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Matrix metal proteinase-2 polypeptide inhibitors and application thereof

A peptide inhibitor and matrix metal technology, applied in the field of matrix metalloproteinase-2 peptides, can solve the problems of side effects, lack of specificity of small molecule inhibitors, etc.

Inactive Publication Date: 2013-09-04
QINGDAO MUNICIPAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation and use of macromolecular inhibitors has limited their development, for example recombinant human matrix proteinase tissue inhibitor of metalloproteinase-3 has an in vivo half-life of only 4 minutes
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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  • Matrix metal proteinase-2 polypeptide inhibitors and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Chemical Synthesis of Peptides

[0019] The peptides were synthesized using Fmoc chemistry. The synthesis reaction proceeds from the C-terminus to the N-terminus. There are free amino groups on the Rink medium (available from Advanced ChemTech), and Arg, Asn, Thr, Thr and Pro are connected in sequence. 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 unlinked free amino groups for 10 min. After each amino acid ligation reaction and before the next amino acid is ligated, the Fmoc-group on the medium must be removed, and the Fmoc-group is removed using dimethylformamide containing 20% ​​piperidine, which takes 15 minutes. Finally, after all amino acid residues are linked sequentially, the peptide is cl...

Embodiment 2

[0023] IC50 values ​​of polypeptide inhibitors against several target enzymes in vitro: matrix metalloproteinase-3, -8, -2 and tumor necrosis factor releasing enzyme (both purchased from sigma company).

[0024] Recombinant human matrix metalloproteinase-2 was expressed by E. coli cells. The enzyme was activated with 0.01 μM matrix metalloproteinase-3 active site in 100 mM Tris / HCl, pH 7.4, 100 mM NaCl, 10 mM CaCl 2 and 0.01% Tween-20. The concentration of MMP-2 upon activation was 72 ng / μl (1 μM). 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=328 nm, detection wavelength=392 nm). All detections were carried out in 100 μl reaction system at 37°C. The measured IC50 value was 54.97 μmol.

[0025] Recombinant human matrix metalloproteinase-8 is detected similarly to that of -2 and uses the same fluorogenic substrate. Reactions were...

Embodiment 3

[0029] Testing the Viability of Peptide Inhibitors in Vivo Using an Endotoxic Shock Model

[0030] Before establishing the endotoxic shock model, we first determined the LD50 of LPS (E. coli 0111: B4) mice to be 50 μg per mouse, and we used 100 μg per mouse in the experiment, so that the control group All mice died. A positive control experiment was done 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 LPS injection. By making a Kaplan-Meier survival curve, it was found that Regasepin2 can effectively protect mice injected with 100 μg and improve the survival rate. After successfully establishing an endotoxin shock model in mice, the model was used to detect the in vivo activity of Pro-Thr-Thr-Asn-Arg. The mice in the control group (12) were injected with 100 μg of LPS, while the mice in the polypeptide inhibitor group (12) were injected...

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Abstract

The invention relates to the field of medicine, and particularly relates to polypeptides capable of inhibiting matrix metal proteinase-2 and tumor necrosis factor liberase and relieving damage of acute inflammation response on organisms. The sequence of the polypeptide is Pro-Thr-Thr-Asn-Arg which is a brand-new sequence. The inhibitors can inhibit the activities of the matrix metal proteinase-2 and tumor necrosis factor liberase in vitro on the 1 micromole level, and increase the survival rate of endotoxin shock mice in an in-vivo test, thereby having potential new drug development value.

Description

technical field [0001] The invention relates to a matrix metalloproteinase-2 polypeptide and its application, in particular to a polypeptide capable of inhibiting matrix metalloproteinase-2 and reducing acute inflammatory response damage to the body. [0002] Background technique [0003] Sepsis has always been a serious medical problem. Early sepsis without timely and effective treatment can progress to severe sepsis and septic shock, with mortality rates of 20%-30% and 40%-70%, respectively. The medical field has been researching effective treatment methods, such as early symptomatic treatment, activated protein C, corticosteroid treatment and so on. 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-2 (MMP-2) plays an important role in endotoxin shock. [0004] Matrix metalloproteinases are a group of more than 20 structurally similar and functionally...

Claims

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Application Information

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IPC IPC(8): C07K7/06A61K38/08A61P29/00
Inventor 罗瑞雪
Owner QINGDAO MUNICIPAL HOSPITAL
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