Stable polypeptide protein covalent inhibitor of papain-like protease PLpro targeting 2019 novel coronavirus

A papain, coronavirus technology, applied in the field of bioengineering, can solve problems such as poor pneumonia

Active Publication Date: 2021-12-03
SHENZHEN BAY LAB PINGSHAN TRANSLATIONAL MEDICINE CENT +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a stable polypeptide protein covalent inhibitor targeting 2019 novel coronavirus protein PLpro and its application. The described targeting 2019 novel coronavirus protein PLpro Stable polypeptide protein covalent inhibitors and their uses should solve the technical problem that the drugs in the prior art are not effective in treating pneumonia caused by new coronaviruses

Method used

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  • Stable polypeptide protein covalent inhibitor of papain-like protease PLpro targeting 2019 novel coronavirus
  • Stable polypeptide protein covalent inhibitor of papain-like protease PLpro targeting 2019 novel coronavirus
  • Stable polypeptide protein covalent inhibitor of papain-like protease PLpro targeting 2019 novel coronavirus

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Embodiment 1

[0028] The present invention adopts the sulfonium salt stabilized polypeptide technology reported in the previous literature (D.Wang, M.Yu, et al.Chem.Sci.10, 4966-4972), through the combination of methionine, cysteine ​​and dialkylene on the polypeptide The sulfonium salt cyclic peptide can be reacted with a base-based reagent, which can not only stabilize the polypeptide, but also covalently modify the cysteine ​​on the interaction site of the target protein, and block the papain-like protease PLpro and various non-structural proteins of the virus. Binding, inhibiting the replication of viral genes.

[0029] The present invention provides a series of stable polypeptide protein covalent inhibitors targeting 2019 novel coronavirus (SARS-CoV-2) papain-like protease (Papain-like protease, PLpro). The inventors synthesized a number of different polypeptides, as shown in Table 1.

[0030] Table 1: Molecular sequences of different stable polypeptide covalent inhibitors targeting t...

Embodiment 2

[0032] Preparation and separation and purification steps of the polypeptide of embodiment 2:

[0033] The polypeptide of the present invention is synthesized in solid phase according to the amino acid sequence, which is a conventional technique and will not be repeated here. This example only describes the core steps for preparing the above-mentioned stable polypeptide as follows (taking CM1 as an example):

[0034] Specific operation steps ( figure 1 )for:

[0035](1) Polypeptide solid-phase synthesis: Weigh Rink amide MBHA resin into a peptide tube, add dichloromethane (DCM), and swell with nitrogen gas for 30 minutes. Add 50% (v / v) morpholine in N,N-dimethylformamide (DMF) solution, blow nitrogen gas for 30 minutes, and remove the Fmoc protecting group. After washing the resin alternately with DMF and DCM, the prepared Fmoc-AA-OH (5eq, 0.4M, DMF) solution, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea Fluorophosphate ester (HCTU) (5eq, 0.38M, DMF) solution and N,N-diiso...

Embodiment 3

[0038] Example 3 Polypeptide Molecule and Protein PLpro Covalent in Vitro

[0039] The methionine and cysteine ​​on different polypeptides with fluorescent groups react with dialkylating reagents to form sulfonium salt cyclic peptides and respectively co-incubate with the protein, the protein and the sulfonium salt polypeptide undergo a covalent reaction, and the protein can be seen Fluorescent display on the strip ( figure 2 ), indicating that CM1-8 polypeptide can covalently react with PLpro protein, while general linear peptides cannot produce covalent linkage.

[0040] We choose the peptide EM-C as an example to study reaction kinetics and stoichiometry studies. FAM-labeled peptide EM-C (10 μM) was reacted with SARS-CoV-2 PLpro (5 μM) in PBS buffer for different times (0.5, 1, 2, 3, 4 hours). The response was dose-dependent. Kinetic and stoichiometric studies clearly show the high efficiency of binding ( image 3 A, B).

[0041] The polypeptide has good specificity, ...

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Abstract

The invention provides a stable polypeptide protein covalent inhibitor of papain-like protease PLpro targeting 2019 novel coronavirus. The invention also provides application of the stable polypeptide protein covalent inhibitor in preparing a drug for inhibiting the enzyme activity of the papain-like protease PLpro. The invention adopts a method of reacting methionine-cysteine on polypeptide with a dialkylating reagent to form a single sulfonium salt to stabilize sulfonium salt cyclopeptide targeting PLpro. The invention adopts a strategy of coupling the sulfonium salt stable polypeptide with small molecules to invent the novel stable polypeptide protein covalent inhibitor of the papain-like protease PLpro. The polypeptide protein covalent inhibitor provided by the invention can effectively inhibit the activity of the papain-like protease PLpro, thereby blocking the immune escape reaction generated by cutting ISG15 by the papain-like protease PLpro.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a stable polypeptide protein covalent inhibitor, specifically a stable polypeptide protein covalent inhibitor targeting 2019 novel coronavirus papain-like protease PLpro and its application. Background technique [0002] The causative agent of COVID-19 is a new type of coronavirus called SARS-CoV-2. This coronavirus is highly contagious and pathogenic, and has spread widely around the world since it was first discovered in December 2019. So far, it has infected more than 200 million people and killed more than 4 million people. The incubation period of COVID-19 infection ranges from 2-14 days and can be as long as 24 days. These longer incubation periods, due to their transmissibility and asymptomatic nature, are responsible for the high number of infections. The growing number of COVID-19 cases reflects the seriousness of the current situation and the need for effective solutions, y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/103A61K38/07A61K47/42A61P31/14
CPCC07K5/101A61K38/07A61K47/42A61P31/14
Inventor 李子刚尹丰刘娜章亦弛
Owner SHENZHEN BAY LAB PINGSHAN TRANSLATIONAL MEDICINE CENT
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