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CN117159558A - Application of tripterine in preparation of Japanese encephalitis virus resistant medicament - Google Patents

Application of tripterine in preparation of Japanese encephalitis virus resistant medicament Download PDF

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Publication number
CN117159558A
CN117159558A CN202210585170.5A CN202210585170A CN117159558A CN 117159558 A CN117159558 A CN 117159558A CN 202210585170 A CN202210585170 A CN 202210585170A CN 117159558 A CN117159558 A CN 117159558A
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CN
China
Prior art keywords
japanese encephalitis
tripterine
encephalitis virus
ns2b
jev
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Pending
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CN202210585170.5A
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Chinese (zh)
Inventor
杨海涛
张兵
王泽方
刘祥
封守琴
肖云杰
朱倩
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TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOMEDICINE
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TIANJIN INTERNATIONAL JOINT ACADEMY OF BIOMEDICINE
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Priority to CN202210585170.5A priority Critical patent/CN117159558A/en
Publication of CN117159558A publication Critical patent/CN117159558A/en
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    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention provides an application of tripterine in preparation of a Japanese encephalitis virus resistant medicament, which aims at JEV NS2B-NS3 protease targets in Japanese encephalitis virus, uses the tripterine as an inhibitor of the protease, has good inhibition activity on JEV NS2B-NS3 protease in Japanese encephalitis virus, has various carriers and administration modes, and is convenient to use and easy to obtain.

Description

Application of tripterine in preparation of Japanese encephalitis virus resistant medicament
Technical Field
The invention belongs to the technical field of pharmacy, and particularly relates to application of tripterine in preparation of a Japanese encephalitis virus resistant medicament.
Background
Japanese encephalitis (JE, japanese encephalitis) is one of the most important epidemic and sporadic encephalitis in tropical areas of Asia. Since the first case in 1871 was found in japan, the geographical area of japanese encephalitis virus (JEV, japanese encephalitis virus) transmission has gradually increased with the development of decades until the whole asia is covered. It is estimated that 30 hundred million people live in JEV epidemic areas, including china, india and peninsula southeast asia. About 70,000 cases of human japanese encephalitis infection are reported each year, and no effective reporting system has been established in most countries with high japanese encephalitis incidence, so that the actual incidence should be higher than the statistical incidence, and thus JEV is far more harmful than what we see. The natural circulatory transmission pathway of JEV involves many vertebrate hosts, culex trilobus as a transmission medium, and pigs and birds can be considered as two more important amplification hosts. Patients with the onset of illness are manifested as blurred consciousness, fever, nausea, vomiting, headache, seizures and intracranial neurological symptoms including facial paralysis, ptosis and abnormal eye movement, and can develop flaccid paralysis, which usually affects the lower limbs, severe cases spread to the extremities, and serious sequelae such as paralysis of the extremities can be caused after illness. Since there is no specific drug against encephalitis B virus at present, the incidence rate is still high and constant under the condition that the JEV vaccination rate is continuously improved, and the JEV vaccination rate is a public health problem which needs to be faced in Asian areas. Therefore, the research on specific drugs of Japanese encephalitis virus is very important at present.
The N-terminal domain of the JEV NS3 protein and the cofactor NS2B form a protease that hydrolyzes the viral polyprotein polymer. NS2B comprises a hydrophilic region, the central region of which comprises a β -barrel which, for its stability, is folded around the β -barrel of the NS3 protease. Upon substrate binding, NS2B undergoes conformational changes, affecting the stability of the β -hairpin, making it part of the active site. Active NS2B-NS3 is necessary for hydrolytic cleavage at the junction of NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS 5. In addition, it can also act as a hub for the formation of viral replication complexes, affecting the pathogenic mechanisms of the virus and the immune response of the host. Thus, JEV NS2B-NS3 can be used as an important target for the research of Japanese encephalitis virus inhibitors.
The tripterine, the English name is Celastrol, is derived from root bark of a traditional Chinese medicine tripterygium wilfordii, is a popular natural compound in recent years, has anti-inflammatory activity in various researches, protects the central nervous system, has anti-diabetes, cardiovascular protection, anticancer and antifungal effects, and has great therapeutic potential. However, the application of tripterine in resisting Japanese encephalitis virus infection is not reported so far.
Disclosure of Invention
The invention aims at solving the problem that the tripterine is used as an inhibitor of the protease aiming at JEV NS2B-NS3 protease target spot in Japanese encephalitis virus, and provides the application of the tripterine in preparing Japanese encephalitis virus resistant medicaments.
In order to solve the technical problems, the invention adopts the following technical scheme:
the application of tripterine in preparing a Japanese encephalitis virus resistant medicament comprises the following steps of:
further, the anti-Japanese encephalitis virus medicament comprises the tripterine and one or more pharmaceutically acceptable carriers.
Further, the carrier comprises 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.
Further, the anti-Japanese encephalitis virus drug is selected from the group consisting of granules, powder, syrup, tablets, pills, suppositories, and combinations thereof.
Further, the mode of administration of the anti-Japanese encephalitis virus drug includes oral administration, injection, implantation, external application, spraying, inhalation or a combination thereof.
The invention provides an application of tripterine in preparation of anti-Japanese encephalitis virus medicines, which aims at JEV NS2B-NS3 protease targets in Japanese encephalitis viruses, uses the tripterine as an inhibitor of the protease, has good inhibition activity on JEV NS2B-NS3 protease in Japanese encephalitis viruses, and has various carriers and administration modes, and is convenient to use and easy to obtain.
The tripterine CAS number of the invention is 34157-83-0 and is purchased from Shanghai Tao Su Biochemical technology Co., ltd.
Drawings
FIG. 1 is a schematic illustration of the inhibitory effect of celastrol on JEV NS2B-NS3 protease of Japanese encephalitis virus according to an embodiment of the present invention;
FIG. 2 shows the inhibitory effect of tripterine on JEV NS2B-NS3 protease of Japanese encephalitis virus according to one embodiment of the present invention 50 Schematic measurement.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Referring to fig. 1-2, the application of tripterine in preparing anti-japanese encephalitis virus drugs is provided in the embodiment of the invention, and the specific implementation manner is as follows:
1. expression and purification of JEV NS2B-NS3 protease;
(1) The reference constructs a recombinant pET-15B plasmid containing the gene encoding JEV NS2B-NS3pro and transformed into E.coli competent cell BL21 (DE 3).
(2) Positive monoclonal colonies were picked on plates containing ampicillin resistance, and when cultured in LB liquid medium (50 mg/L ampicillin) at 37℃until the OD600 of the bacterial liquid became about 0.6, isopropyl thiogalactoside was added to a final concentration of 0.5mM, and protein expression was induced at 16℃for 16-18 hours.
(3) Centrifuging at 3500rpm for 15min by a floor centrifuge to collect thalli, washing the precipitated thalli twice, re-suspending the thalli in a bacteria breaking buffer solution (20mM Tris,300mM NaCl,pH 7.5), and carrying out bacteria breaking treatment by using an ultrasonic cell disruption instrument; after crushing, the mixture was centrifuged at 10000rpm for 60min using a low-temperature high-speed centrifuge, and the supernatant was collected.
(4) And pouring the obtained crude protein supernatant into an affinity chromatographic column containing Ni-NTA filler, and eluting by using buffers containing different imidazole concentrations to obtain more uniform protein.
(5) Further purifying by anion exchange chromatography to obtain target protein with charge uniformity.
(6) Further purification was performed by gel filtration chromatography to increase the purity of the target protein and to observe the molecular weight of the protein to determine whether it was a polymer.
Screening JEV NS2B-NS3pro inhibitors;
Bz-NKRR-AMC polypeptide compound (available from Shanghai Jier Biochemical Co., ltd.) with purity of 95% was used as a substrate; kinetic curves were determined by fluorescence resonance energy transfer. The instrument for measuring the fluorescence intensity isPro full-wavelength multifunctional microplate detector, corresponding incident and emission wavelengths are 355nm and 460nm, respectively.
The protein buffer fraction was 20mM Tris,30% glycerol, 0.5mM ChAPS,pH 9.0. JEV NS2B-NS3pro (final concentration 2. Mu.M) was prepared with buffer, 89. Mu.L of protein solution was added with 1. Mu.L of compound dissolved in 95% DMSO (final concentration 20. Mu.M), left to stand at 37℃for 5min, 10. Mu.L of Bz-NKRR-AMC (final concentration 20. Mu.M) was added, and shaking at 654rpm was carried out for 10s, and fluorescence readings were recorded every 30 s. The negative control test uses 95% dmso instead of the compound sample, and the other experimental conditions are the same.
The enzymatic kinetics was measured using an microplate reader and the slope of the first 300s was analyzed as the initial rate of the enzymatic reaction. Set V 0 At the initial speed without inhibitor, V i Is the initial speed of adding inhibitor. Calculating each compound according to the initial velocityInhibition Ir (Inhibition Rate, ir) of the substance against JEV NS2B-NS3pro (1-V i /V 0 ) Residual Activity Ra (Residual Activity, ra) (V i /V 0 )。
For compounds with an inhibition rate Ir >70%, re-screening and fluorescence quenching experiments are required to eliminate false positive results generated by misoperation. 89. Mu.L of JEV NS2B-NS3pro solution (final concentration 2. Mu.M) was added to the system and reacted with 10. Mu.L of Bz-NKRR-AMC (final concentration 20. Mu.M) for a sufficient period of time to complete the reaction of the substrate, the fluorescence value was recorded as Q1, and 1. Mu.L of the inhibitor was added to the microplate, and the current fluorescence value was recorded as Q2. Then by the formula: qr= (Q1-Q2)/Q2 is 100%, and the fluorescence quenching rate Qr is calculated. If the fluorescence quenching rate is less than 20%, false positives can be eliminated.
3. Tripterine IC 50 Is measured;
assay of Tripterine IC 50 At this time, the protein JEV NS2B-NS3pro required for the experiment was diluted to a final concentration of 2. Mu.M, and the substrate Bz-NlKRR-AMC was prepared to a final concentration of 20. Mu.M. The inhibitor concentrations were then roughly set to 14 based on the results obtained by the preliminary screening, 100. Mu.M, 50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.25. Mu.M, 3.12. Mu.M, 1.56. Mu.M, 0.78. Mu.M, 0.39. Mu.M, 0.19. Mu.M, 0.05. Mu.M, 0.025. Mu.M, 0.012. Mu.M, and 0.006. Mu.M, respectively. JEV NS2B-NS3pro and tripterine are uniformly mixed and incubated for 10min at 37 ℃, 10 mu L of Bz-NKRR-AMC is added, and a change curve of fluorescence value and time is recorded by an enzyme-labeling instrument. Analyzing JEV NS2B-NS3pro reaction initial rate by using Graphpad prism 8.0 software to obtain a relationship curve of the inverse value of tripterine concentration and the inhibition rate Ir, and finally obtaining the IC 50 Values.
The invention relates to the technical field of pharmacy, in particular to application of tripterine in preparation of anti-Japanese encephalitis virus drugs, and the inhibition rate Ir of tripterine in inhibiting proteolytic cleavage of NS2B-NS3 protease in Japanese encephalitis virus>70%, IC was measured at a substrate concentration of 20. Mu.M 50 The preparation method is 3.42+/-0.75 mu M, has great application potential in preparing small molecule inhibitors of NS2B-NS3 protease in Japanese encephalitis virus, and has hope of becoming a potential medicine for resisting Japanese encephalitis virus infection.
The invention has the advantages and beneficial effects that:
the invention provides an application of tripterine in preparation of anti-Japanese encephalitis virus medicines, which aims at JEV NS2B-NS3 protease targets in Japanese encephalitis viruses, uses the tripterine as an inhibitor of the protease, has good inhibition activity on JEV NS2B-NS3 protease in Japanese encephalitis viruses, and has various carriers and administration modes, and is convenient to use and easy to obtain.
It is to be understood that the above-described embodiments of the present invention are merely illustrative of or explanation of the principles of the present invention and are in no way limiting of the invention. Accordingly, any modification, equivalent replacement, improvement, etc. made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. Furthermore, the appended claims are intended to cover all such changes and modifications that fall within the scope and boundary of the appended claims, or equivalents of such scope and boundary.

Claims (5)

1. The application of the tripterine in preparing the anti-Japanese encephalitis virus medicament is characterized in that the tripterine has a molecular structural formula as follows:
2. the use according to claim 1, characterized in that: the anti-Japanese encephalitis virus medicament comprises the tripterine and one or more pharmaceutically acceptable carriers.
3. The use according to claim 2, characterized in that: the carrier comprises 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.
4. A use according to any one of claims 1-3, characterized in that: the anti-Japanese encephalitis virus drug is selected from granule, powder, syrup, tablet, pill, suppository or their combination.
5. The use according to claim 4, characterized in that: the administration mode of the anti-Japanese encephalitis virus drug comprises oral administration, injection, implantation, external application, spraying, inhalation or a combination thereof.
CN202210585170.5A 2022-05-27 2022-05-27 Application of tripterine in preparation of Japanese encephalitis virus resistant medicament Pending CN117159558A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210585170.5A CN117159558A (en) 2022-05-27 2022-05-27 Application of tripterine in preparation of Japanese encephalitis virus resistant medicament

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210585170.5A CN117159558A (en) 2022-05-27 2022-05-27 Application of tripterine in preparation of Japanese encephalitis virus resistant medicament

Publications (1)

Publication Number Publication Date
CN117159558A true CN117159558A (en) 2023-12-05

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