CN111514148A - Application of Letermovir in preparation of SARS-CoV-2 novel coronavirus inhibitor - Google Patents

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an application of Letermovir in preparation of a novel SARS-CoV-2 coronavirus inhibitor. According to the invention, through research, Letermovir can be combined with an RBD region combined with an ACE-2 receptor on a spike protein S1 of SARS-CoV-2 novel coronavirus to block the virus from entering cells, so that the virus replication is inhibited. Therefore, Letermovir can be used as an inhibitor of SARS-CoV-2 novel coronavirus, provides a new thought for preventing and/or treating SARS-CoV-2 novel coronavirus, provides a theoretical basis for development of clinical tests, and has important medical research value.

Description

Application of Letermovir in preparation of SARS-CoV-2 novel coronavirus inhibitor

Technical Field

The invention relates to the technical field of medicines, in particular to application of Letermovir in preparation of a novel SARS-CoV-2 coronavirus inhibitor.

Background

Highly pathogenic coronavirus infection has become a public health concern over the last decade. Such as severe acute respiratory syndrome (SARS, 2002-2004), middle east respiratory syndrome (MERS, 2012-to-date), SARS-CoV-2 (COVID-19), each disease has a tremendous impact on human health and economic development. SARS-CoV-2 is reported to be a new member of the genus Beta coronavirus (Betacononavirus, Beta-CoV), and is closely related to Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV) and several bat coronaviruses.

Because there are no effective drugs and vaccines, the research and development of new SARS-CoV-2 coronavirus medicines is of great significance, and in the war without smoke, many scientific research institutions and pharmaceutical companies are dedicated to research and develop new coronavirus vaccines and therapeutic medicines. Therefore, the development of a novel therapeutic drug for SARS-CoV-2 coronavirus has great social and economic significance.

Letermovir (Letermovir) obtained us FDA approval for marketing in 2017 at 11 months, indicated for prevention of CMV viral infection, the drug acted directly on the target DNA terminase complex, and the drug exerted inhibition of the target effect causing problems with viral genome replication (chenyichuan, compilation, 2018). The pharmacological mechanism of Letermovir belongs to a novel antiviral mechanism and is also an orphan drug which is given FDA priority approval. At present, no report about whether the Latemmovir can inhibit SARS-CoV-2 is found, and the action mechanism is not clear.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the application of Letermovir in preparing a novel SARS-CoV-2 coronavirus inhibitor. Letermovir can bind to RBD region of spike protein S1 of SARS-CoV-2 novel coronavirus which is combined with ACE-2 receptor, and block virus from entering cells, thereby inhibiting virus replication.

Specifically, the invention provides an application of Letermovir in preparing a preparation for inhibiting the nucleic acid proliferation of SARS-CoV-2 novel coronavirus.

Specifically, the invention provides an application of Letermovir in preparing a preparation for blocking SARS-CoV-2 novel coronavirus from entering cells.

Specifically, the invention provides an application of Letermovir in inhibiting the spike protein S1 recognition receptor ACE-2 of SARS-CoV-2 novel coronavirus.

Specifically, the invention provides an application of Letermovir in preparing a medicament for preventing and/or treating SARS-CoV-2 novel coronavirus pneumonia infection.

Another object of the present invention is to provide a medicament for preventing and/or treating SARS-CoV-2 novel coronavirus pneumonia infection, which comprises Letermovir.

Preferably, the blood concentration of the Letermovir is 5-20 mu mol/L.

More preferably, the blood concentration of Letermovir is 10. mu. mol/L.

The receptor ACE-2 is recognized by the spike protein spike (S1) due to the entry of the novel SARS-CoV-2 coronavirus into the cell. Therefore, small molecule inhibitors that inhibit spike interaction with ACE-2 targets may be useful in the prevention and/or treatment of novel SARS-CoV-2 coronavirus. The inventor carries out virtual molecular docking on an ACE-2 receptor binding region on an S protein and carries out scoring by an AI scoring system by all FDA approved old drugs (2514, a drug bank database) to obtain 19 candidate old drugs, tests and screens various old drugs, and finds that Letermovir can be used as an effective 2019 novel coronavirus inhibitor.

The structural formula of Letermovir is shown in figure 1. Chinese name: letemmovir. English name: letermovir. The molecular formula is as follows: c₂₉H₂₈F₄N₄O₄. Molecular weight: 572.5. CAS number: 917389-32-3.

The inventor finds that Letermovir can bind to an RBD region which is positioned on a spike protein S1 of SARS-CoV-2 novel coronavirus and is combined with an ACE-2 receptor, blocks the virus from entering cells, and is a brand new mechanism inhibitor of SARS-CoV-2 novel coronavirus. Letermovir inhibits viral nucleic acid proliferation at the cellular level by 77.33% at 10. mu.M.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, researches show that Letermovir can be used as an inhibitor of SARS-CoV-2 novel coronavirus, and provides a new thought for preventing and/or treating SARS-CoV-2 novel coronavirus; letermovir is used as an old medicine for new application, has no safety problem, provides a theoretical basis for the development of clinical tests, and has important medical research value.

Drawings

FIG. 1 is a structural formula of Letermovir.

FIG. 2 shows the result of affinity determination of Letermovir for the RBD region of SARS-CoV-2 novel coronavirus S protein.

Detailed Description

It should be noted that the raw materials used in the present invention are all common commercial products, and the sources thereof are not particularly limited. The following raw material sources are exemplary.

First, experiment reagent and consumable

Recombinant 2019nCOVSpike RBD protein (KACTUS, Cat. COV-VM5 BD);

protein labeling kit (Cat # MO-L011);

capillary loading (Cat # MO-K022);

DMSO (Biyuntian biotechnology, Cat number ST-1276-;

DMEM medium (Gibco, cat # 12430-;

CCK-8 kit (Biyuntian biotechnology, cat # C0039);

nucleic acid extraction kit (Roche, Cat. 03038505001);

2019 novel coronavirus nucleic acid detection kit (Shanghai Berger, Cat. SJ-HX-226-2).

Second, main instrument

QuantStudioDx fluorescent quantitative PCR instrument, manufacturer ABI, No. 04-620;

MagNA Pure LC2.0 full-automatic nucleic acid extractor, manufacturer Roche;

carbon dioxide incubator (GALAXYS), manufacturer RsBiotech, serial number 04-0303;

SARS-CoV-2 virus strain

The novel coronavirus virus strain is identified by a BSL-3 laboratory of the Jiangsu provincial disease prevention and control center through separation and culture laboratories, and the strain adopted in the experiment is numbered as follows: BetacoV/JS03/human/2020(EPI _ ISL _ 411953).

Tetrakis, Letermovir compound

IUPAC name: 2- [ ((4S) -8-fluoro-2- [4- (3-methoxyphenyl) piperazin-1-yl]-3- [ 2-methoxy-5- (trifluoromethyl) phenyl]-4H-quinazolin-4-yl]Acetic acid. English name: 2- [ (4S) -8-fluoro-2- [4- (3-methoxyphenyl) piperazin-1-yl]- 3-[2-methoxy-5-(trifluoromethyl)phenyl]-4H-quinazolin-4-yl]and (4) acetic acid. Molecular weight 572.5. The chemical formula is as follows: c₂₉H₂₈F₄N₄O₄. Purity: 99.85 percent. Storage conditions are as follows: -20 ℃ (powder); -80 ℃ (solvent). The goods number is: t5396. The compound samples and related information were provided by Shanghai ceramic Biotechnology, Inc.

EXAMPLE 1 microcalorimetric phoroptometer (MST) for determining direct binding of Letermovir to RBD region of SARS-CoV-2 novel coronavirus S protein

1. Principle of

The technology is based on the thermophoresis phenomenon of biomolecules, MST uses infrared laser to carry out local heating to cause the molecules to move directionally, and then the directional movement of particles in a microscopic temperature gradient is analyzed through fluorescence. Changes in the size, charge and hydration layer of the biomolecule due to binding can be detected.

2. Applications of

(1) The process of combining the single-chain antibody with the human antigen protein;

(2) interaction of small molecule compounds with model proteins;

(3) the in vitro synthetic protein interacts with the conjugate molecule;

(4) the stroke of the stereo helical structure of the DNA/RNA molecule;

(5) inhibitory effects of small molecule inhibitors on protein kinases;

(6) calcium ions and protamine left-label/non-label;

(7) measurement of thermodynamic constants of protein-bound small molecules;

(8) stoichiometric measurement of biotin binding avidin.

3. Experimental methods

According to the protein labeling kit instructions, 140. mu.L of 10. mu.M protein solution + 15.56. mu.L of NHS were mixed in half with the protein solution and the drug solution and diluted in a two-fold concentration gradient, 10 concentrations were selected and about 6. mu.L of the solution was aspirated through each capillary.

4. Results of the experiment

Letermovir, experiment selected 25. mu.M as the highest concentration, followed by 10 concentration gradients with a signal-to-noise ratio of 14.0, fitted with a Kd of 193nM (as shown in FIG. 2).

Example 2Letermovir in vitro anti-SARS-CoV-2 Activity Effect

First, biological safety requirements

Letermovir in vitro anti-SARS-CoV-2 activity evaluation experiment involves virus culture, all performed in the Biosafety level three (BSL-3) laboratory, as required by the national Weijian Commission "Biosafety guidelines for New coronavirus laboratories (second edition). Nucleic acid extraction using virus cultures, addition of a lysing or inactivating agent is performed under laboratory and protective conditions of the same scale as virus cultures. Operations such as nucleic acid detection after inactivation of live viruses by a reliable method are performed in the biosafety secondary laboratory (BSL-2).

Second, experimental environment

Room temperature: 22 ℃, relative humidity: and 55 percent.

Third, Experimental methods

1. Experiment grouping

(1) Letermovir 10. mu.M group; (2) a DMSO control group; (3) a virus control group to assess whether DMSO has an effect on virus growth; (4) and a cell control group for observing whether the cell growth is normal.

2. Dilution of drugs

Letermovir added with cell maintenance solution according to the ratio of 1:1000 to obtain 10 mu mol/L application solution. The DMSO is diluted according to the dilution ratio of the corresponding drug.

3. Experimental procedure

(1) Cell preparation and drug pretreatment

Vero-E6 cells were individually assigned the 1 × 10⁴Inoculating cells/well in 96-well culture plate, culturing with DMEM containing 10% fetal calf serum for 16 hr to 80% slice, sucking out cell culture solution from each well, washing cells 1 time with sterile PBS, adding different diluted cell maintenance solution (50 μ l/well) into each well according to experimental groups, setting up 4 multiple wells in each group, placing at 37 deg.C and 5% CO₂Pretreating in an incubator for 1 h. Only 50. mu.l of cell maintenance solution was added to the virus control group and the cell control group.

(2) Viral infection and culture

After 1 hour of drug pretreatment, 2. mu.l of SARS-CoV-2 strain was added to each well in addition to the cell control group so that the Multiplicity of viral infection was 0.05(Multiplicity of infection, MOI ═ 0.05), and the mixture was left at 37 ℃ and 5% CO₂Adsorbing for 2 hours in an incubator. The virus-containing medium was discarded after adsorption, and 200. mu.l of the corresponding fresh medium was added to each group at 37 ℃ in 5% CO₂Culturing in an incubator for 48 h. 120. mu.l of culture supernatant was aspirated from each well and inactivated at 56 ℃ for 30 min. After inactivation, 100. mu.l of each well was added to the lysis solution in the reagent tank containing the nucleic acid extraction reagent. And sterilizing the outer surface of the reagent tank, and transferring the reagent tank to a BSL-2 laboratory for virus nucleic acid extraction and gene detection.

(3) Nucleic acid extraction

Extracting virus nucleic acid by referring to the operation instructions of the full-automatic nucleic acid extractor and the extraction kit.

(4) Fluorescent quantitative PCR detection

Configuring a PCR reaction system according to the specification of the novel Shanghai Berger 2019 coronavirus nucleic acid detection kit, wherein the specific reaction system comprises: qRT-PCR reaction liquid 6 mul, qRT-PCR enzyme mixed liquid 2 mul, primer probe 2 mul and template 2.5 mul; the reaction parameters are as follows: 50 ℃ 10min, 95 ℃ 5min, 40cycles of: 10s at 95 ℃ and 40s at 55 ℃ (FAM channel and VIC channel fluorescence signals are collected at this step); the level of virus replication was reflected by measuring the level of transcription of the SARS-CoV-2 viral genes (ORF1ab and N). Calculation 2 from the CT value given by the PCR instrument^-△CTThe value represents the relative virus content of the experimental group to the control group, and the virus replication inhibition ratio (%) (1-2)^-△CT)×100％。

4. Results of the experiment

The fluorescent quantitative PCR detection result shows that the inhibition rate of Letermovir on the virus growth is 77.33% when the concentration of Letermovir in Vero-E6 cells is 10 mu M. The raw data are shown in table 1.

TABLE 1 Primary data of inhibition experiment of Letermovir against SARS-CoV-2 coronavirus

And (4) conclusion: letermovir has inhibitory effect on virus growth at a concentration of 10. mu.M in Vero-E6 cells.

Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.

Claims (8)

1. The application of Letermovir in preparing SARS-CoV-2 coronavirus inhibitor is disclosed.
2. Use of Letermovir in the preparation of a formulation for inhibiting the nucleic acid proliferation of a novel SARS-CoV-2 coronavirus.
3. Use of Letermovir for the preparation of a preparation for blocking the entry of SARS-CoV-2 novel coronavirus into a cell.
4. Use of Letermovir for inhibiting recognition of ACE-2 receptor by spike protein S1 of SARS-CoV-2 novel coronavirus.
5. The application of Letermovir in preparing medicine for preventing and/or treating SARS-CoV-2 new type coronavirus pneumonia infection.
6. 6. A medicament for the prevention and/or treatment of SARS-CoV-2 novel coronavirus pneumonia infection, said medicament comprising Letermovir.
7. 7. The medicament of claim 6, wherein the blood level of Letermovir is 5-20 μmol/L.
8. 8. The medicament of claim 7, wherein the blood level of Letermovir is 10 μmol/L.

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