CN117159517A - Application of hypericin in preparation of anti-St.Louis encephalitis virus infection medicine - Google Patents
Application of hypericin in preparation of anti-St.Louis encephalitis virus infection medicine Download PDFInfo
- Publication number
- CN117159517A CN117159517A CN202210580833.4A CN202210580833A CN117159517A CN 117159517 A CN117159517 A CN 117159517A CN 202210580833 A CN202210580833 A CN 202210580833A CN 117159517 A CN117159517 A CN 117159517A
- Authority
- CN
- China
- Prior art keywords
- encephalitis virus
- hypericin
- louis encephalitis
- ns2b
- protease
- 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.)
- Pending
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention provides an application of hypericin in preparing a medicine for resisting St.Louis encephalitis virus infection. The beneficial effects of the invention are as follows: the hypericin has remarkable inhibition effect on the activity of the NS2B-NS3 protease in the St.Louis encephalitis virus, can be used as an inhibitor of the NS2B-NS3 protease in the St.Louis encephalitis virus, and is expected to become a potential drug for resisting St.Louis encephalitis virus infection.
Description
Technical Field
The invention belongs to the technical field of St.Louis encephalitis pharmacy, and particularly relates to application of hypericin in preparation of a medicine for resisting St.Louis encephalitis virus infection.
Background
The st.Louis encephalitis virus (Saint Louis encephalitis virus, SLEV) is a pathogenic virus causing st.Louis encephalitis, and is transmitted to human beings mainly through the bite of culex, and can cause symptoms such as fever, headache, sore throat, muscle pain and the like. The incubation period of st.Louis encephalitis is about one week, most cases are asymptomatic or have influenza-like symptoms such as fatigue, headache, nausea, vomiting and body pain, some patients have severe encephalitis symptoms including stiff neck, blurred consciousness and localized neurological conditions, serious patients may have shock, and some patients have sequelae.
St.Louis encephalitis virus (Saint Louis encephalitis virus, SLEV) is a positive strand RNA virus, closely related to Japanese encephalitis virus, poweria virus and West Nile virus, which is prevalent mainly in North America and south America, and is prevalent and distributed mainly in the Mircubi river and Ohio river basin and in California, florida and Dezhou, etc., which regularly outbreaks and prevails in the West Mircubi river basin and in the area of the gulf of Mexico coast, with sporadic cases reported in the United states in Caribbean, canada, mexico and Central America. In temperate regions, transmission occurs mainly in late summer and early autumn, but in warmer climates, transmission occurs throughout the year. The greatest prevalence of st Louis encephalitis occurred in 1975, reporting nearly 2 thousand cases, mainly in the middle of the Mitsubishi river basin. In recent years, with global warming and rapid population growth and flow, there is a great pressure for the input of St.Louis encephalitis virus in China. Therefore, we need to find drug targets in the st.Louis encephalitis virus, and further develop drugs against st.Louis encephalitis virus against these targets.
The St.Louis encephalitis virus is a single-stranded RNA virus with a genome length of about 11kb, three structural proteins are respectively core shell protein, membrane protein and envelope protein, and seven non-structural proteins are respectively NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The main function of NS3 is to process the polyprotein encoded by the viral genome, which plays an important role in viral replication and assembly. Studies have shown that NS3 protease activity is low and that NS3 protease activity can be greatly increased under conditions of binding to NS 2B. Therefore, the NS2B-NS3 protease is a very important drug target against St.Louis encephalitis virus, so that the screening of inhibitors against the NS2B-NS3 protease has great significance for the research and development of related drugs for St.Louis encephalitis virus infection.
Hypericin, named Hypericin, is an extract of Hypericum perforatum, has effects of resisting dengue virus, coronavirus, swine fever virus and highly pathogenic avian influenza virus, and also has anti-tumor effect. However, the use of hypericin against infection with St.Louis encephalitis virus has not been reported so far.
Disclosure of Invention
The first object of the invention is: the application of hypericin in preparing the anti-St.Louis encephalitis virus infection medicine is provided, the hypericin can be used as a small molecular inhibitor of NS2B-NS3 protease in St.Louis encephalitis virus on a molecular level, and a control experiment is established to show that the hypericin has a strong inhibition effect on the NS2B-NS3 protease in St.Louis encephalitis virus, so that the hypericin can be used as a potential medicine for inhibiting St.Louis encephalitis virus infection.
Another object of the invention is: a medicament for treating infection by the st.
In order to solve the technical problems, the invention adopts the following technical scheme: the hypericin is a small molecule inhibitor of the NS2B-NS3 protease in St.Louis encephalitis virus.
Preferably, the hypericin has a structural formula:
a medicament for the treatment of infection by a st.
Preferably, the carrier comprises one or more of surfactant, binder, disintegrating agent, diluent, excipient, absorption enhancer, wetting agent, adsorption carrier, lubricant, filler and synergist which are conventional in pharmaceutical field.
Preferably, the pharmaceutical preparation is a granule, powder, syrup, tablet, pill or suppository.
By adopting the technical scheme, the hypericin has good inhibition activity on the NS2B-NS3 protease in the St.Louis encephalitis virus, so the hypericin can be used as a small molecule inhibitor of the NS2B-NS3 protease in the St.Louis encephalitis virus, and further can be used as a potential drug for inhibiting St.Louis encephalitis virus infection, and the St.Louis encephalitis virus infection is treated by a new way.
Drawings
FIG. 1 is a schematic representation of the inhibition of NS2B-NS3 protease in St.Louis encephalitis virus by hypericin according to the present invention
FIG. 2 is a diagram showing the IC of hypericin to NS2B-NS3 protease in St.Louis encephalitis virus according to the embodiment of the present invention 50 Schematic of measurement of (2)
Detailed Description
The invention is further illustrated by the following examples and figures:
unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments and comparative examples only and is not intended to be limiting of the scope of the present invention. It should be specifically noted that there may be various names for the same organic structure, so long as the structure belongs to the protection object of the present patent within the scope of the present patent.
Unless otherwise defined, the starting materials, reagents, etc. in the following examples and comparative examples are commercially available or may be prepared according to the reported methods.
First, expression and purification of NS2B-NS3 protease in St.Louis encephalitis virus:
s1: pET22B (+) vector containing gene encoding St.Louis encephalitis virus NS2B-NS3 protein was transformed into E.coli BL21 (DE 3) thallus, and positive clones were screened with LB solid medium (containing 50mg/L ampicillin);
s2: selecting positive clone on solid culture medium, culturing at 37deg.C for 6 hr, transferring into 800mL LB culture medium (containing 50mg/L ampicillin), adding 0.2mM IPTG when its absorbance at 600nm reaches 0.6, and culturing at 16deg.C for 18 hr;
s3: after collecting E.coli by centrifugation at 3500rpm for 20min, high pressure disruption was performed; centrifuging the bacteria-destroying liquid for 1h at 18000rpm, and collecting the upper clarified liquid;
s4: adding the supernatant into Ni-NTA affinity chromatographic column pre-equilibrated with bacteria-destroying buffer (50 mM Tris-HCl,0.3M NaCl,pH 9.0) to make target protein and Ni fully combined, and enriching target protein;
s5: washing out unbound hetero-protein with 20mM imidazole-containing bacteria-destroying buffer solution, wherein when the color of the effluent liquid is not changed in the detection of Coomassie brilliant blue G250, most hetero-protein is washed out; the holy lewis encephalitis virus NS2B-NS3 protein is eluted with a 100mM imidazole-containing sterile buffer, then concentrated to 1mL with a 10kD concentration tube and further concentrated by exchange, and further purified by anion exchange chromatography and exclusion chromatography to obtain the target protein with charge uniformity.
Obtaining purified St.Louis encephalitis virus NS2B-NS3 protein, and measuring the activity of St.Louis encephalitis virus NS2B-NS3 protease:
fluorogenic substrate Bz-Nle-KRR-AMC (available from Shanghai Jier Biochemical Co., ltd.) with a purity of more than 95%; subsequently, the fluorescence intensities at different times of the reaction were detected with an Infinite M1000 Pro detector, the wavelength of the excitation light was set to 360nm, and the wavelength of the emission light was set to 460nm.
The protein buffer composition includes: 50mM Tris-HCl,25% glycerol, pH 9.0 was used to dilute the purified St.Louis encephalitis virus NS2B-NS3 protein.
The activity is determined by the following steps:
s1: the protease of St.Louis encephalitis virus NS2B-NS3 (final concentration 400 nM) was prepared with the above protein buffer, and a compound (final concentration 20. Mu.M) dissolved in 95% DMSO (dimethyl sulfoxide) was added, and incubated at 37℃for 10min, followed by rapid addition of fluorogenic substrate Bz-Nle-KRR-AMC at a substrate concentration of 30. Mu.M; vibrating the liquid for 10s at the same time with the rotating speed of 300rpm to uniformly combine the liquid, and recording a reading every 30s to measure 1500s;
meanwhile, a negative control test is designed, no inhibitor is added to the negative control, and other experimental conditions are the same.
S2: preparing an enzyme activity force curve of the St.Louis encephalitis virus NS2B-NS3 protein by taking time as an X axis and taking a fluorescence value as a Y axis; the rate of the enzymatic reaction was analyzed for the previous 300s using GraphPad prism5.0 software, based on the fluorescence intensity values shown by the instrument and the time of the reaction.
S3: set V 0 For the initial rate of the enzymatic reaction without addition of compound, V i Is the initial rate of the enzymatic reaction of the added compound; based on the Rate of the enzymatic reaction, the Inhibition ratio Ir (Inhibition Rate, ir) of each inhibitor was calculated (1-V i /V 0 )。
The inhibition effect of the compound on the St.Louis encephalitis virus NS2B-NS3 protease can be judged by comprehensively considering the residual activity percentage and the fluorescence quenching rate of the compound, but because the system is mainly screened by fluorescence intensity, the compound with fluorescence or similar to AMC can interfere with the system, in addition, the compound with quenching group can quench the fluorescence of the system to cause false positive, so that the compound with quenching group needs to be re-screened;
for the compound with the inhibition rate of more than 70%, in order to eliminate the possibility of false positive caused by misoperation, a fluorescence quenching experiment is designed to re-screen the compound, the St.Louis encephalitis virus NS2B-NS3 protein and Bz-Nle-KRR-AMC are reacted for a corresponding time, and the fluorescence value reaches a maximum value and is set as P 1 Adding the inhibitor with the same volume as the blank group into the ELISA plate, and detecting the fluorescence value of the inhibitor to be P 2 The method comprises the steps of carrying out a first treatment on the surface of the Then the fluorescence values of the two times are calculated according to the formula
(P r ＝P 1 -P 2 )/P 2 *100％
Calculating the fluorescence quenching rate P r The method comprises the steps of carrying out a first treatment on the surface of the If the fluorescence quenching rate isGreater than 20%, may be judged as a false positive compound, and may be excluded; if the fluorescence quenching rate is less than 20%, a positive result can be determined.
As shown in the schematic diagram of the inhibition effect of hypericin on NS2B-NS3 protease in St.Louis encephalitis virus in FIG. 1, the inhibition rate Ir of hypericin on St.Louis encephalitis virus NS2B-NS3 protein is more than 95%, and the fluorescence quenching rate in the fluorescence quenching experiment is less than 20%, by setting up a control experiment, it can be determined that hypericin has a strong inhibition effect on the NS2B-NS3 protease in St.Louis encephalitis virus, hypericin can be used as an inhibitor to inhibit St.Louis encephalitis virus NS2B-NS3 protein, and the inhibition rate is high.
Hypericin IC 50 Is determined by:
in measuring IC 50 When the test was performed, the St.Louis encephalitis virus NS2B-NS3 protein was first prepared at a final concentration of 400nM, and then the substrate Bz-Nle-KRR-AMC was prepared in 95% DMSO to a final concentration of 30. Mu.M.
Then roughly setting 11 inhibitor concentrations according to the primary screening result, wherein the hypericin concentrations are respectively 100 mu M, 40 mu M, 20 mu M, 10 mu M, 5 mu M, 2.5 mu M, 1.25 mu M, 0.63 mu M, 0.31 mu M, 0.16 mu M, 0.08 mu M and 0 mu M;
firstly adding protein into an ELISA plate, incubating the protein with an inhibitor in an ELISA apparatus at 37 ℃ for 30min, rapidly adding 10 mu L of substrate, and recording time and fluorescence change curve; obtaining the initial rate of the protease fluorescence reaction by using Graphpad prism5.0 software, fitting a dose-response relation curve of the compound concentration and the residual activity, and finally obtaining the IC 50 Values.
FIG. 2 hypericin IC for NS2B-NS3 protease in St.Louis encephalitis virus 50 As shown in the measurement schematic diagram of (1), the inhibition rate of hypericin on NS2B-NS3 protein in inhibiting St.Louis encephalitis virus is more than 95%, and the final measurement of hypericin is IC 50 The value is 2.69 plus or minus 0.86 mu M, has great application potential in preparing small molecule inhibitors of NS2B-NS3 protease in resisting St.Louis encephalitis virus, and has potential to be used as potential medicines for resisting St.Louis encephalitis virus infectionIt is desirable.
According to the above experiments, hypericin can be used as an inhibitor of NS2B-NS3 protease in St.Louis encephalitis virus, and can be used as a medicament for treating St.Louis encephalitis virus infection, and the active ingredient of the hypericin comprises hypericin and one or more pharmaceutically acceptable carriers.
The carrier comprises one or more of surfactant, adhesive, disintegrating agent, diluent, excipient, absorption promoter, wetting agent, adsorption carrier, lubricant, filler and synergist which are conventional in pharmaceutical field. The preparation of the medicine is granule, powder, syrup, tablet, pill or suppository. The administration route can be oral administration, inhalation administration, skin and mucous membrane administration, rectal administration and injection administration.
The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention. And the technical terms and other materials referred to in the present invention are only for clearly illustrating the advantages and effects of the present invention, and should not be taken as limitations of the innovations of the present invention. The above embodiments are described in detail for the practical application of the present invention, but the embodiments are not limited to the patent scope of the present invention, and all the modifications and substitutions made by those skilled in the art on the basis of the present invention are included in the scope of the present invention.
Claims (6)
1. The application of hypericin in preparing medicine for resisting St.Louis encephalitis virus infection is provided.
2. Use of hypericin according to claim 1, characterized in that: the hypericin is a small molecule inhibitor of the NS2B-NS3 protease in St.Louis encephalitis virus.
3. Use of hypericin according to claim 1, characterized in that: the hypericin has the structural formula:
4. a medicament for treating infection by a holy lewis encephalitis virus, characterized in that: comprising hypericin according to claim 1 and one or more pharmaceutically acceptable carriers.
5. A medicament for the treatment of infection by the st. The carrier comprises one or more of a surfactant, a binder, a disintegrating agent, a diluent, an excipient, an absorption enhancer, a wetting agent, an adsorption carrier, a lubricant, a filler and a synergistic agent.
6. A medicament for the treatment of infection by the st. The preparation of the medicine is granule, powder, syrup, tablet, pill or suppository.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210580833.4A CN117159517A (en) | 2022-05-26 | 2022-05-26 | Application of hypericin in preparation of anti-St.Louis encephalitis virus infection medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210580833.4A CN117159517A (en) | 2022-05-26 | 2022-05-26 | Application of hypericin in preparation of anti-St.Louis encephalitis virus infection medicine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117159517A true CN117159517A (en) | 2023-12-05 |
Family
ID=88928570
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210580833.4A Pending CN117159517A (en) | 2022-05-26 | 2022-05-26 | Application of hypericin in preparation of anti-St.Louis encephalitis virus infection medicine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117159517A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119548482A (en) * | 2024-12-04 | 2025-03-04 | 南方医科大学 | New use of hypericin in the preparation of medicine for preventing or treating Zika virus infection |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990007876A1 (en) * | 1989-01-19 | 1990-07-26 | New York University | Biological fluids purification systems |
| EP1321463A1 (en) * | 2001-12-21 | 2003-06-25 | Shire Biochem Inc. | Thiazole derivatives and their use for the treatment or prevention of Flavivirus infections |
-
2022
- 2022-05-26 CN CN202210580833.4A patent/CN117159517A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990007876A1 (en) * | 1989-01-19 | 1990-07-26 | New York University | Biological fluids purification systems |
| EP1321463A1 (en) * | 2001-12-21 | 2003-06-25 | Shire Biochem Inc. | Thiazole derivatives and their use for the treatment or prevention of Flavivirus infections |
Non-Patent Citations (1)
| Title |
|---|
| ATSUSHI FURUTA等: "Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase", 《INT. J. MOL. SCI.》, vol. 16, 7 August 2015 (2015-08-07), pages 18439 - 18453 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119548482A (en) * | 2024-12-04 | 2025-03-04 | 南方医科大学 | New use of hypericin in the preparation of medicine for preventing or treating Zika virus infection |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dash et al. | Reemergence of dengue virus type-3 (subtype-III) in India: implications for increased incidence of DHF & DSS | |
| CN116236580B (en) | Application of old drugs such as Auranofin and their combination in combating single positive strand RNA viruses | |
| US20040259934A1 (en) | Inhibiting Coronaviridae viral replication and treating Coronaviridae viral infection with nucleoside compounds | |
| CN115466225B (en) | An amide compound and its preparation method, pharmaceutical composition and use | |
| UA121119C2 (en) | DERIVATIVES OF MONO- OR DYSUBSTITUTED INDOLES AS INHIBITORS OF DENGE VIRUS REPLICATION | |
| Borowski et al. | NTPase/helicase of Flaviviridae: inhibitors and inhibition of the enzyme. | |
| Wang et al. | Bioactivity-guided screening identifies pheophytin a as a potent anti-hepatitis C virus compound from Lonicera hypoglauca Miq. | |
| Dai et al. | Structure-based design, synthesis and biological evaluation of peptidomimetic aldehydes as a novel series of antiviral drug candidates targeting the SARS-CoV-2 main protease | |
| Sun et al. | Specific inhibition of bovine viral diarrhea virus replicase | |
| CN117159517A (en) | Application of hypericin in preparation of anti-St.Louis encephalitis virus infection medicine | |
| CN117159516A (en) | Application of Enbenoic acid in preparation of anti-St.Louis encephalitis virus infection drug | |
| CN113876755A (en) | Potential application of avasimibe in resisting infection of St.Louis encephalitis virus | |
| CN117159566A (en) | Application of fondaparinux sodium in preparation of anti-St.Louis encephalitis virus infection medicines | |
| CN118416063A (en) | Application of a small molecule compound targeting African swine fever virus E165R protein | |
| CN117159567A (en) | Application of corilagin in preparation of anti-St.Louis encephalitis virus medicines | |
| CN113880820B (en) | Butenolide derivative and application thereof, and pharmaceutical composition | |
| CN117159557B (en) | Application of zinc pyrithione in preparation of medicines for treating Japanese encephalitis virus infection | |
| CN117180255A (en) | Application of zinc undecylenate in the preparation of anti-dengue virus drugs | |
| CN114306339A (en) | 3CLpro protease inhibitor and its antiviral application | |
| CN119818487B (en) | New application of eltrombopag | |
| Zhou et al. | The in vitro and in vivo antiviral effects of aloperine against Zika virus infection | |
| CN109897053B (en) | Resindol A, its preparation method and anti-hepatitis C virus use | |
| KR102813837B1 (en) | RNA aptamer that specifically binds to zika virus | |
| CN107898784B (en) | Application of eltrombopag ethanolamine in resisting mycobacterium tuberculosis infection | |
| Wang et al. | Discovery of a potent covalent inhibitor that unusually distorts the catalytic dyad of SARS-CoV-2 main protease |
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 |