KR20220037944A - Phamaceutical composition for preventing or treating Coronavirus infectious disease comprising Coronavirus G-quadruplex ligands as an active ingredient - Google Patents

Abstract

The present invention relates to a composition containing a compound conjugated to G4 structures of coronaviruses for prevention or treatment of coronavirus infectious diseases. The composition according to the present invention can be conjugated to G4 structures of coronaviruses to stabilize the G4 structures, reduce the protein expression of coronaviruses, and effectively inhibit cell infection by viruses, and therefore the compound is expected to be used in the prevention or treatment of coronavirus infectious diseases.

Description

A pharmaceutical composition for preventing or treating coronavirus infection comprising a compound that binds to the G4 structure of the coronavirus as an active ingredient as an active ingredient

The present invention relates to a composition for preventing or treating a coronavirus infection comprising a compound that binds to the G4 structure of a coronavirus.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or 2019-nCoV), which caused a global coronavirus infection 19 (COVID-19) pandemic, mainly in Wuhan, China, as of July 20, 2021 There were 190,877,071 confirmed cases, and 4,095,650 deaths were reported. Accordingly, there is a worldwide demand for treatment and prevention methods.

SARS-CoV-2 (2019-nCoV) is known to penetrate by using angiotensin-converting enzyme 2 (ACE2; angiotensin-converting enzyme 2) on the human cell surface as a receptor. ACE2 is mainly distributed widely in the lungs, arterial vessels, kidneys, heart, and small intestine, and thus spreads through the respiratory tract and is known to cause Sever Acute Respiratory Syndrome (SARS). In the case of SARS-CoV-2 (2019-nCoV), it is understood that several subunits of a spike-shaped protein called the spike(S) protein interact with ACE2 in the host cell to infect it. Most of the treatments currently under development are made using small-molecule substances that bind to viral spike proteins, proteolytic enzymes, or nucleic acid polymerases.

Numerous SARS-CoV-2 strains are emerging and circulating around the world through the COVID-19 pandemic. Most of these variants represent mutations in the SARS-CoV-2 spike protein, such as the N501Y, E484K and D614G mutations. Although the spike protein is the target of most COVID-19 vaccines, many variants have high transmission rates and have the ability to evade neutralizing antibodies. Prophylactic vaccines and drugs that target viral proteins are fundamental methods to combat viral infections, but targeting regions of the genome that are conserved within the same virus family may provide new opportunities to develop novel antiviral therapies. .

DNA in a cell generally has a double helix structure, but it is known that it has a variety of structures during a specific cellular environment or cellular action. Among these special structures, a quadruple helix structure made by four strands of DNA or RNA is well known, and such a structure is called a G4 (G-quadruplex) or G4 structure. The G4 structure is well formed when guanines are consecutive in DNA or RNA bases. Guanines of DNA strands combine to form a square plate structure, and these plates are stacked one after another to form a stable hexahedral knot. The G4 structure is known to regulate gene expression, DNA replication, or protein translation processes.

Under this background, the present inventors effectively inhibited the expression of major proteins of the coronavirus through stabilization of the G4 structure by binding with a compound while studying 25 G4 structures present in the coronavirus and effectively inhibited cell infection of the virus By confirming that it can be inhibited, the present invention was completed.

Republic of Korea Patent Publication No. 10-2021-0014601 (2021.02.09.)

The present inventors effectively inhibited the expression of major proteins of the coronavirus and effectively inhibited viral cell infection through stabilization of the coronavirus G4 structure by binding with a compound while studying the 25 G4 structures present in the coronavirus By confirming that it can, the prevention or treatment effect of coronavirus infection was confirmed, and the present invention was completed based on this.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating coronavirus infection, comprising a compound binding to the G4 structure of coronavirus or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving coronavirus infection, comprising as an active ingredient a compound binding to the G4 structure of the coronavirus or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a quasi-drug composition for preventing or inhibiting coronavirus infection, comprising a compound that binds to the G4 structure of coronavirus as an active ingredient.

Another object of the present invention is to provide an antiviral composition for coronavirus, comprising a compound that binds to the G4 structure of coronavirus as an active ingredient.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned may be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. There will be.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection, comprising a compound binding to the G4 structure of coronavirus or a pharmaceutically acceptable salt thereof as an active ingredient .

In addition, it provides a food composition for preventing or improving coronavirus infection, comprising as an active ingredient a compound binding to the G4 structure of coronavirus or a pharmaceutically acceptable salt thereof.

In addition, the present invention provides a quasi-drug composition for preventing or inhibiting coronavirus infection, comprising a compound that binds to the G4 structure of coronavirus as an active ingredient.

In addition, the present invention provides an antiviral composition for coronavirus, comprising a compound that binds to the G4 structure of the coronavirus as an active ingredient.

In addition, the present invention provides a method for preventing or treating a coronavirus infection, comprising administering the composition to an individual.

In addition, the present invention provides a use for preventing or treating coronavirus infection of a composition comprising a compound or pharmaceutically acceptable salt that binds to the G4 structure of coronavirus as an active ingredient.

The present invention also provides the use of a compound that binds to the G4 structure of a coronavirus for the manufacture of a medicament for the prevention or treatment of coronavirus infection.

In one embodiment of the present invention, the compound may be at least one selected from the group consisting of compounds represented by the following Chemical Formulas 1 to 7, but is not limited thereto.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

In another embodiment of the present invention, the coronavirus is human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-) NL63, New Heaven coronavirus), human coronavirus HKU1, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) may be selected from the group consisting of at least one selected from the group consisting of, but It is not limited.

In another embodiment of the present invention, the compound may be characterized in stabilizing the G4 structure of the coronavirus, but is not limited thereto.

In another embodiment of the present invention, the compound inhibits the expression of one or more proteins selected from the group consisting of coronavirus proteins Nsp1 (non-structural protein 1), Nsp3 (non-structural protein 3), and nucleocapsids. It may be characterized, but is not limited thereto.

In another embodiment of the present invention, the compound may be characterized in that it inhibits the production of coronavirus virions, but is not limited thereto.

In another embodiment of the present invention, the quasi-drug may be characterized in that at least one selected from the group consisting of disinfectant cleaner, shower foam, gargrin, wet tissue, detergent soap, hand wash and ointment, but is not limited thereto.

Since the compound according to the present invention binds to the G4 structure of the coronavirus to stabilize the G4 structure and reduce the protein expression of the coronavirus, as well as effectively inhibit virus cell infection, it can be used to prevent or treat coronavirus infection. can be used

1 shows the distribution of genes and G4 of SARS-CoV-2.
Figures 2a and 2b show the CD spectra of 25 G4s present in coronavirus. The x-axis represents the wavelength (nm), and the y-axis represents the ellipticity (mdeg).
Figures 3a and 3b show the thermal melting curves of 25 G4s present in coronavirus. The x-axis shows the wavelength (nm), and the y-axis shows the normalized ellipticity (mdeg).
4 shows CD spectra and thermal melting curves of WT-G-353, WT-G-644 and WT-G-3467 and Mut-G-353, Mut-G-644 and Mut-G-3467.
5A and 5B show the CD spectra of 25 G4 present in coronavirus with or without G4 binding compounds CX-3543, PDS and PhenDC3 binding.
6A and 6B show the thermal melting curves of 25 G4 present in coronavirus with or without G4 binding compounds CX-3543, PDS and PhenDC3 binding.
Figure 7a shows the cell viability of HEK293T cells when PDS is treated with 0.1, 0.5, 1, 2.5, 5.5, 7.5, 10, 25, 50, 75 μM. The x-axis represents the concentration (μM), and the y-axis represents the survival rate.
Figure 7b shows the cell viability when Vero cells are treated with 0.1, 0.5, 1, 2.5, 5.5, 7.5, 10, 25, 50, 75 μM of PDS. The x-axis represents the concentration (μM), and the y-axis represents the survival rate.
Figure 7c shows the cell viability of HEK293T cells when PhenDC3 is treated with 0.1, 0.5, 1, 2.5, 5.5, 7.5, 10, 25, 50, 75 μM. The x-axis represents the concentration (μM), and the y-axis represents the survival rate.
Figure 7d shows the cell viability when Vero cells are treated with PhenDC3 0.1, 0.5, 1, 2.5, 5.5, 7.5, 10, 25, 50, 75 μM. The x-axis represents the concentration (μM), and the y-axis represents the survival rate.
8 shows T7-WT-G-353, T7-WT-G-644, T7-WT-G-3467, T7-Mut-G-353, T7-Mut-G-644 and T7-Mut-G-3467 shows the results of gradient PCR performed at 61, 63, 65, 67, and 69 ° C. M represents a 1 kb DNA marker ranging from 250 bp to 10 kb.
9a to 9c are IVT analysis results when treated with 5, 15, 25, 50 μM of G4 binding compound PDS in N1-WT-G-353, N1-WT-G-644, N1-WT-G-3467 It is shown as a graph of the band intensity of the Western blot. The x-axis represents the concentration (μM), and the y-axis represents the band intensity.
10a to 10c show IVT analysis results when the G4 binding compound PhenDC3 was treated with 5, 15, 25, 50 μM in N1-WT-G-353, N1-WT-G-644, N1-WT-G-3467. It is shown as a graph of the band intensity of the Western blot. The x-axis represents the concentration (μM), and the y-axis represents the band intensity.
Figures 11a to 11b show that in N1-WT-G-353, N1-WT-G-644, N1-WT-G-3467, when the G4 binding compound PDS was treated with 5, 10, 15, 20, 25, 50 μM, The results of the IVT analysis are shown as Western blots.
11c is a Western blot showing the results of IVT analysis when N1-WT-G-3467 was treated with 5, 15, 25, and 50 μM of the G4 binding compound PDS.
11D is a Western blot showing the results of IVT analysis when N1-Mut-G-3467 was treated with 5, 15 25, and 50 μM of the G4 binding compound PDS.
12A to 12B are Western blots showing the IVT analysis results when the G4-binding compound PhenDC3 was treated with 5, 10, 15, 20, 25, 50 μM in N1-WT-G-353 and N1-WT-G-644. will be.
12c is a Western blot showing the results of IVT analysis when N1-WT-G-3467 was treated with the G4-binding compound PhenDC3 at 5, 15, 25, and 50 μM.
12D is a Western blot showing the results of IVT analysis when N1-Mut-G-3467 was treated with the G4-binding compound PhenDC3 at 5, 15, 25, and 50 μM.
13 is a dose-response curve analysis of the antiviral activity against N protein expression in Vero cells infected with SARS-CoV-2 according to the concentrations of the G4 binding compounds NMM, PDS, PhenDC3, and BRACO19, and inhibition of SARS-CoV-2 infection. it has been shown ● indicates the SARS-CoV-2 inhibition of infection of the compound. ■ denotes the cytotoxicity of each compound, and the number of cells in each well was normalized to the average number of wells in the mock group and marked as 'cell number to mock' on the graph.
Figure 14a shows the antiviral activity against virion production in the culture medium of Vero cells infected with SARS-CoV-2 as a result of analyzing the dose-response curve of SARS-CoV-2 infection inhibition according to the PDS concentration of the G4-binding compound. The x-axis shows the concentration (log μM), and the y-axis shows the infection inhibition rate (%) of SARS-CoV-2.
Figure 14b shows the antiviral activity on virion production in the culture medium of Vero cells infected with SARS-CoV-2 as a result of analyzing the dose-response curve of SARS-CoV-2 infection inhibition according to the concentration of the G4-binding compound PhenDC3. The x-axis shows the concentration (log μM), and the y-axis shows the infection inhibition rate (%) of SARS-CoV-2.
15 shows the antiviral activity on virion production in the culture medium of Vero cells infected with SARS-CoV-2 as a result of analyzing the dose-response curve of SARS-CoV-2 infection inhibition according to the NMM concentration of the G4-binding compound. The x-axis shows the concentration (log μM), and the y-axis shows the infection inhibition rate (%) of SARS-CoV-2.
16 is a dose-response curve of HCoV-OC43 virus infection inhibition according to the concentration of the G4-binding compound NMM, TMPyP2, TMPyP4, or CX3543 for antiviral activity against virion production in a culture medium of HCT-8 cells infected with HCoV-OC43 virus. It is presented as an analysis result. The x-axis shows the concentration (log μM), and the y-axis shows the infection inhibition rate (%) and cell viability (%) of HCoV-OC43 virus. The table in FIG. 16 shows the IC ₅₀ values of each G4 binding compound. Dark red ● indicates the cytotoxicity of each compound in HCT-8 cells, and the cytotoxicity by concentration of each compound is expressed as the relative cell viability (%) on the graph by normalizing the G4-binding compound treatment group to DMSO or distilled water control. did.

The inventors of the present invention completed the present invention by confirming that the compound can reduce the protein expression of the coronavirus by binding to the coronavirus G4 structure and stabilizing the G4 structure.

In one embodiment of the present invention, it was confirmed that the compounds PhenDC3 and PDS stabilize G4 through the CD spectrum and the thermal melting curve according to the presence or absence of the G4 binding compound (see Example 4).

In another example of the present invention, it was confirmed that there was no cytotoxicity of the compounds PhenDC3 and PDS in HEK293T and Vero cells (see Example 5).

In another embodiment of the present invention, inhibition of Nsp1 and Nsp3 expression of the G4-binding compound PDS and PhenDC3 was confirmed through IVT (In-Vitro transcriotion/trnaslation) analysis (see Example 7).

In another embodiment of the present invention, antiviral activity against N protein expression according to G4 binding compounds (NMM, PDS, PhenDC3, BRACO19) in cells infected with SARS-CoV-2 was confirmed (see Example 8).

In another embodiment of the present invention, antiviral activity against virion production according to G4 binding compounds (PDS, PhenDC3, NMM) was confirmed in SARS-CoV-2 infected cells (see Example 9).

In another embodiment of the present invention, antiviral activity against virion production according to G4 binding compounds (NMM, TMPyP2, TMPyP4, CX3543) in cells infected with HCoV-OC43 virus was confirmed (see Example 10).

It was confirmed that the compound according to the present invention stabilizes the coronavirus G4 structure and reduces the protein expression of the coronavirus through the results of Examples as described above, and the compound can be used for the prevention or treatment of coronavirus infection.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating coronavirus infection, comprising a compound that binds to the G4 structure of coronavirus as an active ingredient.

In the present invention, the compound may be characterized in that at least one selected from the group consisting of compounds represented by the following Chemical Formulas 1 to 7, but is not limited thereto.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

In the present invention, the compound of Formula 1 may be named PhenDC3, and 2-N,9-N-bis(1-methylquinolin-1-ium-3-yl)-1,10-phenanthroline-2,9- It can have the IUPAC name of dicarboxamide. Also, the molecular weight may be 550.6, and the chemical formula may be C ₃₄ H ₂₆ N ₆ O ₂ .

In the present invention, the compound of Formula 2 may be named pyridostatin (PDS), and 4-(2-Aminoethoxy)-N2,N6-bis(4-(2-aminoethoxy)quinolin-2-yl)pyridine-2 It may have the IUPAC name of ,6-dicarboxamide. In addition, the molecular weight is 596.6, the chemical formula is C ₃₁ H ₃₂ N ₈ O ₅ , the boiling point may be 753.8±60.0°C, and the flash point may be 409.7±32.9°C.

In the present invention, the compound of Formula 3 may be named N-Methyl Mesoporphyrin IX (NMM9), and 3-[18-(2-carboxyethyl)-7,12-diethyl-3,8,13,17,22 -pentamethyl-23H-porphyrin-2-yl]propanoic acid may have an IUPAC name. In addition, the molecular weight may be 580.7, the chemical formula is C ₃₅ H ₄₀ N ₄ O _{4 , and the} density may be 1.26 g/cm ³ .

In the present invention, the compound of Formula 4 may be referred to as BRACO-19, N-[9-[4-(dimethylamino)anilino]-6-(3-pyrrolidin-1-ylpropanoylamino)acridin-3-yl] It can have the IUPAC name of -3-pyrrolidin-1-ylpropanamide. In addition, the molecular weight is 593.8, the chemical formula is C ₃₅ H ₄₃ N ₇ O _{2 , the} boiling point is 854.95 ℃, the flash point is 470.857 ℃, the density may be 1.275 g / cm ³ .

In the present invention, the compound of Formula 5 may be named TMPyP2, and has an IUPAC name of 5,10,15,20-tetrakis(1-methylpyridin-1-ium-2-yl)-21,23-dihydroporphyrin. can In addition, the molecular weight may be 678.8, and the chemical formula may be C ₄₄ H ₃₈ N ₈ ⁴⁺ .

In the present invention, the compound of Formula 6 may be named TMPyP4, and has an IUPAC name of 5,10,15,20-tetrakis(1-methylpyridin-1-ium-4-yl)-21,23-dihydroporphyrin. can In addition, the molecular weight may be 678.8 and the chemical formula may be C ₄₄ H ₃₈ N ₈ ⁴⁺ .

In the present invention, the compound of Formula 7 may be named CX 3543 and 15-fluoro-N-[2-[(2S)-1-methylpyrrolidin-2-yl]ethyl]-18-oxo-14-(3 -pyrazin-2-ylpyrrolidin-1-yl)-12-oxa-1-azapentacyclo[11.7.1.0 ^2,11 .0 ^4,9 .0 ^17,21 ]henicosa-2,4,6,8,10,13 (21),14,16,19-nonaene-19-carboxamide may have the IUPAC name. In addition, the molecular weight is 604.673, the chemical formula is C ₃₅ H ₃₃ FN ₆ O ₃ , and the boiling point may be 845.3±65.0°C.

In the present invention, the coronavirus may be severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2 or 2019 novel coronavirus or 2019-nCoV), but is not limited thereto.

As used herein, the term "coronavirus" corresponds to a species belonging to the genus of viruses included in the order Nidoviridae, Coronaviridae, Subfamily Coronavirae, or Torovirinae. Coronaviruses are viruses wrapped in +ssRNA and helically symmetrical nucleopepsids. In addition, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the seventh coronavirus that infects humans so far, and the rest are human coronavirus 229E (HCoV-229E) and human coronavirus OC43 (HCoV-OC43). , severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63, New Haven coronavirus), human coronavirus HKU1, Middle East respiratory syndrome coronavirus (MERS-CoV). Proteins that contribute to the overall structure of coronaviruses are the spike, envelope and nucleocapsid. In the case of SARS coronavirus, an established bind receptor domain on the spike (S) mediates the attachment of the virus to its cellular receptor, angiotensin converting enzyme 2 (ACE2). Some coronaviruses (particularly members of the betacoronavirus subfamily) also have short spikes like proteins called antibody esterases. Coronaviruses can cause viral pneumonia or secondary bacterial pneumonia, as well as direct viral bronchitis or secondary bacterial bronchitis. The human coronavirus, discovered in 2003, is the severe acute respiratory syndrome coronavirus (SARS-CoV), which causes severe acute respiratory syndrome (SARS), which causes upper and lower respiratory tract infections.

In addition, the term "SARS-CoV-2" or "2019-nCoV" used in the present invention refers to a novel coronavirus, and refers to strains of SARS and MERS as RNA viruses. SARS-CoV-2 shares about 79.7% sequence identity with SARS and about 50% sequence identity with MERS. However, in contrast to SARS and MERS, the spike glycoprotein of 2019-nCoV forms a structure in which one RBD domain protrudes upward, resulting in 100 to 1,000 times more It shows strong bonding power. This strong binding force makes it easier to penetrate into the cell and acts as a cause to increase the infectious power.

In the present invention, the coronavirus infection may be a coronavirus respiratory infection disease. The viral respiratory infection diseases include cough, sneezing, headache, stuffy nose, sore throat, diarrhea, discoloration of fingers or toes, conjunctivitis, high fever, wheezing, bronchitis, bronchiolitis, pneumonia, asthma, loss of smell and taste, and respiratory failure. symptoms may be present. When the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), fever and respiratory symptoms (cough, sore throat, dyspnea) are the main symptoms, along with headache, muscle pain, hemoptysis and nausea, Symptoms may include chills, chest pain, and diarrhea. In addition, the coronavirus infection may be coronavirus infection 19 (COVID-19).

In the present invention, the virus can modulate its genome through replication and infection for survival. The viral RNA genome possesses various regions such as structural domains, internal ribosome entry sites (IRES), polycistronic mRNAs, overlapping reading frames, and RNA pseudoknot structures to control expression and replication of the genome. For example, the folding of certain regions of genomic RNA into stable non-canonical structures can act as obstacles to viral RNA metabolism.

As used herein, the term "spike protein" or "S protein" refers to a coronavirus surface protein.

As used herein, the term “G4 structure” or “G4 (G-quadruplex)” is a 4-stranded nucleic acid secondary structure formed from a DNA or RNA sequence rich in guanine (G, guanine). The G4 structure is widely distributed in both DNA and RNA virus genomes. For example, Epstein Barr Virus (EBV), Zika Virus (ZIKV), Hepatitis C Virus (HCV), Human Papilloma Virus (HPV), Human Herpes Simplex Virus (HSV), Ebola Virus, Human Immunodeficiency Virus-1 (HIV) -1), influenza virus (H1N1) and human cytomegalovirus (HCMV), as well as long terminal repeat regions of retroviruses and lentiviruses. Recently, bioinformatics analysis of whole genomes and transcripts obtained using G4 specific antibodies and compounds revealed the genetic location of G4. DNA-G4 plays an important role in telomeres, double-strand breaks in mitosis and meiosis, transcription initiation sites and origins of replication. RNA-G4 coordinates many steps of RNA metabolism, from splicing, RNA processing and transport, to mRNA translation (Int. J. Mol. Sci. 2019, 20, 2884).

In the present invention, the compound may be a substance that stabilizes G4, but is not limited thereto. For example, the Braco-19 is a compound that stops virus entry and replication by showing inhibitory activity on the expression of the G4 harboring gene in Nipah virus. In addition, the CX-3543 is the only G4-binding compound that has passed the in vivo phase 2 clinical trial for carcinoid and neuroendocrine tumors. In addition, the PDS is a compound known to reduce gene expression by stabilizing the conserved G4 in EBV, pseudorabies virus and herpes virus miRNA. In addition, the PhenDC3 is a compound known to inhibit HCV and EBV virus replication in cells without cytotoxicity.

In the present invention, the compound may be characterized in that it inhibits the expression of one or more proteins selected from the group consisting of coronavirus proteins Nsp1 (non-structural protein 1), Nsp3 (non-structural protein 3), and nucleocapsid. , but is not limited thereto.

As used herein, the term “Nsp1 (non-structural protein 1)” is the most important virulence factor that plays various roles in interfering with viral replication and host immune response. In SARS-CoV-2, Nsp1 contains G4-353 and G4-644 in an RNA open reading frame (ORF) with a length of 540 bp. Nsp1 mainly targets type I interferon expression and antiviral signaling pathways to inhibit host mRNA translation and suppress host innate immune function.

As used herein, the term “Nsp3 (non-structural protein 3)” is the largest multi-domain gene (5.83 Kbp) in SARS-CoV-2 with various functions. The Nsp3-MAC1 domain is 618 bp and includes G4-3467. The Nsp3-MAC1 domain counteracts the host's innate immunity through antiviral de-ADPribosylating activity catalyzed by poly(ADP-ribose) polymerases (PARPs). De-mono-ADP-ribosylation of STAT1 by the Nsp3-Mac1 domain may trigger the inflammatory cytokine storm observed in severe cases of COVID-19.

The term “nucleocapsid (N)” used in the present invention plays an important role in viral packaging of coronavirus, viral nucleation, and synthesizing viral RNA. As one of the structural proteins, it is a protein that has multiple functions and plays an important role in the viral life cycle, making it a good target for inhibiting or detecting coronavirus.

In the present invention, the compound may be characterized in that it inhibits the production of coronavirus virions, but is not limited thereto.

As used herein, the term “virion” refers to an intact viral particle composed of a capsid and a nucleic acid. In general, it refers to an infectious virus particle. Thus, the production of virions is associated with the smoothness of viral replication.

The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, at least one selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a humectant, a film-coating material, and a controlled-release additive.

The pharmaceutical composition according to the present invention can be prepared according to a conventional method, respectively, in powders, granules, sustained-release granules, enteric granules, liquids, eye drops, elsilic, emulsions, suspensions, alcohols, troches, fragrances, and limonaade. , tablets, sustained release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusates, Warnings, lotions, pasta, sprays, inhalants, patches, sterile injection solutions, or external preparations such as aerosols can be formulated and used, and the external preparations are creams, gels, patches, sprays, ointments, warning agents , lotion, liniment, pasta, or cataplasma.

Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid as additives for tablets, powders, granules, capsules, pills, and troches according to the present invention Calcium monohydrogen, calcium sulfate, sodium chloride, sodium hydrogen carbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methyl cellulose, sodium carboxymethyl cellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methyl Cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, excipients such as Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch powder, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. Hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, silicic anhydride, 1-hydroxy Propyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodite, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, polyethylene glycol 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, A lubricant such as sodium chloride, sodium acetate, sodium oleate, dl-leucine, light silicic anhydride; may be used.

As additives for the liquid formulation according to the present invention, water, diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

In the syrup according to the present invention, a sucrose solution, other sugars or sweeteners may be used, and if necessary, a fragrance, colorant, preservative, stabilizer, suspending agent, emulsifying agent, thickening agent, etc. may be used.

Purified water may be used in the emulsion according to the present invention, and if necessary, an emulsifier, preservative, stabilizer, fragrance, etc. may be used.

Suspending agents such as acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, HPMC 2910 may be used in the suspending agent according to the present invention. and, if necessary, surfactants, preservatives, stabilizers, colorants, and fragrances may be used.

The injection according to the present invention includes distilled water for injection, 0.9% sodium chloride injection solution, ring gel injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG (PEG), lactated ring gel injection solution, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; Solubilizing aids such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tweens, nijeongtinamide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, buffers such as albumin, peptone and gum; isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ), carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), ethylenediaminetetraacetic acid; sulphating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, acetone sodium bisulfite; analgesic agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; suspending agents such as SiMC sodium, sodium alginate, Tween 80, or aluminum monostearate.

The suppository according to the present invention includes cacao fat, lanolin, witepsol, polyethylene glycol, glycerogelatin, methyl cellulose, carboxymethyl cellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lanet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolene (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Butyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium, A, AS, B, C, D, E, I, T, Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository IV type (AB, B, A, BC, BBG, E, BGF, C, D, 299), supostal (N, Es), Wecobi (W, R, S, M, Fs), tester triglyceride base (TG-95, MA, 57) and The same mechanism may be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration can be contemplated, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal It can be administered according to internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, and the like.

The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with several related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.

In the present invention, "individual" means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cattle, etc. means the mammals of

In the present invention, "administration" means providing a predetermined composition of the present invention to an individual by any suitable method. In the present invention, "prevention" means any action that inhibits or delays the onset of a desired disease, "Treatment" means any action in which a desired disease and metabolic abnormalities are improved or changed beneficially by administration of the pharmaceutical composition according to the present invention, and "improvement" refers to the purpose of It refers to any action that reduces the disease-related parameters, for example, the severity of symptoms.

In addition, it provides a food composition for preventing or improving coronavirus infection, comprising as an active ingredient a compound binding to the G4 structure of coronavirus or a pharmaceutically acceptable salt thereof.

When the compound binding to the G4 structure of the coronavirus of the present invention is used as a food additive, the compound may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, in the production of food or beverage, the compound of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health functional foods in the ordinary sense.

The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, as in a conventional beverage. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as taumartin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, Carbonating agents used in carbonated beverages, etc. may be contained. In addition, the composition of the present invention may contain the pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention provides a quasi-drug composition for preventing or inhibiting coronavirus infection, comprising a compound that binds to the G4 structure of coronavirus as an active ingredient.

In the "quasi-drug composition for preventing or suppressing coronavirus infection" according to the present invention, the quasi-drug is a preparation used for sterilization, insecticide, and similar uses for the prevention of infectious diseases described in Article 2, No. 7 (c) of the Pharmaceutical Affairs Act, human or animal It may mean a repellent, exterminant, inhibitor, insecticide, or attractant insecticide for flies and mosquitoes used for the health of people.

In addition, the quasi-drugs may include external preparations for skin and personal care products. For example, it may be a disinfectant cleaner, shower foam, gargrin, wet tissue, detergent soap, hand wash, or ointment, but is not limited thereto.

When the quasi-drug composition according to the present invention is used as a quasi-drug additive, the composition may be added as it is or used together with other quasi-drugs or quasi-drug ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be suitably determined according to the purpose of use.

The quasi-drug composition of the present invention may be prepared in the form of, for example, a general emulsified formulation and a solubilized formulation. For example, it may have formulations such as emulsions, creams, ointments, sprays, oil gels, gels, oils, aerosols, and smokers such as lotions, but is not limited as long as it exhibits the pest control inducing effect of the present invention. . In addition, in the quasi-drug composition, oil, water, surfactant, humectant, lower alcohol having 1 to 4 carbon atoms, thickener, chelating agent, pigment, preservative or fragrance, etc., which are generally formulated in quasi-drug composition in each formulation, are appropriately blended as needed. can be used by

The present invention provides a composition for antiviral against coronavirus, comprising a compound that binds to the G4 structure of coronavirus as an active ingredient.

In the present invention, "antiviral" means to weaken or eliminate the action of a virus that invades the body by inhibiting the proliferation of the virus in the body, and more specifically, the nucleic acid synthesis process of the virus, the gene expression process, or It means to inhibit the proliferation of a virus by inhibiting the viral replication process, etc., and the present invention targets a coronavirus

In the present invention, the antiviral composition is human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV- Antiviral against NL63, New Haven coronavirus), human coronavirus HKU1, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2 or 2019 novel coronavirus or 2019-nCoV) may have activity.

The terms used in the present invention have been selected as currently widely used general terms as possible while considering the functions in the present invention, but these may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Test method]

1. Preparation of oligonucleotides

According to the manufacturer's protocol, a 100 μM stock solution was prepared by dissolving RNA oligonucleotides (Bioneer, Korea) in 200 μl of nuclease-free water. The RNA G4 oligonucleotides used in the present invention are shown in Table 1 below.

2. Circular dichroism (CD) spectral measurement

15 µM of RNA oligonucleotides were annealed in a buffer containing 10 mM Tris-HCl (pH 7.5) and 100 mM KCl in a thermoblock (denatured at 95 °C for 10 min followed by cooling at 0.5 °C/min to 4 °C). Samples were stored overnight at 4 °C prior to performing the experiment. CD spectra were measured between 220 and 320 nm wavelength using a 1 mm quartz cuvette, at 20 °C, a scan rate of 100 nm/min, a data pitch of 1 nm, and a bandwidth of 1 nm. In addition, each spectrum was measured three times and averaged. CD experiments were performed on a Jasco J-810 CD spectropolarimeter equipped with a Jasco CDC-426F Peltier temperature controller.

3. CD Thermal Melting Analysis

For CD melting the samples were heated at 25-95°C with a data pitch of 2°C/min and 0.5°C. CD melting assays were performed with and without G4 binding compounds. The ratio of RNA:G4-binding compound was set to a molar ratio of 1:2. 15 μM of RNA G4 was treated with 30 μM of G4 binding compound and incubated in a buffer containing 10 mM Tris-HCl (pH 7.5) and 100 mM KCl at room temperature for 30 minutes. The ellipticity was measured at 265 nm. The data were then normalized and plotted in OriginPro 2017 to calculate Tm values.

4. Cell Culture

Human embryonic kidney (HEK293T) cells and Vero cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Medium) and incubated in a humidified incubator at 37 °C, 5% CO ₂ .

5. Construction of peYFP-N1 reporter vector for in vitro expression

The pEYFP-N1 reporter (Promega) vector was generated by cloning the eYFP coding region and in-frame target genes (WT-G4-353, 644, 3467) to encode the eYFP fusion protein. Primers used for PCR amplification cloning are shown in Table 2 below. Target gene inserts for WT G4-353, 644, 3467 were synthesized by PUC-GW-Amp (donor plasmid) (Cosmogenetech), subcloned between the Xho1/BamH1 restriction sites of the recipient plasmid pEYFP-N1 vector, and then It was transformed into tant E. coli DH5α cells. The named recombinant plasmids (N1-WT-G4-353, 644 and 3467) were confirmed by sequencing, respectively.

6. Site-Directed Mutagenesis

A PCR reaction was performed according to the protocol (Stratagene) of QuikChange site-directed mutagenesis to introduce site-directed mutations disrupting G4 formation in G4-353, G4-644, and G4-3467. Primers used for mutagenesis are shown in Table 2 above. After mutagenesis, the wild-type vector backbone was digested with Dpnl overnight at 37°C and transformed into DH5α competent cells. The obtained positive clone was confirmed by sequencing. The named mutant G4 plasmids (N1-Mut-G4-353, 644 and 3467) were confirmed by sequencing.

7. Natural polyacrylamide gel electrophoresis

Native polyacrylamide gel electrophoresis was performed on 15% polyacrylamide gel in 1X TBE buffer containing 50 mM KCl at 80V, 4°C for 3 hours. The gel was stained with SYBR Gold nucleic acid dye and observed with a Bio-Rad Gel Doc UV-transilluminator.

8. Cell viability assay

Cytotoxicity was analyzed using the EZ Cytox assay kit (DogenBio, Korea). HEK293T and Vero cells were placed in a 96-well-plate at a density of 2×10 ⁴ cells/well in a medium volume of 100 μl/well and incubated at 37° C. for 24 hours. After 24 hours, cells were treated with various concentrations of G4 binding compounds (0.1, 0.5, 1, 2.5, 5.5, 7.5, 10, 25, 50, 75 μM) and incubated at 37°C for 24 or 48 hours. Cell viability (%) was calculated by measuring the absorbance value at 455 nm (Luminescence multi-mode microplate reader Synergy™ Neo2) using Equation 1 below. All experiments were performed in triplicate and cells not treated with the compound were used as controls. DMEM with EZ Cytox reagent was used as a blank.

[Equation 1]

Cell viability (%) = [(A _{455 (test sample)} - A _{455 (Blank)} ) /(A _{455 (control sample)} - A _{455 (Blank)} )]*100

9. Cell Transfection

Transfection was performed in HEK293T and Vero cells according to the manufacturer's protocol (TurboFect™ Transfection Reagent, Invitrogen, USA). 1.5×10 ⁵ cells or 7.5×10 ⁴ cells were placed in 2ml DMEM medium in a 6 well-plate, respectively, and incubated at 37°C. After 24 hours, transfection was carried out according to the manufacturer's instructions. After 10 hours, the compounds were treated by replacing the medium with or without G4 binding compound. Thereafter, the cells were cultured for 24 or 48 hours, and the cells were harvested for further experiments.

10. In-Vitro transcription/translation (IVT) analysis

The WT-G4 and Mut-G4 sequences were PCR amplified with specific primers designed with the T7 promoter region. The PCR product was 1Kbp or less in length, and included the T7 promoter region and the eGFP gene. Primers used for IVT are shown in Table 2 above. The PCR product was transcribed and translated using the TNT ^® Quick Coupled Transcription/Translation System (Promega), and the PCR-DNA template (0.5 µg) was translated in 50 µl reaction mixture containing 1 mM methionine at 30 °C for 90 min. . The translated product was lysed using 12% SDS-PAGE and analyzed by Western blot.

11. Western Blot

Cells were lysed in 1X-RIPA buffer. The isolated protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon®-P PVDF membrane for Western blotting, Merck KGaA, Darmstadt, Germany) using a Bio-Rad semi-dry transfer system. Non-specific protein binding was blocked with 5% nonfat milk in Tris buffered saline containing 0.05% Tween-20 (TBS-T) buffer for 1 h. Thereafter, the membrane was washed with TBST buffer and probed at 4ºC for 1 hour using a mouse monoclonal anti-EYFP antibody (MAB 8759, Abnova), a primary antibody diluted 1:5000. Thereafter, at room temperature for 1 hour, HRP-bound secondary goat-anti-mouse antibody (SC-2031, Santa Cruz Biotechnology, South Korea) diluted 1:5000 was used for protein detection. Bands were detected using an enhanced chemiluminescence (ECL) western blot substrate by a luminescence image analyzer LAS3000. EYFP and intensity were normalized to the housekeeping gene GAPDH band intensity. Band intensities were quantified using ImageJ.

12. Statistical Analysis

The data of experiments 10 to 11 were analyzed using GraphPad Prism software version 8.3.0. One way ANOVA test was used to determine significant differences between drug-untreated and treated samples by EYFP translational inhibition. Data were expressed as mean±SEM or mean±SD. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

13. SARS-CoV-2 Dose Responsive Curve (DRC) Analysis-N Protein Expression Related

13-1. Viruses and cell lines

SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prevention (KCDC), and Vero cells were obtained from ATCC (ATCC-CCL81).

13-2. reagent

Chloroquine, lopinavir, and remdesivir used as reference compounds were purchased from Sigma-Aldrich, SelleckChem, and MedChemExpress, respectively. A primary antibody specific for anti-SARS-CoV-2 nucleocapsid (N) protein was purchased from Sino Biological, and secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG and Hoechst 33342, were purchased from Molecular Probes.

13-3. Dose-response curve (DRC) analysis by immunofluorescence method

Vero cells were seeded in 384 well-plates at a cell density of 1.2x10 ⁴ cells/well in DMEM supplemented with 2% FBS. After 24 hours, cells were treated with G4 binding compounds (NMM, BRACO19, PDS and PhenDC3) prepared at 10 points by 2-fold serial dilution in DMSO at concentrations ranging from 0.08 to 50 μM. As positive controls, chloroquine diphosphate (C6628, Sigma-Aldrich, St. Louis, MO), lopinavir (S1380, SelleckChem, Houston, TX), and remdesivir (HY-104077, MedChemExpress, Monmouth Junction, NJ), which have well-known antiviral activity, were was used. About 1 hour later, cells were infected with SARS-CoV-2 (0.0125 MOI) in the BSL3 facility and incubated at 37° C. for 24 hours. After fixing the cells with 4% paraformaldehyde (PFA; paraformaldehyde), the cells were permeabilized. Thereafter, cells were stained with anti-SARS-CoV-2 nucleocapsid (N) primary antibody, and Alexa Fluor 488 goat anti-rabbit IgG secondary antibody and Hoechst 33342. Fluorescence images of infected cells were obtained using a large-capacity image analysis instrument, Operetta (Perkin Elmer).

13-4. image analysis

The acquired images were analyzed using Columbus software. The total number of cells per well was calculated as the number of Hoechst stained nuclei, and the number of infected cells was calculated as the number of cells expressing the viral N protein. Infection ratio (infection ratio) was calculated as (number of cells expressing N protein) / (total number of cells). The infectivity per well was normalized to the average infectivity of wells containing uninfected cells (mock) and the average infectivity of wells containing infected cells treated with 0.5% DMSO (v/v) in the same plate. Response curves and IC ₅₀ , CC ₅₀ values according to compound concentrations were derived using Equation 2 below of XLFit 4 (IDBS) software.

[Equation 2]

Y = Bottom +(Top-Bottom)/(1 + (IC ₅₀ /X) ^Hillslope )

All IC ₅₀ and CC ₅₀ values were calculated from a fitted dose-response curve obtained through two replicates, and selectivity index (SI) values were calculated as CC ₅₀ /IC ₅₀ .

14. SARS-CoV-2 Dose Responsive Curve (DRC) analysis-related to virion production

Vero cells were seeded in 12 well-plates at a cell density of 2x10 ⁵ cells/well. After 24 hours, cells were treated with G4 binding compounds (PDS, PhenDC3 and NMM) prepared at 10-point 2-fold serial dilutions at concentrations ranging from 0.08 to 50 μM for 1 hour in the BSL3 facility. Cells were washed once with PBS and then infected with SARS-CoV-2 (0.01 MOI) for 1 h. After removing the virus inoculum and washing the cells once with PBS, they were replaced with 2% FBS DMEM medium containing the same G4 binding compound as above at each concentration and incubated for 24 or 48 hours. DMSO and distilled water vehicle were also added as controls for each compound. At 24 or 48 hours post infection, viral supernatants were collected and total RNA was extracted via automated nucleic acid purification using a PROMEGA Maxwell ^® RSC48 instrument. RT-qPCR was performed using primers specific for SARS-CoV-2 RdRp in duplicate per sample using the THERMO QuantStudio™ 3 system. The viral genome RNA transcription level was analyzed by the ΔΔCt method, and the relative inhibition of infection was calculated by normalizing the G4-binding compound-treated group to DMSO or distilled water control. IC ₅₀ for each compound was derived using the following Equation 3 of Graphpad Prism 5.0 software.

[Equation 3]

Y = Bottom + (Top-Bottom)/(1 + (IC ₅₀ /X) ^Hillslope )

15. Analysis of HCoV-OC43 Dose Responsive Curve (DRC) - Related to virion production

HCT-8 cells, a human colorectal cancer cell line, were seeded in 12 well-plates at a cell density of 2x10 ⁵ cells/well. For 1 hour after 24 hours, cells were treated with G4-binding compounds (CX3543, NMM, TMPyP4 and TMPyP2) prepared at 10-point dilutions by 2-fold serial dilutions at concentrations ranging from 0.08 to 50 μM. Cells were washed once with PBS and then infected with HCoV-OC43 (0.05 MOI) for 1 h. After removing the virus inoculum and washing the cells once with PBS, they were replaced with 2% FBS DMEM medium containing the same G4 binding compound as above at each concentration and incubated for 72 hours. DMSO and distilled water vehicle were also added as controls for each compound. After 72 hours of infection, the viral supernatant was collected and the titer of the infectious virus was quantified through a plaque assay.

For the plaque assay, two wells of Vero cells inoculated in 12 well-plates the day before, and virus supernatant serially diluted 10-fold were inoculated in 2 wells each, and removed after 1 hour of incubation. Plaque assay overlay media containing 0.5% methylcellulose was put in DMEM culture medium and cultured for 5-6 days. After 5-6 days of culture, the overlay media was removed, and the cells were fixed and stained with a solution containing 2% crystal violet staining solution in 20% ethanol to count the number of plaques and measure the virus titer. The relative inhibition of infection was calculated by normalizing the G4-binding compound treatment group to the DMSO or distilled water control group in the virus titer of each sample. IC ₅₀ for each compound was derived using Equation 3 of Graphpad Prism 5.0 software.

For cytotoxicity in HCT-8 cells, cells inoculated in 96-well-plates were treated with the G4 binding compound prepared at the same concentration as in the above dose analysis experiment, and 72 hours later, MTT (3-(3-( 10 μl of 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was treated at 37° C. for 3 hours in each well. After treatment, the supernatant was removed, the cells were washed once with PBS, and then 100 μl DMSO was added and further treated in the dark for 1 hour, and then the absorbance at a wavelength of 540 nm was measured and analyzed. For the cytotoxicity by concentration of each compound, the relative cytotoxicity was calculated by normalizing the G4-binding compound-treated group to DMSO or distilled water control.

[Example]

Example 1. Confirmation of G4 of SARS-CoV-2 genome through bioinformatics analysis

The QGRS mapper was applied to the SARS-CoV-2 genome with a maximum length of 30 bp, and 25 G4s with a minimum G group length of 2 and a loop size of 0 to 36 were identified. The results are shown in FIG. 1 .

As shown in FIG. 1 , G4 was located in the open reading frame region of ORF-1ab, spike (S), ORF3a, membrane (M) and nucleocapsid (N) genes. G4 also appeared at a higher density in the genomic region encoding Nsps (encoded by ORF1 ab) and at a lower density in the genomic region encoding structural proteins. In addition, G4 with a G group length of 3 or 4 was not found in the genome of SARS-CoV-2.

Example 2. Stable G4 structure formation of predicted G4 in SARS-CoV-2

To confirm the formation and structural stability of G4, a biophysical study was performed for all G4 predicted in SARS-CoV-2 using the RNA oligonucleotide corresponding to G4. Circular dichroism (CD) spectra were measured to confirm the formation of the G4 structure, and the structural stability of G4 was confirmed through CD thermal melting analysis. The results are shown in Figs. 2a and 2b, and Figs. 3a and 3b.

As shown in FIGS. 2A and 2B , the CD spectra of all SARS-CoV-2 G4s confirmed the G4 topology characterized by a strong positive peak at about 265 nm and a negative peak at about 240 nm.

In addition, as shown in Figures 3a and 3b, SARS-CoV-2 G4 exhibited a Tm in the range of 50 ~ 75 ℃. This means that the predicted G4 sequence in SARS-CoV-2 can fold into a stable G4 structure in vitro.

Example 3. Instability of G4 following introduction of site-directed mutations for Nsp1 and Nsp3

G4 (G4-353, G4-644, G4-3467) located in Nsp1 and Nsp3 showed the highest ΔTm when treated with PDS and PhenDC3 as shown in Table 4 below and was selected as a mutation target because it has important biological functions. . To visualize the effect of SARS-CoV-2 G4 targeting on Nsp1 and Nsp3, guanine (G) highlighted in red in the WT-G4 sequence was mutated to adenine (A) to create Mut-G4, which disrupts G4 formation. This is shown in Table 3 below. Thereafter, CD spectra of WT and Mut G-353, G-644, and G-3467 were confirmed. The results are shown in FIG. 4 .

As shown in Fig. 4, compared with the CD spectrum of WT-G4, Mut-G4 showed that the maximum value shifted from about 260 nm to about 270 nm and the G4 topology was disrupted. In addition, in CD thermal melting analysis, Mut-G4 showed a lower Tm value than WT-G4. This indicates the instability of G4 in Mut-G4.

Example 4. SARS-CoV-2 G4 stability following treatment with G4 binding compound

15 μM of RNA oligonucleotides were annealed in 10 mM Tris-HCl (pH 7.5) and 100 mM KCl. After that, 30 μM of the compound was treated and incubated. Next, 25 G4s present in SARS-CoV2 were identified through the spectra characteristics emitted through the circular dichroism analyzer. Each spectrum is the average of three measurements. The results are shown in Figures 5a and 5b.

As shown in FIGS. 5A and 5B , when the G4 binding compound was treated, the CD spectrum had a positive peak at about 265 nm and a negative peak at about 240 nm, indicating no significant change in the spectrum of Example 2. This means that the G4 binding compound does not alter the G4 topology.

In addition, 15 µM of RNA G4 was treated with 30 µM of compound and incubated, and the presence or absence of a G4-binding compound was varied to obtain a CD thermal melting curve of RNA G4 (15 µM). In addition, the effect of the G4-binding compound on the stability of SARS-CoV-2 G4 was confirmed by measuring the difference in melting temperature (ΔTm). The results are shown in Figures 6a and 6b.

As shown in FIGS. 6A and 6B , ΔTm of CX-3543 did not show a noticeable change, whereas ΔTm of PhenDC3 and PDS was larger than that of CX-3543. This means that compared to CX-3543, PhenDC3 and PDS significantly stabilized most of G4. Detailed numerical values are shown in Table 4 below. In addition, the comparison of ΔTm between each compound is shown in Table 5 below.

As shown in Table 5, among the 25 G4 predicted in SARS-CoV-2, PDS had ΔTm>20°C in 36% of G4, whereas PhenDC3 had ΔTm>20°C in 72% of G4. This means that the proportion of G4 stabilized by the compound is higher in PhenDC3 than in PDS.

Example 5. Confirmation of cytotoxicity

The toxicity of PDS and PhenDC3 was confirmed in HEK293T cells and Vero cells. The results are shown in FIGS. 7A to 7D .

7A and 7C , PDS in HEK293T cells was safe to use up to 75 μM, but PhenDC3 showed a 25% decrease in viability after 48 hours at 75 μM. In addition, as shown in FIGS. 7B and 7D , no cytotoxicity was observed for PDS and PhenDC3 up to 75 μM after 48 hours in Vero cells.

Example 6. PCR amplification of WT-G4 and Mut-G4 containing T7 promoter

For In-Vitro transcriotion/trnaslation (IVT) analysis, PCR amplification of N1-WT-G4-353, 644, 3467 and N1-Mut-G4-353, 644, 3467 with T7 promoter region and T7-WT-G4- 353, 644, 3467 and T7-Mut-G4-353, 644, 3467. The results are shown in FIG. 8 .

As shown in FIG. 8 , a clear single band of the amplified product eluting from the gel at 65° C. was observed.

Example 7. In-Vitro transcriotion/trnaslation (IVT) analysis

IVT analysis confirmed the effects of various concentrations of G4 binding compounds on translation. If translation is inhibited, it means that the compound is effective in stabilizing the G4 structure. To visualize this, an enhanced yellow fluorescent protein (EYFP) reporter system was constructed, and 48 hours after compound treatment, cells were harvested and EYFP expression was analyzed by Western blot. The results are shown in FIGS. 9A to 9C, 10A to 10C, 11A to 11D, and 12A to 12D.

As shown in FIGS. 9A and 10A, 11A and 12A , when PDS and PhenDC3 were treated at 50 μM in N1-WT-G4-353, respectively, EYFP expression was reduced by 65% and 90%. Concentrations below 50 μM were not effective in inhibiting EYFP reporter expression.

As shown in FIGS. 9b and 10b, 11b and 12b, when PDS was treated with 25 and 50 μM in N1-WT-G4-644, EYFP expression was reduced by 80-90%, but even when PhenDC3 was treated with 5 μM EYFP expression was reduced by 50%.

As shown in FIGS. 9c and 10c, 11c and 12c, when PDS was treated with 15 to 50 μM in N1-WT-G4-3467, EYFP expression was inhibited by 60% and no change was seen with increasing drug concentration. . When PhenDC3 was treated with 50 μM, it showed a concentration-dependent decrease, along with a 70% decrease in EYFP expression.

11d and 12d, when N1-Mut-G4-3467 was treated with PDS and PhenDC3, EYFP expression was not inhibited in a concentration-dependent manner.

These results suggest that PDS and PhenDC3 inhibit the translation of Nsp1 and Nsp3 mRNA by stabilizing the structure of coronavirus G4. It also means that PhenDC3 has a higher effect in stabilizing SARS-CoV-2 G4 than PDS.

Example 8. Confirmation of antiviral efficacy of G4 binding compound against SARS-CoV-2 - related to N protein expression

The N protein is one of the structural proteins and plays an important role in the viral packaging of coronaviruses, viral nucleation, and viral RNA synthesis. To confirm the antiviral activity of the compound, the level of expression of N protein in infected cells was analyzed.

As shown in FIG. 13 , among the G4-binding compounds, PDS and PhenDC3 were most effective in inhibiting SARS-CoV-2 infection with IC ₅₀ of 9.45 μM and 23.21 μM, respectively. In addition, NMM and BRACO19 inhibited SARS-CoV-2 infection at high concentrations. In particular, the IC ₅₀ of PDS is similar to positive controls such as cholorquine (9.63 μM), remdesivir (8.78 μM), and lopinavir (12.13 μM), indicating that PDS exhibits strong antiviral activity against SARS-CoV-2.

Example 9. Confirmation of antiviral efficacy of G4 binding compound against SARS-CoV-2 - related to virion production

A virion is an intact viral particle composed of a capsid and a nucleic acid, and the production of virions is related to the smoothness of viral replication. To confirm the antiviral efficacy of the compound, virion production was analyzed by quantifying the RNA genome of SARS-CoV-2 virus present in the supernatant.

As shown in FIG. 14 , PDS exhibited strong antiviral activity against virion production with IC ₅₀ values of 1.45 μM and 2.77 μM, respectively, at 24 and 48 hours after infection. PhenDC3 also inhibited SARS-CoV-2 virion production and IC ₅₀ values were estimated to be 24.47 μM and 23.17 μM at 24 and 48 hours post infection, respectively. This suggests that, as in Example 8, PDS and PhenDC3 are potent in inhibiting SARS-CoV-2 replication in Vero cells.

As shown in FIG. 15 , the IC ₅₀ of NMM was similar to that _of PhenDC3, and NMM was effective in inhibiting the virion production of SARS-CoV-2 in Vero cells.

Taken together, this means that the G4-binding compounds PDS, PhenDC3 and NMM have excellent inhibitory effects on SARS-CoV-2 replication by stabilizing the G4 structure in the SARS-CoV-2 transcript.

Example 10. Confirmation of antiviral efficacy of G4 binding compound against HCoV-OC43 virus-related to virion production

A virion is an intact viral particle composed of a capsid and a nucleic acid, and the production of virions is related to the smoothness of viral replication. In order to confirm the antiviral efficacy of the G4 binding compound, virion production was analyzed by quantifying the infectious virus present in the supernatant using a plaque assay.

As shown in FIG. 16 , NMM exhibited strong antiviral activity against virion production with an IC ₅₀ value of 2.10 μM after infection. The G4-binding compounds TMPyP2, TMPyP4 and CX3543 also inhibited virion production of HCoV-OC43 virus, and IC ₅₀ values were estimated to be 11.7 μM, 2.80 μM, and 1.40 μM, respectively, after infection. This suggests that CX3543, NMM, TMPyP4 and TMPyP2 are potent in inhibiting HCoV-OC43 virus replication in Vero cells.

This means that the G4-binding compounds NMM, TMPyP2, TMPyP4 and CX3543 have an excellent effect of inhibiting HCoV-OC43 virus replication by stabilizing the G4 structure of the HCoV-OC43 virus.

Therefore, it is expected that the compound binding to the G4 structure of the present invention can be used to treat coronavirus infections including SARS-CoV-2 and HCoV-OC43 viruses.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Pharmaceutical composition for preventing or treating Coronavirus infectious disease comprising Coronavirus G-quadruplex ligands as an active ingredient <130> MP20-182P1 <150> KR 10-2020-0120799 <151> 2020-09-18 <160> 44 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> G-353 <400> 1 ggcuuuggag acuccgugga ggagg 25 <210> 2 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> G-644 <400> 2 gguaauaaag gagcuggugg 20 <210> 3 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> G-1463 <400> 3 gguggucgca cuauugccuu uggagg 26 <210> 4 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> G-1574 <400> 4 gguguuguug gagaagguuc cgaagg 26 <210> 5 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> G-2714 <400> 5 ggcggugcac caacaaaggu uacuuuugg 29 <210> 6 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> G-3467 <400> 6 ggaggaggug uugcagg 17 <210> 7 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> G-4162 <400> 7 gguuauaccu acuaaaaagg cuggugg 27 <210> 8 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> G-4261 <400> 8 ggguuuaaau gguuacacug uagaggagg 29 <210> 9 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> G-8687 <400> 9 ggauacaagg cuauugaugg ugg 23 <210> 10 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> G-10267 <400> 10 ggcugguaau guucaacuca ggguuauugg 30 <210> 11 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> G-13385 <400> 11 gguaugugga aagguuaugg 20 <210> 12 <211> 28 <212> RNA <213> Artificial Sequence <220> <223> G-14947 <400> 12 gguuuuccau uuaauaaaug ggguaagg 28 <210> 13 <211> 27 <212> RNA <213> Artificial Sequence <220> <223> G-15208 <400> 13 ggaacaagca aauucuaugg ugguugg 27 <210> 14 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> G-15448 <400> 14 ggcgguucac uauauguuaa accaggugg 29 <210> 15 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> G-18296 <400> 15 ggauuggcuu cgaugucgag ggg 23 <210> 16 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> G-22316 <400> 16 ggugauucuu cuucagguug gacagcugg 29 <210> 17 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> G-24215 <400> 17 gguuggaccu uuggugcagg 20 <210> 18 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> G-24268 <400> 18 ggcuuauagg uuuaaugga uugg 24 <210> 19 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> G-25197 <400> 19 ggccauggua cauuuggcua gg 22 <210> 20 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> G-25951 <400> 20 gggguuaua cugaaaaaug ggaaucugg 29 <210> 21 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> G-26746 <400> 21 ggaucaccgg uggaauugcu aucgcaaugg 30 <210> 22 <211> 29 <212> RNA <213> Artificial Sequence <220> <223> G-28781 <400> 22 ggcuucuacg cagaagggag cagaggcgg 29 <210> 23 <211> 15 <212> RNA <213> Artificial Sequence <220> <223> G-28903 <400> 23 ggcuggcaau ggcgg 15 <210> 24 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> G-29123 <400> 24 ggaaauuuug gggaccagg 19 <210> 25 <211> 30 <212> RNA <213> Artificial Sequence <220> <223> G-29234 <400> 25 ggcugguaau guucaacuca ggguuauugg 30 <210> 26 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> WT G4-353 (Fwd) <400> 26 tatagctcga gatggagagc cttgtcc 27 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> WT G4-353 (Rev) <400> 27 tatacggatc cacatgacca tgaggtgc 28 <210> 28 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> WT G4-644 (Fwd) <400> 28 tatacctcga gatggttgag ctggtagc 28 <210> 29 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> WT G4-644 (Rev) <400> 29 tatacggatc cccgttaagc tcacgc 26 <210> 30 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> WT G4-3467 (Fwd) <400> 30 gataactcga gatggaactt acaccagttg ttcagac 37 <210> 31 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> WT G4-3467 (Rev) <400> 31 aatataggat ccggaatctc agcgatcttt tgttc 35 <210> 32 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Mut G4-353 (Fwd) <400> 32 cgcgacgtgc tcgtacgtga ctttgaagac tccgtagaag aagtc 45 <210> 33 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Mut G4-353 (Rev) <400> 33 gacttcttct acggagtctt caaagtcacg tacgagcacg tcgcg 45 <210> 34 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Mut G4-644 (Fwd) <400> 34 cggtaataaa agagctagta gccatagtta cggcgccg 38 <210> 35 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Mut G4-644 (Rev) <400> 35 cggcgccgta actatggcta ctagctcttt tattaccg 38 <210> 36 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Mut G4-3467 (Fwd) <400> 36 catggaagaa gtgttgcaag agccttaaat aaggctac 38 <210> 37 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Mut G4-3467 (Rev) <400> 37 gtagccttat ttaaggctct tgcaacactt cttccatg 38 <210> 38 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> T7-G4-353 (Fwd) <400> 38 agtaatacga ctcactatag ggatggagag ccttgtcc 38 <210> 39 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> T7-G4-644 (Fwd) <400> 39 agtaatacga ctcactatag ggatggttga gctggtagca g 41 <210> 40 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> T7-G4-3467 (Fwd) <400> 40 agtaatacga ctcactatag ggatggaact tacaccagtt gttc 44 <210> 41 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> T7-EYFP (Rev) <400> 41 ttacttgtac agctcgtcca tg 22 <210> 42 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> Mut-G-353 <400> 42 gacuuugaag acuccguaga agaag 25 <210> 43 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> Mut-G-644 <400> 43 gguaauaaaa gagcuaguag 20 <210> 44 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> Mut-G-3467 <400> 44 ggaagaagug uugcaag 17

Claims (14)

A pharmaceutical composition for preventing or treating coronavirus infection, comprising a compound binding to the G4 structure of coronavirus or a pharmaceutically acceptable salt thereof as an active ingredient.
According to claim 1,
The compound is a pharmaceutical composition for preventing or treating coronavirus infection, characterized in that at least one selected from the group consisting of compounds represented by the following Chemical Formulas 1 to 7.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

According to claim 1,
The coronavirus is human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63, New Haven coronavirus) , Human coronavirus HKU1, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2), characterized in that at least one selected from the group consisting of, for preventing or treating coronavirus infection pharmaceutical composition.
According to claim 1,
The compound is a pharmaceutical composition for preventing or treating coronavirus infection, characterized in that it stabilizes the G4 structure of the coronavirus.
According to claim 1,
The compound is characterized in that it inhibits the expression of one or more proteins selected from the group consisting of the coronavirus protein Nsp1 (non-structural protein 1), Nsp3 (non-structural protein 3) and nucleocapsid. Prevention or A therapeutic pharmaceutical composition.
According to claim 1,
The compound is characterized in that it inhibits the production of coronavirus virions, a pharmaceutical composition for preventing or treating coronavirus infection.
A food composition for preventing or improving coronavirus infection, comprising as an active ingredient a compound binding to the G4 structure of coronavirus or a pharmaceutically acceptable salt thereof.
8. The method of claim 7,
The compound is a food composition for preventing or improving coronavirus infection, characterized in that at least one selected from the group consisting of compounds represented by the following Chemical Formulas 1 to 7.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

A quasi-drug composition for preventing or inhibiting coronavirus infection, comprising as an active ingredient a compound that binds to the G4 structure of coronavirus.
10. The method of claim 9,
The compound is a quasi-drug composition for preventing or inhibiting coronavirus infection, characterized in that at least one selected from the group consisting of compounds represented by the following Chemical Formulas 1 to 7.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

10. The method of claim 9,
The quasi-drug composition is a quasi-drug composition for preventing or inhibiting coronavirus infection, characterized in that at least one selected from the group consisting of disinfectant cleaner, shower foam, gargrin, wet tissue, detergent soap, hand wash and ointment.
A composition for antiviral against coronavirus, comprising as an active ingredient a compound that binds to the G4 structure of coronavirus.
13. The method of claim 12,
The compound is an antiviral composition for coronavirus, characterized in that at least one selected from the group consisting of compounds represented by the following Chemical Formulas 1 to 7.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

13. The method of claim 12,
The coronavirus is human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63, New Haven coronavirus) , Human coronavirus HKU1, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2), characterized in that at least one selected from the group consisting of, antiviral for coronavirus composition.

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