CN113018421B - Use of cholesterol-25-hydroxylase and enzymatic product thereof in the manufacture of a medicament for inhibiting a novel coronavirus - Google Patents

Abstract

The invention belongs to the field of chemical medicines, and relates to application of cholesterol-25-hydroxylase and an enzymatic product thereof in preparing a medicine for inhibiting novel coronavirus. In vitro cell experiments, either over-expression of cholesterol-25-hydroxylase (CH25H) or administration of synthetic 25-hydroxycholesterol (25HC) can well inhibit SARS-CoV-2 infection with good safety, and both can be used for preparing medicines for inhibiting SARS-CoV-2.

Description

Use of cholesterol-25-hydroxylase and its enzymatic product in the preparation of drugs for inhibiting novel coronavirus

technical field

The invention belongs to the field of chemical medicines, in particular to the use of cholesterol-25-hydroxylase and its enzymatic product 25-hydroxycholesterol in the preparation of medicines for inhibiting novel coronavirus (SARS-CoV-2).

Background technique

Coronaviruses are non-segmented single-stranded positive-stranded RNA viruses with an envelope and have a wide range of animal hosts. SARS-CoV and MERS-CoV derived from zoonotic diseases can cause death in humans and broke out in 2002 and 2012, respectively (Zaki A.M., van Boheemen S., Bestebroer T.M., et al., Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia[J], N.Engl.J.Med., 2012, 367(19): 1814-1820; Cui J., Li F., Shi Z.L., Origin and evolution of pathogenic coronaviruses[J] , Nat. Rev. Microbiol., 2019, 17(3):181-192). In 2019, a novel coronavirus (SARS-CoV-2), a novel coronavirus similar to SARS coronavirus, was detected from patients with pneumonia and was considered to be the pathogen of this pneumonia outbreak. The clinical manifestations of the patients are as follows: more than 90% of patients have fever, 80% of patients have dry cough, 20% of patients have shortness of breath, 15% of patients have dyspnea, and the most important manifestations are leukocytes and lymphocytes. reduction of cells. Previous studies have found that drugs such as remdesivir and chloroquine phosphate can effectively inhibit virus replication (Wang M., Cao R., Zhang L., et al., Remdesivir and chloroquine effectively inhibit therecently emerged novel coronavirus (2019-nCoV) in vitro[J], Cell Res., 2020, 30:269-271), and related research has also been carried out in clinical practice, and related vaccines are also being developed, but at present No drug or vaccine has yet been approved for widespread use.

Innate immunity is an important defense line for the host to resist the invasion of pathogens, and an important part of it is interferon (Interferon, IFN) and a variety of interferon stimulated genes (ISGs) stimulated by it. Cholesterol-25-hydroxylase (CH25H) is a member of ISGs, which can catalyze the conversion of cholesterol into 25-hydroxycholesterol (25-Hydroxycholesterol, 25HC), 25HC is a naturally occurring oxidizer in the human body. Type sterols (Holmes R.S., Vandeberg J.L., Cox L.A., Genomics and proteomics of vertebrate cholesterol ester lipase(LIPA) and cholesterol 25-hydroxylase (CH25H)[J].3Biotech.,2011,1(2):99-109) . Our previous studies on the antiviral effects of CH25H and 25HC showed that both overexpression of CH25H in cells and in vitro synthesis of 25HC can very strongly inhibit RNA viruses (such as HIV, VSV and Ebola virus) and DNA Replication of viruses such as HSV-1 and MHV68 (Liu S.Y., Aliyari R., Chikere K., et al., Interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol[J] , Immunity, 2013, 38(1):92-105). However, whether CH25H and 25HC can fight SARS-CoV-2 infection has not been investigated so far.

SUMMARY OF THE INVENTION

Invention to solve problem

In view of the fact that there is no definite research result to prove whether CH25H and 25HC can fight against SARS-CoV-2 infection, the present invention proves for the first time through in vitro experiments that either overexpression of CH25H or administration of synthetic 25HC can inhibit the infection of SARS-CoV-2. Infect.

solution to the problem

In one aspect, the present invention provides the use of cholesterol-25-hydroxylase (CH25H) in the preparation of a drug for inhibiting novel coronavirus (SARS-CoV-2).

Meanwhile, the present invention provides the use of the pharmaceutical composition comprising cholesterol-25-hydroxylase (CH25H) in the preparation of a medicine for inhibiting novel coronavirus (SARS-CoV-2).

Meanwhile, the present invention provides cholesterol-25-hydroxylase (CH25H) or a pharmaceutical composition comprising the same for inhibiting novel coronavirus (SARS-CoV-2).

Meanwhile, the present invention provides a method for inhibiting novel coronavirus (SARS-CoV-2), which comprises administering an effective amount of cholesterol-25-hydroxylase (CH25H) or a pharmaceutical composition comprising the same to individual needs.

In another aspect, the present invention provides the use of 25-hydroxycholesterol (25HC) in the preparation of a medicament for inhibiting novel coronavirus (SARS-CoV-2).

Meanwhile, the present invention provides the use of a pharmaceutical composition comprising 25-hydroxycholesterol (25HC) in the preparation of a medicine for inhibiting novel coronavirus (SARS-CoV-2).

Meanwhile, the present invention provides 25-hydroxycholesterol (25HC) or a pharmaceutical composition comprising the same for inhibiting novel coronavirus (SARS-CoV-2).

Meanwhile, the present invention provides a method for inhibiting novel coronavirus (SARS-CoV-2), comprising administering an effective amount of 25-hydroxycholesterol (25HC) or a pharmaceutical composition comprising the same to an individual in need thereof .

effect of invention

The inventors of the present invention unexpectedly found in in vitro experiments that either overexpression of CH25H or administration of synthetic 25HC can well inhibit the infection of SARS-CoV-2 with good safety, and both can be used to prepare inhibitor Drugs for SARS-CoV-2.

Description of drawings

Figure 1 shows the safety of 25-hydroxycholesterol in vitro.

Figure 2 shows the SARS-CoV-2 inhibitory effect of cholesterol-25-hydroxylase overexpression in vitro.

Figure 3 shows the SARS-CoV-2 inhibitory effect of 25-hydroxycholesterol in vitro.

Figure 4 shows the inhibitory effect of 25-hydroxycholesterol on SARS-CoV-2 protein expression in in vitro immunoblotting experiments.

Figure 5 shows the inhibitory effect of 25-hydroxycholesterol on SARS-CoV-2 protein expression in an in vitro immunofluorescence staining experiment.

Detailed ways

The technical solutions in the present invention will be further explained or illustrated below in conjunction with specific embodiments. Unless otherwise specified, the instruments, materials or reagents used in the following examples can be obtained by conventional commercial means.

Example 1: Safety assessment of 25HC on cells.

The specific experimental process is as follows:

1) Vero cells were seeded into a 96-well cell culture plate at a concentration of 10,000/well, and cultured overnight at 37°C under 5% CO ₂ .

2) When the cells grow to 50% monolayer, add DMEM containing 2% FBS of different concentrations of drugs (the initial concentration is 400 μM, with a 3-fold dilution gradient, and the drugs to be tested are doubling dilution, a total of 8 concentrations). Maintenance solution, 100 μL/well, 3 replicate wells for each concentration. The culture was continued, and the cell status was observed under a microscope every day.

3) On the 4th day of dosing, add 20 μL of MTS solution, incubate at 37°C under 5% CO ₂ for 1 h, and measure the OD490 value.

4) Using the formula (1-(OD of drug group-OD of culture medium)/(OD of cell control group-OD of culture medium))×100%, calculate the toxicity of different concentrations of drugs to cells (%Cytoxicity), using Graphpad Prism 7 software S-fit analysis was performed on the data to calculate the _CC50 of the drug.

As shown in Figure 1, when 25HC was used at the highest concentration of 400 μM, the toxicity to Vero cells was still less than 50%, which was much higher than the effective inhibitory concentration of 25HC on SARS-CoV-2, and was relatively safe in use. .

Example 2: Inhibition of SARS-CoV-2 by CH25H overexpression in vitro.

The specific experimental process is as follows:

Vero cells were plated into 24-well plates, and 18 h later, 800 ng of green fluorescent protein (GFP) control plasmid and CH25H expression plasmid were transfected into Vero cells. After 12 h, SARS-CoV-2 was infected with 100 TCID _50. After 1 h of infection, the medium was discarded and replaced with fresh 2% FBS DMEM maintenance solution. After 24 hours, the supernatant was collected and subjected to fluorescence quantitative PCR to detect the virus copy number.

As shown in Figure 2, after overexpression of CH25H, it can inhibit the viral RNA copy number in the cell supernatant, which is significantly better than that of GFP.

Example 3: Half maximal effective concentration ( _EC50 ) determination of 25HC against SARS-CoV-2.

The specific experimental process is as follows:

The EC ₅₀ of the drug was determined by nucleic acid quantification. Specifically as follows, Vero cells were seeded in a 48-well plate one day in advance. Drugs were formulated at 300, 100, 33, 11, 3.67, 1.22 and 0.47 μM with 2% FBS in DMEM maintenance solution, and an ethanol control group was established. Discard the cell culture supernatant, add different concentrations of drugs (250 μL/well), 3 replicate wells for each drug concentration, and place them in a 37° C., 5% CO ₂ incubator for cultivation. After 12 hours, the medicated medium was discarded, 20TCID ₅₀ of SARS-CoV-2 was added to infect for 1 hour, and then replaced with medicated medium to continue culturing. Two days after infection, 50 μL of cell supernatant was taken from each well to extract nucleic acid, and the viral load was detected by quantitative RT-PCR, and EC ₅₀ was calculated.

The ordinate in Figure 3 represents the infection rate (% infection), which means the ratio of the viral load of the drug group to the viral load of the ethanol control group. It can be seen that the half-maximum effective concentration of 25HC is only 3.675 μM, showing that 25HC can significantly inhibit SARS-CoV-2 at a very low micromolar level, and has a good ability to inhibit virus infection.

Example 4: Western blot experiment of 25HC against SARS-CoV-2.

The specific experimental process is as follows:

Vero cells were seeded in 12-well plates. Drugs were formulated at 100, 33 and 11 μM with 2% FBS in DMEM maintenance solution, and an ethanol control group was established. Discard the cell culture supernatant, add different concentrations of drugs, and place in a 37°C, 5% CO ₂ incubator for culture. After 12 h, the medicated medium was discarded, SARS-CoV-2 at 20 TCID ₅₀ was added to infect for 1 h, and then replaced with medicated medium to continue culturing. 2 days after infection, the cells were harvested, lysed with cell lysate, and the cell proteins were extracted for immunoblotting experiments. The expression of SARS-CoV-2 protein was detected by using coronavirus S2 protein antibody, and dehydrated with glyceraldehyde-3-phosphate. Hydrogenase (GAPDH) was used as an internal reference protein.

As shown in Figure 4, 25HC was able to inhibit the expression of SARS-CoV-2 protein, and the inhibitory effect was enhanced with the increase of dose, indicating that 25HC had a better ability to inhibit the production of viral proteins.

Example 5: Immunofluorescence staining experiment of 25HC on SARS-CoV-2.

The specific experimental process is as follows:

Vero cells were seeded in 96-well plates. Drugs were formulated at 100, 33 and 11 μM with 2% FBS in DMEM maintenance solution, and an ethanol control group was established. Discard the cell culture supernatant, add different concentrations of drugs, and place in a 37°C, 5% CO ₂ incubator for culture. After 12 h, the medicated medium was discarded, SARS-CoV-2 at 20 TCID ₅₀ was added to infect for 1 h, and then replaced with medicated medium to continue culturing. 2 days after infection, cells were fixed with tissue fixative, Triton X-100 permeabilized, and incubated with rabbit polyclonal antibody against 2019-nCoV S protein as primary antibody, goat anti-rabbit green fluorescent antibody as secondary antibody, and 4' , 6-diamidino-2-phenylindole (DAPI) staining of nuclei, observed and photographed under a fluorescence microscope.

As an intuitive reflection of fluorescence intensity, the density of relatively bright spots in Figure 5 can reflect the inhibitory effect of 25HC on SARS-CoV-2 protein expression. According to the density of bright spots in Figure 5, it can be seen that 25HC can better inhibit viral proteins. expression, and the inhibitory effect increased with increasing dose.

Claims (4)

1. Use of cholesterol-25-hydroxylase in the manufacture of a medicament for inhibiting the novel coronavirus SARS-CoV-2.

2. Use of a pharmaceutical composition comprising cholesterol-25-hydroxylase in the manufacture of a medicament for inhibiting the novel coronavirus SARS-CoV-2.

Use of 25-hydroxycholesterol in the manufacture of a medicament for inhibiting the novel coronavirus SARS-CoV-2.

4. Use of a pharmaceutical composition comprising 25-hydroxycholesterol in the manufacture of a medicament for inhibiting the novel coronavirus SARS-CoV-2.

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