Application of small molecule compound in preparation of anti-coronavirus medicine

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of small molecule compounds with the structural formula shown in the specification in preparation of anti-coronavirus drugs, the small molecule compounds provided by the invention have HCoV-OC43 resisting activity, and the half cytotoxicity (CC50) of the two small molecule compounds on A549 cells is greater than 500 mu M; the median inhibitory concentrations (IC50) in A549 cells infected by HCoV-OC43 are respectively 30.88 [mu] M and 20.50 [mu] M, and the SI values in the A549 cells infected by HCoV-OC43 are respectively 16.19 and 24.39; the two small molecule compounds can inhibit replication and release of HCoV-OC43 at the nucleic acid level, the inhibition effect is dose-dependent, and the invention provides an effective potential antiviral drug for prevention and treatment of coronavirus infectious diseases; # imgabs 0 #, and # imgabs 1 #.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of two small molecule compounds in the preparation of anti-coronavirus drugs. Background Art

[0002] Coronaviruses (CoVs) are widely distributed in nature, with a host range that includes animals and humans. Coronaviruses have caused epidemics many times, causing profound impacts on human survival and development. CoVs belong to the order Nidovirales, family Coronaviridae, and subfamily Coronavirinae. They are single-stranded positive-sense RNA viruses with an envelope structure and a non-segmented linear genome. The subfamily Coronavirinae is further divided into four genera, namely Alpha-coronavirus (α-CoVs), Beta-coronavirus (β-CoVs), Gamma-coronavirus (γ-CoVs), and Delta-coronavirus (δ-CoVs). Among them, α-CoVs and β-CoVs mainly infect mammals, while γ-CoVs and δ-CoVs mainly infect birds and a few mammals. The seven known human coronaviruses (HCoVs) that can infect humans include three highly pathogenic coronaviruses, namely SARS-CoV, MERS-CoV and SARS-CoV-2, and four low pathogenic coronaviruses, namely HCoV-NL63, HCoV-229E, HCoV-OC43 and HCoV-HKU1. The four low pathogenic coronavirus infections mainly cause common cold symptoms, which are seasonally prevalent. Infections with immunocompromised infants and the elderly can lead to severe lower respiratory tract diseases and encephalitis. HCoV-OC43, which belongs to the same genus as SARS-CoV-2, is considered to be the most common strain causing mild coronavirus-related colds, and has the highest detection rate among the four low pathogenic coronaviruses. At present, the symptoms of SARS-CoV-2 infection are relatively mild, similar to those of low pathogenic coronavirus infection, but due to the restrictions of the conditions of the Level 3 biosafety laboratory, in-depth research on SARS-CoV-2 has been hindered to a certain extent. Studies have shown that the homology of the whole genome sequence between SARS-CoV-2 and HCoV-OC43 is 53.1%. HCoVOC43 has highly homologous motifs required for viral replication and pathogenicity, and the study of HCoV-OC43 can be carried out in a Level 2 biosafety laboratory. Therefore, HCoV-OC43 is considered an important model virus for coronavirus research. Using HCoV-OC43 to study anti-coronavirus drugs will provide an important reference for the prevention and control of coronavirus infection. Summary of the invention

[0003] The present invention provides the use of small molecule compounds ZP-91 and ZP-96 in the preparation of anti-coronavirus drugs. The structural formulas of the small molecule compounds ZP-91 and ZP-96 are as follows: .

[0004] The coronavirus is HCoV-OC43.

[0005] The ingredients (or active ingredients) of the drug of the present invention are the above-mentioned small molecule compounds, and one or more excipients acceptable in product production may be added to improve the absorption effect of the active ingredients or facilitate use, such as capsules or pills, powders, tablets, granules, oral liquids and injections, etc., that is, suitable dosage forms.

[0006] The present invention selects HCoV-OC43, which can infect humans, as a representative virus for research. The cytotoxicity of small molecule compounds ZP-91 and ZP-96 in A549 cells was evaluated based on cell viability detection, and the inhibitory effects of these two small molecule compounds on viral replication and release in A549 cells infected with HCoV-OC43 were evaluated based on viral nucleic acid detection. The results showed that the half cytotoxicity (CC 50 ) requires a concentration greater than 500 μM, and the half inhibitory concentration (IC 50 ) were 30.88 μM and 20.50 μM, respectively, and the selectivity index SI values ​​were 16.19 and 24.39, respectively, indicating that the two small molecule compounds were low in toxicity and high in efficiency in this cell model. From the nucleic acid level, both small molecule compounds can inhibit the replication and release of HCoV-OC43, and the inhibitory effect is dose-dependent.

[0007] Advantages and technical effects of the present invention: 1. The small molecule compounds ZP-91 and ZP-96 have low cytotoxicity and can inhibit HCoV-OC43 replication in a dose-dependent manner; 2. Small molecule compounds have the advantages of small molecular weight, short R&D cycle and low production cost, and have broad application prospects in clinic and market. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The results of HCoV-OC43 titer determination; Figure 2 Schematic diagram of the results of cytotoxicity and viral inhibition experiments of small molecule compounds ZP-91 (Figure A), ZP-96 (Figure B) and positive control remdesivir (RDV) (Figure C) in A549 cells; the blue line in the figure is the cytotoxicity result, and the red line is the viral inhibition result; Figure 3The figure below is a schematic diagram of the experimental results of the inhibition of HCoV-OC43 replication by the small molecule compounds ZP-91 and RDV in the supernatant of virus-infected cells; the figure on the left shows the effects of the above drugs on the virus replication level; the figure on the right shows the inhibition rate of the corresponding drugs on the HCoV-OC43 replication level; Figure 4 The figure below is a schematic diagram of the experimental results of the inhibition of HCoV-OC43 replication by the small molecule compounds ZP-91 and RDV in virus-infected cells; the figure on the left shows the effects of the above drugs on the virus replication level; the figure on the right shows the inhibition rate of the corresponding drugs on the HCoV-OC43 replication level; Figure 5 The figure below is a schematic diagram of the experimental results of the inhibition of HCoV-OC43 replication by the small molecule compounds ZP-96 and RDV in the supernatant of infected cells; the figure on the left shows the effects of the above drugs on the replication level of the virus; the figure on the right shows the inhibition rate of the corresponding drugs on the replication level of HCoV-OC43; Figure 6 The figure below is a schematic diagram of the experimental results of the inhibition of HCoV-OC43 replication by the small molecule compounds ZP-96 and RDV in infected cells; the figure on the left shows the effects of the above drugs on the replication level of the virus; the figure on the right shows the inhibition rate of the corresponding drugs on the replication level of HCoV-OC43; The experimental results were expressed as mean ± standard error and analyzed by Two-way ANOVA (or mixed model); compared with the OC43 group, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. DETAILED DESCRIPTION

[0009] In order to more clearly illustrate the technical solution of the present invention, the present invention will be described in detail in conjunction with specific embodiments. However, the protection scope of the present invention is not limited to the above contents. The experimental methods used in this embodiment are conventional methods unless otherwise specified, and the materials and reagents used are commercially available unless otherwise specified; the coronavirus described in the embodiment is a low pathogenic coronavirus HCoV-OC43 (VR-1558); The preparation of small molecule compounds ZP-91 and ZP-96 in the following examples was prepared according to the method in Pan Zhou, Biao Hu, Lingling Lu, Rong Huang & Fuchao Yu. (2017). One-Pot Synthesis of Functionalised 4-Spiro-1,4-Dihydropyridines Via [1+2+1+2]-Cyclisation. Journal of Chemical Research (9), 513-516.

[0010] The small molecule compounds ZP-91 and ZP-96 powders in the following examples were dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a storage concentration of 50 mM, and the completely dissolved drugs were stored in a refrigerator at 4°C for subsequent experiments.

[0011] Example 1: HCoV-OC43 culture and titer determination 1. Amplification of HCoV-OC43 (VR-1558) strain using HRT-18 cells The well-growing HRT-18 cells were inoculated into T25 culture flasks and cultured overnight. When the confluence of the monolayer cells in the culture flask reached 80%, the supernatant was discarded, the flask was washed twice with PBS, 2 mL of the diluted virus solution was inoculated on the cell surface, rotated to cover the entire culture flask, and incubated in a constant temperature incubator at 34°C and 5% CO2 for 2 h. After the incubation, the RPMI-1640 medium (RP2) containing 2% serum was added to 5 mL and continued to be cultured. The cytopathic effect (CPE) was observed every day. When the degree of cytopathic effect in the culture flask reached more than 75%, the cell culture flask was placed in a -80°C refrigerator and repeatedly frozen and thawed. The culture flask was taken out and thawed on ice. After it was completely thawed, the cells and supernatant were collected, centrifuged at 4°C and 3000 rpm / min for 5 min, the supernatant was transferred to a new 15 mL centrifuge tube, mixed, and dispensed into sterile cryopreservation tubes at 1 mL per tube and stored in a -80°C refrigerator.

[0012] 2. HCoV-OC43 titer determination Well-growing A549 cells were inoculated into a 96-well cell culture plate (flat bottom), and 100 μL of cell suspension was added to each well. The cell density was 2.0×10 3 The virus solution was diluted 10-fold with serum-free DMEM medium to 8 concentration gradients (the virus solution dilution factor was 10). -7~10), 100 μL of diluted virus dilution was inoculated into each well, serum-free DMEM medium was used as blank control, and the cells were cultured in a constant temperature incubator at 34°C and 5% CO2 for 2 h. After the incubation, an equal amount of DMEM medium (D2) containing 2% serum was replaced for each well to continue the culture. On the third day, 100 μL of D2 was added to each well, and the cells were cultured for 5 days. The cytopathic effect (CPE) was observed and recorded. On the 7th day, the CCK-8 kit was used to detect cell viability. The CCK-8 reagent and serum-free DMEM medium were mixed at a ratio of 1:10, and the original medium in the cell plate was discarded. 100 μL of the prepared detection solution was added to each well, and the cells were placed in a constant temperature incubator at 37°C and 5% CO2 for incubation for 10 min. The absorbance at 450 nm was detected using an enzyme reader, and the half-cell infectious dose (TCID) of the virus was calculated using the Reed-Muench method. 50 ); The results are as follows Figure 1 As shown, the TCID of HCoV-OC43 50 For 10 -5 / 100μL; means 100μL is inoculated into each well 10 x The virus solution diluted 100 times can cause CPE in 50% of the cells in the well. The virus solution was stored in a -80℃ refrigerator for subsequent experiments.

[0013] Example 2: Screening for small molecule drugs with anti-HCoV-OC43 activity in a small molecule compound library with unknown activity A549 cells with good growth status were subcultured at a ratio of 1:3 and plated in a 96-well cell culture plate (flat bottom). 100 μL of cell suspension was added to each well and the cell density was adjusted to 2.0×10 3 / well. When the cell confluence in the well reaches 70-80%, the virus can be inoculated. Take out the virus solution from the -80℃ refrigerator and dilute the virus solution to 100 TCID with serum-free DMEM medium. 50 100 μL of virus solution was inoculated into each well and incubated in a cell culture incubator at 34°C and 5% CO2 for 2 h. After the incubation, the supernatant was discarded and the drug-containing medium D2 with a working concentration of 50 μM was added. The cells were placed in a cell culture incubator at 34°C and 5% CO2 for further culture. CPE was observed and recorded every day. After 7 days, the cell viability was detected using a CCK-8 kit. The CCK-8 reagent was mixed with serum-free DMEM medium at a ratio of 1:10. The original medium in the cell plate was discarded. 100 μL of the prepared detection solution was added to each well and the cells were placed in a constant temperature incubator at 37°C and 5% CO2 for 10 min. The absorbance at 450 nm was detected using an ELISA reader to calculate the inhibition rate of the drug against the virus. Inhibition rate = (1-(OD value of the drug treatment group-OD value of the infection group) / (OD value of the control group-OD value of the infection group)) × 100%.

[0014] The inhibition rates of the small molecule compounds (50 μM) with unknown activity on the HCoV-OC43 virus are shown in Table 1, among which the small molecule compounds ZP-91 and ZP-96 can achieve 100% inhibition of the virus.

[0015] Table 1 Virus inhibition rate of small molecule compounds .

[0016] Example 3: Cytotoxicity test of small molecule compounds ZP-91 and ZP-96 on A549 cells The cytotoxicity of small molecule compounds ZP-91 and ZP-96 on A549 cells was detected by CCK-8 method. A549 cells with good growth status were inoculated into 96-well cell culture plates (flat bottom), 100 μL of cell suspension was added to each well, and the cell density was adjusted to 2.0×10 3 / well. When the cell confluence in the well reaches 90%, the drug can be added. Discard the supernatant, wash once with PBS, dilute the small molecule compounds ZP-91 and ZP-9 to 500μM with D2, and dilute 8 concentration gradients (500μM, 250μM, 125μM, 62.5μM, 31.25μM, 15.625μM, 7.8125μM, 3.90625μM) in turn with 500μM as the initial concentration, add 100μL of drug-containing culture medium to each well, use D2 as the blank control, and D2 containing 5‰ DMSO as the solvent control, with 6 replicates in each group. After continued cultivation in a 37°C, 5% CO2 constant temperature incubator for 48 hours, CPE was observed and recorded, and cell viability was detected using a CCK-8 kit. CCK-8 reagent and serum-free DMEM medium were mixed at a ratio of 1:10, the original medium in the cell plate was discarded, 100 μL of the prepared detection solution was added to each well, and the cells were placed in a 37°C, 5% CO2 constant temperature incubator for incubation for 10 minutes. The absorbance at 450 nm was detected using an ELISA instrument, and the drug CC was calculated using the Reed-Muench method. 50 , C.C. 50 =(1-(OD value of drug group / OD value of solvent control group))×100%.

[0017] The results are shown in Table 2 and Figure 2 , CC of small molecule compounds ZP-91 and ZP-96 in A549 cell model 50 Both were greater than 500 μM, indicating that the two small molecule compounds were less toxic to the cells and their cytotoxicity was less than that of the control drug RDV (CC 50 was 163.1 μM), which means that ZP-91 and ZP-96 have good safety.

[0018] Example 4: Inhibitory effects of small molecule compounds ZP-91 and ZP-96 on HCoV-OC43 replication The half-maximal inhibitory concentrations of small molecule compounds ZP-91 and ZP-96 were determined by CCK-8 method. A blank control group, a single drug group, a drug treatment group, and a single virus group were set up, with 4 replicates in each group. A549 cells with good growth status were inoculated in a 96-well cell culture plate (flat bottom), 100 μL of cell suspension was added to each well, and the cell density was adjusted to 2.0×10 3 When the cell confluence of the monolayer in the 96-well plate reaches 70-80%, the virus can be inoculated, the supernatant is discarded, and the cells are washed once with PBS. The virus group and the drug treatment group are added with serum-free DMEM medium diluted to 100 TCID 50 100 μL of virus solution was added to each well, and the other groups were added with an equal amount of serum-free DMEM medium, and placed in a 34°C, 5% CO2 constant temperature incubator for 2 h. After the incubation, the supernatant was discarded, and the drug-only group and the drug-treated group were added with drug-containing medium diluted with D2 (with 50 μM as the initial concentration for serial dilution); the other groups were added with an equal amount of D2 and placed in a 34°C, 5% CO2 constant temperature incubator for further culture for 5 days, and CPE was observed and recorded. After 7 days, the cell viability was detected using the CCK-8 kit, and the CCK-8 reagent and serum-free DMEM medium were mixed at a ratio of 1:10, and the original medium in the cell plate was discarded. 100 μL of the prepared detection solution was added to each well, and the cells were placed in a 37°C, 5% CO2 constant temperature incubator for incubation for 10 min. The absorbance at 450 nm was detected using an enzyme reader, and the drug IC was calculated using the Reed-Muench method. 50 IC 50 = (1-(OD value of drug treatment group-OD value of virus group) / (OD value of blank control group-OD value of virus group)) × 100%; Calculate the selection index (SI) = CC 50 / IC 50 ; Table 2 CC of ZP-91, ZP-96 and RDV 50 、IC 50 and SI values

[0019] The experimental results are shown in Table 2. In A549 cells infected with HCoV-OC43, the IC 50 The value is 30.88 μM, and the IC of ZP-96 50 The value was 20.50 μM, and the SI values ​​were 16.19 and 24.39, respectively; the SI values ​​of the two small molecule compounds and the control drug were greater than 2 in the A549 cell model infected with HCoV-OC43, indicating that the two small molecule compounds have potential anti-HCoV-OC43 activity.

[0020] Example 5: Nucleic acid level detection of the inhibitory effect of drugs on HCoV-OC43 A549 cells with good growth status were inoculated into 12-well cell culture plates (flat bottom), 1 mL of cell suspension was added to each well, and the cell density was adjusted to 3.0×10 5 Each well was set up with virus group, drug treatment group, blank control group and solvent control group, with two replicates in each group. The virus solution was diluted to 100 TCID in serum-free DMEM medium. 50 When the cell confluence in the well plate reaches 70%-80%, the virus can be inoculated, the original culture medium is discarded, and the cells are washed once with PBS. Each well of the virus group and drug treatment group is inoculated with 1 mL of infection dose of 100 TCID 50 The virus solution was added, and the remaining components were added with an equal amount (1 mL) of serum-free DMEM culture medium, and incubated in a constant temperature incubator at 34°C and 5% CO2 for 2 h; the small molecule compounds were diluted with D2 into 4 concentration gradients (50 μM, 25 μM, 12.5 μM, and 6.25 μM) for use. After the incubation, 1 mL of D2 was added to each well of the virus group and the blank control group; 1 mL of the diluted drug-containing culture medium was added to the drug treatment group; an equal amount of D2 containing 5‰ DMSO was added to the solvent group, and the cells were placed in a constant temperature incubator at 34°C and 5% CO2 for continued culture for 72 h. The CPE was observed and recorded every day. After the culture was completed, the cell culture supernatant and cells were collected for the following tests.

[0021] Detection of viruses in cell culture supernatants by absolute quantitative PCR n Gene copy number. The cell culture supernatant was sequentially pipetted into 1.5 mL enzyme-free EP tubes, centrifuged at 8000 rpm / min for 5 min, and 140 μL was taken to extract viral RNA using the Omega EZNA viral RNAextraction R6874-02 kit. The reaction system was configured according to the instructions of the Acridine AG11713 kit (as shown in Table 4). The viral nucleocapsid protein (n) gene copy number was detected by qRT-PCR, and the inhibition rate and IC of small molecule compounds against HCoV-OC43 were calculated. 50 , inhibition rate (%) = 1-(drug treatment group n Gene copy number / virome n gene copy number) × 100%, the primer and probe sequences are shown in Table 3, and the qRT-PCR reaction program is reverse transcription at 42°C for 5 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, and a total of 39 cycles of denaturation, annealing and extension; Table 3 Primer and probe sequences for detecting HCoV-OC43 n gene

[0022] Table 4 Acryl (AG11713) reaction system

[0023] The level of viral replication in cells was detected by relative quantitative PCR. Wash the cells in the 12-well plate twice with pre-cooled PBS, add 1 mL of Trizol lysis solution to each well and lyse the cells on ice for 10 minutes, blow the cells until they are completely detached, and collect them in order into enzyme-free 1.5 mL enzyme-free EP tubes, and extract total cell RNA according to the Trizol reagent instructions. The specific experimental method for extracting total cell RNA by Trizol is as follows: Add 200 μL of chloroform to each tube of sample and shake vigorously for 15 seconds, incubate at room temperature for 15 minutes, and centrifuge at 4°C and 13,000 rpm / min for 15 minutes; transfer the supernatant to a new enzyme-free EP tube, add 500 μL of isopropanol, mix by inversion, incubate at room temperature for 10 minutes, and centrifuge at 4°C and 13,000 rpm / min for 15 minutes; discard the supernatant, retain the white precipitate, add 1 mL of 75% ethanol (anhydrous ethanol + DEPC water, freshly prepared and used) to wash the precipitate twice, and centrifuge at 4°C and 13,000 rpm / min for 5 minutes; discard the supernatant, retain the precipitate, open the lid, and let it dry; add 20 μL of RNase water and dissolve the precipitate on ice for 15 minutes; measure the RNA concentration by UV spectrophotometer; configure the reaction system according to the instructions of the Acry AG11728 kit (as shown in Table 6), and reverse transcribe the sample into cDNA. Configure the reaction system according to the instructions of the Vazyme Q311 kit (as shown in Table 7), and detect viruses by qRT-PCR n Gene mRNA expression levels, GAPDH was used as the internal reference gene, and the primer sequences were shown in Table 5; 2 -ΔΔ Ct Methods Calculation of inhibition rate and IC of small molecule compounds against HCoV-OC43 50 , inhibition rate (%) = 1-(virus in drug treatment group n Gene mRNA expression level / virome n The reverse transcription reaction program was: 37℃ 15min, 85℃ 5s, 4℃ ∞; the qRT-PCR reaction program was: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s, 60℃ annealing and extension for 20s, denaturation, annealing and extension for 40 cycles; melting section was 95℃ 15s, 60℃ 1min, 95℃ 15s; Table 5 Primer sequences for detecting HCoV-OC43 mRNA

[0024] Table 6 Acryl AG11728 reaction system

[0025] Table 7 Vazyme Q311 reaction system

[0026] Results Figure 3-6 Both small molecule compounds can inhibit the replication level of HCoV-OC43 in cells and supernatants, and the inhibition is dose-dependent. In addition, ZP-96 has strong anti-HCoV-OC43 activity at 50 μM, 25 μM and 12 μM, and the virus inhibition rate is greater than 90%.

Claims

1. Application of small molecule compounds with structural formulas as shown in Formula I and Formula II in the preparation of anti-coronavirus drugs; Formula I ; Formula II .

2. The use according to claim 1, characterized in that :The coronavirus is HCoV-OC43.

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