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CN-122024834-A - Method for establishing whole system for screening anti-new coronavirus drug

CN122024834ACN 122024834 ACN122024834 ACN 122024834ACN-122024834-A

Abstract

The invention provides a method for establishing a whole system for screening anti-novel coronavirus drugs, which realizes systematic control over the whole life cycle of the viruses and solves the limitation of a single target model by targeting a plurality of key links such as virus invasion (pseudo-virus model), rdRp activity (replication transcription), mpro/3CLpro activity (polyprotein cleavage) and the like. By constructing a plurality of novel crown variant pseudovirus models and verifying the inhibiting effect of the drug on different strains (such as inhibiting the invasion of multiple strains by drug-containing serum and monomer components), the immune escape problem of variant strains is effectively solved, and the universality of a screening system is improved. The Mpro/3CLpro inhibitor screening system is established by a Fluorescence Resonance Energy Transfer (FRET) technology, the inhibition activity of the drug to the target is directly detected, the coverage of key antiviral targets is supplemented, and the screening comprehensiveness is enhanced.

Inventors

  • ZHOU MINGQIAN
  • WEN CHENGPING
  • XU XIAOYING
  • ZHANG XIAFENG
  • DONG YUZHU
  • WANG XUEYAN

Assignees

  • 浙江中医药大学

Dates

Publication Date
20260512
Application Date
20251203

Claims (10)

  1. 1. A method for establishing a whole system for screening anti-new coronavirus drugs, which is characterized by comprising the following steps: (1) Constructing a new coronavirus pseudovirus system, namely co-transfecting 293T cells by using recombinant vesicular stomatitis virus (rVSV-delta G) and SARS-CoV-2 spike protein (S protein) expression plasmid, and packaging to obtain pseudoviruses containing different new coronavirus variant S proteins; (2) Establishing a virus invasion path screening model: a. The TMPRSS 2-mediated membrane fusion pathway was simulated using Caco-2 cells; b. The CTSL mediated endocytic pathway was simulated using Vero cells; c. detecting the infection efficiency of the pseudovirus by a luciferase reporting system or a fluorescence microscope, and evaluating the inhibition effect of the drug on virus invasion; (3) Establishing an RNA-dependent RNA polymerase (RdRP) activity screening model: a. Cotransfecting 293T cells with nsp7, nsp8, nsp12 expression plasmids and a Gaussian luciferase (Gluc) reporter plasmid; b. detecting Gluc activity change, and evaluating the inhibition effect of the drug on RdRP activity; (4) A master protease (Mpro/3 CLpro) activity screening model was established: a. Using a Fluorescence Resonance Energy Transfer (FRET) based protease activity detection kit; b. The inhibitory effect of the drug on the activity of the Mpro/3CLpro protease was examined.
  2. 2. The method of claim 1, wherein in step (1), the SARS-CoV-2S protein expression plasmid comprises one or more strains selected from the group consisting of SARS-CoV-2, BA.4/5, BQ.1.1, EG.5.1, BA.2, XBB.1.5 and JN.1, wherein packaging of the pseudovirus is effected by infection of rVSV- ΔG after transfection of the S protein expression plasmid into 293T cells.
  3. 3. The method of claim 1, wherein in step (2), the drug comprises a drug-containing serum or monomeric compound of a decoction of a traditional Chinese medicine, wherein: a. The Chinese medicine is selected from one or more of Glycyrrhrizae radix, rhizoma Polygoni Cuspidati, herba asari, rhizoma Atractylodis, atractylodis rhizoma, rhizoma Ligustici Chuanxiong, rhizoma Dioscoreae, semen Alpinae Katsumadai, fructus Foeniculi, and flos Lonicerae; b. the monomer compound is selected from one or more of glycyrrhizic acid, alpha-asarone, atractyloside A, polydatin, luteolin, resveratrol, ferulic acid, chlorogenic acid, palmitic acid and catechin.
  4. 4. The method of claim 1, wherein in step (2), the drug is screened at a non-toxic concentration in Caco-2 cells and/or Vero cells, as determined by detecting cell viability by the CCK-8 method.
  5. 5. An anti-novel coronavirus combination selected by the method of any one of claims 1-4, comprising one or a combination of the following: (a) Medicated serum of Glycyrrhrizae radix, rhizoma Polygoni Cuspidati, herba asari, and rhizoma Atractylodis; (b) One or more of glycyrrhizic acid, alpha-asarone, atractyloside A, polydatin and luteolin; (c) Serum containing medicine of Atractylodis rhizoma, rhizoma Ligustici Chuanxiong, semen Tsaoko, flos Lonicerae, rhizoma Atractylodis, rhizoma Dioscoreae and fructus Foeniculi; (d) One or more of ferulic acid, chlorogenic acid, palmitic acid, and catechin; (e) One or more of polydatin, resveratrol and luteolin.
  6. 6. The anti-neocoronavirus combination of claim 5, wherein the combination is for inhibiting invasion of a neocoronavirus into a host cell by TMPRSS2 mediated membrane fusion pathway and/or CTSL mediated endocytosis pathway.
  7. 7. The anti-novel coronavirus combination of claim 5, wherein the combination is for inhibiting the activity of novel coronavirus RNA-dependent RNA polymerase (RdRP).
  8. 8. The anti-novel coronavirus combination of claim 5, wherein the combination is for inhibiting activity of a novel coronavirus main protease (Mpro/3 CLpro).
  9. 9. A pharmaceutical formulation comprising an anti-neocoronavirus combination of any one of claims 5-8 and a pharmaceutically acceptable carrier or adjuvant.
  10. 10. The use of an anti-novel coronavirus combination as claimed in any one of claims 5 to 8 for the preparation of a medicament for the prophylaxis and/or treatment of a novel coronavirus infection, characterized in that the anti-novel coronavirus combination is selected from the following targeted combinations according to the invasion mechanisms of different novel coronavirus variants: Aiming at the dominant strain of the membrane fusion pathway, glycyrrhizic acid, atractyloside A and polydatin are matched with drug-containing serum of liquorice, polygonum cuspidatum, asarum and atractylode; Aiming at endocytic pathway dominant strains, alpha-asarone, atractyloside A and luteolin are matched with medicine-containing serum of asarum and atractylode, and medicine-containing serum of grass nuts and honeysuckle; adding RdRp inhibitor comprising ferulic acid, chlorogenic acid, palmitic acid and catechin for viral replication, and adding drug-containing serum of Atractylodis rhizoma, rhizoma Ligustici Chuanxiong, fructus Tsaoko kernel, flos Lonicerae and rhizoma Atractylodis and drug-containing serum of fructus Foeniculi; the combination of the drug-containing serum of tsaoko cardamon, honeysuckle and rhizoma atractylodis and the drug-containing serum of Chinese yam and fennel are added aiming at inhibiting the virus protease, and are used for inhibiting the activity of Mpro/3CLpro protease.

Description

Method for establishing whole system for screening anti-new coronavirus drug Technical Field The invention relates to the technical field of bioengineering, in particular to a method for establishing a whole system for screening anti-new coronavirus drugs and an anti-new coronavirus combined drug screened by the method. Background SARS-CoV-2 is classified as a biosafety tertiary (BSL-3) pathogen, making the relevant experiment relatively complex and time-consuming, its stringent requirements on scientific research conditions (P3 laboratory), hampering basic research on its biological properties and development of effective antiviral drugs. In order to attract more scientists to study SARS-CoV-2 and improve the development efficiency of anti-SARS-CoV-2 virus drugs, a non-infectious and relatively safe experimental system is urgently needed to be established in a P2 laboratory to support the development and evaluation of anti-virus drugs. The current anti-SARS-CoV-2 virus drug screening system which can be applied in P2 laboratory mainly comprises computer-aided virtual screening, surface plasma resonance technology detection aiming at specific targets, high-throughput screening based on fluorescence polarization and the like, but the experiments lack biological verification. Cell experiments mainly involve the invasion of pseudoviruses, screening of viral RNA-dependent RNA polymerase inhibitors, etc., but these experiments are directed to only a single target. This model, which is directed at only a single target, cannot replicate the complex replication cycle of the virus in humans and the synergy between the components. Rapid variation of SARS-CoV-2, particularly as it is concentrated in structural changes in its spike protein (S protein), constitutes a continuing challenge for epidemic prevention and control. The S protein is the primary target of most neutralizing antibody drugs and vaccines. When a mutation occurs at a critical site, the neutralizing efficacy of an antibody induced by a vaccine developed based on the original strain may be reduced, i.e., an "immune escape" phenomenon may occur. Thus, the global monitoring agency assesses the potential risk of new variants by continually tracking mutations in the S protein and directs iterative updating of vaccine and therapeutic strategies. Disclosure of Invention In order to solve the technical problems in the prior art, the invention provides the following technical scheme: In one aspect, there is provided a method comprising the steps of: (1) Constructing a new coronavirus pseudovirus system, namely co-transfecting 293T cells by using recombinant vesicular stomatitis virus (rVSV-delta G) and SARS-CoV-2 spike protein (S protein) expression plasmid, and packaging to obtain pseudoviruses containing different new coronavirus variant S proteins; (2) Establishing a virus invasion path screening model: a. The TMPRSS 2-mediated membrane fusion pathway was simulated using Caco-2 cells; b. The CTSL mediated endocytic pathway was simulated using Vero cells; c. detecting the infection efficiency of the pseudovirus by a luciferase reporting system or a fluorescence microscope, and evaluating the inhibition effect of the drug on virus invasion; (3) Establishing an RNA-dependent RNA polymerase (RdRP) activity screening model: a. Cotransfecting 293T cells with nsp7, nsp8, nsp12 expression plasmids and a Gaussian luciferase (Gluc) reporter plasmid; b. detecting Gluc activity change, and evaluating the inhibition effect of the drug on RdRP activity; (4) A master protease (Mpro/3 CLpro) activity screening model was established: a. Using a Fluorescence Resonance Energy Transfer (FRET) based protease activity detection kit; b. The inhibitory effect of the drug on the activity of the Mpro/3CLpro protease was examined. Preferably, in step (1), the SARS-CoV-2S protein expression plasmid comprises one or more strains selected from the group consisting of SARS-CoV-2, BA.4/5, BQ.1.1, EG.5.1, BA.2, XBB.1.5, and JN.1, wherein packaging of the pseudovirus is effected by infection of rVSV- ΔG after transfection of the S protein expression plasmid into 293T cells. Preferably, in step (2), the medicament comprises a medicament-containing serum or monomeric compound of a decoction of a traditional Chinese medicine, wherein: a. The Chinese medicine is selected from one or more of Glycyrrhrizae radix, rhizoma Polygoni Cuspidati, herba asari, rhizoma Atractylodis, atractylodis rhizoma, rhizoma Ligustici Chuanxiong, rhizoma Dioscoreae, semen Alpinae Katsumadai, fructus Foeniculi, and flos Lonicerae; b. the monomer compound is selected from one or more of glycyrrhizic acid, alpha-asarone, atractyloside A, polydatin, luteolin, resveratrol, ferulic acid, chlorogenic acid, palmitic acid and catechin. Preferably, in step (2), the drug is screened in Caco-2 cells and/or Vero cells at a non-toxic concentration determined by detecting cell viability by CCK-8 method. In another aspect, there is p