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Substrate metal prolease-9 polypeptide inhibitor 1and application thereof

A technology of peptide inhibitors and matrix metals, applied in peptide/protein components, peptides, anti-inflammatory agents, etc., can solve problems such as side effects and lack of specificity of small molecule inhibitors

Inactive Publication Date: 2013-02-13
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many successful small molecule inhibitors, such as Marimastat, can inhibit the activity of matrix metalloproteinases at the nanomolar level, but small molecule inhibitors lack specificity. produce side effects

Method used

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  • Substrate metal prolease-9 polypeptide inhibitor 1and application thereof
  • Substrate metal prolease-9 polypeptide inhibitor 1and application thereof
  • Substrate metal prolease-9 polypeptide inhibitor 1and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Chemical Synthesis of Peptides

[0024] The peptides were synthesized using Fmoc chemistry. The synthesis reaction is carried out from the C-terminus to the N-terminus. There are free amino groups on the Rink medium (available from Advanced ChemTech Company), and Glu, Gly, D-Arg, D-Bip, Cys, Arg and Pro are connected sequentially. During each ligation step, the amino acid residues are activated, and the activation mixture contains 4 times as many HBTU, HOBt, DIEA and Fmoc-amino acids as there are free amino groups on the medium. After each amino acid linking reaction, a mixture of pyridine / acetic acid / N-methylimidazole (4:1:0.5) was used to block the unlinked free amino group for 10 minutes. After each amino acid linking reaction and before the next amino acid linking, the Fmoc-group on the medium should be removed, and the Fmoc-group should be removed using dimethylformamide containing 20% ​​piperidine, which takes 15 minutes. Finally, after all amino acid residues a...

Embodiment 2

[0027] IC50 values ​​of peptide inhibitors against several target enzymes (matrix metalloproteinase-3, -8, -9 and tumor necrosis factor releasing enzyme) in vitro.

[0028] Recombinant human matrix metalloproteinase-9 was expressed by Sf9 insect cells. The enzyme was activated with 0.01 μM matrix metalloproteinase-3 active site, and the buffer used was 100 mM Tris / HCl, pH 7.4, 100 mM NaCl, 10 mM CaCl 2 and 0.01% Tween-20. The concentration of matrix metalloproteinase-9 during activation was 92ng / μl (1μM). Enzyme activity is detected by cleaving the fluorescently generated peptide substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH 2 And detect the generated fluorescence value (excitation wavelength=328nm, detection wavelength=392nm). All assays were performed in 100 μl reactions at 37°C.

[0029] Recombinant human matrix metalloproteinase-8 is detected similarly to -9 and uses the same fluorogenic substrate. The reaction was also carried out in a 100 μl reaction system at 37°C. ...

Embodiment 3

[0033] Testing the Viability of Peptide Inhibitors in Vivo Using an Endotoxic Shock Model

[0034] Before establishing the endotoxic shock model, we first determined the LD50 of LPS (E.coli 0111: B4) mice as 50 μg per mouse, and we used 100 μg per mouse in the experiment, so that the mice in the control group Rats can all die. In our previous scientific research work, we found that Regasepin2 (another peptide inhibitor, published) can improve the survival rate of C57BL / 6 mice during endotoxin shock. In order to establish endotoxin shock model in our mice, we did a positive control experiment with Regasepin2. The mice in the control group were injected with 100 μg of LPS, while the mice in the Regasepin2 experimental group were injected with 0.7 mg of polypeptide 5 minutes after the injection of LPS. By making the Kaplan-Meier survival curve, it was found that Regasepin2 can effectively protect the mice injected with 100 μg and improve the survival rate ( Figure 7 ). After...

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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 [0001] The invention relates to a matrix metalloproteinase-9 polypeptide inhibitor 1 and its application, in particular to a polypeptide capable of inhibiting matrix metalloproteinase-9 and reducing the destruction of the body caused by acute inflammatory reactions. Background technique [0002] Sepsis has always been a serious medical problem. Early sepsis can progress to severe sepsis and septic shock without timely and effective treatment, and the mortality rates of the two are 20%-30% and 40%-70%, respectively. The medical community has been studying effective treatments, such as early symptomatic treatment, activated protein C, and corticosteroid treatment. In recent years, with the deepening of medical research, great progress has been made in the understanding and treatment of sepsis. Among them, matrix metalloproteinase-9 (MMP-9) plays an important role in endotoxin shock. [0003] Matrix metalloproteinases are a group of more than 20 structurally ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06A61K38/08A61P29/00
Inventor 邱郑胡加亮李伟光
Owner CHINA PHARM UNIV
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