CN102268068A - Substrate metal prolease-9 polypeptide inhibitor 1and application thereof - Google Patents
Substrate metal prolease-9 polypeptide inhibitor 1and application thereof Download PDFInfo
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- CN102268068A CN102268068A CN2011101827376A CN201110182737A CN102268068A CN 102268068 A CN102268068 A CN 102268068A CN 2011101827376 A CN2011101827376 A CN 2011101827376A CN 201110182737 A CN201110182737 A CN 201110182737A CN 102268068 A CN102268068 A CN 102268068A
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Abstract
The invention relates to the field of medicaments, in particular to a polypeptide which has the effects of inhibiting substrate metal prolease-9 and tumor necrosis factor liberase and relieving the damage of an acute inflammatory reaction to an organism. The sequence of the polypeptide is Pro-Arg-Cys-(D-Bip)-(D-Arg)-Gly-Glu which is a brand new sequence (D-Bip is D-type diphenyl alanine, and D-Arg is D-type arginine). The polypeptide can be used for inhibiting the activities of the substrate metal prolease-9 and the tumor necrosis factor liberase on 1 micromole level in vitro and increasing the survival rate of an endotoxic shock mouse in an in-vivo test, and has a potential new medicament development value.
Description
Technical field
The present invention relates to matrix metalloproteinase-9 peptide inhibitor 1 and application thereof, be specifically related to have inhibition matrix metalloproteinase-9, have the acute inflammatory reaction of alleviating body destructive polypeptide.
Background technology
Pyemia (sepsis) is a serious medical problem always.Early stage pyemia can be made progress without timely and effective treatment and is severe pyemia and pyemia shock, and both mortality ratio are respectively 20%～30% and 40%～70%.Medical circle is being studied effective treatment means always, as early stage symptomatic treatment, activated protein c, cortin treatment etc.In recent years along with the going deep into of medical research, the understanding and the treatment of pyemia obtained remarkable progress.Wherein, matrix metalloproteinase-9 (MMP-9) is being played the part of important role in endotoxin shock.
Matrix metalloproteinase is one group and is made up of 20 several structural similitudies protein incision enzyme relevant with function.The physiological function that they can be brought into normal play, ovum implantation for example, bone growth and allelotaxis etc.; These enzymes can also be brought into play pathological effect in wound healing and the tumour cell transfer such as inflammation.Matrix metalloproteinase is more regarded as the regulatory factor of these pathologic processes.Two kinds of gelatinases are arranged in matrix metalloproteinase family, and wherein, gelatin enzyme A/matrix metalloproteinase-2 can constant expression in most histocytes, to keep histiocytic basic function; And gelatinase B/ matrix metalloproteinase-9 is at TNF-α, a large amount of abduction deliverings under the effect of the extracellular factor of IL-6 etc.The enzyme activity that gives expression to the outer matrix metalloproteinase-9 of born of the same parents can be regulated by the activation of proenzyme and the restraining effect of organized enzyme again.
Neutrophil leucocyte is a white corpuscle the abundantest in the recycle system, and when body was subjected to infectation of bacteria, neutrophil leucocyte can be reacted rapidly, at first entered infected position, has constituted the first line of defence of body immune system reply infectation of bacteria.Store the enzyme that produces in advance not of the same race in the cell granulations of neutrophil leucocyte, comprise elastoser (elastase), matrix metalloproteinase-8 (MMP-8) and matrix metalloproteinase-9 (MMP-9) etc.Matrix metalloproteinase-8 can cut collagen protein (collagen), and matrix metalloproteinase-9 can continue the collagen protein of sex change or gelatin are cut into littler fragment.Matrix metalloproteinase-9 itself also has the vigor of cutting VI collagen type, and the VI collagen type is the main component of blood vessel stratum basale.Different with other cell, neutrophil leucocyte is not expressed matrix metalloproteinase-2 and matrix metalloproteinase supressor-1 (TIMP-1).This makes neutrophil leucocyte be subjected to such as LPS, during the stimulation of IL-8 and PMA etc., discharges born of the same parents' endoparticle rapidly, and the oxyradical that has that the matrix metalloproteinase class that discharges can be discharged simultaneously activates, and has higher concentration in the part.These enzymes may cause very big destruction to body under pathological conditions except having normal sterilizing function.The intracellular toxin model is exactly a good example, behind high dosage intravenous injection LPS (8mg/kg mouse), neutrophil leucocyte in the mouse recycle system can discharge the endocorpuscular content of born of the same parents rapidly, the concentration of the matrix metalloproteinase in the blood-9 just can reach peak value in the time of 30 minutes, its enzyme activity can be brought into play such as effects such as destruction blood vessel stratum basalees, and has advanced the development of shock symptom.This point can be proved by the restraining effect of matrix metalloproteinase-9 disappearance (knockout) mouse to endotoxin shock.So matrix metalloproteinase-9 can be used as the main target spot based on the inflammatory reaction of neutrophil leucocyte.
The inhibitor of matrix metalloproteinase-9 comprises macromole and small molecules.The preparation of macromole inhibitor and use have limited their development, and for example the transformation period has only 4 minutes in the body of recombinant human metal matrix proteolytic enzyme tissue inhibiting-3.A lot of successful micromolecular inhibitors, Marimastat for example, the vigor that can on the nmole level, suppress matrix metalloproteinase, but micromolecular inhibitor lacks specificity, if can have side effects in the medium-term and long-term specific matrix metallo-proteinase inhibitor of shortage of using of chronic disease.Thereby, from matrix metalloproteinase application of enzyme inhibitors angle, be correct selection as research object with acute inflammatory reaction.In the acute reaction phase, body can tolerate short-term, metal medium proteolytic enzyme-9 wide spectrum inhibitor is to the inhibition of normal physiological function, makes the physiological structure of critical tissue's organ avoid destroying simultaneously, increased chances of survival.
Synthesized peptide inhibitor by chemical process.This peptide inhibitor can optionally suppress matrix metalloproteinase-8; matrix metalloproteinase-9 and tumour necrosis factor discharge the vigor of enzyme; detecting with the intracellular toxin model in the experiment of vigor in the peptide inhibitor body; peptide inhibitor can make mouse keep away the peak period (haemoconcentration reached peak value in 30 minutes) of blood matrix metalloproteinase-9 behind the injection LPS and suppress the release (1 hour time haemoconcentration reach peak value) of tumour necrosis factor (TNF-α); thereby effectively protected mouse; spend the acute inflammatory reaction phase, increased the survival rate of mouse.
At present, the matrix metallo-proteinase inhibitor of developing has in the world entered II phase clinical (multiple sclerosis, rheumatoid arthritis etc.) and three phases clinical (periodontitis etc.).Five peptide inhibitors in this patent have proved in acute inflammatory reaction effective, have the prospect of developing in other inflammatory models.
Summary of the invention
Goal of the invention
The invention provides brand-new sequence, this sequence matrix metalloproteinase-9 inhibitor has better curative effect to acute inflammation.
Technical scheme
Matrix metalloproteinase-9 peptide inhibitor 1 is characterized in that its sequence is Pro-Arg-Cys-(D-Bip)-(D-Arg)-Gly-Glu, sees Seq NO.1, and wherein D-Bip is a D-type diphenylprop propylhomoserin, and D-Arg is a D-type arginine.
The application of matrix metalloproteinase-9 peptide inhibitor 1 in preparation treatment inhibition acute inflammation medicine.
Beneficial effect
Utilize solid-phase synthesis chemosynthesis matrix metalloproteinase-9 peptide inhibitor 1, this polypeptide has brand-new sequence, but this polypeptide vitro inhibition matrix metalloproteinase-9 ,-8 ,-3 and tumour necrosis factor discharge the enzyme activity of enzyme.In endotoxin shock model experiment successful increase the survival rate of mouse.In acute inflammatory reaction, neutrophil leucocyte discharges a large amount of enzymes and oxonium ion is arranged, and comprising matrix metalloproteinase-8 and matrix metalloproteinase-9, these enzymes can cut and destroy body tissue, cause damage; Mononuclear macrophage expressing tumor necrosin discharges enzyme, and this kind of enzyme can cut and release tumor necrosis factor, and tumour necrosis factor is the core cytokine of inflammatory reaction, and the effect of exacerbate inflammation reaction is arranged.The peptide inhibitor that we find can suppress matrix metalloproteinase-9 simultaneously ,-8 and tumour necrosis factor discharge the enzyme activity of enzyme, thereby alleviated inflammatory reaction, protection body and the destruction that reduces inflammation.
Description of drawings
The synthetic back of the accompanying drawing 1 chemiluminescent polypeptide method elution curve of HPLC purifying.
Accompanying drawing 2 matrix metalloproteinases-9 cutting fluorescence produces the kinetic curve of polypeptide.
Accompanying drawing 3 peptide inhibitors are to the inhibition curve of matrix metalloproteinase-8.
Accompanying drawing 4 peptide inhibitors are to the inhibition curve of matrix metalloproteinase-9.
Accompanying drawing 5. peptide inhibitors discharge the inhibition curve of enzyme to tumour necrosis factor.
Accompanying drawing 6. peptide inhibitors are to the inhibition curve of matrix metalloproteinase-3.
Accompanying drawing 7. peptide inhibitors are to the therapeutic action of acute inflammatory reaction.
Embodiment
The chemical synthesis process of polypeptide
Polypeptide is synthetic with the Fmoc chemical process.Building-up reactions is carried out to the N end from the C end, free amino group is arranged, the Glu that is linked in sequence, Gly, D-Arg, D-Bip, Cys, Arg and Pro on the Rink medium (can buy in Advanced ChemTech company).In each step connection procedure, amino-acid residue all will activate, and the HBTU of 4 times of free amino groups on medium is arranged in the activator mixture, HOBt, DIEA and Fmoc-amino acid.After each amino acid whose ligation, all use the mixture of a pyridine/acetic acid/N-Methylimidazole (4: 1: 0.5) to seal the free amino group that does not connect, capping 10min.After each amino acid whose ligation, next amino acid all will remove the Fmoc-group on the medium before connecting, and goes the Fmoc-group to use the dimethyl formamide that contains 20% piperidines, needs 15 minutes.At last, after all amino-acid residues were linked in sequence, polypeptide cut down from medium with 98% trifluoroacetic acid, and cutting was at room temperature carried out 2 hours.
Use above-mentioned electrochemical conditions and can synthesize and obtain polypeptide, sequence is Pro-Arg-Cys-(D-Bip)-(D-Arg)-Gly-Glu, and this sequence is brand-new sequence.Fig. 1 is that polypeptide is from the elution curve of medium cutting back with the HPLC purifying.
Peptide inhibitor external to several target spot enzymes (matrix metalloproteinase-3 ,-8 ,-9 and tumour necrosis factor discharge enzyme) the IC50 value.
Recombinant human matrix metalloproteinase-9 is by the Sf9 insect cell expression. and this enzyme activates with matrix metalloproteinase-3 avtive spot of 0.01 μ M, and used damping fluid is 100mM Tris/HCl, and pH 7.4,100mM NaCl, 10mM CaCl
2And 0.01%Tween-20.The concentration of matrix metalloproteinase during activation-9 is 92ng/ μ l (1 μ M).The detection of enzyme activity is to produce peptide substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH by cutting fluorescence
2And the fluorescent value realization of detection generation (excitation wavelength=328nm, the detection wavelength=392nm).All detections are carried out in the 100 μ l reaction systems down at 37 ℃.
The detection of recombinant human matrix metalloproteinase-8 and-9 detection type like and use same fluorescence to produce substrate.Reaction also is to carry out in the 100 μ l reaction systems down at 37 ℃.In reaction system, adding 20 μ l recombinant human matrix metalloproteinases-8 (2ng/ μ l) during detection. the final concentration of substrate is 10 μ M.
Recombinant human matrix metalloproteinase-3 usefulness another one fluorescence produces substrate Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys (Dnp)-NH
2Detect (excitation wavelength=320nm, emission wavelength=405nm).Being reflected at 37 ℃ carries out in the 100 μ l reaction systems down.The final concentration that adds 10 μ l matrix metalloproteinases-3 storage liquid (1ng/ μ l) substrate during the reaction beginning in reaction system is 10 μ M.
The vigor that tumour necrosis factor discharges enzyme produces substrate Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Ser-Arg-NH by cutting fluorescence and detects (excitation wavelength=320nm, emission wavelength=405nm).Being reflected at 37 ℃ carries out in the 100 μ l reaction systems down.The final concentration that adds 4 μ l matrix metalloproteinases-3 storage liquid (1ng/ μ l) substrate during the reaction beginning in reaction system is 10 μ M.
Embodiment 3
With vigor in the body of endotoxin shock model detection peptide inhibitor
Before setting up endotoxin shock model, the LD50 that we have at first measured LPS (E.coli 0111:B4) small white mouse is every mouse of 50 μ g, and we have adopted every mouse of 100 μ g in experiment, and like this, mice in control group can be all dead.In the scientific effort of our front, find that Regasepin2 (an other peptide inhibitor, open) can improve the survival rate of C57BL/6 mouse in the endotoxin shock process.In order to set up endotoxin shock model on our small white mouse, we have done a positive control experiment with Regasepin2.Control group mice is injected 100 μ g LPS, and the mouse of Regasepin2 experimental group is after injection LPS 5 minutes, and every mouse is injected the polypeptide of 0.7mg again.Find that by making the Kaplan-Meier survival curve Regasepin2 can effectively protect the mouse of having injected 100 μ g, improved survival rate (Fig. 7).Successfully on small white mouse, set up after the endotoxin shock model, detect the interior vigor of body of Pro-Arg-Cys-(D-Bip)-(D-Arg)-Gly-Glu with this model.Control group mice (12) injection 100 μ g LPS, and peptide inhibitor group mouse (12) is behind injection LPS 5min, every injected in mice 0.7mg peptide inhibitor.Analyze with the Kaplan-Meier survival curve, polypeptide Pro-Arg-Cys-(D-Bip)-(D-Arg)-Gly-Glu can protect small white mouse effectively, improves the survival rate of endotoxin shock mouse.These data show, suppress MMP-9 and TACE vigor and can effectively improve the survival rate of endotoxin shock mouse.
Claims (2)
1. matrix metalloproteinase-9 peptide inhibitor 1, it is characterized in that its sequence is Pro-Arg-Cys-(D-Bip)-(D-Arg)-Gly-Glu.
2. matrix metalloproteinase-9 peptide inhibitor 1 suppresses the application in the acute inflammation medicine in preparation, treatment.
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|---|---|---|---|
| CN 201110182737 CN102268068B (en) | 2011-07-01 | 2011-07-01 | Substrate metal prolease-9 polypeptide inhibitor 1and application thereof |
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| CN 201110182737 CN102268068B (en) | 2011-07-01 | 2011-07-01 | Substrate metal prolease-9 polypeptide inhibitor 1and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102746380A (en) * | 2012-07-25 | 2012-10-24 | 中国药科大学 | Application of angiogenesis inhibitor polypeptide to preparation of medicine for treating tumor and rheumatoid arthritis |
| CN103739676A (en) * | 2013-12-31 | 2014-04-23 | 罗瑞雪 | Application of polypeptide capable of inhibiting immunoglobulin K light chain gene enhancer in medicaments for treating acute inflammation |
| CN108484729A (en) * | 2018-03-23 | 2018-09-04 | 中国药科大学 | A kind of peptide inhibitor of matrix metalloproteinase 9 and its application |
| WO2021037292A3 (en) * | 2019-08-27 | 2021-04-22 | 南京安吉生物科技有限公司 | Polypeptide and use thereof |
-
2011
- 2011-07-01 CN CN 201110182737 patent/CN102268068B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| JIALIANG HU,ET AL: "Inhibition of Lethal Endotoxin Shock with an L-Pyridylalanine Containing Metalloproteinase Inhibitor Selected by High-Throughput Screening of a New Peptide Library", 《COMBINATORIAL CHEMISTRY & HIGH THROUGHPUT SCREENING》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102746380A (en) * | 2012-07-25 | 2012-10-24 | 中国药科大学 | Application of angiogenesis inhibitor polypeptide to preparation of medicine for treating tumor and rheumatoid arthritis |
| CN103739676A (en) * | 2013-12-31 | 2014-04-23 | 罗瑞雪 | Application of polypeptide capable of inhibiting immunoglobulin K light chain gene enhancer in medicaments for treating acute inflammation |
| CN103739676B (en) * | 2013-12-31 | 2015-10-14 | 顾祥茂 | Apply in a kind of Immunosuppression sphaeroprotein K light chain gene enhanser polypeptide therapeutic acute inflammation medicine |
| CN108484729A (en) * | 2018-03-23 | 2018-09-04 | 中国药科大学 | A kind of peptide inhibitor of matrix metalloproteinase 9 and its application |
| CN108484729B (en) * | 2018-03-23 | 2019-09-17 | 中国药科大学 | A kind of peptide inhibitor of matrix metalloproteinase 9 and its application |
| WO2021037292A3 (en) * | 2019-08-27 | 2021-04-22 | 南京安吉生物科技有限公司 | Polypeptide and use thereof |
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| CN102268068B (en) | 2013-02-13 |
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