Use of zinc undecylenate in the preparation of anti-dengue virus drugs

Abstract

The application provides application of zinc undecylenate in preparation of a drug for resisting dengue virus, and uses zinc undecylenate as an inhibitor of NS2B-NS3 protease in the dengue virus, wherein the zinc undecylenate has good inhibitory activity on the NS2B-NS3 protease in the dengue virus, and the zinc undecylenate has various carriers and administration modes, is convenient to use, and is easy to obtain.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of zinc undecenoate in the preparation of anti-dengue virus drugs. Background Technology

[0002] Dengue virus (DENV) is a single-stranded RNA virus. It is transmitted by Aedes mosquitoes and is mainly distributed in tropical and subtropical regions. Infection causes dengue fever, with clinical manifestations including headache, rash, muscle pain, nausea, and vomiting. Most dengue fever cases are self-limiting, but some can develop into life-threatening severe dengue fever, characterized by increased vascular permeability, plasma leakage, and in severe cases, hypovolemic shock, tissue damage, and multiple organ failure. It has been reported that dengue virus is widespread in 125 countries worldwide, infecting approximately 390 million people annually, of whom 96 million develop clinical symptoms, and about 20,000 die. Global warming has accelerated the geographical expansion of Aedes mosquitoes, increasing their reproductive and biting rates, and accelerating viral replication. This has led to a further expansion of the dengue virus's spread, posing a significant threat to human health. All four serotypes of dengue virus (DENV 1, 2, 3, and 4) can cause varying degrees of disease. Of the four serotypes, DENV1 and DENV2 are the most common types associated with outbreaks. Current vaccines do not provide comprehensive protection against all four serotypes, and due to antibody-dependent enhancement, vaccination may increase the risk of children developing severe dengue fever later in life. Currently, dengue treatment is primarily adjunctive therapy, and no specific antiviral drugs have been approved.

[0003] The dengue virus genome encodes a polyprotein that is cleaved by viral and host proteases, producing three structural proteins (C, prM / M, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The structural proteins form the viral capsid, while the non-structural proteins are mainly involved in the regulation of RNA replication, viral assembly, and the host immune response.

[0004] The NS3 protein plays a crucial role in the dengue virus life cycle. Its N-terminus contains a serine protease domain, but its structure is unstable and requires the assistance of the NS2B cofactor to fold correctly and exert its activity. The NS2B-NS3 protease mediates post-translational cleavage of viral polyproteins, which are critical steps in viral maturation. Therefore, targeting this protein is of great significance for the development of drugs against dengue virus.

[0005] Zinc undecylenate, also known as undecylenate, possesses excellent emulsifying and bactericidal properties. It can be used as a co-emulsifier in creams and ointments, and also for treating fungal infections. For example, zinc undecylenate ointment can be used to treat tinea manuum, tinea pedis, tinea corporis, and tinea cruris. In addition, studies have reported its good inhibitory effects on herpes simplex virus and Zika virus. However, to date, there are no reports on the application of zinc undecylenate in treating dengue virus infection. Summary of the Invention

[0006] The problem this invention aims to solve is to target the NS2B-NS3 protease in dengue virus and use zinc undecenoate as an inhibitor of this protease, thus providing the application of zinc undecenoate in the preparation of anti-dengue virus drugs.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] The application of zinc undecenoate in the preparation of anti-dengue virus drugs, wherein the molecular structural formula of zinc undecenoate is:

[0009]

[0010] Furthermore, the anti-dengue virus drug includes zinc undecenoate and one or more pharmaceutically acceptable carriers.

[0011] Furthermore, the carrier includes diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, lubricants, and synergists.

[0012] Furthermore, the anti-dengue virus drug is selected from granules, powders, syrups, tablets, pills, suppositories, or combinations thereof.

[0013] Furthermore, the anti-dengue virus drug can be administered orally, by injection, implantation, external application, spray, inhalation, or a combination thereof.

[0014] This invention provides the application of zinc undecenoate in the preparation of anti-dengue virus drugs. Targeting the NS2B-NS3 protease in dengue virus, zinc undecenoate is used as an inhibitor of this protease. Zinc undecenoate has good inhibitory activity against the NS2B-NS3 protease in dengue virus, and it has multiple carriers and administration methods, making it convenient to use and easy to obtain.

[0015] The zinc undecenoate involved in this invention has CAS number 557-08-4 and was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Attached Figure Description

[0016] Figure 1This is a schematic diagram illustrating the inhibitory effect of zinc undecenoate on dengue virus NS2B-NS3 protease according to an embodiment of the present invention.

[0017] Figure 2 The IC50 value of zinc undecenoate as an embodiment of the present invention for inhibiting dengue virus JEV NS2B-NS3 protease is shown in the figure. 50 Schematic diagram of the measurement. Detailed Implementation

[0018] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0019] Please see the appendix Figure 1 -Appendix Figure 2 This invention provides the application of zinc undecenoate in the preparation of anti-dengue virus drugs, and the specific implementation method is as follows:

[0020] 1. Expression and purification of dengue virus NS2B-NS3 protease (DENV1 NS2B-NS3pro)

[0021] (1) The recombinant NS2B-NS3pro containing only the hydrophilic region of the NS2B center is sufficient to produce a soluble and active protease. Therefore, the nine amino acids Gly4-Ser-Gly4 were used as linkers to construct the NS2B-NS3 fusion protein. The pET-15b(+) recombinant plasmid containing the DENV1NS2B-NS3pro gene was transformed into competent cells and cultured overnight on ampicillin-containing plates.

[0022] (2) Select positive clones with good growth status from the plate, and culture them in a shaker at 37℃ and 220rpm for 6h. Then, transfer them to 800mL of medium containing ampicillin for expansion culture for 6h. After that, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5mM and induce expression in a shaker at 16℃ and 220rpm for 16-18h.

[0023] (3) Centrifuge the collected bacterial solution at 3500 rpm for 30 min, discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells and break them up. Then centrifuge the broken bacterial solution at 18000 rpm for 40 min and collect the supernatant for subsequent operations.

[0024] (4) First, the protein was initially purified. The supernatant was placed in a nickel column and suspended using a vertical suspension apparatus for 1-2 hours to ensure it was fully bound to the nickel medium. The protein was then washed with lysis buffers containing 20 mM imidazole and 200 mM imidazole, respectively. The initially purified protein was concentrated and then further purified by ion exchange chromatography and gel filtration chromatography to obtain a protein sample with high purity and good charge and size uniformity for subsequent experiments.

[0025] 2. Screening for inhibitors of DENV1 NS2B-NS3 protease

[0026] Using Bz-Nle-Lys-Arg-Arg-AMC with a purity greater than 95% as a substrate, enzyme activity was measured and inhibitors were screened using the principle of fluorescence resonance energy transfer. The excitation and emission wavelengths were set to 380 nm and 460 nm, respectively.

[0027] The protein buffer used in the experiment consisted of 20 mM Tris, 30% glycerol, 1 mM CHAPS, and pH 9.0. DENV1 NS2B-NS3pro was diluted to a specific concentration (final concentration 100 nM) using the buffer. 89 μL of diluted DENV1 NS2B-NS3pro and 1 μL of the test compound (final concentration 20 μM) were added to a black 96-well plate. The plate was incubated at 37°C for 10 min, and then 10 μL of substrate (final concentration 20 μM) was quickly added to initiate the reaction. A negative control without the compound was set up by replacing it with 1 μL of 95% dimethyl sulfoxide (DMSO). Fluorescence values ​​were read every 30 seconds, and the data from the first 300 seconds were used to calculate the initial rate of the enzyme reaction. V0 was defined as the initial rate of the enzyme reaction without the compound, and V... i This represents the initial rate of the enzymatic reaction when the compound is added. According to the formula IR = (1 - V) / (1 - V) i / V0)*100% is used to calculate the inhibition rate (IR) of each compound, and effective compounds are initially screened out.

[0028] Compounds with inhibition rates greater than 70% were re-screened, with multiple parallel groups set up. Compounds that were still effective after re-screening were subjected to fluorescence quenching experiments to eliminate false positives.

[0029] 3. Zinc undecenoate IC 50 The determination;

[0030] Zinc undecenoate was diluted with 95% DMSO to achieve final concentrations of 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.3125 μM, 0.1563 μM, 0.0782 μM, and 0.0391 μM in the reaction system. 89 μL of diluted DENV1NS2B-NS3 pro and 1 μL of inhibitor (final concentration 20 μM) were added to a black 96-well plate, and the plate was incubated at 37°C for 10 min. Then, 10 μL of substrate (final concentration 20 μM) was quickly added to initiate the reaction. A negative control without the compound was included. Fluorescence values ​​were read every 30 s. Data were processed using Graphpad Prism 5.0 software, and a curve showing the change in fluorescence increase over time was plotted with time on the x-axis and fluorescence increase value on the y-axis. Then, the initial rate of the enzyme-catalyzed reaction was calculated using the data from the first 300 seconds. The inhibition rate was further calculated using the formula, and a curve was fitted between the logarithm of the inhibitor concentration and the inhibition rate to obtain the IC50. 50 value.

[0031] This invention relates to the field of pharmaceutical technology, specifically the application of zinc undecenoate in the preparation of anti-dengue virus drugs. Zinc undecenoate exhibits an inhibition rate of over 90% against DENV1 NS2B-NS3 pro, and its IC50 at a substrate concentration of 20 μM is [missing information]. 50 The concentration is 1.15±0.08 μM, which has great potential for application in the preparation of small molecule inhibitors of NS2B-NS3 protease in dengue virus and is expected to become a potential drug for dengue virus infection.

[0032] The advantages and beneficial effects of this invention are:

[0033] This invention provides the application of zinc undecenoate in the preparation of anti-dengue virus drugs. Targeting the DENV1 NS2B-NS3 protease in dengue virus, zinc undecenoate is used as an inhibitor of this protease. Zinc undecenoate has good inhibitory activity against the DENV1 NS2B-NS3 protease in dengue virus, and it is convenient to use and easy to obtain due to the availability of various carriers and administration methods.

[0034] It should be understood that the specific embodiments described above are merely illustrative or explanatory of the principles of the invention and do not constitute a limitation thereof. Therefore, any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and scope of the invention should be included within the protection scope of the invention. Furthermore, the appended claims are intended to cover all variations and modifications falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. The application of zinc undecenoate in the preparation of anti-dengue virus drugs, characterized in that, The molecular structural formula of zinc undecenoate is: ； Zinc undecenoate was used as an inhibitor of the NS2B-NS3 protease in dengue virus.

2. The application according to claim 1, characterized in that: The anti-dengue virus drug includes zinc undecenoate and one or more pharmaceutically acceptable carriers.

3. The application according to claim 2, characterized in that: The carrier includes diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, lubricants, and synergists.

4. The application according to any one of claims 1-3, characterized in that: The anti-dengue virus drug is selected from granules, powders, syrups, tablets, pills, suppositories, or combinations thereof.

5. The application according to claim 4, characterized in that: The administration methods of the anti-dengue virus drugs include oral, injection, implantation, external application, spray, inhalation, or combinations thereof.

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