CN117159557A - Application of zinc pyrithione in preparation of medicines for treating Japanese encephalitis virus infection - Google Patents
Application of zinc pyrithione in preparation of medicines for treating Japanese encephalitis virus infection Download PDFInfo
- Publication number
- CN117159557A CN117159557A CN202210578711.1A CN202210578711A CN117159557A CN 117159557 A CN117159557 A CN 117159557A CN 202210578711 A CN202210578711 A CN 202210578711A CN 117159557 A CN117159557 A CN 117159557A
- Authority
- CN
- China
- Prior art keywords
- japanese encephalitis
- encephalitis virus
- zinc pyrithione
- virus infection
- ns2b
- 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.)
- Granted
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
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides application of zinc pyrithione in preparing a medicine for treating Japanese encephalitis virus infection. The beneficial effects of the invention are as follows: the zinc pyrithione has remarkable inhibition effect on the activity of the NS2B-NS3 protease in Japanese encephalitis virus, can be used as an inhibitor of the NS2B-NS3 protease in Japanese encephalitis virus, and is expected to become a potential medicine for resisting Japanese encephalitis virus infection.
Description
Technical Field
The invention belongs to the technical field of Japanese encephalitis resisting medicaments, and particularly relates to application of zinc pyrithione in preparation of a medicament for treating Japanese encephalitis virus infection.
Background
Japanese encephalitis (JE, japanese encephalitis) is an acute infectious disease caused by Japanese encephalitis virus (JEV, japanese encephalitis virus). Japanese encephalitis has high mortality and disability rate, and is one of the main infectious diseases threatening the health of people, especially children. The summer and autumn are the peak season of disease, and the distribution of popular areas is closely related to the distribution of medium mosquitoes. China is a epidemic area of Japanese encephalitis, and has seen pandemics in the beginning of the 60 s and 70 s of the 20 th century, and the incidence rate is greatly reduced with the increase of the vaccination rate, but nearly ten thousand cases are reported every year.
JEV is transmitted primarily through mosquito bites, with culex trichinensis being the primary vehicle of its transmission. The mosquitoes can carry viruses to live through winter and can pass through eggs, so the mosquitoes are not only transmission media, but also long-term storage hosts. Pig and aquatic bird animals serve as amplification hosts of JEV, and can rapidly develop into hyperviremia after JEV infection, and are transmitted to mosquitoes again. Most of infected patients have no obvious symptoms at the early stage, and then high fever with symptoms of listlessness, nausea, vomiting and the like can occur, and serious patients can develop convulsions, coma, paralysis and the like. JEV can cause extensive lesions in the brain parenchyma, with lesions of the cerebral cortex, brainstem and basal nuclei being most pronounced, presenting a great challenge to the patient for post-healing recovery. Since there is no specific drug for treating Japanese encephalitis, doctors can only treat Japanese encephalitis by symptomatic treatment such as antiviral, anti-infective, cooling, dehydration and the like, research on inhibitors against JEV is an important task at present.
JEV belongs to the genus Flaviviridae, an RNA enveloped virus, with a single-stranded forward RNA genome of approximately 11kb in length. The open reading frame in the JEV genomic RNA encodes a polyprotein precursor of about 3432 amino acids that is subsequently cleaved by its own NS3 protease or host protease into three structural proteins and seven non-structural proteins. The JEV NS3 protease is highly conserved in JEV genotypes, having two domains, an N-terminal protease domain and a C-terminal RNA helicase domain, respectively. Whereas the JEV NS2B protein is a cofactor for the NS3 protein, and is associated with NS3 stability and substrate recognition. The N-terminal protease domain (180 residues) of the NS3 protein directly interacts with a hydrophilic domain of about 40 amino acids in the center of NS2B to form an active serine protease. Whereas active NS2B-NS3 protease is essential for viral polyproteinolysis processes, playing a vital role in viral replication and pathogenesis. Thus, NS2B-NS3 can be an important target for studying inhibitors of japanese encephalitis virus.
Zinc pyrithione, named as Zincpyrithione, is an excellent anti-flaking agent and anti-seborrheic agent, can effectively kill dandruff-producing fungi, has the effects of relieving itching, removing dandruff, reducing alopecia and delaying the occurrence of grey hair, and is a high-efficiency and safe antipruritic anti-dandruff agent; meanwhile, the antibacterial agent can also be used as an excellent, broad-spectrum, low-toxicity and environment-friendly antibacterial agent for fungi and bacteria, and can be widely applied to the fields of civil paint, adhesive, carpet and the like. However, to date, the use of zinc pyrithione in combating Japanese encephalitis virus infection has not been reported.
Disclosure of Invention
The first object of the invention is: the application of the zinc pyrithione in preparing a medicine for treating Japanese encephalitis virus infection is provided, the zinc pyrithione can be used as a small molecule inhibitor of NS2B-NS3 protease in Japanese encephalitis virus, and a negative control is established on the molecular level, so that the zinc pyrithione has good inhibitory activity on the Japanese encephalitis virus NS2B-NS3 protease, and therefore the compound is hopefully a potential medicine for inhibiting Japanese encephalitis virus infection.
Another object of the invention is: a medicine for treating Japanese encephalitis virus infection is provided.
In order to solve the technical problems, the invention adopts the following technical scheme: application of zinc pyrithione in preparing medicine for treating Japanese encephalitis virus infection is provided.
Preferably, the zinc pyrithione is a small molecule inhibitor of the NS2B-NS3 protease in Japanese encephalitis virus.
Preferably, the structural formula of the zinc pyrithione is:
a medicament for treating japanese encephalitis virus infection comprising the zinc pyrithione of claim 1 and one or more pharmaceutically acceptable carriers.
Preferably, the carrier comprises one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant and a synergist.
Preferably, the pharmaceutical preparation is an injection, a tablet, a pill, a capsule, a suspension or an emulsion.
According to the technical scheme, the zinc pyrithione can be used as a small molecule inhibitor of the NS2B-NS3 protease in Japanese encephalitis virus, has a remarkable inhibiting effect on the activity of the NS2B-NS3 protease of Japanese encephalitis virus, can further develop potential medicines, and can treat Japanese encephalitis virus through a new way.
Drawings
FIG. 1 is a schematic diagram showing the inhibition effect of zinc pyrithione on Japanese encephalitis virus NS2B-NS3 protease according to the embodiment of the invention
FIG. 2 is a graph showing the inhibitory effect of zinc pyrithione on Japanese encephalitis virus NS2B-NS3 protease according to the embodiment of the invention 50 Schematic measurement diagram
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 Japanese encephalitis virus NS2B-NS3 protease:
s1: obtaining the gene sequences of JEV NS2B and NS3 from NCBI website, reference methods utilize G 4 SG 4 The linker connects the two protein genes, constructs the two protein genes on a pET-15b vector, obtains recombinant plasmids for sequencing identification, and then converts the plasmids with correct sequencing into E.coli BL21 (DE 3) cells.
S2: the transformed BL21 (DE 3) cells are coated on a plate containing ampicillin resistance, a monoclonal colony is picked up to a small test tube LB culture medium for activation culture for 5-6h after overnight culture in an incubator, and the culture medium is transferred to a large bottle LB culture medium for expansion culture, and the culture medium is cultured at 37 ℃ until the bacterial liquid OD is obtained 600 About 0.6, the incubator is cooled to 16 ℃, and isopropyl thiogalactoside is added for induction expression for 16-18h.
S3: transferring the induced bacterial liquid to a bacterial collecting barrel, centrifuging by using a large-sized floor centrifuge, collecting bacterial bodies, pouring out supernatant, re-suspending the precipitated bacterial bodies in a bacterial breaking buffer solution (50mM Tris,300mM NaCl,pH 7.5), performing ultrasonic bacterial breaking by using an ultrasonic cell breaker, and finally centrifuging by using a low-temperature high-speed centrifuge at 4 ℃ and 10000 rpm.
S4: pouring the obtained supernatant into an affinity chromatography column containing Ni-NTA filler, eluting with low-concentration and high-concentration imidazole buffer solution to obtain target protein, and performing polyacrylamide gel electrophoresis verification.
S5: further purified by anion exchange chromatography and size exclusion chromatography to obtain a target protein having high purity and charge uniformity.
The purified Japanese encephalitis NS2B-NS3 protein is obtained, and a stable inhibitor screening system is established for the Japanese encephalitis NS2B-NS3 protein based on the fluorescence resonance energy transfer principle:
preparation of fluorogenic substrate: bz-Nle-Lys-Arg-Arg-AMC polypeptide compound (available from Shanghai Jier Biochemical Co., ltd.) having a purity of 95% was used as a fluorogenic substrate; subsequently determining the enzyme activity buffer composition as 20mM Tris,30% glycerol, 0.5mM CHAPS,pH 9.0, for diluting the purified Japanese encephalitis NS2B-NS3 protein;
the screening system was 100. Mu.L, comprising 89. Mu.L JEV NS2B-NS3 pro solution (final concentration 2. Mu.M), 1. Mu.L compound (final concentration 20. Mu.M) and 10. Mu.L fluorogenic substrate (final concentration 20. Mu.M); the instrument for measuring the fluorescence intensity is an Infinite M1000 Pro full-wavelength multifunctional microplate detector, and the corresponding incident wavelength and emission wavelength are 355nm and 460nm respectively during detection.
The screening steps are as follows:
s1: firstly, 89 mu L of protein solution and 1 mu L of compound are added into a 96-well black ELISA plate, incubation is carried out for 5min at 37 ℃, then 10 mu L of fluorogenic substrate is rapidly added, and fluorescence monitoring is carried out by using an ELISA instrument;
negative control experiments were also designed and 1 μl of compound was replaced with 1 μl of 95% dmso, the other experimental conditions being unchanged.
S2: measuring enzyme kinetic curve with enzyme marker, analyzing slope of 300s as initial speed of enzymatic reaction, and setting V 0 At the initial speed without inhibitor, V i Is the initial speed of adding inhibitor; according to the initial velocity, calculating the Inhibition Rate Ir (Inhibition Rate, ir) (1-V) of each compound on JEV NS2B-NS3 pro i /V 0 ) Residual Activity Ra (Residual Activity, ra) (V i /V 0 )。
S3: for inhibition rate Ir>70% of the compounds need to be re-screened and subjected to a fluorescence quenching experiment, so that false positive results generated by misoperation are eliminated. Firstly, 89 mu L of protein solution is added into a 96-hole black ELISA plate, incubation is carried out for 5min at 37 ℃, then 10 mu L of fluorescent substrate is rapidly added, fluorescence monitoring is carried out by using an ELISA reader until the fluorescence intensity does not change obviously any more, and the fluorescence value is recorded to be Q at the moment 1 Then 1 mu L of inhibitor is added into the ELISA plate, and the current fluorescence value is recorded as Q 2 . Then by the formula:
Q r ＝(Q 1 -Q 2 )/Q 2* 100％
calculating the fluorescence quenching rate Q r The method comprises the steps of carrying out a first treatment on the surface of the If the fluorescence quenching rate is less than 20%, false positives can be eliminated; if the fluorescence quenching rate is larger than or equal toAt 20%, false positives were found.
As shown in fig. 1, substitution of zinc pyrithione, the inhibition rate Ir of zinc pyrithione to JEV NS2B-NS3 pro is >90%, and the fluorescence quenching rate in the fluorescence quenching experiment is less than 20%, it can be determined that zinc pyrithione can act as an inhibitor to inhibit japanese encephalitis protein, and the inhibition rate is higher, so that a better effect can be achieved.
Zinc pyrithione IC 50 And (3) measuring:
first, the protein JEV NS2B-NS3 pro required for the experiment was diluted to a final concentration of 2. Mu.M, and then a substrate Bz-Nle-Lys-Arg-Arg-AMC was prepared to a final concentration of 20. Mu.M.
The final concentration of inhibitor was then roughly set to 17 based on the results obtained by preliminary screening of the screening system, 320. Mu.M, 160. Mu.M, 80. Mu.M, 40. Mu.M, 20. Mu.M, 5. Mu.M, 2.5. Mu.M, 1.25. Mu.M, 0.625. Mu.M, 0.312. Mu.M, 0.156. Mu.M, 0.078. Mu.M, 0.039. Mu.M, 0.019. Mu.M, 0.010. Mu.M, 0.005. Mu.M, 0.002. Mu.M, respectively;
JEV NS2B-NS3 pro was first mixed with zinc pyrithione at various concentrations, incubated at 37℃for 10min, followed by addition of 10. Mu.L of fluorogenic substrate, and the change in fluorescence value with time was recorded by an enzyme-labelling instrument. Analyzing JEV NS2B-NS3 pro reaction initial rate by using Graphpad prism 8.0 software to obtain a relationship curve of a zinc pyrithione concentration inverse value and an inhibition rate Ir, and finally obtaining the IC 50 Values.
As shown in FIG. 2, IC was measured at a substrate concentration of 20. Mu.M 50 The molecular weight of the protease is 0.53+/-0.21 mu M, has great application potential in preparing a small molecule inhibitor of NS2B-NS3 protease in Japanese encephalitis virus, and has hope of becoming a potential medicament for resisting Japanese encephalitis virus infection.
According to the above experiments, the zinc pyrithione can be used as an inhibitor of Japanese encephalitis virus NS2B-NS3 protease, so that a medicament for preventing or treating Japanese encephalitis virus infection can be prepared, wherein the active ingredient of the medicament is zinc pyrithione, and the medicament comprises the zinc pyrithione and one or more pharmaceutically acceptable carriers.
The carrier comprises diluent, excipient, filler, adhesive, wetting agent, disintegrating agent, absorption promoter, surfactant, adsorption carrier, lubricant and synergist which are conventional in pharmaceutical field. The medicine can be made into injection, tablet, pill, capsule, suspension or emulsion. The administration route can be oral, percutaneous, intravenous or intramuscular injection.
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. Application of zinc pyrithione in preparing medicine for treating Japanese encephalitis virus infection is provided.
2. Use of zinc pyrithione according to claim 1, characterized in that: the zinc pyrithione is a small molecule inhibitor of NS2B-NS3 protease in Japanese encephalitis virus.
3. Use of zinc pyrithione according to claim 1, characterized in that: the structural formula of the zinc pyrithione is as follows:
4. a medicament for treating infection by japanese encephalitis virus, characterized in that: comprising the zinc pyrithione of claim 1 and one or more pharmaceutically acceptable carriers.
5. The drug for treating Japanese encephalitis virus infection according to claim 4, wherein: the carrier comprises one or more of diluent, excipient, filler, adhesive, wetting agent, disintegrating agent, absorption promoter, surfactant, adsorption carrier, lubricant and synergist.
6. The drug for treating Japanese encephalitis virus infection according to claim 4, wherein: the preparation of the medicine is injection, tablet, pill, capsule, suspending agent or emulsion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210578711.1A CN117159557B (en) | 2022-05-26 | 2022-05-26 | Application of zinc pyrithione in preparation of medicines for treating Japanese encephalitis virus infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210578711.1A CN117159557B (en) | 2022-05-26 | 2022-05-26 | Application of zinc pyrithione in preparation of medicines for treating Japanese encephalitis virus infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117159557A true CN117159557A (en) | 2023-12-05 |
| CN117159557B CN117159557B (en) | 2025-11-28 |
Family
ID=88928547
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210578711.1A Active CN117159557B (en) | 2022-05-26 | 2022-05-26 | Application of zinc pyrithione in preparation of medicines for treating Japanese encephalitis virus infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117159557B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008127291A2 (en) * | 2006-10-10 | 2008-10-23 | Los Alamos National Security, Llc | Advanced drug development and manufacturing |
| CN113876794A (en) * | 2021-09-23 | 2022-01-04 | 天津国际生物医药联合研究院 | Potential application of punicalagin in resisting Japanese encephalitis virus infection |
| CN113876755A (en) * | 2021-09-23 | 2022-01-04 | 天津国际生物医药联合研究院 | Potential application of avasimibe in resisting infection of St.Louis encephalitis virus |
-
2022
- 2022-05-26 CN CN202210578711.1A patent/CN117159557B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008127291A2 (en) * | 2006-10-10 | 2008-10-23 | Los Alamos National Security, Llc | Advanced drug development and manufacturing |
| CN113876794A (en) * | 2021-09-23 | 2022-01-04 | 天津国际生物医药联合研究院 | Potential application of punicalagin in resisting Japanese encephalitis virus infection |
| CN113876755A (en) * | 2021-09-23 | 2022-01-04 | 天津国际生物医药联合研究院 | Potential application of avasimibe in resisting infection of St.Louis encephalitis virus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117159557B (en) | 2025-11-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Abd Ellah et al. | Nanomedicine as a promising approach for diagnosis, treatment and prophylaxis against COVID-19 | |
| Shi et al. | The preclinical inhibitor GS441524 in combination with GC376 efficaciously inhibited the proliferation of SARS-CoV-2 in the mouse respiratory tract | |
| CN113289018B (en) | Application of old drugs such as auranofin and its composition in anti-single positive-strand RNA virus | |
| Wang et al. | Current understanding of middle east respiratory syndrome coronavirus infection in human and animal models | |
| CN114177176B (en) | Application of compound cyproheptadine hydrochloride in preparation of drug for preventing or treating African swine fever | |
| CN115463144A (en) | Novel coronavirus inhibitor and preparation method and application thereof | |
| CN117159557B (en) | Application of zinc pyrithione in preparation of medicines for treating Japanese encephalitis virus infection | |
| CN112618547B (en) | Application of a kind of quinoline compound in the preparation of anti-coronavirus preparation | |
| CN105018512A (en) | Prokaryotic expression recombinant plasmid pET-IFN gamma, interferon and application | |
| CN119954903A (en) | A group of polypeptides with anti-polysaccharide blue ear disease virus activity and their application | |
| US20210386725A1 (en) | Method for inhibiting coronavirus infection and replication | |
| CN116019805B (en) | Application of wogonin in resisting porcine epidemic diarrhea virus | |
| CN113143897B (en) | Application of 4-aminobenzophenone | |
| WO2024032468A1 (en) | Preparation and use of recombinant five-component sars-cov-2 trimer protein vaccine capable of inducing broad-spectrum neutralization activity | |
| Hua et al. | Infectious hematopoietic necrosis virus truncated G protein effect on survival, immune response, and disease resistance in rainbow trout | |
| CN118453624A (en) | Application of compound taking I73R as target spot in preparation of African swine fever virus resistant medicine | |
| CN113143918A (en) | Application of boceprednol in treating or improving novel coronavirus pneumonia | |
| CN114306339A (en) | 3CLpro protease inhibitor and its antiviral application | |
| CN117159517A (en) | Application of hypericin in preparation of anti-St.Louis encephalitis virus infection medicine | |
| CN118903475B (en) | Application of recombinant protein rMmm604 in preventing infection of Mycoplasma mycoides subsp. | |
| CN111184708A (en) | Application of silver salt of fumaric acid monoethyl ester in resisting novel coronavirus infection | |
| CN107216325B (en) | Naphthyridone compound, its preparation method and application | |
| CN117159516A (en) | Application of Enbenoic acid in preparation of anti-St.Louis encephalitis virus infection drug | |
| CN115531382B (en) | Use of quinoline thiazolinone derivative Ro-3306 in treatment of beta-coronavirus infection | |
| CN117180253A (en) | Application of chlorcyanosamide in the preparation of anti-Japanese encephalitis virus drugs |
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 |