CN113952341A - Application of small molecular compound CB-5083 in preparation of anti-SARS-CoV-2 medicine - Google Patents
Application of small molecular compound CB-5083 in preparation of anti-SARS-CoV-2 medicine Download PDFInfo
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- CN113952341A CN113952341A CN202111227573.4A CN202111227573A CN113952341A CN 113952341 A CN113952341 A CN 113952341A CN 202111227573 A CN202111227573 A CN 202111227573A CN 113952341 A CN113952341 A CN 113952341A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
Abstract
The invention discloses an application of a small molecular compound CB-5083 in preparing a medicine for resisting severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), wherein the medicine for resisting SARS-CoV-2 is a medicine which takes CB-5083 as a unique active component or contains CB-5083, and the medicine for resisting SARS-CoV-2 is a medicine for preventing or treating SARS-CoV-2 infection. The invention uses susceptible cell line of SARS-CoV-2, including Vero E6 of African green monkey kidney cell and Calu-3 of human lung adenocarcinoma cell, to detect the anti-SARS-CoV-2 activity of CB-5083. The experimental result shows that CB-5083 can effectively inhibit the infection of SARS-CoV-2 to the susceptible cells, has small cytotoxicity, is hopeful to be used as an effective anti-SARS-CoV-2 infection medicine, and has application prospect.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an application of a small molecular compound CB-5083 in preparation of a medicine for resisting SARS-CoV-2.
Background
The small molecular compound CB-5083 is an oral p97 AAAATPase inhibitor with bioactivity, is approved by the FDA of the United states for clinical use, and has a remarkable treatment effect on colon cancer cells. There is no literature reporting its role in anti-SARS-CoV-2.
The chemical structural formula of CB-5083 is as follows (CAS No. 1542705-92-9):
severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is a newly-appeared pathogen of high-infection and high-pathogenicity infectious diseases, and is mainly transmitted through respiratory tract by means of droplet, close contact and the like. The disease caused by SARS-CoV-2 infection is called coronavirus disease 2019(COVID-19), which often includes lower respiratory tract infection, namely viral pneumonia, and is mainly manifested by fever, dry cough, hypodynamia and the like, and a few patients are accompanied by upper respiratory tract and digestive tract symptoms such as nasal obstruction, watery nasal discharge, diarrhea and the like. Severe cases often develop dyspnea after 1 week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis and hemorrhagic coagulation dysfunction, multiple organ failure, and the like. Reidesciclovir (Remdesivir) is an adenosine analog, a broad-spectrum antiviral agent, and has inhibitory activity against a variety of RNA viruses, including Ebola and coronavirus. Although Reidesciclovir has received the U.S. food and drug administration's grant for the urgent use of COVID-19, its clinical efficacy is limited. In view of this, the search and development of drugs effective against SARS-CoV-2 infection are important tasks in the world of biomedicine, and are urgent needs for protecting human health.
Disclosure of Invention
The invention aims to provide application of CB-5083 in preparing anti-SARS-CoV-2 medicaments.
The chemical structural formula of the CB-5083 is as follows:
the invention provides an application of a small molecular compound CB-5083 in preparing a medicine for resisting SARS-CoV-2.
The application of CB-5083 in preparing anti-SARS-CoV-2 medicine is characterized in that: the anti-SARS-CoV-2 medicine is medicine for preventing or treating SARS-CoV-2 infection.
The application of CB-5083 in preparing anti SARS-CoV-2 infection medicine is characterized in that: the anti-SARS-CoV-2 medicine is CB-5083 as the only active component, or a medicine composition containing CB-5083.
The application of CB-5083 in preparing anti SARS-CoV-2 infection medicine is characterized in that: the content of CB-5083 in the anti-SARS-CoV-2 medicine is 0.1-99 wt%.
The application of CB-5083 in preparing anti-SARS-CoV-2 medicine is characterized in that: the pharmaceutical preparation is at least one of capsules, suspensions, tablets, powders, emulsions, solutions, syrups or injections.
The application of CB-5083 in preparing anti-SARS-CoV-2 medicine is characterized in that: the administration route of the pharmaceutical preparation is oral administration, injection or respiratory inhalation.
The invention utilizes an experimental operation system of SARS-CoV-2 infected susceptible cells to screen candidate micromolecule medicaments capable of inhibiting SARS-CoV-2 infection from a clinically approved medicament micromolecule library, screens CB-5083 which can effectively inhibit the infection of SARS-CoV-2 to Vero E6 of African green monkey kidney cells and Calu-3 of human lung adenocarcinoma cells, has small cytotoxicity, can be used as a potential anti-SARS-CoV-2 medicament, and has application prospect.
Drawings
FIG. 1 shows the inhibitory effect of different concentrations of CB-5083 on SARS-CoV-2 infection.
Wherein, A: dose effect analysis of the inhibitory effect of Reidesvir and CB-5083 on SARS-CoV-2 in Vero E6; b: in human lung adenocarcinoma cell Calu-3, the dose effect analysis of the inhibitory effect of Reidesciclovir and CB-5083 on SARS-CoV-2. After 18 hours of treatment, infection with SARS-CoV-2 was detected in each well by immunofluorescence, positive cells in each well were counted by a BioTek staining 5Imaging Reader, and then the inhibition ratio (%) of the drug against infection with SARS-CoV-2 was calculated as 1- (number of positive cells in drug-treated well/number of positive cells in DMSO-treated well) × 100%, and the EC50 value was calculated from the inhibition ratio at each concentration. Each value is expressed as the mean ± standard deviation of 3 biological replicates. Black: the number of cells; gray: the rate of viral infection; EC50(concentration for 50% of maximum effect): half maximal effect concentration.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
CB-5083 used in the examples of the present invention can be purchased commercially.
Example 1
First, experimental medicine, reagent and material
1. A compound: the small molecule drug library (product number: L1300-Z349373) approved for clinical use by the U.S. FDA contains 2580 small molecule compounds, purchased from Selleck, USA, all dissolved in DMSO at a concentration of 10. mu.M. Reidesciclovir (Remdesivir) from Selleck, USA, dissolved in DMSO at a concentration of 10. mu.M.
2. Vero E6 of African green monkey kidney cells and Calu-3 of human lung adenocarcinoma cells were purchased from Shanghai cell institute of Chinese academy of sciences and preserved by the biomedical protective research laboratory of naval medical department of the university of naval medical university of the people's liberation army.
DMEM complete cell culture solution containing 10% fetal bovine serum, 0.03% glutamine, non-essential amino acids, ampicillin and streptomycin 100U/mL, adjusted to pH 7.4.
4. Cell digest, containing 0.25% trypsin, was prepared in phosphate buffered saline.
SARS-CoV-2 virus was isolated and cultured from samples of nasopharyngeal swabs from COVID-19 patients at the department of the third Biosafety level (P3) laboratory of the university of military medical science, the gene sequence of which is shown in GenBank Accession No. MT622319, and all experimental procedures involving SARS-CoV-2 infection were carried out at the department of the university of military medical science P3 laboratory.
6. Rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody was purchased from Beijing Yiqian Shenzhou science and technology, Inc. (Sino Biological # 40143-T62).
Alexa Fluor 488-labeled anti-rabbit IgG is a product of Thermo Fisher, USA.
II, an experimental method:
screening anti-SARS-CoV-2 compound from approved small molecule medicine bank
Vero E6 of Vero cells of Vero, which were cultured in a T25 flask, were inoculated into a 96-well plate at 10000 cells per well in 100. mu.L of medium, and after 12 hours, 50. mu.L of SARS-CoV-2 diluted with the culture medium and 50. mu.L of the small molecule compound diluted with the culture medium were added per well. SARS-CoV-2 virus dose is 1X 103Ffu (focus forming units) (i.e. the amount of virus infecting 1000 Vero E6 cells), final concentration of small molecule compound 5 μ M, repeated 3 wells using 5 μ M of rendesivir (Remdesivir) as positive control and DMSO as negative control, which contained an equal volume of solvent. After 18 hours, the infection of SARS-CoV-2 in each well of cells was detected by immunofluorescence, which was performed as follows: removing culture medium by suction, adding 0.1mL of methanol into each well, placing the culture plate in a refrigerator at-20 deg.C, after 20 minutes, taking out the culture plate, removing methanol by suction, washing each well with Phosphate Buffer Solution (PBS) once, adding 100 μ L PBS containing 3% Bovine Serum Albumin (BSA) (hereinafter referred to as 3% BSA-PBS), placing on a horizontal shaking table, slowly shaking at room temperature for 1 hour, removing 3% BSA-PBS in the culture plate by suction, adding 0.1mL polyclonal antibody containing rabbit anti-SARS-CoV-2 nucleocapsid protein into each well1% BSA-PBS (antibody 500 times diluted), slowly shaking for 1 hour at room temperature, absorbing rabbit polyclonal antibody working solution in the culture plate, washing each well with PBS for 3 times, then adding 0.1mL 1% BSA-PBS (fluorescein antibody 1500 times diluted) containing fluorescein AlexaFluor 488-labeled anti-rabbit IgG, slowly shaking for 1 hour at room temperature in a dark place, absorbing fluorescein antibody working solution in the culture plate, adding 0.1mL DAPI cell nuclear staining solution in each well, slowly shaking for 10 minutes at room temperature in a dark place, absorbing DAPI cell nuclear staining solution in the culture plate, washing each well with PBS for 3 times, counting green fluorescent positive cells of each well of cells with a cell Imaging and analysis system (BioTek staining 5Imaging Reader), then, the inhibition rate (%) of the drug against SARS-CoV-2 infection was calculated as 1- (number of drug-treated well positive cells/number of DMSO-treated well positive cells) × 100%.
And (3) taking the micromolecule drug with the inhibition rate of more than 95% and the difference between the cell nucleus count and the DMSO-treated pore phase of not more than 5% as a candidate antiviral drug, and further determining the cytotoxicity and antiviral activity of the micromolecule drug. The screening results show that: the inhibition rate of the Redcixvir serving as a positive control on SARS-CoV-2 infection is more than 99 percent; 98 compounds in 2580 compounds reach the standard of candidate antiviral drugs, wherein the inhibition rate of CB-5083 to SARS-CoV-2 infection is more than 99%. The published research papers and patent of invention are referred to, and no report that CB-5083 has the activity of resisting SARS-CoV-2 is found.
Cytotoxicity assay for (di) CB-5083
Respectively inoculating Vero E6 cells of African green monkey kidney cells and Calu-3 cells of human lung adenocarcinoma in a 96-well plate, wherein 10000 cells in each well and 100 mu L of DMEM culture solution are cultured, sucking out original culture solution after 12 hours, adding 100 mu L of DMEM culture solution containing CB-5083 with concentration gradient dilution, and the final concentration of CB-5083 is 0, 0.064, 0.32, 1.6, 8, 40 and 100 mu M respectively, and taking DMEM culture solution containing DMSO with equal volume as a control. Three duplicate wells were set for each drug concentration and corresponding volume of DMSO, placed at 37 deg.C and 5% CO2Culturing in an incubator. After 48 hours, adding 100 mu L of CellTiter-Glo luminescence method cell activity detection reagent into each hole, incubating for 20min at room temperature, and detecting chemiluminescence of each hole cell on an enzyme labeling instrumentValues (chemiluminescence values represent cell activity). And calculating the ratio of the mean chemiluminescence values of the cells of the drug-treated wells to the chemiluminescence values of the cells of the wells containing the corresponding volume of DMSO solvent, wherein the closer the ratio is to 1, the smaller the influence of the drug on the cell growth is, and the ratio of more than 0.9 is considered to be insignificant.
The results are shown in Table 1, where CB-5083 is not significantly toxic to both cell lines at concentrations less than or equal to 40. mu.M, i.e., cell proliferation is not significantly affected by CB-5083 at these concentrations. When the concentration of CB-5083 is equal to or higher than 100 mu M, the growth of two cells is inhibited to some extent, and the result shows that the cytotoxicity of CB-5083 is lower.
TABLE 1 toxicity (chemiluminescence ratio) of different concentrations of CB-5083 to Vero E6 and Calu-3 cell lines
(III) CB-5083 Activity test against SARS-CoV-2 infection
Vero E6 of Vero cells of Vero, which were cultured in a T25 cell culture plate, and Calu-3 of human lung adenocarcinoma cells were inoculated into a 96-well plate, 10000 cells per well, 100. mu.L of medium were added to each well, and after 12 hours, SARS-CoV-2 (1X 10. mu.L) diluted in 50. mu.L of DMEM medium was added to each well3FFU), and CB-508350 μ L (diluted with DMEM medium) serially diluted two-fold starting from a maximum concentration of 20 μ M to a minimum concentration of 0.0625 μ M, with reidsivir (Remdesivir) as a positive control and DMSO as a negative control containing an equal volume of solvent as a negative control, each concentration being repeated for 3 wells. After 18 hours, the cells in each well were tested for infection with SARS-CoV-2 by immunofluorescence as described above. The green fluorescence positive cells per well were counted using a cell Imaging and analysis system (BioTek staining 5Imaging Reader), and then the inhibition rate (%) of the drug against SARS-CoV-2 infection was calculated as 1- (number of drug-treated well positive cells/number of DMSO-treated well positive cells) × 100%. EC50 values were calculated from the inhibition ratios at each concentration. As shown in FIG. 1, the EC50 of Reidesciclovir for inhibiting SARS-CoV-2 in Vero E6 cell is 0.7741. mu.M, and for inhibiting SARS-CoV-2 in Calu-3 cellEC50 for SARS-CoV-2 was 0.6403. mu.M; CB-5083 the EC50 for inhibiting SARS-CoV-2 in Vero E6 cell is 0.5758. mu.M, and the EC50 for inhibiting SARS-CoV-2 in Calu-3 cell is 0.6763. mu.M. The results show that CB-5083 can effectively inhibit SARS-CoV-2 from infecting Vero E6 and Calu-3 cells.
(IV) identification of the stage of action of CB-5083 in inhibiting SARS-CoV-2 infection
Inoculating Vero E6 Vero and Calu-3 to 96-well plate with 10000 cells per well and 100 μ L culture medium, adding 80 μ LSARS-CoV-2(2 × 10) per well after 12 hr3FFU), the culture supernatant was aspirated after 1 hour, and the wells were washed three times with PBS buffer. 20 μ L of CB-5083 at a concentration of 50 μ M (i.e., CB-5083 at a final concentration of 10 μ M, and CB-5083 at 0 hr, with the addition of virus and CB-5083) were added at 0, 1, 2, 4, 6 and 8 hr (hpi) post-infection, respectively, and the infection of SARS-CoV-2 in the cells of each well was detected by immunofluorescence at 10 hr post-infection, as described above. The green fluorescence positive cells per well were counted using a cell Imaging and analysis system (BioTek staining 5Imaging Reader), and then the inhibition rate (%) of the drug against SARS-CoV-2 infection was calculated as 1- (number of positive cells in drug-treated wells/number of positive cells in DMSO-treated wells) 100%.
The results are shown in Table 2, and the addition of CB-5083 4 hours after SARS-CoV-2 infection of Vero E6 and Calu-3 cells can still obviously inhibit virus infection, indicating that CB-5083 may act on the replication stage of the virus.
TABLE 2 Effect of CB-5083 treatment on different stages of SARS-CoV-2 infection
The above experimental results show that CB-5083 can effectively inhibit SARS-CoV-2 infection and mainly acts on virus replication stage.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. Application of a small molecular compound CB-5083 in preparing anti-SARS-CoV-2 medicines.
2. The use of CB-5083 of claim 1 in the preparation of a medicament against SARS-CoV-2, wherein: the anti-SARS-CoV-2 medicine is medicine for preventing or treating SARS-CoV-2 infection.
3. The use of CB-5083 of claim 2 in the manufacture of a medicament for the treatment of SARS-CoV-2 infection, wherein: the anti-SARS-CoV-2 medicine is CB-5083 as the only active component, or a medicine composition containing CB-5083.
4. The use of CB-5083 of claim 3 in the manufacture of a medicament for the treatment of SARS-CoV-2 infection, wherein: the content of CB-5083 in the anti-SARS-CoV-2 medicine is 0.1-99 wt%.
5. The use of CB-5083 of claim 4 in the preparation of a medicament against SARS-CoV-2, wherein: the pharmaceutical formulation is at least one of a capsule, a suspension, a tablet, a powder, an emulsion, a solution, a syrup, or an injection.
6. The use of CB-5083 of claim 5 in the preparation of a medicament against SARS-CoV-2, wherein: the administration route of the pharmaceutical preparation is oral administration, injection or respiratory inhalation.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106177961A (en) * | 2016-08-18 | 2016-12-07 | 广州威溶特医药科技有限公司 | The application in preparing antitumor drug of VCP inhibitor and oncolytic virus |
| WO2021130638A1 (en) * | 2019-12-24 | 2021-07-01 | Carna Biosciences, Inc. | Diacylglycerol kinase modulating compounds |
| WO2021232050A1 (en) * | 2020-05-11 | 2021-11-18 | Board Of Regents, The University Of Texas System | Natural killer cell immunotherapy for the treatment or prevention of sars-cov-2 infection |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106177961A (en) * | 2016-08-18 | 2016-12-07 | 广州威溶特医药科技有限公司 | The application in preparing antitumor drug of VCP inhibitor and oncolytic virus |
| WO2021130638A1 (en) * | 2019-12-24 | 2021-07-01 | Carna Biosciences, Inc. | Diacylglycerol kinase modulating compounds |
| WO2021232050A1 (en) * | 2020-05-11 | 2021-11-18 | Board Of Regents, The University Of Texas System | Natural killer cell immunotherapy for the treatment or prevention of sars-cov-2 infection |
Non-Patent Citations (1)
| Title |
|---|
| CHANGJIANG LIU ET AL.: "On Drug-Membrane Permeability of Antivirals for SARS-CoV‑2", 《THE JOURNAL OF PHYSICAL CHEMISTRY LETTERS》 * |
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