CN118717980A - ASGR1 as a drug target for the treatment of SARS-CoV-2 infection - Google Patents

Abstract

The present invention discloses the application of ASGR1 as a drug target in the treatment of SARS-CoV-2 infection. Studies have found that transient expression of ASGR1 can lead to SARS-CoV-2 invasion, and SARS-CoV-2 infection can be effectively prevented or treated by inhibiting the binding of SARS-CoV-2 S protein to ASGR1. Therefore, the present invention provides a new idea for the treatment of SARS-CoV-2 infection.

Description

ASGR1 as a drug target for the treatment of SARS-CoV-2 infection

Technical Field

The present invention relates to the field of biomedicine, and in particular to an application of ASGR1 as a drug target in the treatment of SARS-CoV-2 infection.

Background Art

SARS-CoV-2 is a highly contagious human pathogen that can cause fever, cough, severe respiratory disease, and fatal organ failure. SARS-CoV-2 is a member of the betacoronavirus genus and is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV) and several bat coronaviruses.

Host cell receptors are key determinants of viral tropism and pathogenesis. Viruses (such as HIV, HBV, and coronaviruses) can interact with the host through multiple receptors, leading to viral attachment, invasion, or specific host responses. The spike protein (S) on the surface of coronaviruses plays a central role in binding to host receptors. Both SARS-CoV and SARS-CoV-2 utilize ACE2 as the primary entry receptor (3-5), and ACE2 is currently the only widely recognized receptor for SARS-CoV-2. However, the tissue specificity of ACE2 expression makes it difficult to explain the multi-organ tropism of SARS-CoV-2, and ACE2 itself cannot explain the differences in clinical manifestations between SARS-CoV and SARS-CoV-2; these suggest that other receptors exist to mediate the entry process of SARS-CoV-2. In addition, even if the receptor is not involved in the viral entry process, the viral host interaction it mediates can promote other host responses, such as inducing cytokine secretion, cell apoptosis, and stimulation of immune responses, or altering viral budding and release, thereby promoting viral pathogenesis. Therefore, systematic analysis of the host cell receptor repertoire of SARS-CoV-2 is very important for basic and clinical research on the new coronavirus.

Receptors screened from virus-susceptible cell lines are limited to the membrane proteins specifically expressed by the cells. We previously used a flow cytometry-based method to study ligand-receptor interactions; the principle is to incubate cells expressing receptors with labeled ligands, and then label and detect them with anti-label antibodies. This method more realistically simulates ligand-receptor interactions under physiological conditions, but because it is time-consuming and labor-intensive, it is often used to confirm newly discovered ligand-receptor interactions or small-scale interaction screening. Based on this method, we developed a genome-wide secretome interaction screening platform that covers almost all human membrane proteins (a total of 5054 species, accounting for 91.6% of all human membrane proteins). The system can screen virus-related proteins regardless of viral tropism, thereby obtaining almost all related receptors.

We used this platform to systematically screen the SARS-CoV-2S protein and perform functional analysis of the obtained receptors to obtain new therapeutic targets for SARS-CoV-2 infection.

Summary of the invention

In view of the existing shortcomings described in the above background, the purpose of the present invention is to provide an application of targeting KREMEN1 and ASGR1 in the treatment of SARS-CoV-2 infection, so as to provide a new idea for solving the existing technology for treating SARS-CoV-2 infection.

To achieve the above-mentioned and other related purposes, the present invention provides, on the one hand, the use of KREMEN1 as a drug target in in vitro screening of SARS-CoV-2 therapeutic drugs.

The method for in vitro screening of SARS-CoV-2 therapeutic drugs may optionally include: conducting an in vitro viral infection experiment on candidate drugs to screen out drugs that can enhance cell resistance to viral infection.

The therapeutic drug can inhibit or block the binding of KREMEN1 to the S protein of SARS-CoV-2.

Another aspect of the present invention provides use of a KREMEN1 inhibitor in the preparation of a medicament for preventing and/or treating SARS-CoV-2.

The drug uses KREMEN1 as a drug target.

The drug can inhibit or block the binding of KREMEN1 to the S protein of SARS-CoV-2.

The KREMEN1 inhibitor refers to a substance that has an inhibitory effect on KREMEN1 and can reduce the content of KREMEN1 in cells. The KREMEN1 inhibitor is a small molecule inhibitor or a KREMEN1 antibody.

Preferably, when the KREMEN1 inhibitor is a small molecule inhibitor, it is Suramin Sodium (CAS129-46-4), the molecular formula of which is C ₅₁ H ₃₄ N ₆ Na ₆ O ₂₃ S ₆ , and the structural formula is as shown in formula (I):

Preferably, when the KREMEN1 inhibitor is a KREMEN1 antibody, the heavy chain CDR1, CDR2, and CDR3 of the KREMEN1 antibody are shown in SEQ ID NOs: 1 to 3, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 4 to 6, respectively.

The inhibitory effect on KREMEN1 includes but is not limited to: inhibiting the activity of KREMEN1 or inhibiting the transcription or expression of KREMEN1 gene. For example, compared with the control group, the KREMEN1 inhibitor can reduce the content of KREMEN1 in the cell by 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% without affecting other functions of the cell. The KREMEN1 inhibitor can be an antibody or a small molecule compound. The antibody refers to a peptide or protein that can bind to KREMEN1. The KREMEN1 inhibitor can also be a compound that reduces or inhibits the expression or transcription of the KREMEN1 gene, including but not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins or interfering lentiviruses. The nucleic acid includes but is not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA or short hairpin RNA (shRNA) prepared by endoribonuclease III.

Whether a drug can inhibit KREMEN1 activity can be determined using existing technologies, including isotope labeling assays.

Existing technologies can also be used to determine whether a drug can inhibit the transcription or expression of the KREMEN1 gene. For example, cells that normally express KREMEN1 are provided, the cells are cultured in the presence of the drug to be tested or a vector carrying the drug to be tested, and whether the transcription or expression level of KREMEN1 changes.

The use of KREMEN1 inhibitors in the preparation of drugs for preventing or treating SARS-CoV-2 specifically refers to: using KREMEN1 inhibitors as the main active ingredient of the drug for the preparation of drugs for preventing or treating SARS-CoV-2.

Another aspect of the present invention provides a medicament for preventing and/or treating SARS-CoV-2 infection, comprising a therapeutically effective amount of a KREMEN1 inhibitor.

The KREMEN1 inhibitor refers to a compound that has an inhibitory effect on KREMEN1. Preferably, the KREMEN1 inhibitor is Suramin Sodium (CAS129-46-4), the molecular formula is C ₅₁ H ₃₄ N ₆ Na ₆ O ₂₃ S ₆ , and the structural formula is shown in formula (I). The inhibitory effect on KREMEN1 includes but is not limited to: inhibiting the activity of KREMEN1 or inhibiting the gene transcription or expression of KREMEN1. The KREMEN1 inhibitor can be an antibody or a small molecule compound. When the KREMEN1 inhibitor is a small molecule inhibitor, its structure is shown in formula (I). When the KREMEN1 inhibitor is a KREMEN1 antibody, the heavy chain CDR1, CDR2, and CDR3 of the KREMEN1 antibody are shown in sequences SEQ ID NO: 1 to 3, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in sequences SEQ ID NO: 4 to 6, respectively.

KREMEN1 Antibody K33:

Heavy chain: CDR1: GYTFTGYG (SEQ ID NO: 1); CDR2: IYPRSGNT (SEQ ID NO: 2); CDR3: SRYYGPKGFDY (SEQ ID NO: 3);

Light chain: CDR1: ESVDNYGISF (SEQ ID NO: 4); CDR2: AAS (SEQ ID NO: 5); CDR3: QQSKEVPYT (SEQ ID NO: 6).

Another aspect of the present invention provides a method for preventing and/or treating SARS-CoV-2 infection, comprising administering a KREMEN1 inhibitor to a subject.

The object can be a mammal or a virus-infected cell of a mammal. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. The primate is preferably a monkey, an ape or a human. The virus-infected cell can be an in vitro infected cell.

The subject may be a patient infected with SARS-CoV-2 or an individual infected with SARS-CoV-2 who is expecting treatment. Or the subject may be an infected viral cell of a patient infected with SARS-CoV-2 or an individual infected with SARS-CoV-2 who is expecting treatment.

The KREMEN1 inhibitor can be administered to a subject before, during, or after receiving treatment for viral infection. The KREMEN1 inhibitor can be an antibody or a small molecule compound. Preferably, the KREMEN1 inhibitor is Suramin Sodium (CAS129-46-4), with a molecular formula of C ₅₁ H ₃₄ N ₆ Na ₆ O ₂₃ S ₆ , and a structural formula as shown in formula (I). When the KREMEN1 inhibitor is a KREMEN1 antibody, the heavy chain CDR1, CDR2, and CDR3 of the KREMEN1 antibody are respectively shown in sequences SEQ ID NO: 1 to 3, and the light chain CDR1, CDR2, and CDR3 are respectively shown in sequences SEQ ID NO: 4 to 6.

On the other hand, the present invention provides the use of ASGR1 as a drug target in in vitro screening of SARS-CoV-2 therapeutic drugs.

The method for in vitro screening of SARS-CoV-2 therapeutic drugs may optionally include: performing an in vitro viral infection experiment on candidate drugs to screen out drugs that can enhance cell survival.

The therapeutic drug can inhibit or block the binding of ASGR1 to the S protein of SARS-CoV-2.

Another aspect of the present invention provides use of an ASGR1 inhibitor in the preparation of a medicament for preventing and/or treating SARS-CoV-2.

The drug uses ASGR1 as a drug target.

The drug can inhibit or block the binding of ASGR1 to the S protein of SARS-CoV-2.

The ASGR1 inhibitor refers to a substance that has an inhibitory effect on ASGR1 and can reduce the content of ASGR1 in cells. The ASGR1 inhibitor is a small molecule inhibitor or an ASGR1 antibody.

Preferably, when the ASGR1 inhibitor is an ASGR1 antibody, the heavy chain CDR1, CDR2, and CDR3 of the ASGR1 antibody are shown in SEQ ID NOs: 7 to 9, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 10 to 12, respectively.

ASGR1 Antibody S23:

Heavy chain: CDR1: RYTFTDYN (SEQ ID NO: 7); CDR2: ITPNNGGT (SEQ ID NO: 8); CDR3: ARKGGYFDV (SEQ ID NO: 9);

Light chain: CDR1: SSVSY (SEQ ID NO: 10); CDR2: RTSN (SEQ ID NO: 11); CDR3: QQYHSYPLT (SEQ ID NO: 12).

The inhibitory effect on ASGR1 includes but is not limited to: inhibiting the activity of ASGR1 or inhibiting the transcription or expression of ASGR1 gene. For example, compared with the control group, the ASGR1 inhibitor can reduce the ASGR1 content in the cell by 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% without affecting other functions of the cell. The ASGR1 inhibitor can be an antibody or a small molecule compound. The antibody refers to a peptide or protein that can bind to ASGR1. The ASGR1 inhibitor can also be a compound that reduces or inhibits the expression or transcription of the ASGR1 gene, including but not limited to: nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins or interfering lentiviruses. The nucleic acid includes but is not limited to: antisense oligonucleotides, double-stranded RNA (dsRNA), ribozymes, small interfering RNA or short hairpin RNA (shRNA) prepared by endoribonuclease III.

Whether a drug can inhibit ASGR1 activity can be determined using existing technologies, including isotope labeling assays.

Existing technologies can also be used to determine whether a drug can inhibit ASGR1 gene transcription or expression. For example, cells that normally express ASGR1 are provided, the cells are cultured in the presence of a drug to be tested or a vector carrying the drug to be tested, and whether ASGR1 transcription or expression level changes.

The use of ASGR1 inhibitors in the preparation of drugs for preventing or treating SARS-CoV-2 specifically refers to: using ASGR1 inhibitors as the main active ingredient of the drug for the preparation of drugs for preventing or treating SARS-CoV-2.

Another aspect of the present invention provides a medicament for preventing and/or treating SARS-CoV-2 infection, wherein the medicament comprises a therapeutically effective amount of an ASGR1 inhibitor.

The ASGR1 inhibitor refers to a compound that has an inhibitory effect on ASGR1. The ASGR1 inhibitor is a small molecule inhibitor or an ASGR1 antibody. Preferably, when the ASGR1 inhibitor is an ASGR1 antibody, the heavy chain CDR1, CDR2, and CDR3 of the ASGR1 antibody are shown in the sequences SEQ ID NOs: 7 to 9, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in the sequences SEQ ID NOs: 10 to 12, respectively.

The inhibitory effect on ASGR1 includes but is not limited to: inhibiting the activity of ASGR1 or inhibiting the gene transcription or expression of ASGR1.

Another aspect of the present invention provides a method for preventing and/or treating SARS-CoV-2 infection, comprising administering an ASGR1 inhibitor to a subject.

The object can be a mammal or a virus-infected cell of a mammal. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. The primate is preferably a monkey, an ape or a human. The virus-infected cell can be an in vitro infected cell.

The subject may be a patient infected with SARS-CoV-2 or an individual infected with SARS-CoV-2 who is expecting treatment. Or the subject may be an infected viral cell of a patient infected with SARS-CoV-2 or an individual infected with SARS-CoV-2 who is expecting treatment.

The ASGR1 inhibitor can be administered to the subject before, during or after receiving treatment for viral infection. The ASGR1 inhibitor is a small molecule inhibitor or an ASGR1 antibody. Preferably, when the ASGR1 inhibitor is an ASGR1 antibody, the heavy chain CDR1, CDR2 and CDR3 of the ASGR1 antibody are shown in SEQ ID NOs: 7 to 9, respectively, and the light chain CDR1, CDR2 and CDR3 are shown in SEQ ID NOs: 10 to 12, respectively.

Another aspect of the present invention provides the use of KREMEN1 and ASGR1 combined as drug targets in in vitro screening of SARS-CoV-2 therapeutic drugs.

The method for in vitro screening of SARS-CoV-2 therapeutic drugs may optionally include: performing an in vitro viral infection experiment on candidate drugs to screen out drugs that can enhance cell survival.

The therapeutic drug can inhibit or block the binding of KREMEN1 and ASGR1 to the S protein of SARS-CoV-2.

Another aspect of the present invention is to provide a target antibody for preventing and/or treating SARS-CoV-2, wherein the target antibody is a KREMEN1 antibody, and/or the target antibody is an ASGR1 antibody.

The heavy chain CDR1, CDR2, and CDR3 of the KREMEN1 antibody are shown in SEQ ID NOs: 1 to 3, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 4 to 6, respectively;

The heavy chain CDR1, CDR2, and CDR3 of the ASGR1 antibody are shown in SEQ ID NOs: 7 to 9, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 10 to 12, respectively.

Another aspect of the present invention is to provide the use of the target antibody of SARS-CoV-2 as described above in the preparation of a drug for preventing and/or treating SARS-CoV-2.

Another aspect of the present invention provides the use of a KREMEN1 inhibitor and an ASGR1 inhibitor in combination in the preparation of a medicament for preventing and/or treating SARS-CoV-2.

The KREMEN1 inhibitor refers to a substance that has an inhibitory effect on KREMEN1, including a small molecule inhibitor or a KREMEN1 antibody. Preferably, when the KREMEN1 inhibitor is a small molecule inhibitor, it is Suramin Sodium (CAS129-46-4), the molecular formula is C ₅₁ H ₃₄ N ₆ Na ₆ O ₂₃ S ₆ , and the structural formula is shown in formula (I). Preferably, when the KREMEN1 inhibitor is a KREMEN1 antibody, the heavy chain CDR1, CDR2, and CDR3 of the KREMEN1 antibody are shown in the sequences SEQ ID NO: 1 to 3, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in the sequences SEQ ID NO: 4 to 6, respectively.

The inhibitory effect on KREMEN1 includes but is not limited to: inhibiting the activity of KREMEN1 or inhibiting the gene transcription or expression of KREMEN1. The KREMEN1 inhibitor can be an antibody or a small molecule compound.

Whether a drug can inhibit KREMEN1 activity can be determined using existing technologies, including isotope labeling assays.

Existing technologies can also be used to determine whether a drug can inhibit the transcription or expression of the KREMEN1 gene. For example, cells that normally express KREMEN1 are provided, the cells are cultured in the presence of the drug to be tested or a vector carrying the drug to be tested, and whether the transcription or expression level of KREMEN1 changes.

The use of KREMEN1 inhibitors in the preparation of drugs for preventing or treating SARS-CoV-2 specifically refers to: using KREMEN1 inhibitors as the main active ingredient of the drug for the preparation of drugs for preventing or treating SARS-CoV-2.

The ASGR1 inhibitor refers to a substance that has an inhibitory effect on ASGR1, and the ASGR1 inhibitor is a small molecule inhibitor or an ASGR1 antibody. Preferably, when the ASGR1 inhibitor is an ASGR1 antibody, the heavy chain CDR1, CDR2, and CDR3 of the ASGR1 antibody are shown in SEQ ID NOs: 7 to 9, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 10 to 12, respectively.

The inhibitory effect on ASGR1 includes but is not limited to: inhibiting the activity of ASGR1 or inhibiting the gene transcription or expression of ASGR1. The ASGR1 inhibitor can be an antibody or a small molecule compound.

Whether a drug can inhibit the activity of ASGR1 can be determined by using existing technologies, including isotope labeling assay.

Existing technologies can also be used to determine whether a drug can inhibit ASGR1 gene transcription or expression. For example, cells that normally express ASGR1 are provided, the cells are cultured in the presence of a drug to be tested or a vector carrying the drug to be tested, and whether ASGR1 transcription or expression level changes.

In the present invention, the KREMEN1 inhibitor and the ASGR1 inhibitor can also be used in combination with an ACE2 inhibitor. The combined use includes the combined use of the KREMEN1 inhibitor and the ACE2 inhibitor; or, the combined use of the ASGR1 inhibitor and the ACE2 inhibitor; or, the combined use of the KREMEN1 inhibitor, the ASGR1 inhibitor and the ACE2 inhibitor. Among them, the ACE2 inhibitor is a small molecule inhibitor or an ACE2 antibody; when the ACE2 inhibitor is an ACE2 antibody, the ACE2 inhibitor is Ab-414.

Another aspect of the present invention provides a drug for preventing and/or treating SARS-CoV-2 infection, wherein the drug comprises a therapeutically effective amount of a KREMEN1 inhibitor and/or an ASGR1 inhibitor, and the drug comprises or does not comprise an ACE2 inhibitor.

The KREMEN1 inhibitor refers to a substance that has an inhibitory effect on KREMEN1, including a small molecule inhibitor or a KREMEN1 antibody. Preferably, when the KREMEN1 inhibitor is a small molecule inhibitor, it is Suramin Sodium (CAS129-46-4), the molecular formula is C ₅₁ H ₃₄ N ₆ Na ₆ O ₂₃ S ₆ , and the structural formula is shown in formula (I). Preferably, when the KREMEN1 inhibitor is a KREMEN1 antibody, the heavy chain CDR1, CDR2, and CDR3 of the KREMEN1 antibody are shown in the sequences SEQ ID NO: 1 to 3, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in the sequences SEQ ID NO: 4 to 6, respectively.

The inhibitory effect on KREMEN1 includes but is not limited to: inhibiting the activity of KREMEN1 or inhibiting the gene transcription or expression of KREMEN1. The KREMEN1 inhibitor can be an antibody or a small molecule compound.

The ASGR1 inhibitor refers to a substance that has an inhibitory effect on ASGR1. The ASGR1 inhibitor is a small molecule inhibitor or an ASGR1 antibody. Preferably, when the ASGR1 inhibitor is an ASGR1 antibody, the heavy chain CDR1, CDR2, and CDR3 of the ASGR1 antibody are shown in the sequences SEQ ID NO: 7 to 9, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in the sequences SEQ ID NO: 10 to 12, respectively.

The inhibitory effect on ASGR1 includes but is not limited to: inhibiting the activity of ASGR1 or inhibiting the gene transcription or expression of ASGR1.

The ACE2 inhibitor refers to a substance that has an inhibitory effect on ACE2. The ACE2 inhibitor is a small molecule inhibitor or an ASGR1 antibody. Preferably, when the ACE2 inhibitor is an ACE2 antibody, the ASGR1 antibody is Ab-414.

The inhibitory effect on ACE2 includes but is not limited to: inhibiting the activity of ACE2 or inhibiting the gene transcription or expression of ACE2.

Another aspect of the present invention provides a method for preventing and/or treating SARS-CoV-2 infection, comprising administering to a subject a KREMEN1 inhibitor and/or an ASGR1 inhibitor, with or without administering an ACE2 inhibitor.

The KREMEN1 inhibitor includes a small molecule inhibitor or a KREMEN1 antibody. Preferably, when the KREMEN1 inhibitor is a small molecule inhibitor, the KREMEN1 inhibitor is Suramin Sodium (CAS129-46-4), the molecular formula is C ₅₁ H ₃₄ N ₆ Na ₆ O ₂₃ S ₆ , and the structural formula is shown in formula (I). Preferably, when the KREMEN1 inhibitor is a KREMEN1 antibody, the heavy chain CDR1, CDR2, and CDR3 of the KREMEN1 antibody are shown in sequences SEQ ID NO: 1 to 3, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in sequences SEQ ID NO: 4 to 6, respectively.

The ASGR1 inhibitor refers to a substance that has an inhibitory effect on ASGR1, and the ASGR1 inhibitor is a small molecule inhibitor or an ASGR1 antibody. Preferably, when the ASGR1 inhibitor is an ASGR1 antibody, the heavy chain CDR1, CDR2, and CDR3 of the ASGR1 antibody are shown in SEQ ID NOs: 7 to 9, respectively, and the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 10 to 12, respectively.

The ACE2 inhibitor refers to a substance that has an inhibitory effect on ACE2. The ACE2 inhibitor is a small molecule inhibitor or an ASGR1 antibody. Preferably, when the ACE2 inhibitor is an ACE2 antibody, the ASGR1 antibody is Ab-414.

The object can be a mammal or a virus-infected cell of a mammal. The mammal is preferably a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. The primate is preferably a monkey, an ape or a human. The virus-infected cell can be an in vitro infected cell.

The object may be a patient infected with SARS-CoV-2 or an individual infected with SARS-CoV-2 who is expected to be treated. Or the object may be an infected viral cell of a patient infected with SARS-CoV-2 or an individual infected with SARS-CoV-2 who is expected to be treated.

The KREMEN1 inhibitor and ASGR1 inhibitor can be administered to a subject before, during, or after receiving treatment for a viral infection.

Another aspect of the present invention provides a method for screening SARS-CoV-2 therapeutic drugs, the method comprising: using KREMEN1 and/or ASGR1 as drug targets, and searching for substances that can inhibit or block the binding of KREMEN1 and/or ASGR1 to the S protein of SARS-CoV-2 as candidate drugs.

Furthermore, the method comprises: applying the drug to be selected to the cells in vitro, and detecting the content of KREMEN1 and/or ASGR1 in the cells after co-culture.

The cells are cells that can normally express KREMEN1 and/or ASGR1. The cells can be from mammals. The experimenter can determine whether the drug is a drug with therapeutic significance by detecting the content of KREMEN1 and/or ASGR1 after co-culture. Generally speaking, compared with the control group, a drug that can reduce the content of ASGR1 and/or ASGR1 by 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% can be determined as a drug with therapeutic significance.

In the present invention, the drug for preventing and/or treating SARS-CoV-2 means that the drug can be used only for preventing SARS-CoV-2, or can be used only for treating SARS-CoV-2, or can be used for preventing and treating SARS-CoV-2.

As described above, the use of KREMEN1 and ASGR1 of the present invention in treating SARS-CoV-2 infection has the following beneficial effects:

The study found that transient expression of ASGR1 or KREMEN1 can lead to SARS-CoV-2 invasion, but has no such effect on SARS-CoV and MERS-CoV. The expression spectrum of ASGR1 and KREMEN1, as well as ACE2, was analyzed and associated with SARS-CoV-2 sensitivity from the cellular to the tissue level, systematically simulating the interaction between the host and SARS-CoV-2, providing a possible explanation for the symptoms associated with COVID-19 disease, and providing useful resources for studying the tropism and pathogenesis of SARS-CoV-2, as well as developing new targets for drugs and antibodies against COVID-19.

BRIEF DESCRIPTION OF THE DRAWINGS

In Figure 1, A shows a schematic diagram of the secretome interaction screening (SIP) for the SARS-CoV-2 S protein; B shows the S protein binding receptor obtained by SIP screening; C shows the binding status of the obtained receptor with S-ECD shown by flow cytometry analysis.

FIG2 shows the determination of the dissociation constants of the interaction between different receptors and SARS-CoV-2S-ECD.

Figure 3 shows that after 293e expressing different receptors was incubated with NTD-hFc, RBD-hFc, S2-hFc or control hFc protein and labeled with anti-hFc antibody, the binding of receptors to different S protein domains was detected by flow cytometry; the upper right figure shows that the protein concentrations of each S domain in the experiment are relatively uniform and comparable (anti-hFc antibody Western blot detection).

Figure 4 shows that the S-binding receptors identified by SIP were transiently transfected and expressed in ACE2-KO 293T cells, and then infected with pseudotyped SARS-CoV-2, SARS-CoV and MERS-CoV viruses, respectively, and the luciferase activity was measured relative to cells transfected with the empty vector 60 hours after infection.

FIG5 shows the interaction of S-ECD with full-length KREMEN1 or ASGR1 detected by co-immunoprecipitation.

Figure 6 shows that ACE2-KO 293T cells transfected with KREMEN1, ASGR1 or ACE2 were infected with live SARS-CoV-2 virus from patients. The figure shows that 72 hours after SARS-CoV-2 infection, cells were labeled with anti-SARS-CoV-2N protein antibodies for immunofluorescence observation (A) and flow cytometry (B); and SARS-CoV-2N protein primers were used to perform qPCR quantitative analysis of the virus titer in the supernatant (C).

Figure 7 A shows the distribution of ACE2, ASGR1, KREMEN1 and SARS-CoV-2 in different cell populations in the upper respiratory tract of COVID-19 patients. B shows the overlap of ASK expression levels and virus infection patterns in different cell populations. C shows the ASK expression level of SARS-CoV-2 positive cells. D shows the correlation analysis of virus sensitivity and ASK receptors alone or in combination in the total cell population, epithelial cell population and immune cell population in the upper respiratory tract of COVID-19 patients.

Figure 8 A shows the proportion of cells expressing each receptor alone or in combination in ASK-expressing cells in the upper respiratory tract of COVID-19 patients. Figure 8 B shows the correlation analysis between virus sensitivity and ASK receptors alone or in combination in epithelial cell subpopulations (ciliary cells and secretory cells) and immune cell subpopulations (macrophages) in the upper respiratory tract of COVID-19 patients.

Figure 9 A shows the comparable expression levels of ASK receptors in different tissues of the human body (data from public databases, comparable expression level = mRNA level/Kd of the receptor's interaction with S protein). B shows the cluster correlation analysis of virus sensitivity and comparable expression levels of ASK receptors in SARS-CoV-2 positive tissues.

Figure 10 shows that SARS-CoV-2 invasion in HTB-182 and Li7 cells does not rely on ACE2 receptors. A shows that different human lung and liver cell lines were infected with SARS-CoV-2 pseudoviruses, and cell lines with significant infection were marked in red (including NCI-H1944, NCI-H23, Calu1, NCI-H661, NCI-H1650, HTB182, Calu3, Li7, HepG2, Hep 3B2.1-7, Huh-7). B shows that during the SARS-CoV-2 pseudovirus infection, ACE2 neutralizing antibodies were added to detect whether the virus invasion depends on ACE2 receptors.

Figure 11 shows that in HTB-182 cells, interfering with KREMEN1 significantly inhibited pseudotype SARS-CoV-2 virus (SARS-CoV-2 pseudovirus) infection; in Li-7 cells, interfering with ASGR1 significantly inhibited pseudotype SARS-CoV-2 virus infection. A shows the effect of shRNA interference against ACE2, KREMEN1 and ASGR1. B shows the effect on pseudotype SARS-CoV-2 virus infection after interfering with ACE2, KREMEN1 and ASGR1 gene expression in Calu-3 cells. C shows the effect on pseudotype SARS-CoV-2 virus infection after interfering with ACE2, KREMEN1 and ASGR1 gene expression in HTB-182 cells. D shows the effect on pseudotype SARS-CoV-2 virus infection after interfering with ACE2, KREMEN1 and ASGR1 gene expression in Li-7 cells.

Figure 12 shows that in HTB-182 cells, interference with KREMEN1 significantly inhibited SARS-CoV-2 live virus infection (A); in Li-7 cells, interference with ASGR1 significantly inhibited SARS-CoV-2 live virus infection (B).

Figure 13 shows that ASGR1 monoclonal antibody S23 and KREMEN1 monoclonal antibody K33 can specifically block the binding of receptors to S protein and inhibit the invasion of pseudotype SARS-CoV-2 virus mediated by related receptors. A shows the Kd of S23 and K33 antibodies to ASGR1 and KREMEN1 antigens respectively detected by Elisa. B shows that S23 and K33 specifically block the binding of ASGR1 and KREMEN1 to S protein respectively. C shows that S23 specifically inhibits the invasion of pseudotype SARS-CoV-2 virus into 293T-ASGR1 cells, and K33 specifically inhibits the invasion of pseudotype SARS-CoV-2 virus into 293T-KREMEN1 cells. D shows that ASGR1 antibody S23 specifically inhibits the invasion of pseudotype SARS-CoV-2 virus into Li7 cells (IC50 = 4.254ug/ml). E shows that KREMEN1 antibody K33 specifically inhibits the invasion of pseudotype SARS-CoV-2 virus into HTB-182 cells (IC50 = 2.439ug/ml).

Figure 14 shows that the antibody cocktail that simultaneously blocks the binding of S protein to three ASK receptors (ACE2, ASGR1, KREMEN1) is more effective in inhibiting SARS-CoV-2 live virus infection of human lung organoids. Schematic diagram of SARS-CoV-2 infected human lung organoids (A), and immunofluorescence of S protein, ACE2, ASGR1 and KREMEN1 after 48 hours of infection (B). C shows the effect of adding antibodies Ab-414 (blocking ACE2-S binding), S23 (blocking ASGR1-S binding), and K33 (blocking KREMEN1-S binding) alone or in combination on SARS-CoV-2 infection of human lung organoids (the final concentration of each antibody is 4ug/ml). Among them, in C, the first figure is the experimental statistics using the lung organoids from Case 1, the second figure is the experimental statistics using the lung organoids from Case 2, and the third figure is the comprehensive statistical result of the experimental data of the lung organoids from Cases 1 and 2; the bar graphs from left to right are Ctrl Ab, Ab-414, S23, K33, ASK (Ab-414+S23+K33, that is, the combination of three antibodies).

Figure 15 shows the screening system for KREMEN1-S protein binding, and the screening of drug small molecules that specifically block KREMEN1-S protein binding. A shows that the obtained positive small molecule Suramin sodium specifically inhibits KREMEN1-S protein binding. B shows the molecular formula and structure of Suramin sodium. C shows that Suramin sodium blocks the pseudotype SARS-CoV-2 virus from invading HTB-182 cells (IC50 = 18.02uM). D shows that Suramin sodium has no significant effect on the invasion of SARS-CoV-2 virus in Calu3 cells.

DETAILED DESCRIPTION

The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

In the present invention, a polypeptide fragment having an amino acid sequence with more than 90% sequence identity (having a sequence identity of more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) with any of the sequences in SEQ ID No. 1 to 12 and having the function of the polypeptide fragment defined by the sequence is also within the protection scope of the present invention. For example, it can be a polypeptide fragment whose function is not changed by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in any of the sequences in SEQ ID No. 1 to 12, or adding one or more amino acids to the N-terminus and/or C-terminus.

The term "identity" as used in the present invention refers to sequence similarity to natural nucleic acid sequences. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

SARS-CoV-2S (expressed and purified in the laboratory, see Examples 1 and 2 for the relevant processes)

293e cells (ThermoFisher)

pCMV-CFP plasmid (CFP sequence was constructed into pCMV6 (OriGene) plasmid)

293T cells (ATCC)

4-3.Luc.R plasmid (NIH, Cat: 3418)

pCMV-receptor-flag (the receptor sequence is constructed into the pCMV6 (OriGene) plasmid)

pLentilox3.7 (Addgene)

pCDNA3.1-S plasmid (the S protein coding sequence was constructed into the pcDNA3.1 (Thermofisher) plasmid)

Vero E6 cells (ATCC)

SARS-CoV-2/MT020880.1 (BSL-3 laboratory of Fudan University)

Calu-3 (lung cancer cell line) (ATCC)

HTB-182 (lung cancer cell line) (ATCC)

Li-7 (liver cancer cell line) (RIKEN Cell Bank)

Example 1 High-throughput screening of 12 human cell surface receptors that bind to the SARS-CoV-2 S protein extracellular domain (S-ECD)

As the largest envelope protein on the surface of SARS-CoV-2 virus, S protein is an important protein that binds to host cell surface receptors and mediates virus invasion of cells. The present invention uses SARS-CoV-2S protein as a target and uses the established secretome interaction screening system to screen and identify almost all human membrane proteins at the whole genome level, and found a total of 12 membrane proteins that can specifically bind to SARS-CoV-2S protein.

Materials and Methods

1-1) 293e cells expressing each membrane protein: The human membrane protein expression library includes 5054 genes (pCMV vector), which was obtained by purchase and self-construction. The expression plasmid of each membrane protein and the pCMV-CFP plasmid (5:1 mass ratio) were transferred into 293e cells by PEI transfection method (plasmid: PEI ratio is 1:3). After 48 hours, the cells were collected for binding experiments (see 1-4);

1-2) Preparation of SARS-CoV-2 S protein extracellular domain (CoV2 S-ECD): Construct a CoV2 S-ECD expression vector (pCMV-S-ECD-hFc) with C-terminal fusion expression of hFc; pCMV-S-ECD-hFc vector was transfected into 293e cells using PEI transfection method (plasmid: PEI ratio is 1:3). After 96 hours, the cell supernatant was collected and filtered with a 0.45um filter for binding experiments (see 1-4);

1-3) Preparation of control hFc: Construct a secretory hFc expression vector (pCMV-hFc); transfer the pCMV-hFc vector into 293e cells using the PEI transfection method (plasmid: PEI ratio is 1:3). After 96 hours, collect the cell supernatant, filter it with a 0.45um filter, and use it for binding experiments (see 1-4);

1-4) Protein binding experiment: Take 200ul CoV2 S-ECD protein supernatant (1-2) or 200ul control hFc protein supernatant (1-3), mix with 10 ⁴ 293e cells expressing the corresponding membrane protein (1-1), incubate on ice for 1hr; centrifuge at 500g for 5min; wash the cells once with 200ul PBS/2% FBS, centrifuge at 500g for 5min; suspend the cells in 50ul antibody staining solution (anti-hFc-AF647, Jackson Lab product, 1ug/ml, diluent is PBS/2% FBS), on ice for 30min, wash once with PBS/2% FBS, suspend the cells in 100ul PBS/2% FBS, and perform flow cytometry analysis.

result:

The schematic diagram of library screening is shown in Figure 1A, and the results are shown in Figure 1B and Figure 1C. The results showed that the control group 293e cells did not bind to S-ECD-hFc; 293e cells expressing ACE2 (a known SARS-CoV-2 receptor) had a specific and high-intensity binding to S-ECD-hFc, indicating that this screening system can be used for the screening of new crown receptors. In the screening of 5054 membrane proteins (accounting for 91.6% of all predicted membrane proteins in the human genome), a total of twelve receptors were found to be able to specifically bind to S-ECD.

Example 2 Measurement of Kd of the interaction between cell surface receptors and SARS-CoV-2 S protein

In order to evaluate the affinity between the cell surface receptors discovered by the present invention and S protein, the present invention further measured the dissociation constant (Kd) of the interaction between these receptors and S protein.

Materials and Methods

2-1) 293e cells expressing receptors: The receptor expression plasmid (pCMV-receptor) and pCMV-CFP plasmid (5:1 mass ratio) were transfected into 293e cells by PEI transfection method (plasmid: PEI ratio was 1:3). After 48 hours, the cells were collected for Kd measurement;

2-2) Purification of S-ECD protein: The pCMV-S-ECD-hFc vector was transferred into 293e cells using the PEI transfection method. After 96 hours, the cell supernatant was collected and filtered with a 0.45 μm filter. The protein was purified using protein A affinity chromatography, and the buffer was replaced with PBS using a PD-10 desalting column (GE). Finally, the protein was concentrated to 1 mg/ml using a protein concentration column (AMICON) for Kd measurement.

2-3) Kd measurement: using the protein purified in 2-2), 200ul of S-ECD protein with 2-fold gradient concentrations (the buffer was PBS/2% FBS, and the highest concentration of S-ECD was 300nM), different concentrations of S-ECD were mixed with ¹⁰⁴ receptor-293e cells (2-1), and protein binding was performed (same steps as 1-4); the amount of CFP+ cells binding to S-ECD at different concentrations (i.e., APC mean fluorescence intensity, MFI) was plotted as a curve, and Kd was calculated using Prism software.

result:

Using ACE-2 as a control, the results showed that the Kd of ACE2 and S-ECD-Fc was 12.4 nM, which was consistent with previous reports, proving the effectiveness of this Kd measurement system; the Kd measurement curves and values of all receptors and S-ECD are shown in Figure 2 and Table 1.

Table 1: Summary of Kd of the interaction between cell surface receptors and S protein

Example 3 Test to determine the specific binding ability of each receptor to the three main domains of S protein

The SARS-CoV-2 S protein extracellular domain (S-ECD) is divided into several main regions: receptor binding domain (RBD), N-terminal domain (NTD) and S2 domain. In order to further study which functional domain on the S protein these human cell surface receptors specifically bind to, the present invention conducted the following experiments:

Materials and Methods

3-1) 293e cells expressing receptors: The receptor expression plasmid (pCMV-receptor) and pCMV-CFP plasmid (5:1 mass ratio) were transferred into 293e cells by PEI transfection method (plasmid: PEI ratio was 1:3). After 48 hours, the cells were collected for Kd test (see 2-3);

3-2) Purification of the three domains of S protein RBD-hFc, NTD-hFc, S2-hFc proteins, and control hFc protein: pCMV-RBD-hFc, pCMV-NTD-hFc, pCMV-S2-hFc or pCMV-hFc and other vector plasmids were transfected into 293e cells by PEI transfection method. After 96 hours, the cell supernatant was collected and filtered with a 0.45um filter; the protein was purified by protein A affinity chromatography, and the buffer was replaced with PBS using a PD-10 desalting column (GE), and finally the protein was concentrated to 1 mg/ml using a protein concentration column (AMICON);

3-3) Protein binding experiment: Take 200ul CoV2 S-ECD protein supernatant (1-2) or 200ul control hFc protein supernatant (1-3), mix with 10 ⁴ 293e cells expressing the corresponding membrane protein (1-1), incubate on ice for 1hr; centrifuge at 500g for 5min; wash the cells once with 200ul PBS/2% FBS, centrifuge at 500g for 5min; suspend the cells in 50ul antibody staining solution (anti-hFc-AF647, Jackson Lab product, 1ug/ml, diluent is PBS/2% FBS), on ice for 30min, wash once with PBS/2% FBS, suspend the cells in 100ul PBS/2% FBS, and perform flow cytometry analysis.

3-4) Analysis method: For comparison, the present invention quantifies the binding ability; that is, for cells expressing the receptor (CFP+ cells), the binding value of its domain protein is divided by the control hFc binding value to obtain the binding multiples of the receptor with different domains. A value less than 2 is considered as non-specific binding.

result:

The results showed that ACE2 only binds to the RBD region of the S protein and does not interact with other regions; this is consistent with previous reports and proves the effectiveness of this experimental system. The binding map of all receptors to different regions of the S protein is shown in Figure 3, and the relevant values are summarized in Table 2.

Table 2: Summary of the binding ability of cell surface receptors to different domains of S protein

Example 4 Determination of the ability of KREMEN1 and ASGR-1 to directly mediate virus invasion of cells by pseudovirus experiments

To prove whether these binding receptors can directly mediate viral invasion, the present invention expressed 12 receptors on ACE-2 knocked-out 293T cells and tested infection with the new coronavirus pseudovirus.

Materials and Methods

4-1) Preparation of pseudovirus: The expression vector pCDNA3.1-S of the full-length S protein of SARS-CoV-2 or SARS, MERS and the backbone plasmid pNL4-3.Luc.R (1:1 mass ratio) were transfected into 293T cells by PEI transfection method (plasmid: PEI ratio was 1:3). After 48 hours, the virus supernatant was collected and stored at -80 degrees for infection experiments;

4-2) Establishment of 293T-ACE2 KO cell line: Establish ACE-2 knockout 293-T cell line by CRISPER-cas9 technology to exclude the background and interference of trace ACE-2 on infection of other receptors;

4-3) Virus infection: 293T cells were transfected with pCMV-receptor plasmids of 12 receptors. After 24 hours, the cells were transferred to 96-well white plates and 50ul of pseudovirus supernatant collected in 4-1) was added. After 48 hours of infection, the luciferase activity in the cells was measured using a luciferase substrate reaction kit (Beyotime, RG051M) and a multi-function microplate reader.

result:

The results are shown in Figure 4. The expression of ACE-2 receptors in ACE2-KO_293T cells can obviously mediate the invasion of SARS-CoV-2 and SARS pseudoviruses, but cannot mediate the invasion of MERS pseudoviruses, which is very consistent with previous reports and proves the effectiveness of this system. Then the other 11 receptors were tested in the same way, and it was found that KREMEN1 and ASGR-1 can also mediate the invasion of SARS-CoV-2 virus into cells, but cannot mediate the invasion of SARS or MERS pseudoviruses; other receptors have no obvious function of mediating virus invasion.

Example 5 Co-immunoprecipitation (Co-IP) experiment to prove the specific interaction between KREMEN1 and ASGR-1 and SARS-CoV-2 S protein

Co-immunoprecipitation is a classic method to further demonstrate the interaction between proteins. Here, this method is used to demonstrate the interaction between two invasion receptors (KREMEN1 and ASGR1) and the S protein of SARS-CoV-2.

Materials and Methods

5-1) 293T cells expressing receptors: Plasmid (pCMV-receptor-flag) expressing the full-length receptor with Flag fusion was transferred into 293T cells by PEI transfection method (plasmid: PEI ratio was 1:3). After 48 hours, cells were collected and lysed with RIPA buffer, centrifuged at 15000 rpm for 15 minutes at 4 degrees, and the supernatant was collected;

5-2) Purification of S-ECD protein: The pCMV-S-ECD-hFc vector was transferred into 293e cells using the PEI transfection method. After 96 hours, the cell supernatant was collected and filtered through a 0.45 μm filter. The protein was purified using protein A affinity chromatography, and the buffer was replaced with PBS using a PD-10 desalting column (GE). Finally, the protein was concentrated to 1 mg/ml using a protein concentration column (AMICON).

5-3) Co-IP: S-ECD protein (final concentration 10ug/ml) was mixed with anti-FLAG beads and cell lysate supernatant, incubated with shaking at 4 degrees overnight, washed 3 times with RIPA buffer, and the beads-bound protein was prepared for western-blot detection.

result:

Two receptor proteins and S-ECD-Fc or hFC proteins expressed by cells can be detected in the mixture before immunoprecipitation (Input). After co-precipitation with Flag beads and multiple washes (IP), the two receptor proteins were significantly enriched. At the same time, S-ECD-Fc protein was also clearly present in the IP product, while the amount of hFC in the control group was very small. This proves that while the two receptor proteins can be enriched by beads co-precipitation, S protein can also be specifically enriched, and there is a specific binding between the receptor and S protein (Figure 5).

Example 6 Determining the ability of KREMEN1 and ASGR-1 to mediate viral invasion of cells through patient-derived SARS-CoV2 live virus infection experiments

To further prove that KREMEN1 and ASGR-1 can directly mediate viral invasion, these two receptors were expressed on ACE2-KO_293T cells and tested for SARS-CoV-2 infection.

Materials and Methods

6-1) Preparation of live virus: SARS-CoV-2/MT020880.1 from patients was amplified on Vero E6 cells, frozen and thawed three times 50 hours after infection, centrifuged at 2500g for 10 minutes, and the virus supernatant was collected and stored in aliquots at -80 degrees. The virus titer was determined by plaque assay on Vero E6 cells;

6-2) Establishment of 293T-ACE KO cell line: Establish ACE-2 knockout 293-T cell line by CRISPER-cas9 technology to exclude the background and interference of trace ACE-2 on infection of other receptors (same as 4-2);

6-3) Virus infection: pCMV-KREMEN1 or pCMV-ASGR-1 plasmid was transfected into 293T-ACE KO cells, and 24 hours later, the cells were plated in 24-well plates, and the virus was added at an MOI of 1. After 48 hours of infection, the cells were immunofluorescently labeled with anti-SARS-CoV-2Nprotein antibody, and the proportion of positive cells was analyzed by fluorescence microscopy and flow cytometry;

At the same time, 48 hours after infection, the cell supernatant was collected; the viral mRNA level in the supernatant was detected by qPCR using SARS-CoV-2 N protein primers (primers: SARS-CoV-2-N-F 5'-GGGGAACTTCTCCTGCTAGAAT-3'

(SEQ ID NO: 13), SARS-CoV-2-N-R 5'-CAGACATTTTGCTCTCAAGCTG-3' (SEQ ID NO: 14)) were used to quantify the virus titer in the supernatant by qPCR.

result:

Immunofluorescence (Figure 6A) and flow analysis results (Figure 6B) show that ACE2-KO 293T cells that are not transfected with any receptors basically do not support live virus infection, while ACE2-KO 293T cells transiently transfected with ACE2 have a high rate of viral infection, proving the effectiveness of this system; similarly, the present invention clearly detected live virus-infected cells in 293T-ACE2 KO cells transfected with KREMEN1 and ASGR-1 (Figure 6A, Figure 6B). The present invention also performed qPCR detection on the release of viral particles in the cell supernatant, and the results showed that the viral particles in the cell supernatant transfected with ACE2, KREMEN1 and ASGR-1 were significantly higher than those in the control group that was not transfected with any receptor (Figure 6C).

Example 7 ACE2, ASGR1 and KREMEN1 receptors are highly correlated with the cell and tissue tropism of clinical SARS-CoV-2

To investigate the relevance of these invasion receptors to clinical susceptibility to SARS-CoV-2, we analyzed the results of recently published single-cell sequencing (scRNA-seq) of the upper respiratory tract of 19 COVID-19 patients and correlated them with the receptor profile. This dataset comes from nasopharyngeal swabs of patients and contains the gene expression and viral infection status of each cell; these cells are mainly composed of epithelial and immune cells.

ACE2 was mainly expressed in epithelial cell populations, consistent with previous reports; while ASGR1 and KREMEN1 were expressed in both cells (Figure 7A). Most receptor-positive cells expressed only one of the entry receptors (88.5%), while the number of cells expressing KREMEN1 was the largest, 5 times more than cells expressing ACE2 or ASGR1 (Figure 7B and Figure 8A). SARS-CoV-2 mainly infects epithelial ciliated and secretory cells and immune non-resident macrophages (nrMa), which are also the main populations expressing ASK receptors. Among SARS-CoV-2-positive cells (V ⁺ cells), only 10.3% expressed ACE-2, indicating that other receptors are likely to mediate invasion (Figure 7C).

Further analysis was performed to determine the correlation between the three receptors ACE2, ASGR1, and KREMEN1 (collectively called ASK) and SARS-CoV-2 sensitivity. Among all cells, the percentage of receptor-positive cells in V+ cells (R ⁺ %) was significantly higher than that in V- cells (R ⁺ %). In epithelial cells, both ACE2 and KREMEN1 were enriched in V ⁺ cells, while in immune cells (D in Figure 7), only ASGR1 was associated with viral susceptibility, especially in macrophages (D in Figure 7 and B in Figure 8). Epithelial ciliated cells and secretory cells are known target cells of SARS-CoV-2; in ciliated cells, ACE2 and KREMEN1 were associated with viral susceptibility, with ACE2 being more significantly correlated; while in secretory cells, only KREMEN1 was significantly correlated with viral susceptibility (B in Figure 8). Whether in all cell populations or cell subsets, the combination of ASKs is generally more significant than a single receptor in terms of correlation with viral infection (D in Figure 7 and B in Figure 8). The above results reflect at the single-cell level that the expression of ASK is closely related to the clinical infection of the virus, and the comprehensive expression level of ASK is closer to the cellular tropism of the new coronavirus.

SARS-CoV-2 shows multi-organ tropism in COVID-19 patients. However, ACE2 is rarely expressed in the brain, liver, peripheral blood (PB) and even in the lungs, so ACE2 alone is difficult to explain the multi-organ tropism of SARS-CoV-2. Therefore, the present invention models the systemic interaction between host and SARS-CoV-2 based on the expression levels of each ASK receptor in various tissues of the human body in the open source database, and evaluates whether ASK can more accurately predict the tissue tropism of SARS-CoV-2. In order to analyze more objectively from the perspective of the ability of receptors to mediate virus binding, the present invention divides the receptor mRNA transcription level by the dissociation constant (Kd) of the receptor binding to the S protein, so that different receptors in the same tissue can be compared, and they can be superimposed and compared between different tissues. The results show that ACE2 and ASGR1 are highly expressed in the gastrointestinal tract and liver, respectively, while KREMEN1 is widely expressed in the human body. In tissues that can be infected by the new coronavirus, at least one ASK receptor is expressed (A in Figure 9). The present invention correlated these receptors with the virus infection rates of various tissues, and the results showed that in terms of correlation, KREMEN1>ACE2>ASGR1, and the comprehensive expression level of ASK was closer to the actual situation of tissue infection than that of a single receptor (B in Figure 9). The above results further reflect that the expression of ASK is related to the clinical infection of the virus at the organ level, and the comprehensive expression level of ASK is closer to the tissue tropism of the new coronavirus.

Example 8 RNAi gene interference experiments determined that targeting KREMEN1 and ASGR-1 can effectively block SARS-CoV-2 from invading cells

In order to verify whether SARS-CoV-2 invasion of cells can be effectively blocked by targeting KREMEN1 and ASGR-1, the present invention conducted RNAi gene interference experiments on endogenous KREMEN1 and ASGR1 at the cellular level and tested their effects on SARS-CoV-2 infection.

Materials and Methods

8-1) RNAi gene interference: shRNA plasmids for KREMEN1, ASGR1 or ACE2 (targeting sequences: ASGR1 shRNA-1, GTCCTGGGAGGAGCAGAAATT (SEQ ID NO: 15); ASGR1 shRNA-2, GAAGGTGAGGAGCTTGAAACC (SEQ ID NO: 16); KREMEN1 shRNA-1, GCACAACTATTGCAGAAATCC (SEQ ID NO: 17), KREMEN1 shRNA-2: GGACCTTAGGGATTGTCATCA (SEQ ID NO: 18); ACE2 shRNA-1: GCAAACGGTTGAACACAATTC (SEQ ID NO: 19), ACE2 shRNA-2: GCTGTTCAGGGATAATCTAAA (SEQ ID NO: 20)) were constructed in the pLentilox3.7 vector. Two shRNA vectors were constructed for each gene. These plasmid vectors were transfected into 293T cells with psPAX2 and MD2G plasmids at a ratio of 4:3:1 by PEI transfection method. After 48 hours, the lentiviral supernatant expressing shRNA was collected, filtered through 0.45um, and polybrene with a final concentration of 4ug/ml was added to infect the target cells and identify by qPCR, thereby obtaining cells with interfered related genes.

8-2) Preparation of SARS-CoV-2 pseudovirus: The steps are the same as 4-1); the expression vector pCDNA3.1-S of the full-length SARS-CoV-2S protein and the backbone plasmid pNL4-3.Luc.R (1:1 mass ratio) were transfected into 293T cells by PEI transfection method (plasmid: PEI ratio is 1:3). After 48 hours, the virus supernatant was collected and stored at -80 degrees for infection experiments;

8-3) SARS-CoV-2 pseudovirus infection: The cells in 8-1) were used to carry out novel coronavirus pseudovirus infection to verify the effect of related gene interference on novel coronavirus infection. The relevant process is the same as 4-3). In order to verify whether the virus infection depends on the ACE2 receptor, the present invention adds ACE2 neutralizing antibody (Sino Biological, Cat#10108-MM37) with a final concentration of 4ug/ml during the infection process.

8-4) Preparation and infection of live virus: Patient-derived SARS-CoV-2/MT020880.1 was amplified on Vero E6 cells, and after repeated freezing and thawing, the cells were centrifuged at 2500 g for 10 minutes, and the virus supernatant was collected and the titer was measured.

The virus was added to the cell culture medium at an MOI of 1. The supernatant was removed after 2 hours, and the cells were washed twice with PBS. After further culture for 48 hours, the cell supernatant was collected. The viral mRNA level in the supernatant was detected by qPCR (N protein primers: SARS-CoV-2-N-F 5'-GGGGAACTTCTCCTGCTAGAAT-3' (SEQ ID NO: 13), SARS-CoV-2-N-R5'-CAGACATTTTGCTCTCAAGCTG-3' (SEQ ID NO: 14)).

result:

The present invention first screens cell lines that can be infected by SARS-CoV-2. Among the 39 cell lines derived from human lungs and liver, 11 cells (including NCI-H1944, NCI-H23, Calu1, NCI-H661, NCI-H1650, HTB182, Calu3, Li7, HepG2, Hep 3B2.1-7, Huh-7) can be clearly infected by SARS-CoV-2 pseudovirus (Figure 10A).

Since ACE2, ASGR1 and KREMEN1 can independently mediate SARS-CoV-2 infection, the present invention uses ACE2 neutralizing antibodies to treat these cells. The results showed that in HTB-182 (lung cancer cell line) and Li-7 (liver cancer cell line), SARS-CoV-2 infection was not regulated by ACE2 receptors (Figure 10B).

Figure 11 shows that in HTB-182 cells, interfering with KREMEN1 significantly inhibits pseudotype SARS-CoV-2 virus (SARS-CoV-2 pseudovirus) infection; in Li-7 cells, interfering with ASGR1 significantly inhibits pseudotype SARS-CoV-2 virus infection. A shows the shRNA interference effect against ACE2, KREMEN1 and ASGR1. B shows the effect on pseudotype SARS-CoV-2 virus infection after interfering with ACE2, KREMEN1 and ASGR1 gene expression in Calu-3 cells. The present invention further uses RNAi gene interference experiments to find that in HTB-182 cells, KREMEN1 gene interference can effectively block SARS-CoV-2 pseudovirus infection (Figure 11C) and SARS-CoV-2 live virus infection (Figure 12A); in Li-7 cells, ASGR1 gene interference can effectively block SARS-CoV-2 pseudovirus infection (Figure 11D) and SARS-CoV-2 live virus infection (Figure 12B). The above results indicate that SARS-CoV-2 relies on different receptors to infect different types of cells, and targeting KREMEN1 and ASGR-1 can effectively block the invasion of SARS-CoV-2 in receptor-dependent cells.

Example 9 Determining by specific antibodies that targeting KREMEN1 and/or ASGR-1 can effectively block SARS-CoV-2 from invading cells

In order to verify whether the binding of KREMEN1 and/or ASGR-1 to S protein can be targeted by specific antibodies, thereby effectively blocking SARS-CoV-2 from invading cells, the present invention develops specific antibodies against these two receptors, screens antibodies that can specifically block the binding of KREMEN1-S and/or ASGR-1-S proteins, and tests their effects on SARS-CoV-2 infection.

Materials and Methods

9-1) Obtaining antibodies: monoclonal antibodies against KREMEN1 or ASGR1 obtained by immunizing mice, monoclonal antibodies against S protein obtained by immunizing animals, or monoclonal antibodies against S protein isolated from patients.

9-2) Antibody screening: Using the KREMEN1/ASGR1-S protein binding experiment (see Example 1 for specific steps), test whether the antibody can block the binding of the S protein to the receptor KREMEN1/ASGR1; for positive antibodies, the present invention tests the blocking effect of different concentrations of antibodies on the S protein-KREMEN1/ASGR1 interaction, thereby obtaining the IC50 of the antibody inhibiting the interaction;

9-3) Experiment on the inhibition of SARS-CoV-2 virus invasion by antibodies: Using the SARS-CoV-2 virus infection experiment (see Example 4 and Example 6 for specific steps), test whether the antibodies screened in 9-2 can block the invasion of SARS-CoV-2 virus into cells; for antibodies that can significantly block virus invasion, the present invention tests the effect of different concentrations of antibodies on blocking virus invasion, thereby obtaining the IC50 of the antibody in inhibiting virus invasion.

result:

Among the mouse monoclonal antibodies of KREMEN1 or ASGR1 obtained by the present invention, the affinity constant Kd of ASGR1 antibody S23 is 0.1236ug/ml, and the Kd of KREMEN1 antibody K33 is 0.0199ug/ml (Figure 13A). The S23 antibody can specifically block the binding of ASGR1 to the S protein, while the K33 antibody can specifically block the binding of KREMEN1 to the S protein (Figure 13B). In 293T cells overexpressing different receptors such as ACE2, ASGR1 and KREMEN1, S23 specifically inhibits SARS-CoV-2 virus infection mediated by ASGR1, and K33 specifically inhibits SARS-CoV-2 virus infection mediated by KREMEN1 (Figure 13C). The present invention further tested the effects of these antibodies on endogenous ASGR1 and KREMEN1-mediated viral infection. The results showed that in ASGR1-dependent Li7 cells, S23 significantly inhibited SARS-CoV-2 virus infection with an IC50 of 4.254 ug/ml; while in KREMEN1-dependent HTB-182 cells, K33 significantly inhibited SARS-CoV-2 virus infection with an IC50 of 2.439 ug/ml (Figure 13D, Figure 13E).

ASGR1 Antibody S23:

Heavy chain: CDR1: RYTFTDYN (SEQ ID NO: 7); CDR2: ITPNNGGT (SEQ ID NO: 8); CDR3: ARKGGYFDV (SEQ ID NO: 9);

Light chain: CDR1: SSVSY (SEQ ID NO: 10); CDR2: RTSN (SEQ ID NO: 11); CDR3: QQYHSYPLT (SEQ ID NO: 12).

KREMEN1 Antibody K33:

Heavy chain: CDR1: GYTFTGYG (SEQ ID NO: 1); CDR2: IYPRSGNT (SEQ ID NO: 2); CDR3: SRYYGPKGFDY (SEQ ID NO: 3);

Light chain: CDR1: ESVDNYGISF (SEQ ID NO: 4); CDR2: AAS (SEQ ID NO: 5); CDR3: QQSKEVPYT (SEQ ID NO: 6).

Example 10 Antibody cocktails simultaneously targeting ACE2/ASGR1/KREMEN1 can more effectively block SARS-CoV-2 infection of human lung organoids

Data before the present invention showed that in different cell types, SARS-CoV-2 infection can be mediated by different ACE2, ASGR1, and KREMEN1. So in theory, targeting these three receptors at the same time will provide better inhibition of viral infection at the organ level of multiple cell types. Therefore, the present invention tests the effects of these different receptor-targeting antibodies alone and in combination on SARS-CoV-2 infection of human lung organoids.

Materials and Methods

10-1) Cultivation of human lung organoids: Non-tumor tissue was isolated from lung tissue of surgical patients and digested into single cells using collagenase II. After centrifugation, the cells were resuspended in Matrigel (Corning) and cultured at 37°C, 5% CO2 using DF12 medium (containing 10mM HEPES (Gibco), 2mM GlutaMAX-1 (Gibco), 500×Primocin (InvivoGen), 1×B27 (Gibco), 1.56mM N-acetylcysteine (Sigma), 10mM nicotinamide (Gibco), 0.5μM A83-01 (Tocris), 10μM Y27632, 50ng/mL EGF (Peprotech), 10ng/mL FGF10 (Peprotech), 1ng/mL FGF2 (Peprotech), 10% in- _house -prepared R-Spondin1, 10% Noggin and 30% Wnt3a).

10-2) SARS-CoV-2 live virus infection of human lung organoids: Patient-derived SARS-CoV-2/MT020880.1 was amplified on Vero E6 cells, and after repeated freezing and thawing, the virus supernatant was collected and titered at 2500g for 10 minutes. The virus was added to the culture medium containing the lung organoids prepared in step 10-1) at an MOI of 1. After 48 hours of infection, the cells were collected for immunofluorescence analysis, and the viral load in the cells was identified by qPCR. The final concentration of various targeted antibodies used during the infection process was 4ug/ml, and the antibodies included: Ab-414 blocks ACE2-S binding (related literature Wan, J., et al. Cell Rep 32, 107918, 2020); S23 blocks ASGR1-S binding; K33 blocks KREMEN1-S binding.

result:

The present invention first detected the infection of SARS-CoV-2 virus on human lung organoids. Immunofluorescence results showed that ACE2/ASGR1/KREMEN1 receptors were expressed in different infected cells (Figure 14A, Figure 14B), further supporting the conclusion of the present invention that in different cells, SARS-CoV-2 virus relies on different invasion receptors.

Furthermore, the present invention uses ACE2 targeting antibody (Ab-414), ASGR1 targeting antibody (S23), and KREMEN1 targeting antibody (K33) for single or combined treatment in this infection system. The present invention used lung organoids from two patients for experiments, and the results showed that these antibodies could significantly inhibit viral infection when treated with single targeted antibodies; among them, Ab-414 had the strongest effect, followed by K33 and S23; and the inhibitory effect of the combined treatment of the above three antibodies was significantly better than that of any single antibody (Figure 14C).

The above results indicate that ASGR1 and KREMEN1 play an important role together with ACE2 in the process of SARS-CoV-2 virus infection of human lung organoids, and the cocktail antibody targeting ACE2/ASGR1/KREMEN1 can more effectively block SARS-CoV-2 infection.

Example 11 SARS-CoV-2 drug screening platform targeting KREMEN1 and/or ASGR-1

In order to screen specific targeted drugs from the perspective of KREMEN1/ASGR1, thereby effectively blocking SARS-CoV-2 from invading cells, the system of the present invention can provide a rapid screening platform for related drug screening and effectively evaluate whether positive candidate drugs block SARS-CoV-2 infection.

Materials and Methods

11-1) Drug screening targeting KREMEN1 and/or ASGR-1 gene expression:

a. Cell line selection: Cell lines that rely on KREMEN1 and/or ASGR1 to cause SARS-CoV2 infection, such as HTB-182 (KREMEN1-dependent) and Li-7 (ASGR1-dependent) cell lines, were used for drug screening.

b. Cells are cultured in 96-well plates, and drugs are added to the cell culture medium (generally, the final drug concentration is 1-10uMol/L) and then cultured; gradient time sampling (24, 48, 72 hours after adding the drug, etc.) is performed, and cell RNA is extracted using reagents such as Trizol and reverse transcribed into cDNA; qPCR is used to detect the expression levels of genes such as KREMEN1 and ASGR1, and the expression levels are compared with those of control-treated cells. Drugs that can significantly reduce the mRNA levels of KREMEN1 and/or ASGR1 by more than 30% (i.e., drug group/control group <0.7) are selected as candidate drugs; c. Verification and IC50 determination: For the above-mentioned candidate drugs, they are individually verified using the same method as above. For candidate drugs that can be repeatedly verified, the present invention tests the inhibitory effects of different concentrations of candidate drugs on KREMEN1/ASGR1 gene expression, thereby obtaining the IC50 (50% effective inhibitory concentration) of the candidate drug in inhibiting gene expression; these candidate drugs are subjected to 11-3) virus testing.

11-2) Drug screening targeting KREMEN1 and/or ASGR-1 binding to S protein:

(For details on the preparation of relevant reagents, see Examples 1 and 2)

a: The expression plasmids of receptor KREMEN1 or ASGR1 (pCMV-KREMEN1 or pCMV-ASGR1) and pCMV-CFP plasmid (5:1 mass ratio) were transfected into 293e cells by PEI transfection method (plasmid:PEI ratio was 1:3). After 48 hours, the cells were collected by centrifugation at 500g for 5min, resuspended in PBS/2% FBS (concentration was 2x10 ⁵ /ml), and placed on ice for the following screening;

b: Using the purified S-ECD-hFc protein, prepare a S-ECD-hFc protein solution with a final concentration of 10ug/ml (the buffer is PBS/2% FBS), and place it on ice for the following screening;

c: Drug screening: In a 96-well plate (U-bottom), prepare a drug-containing solution, set the initial drug concentration to 1-10uMol/L, 50ul/well (buffer is PBS/2% FBS), and place on ice; add 100ul cells expressing KREMEN1 or ASGR1 (from step a) and 50ul S-ECD-hFc protein solution (from step b), pipette to mix, and incubate on ice for 1hr; centrifuge at 500g for 5min; wash the cells once with 200ul PBS/2% FBS, and centrifuge at 500g for 5min; suspend the cells in 50ul antibody staining solution (anti-hFc-AF647, Jackson Lab product, 1ug/ml, diluent is PBS/2% FBS), put on ice for 30min, wash once with PBS/2% FBS, suspend the cells in 100ul PBS/2% FBS, and perform flow analysis;

d. Result analysis: Flow cytometry results were analyzed using Flowjo software to calculate the amount of S-ECD binding (i.e., APC mean fluorescence intensity, MFI) of receptor-expressing cells (CFP+ cells) under different drug treatment conditions; the cells were compared with control-treated cells, and those that could reduce the S-ECD binding level by more than 30% (i.e., drug group/control group <0.7) were selected as candidate drugs;

11-3) Testing the effectiveness of the candidate drugs obtained above in inhibiting the invasion of SARS-CoV-2 virus:

The specific steps of virus preparation and infection are shown in Example 4; in terms of cell lines, the present invention selects HTB-182 cells infected by SARS-CoV-2 dependent on KREMEN1, Li-7 cells infected by SARS-CoV-2 dependent on ASGR1 receptor, and 293T cells stably expressing KREMEN1 and ASGR1, respectively; the cells are passaged into a 96-well white plate, and the virus is infected on the second day, and the candidate drug is added at the same time; 48 hours after infection, the activity of luciferase in the cells is measured using a luciferase substrate reaction kit (Beyotime, RG051M) and a multifunctional microplate reader; for drugs that can significantly inhibit viral invasion (drug group/control group <50%), they are verified using the same method as above, and for drugs that can be repeatedly verified, the effects of different concentrations of drugs on blocking viral invasion are tested, thereby obtaining the IC50 (50% effective inhibition concentration) of the candidate drug in inhibiting SARS-CoV-2 viral invasion.

result:

Using the platform 11-2), the present invention screened the small molecule drug library and obtained a positive small molecule Suramin Sodium, which can specifically inhibit the binding of KREMEN1 to S protein (Figure 15A), and its structure is shown in Figure 15B. Using the effect evaluation system of inhibiting viral infection 11-3), the present invention found that Suramin Sodium specifically blocked the invasion of SARS-CoV-2 virus in KREMEN1-dependent HTB-182 cells, with an IC50 of 18.02uM (Figure 15C); the small molecule had no effect on the invasion of SARS-CoV-2 virus in ACE2-dependent Calu3 cells (Figure 15D), indicating that Suramin Sodium specifically inhibited the invasion of SARS-CoV-2 virus mediated by KREMEN1.

The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims (11)

1. Use of asgr1 as a drug action target in vitro screening of a prophylactic and/or therapeutic drug for SARS-CoV-2.
2. Use of an asgr1 inhibitor for the manufacture of a medicament for the prevention and/or treatment of SARS-CoV-2 infection.
3. 3. The use according to claim 2, wherein the ASGR1 inhibitor is capable of inhibiting or blocking binding of ASGR1 to the S protein of SARS-CoV-2, or wherein the ASGR1 inhibitor is capable of reducing or inhibiting expression or transcription of an ASGR1 gene;

   And/or, the ASGR1 inhibitor is used in combination with a KREMEN1 inhibitor;

   And/or, the ASGR1 inhibitor is used in combination with a KREMEN1 inhibitor and an ACE2 inhibitor;

   Wherein the KREMEN1 inhibitor can inhibit or block the binding of KREMEN1 and S protein of SARS-CoV-2, or the KREMEN1 inhibitor can reduce or inhibit the expression or transcription of KREMEN1 gene;

   The ACE2 inhibitor can inhibit or block the binding of ACE2 to S protein of SARS-CoV-2, or the ACE2 inhibitor can reduce or inhibit the expression or transcription of ACE2 gene.
4. 4. Use according to claim 3, characterized by comprising any one or more of the following features:

   The KREMEN1 inhibitor is selected from small molecule inhibitor, KREMEN1 antibody, antisense oligonucleotide, double-stranded RNA, ribozyme, small interfering RNA or short hairpin RNA prepared by endoribonuclease III;

   The ASGR1 inhibitor is selected from small molecule inhibitor, ASGR1 antibody, antisense oligonucleotide, double-stranded RNA, ribozyme, small interfering RNA or short hairpin RNA prepared by endoribonuclease III;

   the ACE2 inhibitor is selected from a small molecule inhibitor, an ACE2 antibody, or a short hairpin RNA;

   When the KREMEN1 inhibitor is a small molecule inhibitor, the structure is as follows:

   When the KREMEN1 inhibitor is a KREMEN1 antibody, the heavy chain CDR1, CDR2 and CDR3 of the KREMEN1 antibody are respectively shown in sequences SEQ ID NO. 1-3, and the light chain CDR1, CDR2 and CDR3 are respectively shown in sequences SEQ ID NO. 4-6;

   When the ASGR1 inhibitor is an ASGR1 antibody, the heavy chain CDR1, CDR2 and CDR3 of the ASGR1 antibody are respectively shown in sequences SEQ ID NO 7-9, and the light chain CDR1, CDR2 and CDR3 are respectively shown in sequences SEQ ID NO 10-12;

   when the ACE2 inhibitor is an ACE2 antibody, the ACE2 antibody is Ab-414.
5. 5. A target antibody for preventing and/or treating SARS-CoV-2, characterized in that the target antibody is an ASGR1 antibody; the heavy chain CDR1, CDR2 and CDR3 of the ASGR1 antibody are respectively shown in sequences SEQ ID NO 7-9, and the light chain CDR1, CDR2 and CDR3 are respectively shown in sequences SEQ ID NO 10-12.
6. 6. A medicament for the prevention and/or treatment of SARS-CoV-2 infection, characterized in that the medicament comprises a therapeutically effective amount of an ASGR1 inhibitor,

   Or the medicament comprises a therapeutically effective amount of an ASGR1 inhibitor and a KREMEN1 inhibitor,

   Or the medicament comprises a therapeutically effective amount of an ASGR1 inhibitor, a KREMEN1 inhibitor, and an ACE2 inhibitor.
7. 7. The medicament of claim 6, wherein the KREMEN1 inhibitor is capable of inhibiting or blocking the binding of KREMEN1 to the S protein of SARS-CoV-2, or wherein the KREMEN1 inhibitor is capable of reducing or inhibiting the expression or transcription of the KREMEN1 gene;

   The ASGR1 inhibitor can inhibit or block the binding of ASGR1 to S protein of SARS-CoV-2, or the ASGR1 inhibitor can reduce or inhibit the expression or transcription of ASGR1 gene;

   The ACE2 inhibitor can inhibit or block the binding of ACE2 to S protein of SARS-CoV-2, or the ACE2 inhibitor can reduce or inhibit the expression or transcription of ACE2 gene.
8. 8. The medicament according to claim 7, characterized by comprising any one or more of the following features:

   The KREMEN1 inhibitor is selected from small molecule inhibitor, KREMEN1 antibody, antisense oligonucleotide, double-stranded RNA, ribozyme, small interfering RNA or short hairpin RNA prepared by endoribonuclease III;

   The ASGR1 inhibitor is selected from small molecule inhibitor, ASGR1 antibody, antisense oligonucleotide, double-stranded RNA, ribozyme, small interfering RNA or short hairpin RNA prepared by endoribonuclease III;

   the ACE2 inhibitor is selected from a small molecule inhibitor, an ACE2 antibody, or a short hairpin RNA;

   When the KREMEN1 inhibitor is a small molecule inhibitor, the structure is as follows:

   When the KREMEN1 inhibitor is a KREMEN1 antibody, the heavy chain CDR1, CDR2 and CDR3 of the KREMEN1 antibody are respectively shown in sequences SEQ ID NO. 1-3, and the light chain CDR1, CDR2 and CDR3 are respectively shown in sequences SEQ ID NO. 4-6;

   When the ASGR1 inhibitor is an ASGR1 antibody, the heavy chain CDR1, CDR2 and CDR3 of the ASGR1 antibody are respectively shown in sequences SEQ ID NO 7-9, and the light chain CDR1, CDR2 and CDR3 are respectively shown in sequences SEQ ID NO 10-12;

   when the ACE2 inhibitor is an ACE2 antibody, the ACE2 antibody is Ab-414.
9. 9. A method of screening for a therapeutic agent for SARS-CoV-2, the method comprising: ASGR1 is used as a drug target, and a substance capable of inhibiting or blocking the binding of the ASGR1 and the S protein of SARS-CoV-2 or a substance capable of reducing the content of the ASGR1 is searched for as a candidate drug.
10. 10. The method according to claim 9, the method comprising: the candidate drug is applied to the cells in vitro, and the amount of receptor-expressing cells that bind to S-ECD, or the level of ASGR1 in the cells, is measured after co-culturing.
11. 11. The method of claim 10, wherein the agent is capable of reducing the S-ECD binding level by more than 30% or reducing ASGR1 in the cell by at least 50% and is determined to be a therapeutically significant agent.