Use of hypericin in the preparation of a medicament for the treatment of infection by the saint louis encephalitis virus

Abstract

The application provides application of hypericin in preparation of a medicine for resisting St. Louis encephalitis virus infection. The application has the beneficial effect that the hypericin has a significant inhibitory effect on NS2B-NS3 protease activity 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 medicine for resisting St. Louis encephalitis virus infection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology for St. Louis encephalitis, and in particular relates to the application of hypericin in the preparation of drugs for treating St. Louis encephalitis virus infection. Background Technology

[0002] Saint Louis encephalitis virus (SLEV) is the pathogenic virus that causes Saint Louis encephalitis. It is primarily transmitted to humans through the bite of Culex mosquitoes and can cause symptoms such as fever, headache, sore throat, and muscle pain. The incubation period for Saint Louis encephalitis is about one week. Most cases are asymptomatic or present with flu-like symptoms such as fatigue, headache, nausea, vomiting, and body aches. Some patients develop severe encephalitis symptoms, including neck stiffness, confusion, and focal neurological disorders; severe cases can lead to shock, and some patients may experience sequelae.

[0003] Saint Louis encephalitis virus (SLEV) is a positive-sense RNA virus closely related to Japanese encephalitis virus, Poisson virus, and West Nile virus. It is prevalent in North and South America, primarily in the Mississippi and Ohio River basins, as well as California, Florida, and Texas. Outbreaks and epidemics occur regularly in the Mississippi River basin and the Gulf Coast, with sporadic cases reported in the Caribbean, Canada, Mexico, and Central America. In temperate regions, transmission mainly occurs in late summer and early autumn, but in warmer climates, transmission occurs year-round. The largest Saint Louis encephalitis epidemic occurred in 1975, with nearly 2,000 cases reported, mainly in the central Mississippi River states. In recent years, with global warming, rapid population growth, and migration, my country faces significant pressure from imported Saint Louis encephalitis virus. Therefore, we need to identify drug targets for Saint Louis encephalitis virus and develop antiviral drugs targeting these targets.

[0004] St. Louis encephalitis virus (SLV) is a single-stranded RNA virus with a genome of approximately 11 kb, encoding three structural proteins: a core capsid protein, a membrane protein, and an envelope protein, and seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. NS3's main function is to process the polyproteins encoded by the viral genome, playing a crucial role in viral replication and assembly. Studies have shown that the NS3 protease has very low activity, but its activity can be significantly increased when bound to NS2B. Therefore, the NS2B-NS3 protease is a very important drug target for combating SLV, and screening for inhibitors targeting the NS2B-NS3 protease is of great significance for the development of drugs related to SLV infection.

[0005] Hypericin, an extract of St. John's wort, has antiviral activity against dengue virus, coronavirus, classical swine fever virus, and highly pathogenic avian influenza virus, and also exhibits antitumor effects. However, to date, there are no reports on the application of hypericin in combating St. Louis encephalitis virus infection. Summary of the Invention

[0006] The primary objective of this invention is to provide the application of hypericin in the preparation of drugs against St. Louis encephalitis virus infection. Hypericin can be used as a small molecule inhibitor of the NS2B-NS3 protease in St. Louis encephalitis virus at the molecular level. Through controlled experiments, it was found that hypericin has a strong inhibitory effect on the NS2B-NS3 protease in St. Louis encephalitis virus. Therefore, hypericin can be considered a potential drug for inhibiting St. Louis encephalitis virus infection.

[0007] Another object of the present invention is to provide a drug for treating St. Louis encephalitis virus infection.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: the hypericin is a small molecule inhibitor of NS2B-NS3 protease in St. Louis encephalitis virus.

[0009] Preferably, the structural formula of the hypericin is:

[0010]

[0011] A drug for treating St. Louis encephalitis virus infection, comprising hypericin and one or more pharmaceutically acceptable carriers.

[0012] Preferably, the carrier comprises one or more of the following conventional pharmaceutical materials: surfactants, binders, disintegrants, diluents, excipients, absorption enhancers, wetting agents, adsorbents, lubricants, fillers, and synergists.

[0013] Preferably, the drug is prepared as granules, powders, syrups, tablets, pills, or suppositories.

[0014] Using the above-mentioned technical solution, hypericin has good inhibitory activity against the NS2B-NS3 protease in St. Louis encephalitis virus, so it can be used as a small molecule inhibitor of the NS2B-NS3 protease in St. Louis encephalitis virus. Therefore, hypericin can be used as a potential drug to inhibit St. Louis encephalitis virus infection and treat St. Louis encephalitis virus infection through a new approach. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the inhibitory effect of hypericin on NS2B-NS3 protease in St. Louis encephalitis virus according to an embodiment of the present invention.

[0016] Figure 2 This invention relates to the IC50 assay of hypericin against NS2B-NS3 protease in St. Louis encephalitis virus. 50 Schematic diagram of the measurement Detailed Implementation

[0017] The present invention will be further described below with reference to embodiments and accompanying drawings:

[0018] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments and comparative examples only and are not intended to limit the scope of protection of this invention. It should be specifically noted that the same organic structure may have multiple names, and all such structures fall within the scope of this patent.

[0019] Unless otherwise defined, the raw materials, reagents, etc. in the following examples and comparative examples can be obtained commercially or prepared according to reported methods.

[0020] First, the expression and purification of NS2B-NS3 protease from St. Louis encephalitis virus were performed:

[0021] S1: The pET22b(+) vector containing the gene encoding the St. Louis encephalitis virus NS2B-NS3 protein was transformed into Escherichia coli BL21(DE3) cells, and positive clones were screened using LB solid medium (containing 50 mg / L ampicillin).

[0022] S2: Select positive clones from solid culture medium, culture them in a small shaker at 37℃ for 6 hours, and then transfer them to 800 mL of LB medium (containing 50 mg / L ampicillin). When the absorbance at 600 nm wavelength reaches 0.6, add 0.2 mM IPTG (isopropyl thiogalactoside) and culture at 16℃ for 18 hours.

[0023] S3: Collect E. coli by centrifugation at 3500 rpm for 20 min, and then perform high-pressure sterilization; after centrifugation of the sterilized solution at 18000 rpm for 1 h, collect the clear liquid at the top.

[0024] S4: Add the supernatant to a Ni-NTA affinity chromatography column pre-equilibrated with lysis buffer (50mM Tris-HCl, 0.3M NaCl, pH 9.0) to allow the target protein to fully bind to Ni and thus enrich the target protein.

[0025] S5: Wash away unbound contaminating proteins with lysis buffer containing 20mM imidazole. When the color of the effluent does not change as detected by Coomassie Brilliant Blue G250, it indicates that most of the contaminating proteins have been washed away. Elute St. Louis encephalitis virus NS2B-NS3 protein with lysis buffer containing 100mM imidazole, then concentrate to 1mL using a 10kD concentrator tube and change the buffer for further concentration. Further purification is performed using anion exchange chromatography and size exclusion chromatography to obtain the target protein with charge uniformity.

[0026] The purified St. Louis encephalitis virus NS2B-NS3 protein was obtained, and its activity against the St. Louis encephalitis virus NS2B-NS3 protease was determined:

[0027] The fluorescent substrate used was Bz-Nle-KRR-AMC (purchased from Shanghai Jier Biochemical Co., Ltd.) with a purity greater than 95%. The fluorescence intensity at different reaction times was then detected using an Infinite M1000 Pro detector, with the excitation wavelength set to 360 nm and the emission wavelength set to 460 nm.

[0028] The protein buffer components consist of 50 mM Tris-HCl, 25% glycerol, and pH 9.0, and are used to dilute the purified St. Louis encephalitis virus NS2B-NS3 protein.

[0029] The steps for activity assay are as follows:

[0030] S1: Prepare St. Louis encephalitis virus NS2B-NS3 protease (final concentration 400 nM) using the above protein buffer, add the compound dissolved in 95% DMSO (dimethyl sulfoxide) (final concentration 20 μM), incubate at 37°C for 10 min, and quickly add the fluorescent substrate Bz-Nle-KRR-AMC at a concentration of 30 μM; shake the above liquid for 10 s while rotating at 300 rpm to ensure uniform binding, and then record the reading every 30 s for a total of 1500 s;

[0031] A negative control experiment was also designed, in which no inhibitor was added and all other experimental conditions were the same.

[0032] S2: The enzymatic activity kinetic curve of St. Louis encephalitis virus NS2B-NS3 protein was prepared with time as the X-axis and fluorescence value as the Y-axis; the rate of the enzymatic reaction in the first 300 seconds was analyzed using GraphPad Prism 5.0 software based on the fluorescence intensity value displayed by the instrument and the reaction time.

[0033] S3: Set V0 as the initial rate of the enzymatic reaction without the addition of the compound, V i Let V be the initial rate of the enzymatic reaction of the added compound; based on the rate of the enzymatic reaction, calculate the inhibition rate (Ir) of each inhibitor (1-V). i / V0).

[0034] The inhibitory effect of a compound on the St. Louis encephalitis virus NS2B-NS3 protease can be determined by comprehensively considering the residual activity percentage and fluorescence quenching rate. However, since this system mainly screens by fluorescence intensity, compounds that are fluorescent or similar to AMC will interfere with the system. In addition, compounds containing quenching groups may also quench the fluorescence of the system and cause false positives. Therefore, it is necessary to rescreen them.

[0035] For compounds with inhibition rates greater than 70%, to eliminate the possibility of false positives due to operational errors, a fluorescence quenching experiment was designed for rescreening. First, the St. Louis encephalitis virus NS2B-NS3 protein was reacted with Bz-Nle-KRR-AMC for the appropriate time, allowing the fluorescence value to reach a maximum value (P1). Then, the same volume of inhibitor as the blank group was added to the ELISA plate, and its fluorescence value was measured (P2). The fluorescence values ​​from both experiments were then analyzed according to the formula...

[0036] (P r =P1-P2) / P2*100%

[0037] The fluorescence quenching rate P was calculated. r If the fluorescence quenching rate is greater than 20%, it can be determined as a false positive compound and can be excluded; if the fluorescence quenching rate is less than 20%, it can be determined as a positive result.

[0038] like Figure 1The schematic diagram shows the inhibitory effect of hypericin on the NS2B-NS3 protease of St. Louis encephalitis virus. Substituting hypericin into the assay, the inhibition rate (Ir) of hypericin on the NS2B-NS3 protein of St. Louis encephalitis virus is >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 inhibitory effect on the NS2B-NS3 protease of St. Louis encephalitis virus. Hypericin can act as an inhibitor to suppress the NS2B-NS3 protein of St. Louis encephalitis virus, and the inhibition rate is high.

[0039] Compound Hypericin IC 50 Measurement:

[0040] In measuring IC 50 First, the St. Louis encephalitis virus NS2B-NS3 protein required for the experiment was prepared to a final concentration of 400 nM. Then, the substrate Bz-Nle-KRR-AMC was prepared with 95% DMSO to a final concentration of 30 μM.

[0041] Based on the initial screening results, 11 inhibitor concentrations were roughly set, with hypericin concentrations of 100 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.63 μM, 0.31 μM, 0.16 μM, 0.08 μM, and 0 μM.

[0042] First, the protein was added to an ELISA plate and incubated with the inhibitor at 37°C for 30 min in an ELISA reader. Then, 10 μL of substrate was quickly added, and the time and fluorescence change curves were recorded. The initial fluorescence reaction rate of the protease was obtained using Graphpad Prism 5.0 software, and the dose-response curve of compound concentration and residual activity was fitted to finally obtain the IC50 value. 50 value.

[0043] like Figure 2 Hypericin's IC50 against NS2B-NS3 protease in St. Louis encephalitis virus 50 As shown in the schematic diagram, hypericin exhibited an inhibition rate greater than 95% against NS2B-NS3 protein in St. Louis encephalitis virus, and the final measured IC50 value of hypericin was [missing value]. 50 The value was 2.69±0.86μM, which has great potential for application in the preparation of small molecule inhibitors of NS2B-NS3 protease in anti-St. Louis encephalitis virus and is expected to become a potential drug for anti-St. Louis encephalitis virus infection.

[0044] Based on the above experiments, hypericin can be used as an inhibitor of the NS2B-NS3 protease in St. Louis encephalitis virus, and also represents a potential drug for treating St. Louis encephalitis virus infection. Its active ingredient is hypericin, and the drug includes the aforementioned hypericin and one or more pharmaceutically acceptable carriers.

[0045] The carrier includes one or more of the commonly used pharmaceutical agents such as surfactants, binders, disintegrants, diluents, excipients, absorption enhancers, humectants, adsorbents, lubricants, fillers, and synergists. The drug can be formulated as granules, powders, syrups, tablets, pills, or suppositories. Routes of administration may include oral, inhalation, transdermal and mucosal, rectal, and injection.

[0046] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention. Furthermore, the technical terms and other materials involved in the present invention are only for clearly illustrating the advantages and effects of the present invention and should not be considered as limiting the innovativeness of the present invention. The above embodiments are a partial description of the practical application effects of the present invention, and these embodiments are not intended to limit the patent scope of the present invention. All improvements and substitutions made by those skilled in the art based on the present invention fall within the protection scope of the present invention.

Claims

1. The application of hypericin in the preparation of drugs against St. Louis encephalitis virus infection, characterized in that: Hypericin is a small molecule inhibitor of the NS2B-NS3 protease in St. Louis encephalitis virus.

2. The application of hypericin according to claim 1, characterized in that: The structural formula of hypericin is: 。

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