Antiviral pharmaceutical composition comprising errγ inhibitor as active ingredient

Abstract

The present application relates to an antiviral pharmaceutical composition comprising an estrogen-related receptor γ (ERRγ) inhibitor as an active ingredient. The antiviral pharmaceutical composition comprising an ERRγ inhibitor as an active ingredient, according to embodiments of the present application, has an antiviral effect, and in particular, can exhibit the effect of preventing or treating RNA viral infection. Specifically, the ERRγ inhibitor, particularly an ERRγ inverse agonist, inhibits ERRγ activity in cells infected with RNA virus, thereby blocking SREBP1c-mediated fatty acid biosynthesis, and thus can exhibit broad-spectrum antiviral efficacy.

Description

Antiviral pharmaceutical composition comprising an ERRγ inhibitor as an active ingredient

[0001] The present invention relates to an antiviral pharmaceutical composition comprising an ERRγ (estrogen related receptor γ, estrogen related receptor gamma) inhibitor as an active ingredient.

[0002] The COVID-19 pandemic has highlighted the importance of zoonotic viral pathogens, such as influenza A virus (IAV), Ebola virus, and Zika virus. These RNA viruses possess RNA-dependent RNA polymerases that lack exonuclease proofreading, leading to an inherently high mutation rate. This characteristic ultimately leads to the emergence of numerous novel mutations that confer resistance to existing antiviral drugs targeting viral proteins, such as amantadine, which targets the IAV matrix-2 (M2) ion channel, and oseltamivir, which targets the IAV neuraminidase. This highlights the need for the development of broad-spectrum host-targeted antiviral drugs that are less susceptible to viral resistance and effective in treating both existing and emerging viral infections.

[0003] Nuclear receptors (NRs) are members of a large family of transcription factors that regulate and control a wide array of corresponding genes in response to hormonal, metabolic, developmental, and environmental signals. Several viruses are known to utilize NRs to modulate gene expression or optimize the cellular environment to facilitate viral life cycles. Estrogen-related receptors (ERRs) are orphan NRs because no suitable endogenous ligands have yet been identified. Mammals possess three isoforms (ERRα, β, and γ, encoded by Esrra, Esrrb, and Esrrg, respectively). The function of ERRβ is restricted, although not exclusively, to maintaining embryonic stem cell pluripotency in mice. However, ERRα and ERRγ are widely expressed in multiple organs and regulate many similar gene programs, such as energy metabolism and bone homeostasis. ERRγ expression has been reported to be highly inducible and dynamically regulated by membrane receptors that recognize diverse cellular signals. Therefore, ERRγ is considered a key downstream mediator of multiple endocrine and metabolic signals. Infection with the intramacrophage bacterium Salmonella enterica var. Typhimurium (S. typhimurium) increases hepatic ERRγ expression, which in turn activates hepatic hepcidin, a key hormone regulating body iron levels, leading to alterations in host iron homeostasis. Furthermore, an inverse ERRγ agonist significantly improves host survival after multidrug-resistant S. typhimurium infection, suggesting that ERRγ is a targetable host factor that can regulate S. typhimurium infection.

[0004] Meanwhile, lipid metabolism represents a new potential target for antiviral intervention, as lipids and lipid droplets (LDs) play a crucial role in facilitating the life cycle of various viruses at all levels, including intracellular trafficking, replication, and release. Interestingly, recent reports have shown that LDs accumulated early after viral infection are required for an efficient interferon response, but that in later stages of infection, accumulated LDs are degraded by LD-associated lipases. The released lipids, particularly free fatty acids (FFAs), are used for the formation of viral replication compartments, viral particle formation, or energy generation required for viral replication. During the replication of various viruses, the sterol regulatory element-binding protein (SREBP) (encoded by Srebp1) transactivates genes involved in FFA and cholesterol biosynthesis and LD formation. However, how SREBPs are regulated in virus-infected cells remains unknown.

[0005] [Prior Art Literature]

[0006] [Non-patent literature]

[0007] Baek, Y.B. et al. Therapeutic strategy targeting host lipolysis limits infection by SARS-CoV-2 and influenza A virus. Signal Transduct. Target. Ther. 7, 367 (2022).

[0008] The present invention provides an antiviral pharmaceutical composition comprising an ERRγ (estrogen related receptor γ, estrogen related receptor gamma) inhibitor as an active ingredient.

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

[0010] One aspect of the present invention provides an antiviral pharmaceutical composition comprising an ERRγ (estrogen related receptor γ) inhibitor as an active ingredient.

[0011] An antiviral pharmaceutical composition comprising an ERRγ inhibitor according to the embodiments of the present disclosure as an active ingredient has an antiviral effect, and in particular, may exhibit a preventive or therapeutic effect against RNA virus infections. Specifically, an ERRγ inhibitor, particularly an ERRγ inverse agonist, may exhibit broad-spectrum antiviral efficacy by inhibiting ERRγ activity in cells infected with RNA viruses, thereby blocking fatty acid biosynthesis by SREBP1c.

[0012] Figures 1a to 1h are experimental results showing that in vitro and in vivo infection by SARS-CoV-2 and IAV, in one embodiment of the present invention, activates ERRγ and translocates it to the nucleus. (Figures 1a, 1b) Representative confocal images (left) and quantification (right) of sequential changes in ERRγ (white) and SARS-CoV-2 N protein (red) in the cytoplasm and nucleus of Vero E6 cells infected with SARS-CoV-2 KCDC03 strain at an MOI of 0.1 FFU (Figure 1a) and Representative confocal images (left) and quantification (right) of sequential changes in ERRγ (white) and IAV M2 protein (red) in the cytoplasm and nucleus of A549 cells infected with IAV PR8 strain at an MOI of 1 FFU (Figure 1b); (Figures 1c, 1d) by western blot analysis. Quantification of ERRγ in the cytoplasmic fraction, nuclear fraction, and whole cell lysate of Vero E6 cells infected with SARS-CoV-2 KCDC03 strain at an MOI of 0.1 FFU (Fig. 1c) and in the cytoplasmic fraction, nuclear fraction, and whole cell lysate of A549 cells infected with IAV PR8 strain at an MOI of 1 FFU (Fig. 1d); (Fig. 1e, Fig. 1f)10 5 TCID 50 Representative confocal images (left) and quantification (right) of ERRγ (white) and SARS-CoV-2 N protein (red) in alveolar cells from lungs of Syrian hamsters challenged with SARS-CoV-2 KCDC03 strain (Fig. 1e) and 10 3 Representative confocal images (left) and quantification (right) of ERRγ (white) and IAV M2 proteins (red) in bronchial epithelial cells from lungs harvested from mice challenged with the mouse-adapted IAV PR8 strain at 10 PFU (dpc: days post-challenge) (Fig. 1f); (Fig. 1g, 1h) by Western blot analysis. 5 TCID 50Quantification of ERRγ in cytoplasmic fractions, nuclear fractions, and whole cell lysates of lungs of Syrian hamsters challenged with SARS-CoV-2 KCDC03 strain (Fig. 1g) and 10 3 Quantification of ERRγ in cytoplasmic fractions, nuclear fractions and whole cell lysates of lungs of mice challenged with the mouse-adapted IAV PR8 strain at PFU (Fig. 1h).

[0013] FIGS. 2A to 2G are experimental results showing that, in one embodiment of the present invention, SARS-CoV-2 and IAV induce and transactivate ERRγ through the ROS-induced JNK / c-Jun pathway, (FIG. 2A) ROS levels at different time points in SARS-CoV-2-infected Vero E6 cells (MOI = 0.1 FFU) and IAV-infected A549 cells (MOI = 1 FFU) (normalized to the mock infection control); (FIG. 2B) Reduction of ROS levels in IAV-infected A549 cells or SARS-CoV-2-infected Vero E6 cells by N-acetylcysteine ​​(NAC) antioxidant treatment; (FIG. 2C) Reduction of luciferase activity in a plasmid containing intracellular full-length ERRγ-luc by NAC treatment in IAV-infected A549 cells or SARS-CoV-2-infected Vero E6 cells; (FIG. 2d) Decreased ERRγ expression by NAC treatment in IAV-infected and SARS-CoV-2-infected cells (GAPDH was used as a loading control); (Fig. 2e) Phosphorylation of JNK and c-Jun in response to IAV or SARS-CoV-2 infection; (Fig. 2f) Less activation of the site-specific AP1 mutant ERRγ promoter (ERRγ-AP1mut-luc) than the full-length ERRγ promoter (ERRγ-luc) by IAV and SARS-CoV-2 infection; and (Fig. 2g) Chromatin immunoprecipitation analysis showed a significant decrease in IAV and SARS-CoV-2-mediated c-Jun occupancy at the AP1 regulatory element site of the ERRγ promoter by NAC treatment.

[0014] Figures 3a to 3k are experimental results showing that, in one embodiment of the present invention, ERRγ deficiency inhibits IAV replication in vitro and in vivo, (Figure 3a) graphical representation of Western blot results (compared to the scrambled siRNA transfection control, knockdown of ERRγ by transfection with siRNA against ERRγ reduced the expression levels of ERRγ and IAV PB1 proteins in A549 cells infected with IAV PR8 strain at an MOI of 1 FFU); (Figure 3b) representative confocal images of IAV M2 protein (red) and intracellular LDs stained with BODIPY (green) in A549 cells infected with IAV PR8 strain at an MOI of 1 FFU after knockdown of ERRγ; (Figure 3c) reduction in IAV viral genome copy number by knockdown of ERRγ; (Figures 3d to 3f) 10 3 Wild-type (WT) mice and Esrrg after challenge with PFU of the mouse-adapted IAV PR8 strain (n = 16) + / - Comparison of survival rate (Fig. 3d), body weight change (Fig. 3e), and clinical score (Fig. 3f) between mice; (Figs. 3g to 3i)10 3 WT mice and Esrrg after challenge with PFU of the mouse-adapted IAV PR8 strain (n = 5) + / - Comparison of the levels of IAV PB1 protein (Fig. 3g), viral genome replication (Fig. 3h), and viral titer (Fig. 3i) in the lungs of mice; (Fig. 3j, Fig. 3k)10 3 WT mice or Esrrg after challenge with PFU of mouse-adapted IAV PR8 strain + / - Representative images of histological lesion changes in the lungs of mice (Fig. 3j) and immunohistochemical changes in viral antigen distribution (Fig. 3k) are shown.

[0015] Figures 4a to 4j are experimental results showing that, in one embodiment of the present invention, virus-induced ERRγ transactivates SREBP1c. (Figure 4a) Representative confocal images (left) and quantification (right) of SREBP1 (white) and IAV M2 protein (red) in the cytoplasm and nucleus of A549 cells infected with IAV PR8 strain at an MOI of 1 FFU (scale bar = 25 μm); (Figure 4b) Quantification of SREBP1 protein in the cytoplasmic fraction, nuclear fraction, and whole cell lysate of A549 cells infected with IAV PR8 strain at an MOI of 1 FFU by Western blot analysis; (Figure 4c) Quantification of the transcription levels of SREBP1a and SREBP1c genes in A549 cells infected with IAV PR8 strain at an MOI of 1 FFU by RT-qPCR analysis; (Figure 4d) 10 3 Representative confocal images (left) and quantification (right) of SREBP1 (white) and IAV M2 protein (red) in the cytoplasm and nucleus of bronchial epithelial cells from lungs harvested from mice challenged with the mouse-adapted IAV PR8 strain at 10 PFU (scale bar = 25 μm); (Fig. 4e) Western blot analysis. 3 Quantification of SREBP1 in cytoplasmic, nuclear, and whole-cell lysates of lungs collected from mice challenged with the mouse-adapted IAV PR8 strain at 10 PFU; (Fig. 4f) RT-qPCR analysis. 3Quantification of transcription levels of Srebp1a and Srebp1c genes in the lungs of mice challenged with the mouse-adapted IAV PR8 strain at 10 PFU; (Fig. 4g) ERRE-dependent activation of the SREBP1c promoter in IAV-infected or SARS-CoV-2-infected cells. Cells transfected with SREBP1c-luc or SREBP1c-ERREmut-luc plasmids were retransfected with Flag-ERRγ or infected with IAV (MOI = 2 FFU) or SARS-CoV-2 (MOI = 2 FFU), followed by treatment with 20 μM DN200434. (Fig. 4h) Treatment with the ERRγ inverse agonist DN200434 inhibited ERRE-dependent activation of the SREBP1c promoter in IAV or SARS-CoV-2-infected cells (SREBP1c-luc-transfected cells were transfected with Flag-ERRγ or infected with IAV (MOI = 2 FFU) or SARS-CoV-2 (MOI = 2 FFU) and then treated with 20 μM DN200434); (Fig. 4i) Blockade of ERRγ by treatment with 20 μM inverse agonist DN200434 inhibited transcriptional activity of the target SREBP1c gene in cells individually infected with each target virus; and (Fig. 4j) inhibition of ERRγ by treatment with 20 μM DN200434 reduced intracellular triacylglyceride formation compared to mock-treated controls.

[0016] Figures 5a to 5i show, in one embodiment of the present invention, (Figures 5a to 5g) the half maximum inhibitory concentration (IC 50 ) and half of the maximum cytotoxic concentration (CC 50) and the resulting selectivity index (SI) of DN200434 against seven target RNA viruses: SARS-CoV-2 KCDC03 (MOI = 0.1), influenza A virus (IAV) PR8 strain (MOI = 1 FFU), BCoV KWD strain (MOI = 1 FFU), PEDV QIAP1401 strain (MOI = 0.1 FFU), RVA NCDV strain (MOI = 0.1 FFU), PRRSV LMY strain (MOI = 0.1 FFU), and PSaV Cowden strain (MOI = 0.1 FFU) (The virus yield in the cell supernatant was quantified by cell culture immunofluorescence analysis, and the cytotoxicity of DN200434 against each cell line was measured by MTT assay) (Fig. 5h, i). Viral genome copy numbers for five different RNA viruses in infected cells (Fig. 5h) and the effect of DN200434 on the reduction of viral infectivity titer (Fig. 5i).

[0017] Figures 6a to 6r are experimental results showing the in vitro antiviral and anti-inflammatory cytokine effects of an ERRγ inverse agonist (DN200434) in one embodiment of the present invention, (Figures 6a to 6c) dose-dependent reduction in genome copy number (Figure 6a), infectious progeny production (Figure 6b), and protein synthesis (Figure 6c) by treatment of DN200434 in Vero E6 cells (MOI = 0.1 FFU) infected with SARS-CoV-2 strain KCDC03 (closely related to A-lineage viruses, which are the initial Chinese outbreak strains), strain KDCA51463 (closely related to alpha-lineage viruses including the UK variant), and strain KDCA55905 (closely related to beta-lineage strains with South African variants); (Figures 6d to 6f) A549 infected with IAV PR8 (MOI = 0.1 FFU), Dose-dependent reduction in genome copy number (Fig. 6d), infectious progeny production (Fig. 6e), and protein synthesis (Fig. 6f) in MDCK, Vero E6, and Caco-2 cells; (Fig. 6g) Inhibition of double-membrane vesicle (DMV) formation in DN200434-treated SARS-CoV-2-infected cells (MOI = 0.1 FFU) (note that there were relatively fewer perinuclear DMVs (upper and middle panels) or virus particles (lower panels) in DN200434-treated cells compared to vehicle-treated control cells (scale bars: upper panel, 1 um; lower panel, 800 nm)); (Fig. 6h) Inhibition of double-stranded RNA (dsRNA) formation in DN200434-treated SARS-CoV-2-infected cells (note that there were relatively fewer dsRNA-positive fluorescence signals in DN200434-treated cells compared to vehicle-treated control cells (scale bars = 40 μm)); (Fig. 6i, Fig. 6j) SARS-CoV-2 infected cells (MOI = 0) at 24 hpi, respectively.Representative Western blot (left) and quantification (right) of the inhibitory effect of DN200434 on palmitoylation of S protein in SARS-CoV-2 infected cells (MOI = 0.1 FFU) and HA protein in IAV infected cells (MOI = 1 FFU) at 36 hpi and 24 hpi, respectively; (Fig. 6k) Graphical representation of the inhibitory effect of DN200434 on intracellular fatty acid oxidation (FAO) activity in SARS-CoV-2 infected Vero E6 cells (MOI = 0.1 FFU) and IAV infected A549 cells (MOI = 1 FFU) treated with vehicle or DN200434. The MCP-1 level is represented graphically (each eicosanoid and cytokine level was analyzed by ELISA method).

[0018] Figures 7a to 7l are experimental results showing the restoration of free fatty acid profiles and inhibited viral replication by the addition of exogenous fatty acids in one embodiment of the present invention (Figures 7a, 7b) Heat maps showing changes in fatty acids after treatment of SARS-CoV-2 infected Vero E6 cells (MOI = 0.1 FFU) and IAV infected A549 cells (MOI = 1 FFU) with DN200434 (cells were mock infected or infected with the virus and treated with vehicle or 20 μM DN200434. Incubation times varied depending on the virus, 12 and 24 hours for SARS-CoV-2 experiments and 36 hours for IAV experiments. Higher than normal fatty acid concentrations in the rectangles are indicated in red, and lower than normal are indicated in blue. TMS: trimethylsilyl ester. *: representative fatty acids for further analysis); (Figures 7c to 7d) 7h) DN200434 inhibited the synthesis of representative fatty acids induced by SARS-CoV-2 infection (palmitic acid (Fig. 7c), oleic acid (Fig. 7d), α-linoleic acid (Fig. 7e), steric acid (Fig. 7f), arachidonic acid (Fig. 7g), and 13-eicosenoic acid (Fig. 7h)); (Figs. 7i to 7l) Individual supplementation of exogenous palmitic acid, oleic acid, linoleic acid, or arachidonic acid in the FFA-deficient state showed an increase in the viral genome copy number and progeny number of SARS-CoV-2 (Figs. 7i and 7j) and IAV (Figs. 7k and 7l).

[0019] Figures 8a to 8l are experimental results showing the in vivo antiviral effect of an ERRγ agonist (DN200434) against SARS-CoV-2 and IAV in one embodiment of the present invention, (Figure 8a) Syrian hamsters (n = 5) were exposed to 10 5 TCID 50Schematic diagram of twice daily chemical administration for 4 and a half consecutive days after challenge inoculation with SARS-CoV-2 KCDC03 strain; (Fig. 8b) Reduction of SARS-CoV-2-induced macroscopic lung lesions in hamsters by DN200434 treatment; (Fig. 8c) Effect of DN200434 treatment on body weight recovery in each group; (Fig. 8d) Representative images of dose-dependent reduction of LD (green, lower panel) and SARS-CoV-2 replication (red, middle panel) stained with BODIPY in alveolar cells of lung tissues collected from DN200434-treated SARS-CoV-2 challenged hamsters; (Fig. 8e-g) Triacylglycerides (Fig. 8e), viral genome copy number (Fig. 8f), and infectious progeny in lungs collected from DN200434-treated SARS-CoV-2-infected hamsters (n = 5). Graphical representation of dose-dependent reduction in viral titer (Fig. 8g); (Fig. 8h) Representative images of histological lesion changes in the lungs (upper panel) and immunohistochemical SARS-CoV-2 antigen distribution (lower panel); (Fig. 8i)10 5 TCID 50 Schematic illustration of challenge vaccination of Syrian hamsters (n = 5) with SARS-CoV-2 KCDC03 (closely related to the initial Chinese strain), KDCA51463 (alpha strain with UK variant), and KDCA55905 (beta strain with South African variant) followed by individual or combined chemical administration of DN200434 and remdesivir twice daily for 4.5 consecutive days; (Fig. 8j) Reduction of gross lung lesions in Syrian hamsters challenged with each strain with combination therapy of DN200434 and remdesivir; (Fig. 8k)10 3 After challenge with the mouse-adapted IAV PR8 strain of PFU in mice (n = 16), treatment with DN200434 and oseltamivir twice daily for 4 consecutive days, either individually or in combination, followed by a chemical dosing scheme; (Fig. 8l) Survival rate (expressed as a percentage) of mice challenged with IAV is shown.

[0020] FIGS. 9A to 9J are experimental results showing that, in one embodiment of the present invention, virus-induced ERRγ mediates SREBP1c-dependent fatty acid biosynthesis in virus-infected cells, (FIG. 9A) ERRE-dependent activation of the SREBP1c promoter in virus-infected cells (cells were transformed with SREBP1c-luc or SREBP1c-ERREmut-luc plasmids and infected with BCoV KWD strain at an MOI of 0.1 FFU, PEDV QIAP1401 strain at an MOI of 0.1 FFU, RVA NCDV strain at an MOI of 0.1 FFU, PRRSV LMY strain at an MOI of 0.1 FFU, and PSaV Cowden strain at an MOI of 0.1 FFU); (FIG. 9B) Treatment with DN200434, an inverse agonist of ERRγ, induced ERRE-dependent activation of the SREBP1c promoter in virus-infected cells. Inhibition (cells were transformed with SREBP1c-luc plasmid infected with BCoV KWD strain (MOI = 0.1 FFU), PEDV QIAP1401 strain (MOI = 0.1 FFU), RVA NCDV strain (MOI = 0.1 FFU), PRRSV LMY strain (MOI = 0.1 FFU), and PSaV Cowden strain (MOI = 0.1 FFU) or remained mock infected and then treated with DN200434 at a concentration of 20 μM); (Fig. 9c) Graphical representation of western blot results (transfection of ERRγ siRNA decreased SREBP1 protein levels in cells infected with each target virus individually); (Figs. 9d-9f) RT-qPCR analysis of mRNA of genes involved in fatty acid biosynthesis (inhibition of ERRγ by treatment with DN200434 at a concentration of 20 μM was observed compared to the vehicle-treated control. Blockade of mRNA expression of FASN (Fig. 9d), DGAT (Fig. 9e) and SCD1 (Fig. 9f); (Fig. 9g) Inhibition of ERRγ by treatment with DN200434 at a concentration of 20 μM was observed in BCoV KWD strain (MOI = 0) compared to mock-treated virus-infected controls.TAG formation was reduced in cells individually infected with IAV (MOI = 1 FFU), PEDV QIAP1401 strain (MOI = 0.1 FFU), RVA NCDV strain (MOI = 0.1 FFU), PRRSV LMY strain (MOI = 0.1 FFU), and PSaV Cowden strain (MOI = 0.1 FFU); (Fig. 9h) RT-qPCR analysis of mRNA of SREBP1a gene (Blocking ERRγ by treatment with 20 μM inverse agonist DN200434 did not affect transcriptional activity of SREBP1α gene in cells infected with IAV (MOI = 1 FFU) or SARS-CoV-2 (MOI = 0.1 FFU); (Fig. 9i)10. 3 WT mice and Esrrg after challenge with PFU of the mouse-adapted IAV PR8 strain (n = 5) + / - Comparison of TAG levels in the lungs of mice; (Fig. 9j)10 3 WT mice or Esrrg after challenge with mouse-adapted IAV PR8 strain at 10 PFU + / - Representative confocal images of BODIPY-stained LD and IAV M2 proteins (red) in bronchial epithelial cells of mouse lung (scale bar = 25 μm) are shown.

[0021] Figures 10a to 10j are experimental results showing the in vivo antiviral effect of DN200434 against IAV in one embodiment of the present invention, (Figure 10a)10 3Schematic illustration of mice (n = 16) challenged with PFU of the mouse-adapted IAV PR8 strain followed by twice-daily administration of chemicals for 4 consecutive days; (Figs. 10b-10d) Comparison of survival (Fig. 10b), body weight change (Fig. 10c), and clinical score (Fig. 10d) between mock- and DN200434-treated IAV-challenged mice; (Fig. 10e) Representative confocal images of dose-dependent reduction of LD (green) and IAV replication (red) stained with BODIPY in bronchial epithelial cells of lung tissues collected from IAV-challenged mice treated with DN200434; (Figs. 10f-h) Graph surfaces of dose-dependent reductions in TAG (Fig. 10f), viral genome copy number (Fig. 10g), and infectious progeny titer (Fig. 10h) in lungs collected from IAV challenged mice (n=5) treated with DN200434; (Figs. 10i, 10j) Representative images of histological lung lesions (Fig. 10i) and immunohistochemical antigen distribution of IAV (Fig. 10j) showing improvement in histological lung lesions and inhibition of IAV replication by DN200434 treatment.

[0022] FIGS. 11A to 11G are experimental results showing the in vivo anti-eicosanoid and anti-proinflammatory cytokine effects of DN200434 against SARS-CoV-2 and IAV infection in one embodiment of the present invention, wherein (FIGS. 11A to 11G) the graph surfaces represent the levels of LTB4 (FIG. 11A), PGE2 (FIG. 11B), IFN-α (FIG. 11C), IFN-β (FIG. 11D), IL-6 (FIG. 11E), TNF-α (FIG. 11F), and MCP-1 (FIG. 11G) in bronchoalveolar lavage fluid (BALF) collected from vehicle-treated or DN200434-treated hamsters challenged with SARS-CoV-2 or mice challenged with IAV.

[0023] FIGS. 12a to 12e are experimental results showing weight loss and improvement of clinical signs by DN200434 treatment in hamsters challenged with SARS-CoV-2 and mice challenged with IAV in one embodiment of the present invention, (FIGS. 12a to 12c) treatment effects on weight recovery in groups challenged with SARS-CoV-2 KCDC03 strain (FIG. 12a), challenged with KDCA51463 strain (FIG. 12b), and challenged with KDCA55905 strain (FIG. 12c); (FIGS. 12d, 12e) treatment effects on weight recovery (FIG. 12d) and reduction of clinical signs (FIG. 12e) in each group.

[0024] Hereinafter, with reference to the attached drawings, implementation examples and embodiments of the present invention will be described in detail so that those skilled in the art can easily practice the present invention. However, the present invention may be implemented in various different forms and is not limited to the implementation examples and embodiments described herein. In addition, in the drawings, parts irrelevant to the description have been omitted to clearly explain the present invention, and similar parts have been designated with similar drawing reference numerals throughout the specification.

[0025] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0026] The terms "about," "substantially," and the like used in this specification are used in a meaning that is at or close to the numerical value when manufacturing and material tolerances inherent in the meanings mentioned are presented, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosure in which exact or absolute values ​​are mentioned to aid understanding of the present application.

[0027] The terms “step of ~” or “step of ~” as used throughout this specification do not mean “step for ~.”

[0028] Throughout this specification, the term "combination(s) thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0029] Throughout this specification, references to “A and / or B” mean “A or B, or A and B.”

[0030] As used throughout this specification, the term "alkyl" or "alkyl group" includes linear or branched alkyl groups having 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 5 carbon atoms and all possible isomers thereof. For example, the alkyl or alkyl group may be a methyl group (Me), an ethyl group (Et), an n-propyl group ( n Pr), iso-profiler ( i Pr), n-butyl group ( n Bu), iso-butyl group( i Bu), tert-butyl group (tert-Bu, t Bu), sec-butyl group (sec-Bu, sec Bu), n-pentyl group ( n Pe), iso-pentyl group ( iso Pe), sec-pentyl group ( sec Pe), tert-pentyl group ( t Pe), neo-pentyl group ( neo Pe), 3-pentyl group, n-hexyl group, iso-hexyl group, heptyl group, 4,4-dimethylpentyl group, octyl group, 2,2,4-trimethylpentyl group, nonyl group, decyl group, undecyl group, dodecyl group, and isomers thereof, but may not be limited thereto.

[0031] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise stated.

[0032] Throughout this specification, three independent experiments were conducted under identical conditions to increase the reliability of the results. The values ​​for each group were converted to mean ± standard error. The statistical significance of the results was analyzed using a one-way ANOVA test (GraphPad Prism software version 8.4.2), and a p value of 0.05 or higher was considered statistically significant.

[0033] Below, the implementation examples of the present invention are described in detail, but the present invention may not be limited thereto.

[0034] One aspect of the present invention provides an antiviral pharmaceutical composition comprising an ERRγ (estrogen related receptor γ) inhibitor as an active ingredient.

[0035] The inventors of the present invention found that in virus-infected cells, especially in cells infected with RNA viruses, ERRγ increases from the early to mid-stage of infection and translocates into the nucleus, and consequently, ERRγ transcribes sterol regulatory element-binding protein 1c (SREBP1c) to activate fatty acid (FA) biosynthesis, thereby causing reprogramming of host cell physiology essential for virus replication.

[0036] The inventors of the present invention have confirmed from the above findings that inhibition of ERRγ exhibits broad-spectrum antiviral efficacy, and have invented an antiviral pharmaceutical composition comprising the ERRγ (estrogen-related receptor γ) inhibitor of the present invention as an active ingredient. Therefore, throughout the specification of the present invention, the term "ERRγ inhibitor" may be used to collectively refer to all agents that reduce ERRγ expression at the transcriptional level or interfere with ERRγ activity, thereby reducing the expression or activity of ERRγ.

[0037] Throughout this specification, the term "broad spectrum antiviral efficacy" means inhibiting the replication of viruses, particularly all RNA viruses, regardless of whether the viruses have an envelope, whether the genes are positive-sense, negative-sense, or double-sense, and whether the genes are single-stranded linear or segmented.

[0038] In one embodiment of the present invention, the ERRγ inhibitor may inhibit the activity of an ERRγ protein or inhibit the expression of an ERRγ gene.

[0039] In one embodiment of the present invention, the ERRγ inhibitor may exhibit antiviral efficacy by inhibiting the activity of ERRγ in virus-infected cells.

[0040] In one embodiment of the present invention, the ERRγ inhibitor may include siRNA specific to ERRγ.

[0041] In one embodiment of the present invention, the siRNA is a nucleic acid molecule capable of mediating RNA interference or gene silencing, and can suppress the expression of a target gene, and thus can be used as an efficient gene knockdown method or gene therapy method.

[0042] In one embodiment of the present invention, the ERRγ inhibitor may include an ERRγ inverse agonist.

[0043] In one embodiment of the present invention, the ERRγ agonist may include a compound represented by the following chemical formula 1:

[0044] [Chemical Formula 1]

[0045] ,

[0046] In the above chemical formula 1,

[0047] L is (C6-C 20 )arylene, (C3-C 20 )heteroarylene, or (C3-C 20 ) is a fused heterocycle;

[0048] R 1 Silver (C3-C 20 )heterocycloalkyl, (C3-C 20 )heteroaryl, -O-(CH2) m -R 11 , -(CH2) m -R 12 , -NH-(CH2) m -R 13 , -NHCO-(CH2) n -R 14 , or -SiR 16 R 17 -(CH2) m -R 15 and;

[0049] R 11 Inland R 15 are each independently (C3-C 20 ) is heterocycloalkyl;

[0050] R 16 and R 17 are each independently (C1-C 20 ) is alkyl;

[0051] m is an integer from 1 to 3; and

[0052] n is an integer of 0 or 1;

[0053] Ar is (C6-C 20 )aryl or (C3-C 20 ) is heteroaryl,

[0054] The aryl or heteroaryl of the above Ar is hydroxy, halogen, (C1-C 20 )alkyl, halo(C1-C 20 )alkyl, (C1-C 20 )alkoxy, nitro, cyano, -NR 21 R 22 , (C1-C 20 )alkylcarbonyloxy, (C1-C 20 )alkylcarbonylamino, guanidino, -SO2-R 23 and -OSO2-R 24 is substituted or unsubstituted with one or more substituents selected from

[0055] R 21 and R 22 are each independently hydrogen, (C1-C 20 )alkylsulfonyl or (C3-C 20 ) is cycloalkylsulfonyl;

[0056] R 23 and R 24 are each independently (C1-C 20 )alkyl, halo(C1-C 20 )alkyl or (C3-C 20 ) is cycloalkyl;

[0057] R 2 is hydroxy, halogen, (C1-C 20 )alkylcarbonyloxy or (C1-C 20 )alkylsulfonyloxy;

[0058] The above R 1Heterocycloalkyl or heteroaryl of and the above R 11 Inland R 15 Heterocycloalkyl of (C1-C 20 )alkyl, (C3-C 20 )cycloalkyl, (C2-C 20 )alkenyl, amidino, (C1-C 20 )alkoxycarbonyl, hydroxy, hydroxy(C1-C 20 )alkyl and di(C1-C 20 )alkylamino(C1-C 20 ) is substituted or unsubstituted with one or more substituents selected from alkyl, and

[0059] The above heterocycloalkyl and heteroaryl contain one or more heteroatoms selected from N, O and S,

[0060] The above heterocycloalkyl is a saturated or unsaturated mono-, bi- or spirocycle having a carbon atom or nitrogen atom within the ring as a bonding site.

[0061] In one embodiment of the present invention, the ERRγ agonist may include a compound represented by the following chemical formula 2:

[0062] [Chemical Formula 2]

[0063] ,

[0064] In the above chemical formula 2,

[0065] R 1 Silver (C3-C 20 )heterocycloalkyl, (C3-C 20 )heteroaryl, -O-(CH2) m -R 11 , -(CH2) m -R 12 , -NH-(CH2) m -R 13 , -NHCO-(CH2) n -R 14 , or -SiR 16 R 17 -(CH2)m -R 15 and;

[0066] R 11 Inland R 15 are each independently (C3-C 20 ) is heterocycloalkyl;

[0067] R 16 and R 17 are each independently (C1-C 20 ) is alkyl;

[0068] m is an integer from 1 to 3; and

[0069] n is an integer of 0 or 1;

[0070] Ar is (C6-C 20 )aryl or (C3-C 20 ) is heteroaryl,

[0071] The aryl or heteroaryl of the above Ar is hydroxy, halogen, (C1-C 20 )alkyl, halo(C1-C 20 )alkyl, (C1-C 20 )alkoxy, nitro, cyano, -NR 21 R 22 , (C1-C 20 )alkylcarbonyloxy, (C1-C 20 )alkylcarbonylamino, guanidino, -SO2-R 23 and -OSO2-R 24 is substituted or unsubstituted with one or more substituents selected from

[0072] R 21 and R 22 are each independently hydrogen, (C1-C 20 )alkylsulfonyl or (C3-C 20 ) is cycloalkylsulfonyl;

[0073] R 23 and R 24 are each independently (C1-C 20 )alkyl, halo(C1-C 20 )alkyl or (C3-C 20 ) is cycloalkyl;

[0074] R 2 is hydroxy, halogen, (C1-C 20 )alkylcarbonyloxy or (C1-C 20 )alkylsulfonyloxy;

[0075] The above R 1 Heterocycloalkyl or heteroaryl of and the above R 11 Inland R 15 Heterocycloalkyl of (C1-C 20 )alkyl, (C3-C 20 )cycloalkyl, (C2-C 20 )alkenyl, amidino, (C1-C 20 )alkoxycarbonyl, hydroxy, hydroxy(C1-C 20 )alkyl and di(C1-C 20 )alkylamino(C1-C 20 ) is substituted or unsubstituted with one or more substituents selected from alkyl, and

[0076] The above heterocycloalkyl and heteroaryl contain one or more heteroatoms selected from N, O and S,

[0077] The above heterocycloalkyl is a saturated or unsaturated mono-, bi- or spirocycle having a carbon atom or nitrogen atom within the ring as a bonding site.

[0078] In one embodiment of the present invention, the ERRγ antagonist may include, but may not be limited to, DN200434 and / or GSK5182.

[0079] In one embodiment of the present invention, the DN200434 may be represented by the following chemical formula 3:

[0080] [Chemical Formula 3]

[0081] .

[0082] In one embodiment of the present invention, the GSK5182 may be represented by the following chemical formula 4:

[0083] [Chemical Formula 4]

[0084] .

[0085] In one embodiment of the present invention, the pharmaceutical composition may be for the prevention or treatment of RNA virus infection.

[0086] In one embodiment of the present invention, the RNA virus is selected from the group consisting of SARS-CoV-1, SARS-CoV-2, influenza A virus (IAV), influenza B virus, bovine coronavirus (BCoV), porcine epidemic diarrhea coronavirus (PEDV), bovine species A rotavirus (RVA), porcine reproductive and respiratory syndrome virus (PRRSV), porcine sapovirus (PSaV), norovirus (NoV), feline coronavirus (FIPV), Middle East respiratory syndrome coronavirus (MERS-related coronavirus; MERS-CoV), zika virus (ZIKV), dengue virus, human torovirus (HuTV), hepatitis A virus (HAV), and hepatitis C virus. It may include one or more selected from the group consisting of C virus; HCV).

[0087] Here, the term "coronavirus" in this specification refers to four genera of alpha-, beta-, gamma-, and delta-coronavirus in the Orthocoronavirinae subfamily of the Coronaviridae family. Alpha-coronavirus includes PEDV, FIPV, etc., beta-coronavirus includes BCoV, SARS-CoV-1, SARS-CoV-2, MERS-CoV, gamma-coronavirus includes chicken infectious bronchitis virus (IBV), and delta-coronavirus includes human, pig, etc. Coronaviruses are 80-120 nm in size, have spikes on their outer membrane, and bovine coronaviruses in the gamma-coronavirus genus have one more hemagglutinin / esterase protein spike on their outer membrane. The outer membrane contains a positive-sense, single-stranded RNA genome.

[0088] Here, "influenza A virus" refers to a virus of the genus Alphainfluenzavirus within the family Orthomyxoviridae, and is a virus measuring 80-120 nm in size. The virus has hemagglutinin and neuraminidase spikes oriented outward on the surface of its outer membrane, and contains eight segmented negative-sense genome segments within the outer membrane. Influenza A virus infects mammals such as humans, pigs, horses, and dogs, as well as various birds including chickens.

[0089] Here, "influenza B virus" refers to a virus of the genus Betainfluenzavirus within the family Orthomyxoviridae, and is a virus measuring 80 to 120 nm in size. The virus has an outer membrane surface with hemagglutinin and neuraminidase spikes oriented outward, and the outer membrane contains eight segmented negative-sense genome segments. Unlike influenza A viruses, influenza B viruses infect only a small number of animal species, including humans, martens, pigs, and seals.

[0090] Here, "BCoV" is a virus that causes severe diarrhea in calves and adult cattle worldwide.

[0091] Here, "PEDV" is a virus that causes diarrhea in piglets of all ages worldwide and a mortality rate of more than 60% in suckling piglets.

[0092] Here, "rotavirus" generally refers to species A rotavirus (RVA). There are nine genera of Rotavirus within the Reoviridae family: A, B, C, D, F, G, H, I, and J. Of these, species A rotavirus primarily infects humans, while species AI rotavirus causes outbreaks in animals other than humans. Type H has been reported to infect pigs, types D, F, and G in birds, type I in cats, and type J in bats. Rotavirus has a double-stranded genome with 11 segments within three capsid layers measuring 80 nm in size. Rotavirus is a major cause of diarrhea in humans, infants under 5 years of age, and young livestock. For example, the rotavirus may include bovine species A rotavirus (RVA).

[0093] Here, "PRRSV" refers to a virus within the genus Arterivirus within the family Arteriviridae, and is classified into type 1 (European type) and type 2 (North American type). PRRSV has an outer membrane measuring 50-60 nm, with envelope protein and glycoprotein 4 spikes embedded in it, and contains a single-stranded, positive-sense RNA genome of approximately 15 kb in length. PRRSV causes reproductive disorders such as miscarriage and stillbirth in pregnant sows, and interstitial pneumonia in piglets.

[0094] Here, "sapovirus" refers to a virus belonging to the genus Sapovirus in the family Caliciviridae, which is a causative agent of diarrhea in humans, pigs, and minks.

[0095] Here, "FIPV" is a viral agent that causes vasculitis in cats, resulting in severe pleural effusion and ascites, or chronic granulomatous nodules in various organs, including the kidneys.

[0096] Here, "caliciviruses" are viruses within the Caliciviridae family that have a linear, positive-sense, single-stranded RNA genome within a capsid layer measuring 27-40 nm. There are currently 11 genera within the Caliciviridae family: Norovirus, Sapvorius, Vesivirus, Lagovirus, Nebovirus, Recovirus, Valovirus, Babovirus, Nacovirus, Salovirus, and Minovirus.

[0097] Here, "torovirus" is a virus within the Torovirinae subfamily of the Tobaniviridae family. The Torovirinae subfamily includes bovine torovirus (BToV), equine torovirus (EToV), porcine torovirus (PToV), and human torovirus (HuTV). Toroviruses are 100-150 nm in size, and their outer membrane is studded with spikes and hemagglutinin / esterase protein spikes. The outer membrane contains a positive-sense, single-stranded RNA genome.

[0098] In one embodiment of the present invention, the pharmaceutical composition may further comprise an antiviral agent.

[0099] In one embodiment of the present invention, the antiviral agent may include at least one selected from oseltamivir and remdesivir.

[0100] In one embodiment of the present invention, when the antiviral agent is oseltamivir, the volume ratio of the ERRγ agonist and the oseltamivir may be about 2:2 to about 8:2, but may not be limited thereto.

[0101] In one embodiment of the present invention, when the antiviral agent is oseltamivir, the volume ratio of the ERRγ agonist and the oseltamivir may be about 2:2 to about 8:2, preferably about 3:2 to about 7:2, or more preferably about 4:2 to about 6:2, but may not be limited thereto.

[0102] In one embodiment of the present invention, when the antiviral agent is remdesivir, the volume ratio of the ERRγ agonist and the remdesivir may be about 8:1 to about 20:1, but may not be limited thereto.

[0103] In one embodiment of the present invention, when the antiviral agent is remdesivir, the volume ratio of the ERRγ inverse agonist and the remdesivir may be about 8:1 to about 20:1, preferably about 10:1 to about 18:1, or more preferably about 15:1 to about 16:1, but may not be limited thereto.

[0104] Hereinafter, the present invention will be described in more detail using examples. However, the following examples are provided only to help understand the present invention, and the contents of the present invention are not limited to the following examples.

[0105] [Example]

[0106] 1. Materials and Methods

[0107] Cells and viruses

[0108] Vero E6, MDCK, LLC-PK, MA104, A549, Caco-2, MARC-145, and HRT-18G cells were cultured in EMEM, α-MEM, or DMEM at 37°C in 5% CO2. All media were supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. In the present invention, SARS-CoV-2 (strains KCDC03, KDCA51463, and KDCA55905), IAV [PR8 / 34 (H1N1) strain], BCoV (strain KWD20), PEDV (strain QIAP1401), PRRSV (strain LYM), RVA (strain NCDV), and PSaV (strain Cowden) were used.

[0109] 2. Results

[0110] (1) The dynamics of ERRγ expression are common in RNA virus infections.

[0111] Although specific nuclear receptors (NRs) have been reported to exhibit proviral, antiviral, or dual activities depending on the virus type and its target host, the role of estrogen-related receptors (ERRs) in viral infection remains unknown. Before investigating the association between ERRs and viral replication, we assessed the sequential expression levels of ERRs during replication of seven target RNA viruses. In cells infected with SARS-CoV-2 and IAV, ERRγ protein expression exhibited distinct kinetics, gradually increasing and then decreasing during viral replication (Figures 1A and 1B). Notably, ERRγ, which increased in response to viral replication, translocated into the nucleus (Figures 1A to 1D), suggesting that it may play an important role in viral infection. Other target RNA viruses, including influenza A virus, bovine coronavirus (BCoV), porcine epidemic diarrhea coronavirus (PEDV), bovine species A rotavirus (RVA), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine sapovirus (PSaV), also showed similar ERRγ expression patterns in infected cells.

[0112] To determine whether ERRγ dynamics were also observed in virus-infected animals, we examined alveolar pneumocytes from hamsters challenged with SARS-CoV-2 and bronchiolar epithelial cells from mice challenged with IAV. Results revealed that ERRγ expression gradually increased early in infection and then decreased later (Figures 1e and 1f). Importantly, enhanced translocation of ERRγ from the cytoplasm to the nucleus was observed in infected animals (Figures 1e to 1h). Concomitantly, ERRγ mRNA levels in the lungs of SARS-CoV-2- and IAV-infected animals showed a trend similar to the ERRγ protein levels observed in SARS-CoV-2- and IAV-infected cells. Interestingly, ERRα mRNA levels were unaffected during viral replication. These results suggest that the biphasic expression pattern of ERRγ is a common feature of RNA virus infection.

[0113] (2) Upregulation of ERRγ through activation of the JNK / c-Jun signaling pathway induced by ROS.

[0114] Next, the present invention aimed to elucidate the pathway by which viral infection upregulates ERRγ expression. Oxidative stress induced by ROS is a common pathophysiological feature of various viral infections. Notably, ERRγ has been reported to function as a ROS sensor, suggesting that virus-induced ROS induction could potentially upregulate ERRγ. In the present study, IAV-infected cells exhibited a maximum increase in ROS levels at 8 hours post-infection (hpi) and remained elevated until 12 hpi (Fig. 2a). SARS-CoV-2-infected cells exhibited a steady increase in ROS levels up to 12 hpi (Fig. 2a). Furthermore, treatment with the antioxidant N-acetylcysteine ​​(NAC) significantly inhibited virus-induced ROS production (Fig. 2b).

[0115] To determine whether virus-induced ROS actually transactivates ERRγ, cells were either mock-transfected or transfected with a plasmid containing the full-length ERRγ promoter-luciferase gene (ERRγ-luc). Cells under these conditions were infected with IAV or SARS-CoV-2 in the presence or absence of NAC. Both viruses significantly activated the ERRγ promoter and enhanced ERRγ expression, and in both cases, NAC treatment attenuated this activation (Figures 2C and 2D). Since the ERRγ promoter is known to be activated by the JNK / c-Jun signaling pathway, we investigated whether virus-induced ERRγ activation occurred through this pathway. Interestingly, IAV infection or SARS-CoV-2 infection induced phosphorylation of both JNK (p-JNK) and c-Jun (pc-Jun) at 4 and 8 hpi, respectively (Fig. 2e). Furthermore, mutation of the c-Jun binding element AP1 of the ERRγ promoter (ERRγ-AP1mut-luc) reduced ERRγ-luc activity in cells infected with SARS-CoV-2 or IAV (Fig. 2f). Chromatin immunoprecipitation (ChIP) analysis showed that both viruses increased c-Jun occupancy at the AP1 regulatory element site of the ERRγ promoter, an effect that was significantly blocked by NAC treatment (Fig. 2g). These data suggest that ERRγ transactivation induced by virus infection primarily occurs through the ROS / JNK / c-Jun signaling pathway, culminating in c-Jun directly activating ERRγ expression.

[0116] (3) ERRγ deficiency renders mice resistant to IAV replication in vitro and in vivo.

[0117] To directly investigate the role of ERRγ in viral replication, we first knocked down ERRγ in vitro and infected cells with the target virus. Knockdown of ERRγ had a significant inhibitory effect on the expression levels of viral proteins and the number of viral genomes (Figs. 3a to 3c). Subsequently, a loss-of-function approach was used to determine whether ERRγ knockout (KO) mice exhibited resistance to IAV infection. Because homozygous ERRγ-null mice die shortly after birth, heterozygous mice (Esrrg + / - ) was utilized. This Esrrg + / - Mice showed reduced levels of ERRγ expression in the lungs compared to wild-type (WT) controls. Considering the unavailability of double transgenic mice with concurrent ERRγ heterozygosity combined with human ACE2, which is required for SARS-CoV-2 challenge studies, the IAV PR8 / 34 (H1N1) strain was selected as a representative RNA virus that infects WT mice in the present invention. 10 3 Intranasal challenge of WT mice with PFU of the IAV PR8 strain resulted in 100% mortality within 10 days post-challenge, with severe weight loss and clinical signs (Figures 3d–f). In contrast, Esrrg + / -Mice showed a significantly reduced mortality rate (50%), maintained body weight, and exhibited less severe clinical signs (Figures 3d–3f). Furthermore, compared to WT mice, ERRγ deficiency resulted in decreased IAV protein synthesis, viral genome replication, and progeny virus production in the lungs (Figures 3g–3i). Histopathological lung lesions induced by IAV infection in WT mice were significantly improved in ERRγ-deficient mice (Figure 3j), which was attributed to a decrease in IAV antigen-positive cells (Figure 3k). These data strongly suggest that ERRγ promotes RNA virus replication.

[0118] (4) Virus-induced ERRγ transactivates SREBP1c, thereby inducing FA biosynthesis required for viral replication.

[0119] In a previous study, we established ERRγ as a transcriptional regulator of SREBP1c, a key transcription factor that activates FA biosynthesis, and confirmed that its dysregulation contributes to various fatty liver diseases. As previously reported, we confirmed that RNA virus infection can stimulate lipid droplet (LD) formation through lipogenesis in vitro and in vivo. To investigate whether ERRγ regulates viral infection through the regulation of SREBP1c, we first examined the dynamics of SREBP1c levels in virus-infected cells. Similar to the dynamics of ERRγ, we observed that both SREBP1 protein and SREBP1c transcript levels increased early in infection and then decreased later in infection in cells infected with seven target RNA viruses, including SARS-CoV-2 or IAV (Figures 4a-4c). Notably, the increased SREBP1 was subsequently translocated to the nucleus in RNA virus-infected cells. This pattern was also evident in lung samples from mice challenged with IAV (Figures 4d–f) and hamsters challenged with SARS-CoV-2. However, activation of the SREBP1a isoform was not observed in vitro or in vivo (Figures 4c and 4f).

[0120] Next, we sought to determine whether ERRγ directly regulates the SREBP1c gene in virus-infected cells. We transfected cells with a luciferase reporter vector for the human SREBP1c gene promoter (SREBP1c-luc) and subsequently infected the cells with each target RNA virus. SREBP1c promoter activity was significantly enhanced by infection with all seven target viruses, similar to the positive control for ERRγ overexpression (Flag-ERRγ) (Fig. 4g, Fig. 9a). In contrast, activity was significantly reduced in cells infected with a mutated ERR response element (ERRE) in the SREBP1c gene (SREBP1c-ERREmut-luc) (Fig. 4g, Fig. 9a). Furthermore, treatment with DN200434, an ERRγ inverse agonist, significantly suppressed SREBP1c promoter activity in cells infected with each individual virus (Fig. 4h, Fig. 9b). Furthermore, both DN200434 and ERRγ siRNAs reduced SREBP1c expression activated by individual viral infections (Fig. 4i, Fig. 9c). These findings suggest that ERRγ directly transactivates the SREBP1c gene in response to RNA virus infection.

[0121] In addition to ERRγ-mediated transcriptional activation of SREBP1c, we observed that virus infection markedly increased the mRNA expression of lipogenic genes, including FASN (encoding fatty acid synthase), DGAT1 (encoding diacylglycerol O-acyltransferase 1), and SCD1 (encoding stearoyl-CoA desaturase 1) (Figs. 9d-9f), and increased intracellular triacylglycerol (TAG) levels (Figs. 4j, 9g). DN200434 treatment effectively attenuated lipogenic gene activation and lipid accumulation in virus-infected cells (Figs. 4j, 9c-9g). However, the basal level of SREBP1a mRNA in SARS-CoV-2 or IAV-infected cells was not affected by DN200434 treatment (Fig. 9h). In addition, Esrrg + / - We investigated whether the resistance to IAV infection observed in mice could be due to a failure of ERRγ-activated FA biosynthesis. As a result, ERRγ knockout suppressed SREBP1c activation, resulting in reduced TAG levels and intracellular LD formation (Figs. 9i and 9j).

[0122] To investigate the role of SREBP1c in in vivo viral replication, Srebp1c KO mice were used. PCR genotyping and immunoblotting of lung samples from Srebp1c KO mice confirmed that Srebp1c was knocked out. Notably, the 100% mortality observed in WT mice challenged with IAV was reduced to 25% in IAV-infected Srebp1c KO mice. Furthermore, Srebp1c KO mice challenged with IAV showed significantly improved weight loss and clinical signs 4 days after challenge compared to WT mice challenged with IAV. In the absence of SREBP1c protein, viral protein synthesis, genome replication, and progeny production were all significantly reduced, accompanied by a dramatic decrease in LD formation and TAG production. Concurrently, IAV-infected Srebp1c KO mice exhibited reduced histopathological lung lesions, such as bronchopneumonia and interstitial inflammatory infiltrates, and substantially reduced viral replication in bronchioles and alveoli compared to WT IAV-infected mice. Taken together, these findings highlight that a sequential pathway, beginning with ROS activation of ERRγ, followed by activation of SREBP1c, increased FA biosynthesis, and subsequent LD accumulation, is essential for RNA virus replication.

[0123] (5) In vitro broad-spectrum antiviral activity of DN200434

[0124] Previous studies have shown that inhibition of ERRγ inhibits replication of various RNA viruses. Therefore, we evaluated the antiviral efficacy of DN200434, an ERRγ inverse agonist, against in vitro replication of seven target RNA viruses, including SARS-CoV-2, IAV, BCoV, PEDV, RVA, PRRSV, and PSaV. To evaluate the performance, half maximal cytotoxic concentration (CC) was used. 50 ), half maximal inhibitory concentration (IC 50 ), and selectivity index (SI) were measured. Surprisingly, DN200434 showed CC 50 IC for target RNA virus at substantially lower micromolar concentrations 50 The values ​​were shown (Fig. 5). Specifically, IC 50 The values ​​ranged from 1.86 ± 0.80 μM for SARS-CoV-2 to 6.24 ± 1.07 μM for PSaV. These results yielded significant SI values ​​of 50 SI for SARS-CoV-2 and 34 SI for IAV (Fig. 5), suggesting the broad antiviral potential of DN200434.

[0125] In addition, we examined whether DN200434 inhibited the SARS-CoV-2 prototype and its mutant strains. In a dose-dependent manner, DN200434 effectively inhibited genome replication, progeny production, and protein synthesis of the SARS-CoV-2 prototype strain of lineage A and two mutant strains of lineages alpha and beta (Figs. 6a to 6c). At the maximum dose of 20 μM, the viral genome copy numbers and titers of BCoV within the genus Betacoronavirus and PEDV within the genus Alphacoronavirus were 2.0-Log 10The levels were significantly reduced (Figures 5h and 5i). These findings suggest that DN200434 has a broad-spectrum anti-coronavirus effect.

[0126] Furthermore, we investigated the efficacy of DN200434 against IAV, a persistent threat to human health and the potential to cause lethal pandemics. Interestingly, treatment with DN200434 dose-dependently inhibited viral genome replication, progeny production, and protein synthesis in multiple IAV-infected cell lines, including human lung epithelial (A549) and intestinal (Caco-2) cells, canine kidney epithelial (MDCK) cells, and monkey kidney epithelial (Vero E6) cells, demonstrating the anti-influenza virus efficacy of DN200434 in both human and animal cells (Figures 6d-6f). Collectively, these data provide strong evidence for the broad-spectrum antiviral potential of DN200434 in vitro, specifically targeting SARS-CoV-2 and IAV.

[0127] (6) Role of DN200434 in controlling in vitro viral replication and pathogenicity

[0128] Positive-sense RNA viruses rely on cellular lipid components to build membrane structures called viral factories. These structures serve as anchoring scaffolds for viral replication and transcription complexes, such as double-membrane vesicles (DMVs) induced by coronaviruses. Furthermore, FAs play a pivotal role in the palmitoylation of viral proteins, including the SARS-CoV-2 spike (S) protein and IAV hemagglutinin (HA) and ion channel M2 proteins, thereby promoting crucial processes such as viral membrane fusion, assembly, budding, and virulence. Conversely, disrupting FA biosynthesis can disrupt DMV formation and palmitoylation of the SARS-CoV-2 S protein and IAV HA and M2 proteins in SARS-CoV-2-infected cells. We observed by transmission electron microscopy (TEM) and confocal microscopy (Figs. 6g and 6h) that DN200434 treatment significantly reduced the number of perinuclear DMV clusters containing double-stranded RNA of SARS-CoV-2, including viral double-stranded RNA. Furthermore, TEM analysis revealed a significant reduction in the number of progeny virus particles in DN200434-treated cells compared to vehicle-treated virus-infected cells (bottom two panels of Fig. 6g). Furthermore, palmitoylation of the SARS-CoV-2 S protein and IAV HA protein, which is typically observed in vehicle-treated virus-infected cells, was significantly inhibited by DN200434 (Figs. 6i and 6j). Additionally, DN200434 treatment in SARS-CoV-2-infected and IAV-infected cells inhibited energy production via β-oxidation of FFA substrates in mitochondria (Fig. 6k).

[0129] Next, we investigated the role of DN200434 in reducing excessive proinflammatory cytokine secretion, known as a cytokine storm, which contributes to severe infection and increased mortality in patients infected with SARS-CoV-2 and IAV. Proinflammatory cytokines can be directly induced by cytoplasmic sensors responding to viral components, as well as indirectly through the production of eicosanoids, including FA-derived lipid mediators such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4). In the present study, DN200434 treatment significantly reduced the elevated levels of PGE2 and LTB4 observed in SARS-CoV-2 and IAV-infected cells (Figures 6l and 6m). Furthermore, in IAV-infected A549 cells, DN200434 treatment significantly suppressed proinflammatory cytokines, including interferon-α (IFN-α), IFN-β, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), which are normally activated in response to IAV infection (Figs. 6n to 6r). Because Vero cells cannot synthesize IFNs and SARS-CoV-2 proteins act as antagonists for type I and type III IFNs, DN200434 treatment significantly reduced TNF-α, IL-6, and MCP-1 levels, whereas IFN-α and IFN-β levels remained unchanged (Figs. 6n to 6r). These results suggest that DN200434 may reduce virus-associated severe proinflammatory cytokine responses that may significantly impact mortality in patients with COVID-19 and IAV.

[0130] The effect of DN200434 treatment on the FFA profiles of virus-infected cells was investigated using a gas chromatography-flame ionization detector (GC-FID). Selected FFAs that showed statistically significant changes (p<0.05) were used to generate a heatmap. Interestingly, treatment of SARS-CoV-2- or IAV-infected cells with DN200434 restored the levels of selected FFAs to levels comparable to or below those of mock control cells (Figures 7a and 7b). Notably, the synthesis of representative fatty acids induced by SARS-CoV-2 or IAV infection was significantly inhibited by DN200434 (Figures 7c to 7h). To assess the recovery of virus growth under DN200434 treatment, four major FFAs—saturated palmitic acid (PA), monounsaturated oleic acid (OA), polyunsaturated linoleic acid (LA), and polyunsaturated arachidonic acid (AA)—were selected. Supplementation with these individual FFAs at least partially restored genome replication and progeny production of SARS-CoV-2 and IAV in virus-infected and FFA-depleted cells compared to DN200434-treated controls ( Figures 7i-7l ). Collectively, these findings suggest that DN200434 inhibits viral replication compartment formation, viral morphogenesis (palmitoylation), and energy production through disruption of the downstream SREBP1c-mediated FA biosynthetic pathway by inhibiting ERRγ.

[0131] (7) Antiviral activity of DN200434 against SARS-CoV-2 and IAV infections in vivo

[0132] Previous lead compound optimization and pharmacokinetic studies have shown that DN200434 is the most potent ERRγ inverse agonist with a functional IC of 0.006 μM / L activity. 50 , which was 12 times higher than that of the previous lead compound GSK5182. Importantly, DN200434 showed a promising in vitro / in vivo toxicity profile in essential standard discovery studies. The present inventors administered DN200434 to mice (20 mg kg -1 d -1 ) and Syrian hamster (40 kg) -1 d -1 ) was administered intraperitoneally (ip) to allow the compound to circulate in the bloodstream and reach the lungs. Surprisingly, DN200434 did not induce any signs of toxicity in either mice or Syrian hamsters for 14 days. In addition, no gross or histopathological changes were observed in vital organs such as the liver, lungs, kidneys, heart, and spleen of mice and hamsters treated with DN200434.

[0133] The present inventors found that DN200434 was 10% of the SARS-CoV-2 KCDC03 strain belonging to the A lineage of the early Chinese strains. 5 TCID 50 We investigated whether gross lung lesions could be improved in hamsters challenged with SARS-CoV-2. DN200434 at various concentrations was administered intraperitoneally (twice daily) for 4.5 consecutive days to hamsters challenged with SARS-CoV-2. Gross lung lesions were significantly improved in a dose-dependent manner. In particular, DN200434 at 40 mg kg -1 d -1Treatment with DN200434 resulted in an impressive 92% reduction in gross lung lesions compared to the vehicle-treated control group infected with the virus (Figs. 8a and 8b). Next, the antiviral effect of DN200434 against the mouse-adapted IAV PR8 strain (H1N1) in mice was evaluated. While all IAV-infected, vehicle-treated control mice died within 10 days, intraperitoneal administration of DN200434 at various concentrations for 4 consecutive days (twice daily) to IAV-challenged mice resulted in a concentration-dependent improvement in survival, with a dose-dependent increase of 20 mg kg -1 d -1 In , it reached 40% (Figs. 10a and 10b). In addition, DN200434 administration alleviated not only weight loss caused by SARS-CoV-2 and IAV infection, but also clinical signs associated with IAV infection (Figs. 8c, 10c, and 10d).

[0134] To investigate whether the improvement in SARS-CoV-2-induced lung lesions and IAV-induced mortality by DN200434 administration was associated with inhibition of viral replication, lipid production, and LD formation, cytoplasmic lipid droplets and viral antigens were examined in alveolar and bronchial epithelial cells in lung tissue. DN200434 significantly reduced both LDs and viral antigens in these cells (Figs. 8d, 10e). Furthermore, DN200434 administration reduced levels of TAG, a major component of LDs, in lung tissue compared to virus-challenged and vehicle-treated control animals (Figs. 8e, 10f). Furthermore, inhibition of DN200434-induced intracellular LD formation in virus-challenged animals substantially reduced viral genome replication and progeny production (Figs. 8f and 8g, 10g and 10h). This inhibition of viral replication effectively ameliorated histopathological lung lesions such as interstitial hypertrophy, alveolar and bronchial epithelial cell necrosis, and pulmonary edema (Fig. 8h, Fig. 10i and 10j).

[0135] When DN200434 was administered intraperitoneally to hamsters challenged with SARS-CoV-2, the levels of LTB4, PGE2, IL-6, TNF-α, and MCP-1 were significantly reduced compared to the vehicle-treated control group after virus challenge, whereas no effect was observed on IFN-α and IFN-β levels (Figures 11a to 11g). Furthermore, in mice challenged with IAV, DN200434 administration significantly suppressed the levels of LTB4, PGE2, IFN-α, IFN-β, IL-6, TNF-α, and MCP-1 compared to the vehicle-treated control group after virus infection (Figures 11a to 11g). These results, consistent with the in vitro assay results, confirm that DN200434 exhibits antiviral and anti-inflammatory cytokine efficacy against SARS-CoV-2 and IAV infections.

[0136] (8) Antiviral synergistic effect of DN200434 combined with remdesivir (SARS-CoV-2) or oseltamivir (IAV)

[0137] Combination therapy of two antiviral agents with different targets at lower concentrations can produce synergistic effects and reduce toxicity compared to high-dose monotherapy. We evaluated the antiviral efficacy of a combination therapy with DN200434, two well-known antiviral agents targeting SARS-CoV-2 RdRp and oseltamivir targeting IAV neuraminidase. In this experiment, hamsters were administered 20 mg / kg of DN200434 and 2.5 mg / kg of remdesivir daily, while mice were administered 10 mg / kg of DN200434 and 2 mg / kg of oseltamivir daily. Encouragingly, neither monotherapy nor combination therapy showed signs of toxicity, such as body weight changes. 40 mg kg -1 d -1DN200434 or 2.5 mg kg -1 d -1 Individual treatments with remdesivir resulted in a 50% and 20% reduction in gross lung lesions, respectively. However, the 20 mg kg -1 d -1 DN200434 and 2.5 mg kg -1 d -1 Administration of the remdesivir combination significantly reduced gross lung lesions in hamsters challenged with the KCDC03 strain (A strain), KDCA51463 strain (alpha strain), and KDCA55905 strain (beta strain) by 80%, 75%, and 78%, respectively, compared to 100% of the control group (Figs. 8i and 8j). In addition, in mice challenged with IAV, 10 mg kg -1 d -1 DN200434 and 2 mg kg -1 d -1 Combination therapy with oseltamivir resulted in a 90% survival rate (Figures 8k and 8l). Simultaneously, the combination therapy effectively suppressed weight loss associated with viral infection and significantly alleviated clinical signs compared to either individual therapy (Figures 12a to 12e). Taken together, our data suggest that combination therapy featuring the host-targeting agent DN200434 along with a drug that directly targets the virus yields synergistic antiviral effects.

[0138] 3. Discussion

[0139] The present invention reports that ERRγ induces lipidomic reprogramming through transcriptional activation of SREBP1c, which in turn provides FFAs that promote the replication of various RNA viruses, including SARS-CoV-2 and IAV. The present invention found that RNA virus infection activates the JNK / c-Jun signaling pathway induced by reactive oxygen species (ROS), which ultimately upregulates ERRγ expression. Virus-induced upregulation and nuclear translocation of ERRγ were shown to directly transactivate SREBP1c gene expression, which in turn induces increased FA biosynthesis and LD formation. Disruption of ERRγ action through treatment with the inverse agonist DN200434 or siRNA knockdown significantly blocked the replication of various RNA viruses by inhibiting SREBP1c-dependent FA biosynthesis and LD formation. These inhibitory effects are manifested, inter alia, by inhibition of viral protein palmitoylation, which is essential for viral replication, reduction in double-membrane vesicle (DMV) formation, and reduction in mitochondrial beta-oxidation-mediated energy production. Finally, administration of DN200434 significantly attenuated gross lung lesions induced by SARS-CoV-2 in hamsters and protected mice from lethal IAV infection by blocking SREBP1c-mediated FA biosynthesis. Collectively, the present invention demonstrates that targeting ERRγ with DN200434, an ERRγ inverse agonist, can protect against COVID-19, IAV infection, and RNA virus diseases that may emerge in the near future.

[0140] ERRγ is known to be transcribed by the JNK / c-Jun signaling pathway induced by ROS in mouse liver. Many viral infections, both in vitro and in vivo, are known to activate ROS. This suggests that ROS induced by viral infection activates the JNK / c-Jun signaling pathway, ultimately transactivating ERRγ. The present invention confirms that ROS activates the JNK / c-Jun signaling pathway in IAV-infected or SARS-CoV-2-infected cells, ultimately transactivating ERRγ. Furthermore, it was shown that ROS activation was inhibited in IAV-infected and SARS-CoV-2-infected cells treated with an antioxidant (NAC), significantly reducing ERRγ promoter activity. This indicates that ERRγ is indeed a ROS sensor in virus-infected cells. Notably, infection with IAV or SARS-CoV-2 induced phosphorylation of JNK and c-Jun, and activation of the ERRγ promoter, but not of the ERRγ promoter harboring a mutation in the AP1 regulatory element site. Furthermore, NAC treatment significantly inhibited c-Jun binding to the AP1 regulatory element site of the ERRγ promoter. This suggests that ROS induced by viral infection activate the JNK / c-Jun signaling pathway, leading to transcriptional activation of ERRγ.

[0141] SREBP and its associated FA and cholesterol biosynthetic pathways play crucial roles in multiple stages of the viral life cycle. However, how SREBP is activated in response to viral replication remained unclear. Previous studies have shown that ERRγ activates the SREBP1c promoter, and we demonstrate here that ERRγ-mediated SREBP1c activation results in the reprogramming of FA biosynthesis, which is essential for viral replication. We found that many RNA viruses activate ERRγ, and that activated ERRγ translocates to the nucleus. Furthermore, ERRγ directly binds to the SREBP1c gene promoter in RNA virus-infected cells, promoting SREBP1c-dependent FA biosynthesis reprogramming in virus-infected cells. In contrast, inhibition of ERRγ inhibited SREBP1c-dependent FA biosynthesis, thereby blocking de novo FA synthesis, thereby reducing the formation of viral replication compartments (DMVs for SARS-CoV-2), viral morphogenesis (protein palmitoylation), and energy production, ultimately inhibiting viral replication. Furthermore, inhibition of ERRγ by DN200434 restored replication of SARS-CoV-2 and IAV upon addition of FA under FFA-deficient conditions. Therefore, we demonstrate that upregulation of ERRγ in response to RNA virus infection directly modulates SREBP1c-dependent FA biosynthesis, thereby inducing reprogramming of host cell physiology essential for viral replication (Figures 11a-g), making ERRγ a potentially important target for therapeutic intervention against various RNA viruses, including SARS-CoV-2 and IAV.

[0142] Recent evidence suggests that LD plays a central role in the antiviral host response through the localization of immune proteins such as viperin, enhancing the innate immune pathway. The latter, in particular, requires an efficient interferon response following initial viral infection or poly I:C treatment. Furthermore, we previously demonstrated that poly I:C treatment activates ERRγ expression via the RIG-I / MDA5 / JNK / AP1 signaling pathway, which in turn directly transactivates type I interferon genes. Thus, while ERRγ has both proviral and antiviral aspects in viral infection, our present invention clearly demonstrates that the proviral aspect of ERRγ is significantly more pronounced than the antiviral aspect. If the antiviral effect of ERRγ is more potent than the proviral effect, then inhibition of ERRγ should result in viral replication. However, inhibition of ERRγ with DN200434 or siRNA inhibited the proliferation of various RNA viruses. DN200434 administration attenuated lung lesions in hamsters challenged with SARS-CoV-2 and protected mice from lethal IAV challenge. Furthermore, resistance to lethal IAV challenge in ERRγ KO mice further supports this hypothesis.

[0143] Additionally, lipogenic enzyme inhibitors such as statins (inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase during cholesterol biosynthesis), TOFA (inhibitors of acetyl-CoA carboxylase during FA biosynthesis), and C75 (inhibitors of FAS during FA biosynthesis) are known to block viral replication. Taken together, these observations indicate that upregulation of ERRγ has a stronger proviral effect than an antiviral effect, and that LDs and lipid components are used as an energy source to drive viral production, viral factories, viral replication compartments, and viral components.

[0144] A surge of proinflammatory cytokines, called a cytokine storm, plays a significant role in exacerbating pneumonia in patients with COVID-19 and influenza, ultimately increasing the severity and mortality of the infected patients. In the present invention, inhibition of ERRγ with DN200434 in vitro and in vivo significantly reduced eicosanoid and cytokine levels, pneumonia severity, and overall mortality compared to untreated controls. Our data suggest that inhibition of eicosanoid and cytokine storms can alleviate lung lesions and reduce mortality through two mechanisms. FFA-derived eicosanoid storms following viral infection itself or endoplasmic reticulum stress response via virus-induced cellular debris can induce cytokine storms. Therefore, DN200434 treatment inhibits SREBP1c-dependent FA biosynthesis, which generates eicosanoids associated with cytokine storms. Infected non-immune cells; Innate immune cells, such as macrophages and dendritic cells, recognize viral genomes and proteins as pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), rapidly inducing an antiviral response through the production of proinflammatory cytokines. In the present invention, DN200434 treatment reduced FA levels in virus-infected cells by blocking ERRγ-mediated SREBP1c-dependent FA biosynthesis. This negatively affects the formation of viral replication compartments, viral morphogenesis, and energy production, which are required for viral replication. Therefore, a second mechanism is that the reduction in the production of viral genomes and proteins as PAMPs by DN200434 treatment may not be sufficient to induce a cytokine storm through PRR-mediated decisions in infected cells or immune cells.Overall, our study results demonstrate that the inhibitory effect of DN200434 on cytokine storm warrants further review as a priority candidate for clinical evaluation in COVID-19 and IAV clinical trials targeting patients with severe pneumonia.

[0145] Conventional antiviral drugs have traditionally been designed to target viral proteins. However, each life cycle of a virus utilizes various host proteins. In particular, host proteins commonly utilized by various viruses can be attractive targets for the development of host-targeted antivirals with broad efficacy. In the present invention, the genome structure of the viruses used is positive-stranded (SARS-CoV-2, BCoV, PEDV, PRRSV, and PSaV), negative-stranded (IAV), or double-stranded (RVA), and the shape is capsid (RVA and PSaV) or envelope (SARS-CoV-2, BCoV, PEDV, PRRSV, and IAV), and the genome arrangement is linear (SARS-CoV-2, BCoV, PEDV, PRRSV, and PSaV) or segmented (IAV and RVA). Therefore, the viruses used in this outbreak may represent major RNA viruses circulating in the past, present, and possibly in the future. Interestingly, we found that DN200434, an ERRγ inverse agonist, possesses broad antiviral activity against various target viruses grown in multiple cell lines. Furthermore, we confirmed that DN200434 is effective against the currently occurring pandemic SARS-CoV-2 and its variants. Nuclear receptors are considered attractive targets for drug development because they are easily modulated by small molecules. In particular, approximately 10% to 20% of currently FDA-approved drugs target nuclear receptors. DN200434, an ERRγ inverse agonist in the present invention, not only shows significant potential for treating a wide range of viruses, but may also provide broader protection against the emergence of drug-resistant virus strains or emerging pandemic strains, including notable examples such as IAV, SARS-CoV, MERS-CoV, and SARS-CoV-2.

[0146] In a previous report, the inventors reported that DN200434 had a functional IC of 0.006 μM / L, which is 12-fold higher than that of GSK5182, an active metabolite of tamoxifen and an FDA-approved drug optimized for estrogen receptor (ER)-positive breast cancer. 50 DN200434 was confirmed to be the most potent ERRγ antagonist with a potent anti-inflammatory cytokine activity. DN200434 was originally developed for the diagnosis and treatment of anaplastic thyroid cancer and exhibited high ERRγ affinity (IC50 = 0.04 μM / L). Furthermore, DN200434 demonstrated in vitro and in vivo absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles. In the current in vivo experiment, animals treated with the maximum dose of DN200434 did not exhibit any clinical, macroscopic, or histopathological adverse effects. Furthermore, DN200434 was detected to reach the lungs in sufficient amounts via the bloodstream. Therefore, DN200434 may have potential clinical applications for the treatment of a wide range of RNA viral diseases.

[0147] Combination therapy is an effective antiviral therapy that reduces toxicity and increases efficacy. Antiviral agents can be classified into two broad mechanisms of action: 1) those targeting viral proteins or nucleic acids, and 2) those targeting essential host factors for viral replication. In the present invention, DN200434 (10 mg kg) was used for the treatment of IAV-infected mice. -1 d -1 ) and oseltamivir (2 mg kg -1 d -1 ) for combination therapy; and DN200434 (20 mg kg) for the treatment of SARS-CoV-2 infected hamsters -1 d -1 ) and remdesivir (2.5 mg kg -1 d -1) significantly reduced single virus replication and significantly reduced IAV-induced mortality and SARS-CoV-2-induced lung lesions. Future clinical trials using dual or triple antiviral agents with DN200434 as the backbone will be needed to treat patients with severe acute viral infections such as COVID-19.

[0148] In summary, the present invention revealed that early transcriptional activation of ERRγ via the ROS / JNK / c-Jun pathway in virus-infected host cells induces SREBP1c-dependent FA biosynthetic reprogramming essential for viral replication. DN200434 treatment specifically reversed virus-induced lipogenic reprogramming, inhibited replication of various RNA viruses in vitro, protected mice from lethal IAV infection, and ameliorated lung lesions in hamsters challenged with SARS-CoV-2 and its variants. Furthermore, dual antiviral therapy with DN200434 and remdesivir for COVID-19 and dual antiviral therapy with DN200434 and oseltamivir for IAV infection enhanced the efficacy in virus-infected mice and hamsters, respectively. Given the affinity, excellent ADMET efficacy, and broad-spectrum antiviral properties of DN200434, DN200434 may serve as a novel broad-spectrum antiviral drug and may be used in the treatment of viral diseases such as COVID-19 and novel pandemic virus infections in the near future.

[0149] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0150] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. An antiviral pharmaceutical composition comprising an ERRγ (estrogen related receptor γ) inhibitor as an active ingredient.

2. In paragraph 1, An antiviral pharmaceutical composition wherein the ERRγ inhibitor inhibits the activity of an ERRγ protein or the expression of an ERRγ gene.

3. In paragraph 1, An antiviral pharmaceutical composition wherein the ERRγ inhibitor exhibits antiviral efficacy by inhibiting the activity of ERRγ in virus-infected cells.

4. In paragraph 1, An antiviral pharmaceutical composition, wherein the ERRγ inhibitor comprises siRNA specific to ERRγ.

5. In paragraph 1, An antiviral pharmaceutical composition, wherein the ERRγ inhibitor comprises an ERRγ inverse agonist.

6. In paragraph 5, An antiviral pharmaceutical composition comprising a compound represented by the following chemical formula 1, wherein the ERRγ agonist is: [Chemical Formula 1] , In the above chemical formula 1, L is (C6-C 20 )arylene, (C3-C 20 )heteroarylene, or (C3-C 20 ) is a fused heterocycle; R 1 Silver (C3-C 20 )heterocycloalkyl, (C3-C 20 )heteroaryl, -O-(CH2) m -R 11 , -(CH2) m -R 12 , -NH-(CH2) m -R 13 , -NHCO-(CH2) n -R 14 , or -SiR 16 R 17 -(CH2) m -R 15 and; R 11 Inland R 15 are each independently (C3-C 20 ) is heterocycloalkyl; R 16 and R 17 are each independently (C1-C 20 ) is alkyl; m is an integer from 1 to 3; and n is an integer of 0 or 1; Ar is (C6-C 20 )aryl or (C3-C 20 ) is heteroaryl, The aryl or heteroaryl of the above Ar is hydroxy, halogen, (C1-C 20 )alkyl, halo(C1-C 20 )alkyl, (C1-C 20 )alkoxy, nitro, cyano, -NR 21 R 22 , (C1-C 20 )alkylcarbonyloxy, (C1-C 20 )alkylcarbonylamino, guanidino, -SO2-R 23 and -OSO2-R 24 is substituted or unsubstituted with one or more substituents selected from R 21 and R 22 are each independently hydrogen, (C1-C 20 )alkylsulfonyl or (C3-C 20 ) is cycloalkylsulfonyl; R 23 and R 24 are each independently (C1-C 20 )alkyl, halo(C1-C 20 )alkyl or (C3-C 20 ) is cycloalkyl; R 2 is hydroxy, halogen, (C1-C 20 )alkylcarbonyloxy or (C1-C 20 )alkylsulfonyloxy; The above R 1 Heterocycloalkyl or heteroaryl of and the above R 11 Inland R 15 Heterocycloalkyl of (C1-C 20 )alkyl, (C3-C 20 )cycloalkyl, (C2-C 20 )alkenyl, amidino, (C1-C 20 )alkoxycarbonyl, hydroxy, hydroxy(C1-C 20 )alkyl and di(C1-C 20 )alkylamino(C1-C 20 ) is substituted or unsubstituted with one or more substituents selected from alkyl, and The above heterocycloalkyl and heteroaryl contain one or more heteroatoms selected from N, O and S, The above heterocycloalkyl is a saturated or unsaturated mono-, bi- or spirocycle having a carbon atom or nitrogen atom within the ring as a bonding site.

7. In paragraph 5, An antiviral pharmaceutical composition comprising a compound represented by the following chemical formula 2, wherein the ERRγ agonist is: [Chemical Formula 2] , In the above chemical formula 2, R 1 Silver (C3-C 20 )heterocycloalkyl, (C3-C 20 )heteroaryl, -O-(CH2) m -R 11 , -(CH2) m -R 12 , -NH-(CH2) m -R 13 , -NHCO-(CH2) n -R 14 , or -SiR 16 R 17 -(CH2) m -R 15 and; R 11 Inland R 15 are each independently (C3-C 20 ) is heterocycloalkyl; R 16 and R 17 are each independently (C1-C 20 ) is alkyl; m is an integer from 1 to 3; and n is an integer of 0 or 1; Ar is (C6-C 20 )aryl or (C3-C 20 ) is heteroaryl, The aryl or heteroaryl of the above Ar is hydroxy, halogen, (C1-C 20 )alkyl, halo(C1-C 20 )alkyl, (C1-C 20 )alkoxy, nitro, cyano, -NR 21 R 22 , (C1-C 20 )alkylcarbonyloxy, (C1-C 20 )alkylcarbonylamino, guanidino, -SO2-R 23 and -OSO2-R 24 is substituted or unsubstituted with one or more substituents selected from R 21 and R 22 are each independently hydrogen, (C1-C 20 )alkylsulfonyl or (C3-C 20 ) is cycloalkylsulfonyl; R 23 and R 24 are each independently (C1-C 20 )alkyl, halo(C1-C 20 )alkyl or (C3-C 20 ) is cycloalkyl; R 2 is hydroxy, halogen, (C1-C 20 )alkylcarbonyloxy or (C1-C 20 )alkylsulfonyloxy; The above R 1 Heterocycloalkyl or heteroaryl of and the above R 11 Inland R 15 Heterocycloalkyl of (C1-C 20 )alkyl, (C3-C 20 )cycloalkyl, (C2-C 20 )alkenyl, amidino, (C1-C 20 )alkoxycarbonyl, hydroxy, hydroxy(C1-C 20 )alkyl and di(C1-C 20 )alkylamino(C1-C 20 ) is substituted or unsubstituted with one or more substituents selected from alkyl, and The above heterocycloalkyl and heteroaryl contain one or more heteroatoms selected from N, O and S, The above heterocycloalkyl is a saturated or unsaturated mono-, bi- or spirocycle having a carbon atom or nitrogen atom within the ring as a bonding site.

8. In paragraph 5, An antiviral pharmaceutical composition, wherein the ERRγ agonist comprises DN200434 and / or GSK5182.

9. In paragraph 1, An antiviral pharmaceutical composition for the prevention or treatment of RNA virus infection.

10. In paragraph 9, The RNA viruses include SARS-CoV-1, SARS-CoV-2, influenza A virus (IAV), influenza B virus, bovine coronavirus (BCoV), porcine epidemic diarrhea coronavirus (PEDV), bovine species A rotavirus (RVA), porcine reproductive and respiratory syndrome virus (PRRSV), porcine sapovirus (PSaV), norovirus (NoV), feline coronavirus (FIPV), Middle East respiratory syndrome coronavirus (MERS-related coronavirus; MERS-CoV), Zika virus (ZIKV), dengue virus, human torovirus (HuTV), hepatitis A virus (HAV), and hepatitis C virus (HCV). An antiviral pharmaceutical composition comprising one or more selected agents.

11. In paragraph 1, An antiviral pharmaceutical composition further comprising an antiviral agent.

12. In paragraph 11, An antiviral pharmaceutical composition comprising at least one antiviral agent selected from oseltamivir and remdesivir.

13. In paragraph 12, If the above antiviral agent is oseltamivir, An antiviral pharmaceutical composition, wherein the volume ratio of the ERRγ agonist and the oseltamivir is 2:2 to 8:

2.

14. In paragraph 12, If the above antiviral agent is remdesivir, An antiviral pharmaceutical composition, wherein the volume ratio of the ERRγ agonist and the remdesivir is 8:1 to 20:1.

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