CN113018421A - Use of cholesterol-25-hydroxylase and enzymatic products thereof for the preparation 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 a 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 enzymatic products thereof for the preparation of a medicament for inhibiting a novel coronavirus

Technical Field

The invention belongs to the field of chemical drugs, and particularly relates to an application of cholesterol-25-hydroxylase and an enzymatic product 25-hydroxycholesterol thereof in preparing a drug for inhibiting a novel coronavirus (SARS-CoV-2).

Background

Coronaviruses (Coronavirus) are nonsegmented, single-stranded, positive-stranded RNA viruses with an envelope, and have a wide range of animal hosts. SARS coronavirus and MERS coronavirus derived from animal infectious diseases can cause death in humans and outbreaks in 2002 and 2012, respectively (Zaki a.m., van Boheemen s, bestbromer t.m., et al, Isolation of a novel coronavir from a man with a pneumonia in Saudi Arabia [ J ], n.engl.j.med.,2012,367(19): 1814-. New coronavirus (SARS-CoV-2), a new coronavirus similar to SARS coronavirus, was detected in pneumonia patients in 2019 and considered as the pathogen of the pneumonia epidemic situation. The clinical manifestations of the patients were as follows: more than 90% of patients present with fever, 80% with dry cough, 20% with shortness of breath, 15% with dyspnea, and most importantly with a reduction in leukocytes and lymphocytes. In the previous studies, drugs such as Reidesvir (Remdesivir) and chloroquine phosphate (chloroquine) can well inhibit the replication of viruses (Wang M., Cao R., Zhang L., et al., Remdesivir and chloroquine effective inhibition of the recombinant expressed novel polynucleotide (2019-nCoV) in vitro [ J ], Cell Res.,2020,30:269-271), and related studies have been clinically carried out, and related vaccines are also developed, but no drug or vaccine is approved to be widely applied at present.

Innate immunity is an important defense line against pathogenic invasion in a host, and an important component of the innate immunity is Interferon (IFN) and various Interferon-stimulated genes (ISGs) produced by the IFN. Cholesterol-25-Hydroxylase (CH 25-Hydroxylase, CH25H) is a member of the ISGs that catalyze the conversion of Cholesterol to 25-Hydroxycholesterol (25-Hydroxycholestrol, 25HC), 25HC being an oxygenated steroid alcohol naturally occurring in the human body (Holmes R.S., Vandeberg J.L., Cox L.A., Genomics and proteomics of versatate cholestrol ester Lipase (LIPA) and cholestrol 25-hydroxycholestase (CH25H) [ J ].3Biotech, 2011,1(2): 99-109). Our previous results of studies on the antiviral effects of CH25H and 25HC show that either overexpression of CH25H in cells or in vitro synthesis of 25HC inhibits replication of RNA (such as HIV, VSV and Ebola) and DNA (such as HSV-1 and MHV68) viruses (Liu S.Y., Aliyari R., Chikere K., et al, interference-induced cholesterol-25-hydroxyzyme branched viral vector expression of 25-hydroxyzyme [ J ], Immunity,2013,38(1):92-105) very strongly. However, the ability of CH25H and 25HC to combat SARS-CoV-2 infection has not been investigated to date.

Disclosure of Invention

Problems to be solved by the invention

Aiming at the fact that the results of no definite research at present prove that CH25H and 25HC can resist the infection of SARS-CoV-2, the invention proves that whether the CH25H is over-expressed or the synthesized 25HC is applied can inhibit the infection of SARS-CoV-2 for the first time through in vitro experiments.

Means for solving the problems

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

Meanwhile, the invention provides application of the pharmaceutical composition containing cholesterol-25-hydroxylase (CH25H) in preparing 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, which is useful for inhibiting a novel coronavirus (SARS-CoV-2).

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

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

Meanwhile, the invention provides an application of the pharmaceutical composition containing 25-hydroxycholesterol (25HC) in preparing a medicine for inhibiting the novel coronavirus (SARS-CoV-2).

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

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

ADVANTAGEOUS EFFECTS OF INVENTION

The inventor of the invention unexpectedly finds that either the over-expression of CH25H or the application of synthetic 25HC can well inhibit the infection of SARS-CoV-2, and the safety is good, and the both can be used for preparing the medicine for inhibiting SARS-CoV-2.

Drawings

FIG. 1 shows the safety of 25-hydroxycholesterol in vitro experiments.

FIG. 2 shows SARS-CoV-2 inhibition of cholesterol-25-hydroxylase overexpression in an in vitro assay.

FIG. 3 shows SARS-CoV-2 inhibition by 25-hydroxycholesterol in an in vitro assay.

FIG. 4 shows the inhibitory effect of 25-hydroxycholesterol on SARS-CoV-2 protein expression in an in vitro immunoblot assay.

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

Detailed Description

The technical solutions of the present invention will be further explained or illustrated with reference to specific examples. Unless otherwise indicated, the instruments, materials, reagents and the like used in the following examples are all available by conventional commercial means.

Example 1: safety assessment of 25HC on cells.

The specific experimental process is as follows:

1) vero cells were seeded at 10000/well in 96-well cell culture plates at 37 ℃ with 5% CO₂Incubated under conditions overnight.

2) When the cells grow to 50% of a monolayer, DMEM maintenance solution containing 2% FBS of different concentrations of drugs (initial concentration is 400 mu M, 3 times of dilution gradient is adopted, the drug to be detected is diluted in a multiple proportion, and the total concentration is 8) is added, 100 mu L/hole, and 3 multiple holes are detected in each concentration. The culture was continued, and the state of the cells was observed under a microscope every day.

3) On day 4, 20. mu.L of MTS solution was added at 37 ℃ with 5% CO₂The cells were incubated for 1h under the conditions, and the OD490 value was measured.

4) Calculating the toxicity of the drugs with different concentrations to the cells (% Cytoxicity) by adopting a formula (1- (drug group OD-culture medium OD)/(cell control group OD-culture medium OD)) × 100 percent, performing S fitting analysis on the data by using Graphpad Prism 7 software, and calculating the CC of the drugs₅₀。

As shown in figure 1, 25HC used at a maximum concentration of 400 μ M still has less than 50% toxicity to Vero cells, which is much higher than the effective SARS-CoV-2 inhibitory concentration of 25HC, and is safer in use.

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

The specific experimental process is as follows:

vero cells were plated in 24-well plates and, 18h later, 800ng of a Green Fluorescent Protein (GFP) control plasmid and CH25H expression plasmid were transfected into Vero cells. After 12h, 100TCID₅₀SARS-CoV-2 was infected, and 1h after infection, the medium was discarded and replaced with fresh 2% FBS in DMEM. And after 24h, collecting the supernatant, and performing fluorescent quantitative PCR to detect the virus copy number.

As shown in fig. 2, CH25H was capable of suppressing the copy number of viral RNA in the cell supernatant after overexpression, and was significantly superior to GFP.

Example 3: half maximal Effective Concentration (EC) of 25HC for SARS-CoV-2₅₀) And (4) measuring.

The specific experimental process is as follows:

determination of drug EC by nucleic acid quantitation₅₀. Vero cells were seeded one day in 48-well plates as follows. The drugs were formulated into 300, 100, 33, 11, 3.67, 1.22 and 0.47 μ M with DMEM maintenance solution of 2% FBS, and ethanol control groups were established. Discard cell culture supernatant, add different concentrations of drug (250. mu.L/well), 3 multiple wells per drug concentration, place at 37 ℃ with 5% CO₂Culturing in an incubator. After 12h, the drug-containing medium was discarded and 20TCID was added₅₀SARS-CoV-2 is infected for 1h, and then replaced by the culture medium containing the medicine for further culture. At 2 days post infection, 50. mu.L of cell supernatant was taken per well to extract nucleic acids, viral load was detected using quantitative RT-PCR, and EC was calculated₅₀。

The ordinate in fig. 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, and it can be seen that the half maximal effective concentration of 25HC is only 3.675 μ M, showing that 25HC can significantly inhibit SARS-CoV-2 at a very low micromolar level, and has a good ability to inhibit viral infection.

Example 4: western blot experiment of 25HC on SARS-CoV-2.

The specific experimental process is as follows:

vero cells were seeded in 12-well plates. The drugs were formulated in DMEM maintenance solution with 2% FBS at 100, 33 and 11 μ M, and ethanol control groups were established. Discarding cell culture supernatant, adding drugs with different concentrations, standing at 37 deg.C and 5% CO₂Culturing in an incubator. After 12h, the drug-containing medium was discarded and 20TCID was added₅₀SARS-CoV-2 is infected for 1h, and then replaced by the culture medium containing the medicine for further culture. At 2 days after infection, cells were harvested, lysed using cell lysates, cellular proteins were extracted, immunoblot experiments were performed, expression of SARS-CoV-2 protein was detected using coronavirus S2 protein antibody, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control protein.

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

Example 5: immunofluorescent staining (immunofluorescent staining) experiment with 25HC for SARS-CoV-2.

The specific experimental process is as follows:

vero cells were seeded in 96-well plates. The drugs were formulated in DMEM maintenance solution with 2% FBS at 100, 33 and 11 μ M, and ethanol control groups were established. Discarding cell culture supernatant, adding drugs with different concentrations, standing at 37 deg.C and 5% CO₂Culturing in an incubator. After 12h, the drug-containing medium was discarded and 20TCID was added₅₀SARS-CoV-2 is infected for 1h, and then replaced by the culture medium containing the medicine for further culture. At 2 days post infection, cells were fixed with tissue fixative, permeabilized with Triton X-100, and after incubation of rabbit polyclonal antibody against the New coronavirus S protein as primary antibody, goat anti-rabbit green fluorescent antibody as secondary antibody, and 4', 6-diamidino-2-phenylindole (DAPI) stained nuclei, photographs were observed under a fluorescent microscope, respectively.

As a visual representation of fluorescence intensity, the density of the relatively bright spots in FIG. 5 reflects the inhibition of SARS-CoV-2 protein expression by 25HC, and as can be seen from the density of the bright spots in FIG. 5, 25HC is able to better inhibit viral protein expression, and the inhibition is enhanced 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.

Images