Pharmaceutical composition for preventing or treating coronavirus infectious diseases comprising cell-permeable peptide-nucleic acid oligomer as active

Abstract

The present invention provides use of a PNA oligomer comprising modified PNA for the prevention or treatment of coronavirus infectious diseases. The PNA oligomer of the present invention can be delive

Description

Pharmaceutical composition for preventing or treating coronavirus infectious disease comprising a cell-penetrating peptide-nucleic acid oligomer as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating a coronavirus infection disease, which comprises a peptide-nucleic acid oligomer targeting the genomic positive(+) and / or negative(-) RNA of a coronavirus as an active ingredient.

[0002] Coronaviruses primarily cause enzootic infections in animals, including birds and mammals, but can also cross the animal-human species barrier and cause zoonotic infections in humans. Coronaviruses cause a variety of diseases, including respiratory, gastrointestinal, and central nervous system infections, in mammals, birds, and humans. Over the past 50 years, a variety of coronaviruses causing human and zoonotic diseases have emerged. Coronaviruses also cause various diseases in livestock, particularly those affecting agriculture. After acute respiratory infections were first reported in chickens raised as livestock in North America in the late 1920s, in 1961, Edward C. Kendall, Malcolm Bynoe, and David Tyrrell identified a coronavirus as a cause of the common cold in animals, naming it coronavirus B814. After many studies, it was first discovered in 1965 in the UK that coronaviruses were one of the causes of respiratory infections in humans, and in 1967, virologist June Almeida and physician David Tyrrell named them coronaviruses and published them in the journal Nature in 1968. Since the 1970s, they have been studied using betacoronavirus, murine hepatitis virus (MHV), and human alphacoronavirus HCoV-229E models for about 20 years. Human coronaviruses (HCoVs), HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1, generally cause mild upper respiratory tract infections, and collectively are associated with 10-30% of common cold cases.Coronaviruses, once known only as one of the causes of the common cold, have undergone several mutations since the outbreak of severe acute respiratory syndrome (SARS) in 2002, and have now developed into SARS-CoV-2, which causes severe acute respiratory syndrome type 2 (COVID-19). This causes new infectious diseases such as severe contagious pneumonia in humans.

[0003] Coronaviruses (CoVs) are spherical, enveloped RNA viruses, approximately 80–125 nm in diameter, with an envelope derived from the host cell's plasma membrane. The viral genome is protected within a helical capsid formed by the nucleocapsid protein (N), which is also surrounded by the envelope. The envelope protrudes from three viral structural proteins: the spike protein (S), the membrane protein (M), and the envelope protein (E), and contains the nucleocapsid protein within the membrane.

[0004] The coronavirus genome is a nonsegmented, single-stranded positive-sense RNA measuring 26–32 kilobases, making it the largest genome among all RNA viruses.

[0005] Meanwhile, antiviral drugs treat viral infections by weakening or eliminating the effects of viruses that have invaded the human body. Antiviral drugs are categorized into those that directly recognize and attack viral proteins, those that inhibit each stage of the viral life cycle to block viral replication, and those that enhance immunity.

[0006] Antisense oligonucleotides (ASOs) that specifically bind to the viral genome can inhibit the synthesis of viral proteins and suppress their proliferation. However, ASOs composed of DNA and / or RNA have low stability and are easily degraded by nucleases present in the body, which limits their therapeutic use. In addition, they require a delivery vehicle to be introduced into cells.

[0007] The present inventors have conducted extensive research to develop a therapeutic agent for coronavirus infectious diseases using a peptide-nucleic acid derivative that can move into cells without a carrier and has high stability, and have completed the present invention.

[0008] The technical problem to be achieved by the present invention is to provide a PNA oligomer including a modified PNA derivative for use in the prevention and / or treatment of coronavirus infectious diseases.

[0009] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0010] In order to solve the above problem, the present invention provides a pharmaceutical composition for preventing or treating coronavirus infection disease, which comprises a peptide nucleic acid (PNA) oligomer represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0011] [Chemical Formula 1]

[0012]

[0013] In the present invention, the PNA oligomer has a length of 10 to 30 mer and can complementarily bind to a portion of the genomic (+) RNA or genomic (-) RNA of a coronavirus.

[0014] Specifically, in the above chemical formula 1, n is an integer between 10 and 30;

[0015] X is one selected from the group consisting of hydrogen [H], formyl [HC(=O)-], aminocarbonyl [NH2-C(=O)-], substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkylacyl, substituted or unsubstituted arylacyl, substituted or unsubstituted alkyloxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted alkylsulfonyl, and substituted or unsubstituted arylsulfonyl;

[0016] Z is one selected from the group consisting of hydroxy[-OH], substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, amino[-NH2], substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino, substituted or unsubstituted alkyl, and substituted or unsubstituted aryl;

[0017] B1, B2, Bn-1, Bn-1 and Bn are each independently selected from a natural nucleotide and an unnatural nucleotide of adenine (A), guanine (G), thymine (T), or cytosine (C);

[0018] At least one of B1 to Bn is a modified PNA, and specifically, at least one of B1 to Bn is independently selected from the group consisting of modified PNAs of the following chemical formulae 2 to 4;

[0019] [Chemical Formula 2]

[0020]

[0021] [Chemical Formula 3]

[0022]

[0023] [Chemical Formula 4]

[0024]

[0025] In the above chemical formulas 2 to 4, j, k, l, and m are each independently an integer between 1 and 16.

[0026] Meanwhile, the pharmaceutical composition of the present invention does not include a carrier for intracellular delivery of the PNA oligomer.

[0027] As one embodiment of the present invention, in the chemical formula 1, X may be Fmoc [(9-fluorenyl)methyloxy]carbonyl], Fethoc [2-(9-fluorenyl)ethyl-1-oxy]carbonyl], acetyl (Ac), benzoyl, or hydrogen, and more specifically, may be Fethoc-.

[0028] As another embodiment of the present invention, in the chemical formula 1, X may be Fethoc, Z may be NH2, j may be 2, k may be 1, l may be 5, and m may be 6.

[0029] In another embodiment of the present invention, the coronavirus may be at least one selected from the group consisting of human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus type 2 (SARS-Cov-2).

[0030] The present invention produces a PNA oligomer complementary to HCoV-OC43 RNA of Table 1 below and confirms its virus proliferation inhibitory effect. As another embodiment of the present invention, the PNA oligomer may be at least one selected from the group consisting of PNA oligomers of Table 1, and the coronavirus may be HCoV-OC43, but the target virus is not limited thereto.

[0031] The present invention produces a PNA oligomer complementary to SARS-CoV-2 RNA of Table 2 below and confirms its virus proliferation inhibitory effect. As another embodiment of the present invention, the PNA oligomer may be at least one selected from the group consisting of PNA oligomers of Table 2, and the coronavirus may be SARS-CoV-2, but the target virus is not limited thereto.

[0032] Meanwhile, the present invention randomly selected a PNA oligomer from among the PNA oligomers in Table 1 and confirmed whether it could be applied to inhibiting SARS-CoV-2 virus proliferation, and as a result, it was confirmed that the selected PNA oligomer formed a complementary binding region containing some mismatch with the SARS-CoV-2 genome and inhibited the expression of the SARS-CoV-2 protein. As another embodiment of the present invention, the PNA oligomer may be complementary to 80% or more of the genomic (+) RNA or part of the genomic (-) RNA of the coronavirus.

[0033] In addition, the present invention provides a method for preventing or treating a coronavirus infection disease, comprising a step of administering the above-described PNA oligomer to a subject, and a method for inhibiting the proliferation of the coronavirus.

[0034] In one embodiment of the present invention, the subject may be a mammal infected with a coronavirus or at risk of exposure to the virus.

[0035] As another embodiment of the present invention, each of the above methods may further comprise a step of detecting a coronavirus in a biological sample isolated from the subject after administration of the PNA oligomer.

[0036] In addition, the present invention provides a method for inhibiting the proliferation of a coronavirus, comprising the step of treating a cell with the above-described PNA oligomer.

[0037] In one embodiment of the present invention, the cell may be a mammalian cell.

[0038] In another embodiment of the present invention, the cell may be a cell infected with a coronavirus.

[0039] As another embodiment of the present invention, the method may further comprise the step of lysing cells and detecting genetic material of the coronavirus after PNA oligomer treatment.

[0040] As another embodiment of the present invention, the method may separately prepare cells that are not treated with PNA oligomers as a control group to confirm the effect of PNA oligomer treatment on inhibiting coronavirus proliferation before PNA oligomer treatment, and the control group cells may be the same cell line as the cells treated with PNA oligomers.

[0041] In another embodiment of the present invention, the method may further comprise the step of lysing cells of the same cell line as the cells treated with the PNA oligomer and the cells not treated with the PNA and quantifying the level of genetic material of the coronavirus.

[0042] The present invention also provides the use of the above-described PNA oligomer for the manufacture of a medicament for the prevention or treatment of coronavirus.

[0043] The present invention provides a PNA oligomer comprising a modified PNA for use in the prevention or treatment of coronavirus infectious diseases. The PNA oligomer of the present invention can be delivered into virus-infected cells without a carrier to inhibit viral proliferation, and thus does not have the side effects caused by carriers for increasing cell permeability of conventional drugs. Furthermore, due to its high binding affinity with the target, it can bind to the target and inhibit viral proliferation even in the presence of a mismatch, and is expected to be used as an effective coronavirus therapeutic agent capable of responding to rapidly mutating viral infectious diseases.

[0044] Figures 1a to 1c are some of the chromatogram results confirming that the designed PNA oligomer was produced as intended. Specifically, Figure 1a is a C18-reverse-phase UPLC chromatogram result of PNA123 before HPLC purification, Figure 1b is a C18-reverse-phase UPLC chromatogram result of PNA123 after HPLC purification, and Figure 1c is an ESI-TOF mass spectrum result of PNA123 after C18-RP prep HPLC.

[0045] Figure 2 shows the results of the titer analysis of Paxlovid in the SARS-CoV-2 virus.

[0046] Figures 3a to 3i and Figures 4a to 4k show the results of potency analysis of PNA oligomers and their combination drugs in SARS-CoV-2 virus.

[0047] Figure 5 shows the results of a Western blot confirming the effect of inhibiting SARS-CoV-2 virus replication using a PNA oligomer complementary to a portion of HCoV-OC43 RNA.

[0048] Coronaviruses are single-stranded RNA viruses that synthesize genomic positive (+) RNA using RNA polymerase to synthesize genomic negative (-) RNA, producing double-stranded RNA. The present inventors attempted to block its replication process using oligonucleotides targeting the coronavirus genome.

[0049] However, oligonucleotides composed of DNA and / or RNA have low stability and are easily degraded by nucleases present in the body, limiting their use in treating viral infections. To overcome this, various types of non-natural oligonucleotides have been developed, and in previous research, the present inventors developed a novel peptide nucleic acid (PNA).

[0050] PNA is a polypeptide with N-(2-aminoethyl)glycine as its backbone, and was invented by Nielsen et al. PNA can bind to complementary nucleic acids, just like DNA or RNA, and the binding affinity of RNA-PNA is stronger than that of RNA-DNA or RNA-RNA. Furthermore, because PNA is structurally very different from DNA, it is not recognized by hepatic transporters that recognize DNA.

[0051] PNA monomer is It has a structure, and in the PNA monomer structure, B is a nucleobase, and the nucleobase may be a natural or non-natural nucleobase, and examples of natural or non-natural nucleobase in the present specification are as follows.

[0052]

[0053]

[0054] In a previous study, the present inventors developed a PNA using a "modified nucleobase" covalently linked to a cationic lipid, and confirmed that the PNA containing the modified nucleobase exhibited high cell membrane permeability, thereby developing a PNA with enhanced cell permeability (PCT / KR2009 / 001256). However, no studies have been conducted on whether the modified PNA can be used to treat viral infections.

[0055] The modified nucleic acid bases used in the present invention to produce PNA with increased cell penetration ability are as follows.

[0056]

[0057]

[0058]

[0059] The above modified nucleotide belongs to the non-natural nucleotide base, and in this specification, the term “modified nucleotide base” may be used interchangeably with the term “non-natural nucleotide base” unless it is necessary to distinguish it from non-natural nucleotides other than the modified nucleotide base. In addition, in this specification, when it is necessary to distinguish between PNA including a modified nucleotide base and PNA not including a modified nucleotide base, the PNA including the modified nucleotide base is called a “modified PNA.”

[0060] The present inventors produced a PNA oligomer of the following chemical formula 1 in which the above-described PNA monomers are covalently linked.

[0061] [Chemical Formula 1]

[0062]

[0063] The PNA oligomer of the above chemical formula 1 was designed to be able to complementarily bind to a portion of the coronavirus genomic RNA of the coronavirus, and the complementary binding region was set to be 10 mer or more.

[0064] Accordingly, in the above chemical formula 1, n is an integer between 10 and 30. This literally means that n is an integer selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30.

[0065] As used herein, the term "complementary binding region" refers to a continuous region including the beginning and end of the bases where the coronavirus genomic RNA and the PNA oligomer form base pairs. Meanwhile, modified PNAs have high binding affinity to the target gene, and even if there is a mismatch in the complementary binding region, the PNA can bind to the target and inhibit viral replication. Therefore, as used herein, "complementary binding" includes complete complementary binding and partial complementary binding with a mismatch of 20% or less, within the range where the PNA derivative can maintain binding affinity to the target RNA. For example, when n=10 in the above chemical formula 1, the PNA oligomer of the above chemical formula 1 may include one or two mismatches in the complementary binding region with a portion of the coronavirus genome RNA, when n=20, the PNA oligomer of the above chemical formula 1 may include one or four mismatches in the complementary binding region with a portion of the coronavirus genome RNA, and when n=30, the PNA oligomer of the above chemical formula 1 may include one or six mismatches in the complementary binding region with a portion of the coronavirus genome RNA. In this case, the mismatches may be continuous and / or discontinuous.

[0066] In the above chemical formula 1, X is one selected from the group consisting of hydrogen [H], formyl [HC(=O)-], aminocarbonyl [NH2-C(=O)-], substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkylacyl, substituted or unsubstituted arylacyl, substituted or unsubstituted alkyloxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted alkylsulfonyl, and substituted or unsubstituted arylsulfonyl.

[0067] In the above chemical formula 1, Z is one selected from the group consisting of hydroxy [-OH], substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, amino [-NH2], substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino, substituted or unsubstituted alkyl, and substituted or unsubstituted aryl.

[0068] Substituents for the description of the PNA derivative of the above chemical formula 1 are described below.

[0069] Examples of the substituted or unsubstituted alkyl group are as follows.

[0070]

[0071]

[0072] Examples of the above substituted or unsubstituted alkyl acyls are as follows.

[0073]

[0074]

[0075] Examples of the above substituted or unsubstituted arylacyl are as follows.

[0076]

[0077] Examples of the substituted or unsubstituted alkylamino and substituted or unsubstituted arylamino are as follows.

[0078]

[0079] Examples of the above substituted or unsubstituted aryl are as follows.

[0080]

[0081] The above substituted or unsubstituted alkylsulfonyl and substituted or unsubstituted arylsulfonyl are as follows.

[0082]

[0083] The substituted or unsubstituted alkylphosphonyl and substituted or unsubstituted arylphosphonyl are as follows.

[0084]

[0085] The above substituted or unsubstituted alkyloxycarbonyl is as follows.

[0086]

[0087] The above substituted or unsubstituted aryloxycarbonyl is as follows.

[0088]

[0089] The above substituted or unsubstituted alkylaminocarbonyl is as follows.

[0090]

[0091] The above substituted or unsubstituted arylaminocarbonyl is as follows.

[0092]

[0093] Additionally, the N- and C-terminals of the above chemical formula 1 may include the following substituents.

[0094]

[0095] Abbreviations for the substituents at the N-terminus and C-terminus are specifically described as follows: "Fmoc-" is an abbreviation for "[(9-fluorenyl)methyloxy]carbonyl]-"; "Fethoc-" is "[2-(9-fluorenyl)ethyl-1-oxy]carbonyl"; "Ac-" is "acetyl-"; "benzoyl-" is "benzenecarbonyl-"; "Piv-" is "pivalyl-"; "Me-" is "methyl-"; "n-Propyl-" is 1-(n-propyl)-"; "H-" is a "hydrido-" group; "p-Toluenesulfonyl" is "(4-methylbenzene)-1-sulfonyl-"; "-Lys-" is the amino acid residue "lysine"; "-Val-" is the amino acid residue "valine"; "-Leu-" is the amino acid residue "leucine"; "-Arg-" is the amino acid residue "arginine"; "-Gly-" is the amino acid residue "glycine"; "[N-(2-Phenylethyl)amino]carbonyl-" is "[N-1-(2-phenylethyl)amino]carbonyl-"; "Benzyl- " is "1-(phenyl)methyl-"; "Phenyl-" is "phenyl-"; "Me-" is "methyl-"; "-HEX-" is "6-amino-1-hexanoyl-", "FAM-" stands for "5, or 6-fluoresceincarbonyl- (mixture of isomers)"; "-NH2" stands for unsubstituted "-amino" group.

[0096] The examples of substituents at the N-terminus and C-terminus of the compound of formula 1 in the present invention are merely intended to illustrate the diversity of permissible substituents and do not limit the scope of the present invention. In particular, the sequence of the PNA oligomer may be more important than the substituents at the N- and / or C-terminus of the PNA oligomer in binding to the target RNA.

[0097] In addition, in the above chemical formula 1, B1, B2, … … , B n-1 and B nare each independently selected from natural and unnatural nucleotide bases of Adenine (A), Guanine (G), Thymine (T), or Cytosine (C), and are B1 to B n At least one of which contained a modified nucleic acid base.

[0098] The present inventors produced a 20-mer PNA oligomer complementary to the target region based on the HCoV-OC43 RNA sequence provided by NCBI (NCBI Reference Sequence: NC_006213) (Table 1).

[0099] PNA 서리고머PNA 이지 (N → C)타경 에서PNA 1Fethoc-GCA(5)-CGC-AA(5)A-TCG(6)-CTC-ACA(5)-AT-NH25' UTRPNA 2Fethoc-TGA-A(5)GC-GGG(6)-ATG-CA(5)C-GCA(5)-CG-NH25' UTRPNA 3Fethoc-TCA-GTG-AA(5)G-CGG(6)-GAT-GCA(5)-CGC-A(5)CG-NH25' UTRPNA 4Fethoc-TCA(5)-GTG-AA(5)G-CGG(6)-GAT-GCA(5)-CGC-A(5)CG-NH25' UTRPNA 5Fethoc-CTA(5)-ACA-A(5)GA-GAT-CA(5)G-TGA-A(5)G-NH25' UTRPNA 6Fethoc-AGA(5)-TCT-A(5)AC-AA(5)G-AGA-TCA(5)-GTG-AA(5)G-NH25' UTRPNA 7Fethoc-C(1O2)TA-ACA-A(5)GA-GAT-CA(5)G-TGA-A(5)G-NH25' UTRPNA 8Fethoc-CTA(5)-ACA-A(5)GA-GAT-CA(5)G-TGA-AG(6)-NH25' UTRPNA 9Fethoc-AGA(5)-TTA-CAA(5)-AAA-GA(5)T-CTA-A(5)C-NH25' UTRPNA 10Fethoc-GTT-TA(5)G-ATT-A(5)CA-AA(5)A-AGA(5)-TCT-AA(5)C-NH25' UTRPNA 11Fethoc-GTT-TA(5)G-ATT-A(5)CA-AA(5)A-AGA(5)-TC-NH25' UTRPNA 12Fethoc-GTT-TA(5)G-ATT-A(5)CA-AA(5)A-AGA-TC(1O2)-NH25' UTRPNA 13Fethoc-G(6)TT-TAG-A(5)TT-ACA-AA(5)A-AGA(5)-TC-NH25' UTRPNA 14Fethoc-GTT-TA(5)G-ATT-A(5)CA-AA(5)A-AGA-TC-NH25' UTRPNA 15Fethoc-G(6)TT-TTT-A(5)TA-AA(5)G-TTT-A(5)GA-TT-NH25' UTRPNA16Fethoc-TTA(5)-CA(5)G-GGA(5)-GTG-GA(5)T-GTT-TT-NH25’ UTRPNA 17Fethoc-G(6)CC-CAC-A(5)AG-CAT-A(5)GA-TTA(5)-CA-NH25’ UTRPNA 18Fethoc-A(5)CA-TC(1O2)T-G(6)AA-TC(1O2)A-A(5)-NH2Nsp3PNA 19Fethoc-TTG-A(5)TT-C(1O2)AG-A(5)TG(6)-T-NH2Nsp3PNA 20Fethoc-AG(6)T-TA(5)T-TA(5)C-A(5)AC(1O2)-T-NH2Nsp3PNA 21Fethoc-AG(6)T-TG(6)T-AA(5)T-AA(5)C-T-NH2Nsp3PNA 22Fethoc-TTT-TTA(5)-AAC-G(6)GG-TTC-G(6)GG-G(6)T-NH2Nsp12PNA 23Fethoc-A(5)CC-CCG-A(5)AC-CCG(6)-TTT-AAA(5)-AA-NH2Nsp12PNA 24Fethoc-A(5)CC-CCG-A(5)AC-CCG(6)-TTT-A(5)AA-A(5)A-NH2Nsp12PNA 25Fethoc-A(5)CC-C(1O2)CG-AA(5)C-CCG(6)-TTT-A(5)AA-A(5)A-NH2Nsp12PNA 26Fethoc-TTT-TTA(5)-AAC-G(6)GG-TTC-G(6)GG-G(6)TA-CGA-NH2Nsp12PNA 27Fethoc-TCG-TA(5)C-CCC-GA(5)A-CCC-G(6)TT-TAA-A(5)AA-NH2Nsp12PNA 28Fethoc-TCG-TA(5)C-CC(1O2)C-GAA(5)-CCC-G(6)TT-TA(5)A-AA(5)A-NH2Nsp12PNA 29Fethoc-TTT-TTA(5)-AAC(1O2)-GGG(6)-TTC-G(6)GG-G(6)T-NH2Nsp12PNA 30Fethoc-TTT-TTA(5)-AAC-G(6)GG-TTC(1O2)-GGG-G(6)T-NH2Nsp12PNA 31Fethoc-A(5)AC(1O2)-CCG(6)-TTT-A(5)AA-A(5)A-NH2Nsp12PNA32Fethoc-TTT-TTA(5)-AA(5)C-G(6)GG(6)-TT-NH2Nsp12PNA 33Fethoc-TTA(5)-AAC-G(6)GG-TTC-G(6)GG-G(6)T-NH2Nsp12PNA 34Fethoc-A(5)TA-G(6)CA-GC(1O2)A-TTA(5)-CCA(5)-TCC(1O2)-TG-NH2Nsp12PNA 35Fethoc-C(1O2)AG-G(6)AT-G(6)GT-A(5)AT-GC(1O2)T-GCT-A(5)T-NH2Nsp12PNA 36Fethoc-G(6)GA-TA(5)A-TC(1O2)C-CA(5)A-CC(1O2)C-ATA(5)-AG-NH2Nsp12PNA 37Fethoc-C(1O2)TT-A(5)TG-G(6)GT-TG(6)G-GA(5)T-TAT-C(1O2)C-NH2Nsp12PNA 38Fethoc-G(6)TA-A(5)TA-G(6)CA-A(5)CA(5)-TT-NH2Nsp14PNA 39Fethoc-A(5)AT-G(6)TT-G(6)CT-A(5)TT-A(5)C-NH2Nsp14PNA 40Fethoc-CA(5)C-TC(1O2)T-AG(6)T-G(6)TC-AA(5)A-TC(1O2)T-AC-NH2Nsp14PNA 41Fethoc-G(6)TA-GA(5)T-TTG(6)-AC(1O2)A-CTA(5)-GAG(6)-TG-NH2Nsp14PNA 42Fethoc-G(6)TG-TG(6)G-AA(5)T-GC(1O2)A-TG(6)T-TTA(5)-TT-NH2Nsp15PNA 43Fethoc-A(5)AT-A(5)AA-C(1O2)AT-G(6)CA-TTC(1O2)-CAC-A(5)C-NH2Nsp15PNA 44Fethoc-ATC-A(5)CA-AGA(5)-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 45Fethoc-ATC-A(5)CA-AGA(5)-TGC-AA(5)T-TTA(5)-GGT-G(6)GT-GC-NH2Nsp15PNA 46Fethoc-CA(5)C-AAG-A(5)TG-C(1O2)AA(5)-TTT-A(5)GG(6)-TGG(6)-TGC-NH2Nsp15PNA47Fethoc-CAA-GA(5)T-GC(1O2)A-A(5)TT-TA(5)G-G(6)TG-G(6)TG-C-NH2Nsp15PNA 48Fethoc-AGA(5)-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 49Fethoc-AGA-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 50Fethoc-AGA-TGC(1O2)-AA(5)T-TTA(5)-GGT-GG(6)T-GC-NH2Nsp15PNA 51Fethoc-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GCA(5)-GT-NH2Nsp15PNA 52Fethoc-TGC(1O2)-AA(5)T-TTA(5)-GG(6)-NH2Nsp15PNA 53Fethoc-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 54Fethoc-TGC-AAT-TTA-GGT-GGT-GC-NH2Nsp15PNA 55Fethoc-TGC-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 56Fethoc-TG(6)C-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 57Fethoc-TG(6)C-AA(5)T-TTA-G(6)GT-G(6)GT-G(6)C-NH2Nsp15PNA 58Fethoc-TGC-AA(5)T-TTA-GGT-GGT-GC-NH2Nsp15PNA 59Fethoc-TGC-AAT-TTA(5)-GGT-GGT-GC-NH2Nsp15PNA 60Fethoc-TGC-AA(5)T-TTA(5)-GGT-GGT-GC-NH2Nsp15PNA 61Acetyl-TGC-AAT-TTA-GGT-GGT-GC-NH2Nsp15PNA 62Acetyl-TGC-AA(5)T-TTA-GGT-GGT-GC-NH2Nsp15PNA 63Acetyl-TGC-AAT-TTA(5)-GGT-GGT-GC-NH2Nsp15PNA 64Acetyl-TGC-AA(5)T-TTA(5)-GGT-GGT-GC-NH2Nsp15PNA65Acetyl-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 66H-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 67Benzoyl-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 68Fethoc-TGC(1O2)-AA(5)T-TTA(5)-GGT-GG(6)T-GC-NH2Nsp15PNA 69Fethoc-GCA(5)-CCA(5)-CC(1O2)T-AAA(5)-TTG(6)-CA-NH2Nsp15PNA 70Fethoc-GCA-CCA(5)-CCT-A(5)AA-TTG-CA-NH2Nsp15PNA 71Fethoc-GCA(5)-CCA(5)-CCT-A(5)AA-TTG-CA-NH2Nsp15PNA 72Fethoc-C(1O2)AA(5)-TTT-A(5)GG(6)-TGG(6)-TG-NH2Nsp15PNA 73Fethoc-TTA(5)-CAC(1O2)-CAA(5)-AGC(1O2)-ATA(5)-AA-NH2Nsp16PNA 74Fethoc-TTT-A(5)TG-C(1O2)TT-TG(6)G-TG(6)T-AA(5)-NH2Nsp16

[0100] In addition, the inventors of the present invention produced a 20-mer PNA oligomer complementary to the target region based on the SARS-CoV-2 RNA sequence provided by NCBI (NCBI Reference Sequence: NC_045512) (Table 2).

[0101] PNA 올리고머PNA 서열 (N → C)타겟 영역PNA 75Fethoc-GG(6)T-ATA(5)-AAC(1O2)-CTT-TA(5)A-T-NH25’ UTRPNA 76Fethoc-GGA(5)-AGG-TA(5)T-AAA(5)-CCT-TTA(5)-AT-NH25’ UTRPNA 77Fethoc-TG(6)T-TAC(1O2)-CTG-G(6)GA-A(5)GG-T-NH25’ UTRPNA 78Fethoc-A(5)CC-TTC(1O2)-CCA-G(6)GT-AA(5)C-A-NH25’ UTRPNA 79Fethoc-TTG-G(6)TT-TG(6)T-TAC-CTG(6)-GGA-A(5)G-NH25’ UTRPNA 80Benzoyl-TTG-G(6)TT-TG(6)T-TAC-CTG(6)-GGA-A(5)G-NH25’ UTRPNA 81Fethoc-TTG-G(6)TT-GG(6)T-TTG-TTA(5)-CCT-G(6)G-NH25’ UTRPNA 82Fethoc-CG(6)A-AA(5)G-TTG(6)-GTT-G(6)GT-T-NH25’ UTRPNA 83Fethoc-AA(5)C-CAA(5)-CCA-A(5)CT-TTC-G(6)-NH25’ UTRPNA 84Fethoc-CTA(5)-CAA-GA(5)G-ATC-GA(5)A-AGT-TG(6)-NH25’ UTRPNA 85Fethoc-ACA(5)-GAT-CTA(5)-CAA-GA(5)G-ATC-GA(5)-NH25’ UTRPNA 86Fethoc-ACA-GA(5)T-CTA-CAA-GA(5)G-ATC-GA-NH25’ UTRPNA 87Fethoc-CTA(5)-CAA(5)-GAG-A(5)TC-G-NH25’ UTRPNA 88Fethoc-CA(5)G-ATC(1O2)-TAC-A(5)AG-AGA(5)-T-NH25’ UTRPNA 89Fethoc-ATC(1O2)-TCT-TG(6)T-AGA(5)-TCT-G(6)-NH25’ UTRPNA 90Fethoc-G(6)TT-CG(6)T-TTA(5)-GAG(6)-AAC(1O2)-AGA(5)-TC-NH25’ UTRPNA91Fethoc-GTT-CG(6)T-TTA(5)-GAG-AA(5)C-AGA(5)-TC-NH25’ UTRPNA 92Fethoc-GTT-C(1O2)GT-TTA(5)-GAG(6)-AAC-A(5)-NH25’ UTRPNA 93Fethoc-TG(6)T-TC(1O2)T-CTA-A(5)AC-GAA(5)-C-NH25’ UTRPNA 94Fethoc-GTT-C(1O2)GT-TTA(5)-GAG(6)-AA(5)-NH25’ UTRPNA 95Fethoc-GTT-C(1O2)GT-TTA(5)-GAG(6)-NH25’ UTRPNA 96Fethoc-GTG(6)-ACA(5)-GCC-A(5)CA-CAG(6)-A-NH25’ UTRPNA 97Fethoc-CA(5)G-TGG-CA(5)A-GAT-AA(5)C-A-NH2Nsp3PNA 98Fethoc-CA(5)G-TGG(6)-CAA-G(6)AT-AA(5)C-A-NH2Nsp3PNA 99Fethoc-CA(5)G-TGG-CA(5)A-GAT-A(5)-NH2Nsp3PNA 100Fethoc-TA(5)T-ATG(6)-TTT-A(5)TA-GTG(6)-ACC-A(5)C-NH2Nsp3PNA 101Fethoc-TA(5)T-ATG-TTT-A(5)TA-GTG-ACC-A(5)C-NH2Nsp3PNA 102Fethoc-TAT-A(5)TG-TTT-ATA-GTG-A(5)CC-AC-NH2Nsp3PNA 103Fethoc-A(5)TG-TTT-A(5)TA-GTG-A(5)-NH2Nsp3PNA 104Fethoc-TA(5)C-ATT-C(1O2)TA-A(5)CC-A(5)TA-NH2Nsp3PNA 105Fethoc-C(1O2)TC-TA(5)T-TA(5)C-G(6)TT-T-NH2Nsp3PNA 106Fethoc-CTC-TA(5)T-TA(5)C-GTT-T-NH2Nsp3PNA 107Fethoc-CTT-G(6)TT-GC(1O2)T-CTA(5)-TTA-CG(6)T-TT-NH2Nsp3PNA 108Fethoc-TTA(5)-CCT-C(1O2)CA(5)-TTA(5)-G-NH2Nsp3PNA109Fethoc-GC(1O2)A-GCA(5)-CTA(5)-CGT-A(5)-NH2Nsp3PNA 110Fethoc-TA(5)A-AA(5)A-CTC(1O2)-TAG(6)-GTA(5)-A-NH2Nsp4PNA 111Fethoc-ATC-A(5)CA-TG(6)T-CTT-G(6)GA-CA(5)G-TA-NH2Nsp5PNA 112Fethoc-CAT-G(6)TC-TTG(6)-GAC-A(5)-NH2Nsp5PNA 113Fethoc-CTA(5)-CCA(5)-CA(5)T-GAA(5)-CC-NH2Nsp5PNA 114Fethoc-CCA(5)-ACA-CTA(5)-CCA-CA(5)T-GAA(5)-CC-NH2Nsp5PNA 115Fethoc-CCA-ACA(5)-CTA-CCA-CA(5)T-GAA-CC-NH2Nsp5PNA 116Fethoc-TA(5)C-AA(5)C-CA(5)A-GC(1O2)T-A-NH2Nsp5PNA 117Fethoc-CA(5)A-AC(1O2)C-CG(6)T-TTA(5)-AAA(5)-AC-NH2Nsp12PNA 118Fethoc-CA(5)A-AA(5)C-CA(5)T-TTC(1O2)-AAG(6)-AC-NH2Nsp12PNA 119Fethoc-TTT-TTA(5)-AAC-G(6)GG-TTT-G(6)CG-G(6)T-NH2Nsp12PNA 120Fethoc-A(5)CC-GCA-A(5)AC-CCG(6)-TTT-AAA(5)-AA-NH2Nsp12PNA 121Fethoc-A(5)CC-GC(1O2)A-AA(5)C-CCG(6)-TTT-AAA(5)-AA-NH2Nsp12PNA 122Benzoyl-A(5)CC-GCA-A(5)AC-CCG(6)-TTT-AAA(5)-AA-NH2Nsp12PNA 123Fethoc-A(5)GC-C(1O2)CT-G(6)TA-TA(5)C-GA(5)C-ATC(1O2)-AG-NH2Nsp12PNA 124Fethoc-A(5)GC-C(1O2)CT-A(5)TA-TA(5)C-GA(5)C-ATC(1O2)-AG-NH2Nsp12PNA125Fethoc-A(5)GC-A(5)CT-A(5)TA-TG(6)C-GC(1O2)C-ATC(1O2)-AG-NH2Nsp12PNA 126Benzoyl-A(5)GC-C(1O2)CT-G(6)TA-TA(5)C-GA(5)C-ATC(1O2)-AG-NH2Nsp12PNA 127Fethoc-GA(5)A-TA(5)A-TA(5)A-GA(5)A-TC(1O2)T-A-NH2Nsp12PNA 128Fethoc-TTA(5)-AAA(5)-CTA-AG(6)T-CTA-G(6)AG-CT-NH2Nsp12PNA 129Fethoc-A(5)GC-TCT-A(5)GA-CTT-A(5)GT-TTT-A(5)A-NH2Nsp12PNA 130Fethoc-TA(5)A-AA(5)C-TA(5)A-GTC(1O2)-TAG-A(5)-NH2Nsp12PNA 131Fethoc-TC(1O2)T-A(5)GA(5)-CTT-AG(6)T-TTT-A(5)-NH2Nsp12PNA 132Fethoc-TA(5)A-AA(5)C-TAA(5)-GTC-T-NH2Nsp12PNA 133Fethoc-A(5)GA-CTT-A(5)GT-TTT-A(5)-NH2Nsp12PNA 35 aFethoc-C(1O2)AG-G(6)AT-G(6)GT-A(5)AT-GC(1O2)T-GCT-A(5)T-NH2Nsp12PNA 134Fethoc-A(5)GC-ATT-A(5)CC-A(5)TC-C-NH2Nsp12PNA 135Fethoc-GGA(5)-TGG-TA(5)A-TG(6)C-T-NH2Nsp12PNA 136Fethoc-TCA(5)-TTG(6)-AAT-C(1O2)AT-AA(5)T-A-NH2Nsp12PNA 137Fethoc-CA(5)C-TCA(5)-ATA(5)-CTT-G(6)AG-C(1O2)AC-A(5)C-NH2Nsp12PNA 138Fethoc-CCA(5)-CC(1O2)T-GG(6)T-TTA(5)-ACA(5)-TAT-A(5)G-NH2Nsp12PNA 139Fethoc-TTA(5)-GCA-TA(5)A-GC(1O2)A-G(6)TT-G(6)TG-G(6)C-NH2Nsp12PNA 140Fethoc-TAA-AG(6)A-AC(1O2)T-GA(5)C-TTA(5)-A-NH2Nsp12PNA 141Fethoc-TTA(5)-AGT-CA(5)G-TTC(1O2)-TTT-A(5)-NH2Nsp12PNA 142Fethoc-ATA(5)-ATA-A(5)AG-AA(5)C-TGA(5)-CTT-A(5)A-NH2Nsp12PNA 143Fethoc-TTA(5)-AGT-CA(5)G-TTC(1O2)-TTT-A(5)TT-AT-NH2Nsp12PNA 144Fethoc-ATA-ATA-A(5)AG-AAC-TGA(5)-CTT-AA-NH2Nsp12PNA 145Fethoc-TTA-AGT-CA(5)G-TTC-TTT-A(5)TT-AT-NH2Nsp12PNA 146Fethoc-TAA(5)-AGA-A(5)CT-GA(5)C-T-NH2Nsp12PNA 147Fethoc-A(5)GT-CA(5)G-TTC(1O2)-TTT-A(5)-NH2Nsp12PNA 148Fethoc-TA(5)C-TAC-A(5)AT-C(1O2)TT-TAA(5)-A-NH2Nsp14PNA149Fethoc-GA(5)C-ATT-G(6)GT-AA(5)C-CCT-A(5)AA-G(6)C-NH2Nsp14PNA 150Fethoc-A(5)TT-GGT-A(5)AC-CCT-A(5)AA-GCT-A(5)T-NH2Nsp14PNA 151Fethoc-ATT-GGT-A(5)AC-CCT-AAA(5)-GCT-AT-NH2Nsp14PNA 152Fethoc-GTA(5)-ACC-CTA(5)-AAG-C-NH2Nsp14PNA 153Fethoc-G(6)TA-A(5)CC-CTA(5)-AAG(6)-C-NH2Nsp14PNA 154Fethoc-CG(6)T-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp14PNA 53 aFethoc-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp14PNA 155Fethoc-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GCT-G(6)T-NH2Nsp14PNA 156Fethoc-TGC-A(5)AT-TTA(5)-GGT-G(6)GT-G(6)CT-GT-NH2Nsp14PNA 157Fethoc-TGC(1O2)-AA(6)T-TTA(6)-GG(5)T-GG(5)T-GC-NH2Nsp14PNA 158Fethoc-TG(6)C(1O2)-A(5)A(5)T-TTA-GGT-GGT-GC-NH2Nsp14PNA 159Fethoc-TGC-AAT-TTA(5)-G(6)G(6)T-G(6)GT-GC-NH2Nsp14PNA 160Fethoc-TGC-AAT-TTA-GGT-G(6)G(6)T-G(6)C(1O2)-NH2Nsp14PNA 161Fethoc-GCA(5)-ATT-TA(5)G-GTG-G-NH2Nsp14PNA 162Fethoc-GTC(1O2)-ATA(5)-GAA(5)-CAA(5)-A-NH2Nsp15PNA 163Fethoc-GCA(5)-AAA-TA(5)T-ACT-CA(5)A-CTG(6)-TG-NH2Nsp16PNA 164Fethoc-GCA-AA(5)A-TAT-ACT-CAA(5)-CTG-TG-NH2Nsp16PNA 165Fethoc-CA(5)A-AAT-A(5)TA-CTC-A(5)-NH2Nsp16PNA 166Fethoc-GA(5)A-CCA(5)-GCA-C(1O2)CA-A(5)A-NH2Nsp16PNA 167Fethoc-A(5)TT-ATT-A(5)GT-GA(5)T-ATG-TA(5)C-GA-NH2Nsp16PNA 168Fethoc-ATT-ATT-A(5)GT-GAT-ATG-TA(5)C-GA-NH2Nsp16PNA 169Fethoc-GTG-A(5)TA-TGT-A(5)CG-A(5)-NH2Nsp16PNA 170Fethoc-TA(5)T-AAA(5)-GAT-A(5)AC-AGA(5)-ACA(5)-TT-NH2Nsp16PNA171Fethoc-TA(5)T-AAA(5)-GAT-A(5)AC-AGA(5)-ACA-TT-NH2Nsp16PNA 172Fethoc-TAT-AAA(5)-GAT-AAC-AGA(5)-ACA-TT-NH2Nsp16PNA 173Fethoc-AA(5)G-ATA(5)-ACA-GA(5)A-C-NH2Nsp16PNA 174Fethoc-TCA(5)-TCA(5)-TCT-GA(5)A-GCA(5)-TTT-TT-NH2Nsp16PNA 175Fethoc-TCA-TCA(5)-TCT-GAA-GCA(5)-TTT-TT-NH2Nsp16PNA 176Fethoc-CA(5)T-CTG-A(5)AG-CA(5)T-T-NH2Nsp16PNA 177Fethoc-ACA(5)-AAC-A(5)TT-G(6)TT-C(1O2)GT-T-NH2Nsp16-S proteinPNA 178Fethoc-CTG(6)-CAC(1O2)-CAA(5)-GTG(6)-ACA(5)-TAG(6)-TG-NH2S proteinPNA 179Fethoc-TG(6)T-GTA(5)-CAA(5)-AAA(5)-CT-NH2S proteinPNA 180Fethoc-GA(5)A-AA(5)A-TTA(5)-AAA(5)-CC-NH2S proteinPNA 181Fethoc-AA(5)C-CTA(5)-TCA-A(5)TT-TG(6)-NH2S proteinPNA 182Fethoc-CTA(5)-ATT-A(5)AT-TG(6)T-TG(6)-NH2S proteinPNA 183Fethoc-CTG(6)-CAG(6)-CTC(1O2)-TAA(5)-TTA-A(5)-NH2S proteinPNA 184Fethoc-CTG(6)-CAG(6)-CTC(1O2)-TAA(5)-NH2S proteinPNA 185Fethoc-CTG(6)-CAG(6)-CTC(1O2)-TAA(5)-TT-NH2ORF3aPNA 186Fethoc-TG(6)T-GC(1O2)T-TA(5)C-AA(5)A-GG(6)C-AC(1O2)G-CT-NH2ORF3aPNA187Fethoc-TG(6)A-GTA(5)-CA(5)T-AA(5)G-TTC(1O2)-GTA(5)-CT-NH2E proteinPNA 188Fethoc-TA(5)C-ATA(5)-AGT-TC(1O2)G-TA(5)-NH2E proteinPNA 189Fethoc-ACA(5)-ATC-G(6)AA-G(6)CG-CA(5)G-TA(5)A-G(6)G-NH2E proteinPNA 190Fethoc-CA(5)A-GA(5)C-TC(1O2)A-CG(6)T-TA(5)A-CA(5)A-TA-NH2E proteinPNA 191Fethoc-A(5)GA-A(5)TT-CA(5)G-A(5)TT-TTT-A(5)AC-A(5)C-NH2E proteinPNA 192Fethoc-TA(5)T-TTA(5)-GTT-C(1O2)GT-TTA(5)-G-NH2E proteinPNA 193Fethoc-CTT-A(5)CT-G(6)TA(5)-CAA(5)-G-NH2M proteinPNA 194Fethoc-AG(6)T-TTG(6)-TTC(1O2)-GTT-TA(5)-NH2ORF8PNA 195Fethoc-TCA(5)-TTT-TA(5)C-CG(6)T-CA(5)C-CA(5)C-CA(5)-NH2N proteinPNA 196Fethoc-GTG(6)-CAA(5)-TTT-G(6)CG-G(6)CC-A(5)AT-G(6)T-NH2N proteinPNA 197Fethoc-TG(6)T-CA(5)T-TCT-C(1O2)CT-AA(5)G-A-NH23’ UTRPNA 198Fethoc-TC(1O2)T-TAG(6)-GAG-A(5)AT-GA(5)C-A-NH23’ UTR

[0102] In the PNA sequences of Tables 1 and 2 above, X(A, G, T, or C) is each a natural nucleotide base, X(p) or X(pOq) is a modified nucleotide base, Fethoc- is [2-(9-fluorenyl)ethyl-1-oxy]carbonyl], and the substituent at the N-terminus may be, in addition to Fethoc-, Fmoc- in the structure below, [(9-fluorenyl)methyloxy]carbonyl]-, acetyl (Ac), benzoyl, or hydrogen.

[0103] , , ,

[0104] ,

[0105]

[0106] Meanwhile, the PNA derivatives in Table 3 below target sequences conserved across multiple coronaviruses. Specifically, PNA35 targets a sequence common to HCoV-OC43 and SARS-CoV-2, and PNA53 targets a sequence common to HCoV-OC43, SARS-CoV-2, and SARS-CoV.

[0107] PNA 35Fethoc-C(1O2)AG-G(6)AT-G(6)GT-A(5)AT-GC(1O2)T-GCT-A(5)T-NH2Nsp12PNA 53Fethoc-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp14

[0108] The present inventors designed and manufactured the PNA oligomers of Tables 1 and 2, performed mass spectrometry, and compared the mass predicted value of the designed PNA oligomer with the mass value of the manufactured PNA, thereby confirming that the designed PNA oligomer was manufactured (see Examples 2-1 and 3-1).

[0109] Next, the inventors of the present invention confirmed whether the manufactured PNA oligomers could inhibit viral replication. First, to confirm whether the manufactured HCoV-OC43 RNA-targeting PNA oligomers could bind to viral genomic RNA and inhibit its replication and translation with high efficiency, qRT-PCR was performed to determine the level of viral genes in the virus culture solution treated with the PNA oligomers. At this time, it was also confirmed whether the enhanced viral RNA replication inhibition effect could be suppressed through a combination of multiple PNA oligomers. For the above efficacy comparison, the commercially available drug PF07321332 was used as a control. As a result, it was confirmed that the vast majority of the manufactured PNA oligomers could inhibit the replication of HCoV-OC43 viral RNA, and further, the combination of PNA oligomers exhibited enhanced virus replication inhibition activity (see Example 2-2).

[0110] Next, we confirmed whether the manufactured SARS-CoV-2 RNA-targeting PNA oligomer could bind to viral genomic RNA and inhibit its replication with high efficiency. At this time, we also confirmed whether the enhanced viral RNA replication and translation inhibition effect could be suppressed through a combination of multiple PNA oligomers. As a result of qRT-PCR experiments, like the HCoV-OC43-targeting PNA oligomer, the SARS-CoV-2-targeting PNA oligomer effectively inhibited the replication of the target viral gene, and in particular, it was confirmed that the combination of multiple PNA oligomers inhibited viral replication more effectively. In addition, as a result of performing qRT-PCR, 20 drugs that inhibited viral replication with high efficiency were selected and titer analysis was performed, and it was confirmed that 19 drugs showed a high viral replication inhibition rate of more than 90% at 1000 nM.

[0111] Meanwhile, the inventors of the present invention sought to determine whether the designed and manufactured PNA oligomers could be universally applied to coronaviruses. They hypothesized that the modified PNAs could inhibit viral replication by maintaining target binding affinity even when mismatches exist in the complementary binding region due to their high binding affinity to the target. Furthermore, they treated a SARS-CoV-2 culture medium with a PNA oligomer whose sequence is 100% complementary to a portion of HCoV-OC43 RNA to determine the level of viral protein expression. As a result, they confirmed that the PNA oligomer targeting HCoV-OC43 inhibited the protein expression of the SARS-CoV-2 genome, which contained some mismatches.

[0112] From the above, the present inventors have produced an oligomer capable of complementary binding to a portion of the coronavirus genome using a modified PNA, and can penetrate into virus-infected cells without a vector and inhibit virus proliferation. Furthermore, the PNA oligomer can operate even when a mismatch is included in the complementary binding region, and thus the present invention can be provided as a universally usable virus therapeutic agent even when a mutation of the coronavirus occurs.

[0113] The compound of formula I of the present invention does not require a separate invasive formulation to facilitate systemic delivery for the intended therapeutic or biological activity. The invasive formulation is described herein as a “carrier” to enhance cell permeability. The compound of formula I is typically dissolved in a phosphate buffer solution or saline solution (PBS) and then administered systemically to exhibit the desired therapeutic (analgesic) or biological activity in target tissues (primarily nerve cells). The compound of the present invention does not require a separate, strongly invasive formulation for systemic therapeutic activity.

[0114] Meanwhile, the in vivo administration of oligonucleotides for disease treatment has presented various challenges, including degradation by blood enzymes, interaction with blood substances, and non-specific delivery to cells. To overcome these challenges, attempts have been made to improve delivery by using nuclease-resistant analogs or linking them with structures such as PEG. Both viral vector-based and non-viral delivery systems have been developed. However, viral vector-based delivery systems face challenges such as immunogenicity induced by the body's immune system, unwanted genome integration, size limitations of the target nucleic acid, difficulty in repeated administration, the risk of complications, and high production costs. Meanwhile, non-viral delivery systems that encapsulate and deliver nucleic acids, such as polymers, lipids, and lipid nanoparticles (LNPs), have utilized lipid-based structures using cholesterol or PEG, and polymer-based structures such as PLGA. However, these systems suffer from toxicity issues and are limited to specific organs. Furthermore, non-natural oligonucleotides used to enhance stability require carriers to penetrate cell membranes.

[0115] The compound of formula (I) of the present invention does not require a separate carrier for cell membrane penetration, and thus inhibits coronavirus replication within cells even when administered systemically. Therefore, the compound of the present invention is useful for safely treating symptoms associated with coronavirus infectious diseases.

[0116] The compound of formula I of the present invention can be used together with a pharmaceutically acceptable acid or base. The pharmaceutically acceptable acid or base includes, but is not necessarily limited to, sodium hydroxide, potassium hydroxide, hydrochloric acid, methanesulfonic acid, citric acid, and trifluoroacetic acid.

[0117] In the present invention, the compound of formula I or a pharmaceutically acceptable salt may be administered together with a pharmaceutically acceptable adjuvant, which may include, but is not limited to, citric acid, hydrochloric acid, tartaric acid, stearic acid, polyethyleneglycol, polypropyleneglycol, ethanol, isopropanol, sodium bicarbonate, distilled water, and a preservative.

[0118] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0119] [Example]

[0120] Example 1. General method for synthesizing PNA oligomers

[0121] PNA monomers with modified nucleobases were synthesized by the method disclosed in the prior art [PCT / KR 2009 / 001256] or by a slight modification thereof. Using Fmoc[{(9-fluorenyl)methoxy}carbonyl]-protected PNA monomers with modified nucleobases and Fmoc-protected PNA monomers with natural nucleobases, PNA oligomers of formula (I) were synthesized in the solid phase by the method disclosed in the prior art [US6,133,444; WO96 / 40685] or by a slight modification thereof, as shown in [Scheme 1]. The solid phase used for the synthesis was mainly H-Rink Amide-ChemMatrix resin purchased from PCAS BioMatrix Inc. (Quebec, Canada). The PNA oligomers synthesized in this way were identified by TOF LC / MS and C 18 - Separated and analyzed using reverse phase UPLC. (Distilled water / acetonitrile or distilled water / methanol, 0.1% TFA) [Figures 1a-1b] are HPLC chromatograms before and after HPLC purification of “PNA123”, and [Figure 1c] is an ESI / TOF / MS spectrum for “PNA123” purified by HPLC, which are provided for the purpose of explaining the PNA oligomers of the present invention and are not intended to limit the scope of the present invention.

[0122] [Reaction Scheme 1] schematically illustrates a typical process of solid-state synthesis, and each reaction process is briefly provided as follows.

[0123] [Activation of H-Rink-ChemMatrix resin] When the Fmoc protecting group is attached to the amine of the resin, a suspension of 0.94 mmol (approximately 200 mg of resin) and 5 mL 20% piperidine / dimethylformamide (DMF) is mixed and vortexed in a Libra tube for 5 minutes, and the DeFmoc solution is filtered off. After washing the resin with 5 mL methylene chloride (MC), 5 mL DMF, and 5 mL DMF for 30 seconds each, the amine of the resin is ready to react with the Fmoc-protected PNA monomer.

[0124]

[0125] [Reaction Formula 1]

[0126]

[0127] [Fmoc removal (DeFmoc)] This applies when the Fmoc protecting group is attached to the amine of the resin. A suspension of 0.94 mmol (approximately 200 mg resin) and 5 mL 20% piperidine / DMF is mixed and vortexed in a Libra tube for 5 minutes, and the DeFmoc solution is filtered to remove it. After washing with 5 mL methylene chloride (MC), 5 mL DMF, and 5 mL DMF for 30 seconds each, the amine of the resin is ready to react with the Fmoc-protected PNA monomer.

[0128] [Coupling with Fmoc-PNA monomer] The amine of the resin is coupled with the Fmoc-PNA monomer as follows. 0.235 mmol PNA monomer, 0.235 mmol HBTU, and 0.2585 mmol DIEA are dissolved in 5 mL anhydrous DMF, and the solution is added to the resin after 2 minutes. The resin suspension is vortexed for 1 hour, the solution is filtered off, and the resin is washed with 5 mL MC, 5 mL DMF, 5 mL MC, 5 mL DMF, and 5 mL DMF for 30 seconds each.

[0129] [Capping] Unreacted amines after the coupling reaction are capped by reacting in 5 mL of a capping solution (a DMF solution of 5% acetic anhydride and 6% 2,6-lutidine) for 5 minutes. After filtering off the capping solution, the resin is washed with 5 mL of MC, 5 mL of DMF, and 5 mL of DMF for 30 seconds each.

[0130] [Introduction of "Fethoc" group to N-terminus] "Fethoc" group is introduced to the N-terminal amine of the resin by reaction with "Fethoc-OSu" under basic coupling conditions, and "Fethoc-OSu" [CAS No. 179337-69-0, C 20 H 17 NO5, MW 351.36] Its chemical structure is as follows.

[0131]

[0132] [Separation from Resin] PNA oligomers bound to the resin are separated from the resin by reacting with 6 mL of cleavage solution (2% triisopropylsilane and 2% trifluoroacetic acid solution in water) for 3 hours. The resin is removed by filtration, and the filtrate is concentrated under low pressure. The residue is treated with ether (diethylether), and the resulting solid is filtered and purified by reverse-phase HPLC.

[0133] [HPLC analysis and purification] The crude PNA oligomer product separated from the resin was purified by C using water / acetonitrile or water / methanol containing 0.1% TFA as the eluent. 18 -Purified by reverse phase HPLC. Figures 1a and 1b show C before and after purification of "PNA 123". 18 -This is an example of a reverse phase UPLC chromatogram.

[0134]

[0135] Synthetic examples of PNA derivatives of formula I

[0136] PNA oligomers that complementarily bind to the genomic RNA of coronavirus were synthesized using the above method with some modifications. These PNA oligomers targeting coronavirus RNA are provided to illustrate the present invention and are not intended to limit the scope of the present invention.

[0137] Tables 1 and 2 above are PNA oligomers targeting HCoV-OC43 RNA or SARS-CoV-2 RNA.

[0138] Table 4 below shows PNA oligomers that are completely complementary to HCoV-OC43 RNA among the manufactured PNA oligomers, and Table 5 shows PNA oligomers that are completely complementary to SARS-CoV-2 RNA among the manufactured PNA oligomers.

[0139] <h2 style=";text-align:left;direction:ltr">UTRPNA 올리고머PNA 서열 (N → C)타겟 영역PNA 2Fethoc-TGA-A(5)GC-GGG(6)-ATG-CA(5)C-GCA(5)-CG-NH25' UTRPNA 3Fethoc-TCA-GTG-AA(5)G-CGG(6)-GAT-GCA(5)-CGC-A(5)CG-NH25' UTRPNA 4Fethoc-TCA(5)-GTG-AA(5)G-CGG(6)-GAT-GCA(5)-CGC-A(5)CG-NH25' UTRPNA 5Fethoc-CTA(5)-ACA-A(5)GA-GAT-CA(5)G-TGA-A(5)G-NH25' UTRPNA 6Fethoc-AGA(5)-TCT-A(5)AC-AA(5)G-AGA-TCA(5)-GTG-AA(5)G-NH25' UTRPNA 7Fethoc-C(1O2)TA-ACA-A(5)GA-GAT-CA(5)G-TGA-A(5)G-NH25' UTRPNA 8Fethoc-CTA(5)-ACA-A(5)GA-GAT-CA(5)G-TGA-AG(6)-NH25' UTRPNA 9Fethoc-AGA(5)-TTA-CAA(5)-AAA-GA(5)T-CTA-A(5)C-NH25' UTRPNA 10Fethoc-GTT-TA(5)G-ATT-A(5)CA-AA(5)A-AGA(5)-TCT-AA(5)C-NH25' UTRPNA 11Fethoc-GTT-TA(5)G-ATT-A(5)CA-AA(5)A-AGA(5)-TC-NH25' UTRPNA 12Fethoc-GTT-TA(5)G-ATT-A(5)CA-AA(5)A-AGA-TC(1O2)-NH25' UTRPNA 13Fethoc-G(6)TT-TAG-A(5)TT-ACA-AA(5)A-AGA(5)-TC-NH25' UTRPNA 15Fethoc-G(6)TT-TTT-A(5)TA-AA(5)G-TTT-A(5)GA-TT-NH25' UTRPNA 16Fethoc-TTA(5)-CA(5)G-GGA(5)-GTG-GA(5)T-GTT-TT-NH25' UTRPNA17Fethoc-G(6)CC-CAC-A(5)AG-CAT-A(5)GA-TTA(5)-CA-NH25’ UTRPNA 22Fethoc-TTT-TTA(5)-AAC-G(6)GG-TTC-G(6)GG-G(6)T-NH2Nsp12PNA 23Fethoc-A(5)AC(1O2)-CCG(6)-TTT-A(5)AA-A(5)A-NH2Nsp12PNA 24Fethoc-A(5)CC-CCG-A(5)AC-CCG(6)-TTT-A(5)AA-A(5)A-NH2Nsp12PNA 25Fethoc-A(5)CC-C(1O2)CG-AA(5)C-CCG(6)-TTT-A(5)AA-A(5)A-NH2Nsp12PNA 26Fethoc-TTT-TTA(5)-AAC-G(6)GG-TTC-G(6)GG-G(6)TA-CGA-NH2Nsp12PNA 27Fethoc-TCG-TA(5)C-CCC-GA(5)A-CCC-G(6)TT-TAA-A(5)AA-NH2Nsp12PNA 28Fethoc-TCG-TA(5)C-CC(1O2)C-GAA(5)-CCC-G(6)TT-TA(5)A-AA(5)A-NH2Nsp12PNA 29Fethoc-TTT-TTA(5)-AAC(1O2)-GGG(6)-TTC-G(6)GG-G(6)T-NH2Nsp12PNA 30Fethoc-TTT-TTA(5)-AAC-G(6)GG-TTC(1O2)-GGG-G(6)T-NH2Nsp12PNA 33Fethoc-TTA(5)-AAC-G(6)GG-TTC-G(6)GG-G(6)T-NH2Nsp12PNA 44Fethoc-ATC-A(5)CA-AGA(5)-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 45Fethoc-ATC-A(5)CA-AGA(5)-TGC-AA(5)T-TTA(5)-GGT-G(6)GT-GC-NH2Nsp15PNA 46Fethoc-CA(5)C-AAG-A(5)TG-C(1O2)AA(5)-TTT-A(5)GG(6)-TGG(6)-TGC-NH2Nsp15PNA 47Fethoc-CAA-GA(5)T-GC(1O2)A-A(5)TT-TA(5)G-G(6)TG-G(6)TG-C-NH2Nsp15PNA48Fethoc-AGA(5)-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 49Fethoc-AGA-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 50Fethoc-AGA-TGC(1O2)-AA(5)T-TTA(5)-GGT-GG(6)T-GC-NH2Nsp15PNA 51Fethoc-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GCA(5)-GT-NH2Nsp15PNA 52Fethoc-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)-NH2Nsp15PNA 53Fethoc-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 54Fethoc-TGC-AAT-TTA-GGT-GGT-GC-NH2Nsp15PNA 55Fethoc-TGC-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 56Fethoc-TG(6)C-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 57Fethoc-TG(6)C-AA(5)T-TTA-G(6)GT-G(6)GT-G(6)C-NH2Nsp15PNA 58Fethoc-TGC-AA(5)T-TTA-GGT-GGT-GC-NH2Nsp15PNA 59Fethoc-TGC-AAT-TTA(5)-GGT-GGT-GC-NH2Nsp15PNA 60Fethoc-TGC-AA(5)T-TTA(5)-GGT-GGT-GC-NH2Nsp15PNA 61Acetyl-TGC-AAT-TTA-GGT-GGT-GC-NH2Nsp15PNA 62Acetyl-TGC-AA(5)T-TTA-GGT-GGT-GC-NH2Nsp15PNA 63Acetyl-TGC-AAT-TTA(5)-GGT-GGT-GC-NH2Nsp15PNA 64Acetyl-TGC-AA(5)T-TTA(5)-GGT-GGT-GC-NH2Nsp15PNA 65Acetyl-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA66H-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp15PNA 68Fethoc-TGC(1O2)-AA(5)T-TTA(5)-GGT-GG(6)T-GC-NH2Nsp15PNA 70Fethoc-GCA-CCA(5)-CCT-A(5)AA-TTG-CA-NH2Nsp15PNA 71Fethoc-GCA(5)-CCA(5)-CCT-A(5)AA-TTG-CA-NH2Nsp15PNA 72Fethoc-C(1O2)AA(5)-TTT-A(5)GG(6)-TGG(6)-TG-NH2Nsp15

[0140] PNA 올리고머PNA 서열 (N → C)타겟 영역PNA 35Fethoc-C(1O2)AG-G(6)AT-G(6)GT-A(5)AT-GC(1O2)T-GCT-A(5)T-NH2Nsp12PNA 53Fethoc-TGC(1O2)-AA(5)T-TTA(5)-GG(6)T-GG(6)T-GC-NH2Nsp14PNA 76Fethoc-GGA(5)-AGG-TA(5)T-AAA(5)-CCT-TTA(5)-AT-NH25’ UTRPNA 79Fethoc-TTG-G(6)TT-TG(6)T-TAC-CTG(6)-GGA-A(5)G-NH25’ UTRPNA 81Fethoc-TTG-G(6)TT-GG(6)T-TTG-TTA(5)-CCT-G(6)G-NH25’ UTRPNA 84Fethoc-CTA(5)-CAA-GA(5)G-ATC-GA(5)A-AGT-TG(6)-NH25’ UTRPNA 88Fethoc-CA(5)G-ATC(1O2)-TAC-A(5)AG-AGA(5)-T-NH25’ UTRPNA 91Fethoc-GTT-CG(6)T-TTA(5)-GAG-AA(5)C-AGA(5)-TC-NH25’ UTRPNA 105Fethoc-C(1O2)TC-TA(5)T-TA(5)C-G(6)TT-T-NH2Nsp3PNA 111Fethoc-ATC-A(5)CA-TG(6)T-CTT-G(6)GA-CA(5)G-TA-NH2Nsp5PNA 120Fethoc-A(5)CC-GCA-A(5)AC-CCG(6)-TTT-AAA(5)-AA-NH2Nsp12PNA 121Fethoc-A(5)CC-GC(1O2)A-AA(5)C-CCG(6)-TTT-AAA(5)-AA-NH2Nsp12PNA 123Fethoc-A(5)GC-C(1O2)CT-G(6)TA-TA(5)C-GA(5)C-ATC(1O2)-AG-NH2Nsp12PNA 129Fethoc-A(5)GC-TCT-A(5)GA-CTT-A(5)GT-TTT-A(5)A-NH2Nsp12PNA 143Fethoc-TTA(5)-AGT-CA(5)G-TTC(1O2)-TTT-A(5)TT-AT-NH2Nsp12PNA157Fethoc-TGC(1O2)-AA(6)T-TTA(6)-GG(5)T-GG(5)T-GC-NH2Nsp14

[0141] Example 2. Analysis of PNA oligomers targeting HCoV-OC43 RNA

[0142] 2-1. Mass structure analysis

[0143] Table 6 below shows the results of mass structure analysis of the manufactured PNA oligomer targeting HCoV-OC43.

[0144] PNA OligomerExact Mass, m / zPNA OligomerExact Mass, m / ztheor. a obs. b theor. a obs. b<h2 style=";text-align:left;direction:ltr">PNA 16007.656007.47PNA 457858.397858.32PNA 26144.666144.72PNA 467513.347513.29PNA 37228.087228.05PNA 476887.026886.98PNA 47328.187328.15PNA 486360.816360.86PNA 56136.686136.69PNA 496260.716260.69PNA 67344.217344.15PNA 506161.66161.64PNA 76133.636133.63PNA 516351.86351.85PNA 86135.686135.67PNA 524611.064611.17PNA 96079.686079.56PNA 535419.375419.41PNA 107269.197269.16PNA 544923.914923.96PNA 116101.666101.54PNA 555322.325322.39PNA 126098.626098.6PNA 565421.425421.49PNA 136100.676100.66PNA 575420.435420.47PNA 152103.636103.69PNA 585024.015024.06PNA 166171.636171.74PNA 595024.015024.07PNA 176031.666031.37PNA 605124.115124.16PNA 226144.646144.67PNA 614729.844729.74PNA 235991.655991.73PNA 624829.944829.97PNA 246091.756091.83PNA 634829.944829.93PNA 256188.816188.91PNA 644930.044930.07PNA 267237.077237.03PNA 655225.35225.25PNA 277066.077066.02PNA 665183.295183.33PNA 287263.227263.14PNA 685320.275320.27PNA 296241.696241.62PNA 704991.124991.14PNA 306142.586142.57PNA 715091.225091.24PNA 335346.335346.34PNA 724611.064611.1PNA 448054.558054.52.

[0145] a) Theoretical value, b) Observed value.

[0146]

[0147] 2-2. Evaluation of the antiviral efficacy of PNA oligomers using qRT-PCR

[0148] We performed an efficacy evaluation to see how much the manufactured PNA oligomer-based drug could block viral gene replication.

[0149] Additionally, to determine whether the antiviral efficacy is enhanced when treated with a combination of PNA oligomers, an experiment was also conducted using a drug (Combi 1) that combined PNA 46 and PNA 8.

[0150] To analyze the level of viral RNA in virus particles present in the virus culture medium and to determine how much the manufactured PNA oligomer-based drug inhibits viral gene replication, qRT-PCR was performed.

[0151] Specifically, HCT8 cells cultured in 6-well plates one day before for drug screening were infected with HCoV-OC43 at a dose of 500 TCID50 using serum-free RPMI medium. Simultaneously with infection, 1 μM of PNA oligomer was treated, and after 30 minutes, the cells were washed twice with serum-free RPMI, and the medium was replaced with RPMI-Complete media (containing 10% FBS and 1% penicillin / streptomycin) and treated with 1 μM of PNA oligomer. On the third day after infection, viral RNA was extracted from 140 μL of the cell supernatant using Qiagen's QIAmp viral RNA mini kit. Virus extraction was performed according to the kit manual. The extracted viral RNA was measured for viral titer by real-time PCR using Takara's One step TB green kit. The primers used are as follows: HCoV-OC43 primer; Nucleoprotein (NP) gene (5'-AGCAACCAGGCTGATGTCAATACC-3', 5'-AGCAGACCTTCCTGAGCCTTCAAT-3'). Real-time PCR conditions are as follows. Temperature Stage 1: Reverse transcription 42℃ 5 min, 95℃ 10 sec, 1 cycle. Stage 2: PCR reaction 95℃ 5 sec, 60℃ 34 sec, 35 cycles. Stage 3: Melting curve analysis 95℃ 15 sec, 65℃ 60 sec, 95℃ 15 sec.

[0152] The results of the antiviral efficacy evaluation of PNA oligomers and their combinations targeting HCoV-OC43 through qRT-PCR-based viral RNA level analysis are shown in Table 7 below.

[0153] PNA Oligomer Inhibition%PNA Oligomer Inhibition%PNA 131.78PNA 4585.13PNA 261.44PNA 4673.15PNA 364.93PNA 4765.28PNA 455.2PNA 4858.95PNA 557.12PNA 4966.92PNA 660.75PNA 5068.55PNA 750.13PNA 511.44PNA 876.47PNA 5228.11PNA 93.36PNA 5356.68PNA 1040.44PNA 5428.03PNA 1133.4PNA 55-2.77PNA 1243.36PNA 562.02PNA 1340.94PNA 5721.56PNA 1529.88PNA 5831.55PNA 1644.97PNA 5934.93PNA 1757.69PNA 6046.93PNA 2258.67PNA 6111.7PNA 2356.12PNA 6224.45PNA 2443.51PNA 6310.36PNA 2532.47PNA 6446.9PNA 2641.64PNA 6552.75PNA 2740.12PNA 6644.26PNA 2870.19PNA 6873.71PNA 2955.58PNA 7023.06PNA 3053.76PNA 7110.76PNA 3330.51PNA 7222.34PNA 4471.17Combi 179.55 PF07321332 a 97.98 a) PF07321332 (Nirmatrelvir, viral protease inhibitor developed by Pfizer)

[0154]

[0155] Example 3. Analysis of PNA oligomers targeting SARS-CoV-2 RNA

[0156] 3-1. Mass structure analysis and target binding strength confirmation

[0157] Next, mass structural analysis was performed to confirm whether the designed SARS-CoV-2 target PNA oligomers were manufactured as intended, and Tm analysis was performed to confirm the binding affinity of each PNA oligomer to the target. In Table 2, each designed PNA oligomer showed a mass value that matched the predicted value and the measured value during design, indicating that they were all manufactured as intended, and Table 8 below provides some of the data. Meanwhile, Table 9 shows some of the results of the Tm measurement experiment to confirm the binding affinity of the PNA oligomer to the target. Since the 20-mer PNA oligomer showed high binding affinity even above 90°C, the Tm value was measured using the 10-mer at the N-terminus and the 10-mer at the C-terminus of the PNA to compare the relative binding affinity of each PNA oligomer.

[0158] The Tm value for the duplex of 10-mer DNA complementary to PNA is too high to be reliably measured in a buffer solution because the buffer solution tends to boil during the Tm measurement. Therefore, the Tm value was measured using a UV / Vis spectrophotometer as follows. In a 15 mL polypropylene falcon tube, 4 μM PNA oligomer and 4 μM 10-mer DNA for the N-terminus were mixed in a buffer solution (pH 7.16, 10 mM sodium phosphate, 100 mM NaCl), incubated at 90 °C for 1 min, and slowly cooled to room temperature. The solution was transferred to a 3 mL quartz cuvette, sealed well, and mounted on an Agilent Cary 100 UV / Visible spectrophotometer. The temperature of the cuvette was increased at a rate of 0.5 or 1.0 ℃ per minute, and the change in absorbance at 260 nm was measured. The temperature at which the rate of change in absorbance was the greatest, i.e., the inflection point, was determined as the Tm value between the PNA oligomer and DNA. The DNA used for Tm measurement was purchased from Bioneer Co., Ltd. (www.bioneer.com, Daejeon, South Korea) and used without separate purification.

[0159] PNA OligomerExact Mass, m / ztheor. a obs. bPNA 35 6317.756317.9PNA 535419.375419.41PNA 766117.666117.7PNA 796144.646144.6PNA 816126.616126.6PNA 846126.676126.66PNA 885001.195001.25PNA 916108.656108.65PNA 1054106.824106.92PNA 1116067.656067.59PNA 1206015.666015.6PNA 1216112.726112.7PNA 1236232.756232.73PNA 1296059.636059.75PNA 1436037.566037.52PNA 1575419.375419.38a) Theoretical value, b) Observed value

[0160] PNA Oligomer Tm Values ​​(℃) 10-mer DNA for N-terminus 10-mer DNA for C-terminus PNA 5368.2 277.02 PNA 7679.2 763.02 PNA 7965.4 280.02 PNA 8159.02 60.02 PNA 8471.02 67.02 PNA 8859.02 69.07 PNA 9151.02 51.02 PNA 12070.4 248.02 PNA 12173.02 5.02 PNA 12377.5 271.07 PNA 12968.00 59.02 PNA 14366.02 52.02

[0161] 3-2. Antiviral efficacy evaluation

[0162] We performed an efficacy evaluation to determine how much the manufactured PNA oligomer-based drug could block viral protein production and gene replication.

[0163] Additionally, to determine whether the antiviral efficacy is enhanced when multiple PNA oligomers are combined and treated, an experiment was also conducted in which a combination of drugs (Multi) was treated as shown in Table 10 below.

[0164] Multi 약물명약물 1약물 2약물 3약물 4Multi 1PNA 35PNA 76 Multi 2PNA 129PNA 76 Multi 3PNA 129PNA 79 Multi 4PNA 129PNA 84 Multi 5PNA 143PNA 76 Multi 6PNA 143PNA 88 Multi 7PNA 53PNA 120 Multi 8PNA 35PNA 120 Multi 9PNA 129PNA 120 Multi 10PNA 143PNA 120 Multi 11PNA 88PNA 120 Multi 12PNA 76PNA 120 Multi 13PNA 81PNA 120 Multi 14PNA 79PNA 120 Multi 15PNA 84PNA 120 Multi 16PNA 91PNA 120 Multi 17PNA 88PNA 121 Multi 18PNA 76PNA 121 Multi 19PNA 81PNA 121 Multi 20PNA 79PNA 121 Multi 21PNA 84PNA 121 Multi 22PNA 91PNA 121 Multi 23PNA 79PNA 123 Multi 24PNA 105PNA 120 Multi 25PNA 111PNA 120 Multi 26PNA 120PNA 53PNA 79 Multi 27PNA 129PNA 53PNA 79 Multi 28PNA 120PNA 129PNA 53PNA 79Multi 29PNA 35PNA 76PNA 120 Multi 30PNA 129PNA 76PNA 120 Multi 31PNA 129PNA 79PNA 120 Multi 32PNA 129PNA 84PNA 120 Multi 33PNA 143PNA 76PNA 120 Multi 34PNA 143PNA 88PNA 120 Multi 35PNA 53PNA 76PNA 120 Multi 36PNA 53PNA 79PNA 123

[0165] 3-2-1. qRT-PCR

[0166] To analyze the level of viral RNA in virus particles present in the virus culture medium to determine how much the manufactured PNA oligomer-based drug inhibits viral gene replication, the infectious virus gene was quantified by performing qRT-PCR in the virus culture medium treated with the test substance (drug) at a concentration of 1000 nM.

[0167] To this end, Vero cells cultured in 12-well plates the day before were pre-incubated for 2 hours in a cell culture medium containing the evaluation drug at a set concentration (1000 nM), then infected with the virus, and the cell culture medium was treated with the evaluation drug at the same concentration. 24 hours after infection, viral RNA was extracted from the virus culture medium and quantified using qPR-PCR to compare and analyze the antiviral efficacy to select therapeutic substances. Paxlovid, used as a positive control, was also treated in the same manner.

[0168] The screening results are shown in Table 10 below.

[0169] Among the 50 PNA and Multi drugs, the 20 drugs indicated in bold in Table 11 were confirmed to have a virus reversal rate equal to or higher than that of Paxlovid (96.03%).

[0170] PNA Oligomer Inhibition%PNA Oligomer Inhibition%PNA 3595.11Multi 1295PNA 5391.53Multi 1377.95PNA 7681.01Multi 1479.61PNA 7988.62Multi 1595.98PNA 8158.46Multi 1685.98PNA 8487.52Multi 1793.29PNA 8879.03Multi 1896.78PNA 9165.14Multi 1994.21PNA 12050.95Multi 2094.82PNA 12171.48PNA 2196.09PNA 12397.44Multi 2295.53PNA 12995.34Multi 2398.49PNA 14348.5Multi 2484.16PNA 15790.07Multi 2582.07Multi 196.94Multi 2697.55Multi 297.84Multi 2797.95Multi 396.57Multi 2898.15Multi 496.51Multi 2998.5Multi 594.16Multi 3097.46Multi 695.83Multi 3196.03Multi 796.31Multi 3296.16Multi 897.81Multi 3395.25Multi 995.67Multi 3497.7Multi 1089.35Multi 3596.5Multi 1180.04Multi 3697.4 Paxlovid96.03

[0171] 3-2-2. Reverse analysis

[0172] In the above Example 3-2-1, the 50% inhibitory concentration (IC50) was calculated for 20 drugs with excellent virus proliferation inhibition efficacy.

[0173] To this end, Vero cells were cultured in 12-well plates and pre-incubated for 2 hours in a cell culture medium containing the drug of interest at each concentration. The cells were then infected with the virus, and the same concentration of the drug of interest was added to the cell culture medium for each well. After 24 hours, viral RNA was extracted from the virus culture medium and quantified using qPR-PCR to analyze the antiviral efficacy. The treatment concentrations of the test substances used in this experiment were set as shown in Table 12 below, and a 2-fold dilution was used.

[0174] Well123456Concentration (nM)100050025012562.531.25Well789101112Concentration (nM)15.637.813.911.950.980.49

[0175] The results of the three repeated experiments are shown in Table 13 and Figures 2 to 3b.

[0176] PNA derivative IC50 (nM)PNA 123263.70Multi 1327.70Multi 2375.20Multi 3434.40Multi 4501.90Multi 7323.60Multi 8342.60Multi 18690.10Multi 21481.10Multi 23287.10Multi 26218.30Multi 27215.10Multi 2861.54Multi 29AmbiguousMulti 30262.60Multi 31316.30Multi 32370.40Multi 34275.70Multi 35432.50Multi 36216.60Paxlovid (PC)-1316.60Paxlovid (PC)-2328.30Paxlovid (PC)-3464.40

[0177] The IC50 of Paxlovid, used as a positive control in each experiment, was confirmed to be 316.6, 328.3, and 464.4 nM, respectively (Fig. 2).

[0178] The IC50 derivation results confirmed that 19 out of 20 species had virus inhibition rates exceeding 90% at 1000 nM. In addition, when the concentration of the test substance was below 250 nM, the inhibition rate did not exceed 50% or the antiviral efficacy disappeared (Figs. 3a to 3i and 4a to 4k).

[0179] Multi 18 was the only drug to demonstrate a virus inhibition rate of less than 90% at 1,000 nM. Furthermore, Multi 28 and Multi 29 showed no dose-dependent pattern, resulting in very low or no IC50 values, resulting in ambiguous values ​​of 61.54 nM and 61.54 nM, respectively. Therefore, IC50 values ​​were not derived for these three substances for revalidation.

[0180] The IC50 values ​​for 17 substances, excluding the three excluded substances, were found to range from 215.1 to 690.1 nM (Table 9). Among them, Multi 27, Multi 36, and Multi 26 were found to have the best IC50 values. As a single drug, PNA 123 also demonstrated a superior IC50 value to Paxlovid.

[0181]

[0182] Example 4. Inhibitory effect of PNA oligomer targeting HCoV-OC43 RNA on SARS-CoV-2 proliferation.

[0183] To determine whether PNA oligomers targeting HCoV-OC43 RNA could inhibit SARS-CoV-2 protein translation even when there is a mismatch in the complementary binding region between the PNA oligomer and the target gene, we examined whether PNA oligomers targeting HCoV-OC43 RNA could inhibit SARS-CoV-2 virus protein translation. To this end, three types of PNA oligomers (PNA69, 53, and 46), designed to be complementary to portions of HCoV-OC43 RNA, were treated with SARS-CoV-2 virus cultures, and Western blotting was performed to determine the level of expression of the viral N protein.

[0184] Specifically, for drug screening, HCT8 cells cultured in 6-well plates one day in advance were treated with 1 μM PNA derivatives while replacing the medium with fresh ones. Myc-NSP14 was then transfected using Lipofectamin 2000 according to the manual. 48 hours after transfection, the cells were harvested, lysed with M-PER lysis solution, centrifuged, and the supernatant was used as a Western sample with 4x sample buffer. Analysis was performed on a 12% SDS-PAGE gel. Anti-myc (cell signaling technology) and anti-β-actin (Merck) antibodies were used. Detection was performed using ECL Detection Reagent (AbClon), and visualization and analysis were performed using Amersham ImageQuant 800. The results are shown in Fig. 5.

[0185]

[0186] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0187] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

[0188] The modified PNA oligomer of the present invention can be used as an effective coronavirus therapeutic agent to combat rapidly mutating viral infections.

Claims

1. A pharmaceutical composition for preventing or treating coronavirus infection, comprising a peptide nucleic acid (PNA) oligomer represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, A pharmaceutical composition wherein the above PNA oligomer is capable of complementary binding to a portion of the genomic (+) RNA or genomic (-) RNA of a coronavirus: [Chemical Formula 1] In the above formula, n is an integer between 10 and 30; X is one selected from the group consisting of hydrogen [H], formyl [HC(=O)-], aminocarbonyl [NH2-C(=O)-], substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkylacyl, substituted or unsubstituted arylacyl, substituted or unsubstituted alkyloxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted alkylsulfonyl, and substituted or unsubstituted arylsulfonyl; Z is one selected from the group consisting of hydroxy[-OH], substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, amino[-NH2], substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino, substituted or unsubstituted alkyl, and substituted or unsubstituted aryl; B1, B2, … … , B n-1 and B n are each independently selected from a natural nucleotide base and an unnatural nucleotide base of adenine (A), guanine (G), thymine (T), or cytosine (C); B1 to B n At least one of the following is independently selected from the group consisting of modified non-natural nucleic acid groups of formulae 2 to 4; [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] In the above chemical formulas 2 to 4, j, k, l, and m are each independently an integer between 1 and 16.

2. In paragraph 1, A pharmaceutical composition, wherein the pharmaceutical composition does not contain a carrier for intracellular penetration of the PNA oligomer.

3. In paragraph 1, A pharmaceutical composition wherein X is Fmoc [(9-fluorenyl)methyloxy]carbonyl], Fethoc [2-(9-fluorenyl)ethyl-1-oxy]carbonyl], acetyl (Ac), benzoyl, or hydrogen.

4. In paragraph 1, The above X is Fethoc [2-(9-fluorenyl)ethyl-1-oxy]carbonyl], The above Z is NH2, The above j is 2, The above k is 1, The above l is 5, The pharmaceutical composition wherein m is 6.

5. In paragraph 1, A pharmaceutical composition, wherein the coronavirus is at least one selected from the group consisting of human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU1 (HCoV-HKU1), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus type 2 (SARS-Cov-2).

6. In paragraph 1, The above coronavirus is human coronavirus OC43 (HCoV-OC43), A pharmaceutical composition, wherein the PNA oligomer is at least one selected from the group consisting of PNA oligomers of Table 1.

7. In paragraph 1, The above coronavirus is severe acute respiratory syndrome coronavirus type 2 (SARS-Cov-2), A pharmaceutical composition, wherein the PNA oligomer is at least one selected from the group consisting of PNA oligomers of Table 2.

8. In paragraph 7, The pharmaceutical composition above comprises PNA 123 [Fethoc-A(5)GC-C(1O2)CT-G(6)TA-TA(5)C-GA(5)C-ATC(1O2)-AG-NH2] of Table 2.

9. In paragraph 1, The above coronavirus is at least one selected from the group consisting of human coronavirus OC43 (HCoV-OC43) and severe acute respiratory syndrome coronavirus type 2 (SARS-Cov-2), A pharmaceutical composition, wherein the PNA oligomer is at least one selected from the group consisting of PNA oligomers of Table 3.

10. In paragraph 1, A pharmaceutical composition wherein the PNA oligomer is at least 80% complementary to a portion of the genomic (+) RNA or genomic (-) RNA of a coronavirus.

11. In paragraph 1, A pharmaceutical composition wherein the PNA oligomer is 90% complementary to a portion of the genomic (+) RNA or genomic (-) RNA of a coronavirus.

12. A method for preventing or treating a coronavirus infection disease, comprising administering to a subject a peptide nucleic acid (PNA) oligomer represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof, A method for prevention or treatment, wherein the above PNA oligomer is capable of complementary binding to a portion of the genomic (+) RNA or genomic (-) RNA of a coronavirus: [Chemical Formula 1] In the above formula, n is an integer between 10 and 30; X is one selected from the group consisting of hydrogen [H], formyl [HC(=O)-], aminocarbonyl [NH2-C(=O)-], substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkylacyl, substituted or unsubstituted arylacyl, substituted or unsubstituted alkyloxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted alkylsulfonyl, and substituted or unsubstituted arylsulfonyl; Z is one selected from the group consisting of hydroxy[-OH], substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, amino[-NH2], substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino, substituted or unsubstituted alkyl, and substituted or unsubstituted aryl; B1, B2, … … , B n-1 and B n are each independently selected from a natural nucleotide base and an unnatural nucleotide base of adenine (A), guanine (G), thymine (T), or cytosine (C); B1 to B n At least one of the following is independently selected from the group consisting of modified non-natural nucleic acid groups of formulae 2 to 4; [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] In the above chemical formulas 2 to 4, j, k, l, and m are each independently an integer between 1 and 16.

13. A method for inhibiting the proliferation of coronavirus, comprising administering to a subject a peptide nucleic acid (PNA) oligomer represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof, A method for inhibiting the proliferation of a coronavirus, wherein the above PNA oligomer can complementarily bind to a portion of the genomic (+) RNA or genomic (-) RNA of the coronavirus: [Chemical Formula 1] In the above formula, n is an integer between 10 and 30; X is one selected from the group consisting of hydrogen [H], formyl [HC(=O)-], aminocarbonyl [NH2-C(=O)-], substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkylacyl, substituted or unsubstituted arylacyl, substituted or unsubstituted alkyloxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted alkylsulfonyl, and substituted or unsubstituted arylsulfonyl; Z is one selected from the group consisting of hydroxy[-OH], substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, amino[-NH2], substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino, substituted or unsubstituted alkyl, and substituted or unsubstituted aryl; B1, B2, … … , B n-1 and B n are each independently selected from a natural nucleotide base and an unnatural nucleotide base of adenine (A), guanine (G), thymine (T), or cytosine (C); B1 to B n At least one of the following is independently selected from the group consisting of modified non-natural nucleic acid groups of formulae 2 to 4; [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] In the above chemical formulas 2 to 4, j, k, l, and m are each independently an integer between 1 and 16.

14. For the manufacture of a drug for the prevention or treatment of coronavirus infection disease, comprising a peptide nucleic acid (PNA) oligomer represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof, The above PNA oligomer is capable of complementary binding to a portion of the genomic (+) RNA or genomic (-) RNA of a coronavirus, for use: [Chemical Formula 1] In the above formula, n is an integer between 10 and 30; X is one selected from the group consisting of hydrogen [H], formyl [HC(=O)-], aminocarbonyl [NH2-C(=O)-], substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted alkylacyl, substituted or unsubstituted arylacyl, substituted or unsubstituted alkyloxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted alkylaminocarbonyl, substituted or unsubstituted arylaminocarbonyl, substituted or unsubstituted alkylsulfonyl, and substituted or unsubstituted arylsulfonyl; Z is one selected from the group consisting of hydroxy[-OH], substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, amino[-NH2], substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino, substituted or unsubstituted alkyl, and substituted or unsubstituted aryl; B1, B2, … … , B n-1 and B n are each independently selected from a natural nucleotide base and an unnatural nucleotide base of adenine (A), guanine (G), thymine (T), or cytosine (C); B1 to B n At least one of the following is independently selected from the group consisting of modified non-natural nucleic acid groups of formulae 2 to 4; [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] In the above chemical formulas 2 to 4, j, k, l, and m are each independently an integer between 1 and 16.

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