A compound with anti-coronavirus activity and its application

Abstract

The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to an application of a compound represented by formula (1) in the preparation of a drug, wherein the drug is a drug for inhibiting coronavirus, preferably, the drug is a drug for treating human coronavirus infection, and the coronavirus is preferably a human alpha coronavirus or a human beta coronavirus.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and in particular, relates to a compound with anti-coronavirus activity and an application thereof. Background Art

[0002] Acute infectious diseases have become a major threat to public health, among which acute respiratory infections are an important cause of morbidity and mortality from infectious diseases worldwide. Human coronavirus is one of the common pathogens that cause acute respiratory infections, which can cause zoonotic diseases and may cause a global pandemic. Since the 21st century, the world has faced three epidemics caused by highly pathogenic coronaviruses, namely severe acute respiratory syndrome (SARS) in 2002, Middle East respiratory syndrome (MERS) in 2012, and COVID-19 caused by the new coronavirus (SARS-CoV-2) in late 2019. [1] COVID-19 has become the fifth global pandemic recorded since the 1918 influenza pandemic. It is also the first coronavirus pandemic in human history, causing unprecedented damage to global health and economic development. [2] Due to the rapid spread of the COVID-19 epidemic and its relatively high mortality rate, it is urgent to develop safe and effective drugs to fight the novel coronavirus. [3] However, there is currently no specific antiviral drug for coronavirus, and the clinical approach is still mainly to combine traditional antiviral drugs with symptomatic treatment, but the efficacy is not certain. [4] . Moreover, coronaviruses have a wide range of hosts and are highly variable, and new variants may continue to emerge, posing a real threat to human health. Therefore, it is necessary to develop anti-coronavirus drugs. [5] .

[0003] Coronavirus is a type of positive-sense single-stranded RNA enveloped virus characterized by spikes on its surface, an RNA genome of approximately 30 kb in length, and a unique replication strategy. [6] The coronavirus is an elliptical particle with a diameter of about 120nm, which is composed of an outer envelope and an inner nucleocapsid. The structural proteins that make up the outer envelope include spike protein, membrane protein, and envelope protein. The inner layer is composed of nucleocapsid protein (NP) that wraps RNA in a spiral symmetric form. [7]. Coronaviruses can infect vertebrates and spread between different host species. Seven coronaviruses are currently known to infect humans. Four of these viruses (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1) can cause mild upper respiratory tract infections, while SARS-CoV, MERS-CoV, and the more recent SARS-CoV-2 can cause severe pneumonia and progress to multiple organ failure and death. [8] In theory, any stage that interferes with the viral life cycle can become a target for antiviral drug discovery.

[0004] Lipids play a vital role in various stages of the viral life cycle. First, lipids are considered to be structural elements of viral and cell membranes. Lipids can act as direct receptors or cofactors on the cell surface to facilitate viral infection of the host. [9] After viral infection, new cytoplasmic membrane structures and compartments are formed in the host, where the viral genome replicates and assembles and may shield the host's innate immune response.

[10] . Second, lipid metabolism can provide the energy required for viral replication. In addition, lipids can determine the proper distribution of viral proteins in the cell, as well as the transport, assembly, and release of viral particles.

[11] . Therefore, regulating host cell lipid metabolism plays an indispensable role in interfering with the viral replication cycle. Small molecule drugs have many advantages such as low cost and convenient administration, and show great advantages in drug development. The structure of small molecule drugs has a certain degree of spatial discreteness, and its chemical properties determine its good drugability and pharmacokinetic properties, which means that small molecule drugs may have the potential to compete with biological agents.

[12] This also makes the development of small molecule drugs against coronaviruses extremely attractive.

[0005] We screened the anti-common coronavirus activity by measuring the degree to which the samples inhibited virus-induced cytopathic effects and obtained several active compounds. After a series of anti-coronavirus pharmacodynamic studies, we determined that the positive compound IMB-C5 had a good inhibitory effect on the replication of HCoV-229E and HCoV-OC43. IMB-C5 is a new PCSK9 small molecule inhibitor that our laboratory screened using the PCSK9 transcription inhibitor screening model. It has a good effect on lowering blood lipids and inhibiting the formation of atherosclerotic plaques.

[13] Therefore, IMB-C5 may affect the replication of coronavirus by participating in the regulation of lipid metabolism, and related research is still in progress.

[0006] [References]

[0007] [1]Raoult D,Zumla A,Locatelli F,et al.Coronavirus infections:Epidemiological,clinical and immunological features and hypotheses.CellStress,2020,4:66-74.

[0008] [2]Liu YC,Kuo RL,Shih SR.COVID-19:The first documented coronaviruspandemic in history.Biomed J,2020,43(4):328-333.

[0009] [3]Guy RK,DiPaola RS,Romanelli F,et al.Rapid repurposing of drugs forCOVID-19.2020,368(6493):829-830.

[0010] [4]Shereen M,Khan S,Kazmi A,et al.COVID-19infection:Origin,transmission,and characteristics of human coronaviruses.Journal of AdvancedResearch,2020,24.

[0011] [5]Rabaan A,Al-Ahmed S,Haque S,et al.SARS-CoV-2,SARS-CoV,and MERS-CoV:a comparative overview.Le infezioni in medicina:rivista periodica dieziologia,epidemiologia,diagnostica,clinica e terapia delle patologieinfettive,2020,28:174-184.

[0012] [6]Fehr AR,Perlman S.Coronaviruses:an overview of their replicationand pathogenesis.Methods Mol Biol,2015,1282:1-23.

[0013] [7]Malik Y.Properties of Coronavirus and SARS-CoV-2.The Malaysianjournal of pathology,2020,42:3-11.

[0014] [8]Shang Z,Chan SY,Liu W,et al.Recent Insights into EmergingCoronavirus:SARS-CoV-2.ACS Infectious Diseases,2020,doi:10.1021 / acsinfecdis.0c00646

[0015] [9]Nagy PD,Strating JR,van Kuppeveld FJ.Building Viral ReplicationOrganelles:Close Encounters of the Membrane Types.PLoS Pathog,2016,12(10):e1005912.

[0016]

[10] Yuan S,Chu H,Chan JF-W,et al.SREBP-dependent lipidomicreprogramming as a broad-spectrum antiviral target.Nature Communications,2019,10(1):120.

[0017]

[11] Yan B,Chu H,Yang D,et al.Characterization of the LipidomicProfile of Human Coronavirus-Infected Cells:Implications for Lipid MetabolismRemodeling upon Coronavirus Replication.Viruses,2019,11:73.

[0018]

[12] Yang Y, Li X, Wang T, et al. Emerging agents that target signaling pathways in cancer stem cells. Journal of Hematology&Oncology, 2020,13.

[0019]

[13] Hong Bin, Wang Li, Chen Xiaofang, Wang Xuelei, Du Yu, Yang Mengxia, Zhang Xiumin, Wu Yanbin, Li Xingxing, Shi Yuanyuan. A drug for lowering blood lipids. Application date: 2019.5.17, Application number: 201910410812.6 Summary of the invention

[0020] The present invention relates to the use of a compound represented by formula (1) in the preparation of a medicine, wherein the medicine is a medicine for inhibiting coronavirus.

[0021]

[0022] Preferably, the coronavirus is a human coronavirus;

[0023] More preferably, the coronavirus is a human alpha coronavirus or a human beta coronavirus.

[0024] Most preferably, the coronavirus is HCoV-229E, HCoV-OC43, or SARS-CoV-2.

[0025] A pharmaceutical composition for treating coronavirus, characterized in that it contains a therapeutically effective amount of a compound of formula (1) and necessary pharmaceutical excipients. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 , toxicity assay of IMB-C5 on different cells.

[0027] Figure 2 , effects of IMB-C5 on the mRNA level of coronavirus HCoV-229E in Huh7 and Huh7.5 cells.

[0028] Figure 3 , the effect of IMB-C5 on dsRNA production during HCoV-229E replication in Huh7 cells.

[0029] Figure 4 , Effect of IMB-C5 on the mRNA level of coronavirus HCoV-OC43 NP in C3A cells.

[0030] Figure 5, the effect of IMB-C5 on the NP protein level of coronavirus HCoV-OC43 in C3A cells.

[0031] Figure 6 , time course experiment of IMB-C5 action on coronavirus. DETAILED DESCRIPTION

[0032] Experimental Materials

[0033] 1. Cell lines and viruses

[0034] Human hepatoma cell line C3A (ATCC, CRL-10741) was stored in our laboratory.

[0035] Human hepatoma cell line Huh7 was preserved in our laboratory.

[0036] Human hepatoma cell line Huh7.5 was preserved in our laboratory.

[0037] African green monkey kidney cells Vero E6 (ATCC, CRL-1586) were stored in our laboratory.

[0038] Human coronavirus HCoV-229E (ATCC, VR-740), stored in this laboratory.

[0039] Human coronavirus HCoV-OC43 (ATCC, VR-1558) was stored in this laboratory.

[0040] The South African variant of human coronavirus SARS-CoV-2 was obtained from Guangdong Provincial Center for Disease Control.

[0041] 2. Compounds

[0042] The positive control drug ribavirin (RBV) injection was purchased from Tianjin Jinyao Group Hubei Tianyao Pharmaceutical Co., Ltd. with batch number 31712252 and specification of 100 mg / ml; the positive control drug Remdesivir (RDV) was purchased from MedChemExpress with item number HY-104077.

[0043] 3. Reverse transcription quantitative PCR (RT-qPCR) primers

[0044] F=Forward primer, R=Reverse primer, P=Probe, NP=Nucleocapsid protein

[0045]

[0046] 4. Antibodies

[0047] Coronavirus NP antibody (mouse monoclonal antibody, MAB9013, Millipore),

[0048] GAPDH antibody (mouse monoclonal antibody, ZB2305, Zhongshan Jinqiao),

[0049] Anti-dsRNA antibody (mouse monoclonal antibody J2, SCICONS),

[0050] FITC (Fluorescein Isothiocyanate)-labeled goat anti-mouse IgG (H+L) (HS211, all-gold).

[0051] Example 1. Cytotoxicity assay of compound IMB-C5

[0052] In order to clarify the possible cytotoxicity of the compound, the CCK-8 method was first used to detect the effect of IMB-C5 administration at different concentrations for 48 hours on the survival rate of different cells. Figure 1 As shown in the figure, when the concentration of IMB-C5 was below 100 μM, the survival rates of the three liver cancer cells (Huh7, Huh7.5 and C3A) were all above 80% of the control group 48 hours after administration, and the cells were in good condition. The half toxic concentration TC of the drug was calculated by using the Reed-Muench formula 50 , IMB-C5 for TC of Huh7, Huh7.5 and C3A cells 50 The concentrations of the compounds were 403.5 μM, 896 μM and 1,093 μM, respectively, indicating that the compounds had low toxicity to the above cells ( Figure 1 ).

[0053] Example 2, IMB-C5 anti-alpha coronavirus HCoV-229E activity determination

[0054] 1. Cytopathic effect (CPE) assay to determine anti-HCoV-229E activity

[0055] (1) The experiment was performed in Huh7 and Huh7.5 cells. The cells were seeded in 96-well culture plates and placed in a 5% CO 2 , culture at 37℃ for about 24h;

[0056] (2) IMB-C5 was diluted with MEM culture medium containing 2% FBS and 1% P / S to obtain samples of 8 doses.

[0057] (3) 100 TCID 50The HCoV-229E virus solution was used to infect the cells, and samples containing different dilutions were added at the same time. Cell control wells and virus control wells were set up at the same time. The cells were incubated at 5% CO 2 , and cultured at 35°C for about 48 hours. When the cytopathic effect (CPE) of the virus control group reached 4+, the cytopathic effect of each group was observed, and the half toxic concentration (TC) of the samples to cells was calculated using the Reed-Muench method. 50 ) and the half inhibitory concentration (IC 50 ), and calculate the selection index (SI = TC 50 / IC 50 ).

[0058] CPE evaluation criteria: The cell death ratio is marked as 4+ (cell death ratio 75% to 100%), 3+ (cell death ratio 50% to 75%), 2+ (cell death ratio 25% to 50%), 1+ (cell death ratio 0 to 25%), and 0+ (all cells survive).

[0059] The results are shown in Table 1. IMB-C5 has good inhibitory activity against HCoV-229E in Huh7 and Huh7.5 cells, which is better than the positive control drug RBV.

[0060] Table 1 Anti-HCoV-229E activity of IMB-C5 in Huh7 and Huh7.5 cells determined by CPE assay

[0061]

[0062] 2. IMB-C5 inhibits coronavirus HCoV-229E at the RNA level

[0063] In the in vitro efficacy test, the effect of IMB-C5 on the mRNA level of coronavirus HCoV-229E NP was first tested ( Figure 2 ).

[0064] Huh7 and Huh7.5 cells were infected with a viral load of MOI = 0.035, and three concentrations of IMB-C5 or 200 μM positive drug RBV were administered at the same time. RNA was extracted 24 hours later and detected by RT-qPCR. Figure 2 As shown in A and B, IMB-C5 can dose-dependently reduce the level of HCoV-229E NP mRNA in both Huh7 and Huh7.5 cells, and its antiviral activity in Huh7 cells is better than that in Huh7.5.

[0065] Then, immunofluorescence experiments were performed to detect the effect of IMB-C5 on HCoV-229E double-strand RNA (dsRNA). Figure 3 ).

[0066] Huh7 cells were infected with a viral load of MOI = 0.035, and gradient concentrations of IMB-C5 or positive drugs were added during infection. After culturing at 35°C for 24 hours, the treated cells were washed three times with PBS, and then 4% paraformaldehyde was added and incubated at room temperature for 15 minutes for fixation. After washing, the cells were permeabilized by incubating with PBS buffer containing 0.5% Triton X-100 at room temperature for 20 minutes. After washing, TBST buffer containing 1% bovine serum albumin (BSA) was added for blocking at room temperature for 1 hour. Anti-dsRNA antibody was added and incubated overnight at 4°C. After washing the cells with PBST buffer, FITC fluorescently labeled secondary antibody was added and incubated at room temperature in the dark for 1 hour. Finally, nuclear staining solution (Hoechst 33342) was added and incubated at room temperature for 10 minutes, and the level of viral dsRNA was observed using a fluorescence microscope. Figure 3 As shown, IMB-C5 also has an inhibitory effect on the production of dsRNA of HCoV-229E during the replication of Huh7 cells. This indicates that IMB-C5 has a good inhibitory effect on the replication of alpha coronavirus HCoV-229E RNA.

[0067] Example 3. Determination of IMB-C5 activity against β-coronavirus HCoV-OC43

[0068] 1. IMB-C5 inhibits HCoV-OC43 at the RNA level

[0069] Using C3A cells as the virus host, the effect of IMB-C5 on the level of coronavirus HCoV-OC43 NP mRNA was detected. Figure 4 ). C3A cells were infected with a viral load of MOI = 0.023, and three concentrations of IMB-C5 or 200 μM positive drug RBV were administered at the same time. RNA was extracted 24 hours later and detected by RT-qPCR. The results showed that IMB-C5 could dose-dependently reduce the level of HCoV-OC43 NP mRNA in C3A cells.

[0070] 2. IMB-C5 inhibits HCoV-OC43 at the protein level

[0071] We further examined the effect of IMB-C5 on the NP protein level of coronavirus HCoV-OC43 ( Figure 5). C3A cells were infected with a viral load of MOI = 0.023, and three concentrations of IMB-C5 or 200μM positive drug RBV were administered at the same time. The level of viral nucleocapsid protein was detected after 24 hours. As shown in the figure, IMB-C5 has a good inhibitory effect on HCoV-OC43, and can inhibit the expression of viral nucleocapsid protein in a dose-dependent manner, and the inhibitory effect of high concentration compound (50μM) is equivalent to that of positive drug 200μM RBV.

[0072] Example 4. IMB-C5 anti-SARS-CoV-2 activity assay

[0073] The activity of IMB-C5 against SARS-CoV-2 was determined by the CPE method. Vero E6 cells were inoculated into 96-well culture plates, cultured overnight at 37°C, and then the culture medium was discarded. The cells were infected with the South African variant of SARS-CoV-2 (MOI = 0.05). The drug was diluted with DMEM medium without FBS and added to the 96-well culture plate. After 1 hour of infection, the culture medium was discarded, and the drug diluted in DMEM medium with 2% FBS was added to the 96-well culture plate according to a concentration gradient and continued to be cultured. When the CPE of the virus control group reached 4+, the degree of cytopathic effect of each group was observed. The results showed that IMB-C5 has inhibitory activity against the South African variant of SARS-CoV-2.

[0074] Table 2 Anti-SARS-CoV-2 activity of IMB-C5 in Vero E6 cells determined by CPE assay

[0075]

[0076] Example 5: IMB-C5 acts on the early stages of coronavirus infection

[0077] The time course experiment was used to study which stage of the coronavirus replication cycle IMB-C5 acts on. 50 μM IMB-C5 was added at different time points after HCoV-OC43 (MOI = 0.28) infected C3A cells, and the viral nucleocapsid protein ( Figure 6 ). The results showed that the inhibitory effect of the drug on the virus was most obvious when it was added at the same time as the virus was infected. The drug had a good antiviral effect when it was given 1 to 4 hours after infection, while the antiviral effect produced by the drug 6 hours after infection and later was greatly weakened. This suggests that IMB-C5 may act on the early stages of coronavirus infection.

[0078] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the protection scope of the present invention.

Claims

1. Use of the compound represented by formula (1) in the preparation of a drug, wherein the drug is a drug for inhibiting coronavirus; The coronavirus is a human alpha coronavirus.

2. The use according to claim 1, characterized in that: The coronavirus is HCoV-229E.

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