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
- Publication number
- CN113546076B CN113546076B CN202011217467.3A CN202011217467A CN113546076B CN 113546076 B CN113546076 B CN 113546076B CN 202011217467 A CN202011217467 A CN 202011217467A CN 113546076 B CN113546076 B CN 113546076B
- Authority
- CN
- China
- Prior art keywords
- verteporfin
- cov
- sars
- novel coronavirus
- novel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- ZQFGRJWRSLZCSQ-ZSFNYQMMSA-N verteporfin Chemical compound C=1C([C@@]2([C@H](C(=O)OC)C(=CC=C22)C(=O)OC)C)=NC2=CC(C(=C2C=C)C)=NC2=CC(C(=C2CCC(O)=O)C)=NC2=CC2=NC=1C(C)=C2CCC(=O)OC ZQFGRJWRSLZCSQ-ZSFNYQMMSA-N 0.000 title claims abstract description 65
- 229960003895 verteporfin Drugs 0.000 title claims abstract description 65
- 241001678559 COVID-19 virus Species 0.000 title claims abstract description 30
- 239000003814 drug Substances 0.000 title claims abstract description 29
- 241000711573 Coronaviridae Species 0.000 claims abstract description 24
- 208000015181 infectious disease Diseases 0.000 claims abstract description 14
- 208000025721 COVID-19 Diseases 0.000 claims description 12
- 208000037847 SARS-CoV-2-infection Diseases 0.000 claims description 10
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 229940079593 drug Drugs 0.000 abstract description 14
- 201000010099 disease Diseases 0.000 abstract description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 12
- 238000002474 experimental method Methods 0.000 abstract description 4
- 241000282552 Chlorocebus aethiops Species 0.000 abstract description 3
- 210000003292 kidney cell Anatomy 0.000 abstract description 3
- 238000012606 in vitro cell culture Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 29
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 238000011529 RT qPCR Methods 0.000 description 6
- 230000000120 cytopathologic effect Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000012404 In vitro experiment Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 208000001528 Coronaviridae Infections Diseases 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 208000000059 Dyspnea Diseases 0.000 description 2
- 206010013975 Dyspnoeas Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 241000315672 SARS coronavirus Species 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- XXSMGPRMXLTPCZ-UHFFFAOYSA-N hydroxychloroquine Chemical compound ClC1=CC=C2C(NC(C)CCCN(CCO)CC)=CC=NC2=C1 XXSMGPRMXLTPCZ-UHFFFAOYSA-N 0.000 description 2
- 229960004171 hydroxychloroquine Drugs 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000002547 new drug Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 241000008904 Betacoronavirus Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 201000002563 Histoplasmosis Diseases 0.000 description 1
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 description 1
- 206010029113 Neovascularisation Diseases 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 108091006197 SARS-CoV-2 Nucleocapsid Protein Proteins 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 229960001997 adefovir Drugs 0.000 description 1
- WOZSCQDILHKSGG-UHFFFAOYSA-N adefovir depivoxil Chemical compound N1=CN=C2N(CCOCP(=O)(OCOC(=O)C(C)(C)C)OCOC(=O)C(C)(C)C)C=NC2=C1N WOZSCQDILHKSGG-UHFFFAOYSA-N 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 208000001309 degenerative myopia Diseases 0.000 description 1
- 230000004340 degenerative myopia Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000011880 melting curve analysis Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 238000002428 photodynamic therapy Methods 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 208000013220 shortness of breath Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- 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
Landscapes
- 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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010324813 | 2020-04-23 | ||
| CN2020103248131 | 2020-04-23 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113546076A CN113546076A (en) | 2021-10-26 |
| CN113546076B true CN113546076B (en) | 2024-04-30 |
Family
ID=78130029
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011217467.3A Active CN113546076B (en) | 2020-04-23 | 2020-11-04 | Application of verteporfin in preparing medicine for resisting novel coronavirus SARS-CoV-2 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113546076B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2020
- 2020-11-04 CN CN202011217467.3A patent/CN113546076B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| Potent antiviral effect of protoporphyrin IX and verteporfin on SARS-CoV-2 infection;Chenjian Gu等;《bioRxiv》;全文 * |
| 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 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113546076A (en) | 2021-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TWI787210B (en) | New antiviral compositions for the treatment of coronavirus-related infections | |
| Huang et al. | Antiviral activities of resveratrol against rotavirus in vitro and in vivo | |
| WO2017010835A1 (en) | Use of radotinib for prevention or treatment of viral respiratory disease | |
| CN116236580A (en) | Application of old medicines such as auranofin and the like and compositions thereof in resisting single positive strand RNA viruses | |
| CN114796177B (en) | Anti-coronavirus medicine and application | |
| CN113546076B (en) | Application of verteporfin in preparing medicine for resisting novel coronavirus SARS-CoV-2 | |
| CN112516132B (en) | Application of salvianolic acid C or pharmaceutically acceptable salt thereof in preparing antiviral drug | |
| CN107308168A (en) | A kind of medicine for suppressing the intracellular duplication of porcine reproductive and respiratory syndrome virus | |
| CN113274375A (en) | Application of metformin and derivatives or pharmaceutically acceptable salts thereof in preparation of drugs for treating coronavirus infection | |
| CN115837025A (en) | Application of berbamine hydrochloride in preparation of drug for preventing or treating African swine fever | |
| CN115381816A (en) | Application of VER50589 in preparing medicine for resisting enterovirus 71 | |
| CN109700823B (en) | Application of telithromycin in resisting ebola virus infection | |
| CN111920801A (en) | Application of epigallocatechin gallate in preparing medicine for treating and/or preventing diseases caused by Ehrlich virus infection | |
| CN109512829A (en) | Luteolin 7-O- glucoside is preparing the application in anti-hepatic-B virus medicine | |
| CN112315959A (en) | Application of porphyrin compound in preparation of anti-coronavirus drugs | |
| CN113230261A (en) | Application of dihydrotanshinone I or pharmaceutically acceptable salt thereof in preparation of anti-coronavirus medicines | |
| CN111568900A (en) | Application of indomethacin in resisting coronavirus infection | |
| CN106619591B (en) | The purposes and pharmaceutical composition of oxetacaine in medicine preparation | |
| CN113750083B (en) | Application of metformin in preparation of medicine for treating hand-foot-and-mouth disease | |
| CN115350181B (en) | Application of small molecular compound in preparation of antiviral infection medicines | |
| CN111529517B (en) | Use of guanidine hydrochloride as a medicament for preventing or treating coronavirus infection | |
| US20230241074A1 (en) | Inhibitors of porcine reproductive and respiratory syndrome virus | |
| CN113546077A (en) | Application of protoporphyrin in preparing medicine for resisting novel coronavirus SARS-CoV-2 | |
| Mendoza | Chloroquine and Hydroxychloroquine for the Treatment of COVID-19 patients: What Every Clinician Should Know | |
| CN106344575B (en) | Application of the alkaloid compound in the drug of preparation treatment viral disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |