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Application of moutan bark glycoside C in preparation of protein disulfide bond isomerase inhibitor

A technology of disulfide bond isomerase and protein, which is applied in the field of biomedicine to achieve the effect of inhibiting collagen-induced platelet aggregation and efficiently inhibiting the activity of protein disulfide bond isomerase

Pending Publication Date: 2022-05-13
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There is no report on the protein disulfide isomerase of paeonolin C in the existing literature; this patent will obtain and confirm the effect of paeonin C on inhibiting protein disulfide isomerase and antiplatelet for the first time

Method used

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  • Application of moutan bark glycoside C in preparation of protein disulfide bond isomerase inhibitor
  • Application of moutan bark glycoside C in preparation of protein disulfide bond isomerase inhibitor
  • Application of moutan bark glycoside C in preparation of protein disulfide bond isomerase inhibitor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Screening for a small molecule inhibitor of protein disulfide bond isomerase.

[0030] experimental method:

[0031] (1) Dried Moutan cortex Chinese medicine decoction pieces at below 50°C, crushed and passed through a No. 4 sieve in the Chinese Pharmacopoeia, and took the sieved Moutan cortex medicinal material powder for later use.

[0032] (2) Add 10 times the weight of ethanol with a weight percentage concentration of 75% to the Moutan Cortex powder, conduct ultrasonic extraction at 20° C. for 30 min, and the ultrasonic frequency is 40 KHz. Centrifuge at 12000rpm for 15min, take the supernatant into an evaporating dish, evaporate it to dryness at 50°C, add 1ml DMSO to redissolve, and prepare the Cortex Moutan extract with a crude drug amount of 1g / ml for future use.

[0033] (3) Dilute the product of step (2) to 10mg / ml, add 0.5ml of the diluted solution to the PDI affinity column, and incubate at 20°C for 2h. After incubation, the "unbound fraction" and the "boun...

Embodiment 2

[0038] Evaluation of the inhibitory effect of a compound of the present invention on protein disulfide bond isomerase.

[0039] Experimental method: Insulin turbidimetry was used to determine the inhibitory activity of compounds on disulfide bond isomerase. Insulin consists of two peptide chains, A and B, connected by two disulfide bonds. In the presence of dithiothreitol (DTT), disulfide bond isomerase can play its role as a reductase to reduce the disulfide bond between the two peptide chains of insulin, and separate the A chain and the B chain. The large B chain will settle down, causing the reaction system to be turbid. Use a microplate reader to measure the turbidity of the reaction system to determine the activity of disulfide bond isomerase.

[0040]In the reaction system, the final concentration of disulfide isomerase is 263nM, the final concentration of insulin is 0.2mM, and the final concentration of DTT is 0.3mM. The reaction is carried out in 100mM potassium phosp...

Embodiment 3

[0046] An analysis of the interaction between a compound of the present invention and protein disulfide bond isomerase.

[0047] Experimental method: use micro-scale thermophoresis analysis (Micro-Scale Thermophoresis, MST), the micro-thermophoresis instrument heats the solution in the capillary by infrared laser, the fluorescent molecules in the solution are subjected to the effect of thermophoresis to present a temperature gradient field, due to the fluorescence The degree of change in the signal is related to the binding of the ligand and the fluorescent molecule, so MST can be used to measure the intermolecular affinity.

[0048] The affinity of fluorescently labeled protein disulfide bond isomerase and its b'-x fragment to paeonidin C was determined at room temperature using MO NT.115 microthermophoresis instrument. His-tagged proteins were labeled using the MO-L018 RED-tris-NTA kit. Dilute the small molecule ligand (paeonidin C) in a gradient in a PCR tube, add the labe...

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Abstract

The invention belongs to the technical field of biological medicine, and discloses application of moutan bark glycoside C in preparation of a protein disulfide isomerase inhibitor. A protein disulfide bond isomerase binding component is obtained through affinity chromatography, and activity determination finds that moutan glycoside C can specifically inhibit protein disulfide bond isomerase (EC50lt; the antithrombotic peptide has the advantages that the antithrombotic peptide has a potential antithrombotic effect and is expected to be developed into drugs for preventing thrombotic diseases, and the effect of inhibiting platelet aggregation is achieved by interfering adhesion and expansion of platelets.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to the application of paeonin C in the preparation of protein disulfide bond isomerase inhibitors. Background technique [0002] Disulfide isomerase (protein disulfide isomerase, PDI) is a class of enzymes in the endoplasmic reticulum (ER), which has redox and isomerase activities, can promote the formation of disulfide bonds in nascent proteins, and make proteins fold correctly , also has molecular chaperone function. PDI is the first member of the PDI family, about 57kDa, composed of 519 amino acids, and has four thioredoxin-like domains, namely a, b, b', a'. The a and a' domains are the catalytic domains of PDI, the b and b' domains are the binding domains, and the hydrophobic pocket on the b' domain is the main substrate-binding region. [0003] Disulfide bond isomerase plays an important role in the process of thrombus formation. Many membrane glycoprotein receptors related...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7048A61P7/02A61K36/71A61K36/65A61K125/00
CPCA61K31/7048A61P7/02A61K36/71A61K36/65
Inventor 余伯阳刘秀峰周娅彤
Owner CHINA PHARM UNIV
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