CN113546076B - Application of verteporfin in preparing medicine for resisting novel coronavirus SARS-CoV-2 - Google Patents
Application of verteporfin in preparing medicine for resisting novel coronavirus SARS-CoV-2 Download PDFInfo
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- 230000000840 anti-viral effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of medicines, relates to a novel medicinal application of verteporfin, and in particular relates to an application of verteporfin in preparing a medicine for resisting novel coronavirus SARS-CoV-2. Experiments prove that the verteporfin can effectively inhibit the infection of novel coronaviruses by intervening in vitro cell culture Vero-E6 (African green monkey kidney cells), can further prepare and treat novel coronavirus diseases caused by the novel coronaviruses, and provides a novel medicament for treating and controlling the novel coronavirus diseases.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a novel medicinal application of verteporfin, and in particular relates to an application of verteporfin in preparing a medicine for resisting novel coronavirus SARS-CoV-2.
Background
Novel coronavirus disease (Corona Virus Disease 2019, covd-19), also known as novel coronavirus infection, is a serious acute respiratory infectious disease. The disease has strong infectivity and high mortality rate. The pathogen responsible for the novel coronavirus disease is the novel coronavirus (SARS-CoV-2). SARS-CoV-2 and the known severe acute respiratory syndrome coronavirus (SARS-CoV), the middle east respiratory syndrome coronavirus (MERS-CoV) are of the genus beta coronavirus belonging to the family Coronaviridae.
Clinical practice shows that the common symptoms of SARS-CoV-2 infection are respiratory tract symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. There is currently no specific treatment for SARS-CoV-2-caused disease. Studies have shown that Ruidexivir and hydroxychloroquine have significant inhibitory effects on SARS-CoV-2 replication in vitro studies. However, clinical studies have found that hydroxychloroquine has no obvious therapeutic effect. While ryposi Wei Zeng has been reported to cure 1 case, in addition, the test results of treating 53 severe patients with ryposi with the same-condition drug show that 68% of severe patients have symptoms relieved after the use of ryposi, but the mortality rate is still 13%, and 60% of patients report side effects. The above studies with adefovir have few cases and there are significant limitations on the data from symptomatic medication.
The research shows that the novel coronavirus can replicate in Vero-E6 cell strain, which leads to obvious cytopathic effect (CPE) such as syncytia, cell shedding and the like, is a universal novel coronavirus cell infection model and is used for separation, culture and pharmacodynamics determination of the novel coronavirus.
Verteporfin, chinese alias: microtipofin, english name: verteporfin. The verteporfin is a second generation porphyrin photosensitizer, can be activated by light (wavelength 689 nm) irradiation, and can be used for photodynamic therapy for treating macular degeneration. Studies show that the medicine can selectively enter into the diseased blood vessel, and active oxygen is generated by non-thermal laser irradiation to occlude the diseased blood vessel, so that leakage of the blood vessel is stopped, and vision is preserved. Verteporfin is suitable for patients with secondary age-related ocular neovascularization, as well as for pathological myopia or ocular histoplasmosis. Heretofore, no report has been made on the use of verteporfin in inhibiting coronavirus, particularly novel coronavirus infection and replication.
Disclosure of Invention
The invention aims at providing a new medicinal application of verteporfin, in particular to an application of verteporfin in preparing a medicine for resisting novel coronavirus SARS-CoV-2, aiming at solving the problem that an effective medicine for resisting SARS-CoV-2 is needed urgently at present.
The invention evaluates the activity of the verteporfin for resisting SARS-CoV-2 through in vitro experiments; the in vitro experiment mainly comprises the measurement of the inhibition of SARS-CoV-2 infection by the verteporfin, the measurement of the cytotoxicity of the medicine and the like, and provides a new application of the verteporfin in the medicine for resisting SARS-CoV-2, and the verteporfin can be used for preparing the medicine for resisting novel coronavirus SARS-CoV-2.
The purpose of the invention is achieved by the following technical scheme Shi Xian:
The invention provides a new application of verteporfin in resisting SARS-CoV-2.
Further, the present invention provides an anti-novel coronavirus drug comprising verteporfin.
The invention screens anti-SARS-CoV-2 medicine, and the result shows that the known chemical substance 'verteporfin' has obvious inhibition effect on SARS-CoV-2 infection on Vero-E6 cell, and further evaluates the activity of verteporfin anti-SARS-CoV-2 through in vitro experiment; the in vitro experiment mainly comprises the measurement of inhibiting SARS-CoV-2 infection by verteporfin, the measurement of cytotoxicity of medicines and the like, and the result shows that the verteporfin can be used for preparing anti-SARS-CoV-2 medicines and treating novel coronavirus diseases caused by SARS-CoV-2. Novel coronavirus diseases (Corona Virus Disease 2019, covd-19), also known as novel coronavirus infections.
The invention discloses a verteporfin, wherein the English name of the verteporfin is Verteporfin, the molecular formula of the verteporfin is C 82H84N8O16, and the molecular structure of the verteporfin is shown as formula 1.
In one embodiment of the invention, the use of verteporfin in the manufacture of a medicament for the treatment of SARS-CoV-2 is provided.
In another preferred embodiment of the invention, verteporfin is used for the preparation of anti-SARS-CoV-2 drugs and for the treatment of novel coronavirus diseases caused by anti-SARS-CoV-2 drugs.
The invention adopts the verteporfin to intervene in vitro cell culture Vero-E6 (African green monkey kidney cells), and experimental results prove that the verteporfin can effectively inhibit the infection of novel coronaviruses, and the verteporfin can be used for preparing and treating novel coronavirus diseases caused by the novel coronaviruses.
Further, the invention provides other novel anti-novel coronavirus medicines using verteporfin as a prodrug, a composition of verteporfin and a pharmaceutically acceptable carrier and the like.
The invention has the following beneficial effects:
Experiments show that the verteporfin can effectively inhibit SARS-CoV-2 infection on Vero-E6 (African green monkey kidney cells) cultured in vitro, provides a new application of verteporfin as an anti-SARS-CoV-2 drug, and provides a new drug for treating and controlling novel coronavirus diseases.
Drawings
FIG. 1 is an experimental result of observation of inhibition of infection of SARS-CoV-2 on Vero-E6 cells by different concentrations of verteporfin by cytopathic effect (CPE).
FIG. 2 is an experimental result of observing the inhibition of infection of SARS-CoV-2 on Vero-E6 cells by different concentrations of verteporfin by indirect immunofluorescence experiments.
FIG. 3 shows the inhibition of SARS-CoV-2 infection by verteporfin by detecting the viral copy number of Vero-E6 cell supernatant by a real-time quantitative PCR (Q-RT-PCR) method.
FIG. 4 shows the toxic effects of different concentrations of verteporfin on Vero-E6 cells.
Detailed Description
The present invention will be further illustrated by the following examples, but the present invention is not limited to these specific embodiments.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology and the like, which are well known to those skilled in the art. These techniques are fully described in, for example, the specifications of Bruce Alberts, cell molecular biology, 5 th edition (2002), or may be carried out according to the instructions provided by the reagent manufacturers.
Example 1
Inhibition of infection of SARS-CoV-2 on Vero-E6 cells by different concentrations of verteporfin was observed by cytopathic effect (CPE):
Inoculating Vero-E6 cell suspension (4×10 4 cells/well) in 96-well plate, pre-culturing the culture plate in incubator for 12 hr, and growing cells on wall;
the treatment is carried out for 1 hour in advance by adding the verteporfin with corresponding concentration, the solvent control is added with DMSO, and the negative control is not added with medicine;
200PFU SARS-CoV-2 virus (GenBank: MT 121215.1) was then added to each well except for the negative control, and after 12 hours of infection, PBS was washed twice, fresh verteporfin-containing medium was added, and after 48 hours of incubation at 37℃cytopathy was observed microscopically.
As shown in FIG. 1, the experimental results show that normal Vero-E6 cells (negative control) grow normally after 48 hours of culture, no cytopathy is observed, the Vero-E6 (DMSO solvent control) infected with SARS-CoV-2 shows obvious cytopathy after 48 hours of culture, obvious cytopathy appears, 0.31 mu M of Vero-E6 cells treated with verteporfin has no obvious cytopathy after 48 hours of infection with SARS-CoV-2, and 0.16 mu M of Vero-E6 cells treated with verteporfin shows cytopathy, which indicates that 0.31 mu M of verteporfin can effectively inhibit SARS-CoV-2 infection.
Example 2
Inhibition of infection of SARS-CoV-2 on Vero-E6 cells by different concentrations of verteporfin was observed by indirect immunofluorescence experiments:
cells were treated as in example 1, washed with PBS 2 times for 5 minutes after 48 hours of viral infection;
adding 4% paraformaldehyde for fixing at room temperature for 15 minutes;
discarding the solution, adding PBS solution containing 0.5% Triton X-100, allowing to act at room temperature for 10 min, and washing with PBS for 2 times each for 5 min;
adding a freshly prepared PBS solution containing 5% BSA, blocking for 1 hour at room temperature, and washing with PBS for 2 times each for 5 minutes;
Adding 1:1000 dilution of mouse anti-N protein (SARS-CoV-2) polyclonal antibody, and reacting at room temperature for 1 hr;
washing with PBS 3 times for 5 minutes each;
Adding FITC-goat anti-mouse IgG secondary antibody diluted by 1:10000, and reacting for 1 hour at room temperature; washing with PBS 3 times for 5 minutes each;
adding DAPI solution for 20 seconds, and washing with PBS for 2 times for 5 minutes each time;
And observing under a fluorescence microscope.
As shown in FIG. 2, the results show that, consistent with the results of example 1, vero-E6 cells were treated with 0.31. Mu.M verteporfin and then infected with SARS-CoV-2, the N protein of SARS-CoV-2 could not be detected in the cells, and the other two groups (DMSO solvent control and 0.16. Mu.M verteporfin treatment) could detect a green positive signal.
Example 3
Real-time quantitative PCR (Q-RT-PCR) method for detecting the inhibition of infection of SARS-CoV-2 by Vero-E6 cell supernatant virus copy number verification of verteporfin:
Treating cells as in example 1;
After 48 hours, cell supernatants were collected for RNA extraction.
And (3) RNA extraction:
100 mu L of cell supernatant is added into 300 mu L of TRIzol lysate, and the mixture is fully mixed and lysed;
200 mu L of chloroform is added for vigorous shaking, and after standing for 2-3 minutes, the mixture is centrifuged for 15 minutes at 4 ℃ and 12000 g;
sucking the supernatant, adding 500 mu L of isopropanol, standing for 10 minutes, and centrifuging at 4 ℃ for 10 minutes by 12000 g;
Washing the RNA precipitate by adding 1mL of 75% ethanol; centrifuging at 4 ℃ for 5 minutes at 7500 g;
Discarding 75% ethanol, drying, and adding 20-50 μl water to dissolve RNA precipitate;
Reverse transcription of RNA:
RNA reverse transcription cDNA was synthesized in one step using the root FastKing (Tiangen, KR 118). To 20. Mu.L of the reaction system, 4. Mu.L of 5X FastKing-RT Supermix, 20-2. Mu.g of RNA was added, and after 15 minutes at 42℃the reaction was carried out, the reaction was carried out at 95℃for 3 minutes.
Real-time quantitative PCR (qrtPCR);
Real-time quantitative PCR was performed using the Tiangen SYBR Green I chimeric fluorescence method (Tiangen, FP 205). To 20. Mu.L of the reaction system, 10. Mu.L of 2X SuperReal PreMix Plus, 1. Mu.L of each primer (10. Mu.M) and 1. Mu.L of the template cDNA were added, and the mixture was then filled with water to 20. Mu.L. The reaction conditions were as follows: pre-denaturation: 15 minutes at 95 ℃; amplification for 40 cycles: 94℃for 10 seconds and 55℃for 20 seconds; 20 seconds at 72 ℃; melting curve analysis was performed. The amplification primers used are specific primers for SARS-CoV-2N protein gene: an upstream primer: 5'-GGGGAACTTCTCCTGCTAGAAT-3'; a downstream primer: 5'-CAGACATTTTGCTCTCAAGCTG-3'.
The results are shown in FIG. 3, and show that with increasing concentration of the drug of verteporfin, the level of viral RNA in the supernatant of Vero-E6 cells infected with SARS-CoV-2 is significantly reduced, indicating that verteporfin has a significant inhibitory effect on SARS-CoV-2 infection, and its half-effective concentration (EC 50) is 0.028. Mu.M.
Example 4
Detecting the toxic effect of verteporfin on Vero-E6 cells:
Vero-E6 cell suspensions (1 x 10 4/well) were seeded in 96-well plates and the plates were placed in an incubator for 12 hours;
the test drugs with different concentrations are added into the culture holes, and the control group is solvent DMSO.
The effect of the drug on cell proliferation was examined after 48 hours. A syngeneic CCK-8 endpoint kit (ck 04) was used. Discard excess medium in wells and add 100 μl of serum-free medium containing 10% cck8 solution per well;
placing the culture plate in an incubator for incubation for 1-4 hours;
The absorbance at 450nm was then measured using a microplate reader.
As shown in FIG. 4, the effect of verteporfin at a concentration of 2.5. Mu.M was less than that of verteporfin, and the effect was not cytotoxic to Vero-E6 cells, and the half-cell toxicity concentration (CC 50) was 10.33. Mu.M. The Selection Index (SI) was 369 (si=cc50/EC 50).
In conclusion, the research of the invention shows that the verteporfin can inhibit the infection of the Vero-E6 cells by SARS-CoV-2; after treatment with low concentration of verteporfin (nM level), SARS-CoV-2 can not infect Vero-E6 cells, thus providing a new antiviral application for verteporfin application; and thus, a new function of the verteporfin in preventing and treating SARS-CoV-2 infection is developed, and a new thought and method are provided for the research and development of new drugs for novel coronavirus diseases.
It should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is for clarity only, and that the skilled artisan should recognize that the embodiments may be combined as appropriate to form other embodiments that will be understood by those skilled in the art.
The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.
Claims (2)
1. The application of the verteporfin in the formula 1 in preparing the medicine for resisting the novel coronavirus SARS-CoV-2 infection, wherein the verteporfin has the English name Verteporfin and the molecular formula of C82H84N8O16,
Formula 1.
2. The use according to claim 1, wherein said verteporfin is resistant to the novel coronavirus by inhibiting infection with the novel coronavirus SARS-CoV-2.
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CN105748468A (en) * | 2016-03-30 | 2016-07-13 | 四川大学 | Application of verteporfin to preparing anti-ovarian cancer medicine and anti-ovarian cancer medicine |
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Protoporphyrin IX and verteporfin potently inhibit SARS-CoV-2 infection in vitro and in a mouse model expressing human ACE2;Chenjian Gu等;《Science Bulletin》;第66卷;全文 * |
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