012933 kinetic analysis Methods 0.000 description 2
- 229960003511 macrogol Drugs 0.000 description 2
- DANUORFCFTYTSZ-BIBFWWMMSA-N nigericin Chemical compound C([C@@H]1C[C@H]([C@H]([C@]2([C@@H](C[C@](C)(O2)C2O[C@@](C)(CC2)C2[C@H](CC(O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C)O1)C)OC)[C@H]1CC[C@H](C)C([C@@H](C)C(O)=O)O1 DANUORFCFTYTSZ-BIBFWWMMSA-N 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- RGCLLPNLLBQHPF-HJWRWDBZSA-N phosphamidon Chemical compound CCN(CC)C(=O)C(\Cl)=C(/C)OP(=O)(OC)OC RGCLLPNLLBQHPF-HJWRWDBZSA-N 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229940018489 pronto Drugs 0.000 description 2
- 239000012521 purified sample Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-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
- JMTMSDXUXJISAY-UHFFFAOYSA-N 2H-benzotriazol-4-ol Chemical compound OC1=CC=CC2=C1N=NN2 JMTMSDXUXJISAY-UHFFFAOYSA-N 0.000 description 1
- YPLZVJKSYBUKBU-UHFFFAOYSA-N 3-amino-4-methylchromen-2-one Chemical compound C1=CC=CC2=C1OC(=O)C(N)=C2C YPLZVJKSYBUKBU-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- GJNDXQBALKCYSZ-RYUDHWBXSA-N Val-Phe Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 GJNDXQBALKCYSZ-RYUDHWBXSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
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- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
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- 230000006957 competitive inhibition Effects 0.000 description 1
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- 238000010511 deprotection reaction Methods 0.000 description 1
- MYRTYDVEIRVNKP-UHFFFAOYSA-N divinylbenzene Substances C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000003301 hydrolyzing effect Effects 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
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000009177 immunoglobulin therapy Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 108010093564 inter-alpha-inhibitor Proteins 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- TZBAVQKIEKDGFH-UHFFFAOYSA-N n-[2-(diethylamino)ethyl]-1-benzothiophene-2-carboxamide;hydrochloride Chemical compound [Cl-].C1=CC=C2SC(C(=O)NCC[NH+](CC)CC)=CC2=C1 TZBAVQKIEKDGFH-UHFFFAOYSA-N 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000006959 non-competitive inhibition Effects 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
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- 159000000001 potassium salts Chemical class 0.000 description 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
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- 159000000000 sodium salts Chemical class 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、医薬、免疫グロブリン作製用のハプテン等と
して有用な新規ペプチドおよび当該ペプチドをはじめと
する後天性免疫不全症候群の病原ウィルス(HIV)の
外膜蛋白関連ペプチド及びその誘導体を有効成分とする
プロテアーゼ阻害剤に関するものである。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel peptide useful as a medicine, a hapten for producing immunoglobulin, etc., and a pathogenic virus (HIV) that causes acquired immunodeficiency syndrome, including the peptide. The present invention relates to a protease inhibitor containing an outer membrane protein-related peptide and a derivative thereof as an active ingredient.
〔従来技術・発明が解決しようとする課題〕プロテアー
ゼ阻害剤は生体内において幅広い酵素系に影響を与え、
たとえば2.性膵炎、ショック、出血時の止血等の治療
等に使用されている。[Prior art/issues to be solved by the invention] Protease inhibitors affect a wide range of enzyme systems in vivo;
For example 2. It is used to treat pancreatitis, shock, and to stop bleeding.
現在用いられている、蛋白性のプロテアーゼ阻害剤では
抗体産生の可能性などの問題点があった。Currently used protein-based protease inhibitors have problems such as the possibility of antibody production.
従って、低分子性で抗体産生による副作用の少ないプロ
テアーゼ阻害剤の出現が待望されているのが実情である
。Therefore, the reality is that the emergence of a protease inhibitor that is low molecular weight and has fewer side effects due to antibody production is eagerly awaited.
本発明者らは、HIVの外被蛋白質(gp120)のア
ミノ酸配列からなるペプチド等のHIVの外膜蛋白関連
ペプチドについて研究をコメた結果、ペプチドにおいて
、後記の式(IV)で表されるアミノ酸配列部位を有し
さえすれば、優れたプロテアーゼ阻害作用を存すること
を見出した。The present inventors conducted research on HIV outer membrane protein-related peptides, such as peptides consisting of the amino acid sequence of HIV coat protein (gp120), and found that in peptides, the amino acid represented by formula (IV) below It has been found that as long as it has the sequence site, it has an excellent protease inhibitory effect.
即ち、HIVに対する中和抗体誘導における抗原決定基
としてすでに公知のアミノ酸配列(Proc。That is, the amino acid sequence (Proc) is already known as an antigenic determinant in inducing neutralizing antibodies against HIV.
Natl、 Acad、 Set、 USA、 FJ4
.3198〜3202 (1988))の中に、タンパ
ク質性トリプシンインヒビターとして広く知られている
ダイズのトリプシンインヒビターおよびボーマン−バー
クインヒビターのそれぞれの反応部位である一Arg−
Ile−および−Lys−3er−というアミノ酸配列
、並びに哺乳類の血清中に存在するプロテアーゼインヒ
ビターの一種であるインター−α−トリプシンインヒビ
クーの反応部位のアミノ酸配列と高い相同性を示すアミ
ノ酸配列を有することを見出し、この部分を含む合成ペ
プチドが生理活性物質として有効ではないかと考えるに
至った。Natl, Acad, Set, USA, FJ4
.. 3198-3202 (1988)), Arg-
It has an amino acid sequence of Ile- and -Lys-3er-, as well as an amino acid sequence that shows high homology with the amino acid sequence of the reaction site of inter-α-trypsin inhibitor, a type of protease inhibitor present in mammalian serum. This led them to consider that a synthetic peptide containing this portion might be effective as a physiologically active substance.
そこで、この部分を含む新規ペプチドを合成するととも
に、当該ペプチドをはじめとしてHIVの外膜蛋白関連
ペプチドのプロテアーゼ阻害効果を検討し、先に
(1)下式(A)のアミノ酸配列を有するペプチドまた
はその薬理学的に許容される塩。Therefore, we synthesized a new peptide containing this part and examined the protease inhibitory effect of this peptide and other HIV outer membrane protein-related peptides. its pharmacologically acceptable salts;
Cys−Thr−Arg−Pro−^5n−Asn−^
5n−Thr−^yg−Lys−3er−Ile−Ar
g−Ile−Gln−Arg−Gly−Pro−Gly
−Arg−Ala−Phe−Val−Thr−ロe−G
ly−Lys−Ile−Gly−^snMet−Arg
−Gln−Ala−His−(:ys (
A )(2)少なくとも下式(B)のアミノ酸配列を有
するペプチドまたはその薬理学的に許容される塩を有効
成分とするプロテアーゼ阻害剤。Cys-Thr-Arg-Pro-^5n-Asn-^
5n-Thr-^yg-Lys-3er-Ile-Ar
g-Ile-Gln-Arg-Gly-Pro-Gly
-Arg-Ala-Phe-Val-Thr-Roe-G
ly-Lys-Ile-Gly-^snMet-Arg
-Gln-Ala-His-(:ys (
A) (2) A protease inhibitor containing at least a peptide having the amino acid sequence of the following formula (B) or a pharmacologically acceptable salt thereof as an active ingredient.
Asn−Asn−Thr−Arg−Lys−5er−I
le−Arg−Ile−Gln−Arg−Gly−Pr
o−Gly−Arg−Ala−Phe−Val−Thr
−Ile−Gly−Lys41e−Giy
(B )を提案している(特願昭63−31
0634号)。Asn-Asn-Thr-Arg-Lys-5er-I
le-Arg-Ile-Gln-Arg-Gly-Pr
o-Gly-Arg-Ala-Phe-Val-Thr
-Ile-Gly-Lys41e-Giy
(B) (Patent application 1986-31)
No. 0634).
本発明者らはこのような状況下に、さらに研究を重ねた
ところ、式(B)のアミノ酸配列の一部である後記式(
IV)で表される、極めて少ないアミノ酸よりなるアミ
ノ酸配列部位を有すれば、プロテアーゼ、就中トリプシ
ンおよびキモトリプシンの活性を極めて有為に阻害する
ことを見出すとともに、後記の式(1)、(n)および
(III)で表される新規ペプチドを創製した。Under these circumstances, the present inventors conducted further research and found that the following formula (
It has been found that having an amino acid sequence region consisting of extremely few amino acids, represented by IV), can extremely significantly inhibit the activity of proteases, especially trypsin and chymotrypsin, and the following formulas (1), (n ) and (III) were created.
本発明はかかる新知見に基づいて完成されたものであり
、その第一番目の発明は下式(1)、(n)〜(I[[
)から選ばれるアミノ酸配列を有する新規ペプチド〔以
下、各々ペプチド(I)、ペプチド(n)およびペプチ
ド(I[[)という〕またはその薬理学的に許容される
塩(以下、単に塩ともいう)である。The present invention was completed based on this new knowledge, and the first invention is based on the following formulas (1), (n) to (I[[
) [hereinafter referred to as Peptide (I), Peptide (n) and Peptide (I [[)] respectively] or a pharmacologically acceptable salt thereof (hereinafter also simply referred to as salt) It is.
1ie−Arg−Ile−Gln−Arg−Gly−P
ro−Gly−Arg−Ala−Phe−Val
’ (1)+1e
−11is−11s−Gly−Pro−Gly−Arg
−Ala−Phe−Val ([1)Val−Phe
−Ala−Arg−Gly−Pro−Gly−Arg−
Gln−lieArg−IIs
(III)第二番目の本発明は、
少なくとも下式(IV)のアミノ酸配列部位を有し、か
つ下式(V)のアミノ酸配列を有しないペプチド〔以下
、このペプチドをペプチド(IV)誘導体という〕また
はその塩を有効成分とするプロテアーゼ阻害剤である。1ie-Arg-Ile-Gln-Arg-Gly-P
ro-Gly-Arg-Ala-Phe-Val
'(1)+1e
-11is-11s-Gly-Pro-Gly-Arg
-Ala-Phe-Val ([1) Val-Phe
-Ala-Arg-Gly-Pro-Gly-Arg-
Gln-lieArg-IIs
(III) The second invention is
Protease inhibition using a peptide having at least the amino acid sequence of the following formula (IV) and not having the amino acid sequence of the following formula (V) [hereinafter, this peptide is referred to as a peptide (IV) derivative] or a salt thereof as an active ingredient It is a drug.
Gly−Pro−Gly−Arg−(■)^sn−As
n−Thr−Arg−Lys−5er−Ile−Arg
−Ile−Gln八rへ−Gly−Pro−Gly−A
rg−Ala−(V )本明細書において、薬理学的に
許容される塩は、低毒性であれば特に制限されるもので
はなく、塩酸塩、酢酸塩、リン酸塩、クエン酸塩、コハ
ク酸塩等の有機酸塩、アルカリ金属塩(ナトリウム塩、
カリウム塩等)等の無機酸塩があげられる。Gly-Pro-Gly-Arg-(■)^sn-As
n-Thr-Arg-Lys-5er-Ile-Arg
-Ile-Gln8r-Gly-Pro-Gly-A
rg-Ala-(V) In the present specification, pharmacologically acceptable salts are not particularly limited as long as they have low toxicity, and include hydrochloride, acetate, phosphate, citrate, and succinate. Organic acid salts such as acid salts, alkali metal salts (sodium salts,
Examples include inorganic acid salts such as potassium salts, etc.
本発明において式(IV)で表されるアミノ酸配列はH
IVの外被蛍白質(gP120)における一部のアミノ
酸配列に該当するものである。しかして、ペプチド(I
V)誘導体は少なくとも上記の式(IV)で表されるア
ミノ酸配列部位を有するペプチドであれば特に制限はな
く、当該アミノ酸配列に付加的に1種以上のアミノ酸が
ペプチド結合したもの、その抗原性を減するためにこれ
らペプチドをポリエチレングリコールまたはそのFaR
体(ポリエチレングリコール、モノメトキシポリエチレ
ングリコール等)にて修飾したペプチド、その末端r−
GIyJまたは/およびrArg−,1部位に水素が付
加されて、当該部分が各々末端アミノ酸となっているも
の等が例示され、上記ペプチド(1)、(II)および
(I[[)、HIVの外膜蛋白由来のもの、これらの修
飾化合物等のHIVO外被蛋白質関連ペプチドが例示さ
れる。In the present invention, the amino acid sequence represented by formula (IV) is H
This corresponds to a part of the amino acid sequence in the coated fluorescent substance (gP120) of IV. However, the peptide (I
V) Derivatives are not particularly limited as long as they have at least the amino acid sequence region represented by the above formula (IV); These peptides can be combined with polyethylene glycol or its FaR to reduce
Peptides modified with polyethylene glycol, monomethoxypolyethylene glycol, etc., whose terminal r-
Examples include peptides (1), (II) and (I[[), HIV Examples include HIVO coat protein-related peptides, such as those derived from outer membrane proteins and modified compounds thereof.
本願発明におけるペプチド(1)、(II)および(I
[[)を含むペプチド(rV) 誘導体はgp120の
分解、通常のペプチド化学において使用されるペプチド
合成法、修飾法等によって製造される。Peptides (1), (II) and (I) in the present invention
Peptide (rV) derivatives containing [
ペプチド合成法としては、固相法、液相法のいずれでも
よい。固相法は、たとえば次のようにして行われる。即
ち、まずアミノ基が保護されたC末端アミノ酸をカルボ
キシル基によって不溶性担体に結合させる。不溶性担体
としては自体既知のものを使用すればよい0次にアミノ
保護基を除去した後、アミノ酸配列に従って順次アミノ
基の保護されたアミノ酸をペプチド結合させて一段ずつ
反応させ全アミノ酸配列を合成した後、不溶性担体を除
去することによって製造される。この際、反応性の官能
基はペプチド合成において既知の保護基によって保護し
ておくことが好ましい。The peptide synthesis method may be either a solid phase method or a liquid phase method. The solid phase method is performed, for example, as follows. That is, first, a C-terminal amino acid with a protected amino group is bonded to an insoluble carrier via a carboxyl group. As the insoluble carrier, a known one can be used. After removing the amino protecting group, the amino acids with protected amino groups were sequentially peptide bonded according to the amino acid sequence and reacted step by step to synthesize the entire amino acid sequence. After that, it is manufactured by removing the insoluble carrier. At this time, it is preferable that the reactive functional group is protected by a known protecting group in peptide synthesis.
かくして製造されたペプチドは、たとえばイオン交換樹
脂、分配クロマトグラフィー、ゲルクロマトグラフィー
、逆相クロマトグラフィー等のペプチド化学において通
常使用される精製方法によって任意の純度のものとして
精製することができる。The peptide thus produced can be purified to any degree of purity by purification methods commonly used in peptide chemistry, such as using ion exchange resins, partition chromatography, gel chromatography, reversed phase chromatography, and the like.
上記のようにして製造されたペプチドは自体既知の方法
によって塩とすることができ、また塩を自体既知の方法
によって加水分解することによって遊離のペプチドが製
造される。The peptide produced as described above can be converted into a salt by a method known per se, and a free peptide can be produced by hydrolyzing the salt by a method known per se.
本明細書において、アミノ酸、ペプチド、保護基等に関
して略号で表示する場合、それらはIUPAC−IUB
Co+u+1ssion on Biologica
l Nomenclatureによる略号あるいは当該
分野における慣用略号に基づくものであり、その例を次
にあげる。In this specification, when amino acids, peptides, protecting groups, etc. are indicated by abbreviations, they are IUPAC-IUB
Co+u+1 session on Biologica
The abbreviations are based on Nomenclature or common abbreviations in the field, examples of which are given below.
Thr:スレオニン ^「g:アルギニンPro
ニブロリン Lys : リジン■e:イソロイ
シン Gln: グルタミンGlyニゲリシン
Ala:アラニンPhe:フェニルアラニン
Val:バリン 旧S:ヒスチジンt−BO
C: t−ブチルオキシカルボニル〔作用・効果〕
ペプチド(1)、(II)および(Ill)を包含する
ペプチド(IV)誘導体およびその塩はヒト、イヌ、ウ
シ、ウマ、マウス、ラット等の哺乳動物に対して優れた
プロテアーゼ阻害活性、特にトリプシン阻害活性、キモ
トリプシン阻害活性を有するものであり、抗炎症剤、抗
腫瘍剤、発癌抑制剤等として、また免疫グロブリン療法
用の抗体作製のハブテン等として有用なものである。Thr: Threonine ^'g: Arginine Pro
Nibroline Lys: Lysine e: Isoleucine Gln: Glutamine Gly nigericin
Ala: Alanine Phe: Phenylalanine Val: Valine Old S: Histidine t-BO
C: t-Butyloxycarbonyl [Action/Effect] Peptide (IV) derivatives including peptides (1), (II) and (Ill) and their salts can be used in mammals such as humans, dogs, cows, horses, mice and rats. It has excellent protease inhibitory activity in animals, especially trypsin inhibitory activity and chymotrypsin inhibitory activity, and is used as an anti-inflammatory agent, antitumor agent, carcinogenesis inhibitor, etc., and as a hubten for producing antibodies for immunoglobulin therapy. It is useful.
本発明のプロテアーゼ阻害剤は、通常ペプチド(rV)
誘導体、その塩の凍結乾燥品として製剤化されて経口的
または非経口的に投与され、その剤型は投与経路等によ
って異なる。The protease inhibitors of the present invention are usually peptide (rV)
The derivative or its salt is formulated as a lyophilized product and administered orally or parenterally, and the dosage form varies depending on the route of administration.
本発明のプロテアーゼ阻害剤の好ましい剤型としては、
たとえば注射剤、カプセル剤、錠剤、クリーム、軟膏剤
、貼付剤、うめこみ剤等が例示され、これらは自体既知
の方法によって製造される。Preferred dosage forms of the protease inhibitor of the present invention include:
Examples include injections, capsules, tablets, creams, ointments, patches, and burying agents, which are manufactured by methods known per se.
たとえば、ペプチド(rv) 誘導体およびその塩を抗
炎症の治療削として使用する場合、その投与量は、たと
えば注射剤の場合、ペプチド(IV)誘導体またはその
塩として1日0.1〜1000s+g/人が一般的であ
り、これを1日1〜数回に分けて投与される。For example, when a peptide (RV) derivative or its salt is used as an anti-inflammatory therapeutic agent, the dosage is, for example, in the case of an injection, 0.1 to 1000 s+g/person per day as the peptide (IV) derivative or its salt. This is generally administered in one to several divided doses a day.
実験例1
トリプシン活性に対するペプチド(1)の阻害効果を、
酵素反応の速度論的解析により示した。Experimental Example 1 The inhibitory effect of peptide (1) on trypsin activity was
This was demonstrated by kinetic analysis of the enzymatic reaction.
市販の精製トリプシンtpg、50g+M)リス−塩酸
塩緩衝液pH8,0,0,15M塩化ナトリウムおよび
基質であるt−ブチルオキシカルボニル−し−フェニル
アラニル−L−セリル−L−アルギニン4−メチル−ク
マリル−7−アミドからなる反応液中に、ペプチド(I
)、または対照として蒸留水を添加し、螢光セル内で2
5°Cで反応させ、励起波長380ns+ 、発光波長
440n−の螢光強度を測定することによって酵素活性
を算出した。50μHのペプチド(1)の存在下でのラ
インライ−バー・パークプロットのパターンを第1図に
示した。ラインライ−バー・パークプロットは、上記の
反応液中で基[1度25.33.3.50.100μM
における酵素活性を、1秒間に酵素が基質と反応し7−
アミノ−4−メチルクマリンを1ピコモル生成させる酵
素単位を1pKatとして算出し、各々の基質濃度の逆
数に対して、算出した酵素活性の逆数をプロットするこ
とにより得た。Commercially available purified trypsin tpg, 50 g + M) Lis-hydrochloride buffer pH 8, 0, 0, 15 M sodium chloride and substrate t-butyloxycarbonyl-phenylalanyl-L-seryl-L-arginine 4-methyl- In a reaction solution consisting of coumaryl-7-amide, peptide (I
), or distilled water was added as a control and 2
The enzyme activity was calculated by reacting at 5°C and measuring the fluorescence intensity at an excitation wavelength of 380 ns+ and an emission wavelength of 440 n-. The Lineleiber-Park plot pattern in the presence of 50 μH of peptide (1) is shown in FIG. Reinleiber-Park plot shows that the group [1 degree 25.33.3.50.100 μM
The enzyme activity in 1 second is 7-
The enzyme unit that produces 1 pmol of amino-4-methylcoumarin was calculated as 1 pKat, and the reciprocal of the calculated enzyme activity was plotted against the reciprocal of each substrate concentration.
この結果からペプチド(1)によるトリプシン活性の阻
害が拮抗型であることがわかる。第1図中、Oはコント
ロール、・は50μMのペプチド(+)存在下での結果
である。This result shows that the inhibition of trypsin activity by peptide (1) is antagonistic. In FIG. 1, O is the control, and * is the result in the presence of 50 μM peptide (+).
実験例2
α−キモトリプシン活性に対するペプチド(1)の阻害
効果を、酵素反応の速度論的解析により示した。Experimental Example 2 The inhibitory effect of peptide (1) on α-chymotrypsin activity was demonstrated by kinetic analysis of the enzymatic reaction.
市販の精製α−キモトリプシン20μg、 50mMト
リス−塩酸塩pH8,0,10mM塩化ナトリウムおよ
び基質であるサクシニル−L−アラニル−し−アラニル
−し−プロリル−し−フェニルアラニル4−メチル−ク
マリル−7−アミドからなる反応液中にペプチド(I)
、または対照として蒸留水を添刺し、実験例1と同様の
方法にてラインライ−バー・バークプロントを得た。5
0μMのペフ。20 μg of commercially available purified α-chymotrypsin, 50 mM Tris-hydrochloride pH 8, 0, 10 mM sodium chloride and the substrate succinyl-L-alanyl-shi-alanyl-shi-prolyl-shi-phenylalanyl 4-methyl-coumaryl-7. - Peptide (I) in the reaction solution consisting of amide
Or, as a control, Rheinliver bark pronto was obtained in the same manner as in Experimental Example 1 except that distilled water was added. 5
0 μM pef.
チド(1)の存在下でのラインライ−バー・パークプロ
ットのパターンを第2図に示した。The Rheinleiber-Park plot pattern in the presence of Tido(1) is shown in FIG.
この結果からペプチド(1)によるα−キモトリプシン
活性の阻害が不拮抗型であることがわかる。第2図中、
○はコントロール、・は50μMのペプチド(1)存在
下での結果である。This result shows that the inhibition of α-chymotrypsin activity by peptide (1) is non-competitive. In Figure 2,
○ indicates the control, and . indicates the result in the presence of 50 μM peptide (1).
実験例3
ペプチド(I)、ペプチド(1)のアミノ酸配列の一部
から成るペプチドおよびペプチド(I)のアミノ酸配列
と異なる配列から成るペプチドについて、トリプシンお
よびα−キモトリプシンに対する阻害定数を求めた。Experimental Example 3 Inhibition constants for trypsin and α-chymotrypsin were determined for peptide (I), a peptide consisting of a part of the amino acid sequence of peptide (1), and a peptide consisting of a sequence different from the amino acid sequence of peptide (I).
阻害定数は、拮抗阻害を示すトリプシンに対してはデイ
クソンプロント、不拮抗阻害を示すαキモトリプシンに
対してはラインライ−バー・パークプロットから算出し
た。デイクソンプロットは、実験例2と同様の反応液中
で、濃度が0.20.40,60I!Mのペプチドを添
加した場合の酵素活性を算出し、各ペプチド濃度に対し
て酵素活性の逆数をプロットすることにより得た。Inhibition constants were calculated from the Dixon Pronto plot for trypsin showing competitive inhibition, and from the Reinleiber-Park plot for α-chymotrypsin showing non-competitive inhibition. The Dixon plot shows that in the same reaction solution as in Experimental Example 2, the concentration was 0.20.40.60I! The enzyme activity when peptide M was added was calculated and obtained by plotting the reciprocal of the enzyme activity for each peptide concentration.
トリプシンおよびα−キモトリプシンに対する阻害定数
を第1表に示した。この結果から、ペプチド(1)のみ
がトリプシン及びα−キモトリプシンの両方に対して著
しい阻害効果を持つことが示された。また、トリプシン
に対しては、アルギニンとリジンの数に依存した阻害で
あるのに対し、α−キモトリプシンに対しては、ペプチ
ド(1)が特異的に阻害していることがわかる。The inhibition constants for trypsin and α-chymotrypsin are shown in Table 1. This result showed that only peptide (1) had a significant inhibitory effect on both trypsin and α-chymotrypsin. Furthermore, it can be seen that the inhibition of trypsin depends on the number of arginine and lysine, whereas the peptide (1) specifically inhibits α-chymotrypsin.
第1表
夏 :イソロイシン R:アルギニン0 :グ
ルタミン G ニゲリシンP ニブロリン
A :アラニンF :フェニルアラニン
V :バリン実験例4
ペプチド(■)、ペプチド(■)、ペプチド(III)
の、キモトリプシン活性に対する阻害効果を調べた。Table 1 Summer: Isoleucine R: Arginine 0: Glutamine G Nigericin P Nibroline
A: Alanine F: Phenylalanine
V: Valine Experimental Example 4 Peptide (■), Peptide (■), Peptide (III)
The inhibitory effect of chymotrypsin on chymotrypsin activity was investigated.
実験例1と同様の方法にて基質濃度を100μMとした
場合のキモトリプシン活性を、ペプチドを加えないコン
トロールの活性を100%とした相対活性で表わした。The chymotrypsin activity when the substrate concentration was set to 100 μM in the same manner as in Experimental Example 1 was expressed as a relative activity, with the activity of the control to which no peptide was added as 100%.
ペプチド(1)、(II)および(III)の濃度依存
性を第3図に示した。The concentration dependence of peptides (1), (II) and (III) is shown in FIG.
この結果からペプチド(TV)誘導体であるペプチド(
II)および(II[)は、ペプチド(I)と類偵のト
リプシン阻害効果を示すことがゎがる。第3図中、・は
ペプチド(I)、ムはペプチド(n)閣はペプチド([
[I)存在下での結果である。From this result, peptide (TV) derivative, peptide (
II) and (II[) are likely to exhibit similar trypsin inhibitory effects to peptide (I). In Figure 3, . is peptide (I), mu is peptide (n), and peptide ([
[I) These are the results in the presence of
実験例5
ペプチド(■)、ペプチド(II)および(III)の
α−キモトリプシン活性に対する阻害効果を調べた。Experimental Example 5 The inhibitory effects of peptide (■), peptides (II) and peptides (III) on α-chymotrypsin activity were investigated.
実験例2と同様の方法にて基質濃度を100μMとした
場合のα−キモトリプシン活性を、ペプチドを加えない
コントロールの活性を100%とした相対活性で表わし
た。ペプチド([)、(U)および(I[l)の濃度依
存性を第4図に示した。The α-chymotrypsin activity when the substrate concentration was set to 100 μM in the same manner as in Experimental Example 2 was expressed as a relative activity with the activity of the control to which no peptide was added as 100%. The concentration dependence of peptides ([), (U) and (I[l) is shown in FIG. 4.
この結果からペプチド(U)および(III)は、ペプ
チド(+)と類似のキモトリプシン阻害効果を示すこと
がわかる。第4図中、・はペプチド(■)、ムはペプチ
ド(■)、■はペプチド(III)存在下での結果であ
る。This result shows that peptides (U) and (III) exhibit similar chymotrypsin inhibitory effects as peptide (+). In FIG. 4, * is the result in the presence of peptide (■), mu is the peptide (■), and ■ is the result in the presence of peptide (III).
比較例1
ペプチド(r)の阻害効果を、α−キモトリプシンを特
異的に阻害する市販のプロテアーゼ合成阻害剤であるL
−1−トシルアミド−2−フェニルエチルクロロメチル
ケトン(TPCK)の効果と比較した。Comparative Example 1 The inhibitory effect of peptide (r) was suppressed by L, a commercially available protease synthesis inhibitor that specifically inhibits α-chymotrypsin.
The effect was compared with that of -1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK).
実験例2と同様の方法にて、基質濃度を50μMとし、
ペプチド(1)の代わりにTPCKを添jltl した
ものとの相対活性を比較した。In the same manner as in Experimental Example 2, the substrate concentration was set to 50 μM,
The relative activity was compared with that in which TPCK was added instead of peptide (1).
ペプチド(1)およびTPCKの濃度依存性を第5図に
示した。The concentration dependence of peptide (1) and TPCK is shown in FIG.
この結果からペプチド(1)の方がTPCKよりも阻害
効果が大きいことがわかる。第5図中、・はペプチド(
1)、OはTPCK存在下での結果である。This result shows that peptide (1) has a greater inhibitory effect than TPCK. In Figure 5, . is a peptide (
1), O is the result in the presence of TPCK.
以下に実施例を挙げて本発明をより具体的に説明するが
、これらは本発明を何ら限定するものではない。EXAMPLES The present invention will be described in more detail with reference to Examples below, but these are not intended to limit the present invention in any way.
なお、以下の実施例で使用されるアミノ酸は、特に言及
しない限り何れもL一体である。Note that all amino acids used in the following examples are L-units unless otherwise specified.
合成例1 ペプチド(1)の合成を行なった。Synthesis example 1 Peptide (1) was synthesized.
ペプチド合成は、アプライド・バイオシステムズ社製ペ
プチドシンセサイザー43OAを用い、固相法により行
なった。即ち、0.5ミリモルのL−Boc−L−バリ
ン結合フェニルアセトアミドメチル樹脂(スチレン−1
%ジビニルベンゼン共重合体)に、N末端をt−Boc
で保護した2ミリモルのt−Bocアミノ酸をN、N−
ジメチルホルムアミド中、1ミリモルN、N−ジシクロ
へキシルカルボジイミドを用いた対称アミノ酸無水物法
で縮合し、ペプチド結合を1個ずつ延長させる逐次延長
法にて行なった。ただし、Gln、Argの縮合につい
ては、N、N−ジメチルホルムアミド中、1ミリモルの
ヒドロキシベンゾトリアゾールを用いた活性エステル法
で2回縮合した。Peptide synthesis was performed by a solid-phase method using Peptide Synthesizer 43OA manufactured by Applied Biosystems. That is, 0.5 mmol of L-Boc-L-valine-bonded phenylacetamidomethyl resin (styrene-1
% divinylbenzene copolymer), the N-terminus was
2 mmol of t-Boc amino acid protected with N,N-
Condensation was carried out by a symmetrical amino acid anhydride method using 1 mmol N,N-dicyclohexylcarbodiimide in dimethylformamide, followed by a sequential extension method in which peptide bonds were extended one by one. However, regarding the condensation of Gln and Arg, condensation was performed twice by an active ester method using 1 mmol of hydroxybenzotriazole in N,N-dimethylformamide.
t−BO(、アミノ酸の側鎖の保護基としては、Arg
では2,4.6−トリメチルベンゼンスルホニル基、L
ysではN −t−ブトキシカルボニル−N−2−ク
ロロベンジルオキシカルボニル基、SerおよびThr
ではベンジル基をそれぞれ用いた。t-BO (, as a protecting group for the side chain of an amino acid, Arg
2,4.6-trimethylbenzenesulfonyl group, L
ys is N-t-butoxycarbonyl-N-2-chlorobenzyloxycarbonyl group, Ser and Thr
In each case, a benzyl group was used.
ペプチドの標準合成プログラムは、脱保護として60%
トリフルオロ酢酸/ジクロロメタンで1分または15分
間、洗浄としてジクロロメタンで1分間を3回、中和と
して10%N、N−ジイソプロピルエチルアミンで1分
間を2回、洗浄としてN、N−ジメチルホルムアミドで
1分間を6回、縮合としてN、N−ジメチルホルムアミ
ド中でN。The standard synthesis program for peptides is 60% deprotection.
trifluoroacetic acid/dichloromethane for 1 min or 15 min, 3 x 1 min with dichloromethane as a wash, 2 x 1 min with 10% N, N-diisopropylethylamine as a neutralization, and 1 x 1 min with N, N-dimethylformamide as a wash. N in N,N-dimethylformamide as condensation for 6 min.
N−ジシクロヘキシカルボジイミドを用いた対称アミノ
酸無水物法を20分間、洗浄としてN、 N−ジメチル
ホルムアミドで1分間を2回およびジクロロメタンで4
回行う、これらの標準合成プログラムは、各アミノ酸に
対して、反応温度および反応時間等が詳細にマイクロコ
ンピュータにより制御されている。Symmetrical amino acid anhydride method with N-dicyclohexycarbodiimide for 20 min, two 1 min washes with N,N-dimethylformamide and 4x 1 min with dichloromethane.
In these standard synthesis programs, which are performed multiple times, the reaction temperature, reaction time, etc. for each amino acid are controlled in detail by a microcomputer.
ペプチド自動合成装置により得られたペプチド樹脂は、
トリフルオロメタンスルホン酸により支持体樹脂および
アミノ酸側鎖の保護基を切断した。The peptide resin obtained by the automatic peptide synthesizer is
The support resin and the protecting groups on the amino acid side chains were cleaved with trifluoromethanesulfonic acid.
即ち、合成ペプチド樹脂1gおよびチオアニソールlI
d、エタンジチオール500μlを室温で10分間撹拌
し、フラスコを氷水で冷却しながらトリフルオロ酢酸を
10−加え、10分間攪拌する。That is, 1 g of synthetic peptide resin and thioanisole II
d. Stir 500 µl of ethanedithiol at room temperature for 10 minutes, add 10% of trifluoroacetic acid while cooling the flask with ice water, and stir for 10 minutes.
次に1jlllのトリフルオロメタンスルホン酸を加え
、フラスコを氷水から取り出し室温で30分間攪拌する
。冷ジエチルエーテルをペプチドの沈澱が出なくなるま
でフラスコに加え、1分間攪拌しテフロンフィルターで
結晶を濾過し、これをさらに少量のトリフルオロ酢酸で
溶解し、冷ジエチルエーテル中に移送し、再びテフロン
フィルターで濾過し、これを2N酢酸等にて溶解し粗精
製ペプチドを得た。Next, 1 ml of trifluoromethanesulfonic acid is added, and the flask is taken out of the ice water and stirred at room temperature for 30 minutes. Add cold diethyl ether to the flask until no more peptide precipitates appear, stir for 1 minute, filter the crystals with a Teflon filter, further dissolve this in a small amount of trifluoroacetic acid, transfer to cold diethyl ether, and pass through the Teflon filter again. The crude peptide was filtered and dissolved in 2N acetic acid or the like to obtain a crudely purified peptide.
粗精製ペプチドは、オクタデシル基を結合させたシリカ
ゲル充填剤から成る逆相カラム(直径21、長さ25C
11)による高速液体クロマトグラフィー(カラム:
YMCR−ODS−5)により精製を行なった。溶出は
移動相としてO,1%トリフルオロ酢酸を用い90%ア
セトニトリルを30%〜70%直線濃度勾配させること
により行なった(流速5d/sin、) 、精製標品の
クロマトグラムは第6図(220nmの紫外吸収で検出
)に示した通りである。The crudely purified peptide was purified using a reverse phase column (diameter 21, length 25C) consisting of a silica gel packing with octadecyl groups bonded to it.
11) high performance liquid chromatography (column:
Purification was performed using YMCR-ODS-5). Elution was performed using O, 1% trifluoroacetic acid as the mobile phase and a linear concentration gradient of 90% acetonitrile from 30% to 70% (flow rate 5 d/sin). The chromatogram of the purified sample is shown in Figure 6 ( (detected by ultraviolet absorption at 220 nm).
精製したペプチドは、1ナノモルを気相シーケンサ−に
より分析した。その結果、分析サイクル1〜12で検出
されたアミノ酸残基は、順に、IIe+Arg、 rl
e、 Gln+Arg+ Gly+ Pro+ GIy
+Arg、 Ala。One nanomole of the purified peptide was analyzed using a gas phase sequencer. As a result, the amino acid residues detected in analysis cycles 1 to 12 are, in order, IIe+Arg, rl
e, Gln+Arg+ Gly+ Pro+ GIy
+Arg, Ala.
Phe、 Vatであり、目的とするペプチド(1)が
得られたことカミ1認された。精製後のペプチドの収量
は110■であった。Phe and Vat, and it was confirmed that the target peptide (1) was obtained. The yield of peptide after purification was 110 μ.
合成例2 ペプチド(II)の合成を行なった。Synthesis example 2 Peptide (II) was synthesized.
ペプチド合成は、合成例1と同様の方法で行なった。加
えて、t−Bocアミノ酸側鎖の保護基は、Hi sで
はジニトロフヱニル基を用いた。Peptide synthesis was performed in the same manner as in Synthesis Example 1. In addition, a dinitrophenyl group was used as the protecting group for the t-Boc amino acid side chain in His.
合成したペプチド樹脂は、Hlsを含むので合成例1と
同様のトリフルオロメタンスルホン酸による切断を行う
前に、次の前処理を行なった。ペプチド樹脂にN、N−
ジメチルホルムアミド24tel、チオフェノールId
を加え、室温で1時間攪拌後、テフロンフィルターにて
吸引濾過し、1〇−ジクロロメタンで3回洗浄および乾
燥させた。Since the synthesized peptide resin contains Hls, the following pretreatment was performed before the cleavage with trifluoromethanesulfonic acid as in Synthesis Example 1. N, N- on peptide resin
Dimethylformamide 24tel, thiophenol Id
After stirring at room temperature for 1 hour, the mixture was suction filtered using a Teflon filter, washed three times with 10-dichloromethane, and dried.
さらに、この樹脂を0°Cに保ちながら、m−フレソー
ル1rId11 ジメチルスルフィド3ffii!、ト
リフルオロ酢酸5d、トリフルオロメタンスルホン酸1
d中で3時間反応させ、テフロンフィルター上で冷ジエ
チルエーテルで洗浄、乾燥後、合成例1と同様の方法に
て切断した。Furthermore, while keeping this resin at 0°C, m-Fresol 1rId11 Dimethyl sulfide 3ffii! , trifluoroacetic acid 5d, trifluoromethanesulfonic acid 1
The reaction mixture was reacted for 3 hours in a Teflon filter, washed with cold diethyl ether on a Teflon filter, dried, and then cut in the same manner as in Synthesis Example 1.
合成例1と同様の方法で高速液体クロマトグラフィーに
よって精製した。精製標品のクロマトグラムは第7図(
220nmの紫外吸収で検出)に示した通りである。It was purified by high performance liquid chromatography in the same manner as in Synthesis Example 1. The chromatogram of the purified sample is shown in Figure 7 (
(detected by ultraviolet absorption at 220 nm).
合成例1と同様の方法で分析した結果分析サイクル1〜
11で検出されたアミノ酸残基はIle。Results of analysis using the same method as Synthesis Example 1 Analysis cycle 1~
The amino acid residue detected in No. 11 is He.
His、 lie、 Gly、 Pro、 Gly、A
rg、 Ala、 Phe、 Vat。His, lie, Gly, Pro, Gly, A
rg, Ala, Phe, Vat.
Thrであり、目的とするペプチド(n)が得られたこ
とが確認された。精製後のペプチドの収量は1801g
であった。It was confirmed that the target peptide (n) was obtained. The yield of peptide after purification was 1801g.
Met.
合成例3 ペプチド(Ill)の合成を行なった。Synthesis example 3 Peptide (Ill) was synthesized.
ペプチド合成は、合成例1と同様の方法で行った。Peptide synthesis was performed in the same manner as in Synthesis Example 1.
合成例1と同様の方法で高速液体クロマトグラフィーに
よって精製した。当1亥クロマトグラムは第8図(22
0nmの紫外吸収で検出)に示した通りである。It was purified by high performance liquid chromatography in the same manner as in Synthesis Example 1. The current chromatogram is shown in Figure 8 (22
(detected by ultraviolet absorption at 0 nm).
合成例1と同様の方法で分析した結果、分析サイクルl
〜12で検出されたアミノ酸残基は、Val。As a result of analysis in the same manner as in Synthesis Example 1, analysis cycle l
The amino acid residues detected at ~12 are Val.
Phe、Ala、 Arg、 Gly、 Pro
+ GIy+ Arg、 Gin、 Ile。Phe, Ala, Arg, Gly, Pro
+ GIy+ Arg, Gin, Ile.
^rL Ileであり、目的とするペプチド(1)が得
られたことが確認された。It was confirmed that the target peptide (1) was obtained.
製剤例1
ペプチド(1)を精製した後、アンプルに注入し凍結乾
燥させ封入して、100■/アンプルの注射用製剤を得
た。Formulation Example 1 After the peptide (1) was purified, it was injected into ampoules, freeze-dried and sealed to obtain an injection preparation of 100 μ/ampule.
製剤例2
マクロゴール軟膏(第8改正日本薬局方)(マクロゴー
ル4000 50g、マクロゴール40050gを混合
したもの)に1gの凍結乾燥ペプチド(1)を混合よく
練り均一として1%軟膏製剤を得た。Formulation Example 2 Macrogol ointment (8th edition Japanese Pharmacopoeia) (mixture of 50 g of Macrogol 4000 and 50 g of Macrogol 4005) was mixed with 1 g of lyophilized peptide (1) and kneaded thoroughly to obtain a 1% ointment preparation. .
製剤例3
製剤例1において、ペプチド(I)に代えてペプチド(
II)を用いて注射用製剤を得た。Formulation Example 3 In Formulation Example 1, peptide (I) was replaced with peptide (I).
II) to obtain an injectable preparation.
製剤例4
製剤例2において、ペプチド(1)に代えてペプチド(
If)を用いて軟膏剤を得た。Formulation Example 4 In Formulation Example 2, peptide (1) was replaced with peptide (
If) was used to obtain an ointment.
製剤例4
製剤例2において、ペプチド(1)に代えてペプチド(
III)を用いて軟膏剤を得た。Formulation Example 4 In Formulation Example 2, peptide (1) was replaced with peptide (
III) to obtain an ointment.
第1図はラインライ−バー・パークプロットによる、本
発明のプロテアーゼ阻害剤〔ペプチド(■)〕のトトリ
シンに対する阻害効果を示すグラフである。
第2図は、ラインライ−バー・パークプロットによる、
本発明のプロテアーゼ阻害剤〔ペプチド(■)〕の〕α
−キモトリプシに対する阻害効果を示すグラフである。
第3図は、ペプチド(1)、(I[I)および(TV)
のトリプシンに対する濃度依存性の阻害効果を示すグラ
フである。
第4図は、ペプチド(1)、(III)および(IV)
のα−キモトリプシンに対する濃度依存性の阻害効果を
示すグラフである。
第5図は、本発明のプロテアーゼ阻害剤〔ペプチド(I
)〕と、市販の合成プロテアーゼ阻害剤(TPCK)の
α−キモトリプシンに対する阻害効果を比較したグラフ
である。
第6閲は、精製したペプチド(1)の高速液体クロマト
グラフィーのチャートである。
第7図は、精製したペプチド(II)の高速液体クロマ
トグラフィーのチャートである。
第8図は、精製したペプチド(II)の高速液体クロマ
トグラフィーのチャートである。
第8図
1/(51(rnM)−1
1/(S) (r!IM)−1
第3図
Ao704 トi/l Ifi M+
第4図
へ〇7°子ド゛i/盲(μM)
第6図
第7図FIG. 1 is a Reinleiber-Park plot graph showing the inhibitory effect of the protease inhibitor of the present invention [peptide (■)] on totricin. Figure 2 shows the Rheinleiber-Park plot.
α of the protease inhibitor [peptide (■)] of the present invention
- is a graph showing the inhibitory effect on chymotrypsis. Figure 3 shows peptides (1), (I[I) and (TV)
1 is a graph showing the concentration-dependent inhibitory effect on trypsin. Figure 4 shows peptides (1), (III) and (IV)
2 is a graph showing the concentration-dependent inhibitory effect of α-chymotrypsin on α-chymotrypsin. FIG. 5 shows the protease inhibitor [peptide (I) of the present invention.
)] and a commercially available synthetic protease inhibitor (TPCK) are graphs comparing the inhibitory effects on α-chymotrypsin. The sixth view is a high performance liquid chromatography chart of purified peptide (1). FIG. 7 is a chart of high performance liquid chromatography of purified peptide (II). FIG. 8 is a chart of high performance liquid chromatography of purified peptide (II). Fig. 8 1/(51(rnM)-1 1/(S) (r!IM)-1 Fig. 3 Ao704 Toi/l Ifi M+ To Fig. 4〇7° child doi/blindness (μM) Figure 6 Figure 7
Claims (2)
列を有するペプチドまたはその薬理学的に許容される塩
。 Ile−Arg−Ile−Gln−Arg−Gly−P
ro−Gly−Arg−Ala−Phe−Val( I
) Ile−His−Ile−Gly−Pro−Gly−A
rg−Ala−Phe−Val(II)Val−Phe−
Ala−Arg−Gly−Pro−Gly−Arg−G
ln−Ile−Arg−Ile(III)(1) A peptide having an amino acid sequence selected from the following formulas (I) to (III) or a pharmacologically acceptable salt thereof. He-Arg-Ile-Gln-Arg-Gly-P
ro-Gly-Arg-Ala-Phe-Val(I
) Ile-His-Ile-Gly-Pro-Gly-A
rg-Ala-Phe-Val(II) Val-Phe-
Ala-Arg-Gly-Pro-Gly-Arg-G
ln-Ile-Arg-Ile(III)
配列部位を有し、かつ式 −Asn−Asn−Thr−Arg−Lys−Ser−
Ile−Arg−Ile−Gln−Arg−Gly−P
ro−Gly−Arg−Ala−(V)のアミノ酸配列
部位を有しないペプチドまたはその薬理学的に許容され
る塩を有効成分とするプロテアーゼ阻害剤。(2) has at least an amino acid sequence site of the formula -Gly-Pro-Gly-Arg-(IV), and has the formula -Asn-Asn-Thr-Arg-Lys-Ser-
He-Arg-Ile-Gln-Arg-Gly-P
A protease inhibitor containing as an active ingredient a peptide that does not have the amino acid sequence ro-Gly-Arg-Ala-(V) or a pharmacologically acceptable salt thereof.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP1135678A JP2782232B2 (en) | 1989-05-29 | 1989-05-29 | Protease inhibitor |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP1135678A JP2782232B2 (en) | 1989-05-29 | 1989-05-29 | Protease inhibitor |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH032195A true JPH032195A (en) | 1991-01-08 |
JP2782232B2 JP2782232B2 (en) | 1998-07-30 |
Family
ID=15157371
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP1135678A Expired - Lifetime JP2782232B2 (en) | 1989-05-29 | 1989-05-29 | Protease inhibitor |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP2782232B2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0626178A1 (en) * | 1993-05-17 | 1994-11-30 | Ciba-Geigy Ag | Use of inhibitors of HIV-protease for the treatment of tumorous diseases |
GR990100345A (en) * | 1999-10-06 | 2001-06-29 | Composite liposomes for use against the aids virus and for the activation of memory lymphocytes |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1988009181A2 (en) * | 1987-05-29 | 1988-12-01 | Tanox Biosystems, Inc. | Monoclonal antibodies neutralizing hiv-1 |
JPH0269194A (en) * | 1988-09-02 | 1990-03-08 | Chemo Sero Therapeut Res Inst | Mixed antigen particle composed of hbc or hbe antigen and hiv-neutralizing epitope |
-
1989
- 1989-05-29 JP JP1135678A patent/JP2782232B2/en not_active Expired - Lifetime
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1988009181A2 (en) * | 1987-05-29 | 1988-12-01 | Tanox Biosystems, Inc. | Monoclonal antibodies neutralizing hiv-1 |
JPH0269194A (en) * | 1988-09-02 | 1990-03-08 | Chemo Sero Therapeut Res Inst | Mixed antigen particle composed of hbc or hbe antigen and hiv-neutralizing epitope |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0626178A1 (en) * | 1993-05-17 | 1994-11-30 | Ciba-Geigy Ag | Use of inhibitors of HIV-protease for the treatment of tumorous diseases |
GR990100345A (en) * | 1999-10-06 | 2001-06-29 | Composite liposomes for use against the aids virus and for the activation of memory lymphocytes |
Also Published As
Publication number | Publication date |
---|---|
JP2782232B2 (en) | 1998-07-30 |
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