Use of embonic acid in the preparation of a medicament for the treatment of infection by the St. Louis encephalitis virus

Abstract

The application provides application of embelin in preparation of a medicine for resisting St. Louis encephalitis virus infection. The application has the beneficial effect that the embelin has 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 treating St. Louis encephalitis, and in particular relates to the application of embenic acid 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 through mosquitoes and birds and can cause symptoms such as fever, headache, sore throat, and muscle pain. Most patients present with fever, headache, and sore throat; some develop severe encephalitis symptoms, including coma. Some patients may experience sequelae. Advanced age is a significant contributing factor to death, with the mortality rate increasing with age.

[0003] Saint Louis encephalitis virus (SLEV) is a positive-sense RNA virus first discovered in the United States in 1933. SLEV particles are approximately 45 nm in diameter, with a raised envelope and agglutinating core on their surface. They can agglutinate erythrocytes in poultry under pH conditions of 6.0-7.9. The virus is prevalent in North and South America, and in much of the Midwestern and Southeastern United States. It is primarily transmitted by mosquitoes in late summer and early autumn, and causes annual public health concern. The prevalence of SLEV is related to temperature and rainfall. SLEV can invade the human central nervous system, causing acute encephalitis with fever. Between 1975 and 1979, more than 2,500 people were infected with SLEV. Since then, outbreaks have occurred approximately every 10 years, each making many people susceptible to the disease. The elderly are most vulnerable to infection. Encephalitis is a severe form of SLEV, with a mortality rate reaching 20%. In recent years, with climate warming, rapid population growth and mobility, this virus has posed a huge threat to the lives and health of all mankind. Therefore, we need to find drug targets in St. Louis encephalitis virus and then develop drugs against St. Louis encephalitis virus targeting these targets.

[0004] St. Louis encephalitis virus (ESV) is a single-stranded RNA virus with a genome length 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 plays a crucial role in ESV replication and its interaction with the host, possessing RNA helicase, nucleoside triphosphatase, and serine protease activities. It is primarily responsible for RNA self-replication and the cleavage and modification of viral polyproteins. The first 180 amino acids of the N-terminus of NS3 contain a conserved domain of a serine protease, but the central hydrophilic domain of NS2B, acting as a cofactor, is essential for NS3's catalytic activity. Therefore, the NS2B-NS3 protease is a very important drug target for anti-ESV drugs, and screening for inhibitors targeting the NS2B-NS3 protease is of great significance for the development of drugs related to ESV infection.

[0005] Embelin, isolated from Japanese purslane, is an inhibitor of X-linked apoptosis inhibitors. However, to date, there are no reports on the application of embelin in the treatment of St. Louis encephalitis virus infection. Summary of the Invention

[0006] The primary objective of this invention is to provide the application of embenic acid in the preparation of drugs against St. Louis encephalitis virus infection. Embenic acid can be used as a small molecule inhibitor of the NS2B-NS3 protease in St. Louis encephalitis virus at the molecular level. Through the establishment of a control experiment, it was found that embenic acid has a strong inhibitory effect on the NS2B-NS3 protease in St. Louis encephalitis virus. Therefore, embenic acid can be used as a potential drug to inhibit 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 embenic acid is a small molecule inhibitor of NS2B-NS3 protease in St. Louis encephalitis virus.

[0009] Preferably, the structural formula of the embenic acid is:

[0010]

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

[0012] Preferably, the carrier includes one or more of surfactants, adhesives, disintegrants, diluents, excipients, absorption promoters, wetting agents, adsorbent carriers, lubricants, fillers, and synergists.

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

[0014] Based on the above technical solution, embenic acid has good inhibitory activity against the NS2B-NS3 protease in St. Louis encephalitis virus, indicating that embenic acid can be used as a small molecule inhibitor of the NS2B-NS3 protease in St. Louis encephalitis virus, thereby enabling the development of potential drugs and the treatment of St. Louis encephalitis virus infection through new pathways. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the inhibitory effect of embenic acid 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 embenic acid 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 20 mM imidazole. When the color of the effluent remains unchanged 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 100 mM imidazole, then concentrate to 1 mL using a 10 kDa concentrator tube and change the buffer for further concentration. Further purification using anion exchange chromatography and size exclusion chromatography yields the target protein with uniform charge.

[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. rIf 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 1 The schematic diagram shows the inhibitory effect of embenic acid on the NS2B-NS3 protease of St. Louis encephalitis virus. Substituting embenic acid into the model, the inhibition rate (Ir) of embenic acid on the NS2B-NS3 protein of St. Louis encephalitis virus is >80%, and the fluorescence quenching rate in the fluorescence quenching experiment is less than 20%. It can be determined that embenic acid can act as an inhibitor to inhibit the NS2B-NS3 protein of St. Louis encephalitis virus, and the inhibition rate is high, which can play a good role.

[0039] Compound Enbenic acid 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 embezzic acid concentrations of 300 μM, 100 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.63 μM, 0.31 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 Enbecic acid's IC50 against NS2B-NS3 protease in St. Louis encephalitis virus 50 As shown in the schematic diagram, embenic acid inhibited the NS2B-NS3 protein in St. Louis encephalitis virus by more than 80%, and the final measured IC50 of embenic acid was [data missing]. 50 The value was 10.48±0.78 μM, which has great potential for application in the preparation of small molecule inhibitors of NS2B-NS3 protease in St. Louis encephalitis virus and is expected to become a potential drug for treating St. Louis encephalitis virus infection.

[0044] Based on the above experiments, it can be seen that embenic acid can act 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 embenic acid, and the drug includes the aforementioned embenic acid and one or more pharmaceutically acceptable carriers.

[0045] The carrier includes one or more of the commonly used surfactants, binders, disintegrants, diluents, excipients, absorption enhancers, humectants, adsorbents, lubricants, fillers, and synergists in the pharmaceutical field. The drug is formulated as granules, powders, syrups, tablets, pills, or suppositories.

[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 embenic acid in the preparation of drugs against St. Louis encephalitis virus infection, characterized in that: The embenic acid is a small molecule inhibitor of the NS2B-NS3 protease in St. Louis encephalitis virus.

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

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