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A stable polypeptide inhibitor derived from lsd1 substrate snail1 and its use

A technology for polypeptide inhibitors and uses, applied in the field of bioengineering, to achieve significant technological progress, increase treatment, and solve the effects of poor selectivity

Active Publication Date: 2021-10-15
PEKING UNIV SHENZHEN GRADUATE SCHOOL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tortorici et al. reported that the peptide derived from SNAIL1 was a reversible LSD1 inhibitor. Suzuki et al. modified the peptide by adding propargylamine, hydrazine or PCPA groups, which had an irreversible inhibitory effect on LSD1 and increased LSD1 to a certain extent. However, it is still an inhibitor targeting FAD in amine oxidase, which inevitably has certain effects on other proteins of the amine oxidase family

Method used

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  • A stable polypeptide inhibitor derived from lsd1 substrate snail1 and its use
  • A stable polypeptide inhibitor derived from lsd1 substrate snail1 and its use
  • A stable polypeptide inhibitor derived from lsd1 substrate snail1 and its use

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Experimental program
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Embodiment 1

[0031] Embodiment 1: the design of polypeptide inhibitor

[0032] The present invention uses a chemical method based on sulfonium salt side chain stabilizing polypeptides to modify the polypeptide targeting LSD1. We select a section of polypeptide sequence (PRSFLVRKP) near the LSD1 selective substrate SNAIL1, because Arg7 of the polypeptide and Cys360 near the active pocket of LSD1 protease Appropriate for using the "sulfonium salt-based stabilization method" to stabilize the polypeptide and enable the covalent binding of the polypeptide as a ligand to the protein recognition without affecting the recognition of the target by the N-terminus of the polypeptide.

[0033] In order to further verify the importance of the peptide sequence, we screened a series of peptide sequences, such as Figure 4 As shown, there are polypeptide sequences of different lengths, sequences of the same length and different amino acid arrangements, and the inhibitory effect of these polypeptide sequen...

Embodiment 2

[0034] Embodiment 2: the synthetic preparation of polypeptide inhibitor

[0035] The solid-phase synthesis of polypeptides involved in the present invention all use Rink Amide MBHA resin, the resin with a loading degree of 0.37mmol / g is used for long peptides, and the resin with a loading degree of 0.54mmol / g is used for short peptides. The specific operation of solid-phase synthesis of peptides is as follows:

[0036] (1) Select Rink Amide MBHA resin with a suitable loading degree and swell with DMF for 30 minutes;

[0037](2) remove the Fmoc protecting group with 50% morpholine (dissolved in DMF), the reaction time is 30 minutes, repeat twice;

[0038] (3) Then use DMF (washing 4 times) and DCM (washing 4 times) to cross-wash respectively;

[0039] (4) Use 4 reaction equivalents of Fmoc-protected amino acids (obtained by conversion of the initial resin load), 3.9 equivalents of HCTU are mixed with DMF to dissolve, and then 8 equivalents of DIPEA are added for activation. T...

Embodiment 3

[0047] Example 3: Research on the Inhibitory Effect of Polypeptide LSD1 Enzyme Activity

[0048] The LSD1-HRP coupling reaction can be used to detect LSD1 activity. The principle is that the by-product H is produced in the process of substrate demethylation catalyzed by LSD1. 2 o 2 , so horseradish peroxidase (HRP) can be used to catalyze H 2 o 2 React with Amplex Red (a dye) to produce Resorufin (a substance that can produce strong fluorescence), and the enzymatic activity of LSD1 can be indirectly obtained by detecting the fluorescence intensity of the product. in the H 2 o 2 is reduced to H 2 In the process of O, Amplex Red acts as an electron donor, which is oxidized into Resorufin for detection of fluorescence. Its maximum excitation wavelength is about 530nm, and its maximum emission wavelength is about 590nm, and the reactants Amplex Red and H 2 o 2 There is no absorption at 530nm and will not interfere with detection. According to the limitations of experimen...

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Abstract

The present invention provides a polypeptide inhibitor, whose amino acid sequence structure is as follows: The present invention also provides the use of the above-mentioned polypeptide inhibitor in the preparation of a drug for inhibiting LSD1 enzyme activity. The polypeptide of the invention can be covalently combined with the cysteine ​​(Cys360) near the active pocket of the LSD1 enzyme, thereby inhibiting the activity of the LSD1 protease, and can be used for anti-tumor treatment in vivo.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a polypeptide, specifically a stable polypeptide inhibitor derived from the LSD1 substrate SNAIL1 and its application. Background technique [0002] Cancer is a major public health problem worldwide, and in China it is the leading cause of death and causes a significant disease burden. The types of tumors and the causes of tumors are different in different genders and ages. The complexity of tumor diseases has brought challenges to human beings to overcome this disease. More and more studies have found that disorders of epigenetic regulation are closely related to various diseases, such as cancer, autoimmune diseases, mental disorders or diabetes. The formation of a variety of tumors is associated with abnormalities in genes controlling epigenetic processes, especially the aberrant expression of LSD1. LSD1 protein plays an important role in the occurrence and development of various t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47A61K38/17A61P35/00
CPCA61K38/00A61P35/00C07K14/4703
Inventor 李子刚尹丰马越
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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