Matrix metalloproteinase-2 polypeptide inhibitor and application
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.
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Embodiment 1
[0017] Chemical Synthesis of Peptides
[0018] 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 Met, Lys, Glu, Gly, Gly, Gly, Gly, Val, Asn, Ser and Trp 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 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 ...
Embodiment 2
[0022] 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).
[0023] Recombinant human matrix metalloproteinase-2 is expressed by E. coli cells. The enzyme is activated with 0.01 μM MMP-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 assays were performed in 100 μl reactions at 37°C. The measured IC50 value is 46.32 μmol.
[0024] Recombinant human matrix metalloproteinase-8 is detected similarly to that of -2 and uses the same fluorogenic substrate. Reactions were also performed in 100 μl reactions...
Embodiment 3
[0028] Testing the Viability of Peptide Inhibitors in Vivo Using an Endotoxic Shock Model
[0029] 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 Trp-Ser-Asn-Val-Gly-Gly-Gly-Gly-Glu-Lys-Met. The mice in the control group (12) were injected with 100 μg of LPS, while the mice in the peptide inhibitor gr...
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