CN103275185B - 基质金属蛋白酶-2多肽抑制剂及其应用 - Google Patents
基质金属蛋白酶-2多肽抑制剂及其应用 Download PDFInfo
- Publication number
- CN103275185B CN103275185B CN201310209064.8A CN201310209064A CN103275185B CN 103275185 B CN103275185 B CN 103275185B CN 201310209064 A CN201310209064 A CN 201310209064A CN 103275185 B CN103275185 B CN 103275185B
- Authority
- CN
- China
- Prior art keywords
- mmp
- ser
- cys
- matrix metal
- polypeptide
- 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 title claims abstract description 32
- 239000003112 inhibitor Substances 0.000 title claims abstract description 26
- 229920001184 polypeptide Polymers 0.000 title abstract description 14
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 14
- 239000011159 matrix material Substances 0.000 title abstract description 6
- 239000002184 metal Substances 0.000 title abstract description 6
- 102100026802 72 kDa type IV collagenase Human genes 0.000 claims description 28
- 101710151806 72 kDa type IV collagenase Proteins 0.000 claims description 28
- 206010040070 Septic Shock Diseases 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 9
- 230000004083 survival effect Effects 0.000 abstract description 9
- 230000006378 damage Effects 0.000 abstract description 5
- 241000699670 Mus sp. Species 0.000 abstract description 4
- 108060008682 Tumor Necrosis Factor Proteins 0.000 abstract description 4
- 208000038016 acute inflammation Diseases 0.000 abstract description 3
- 230000006022 acute inflammation Effects 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 230000004044 response Effects 0.000 abstract description 2
- 108010052014 Liberase Proteins 0.000 abstract 2
- 102000003390 tumor necrosis factor Human genes 0.000 abstract 2
- 238000009509 drug development Methods 0.000 abstract 1
- 238000000338 in vitro Methods 0.000 abstract 1
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 239000002547 new drug Substances 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 20
- 108090000790 Enzymes Proteins 0.000 description 20
- 229940088598 enzyme Drugs 0.000 description 20
- 241000699666 Mus <mouse, genus> Species 0.000 description 19
- 238000006243 chemical reaction Methods 0.000 description 14
- 206010061218 Inflammation Diseases 0.000 description 12
- 230000004054 inflammatory process Effects 0.000 description 12
- 238000001514 detection method Methods 0.000 description 8
- 239000002158 endotoxin Substances 0.000 description 8
- 210000004493 neutrocyte Anatomy 0.000 description 8
- 108050007852 Tumour necrosis factor Proteins 0.000 description 7
- 102000018594 Tumour necrosis factor Human genes 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 6
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 6
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 6
- 206010040047 Sepsis Diseases 0.000 description 6
- 230000001154 acute effect Effects 0.000 description 6
- 108030001564 Neutrophil collagenases Proteins 0.000 description 5
- 102000056189 Neutrophil collagenases Human genes 0.000 description 5
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- 108010035532 Collagen Proteins 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
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 102000004266 Collagen Type IV Human genes 0.000 description 2
- 108010042086 Collagen Type IV Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 206010018873 Haemoconcentration Diseases 0.000 description 2
- 101000627872 Homo sapiens 72 kDa type IV collagenase Proteins 0.000 description 2
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 101710108790 Stromelysin-1 Proteins 0.000 description 2
- 102100030416 Stromelysin-1 Human genes 0.000 description 2
- 108010031374 Tissue Inhibitor of Metalloproteinase-1 Proteins 0.000 description 2
- 102000005353 Tissue Inhibitor of Metalloproteinase-1 Human genes 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000001311 chemical methods and process Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229940121386 matrix metalloproteinase inhibitor Drugs 0.000 description 2
- 239000003771 matrix metalloproteinase inhibitor Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 210000000438 stratum basale Anatomy 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- MCTWTZJPVLRJOU-UHFFFAOYSA-N 1-methyl-1H-imidazole Chemical compound CN1C=CN=C1 MCTWTZJPVLRJOU-UHFFFAOYSA-N 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- QCDFBFJGMNKBDO-UHFFFAOYSA-N Clioquinol Chemical compound C1=CN=C2C(O)=C(I)C=C(Cl)C2=C1 QCDFBFJGMNKBDO-UHFFFAOYSA-N 0.000 description 1
- WYZLWZNAWQNLGQ-FXQIFTODSA-N Cys-Cys-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CS)N WYZLWZNAWQNLGQ-FXQIFTODSA-N 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 206010014824 Endotoxic shock Diseases 0.000 description 1
- 101000990908 Homo sapiens Neutrophil collagenase Proteins 0.000 description 1
- 101000990915 Homo sapiens Stromelysin-1 Proteins 0.000 description 1
- ZNOBVZFCHNHKHA-KBIXCLLPSA-N Ile-Ser-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZNOBVZFCHNHKHA-KBIXCLLPSA-N 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 125000000393 L-methionino group Chemical group [H]OC(=O)[C@@]([H])(N([H])[*])C([H])([H])C(SC([H])([H])[H])([H])[H] 0.000 description 1
- 108010016165 Matrix Metalloproteinase 2 Proteins 0.000 description 1
- 102000000424 Matrix Metalloproteinase 2 Human genes 0.000 description 1
- 102000001776 Matrix metalloproteinase-9 Human genes 0.000 description 1
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 1
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000008468 bone growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- BFPSDSIWYFKGBC-UHFFFAOYSA-N chlorotrianisene Chemical compound C1=CC(OC)=CC=C1C(Cl)=C(C=1C=CC(OC)=CC=1)C1=CC=C(OC)C=C1 BFPSDSIWYFKGBC-UHFFFAOYSA-N 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- OCSMOTCMPXTDND-OUAUKWLOSA-N marimastat Chemical compound CNC(=O)[C@H](C(C)(C)C)NC(=O)[C@H](CC(C)C)[C@H](O)C(=O)NO OCSMOTCMPXTDND-OUAUKWLOSA-N 0.000 description 1
- 229950008959 marimastat Drugs 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- -1 oxonium ion Chemical class 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229960000856 protein c Drugs 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 206010040560 shock Diseases 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000002636 symptomatic treatment Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
基质金属蛋白酶-2多肽抑制剂及其应用。本发明涉及药物领域，具体涉及具有抑制基质金属蛋白酶-2和肿瘤坏死因子释放酶、具有减轻急性炎症反应对机体破坏的多肽。其序列为Ile-Ser-Glu-Cys-Cys-Met-Ser是全新的序列，它们可以在1微摩尔水平上体外抑制基质金属蛋白酶-2和肿瘤坏死因子释放酶的活力，并在体内试验中增加内毒素休克小鼠的生存率，具有潜在的新药开发价值。
Description
技术领域
本发明涉及基质金属蛋白酶-2多肽及其应用，具体涉及具有抑制基质金属蛋白酶-2、具有减轻急性炎症反应对机体破坏的多肽。
背景技术
脓毒血症(sepsis)一直是一个严重的医学问题。早期脓毒血症不经过及时有效治疗可进展为重症脓毒血症和脓毒血症性休克,两者死亡率分别为20%～30%和40%～70% 。医学界一直在研究有效的治疗手段,如早期对症治疗、激活蛋白C、皮质激素治疗等。近年来随着医学研究的深入,对脓毒血症的认识和治疗取得了很大进展。其中，基质金属蛋白酶-2( MMP-2)在内毒素休克中扮演着重要角色。
基质金属蛋白酶是一组由20几种结构相似和功能相关的蛋白内切酶组成。它们可以发挥正常的生理功能，例如卵细胞着床，骨骼生长和器官发育等；这些酶还可以在诸如炎症，伤口愈合和肿瘤细胞转移中发挥病理作用。基质金属蛋白酶被更多的看作是这些病理过程的调节因子。在基质金属蛋白酶家族中，MMP－2 和MMP－9, 是MMP 家族的重要成员，分别编码72kDa 和92kDa 的明胶酶A 和明胶酶B。正常稳定状态下，组织较少合成、分泌MMP－2，在炎症、肠化、增生和损伤等刺激下，细胞内部调控平衡机制紊乱，合成、分泌MMP－2 增多。
中性粒细胞是循环系统中最丰富的白细胞，在机体受到细菌感染时，中性粒细胞可迅速作出反应，首先进入被感染部位，构成了机体免疫系统应对细菌感染的第一道防线。中性粒细胞的细胞颗粒中储存有不同种预先产生的酶类，包括弹性蛋白酶（elastase）, 基质金属蛋白酶-8（MMP-8）和基质金属蛋白酶-2（MMP-2）等。基质金属蛋白酶-8可切割胶原蛋白（collagen）, 基质金属蛋白酶-2可继续把变性的胶原蛋白或明胶切割成更小的片段。基质金属蛋白酶-2本身也有切割Ⅳ型胶原蛋白的活力，Ⅳ型胶原蛋白是血管基底层的主要成分。与其它细胞不同，中性粒细胞不表达基质金属蛋白酶-2和基质金属蛋白酶抑制因子-1 （TIMP-1）。这使得中性粒细胞在受到诸如LPS，IL-8和PMA等的刺激时，迅速释放胞内颗粒，而释放的基质金属蛋白酶类可被同时释放的有氧自由基激活，在局部具有较高的浓度。 这些酶除了具有正常的杀菌功能外，在病理条件下可能会对机体造成很大的破坏。内毒素模型就是一个很好的例子，在高剂量静脉注射LPS后（8mg/kg小鼠），小鼠循环系统中的中性粒细胞可迅速释放胞内颗粒的内容物，血液中的基质金属蛋白酶-2的浓度在30分钟时就可以达到峰值，其酶活力可以发挥诸如破坏血管基底层等作用，并推进了休克症状的发展。这一点可由基质金属蛋白酶-2缺失（knockout）小鼠对内毒素休克的抑制作用证明。所以，基质金属蛋白酶-2可以作为以中性粒细胞为主的炎症反应的主要靶点。
基质金属蛋白酶-2的抑制剂包括大分子和小分子。 大分子抑制剂的制备和使用限制了它们的发展，例如重组人金属基质蛋白酶组织抑制因子-3的体内半衰期只有4分钟。很多成功的小分子抑制剂，例如Marimastat，可以在纳摩尔水平上抑制基质金属蛋白酶的活力，但小分子抑制剂缺乏特异性，如果在慢性疾病中长期使用缺乏特异性的基质金属蛋白酶抑制剂可产生副作用。 因而，从基质金属蛋白酶酶抑制剂的应用角度来看，以急性炎症反应作为研究对象是正确的选择。在急性反应期，机体能够耐受短期的、金属介质蛋白酶-2广谱抑制剂对正常生理功能的抑制，同时使关键组织器官的生理结构免受破坏，增加了生存几率。
通过化学方法合成了多肽抑制剂。这条多肽抑制剂可以选择性的抑制基质金属蛋白酶-8，基质金属蛋白酶-2和肿瘤坏死因子释放酶的活力，在用内毒素模型检测多肽抑制剂体内活力的实验中，多肽抑制剂能够使小鼠避过了注射LPS后血液中基质金属蛋白酶-2的高峰期（30分钟血液浓度达到峰值）并抑制了肿瘤坏死因子（TNF-α）的释放（1小时时血液浓度达到峰值），从而有效的保护了小鼠，度过了急性炎症反应期，增加了小鼠的生存率。
目前，国际上开发出的基质金属蛋白酶抑制剂已进入Ⅱ期临床（多发性硬化症，类风湿性关节炎等）和三期临床（牙周炎等）。本专利中的多肽抑制剂已证明在急性炎症反应中有效，具有在其他炎症模型中开发的前景。
发明内容
发明目的
本发明提供全新的序列，该序列基质金属蛋白酶-2抑制剂，对急性炎症具有很好的疗效。
技术方案
基质金属蛋白酶-2多肽抑制剂4，其特征在于其序列为Ile-Ser-Glu-Cys-Cys-Met-Ser。
基质金属蛋白酶-2多肽抑制剂4在制备治疗抑制急性炎症药物中的应用。
有益效果
利用固相合成法化学合成基质金属蛋白酶-2多肽抑制剂4，该多肽具有全新的序列，该多肽可体外抑制基质金属蛋白酶-2，-8，-3和肿瘤坏死因子释放酶的酶活力。在内毒素休克模型实验中成功的增加了小鼠的生存率。在急性炎症反应中，中性粒细胞释放大量酶和有氧离子，其中包括基质金属蛋白酶-8和基质金属蛋白酶-2，这些酶可以切割并破坏机体组织，造成损伤；单核巨噬细胞表达肿瘤坏死因子释放酶，这种酶可切割并释放肿瘤坏死因子，肿瘤坏死因子是炎症反应的核心细胞因子，有加重炎症反应的作用。我们发现的多肽抑制剂可以同时抑制基质金属蛋白酶-2，-8和肿瘤坏死因子释放酶的酶活力，从而减轻了炎症反应，保护机体并减轻炎症的破坏作用。
附图说明
附图1多肽抑制剂分子构像示意图. 1.异亮氨酸Ile，2.丝氨酸Ser，3.Glu谷氨酸，4.Cys半胱氨酸，5. 半胱氨酸Cys，6.Met甲硫氨酸，7.丝氨酸Ser。
具体实施方式
实施例1
多肽的化学合成方法
多肽用Fmoc化学方法合成。合成反应从C端向N端进行，Rink介质（可在Advanced ChemTech公司购得）上有自由氨基，顺序连接Ser，Met，Cys，Cys， Glu，Ser和Ile。每一步连接过程中，氨基酸残基都要活化，活化混合物中有4倍于介质上自由氨基的HBTU，HOBt，DIEA和Fmoc-氨基酸。每次氨基酸的连接反应之后，都用一个吡啶/醋酸/N-甲基咪唑（4:1:0.5）的混合物来封闭未连接的自由氨基，封闭反应10 min。每次氨基酸的连接反应之后，下一个氨基酸连接之前，都要把介质上的Fmoc-基团去掉，去Fmoc-基团使用含20%哌啶的二甲基甲酰胺，需15分钟。最后，当所有氨基酸残基顺序连接之后，多肽用98%三氟乙酸从介质上切割下来，切割在室温下进行2小时。
应用上述化学条件可合成并获得多肽，序列为Ile-Ser-Glu-Cys-Cys-Met-Ser，该序列为全新序列。
也可委托上海生工合成。
实施例2
多肽抑制剂体外对几种靶点酶：基质金属蛋白酶-3, -8，-2及肿瘤坏死因子释放酶(均购于sigma公司)的IC50值。
重组人基质金属蛋白酶-2由E. coli细胞表达. 该酶用0.01 μM 的基质金属蛋白酶-3活性位点活化，所用的缓冲液为100 mM Tris/HCl, pH 7.4, 100 mM NaCl, 10 mM CaCl2 and 0.01% Tween-20。活化时基质金属蛋白酶-2的浓度为72 ng/μl (1 μM)。酶活力的检测是通过切割荧光产生多肽底物Mca- Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 并检测产生的荧光值实现的(激发波长=328 nm, 检测波长=392 nm). 所有的检测在37°C下100 μl反应体系中进行。测得的IC50值为48.68μmol。
重组人基质金属蛋白酶-8的检测与-2的检测类似并使用同样的荧光产生底物。反应也是在37°C 下100 μl反应体系中进行。检测时向反应体系中加20 μl重组人基质金属蛋白酶-8 (2 ng/μl). 底物的终浓度为10 μM。测得的IC50值为32.65μmol。
重组人基质金属蛋白酶-3 用另外一个荧光产生底物Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH2来检测 (激发波长=320 nm, 发射波长=405 nm)。 反应在37°C下100 μl反应体系中进行。反应开始时向反应体系中加10 μl 基质金属蛋白酶-3存储液 (1 ng/μl)底物的终浓度为10 μM。测得的IC50值为24.12μmol。
肿瘤坏死因子释放酶的活力是通过切割荧光产生底物Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Ser-Arg-NH检测的(激发波长=320 nm, 发射波长=405 nm)。反应在37°C下100 μl反应体系中进行。反应开始时向反应体系中加4 μl 基质金属蛋白酶-3存储液 (1 ng/μl)底物的终浓度为10 μM。测得的IC50值为71.43μmol。
实施例3
用内毒素休克模型检测多肽抑制剂的体内活力
在建立内毒素休克模型之前，我们首先测定了LPS（E. coli 0111：B4）小白鼠的LD50为50 μg每只小鼠，在实验中我们采用了100 μg每只小鼠，这样，对照组的小鼠可全部死亡。用Regasepin2做了一个阳性对照实验。对照组小鼠注射100 μg LPS，而Regasepin2实验组的小鼠在注射LPS 5分钟之后，每只小鼠再注射0.7 mg的多肽。通过制作Kaplan-Meier生存曲线发现Regasepin2可有效的保护注射了100 μg的小鼠，提高了生存率。成功地在小白鼠上建立内毒素休克模型之后，用该模型检测Ile-Ser-Glu-Cys-Cys-Met-Ser的体内活力。对照组小鼠（12只）注射100 μg LPS，而多肽抑制剂组小鼠（12只）在注射LPS 5 min后，每只小鼠注射0.7 mg多肽抑制剂。用Kaplan-Meier生存曲线分析，多肽Ile-Ser-Glu-Cys-Cys-Met-Ser可有效地保护小白鼠，提高内毒素休克小鼠的生存率。这些数据表明，抑制MMP-2和TACE活力可有效提高内毒素休克小鼠的生存率。
SEQUENCE LISTING
<110> 苏州普罗达生物科技有限公司
<120> 基质金属蛋白酶-2多肽抑制剂4及其应用
<130>
<160> 1
<170> Patent In version 3.3
<210> 1
<211> 18
<212> PRT
<213> 人工序列
<400> 1
Ile Ser Glu Cys Cys Met Sert
1 5
Claims (1)
1.基质金属蛋白酶-2多肽抑制剂4，其特征在于其序列为Ile-Ser-Glu-Cys-Cys-Met-Ser。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310209064.8A CN103275185B (zh) | 2013-05-30 | 2013-05-30 | 基质金属蛋白酶-2多肽抑制剂及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310209064.8A CN103275185B (zh) | 2013-05-30 | 2013-05-30 | 基质金属蛋白酶-2多肽抑制剂及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103275185A CN103275185A (zh) | 2013-09-04 |
| CN103275185B true CN103275185B (zh) | 2014-08-13 |
Family
ID=49057826
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310209064.8A Active CN103275185B (zh) | 2013-05-30 | 2013-05-30 | 基质金属蛋白酶-2多肽抑制剂及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103275185B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105504028A (zh) * | 2016-01-30 | 2016-04-20 | 苏州普罗达生物科技有限公司 | 基质金属蛋白酶10抑制多肽及其应用 |
-
2013
- 2013-05-30 CN CN201310209064.8A patent/CN103275185B/zh active Active
Non-Patent Citations (6)
| Title |
|---|
| 于松林等.基质金属蛋白酶-2促进肝纤维化作用免疫组织化学研究.《宁夏医学杂志》.2013,第35卷(第2期),111-112. |
| 基质金属蛋白酶-2与2型糖尿病大血管病变的研究进展;王海娇等;《黑龙江医药科学》;20120229;第36卷(第1期);83-84 * |
| 基质金属蛋白酶-2促进肝纤维化作用免疫组织化学研究;于松林等;《宁夏医学杂志》;20130228;第35卷(第2期);111-112 * |
| 杨文华等.蝎毒多肽提取物对白血病NOD/SCID小鼠MMP2、MMP9表达的影响.《天津医药》.2009,第37卷(第10期),第856-858页. |
| 王海娇等.基质金属蛋白酶-2与2型糖尿病大血管病变的研究进展.《黑龙江医药科学》.2012,第36卷(第1期),83-84. |
| 蝎毒多肽提取物对白血病NOD/SCID小鼠MMP2、MMP9表达的影响;杨文华等;《天津医药》;20091031;第37卷(第10期);第856-858页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103275185A (zh) | 2013-09-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103304634B (zh) | 基质金属蛋白酶-2多肽抑制剂及其应用 | |
| Henikoff et al. | Assembly of variant histones into chromatin | |
| EP1578988B1 (en) | Protein based tnf-alpha variants for the treatment of tnf-alpha related disorders | |
| Goldberg | Hemoglobin degradation | |
| CN102268069B (zh) | 基质金属蛋白酶-9多肽抑制剂4及其应用 | |
| Tu et al. | Progress in the development of matrix metalloproteinase inhibitors | |
| US20070207961A1 (en) | TNF-alpha VARIANTS PROTEINS FOR THE TREATMENT OF TNF-alpha RELATED DISORDERS | |
| US7687461B2 (en) | Treatment of TNF-α related disorders with TNF-α variant proteins | |
| Grabowski et al. | Immunomodulatory Yersinia outer proteins (Yops)–useful tools for bacteria and humans alike | |
| CN102268068B (zh) | 基质金属蛋白酶-9多肽抑制剂1及其应用 | |
| Gu et al. | Evolutionary dynamics and molecular mechanisms of HORMA domain protein signaling | |
| Dókus et al. | Modulators of calpain activity: Inhibitors and activators as potential drugs | |
| CN102268070B (zh) | 基质金属蛋白酶-9多肽抑制剂2及其应用 | |
| Vane | The history of inhibitors of angiotensin converting enzyme | |
| CN103275185B (zh) | 基质金属蛋白酶-2多肽抑制剂及其应用 | |
| CN103254290B (zh) | 基质金属蛋白酶-2多肽抑制剂及其应用 | |
| CN103275184B (zh) | 基质金属蛋白酶-2多肽抑制剂及其应用 | |
| WO1993018794A1 (en) | In vivo peptide therapy | |
| CN102250212B (zh) | 基质金属蛋白酶-9多肽抑制剂5及其应用 | |
| CN102250213B (zh) | 基质金属蛋白酶-9多肽抑制剂3及其应用 | |
| CN104387474B (zh) | 一种肿瘤血管梗塞剂多肽、基因、表达载体及其应用 | |
| CN108484729B (zh) | 一种基质金属蛋白酶9的多肽抑制剂及其应用 | |
| CN103254291B (zh) | 肿瘤坏死因子-α多肽抑制剂及其应用 | |
| CN110540577A (zh) | 一种具有降血压作用的鸭源多肽及其应用 | |
| CN113087767B (zh) | 具有降血压功能的三肽rfy及其用途 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: WHITMAN BIOTECH (NANJING) CO., LTD. Free format text: FORMER OWNER: SUZHOU PULUODA BIOLOG SCIENCE + TECHNOLOGY CO., LTD. Effective date: 20150520 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Chang Junjun Inventor after: Sun Wenyong Inventor before: Luo Ruixue |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 215000 SUZHOU, JIANGSU PROVINCE TO: 210000 NANJING, JIANGSU PROVINCE Free format text: CORRECT: INVENTOR; FROM: LUO RUIXUE TO: CHANG JUNJUN SUN WENYONG |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20150520 Address after: 210000 Building No. 2, No. 108 South Bridge Road, Yuhuatai District, Nanjing, Jiangsu Patentee after: Wittman Biotechnology (Nanjing) Co., Ltd. Address before: High tech Zone Suzhou city Jiangsu province 215000 Chuk Yuen Road No. 209 Patentee before: Suzhou Pu Luo Da Biotechnology Co., Ltd. |
|
| CP03 | Change of name, title or address |
Address after: 210039 Building 2, 108 West Road South Road, Yuhuatai District, Nanjing, Jiangsu Patentee after: Neusoft Whitman Biotechnology (Nanjing) Co., Ltd. Address before: 210000 Building 2, 108 West Road South Road, Yuhuatai District, Nanjing, Jiangsu Patentee before: Wittman Biotechnology (Nanjing) Co., Ltd. |
|
| CP03 | Change of name, title or address |