Application of NEDD8 activator in the preparation of drugs for treating Japanese encephalitis

Abstract

This invention relates to the field of pharmaceutical technology, specifically the application of NEDD8 activator in the preparation of drugs for treating Japanese encephalitis. Using human neuroblastoma cells (SH-SY5Y) as target cells, this invention utilizes activators from a library of ubiquitinated compounds to target and activate key enzymes in the ubiquitination pathway, aiming to identify host factors associated with Japanese encephalitis virus (JEV) infection of SH-SY5Y cells. This helps to understand the mechanism by which JEV invades the central nervous system and causes neuronal cell damage, and also provides new targets for therapeutic drugs against JEV-induced Japanese encephalitis. This invention experimentally discovered that NEDD8 activator (NAE) has the property of inhibiting JEV infection of SH-SY5Y cells. This invention provides the application of NEDD8 activator in the preparation of drugs for treating Japanese encephalitis, offering new targets and treatment strategies for the prevention and treatment of JEV.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of NEDD8 activator in the preparation of drugs for treating Japanese encephalitis. Background Technology

[0002] Japanese encephalitis (JE) is caused by the Japanese encephalitis virus (JEV), which is transmitted by mosquitoes. JEV belongs to the Flaviviridae family and the Flavivirus genus and is a single-stranded positive-sense RNA virus. JEV is neuropathogenic and can cause Japanese encephalitis, which is characterized by inflammation of the brain parenchyma. Severe Japanese encephalitis patients mainly present with symptoms such as high fever, headache, and coma. The mortality rate of those with disease symptoms can be as high as 30%, and about 30%-50% of severe survivors have neurological sequelae such as paralysis and intellectual disability (Ashraf U, Ding Z, Deng S, et al. Pathogenicity and virulence of Japanese encephalitis virus: Neuroinflammation and neuronal cell damage. Virulence. 2021; 12(1):968-980.). Japanese encephalitis is mainly prevalent in East Asia, Southeast Asia, and parts of Oceania. The main control measures are vaccination and mosquito elimination. Although vaccination has significantly reduced the incidence of Japanese encephalitis (JE), the incidence continues to rise. In recent years, the development of anti-JE drugs has been a hot topic in medical and biological research both domestically and internationally. Through the tireless efforts of researchers, several drugs with anti-JE activity have been discovered. These drugs can be broadly classified into two categories based on their properties: first, synthetic antiviral drugs, including nucleic acid-targeted therapeutic agents, nucleic acid analogs, cytokines, flavonoids, and antibiotics; and second, antiviral drugs obtained from natural medicines, including phenolic compounds, arctiinogen, and extracts of some traditional Chinese medicine components. However, the evaluation of the efficacy of these drugs is mainly limited to animal models, cellular, and molecular levels, and further research is needed to determine whether they can be applied clinically.

[0003] Neural precursor cells express developmentally downregulated protein 8 (NEDD8) activating enzyme (NAE), a heterodimer composed of amyloid precursor protein binding protein 1 (APPBP1), also known as NEDD8 activating enzyme E1 subunit 1 (NAE1), and ubiquitin-like modifier activating enzyme 3 (UBA3). NAE is currently the only known NEDD8 activating enzyme, and it is involved in the reaction of ATP and Mg... 2+Under the action of enzymes, NEDD8 can be activated, and then NEDD8 is covalently bound to the substrate protein for modification under the enzymatic cascade catalysis of NEDD8 E2 conjugating enzyme (such as Ubc12) and NEDD8 E3 ligase. This process, which is similar to ubiquitination, specifically covalently links ubiquitin-like protein (UBL) NEDD8 to the substrate protein under the cascade catalysis of NEDD8 activating enzyme, NEDD8 conjugating enzyme and NEDD8 ligase, is called Neddylation (Lin CM, Jiang Z, Gao Z, et al. Small molecules targeting the NEDD8·NAE protein-protein interaction. Chem Sci. 2020; 12(4):1535-1543.). The most studied substrates for neddylation modification are members of the Cullin family, which are scaffold components of Cullin-RING ligases (CRLs). Neddylation-like ubiquitination of Cullin is essential for CRL activation. Neddylation is a post-translational modification that regulates substrate protein activity. It participates in various cellular processes, including degradation of the ubiquitin-proteasome system, cell division, cytoskeleton remodeling, stress response, and neuronal function, by altering substrate structure, stability, and subcellular localization. NAE is a key enzyme in the entire neddylation modification process. Studies have shown that overexpression and activation of NAE and neddylation have been observed in various human solid tumors, hematological malignancies, and neurodegenerative diseases, and are closely related to tumorigenesis and development (Gai W, Peng Z, Liu C, et al. Advances in Cancer Treatment by Targeting the Neddylation Pathway. Front Cell Dev Biol. 2021; 9:653882.).

[0004] VII-31 is a potent and selective small-molecule activator of NAE (Neo-acrylamide), a novel tertiary amide derivative that directly interacts with NAE, enhancing its thermal stability. Studies have shown that VII-31 activates the NEDDylation modification of NAE-Ubc12-Cullin1 through direct interaction with NAE, thereby activating the activity of the SCF E3 ubiquitin ligase composed of SKP1-Cullin1-F-box proteins. This leads to the degradation of its substrates, the anti-apoptotic proteins c-IAP1 and XIAP, ultimately inducing apoptosis in human gastric cancer cells MGC803. Furthermore, cell cycle analysis indicates that VII-31 arrests MGC803 cells in the G2 / M phase, thus inhibiting cell viability. VII-31 has also been shown to inhibit the growth of MGC803 xenograft tumors in mice without significant toxicity (Fu D, Song J, Zhu T, et al. Discovery of novel tertiary amide derivatives as NEDDylation pathway activators to inhibit the tumor progression in vitro and in vivo. Eur J Med Chem. 2020; 192:112153.).

[0005] However, there are currently no reports on the therapeutic effects of NEDD8 activator in Japanese encephalitis. Summary of the Invention

[0006] The purpose of this invention is to provide the application of NEDD8 activator in the preparation of drugs for treating Japanese encephalitis.

[0007] In a first aspect, the present invention provides the use of a NEDD8 activator in the preparation of a drug for treating Japanese encephalitis.

[0008] Furthermore, the NEDD8 activator is VII-31, chemically named N-(4-methoxybenzyl)-2-(thiophen-2-yl)-N-(3,4,5-trimethoxyphenyl)acetamide, with the structural formula shown in Formula I. VII-31 is a novel tertiary amide derivative that can directly interact with NAE, improving the thermal stability of NAE.

[0009]

[0010] Furthermore, the NEDD8 activator is used in the preparation of drugs that inhibit Japanese encephalitis virus infection and cytotoxicity.

[0011] A second aspect of the present invention provides a medicament for treating Japanese encephalitis, comprising:

[0012] (A) An effective amount of NEDD8 activator; and

[0013] (B) Pharmaceutically or immunologically acceptable carriers or excipients.

[0014] Furthermore, the NEDD8 activator is VII-31, whose structural formula is shown in Formula I.

[0015] The advantages of this invention are:

[0016] 1. This invention uses human neuroblastoma cells (SH-SY5Y) as target cells and utilizes activators from a library of ubiquitinated compounds to target and activate key enzymes in the ubiquitination pathway, aiming to identify host factors associated with JEV infection of SH-SY5Y cells. This contributes to understanding the mechanism by which JEV invades the central nervous system and causes neuronal cell damage. Simultaneously, it provides new targets for therapeutic drugs targeting JEV-induced central nervous system infections and pathogenesis. This invention experimentally discovered that NEDD8 activator also possesses inhibitory properties against Japanese encephalitis virus.

[0017] 2. This invention provides the application of NEDD8 activator in the preparation of drugs for treating Japanese encephalitis, providing a new target and therapeutic drug for the prevention and treatment of Japanese encephalitis virus, and has good market value and clinical application prospects. Attached Figure Description

[0018] Figure 1 A shows partial immunofluorescence detection results of screening a library of ubiquitinated compounds using the JEV in vitro cell culture system; B shows the statistical graph of the corresponding viral infection inhibition rate.

[0019] Control: JEV-infected SH-SY5Y cells without any added drugs (blank control group);

[0020] Chloroquine: JEV infection of SH-SY5Y cells with 100uM chloroquine (positive control group);

[0021] Drug group: SH-SY5Y cells infected with JEVs targeting different key enzymes in the ubiquitin-proteasome system at a drug concentration of 5 μM (experimental group).

[0022] Figure 2The graph shows the inhibitory effect of NAE activator VII-31 on viral infection. In the graph, A is a schematic diagram of the inhibition of JEV infection and cytotoxicity detection after using different concentrations of VII-31 on target cells. The main vertical axis represents the amount of JEV virus, and the secondary vertical axis represents the effect on cytotoxicity. B is an immunofluorescence detection graph of the effect of different concentrations of VII-31 on viral infectivity after acting on target cells. Detailed Implementation

[0023] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0024] Example 1

[0025] I. Experimental Materials

[0026] Ubiquitination Compound Library (Cat. No.: HY-L050), purchased from MCE.

[0027] NAE activator VII-31 (Cat. No.: HY-133558), purchased from MCE.

[0028] The human neuroblastoma strain SH-SY5Y was purchased from ATCC, accession number: ATCC CRL-2266.

[0029] II. Experimental Methods

[0030] 1. Real-time quantitative PCR (RT-PCR) for detecting JEV viral load

[0031] 1) Total RNA was extracted from cells in the control and treatment groups using TRIzol. The specific steps are as follows:

[0032] After treating the target cells, discard the culture supernatant. Add 1 ml of TRIzol to the cells and mix thoroughly to lyse the cells at room temperature for 3-5 min. Add 1 / 5 volume of chloroform and mix vigorously manually for 15 s. Centrifuge at 12,000 rpm for 15 min at 4 °C. Collect the upper aqueous phase and transfer it to a new EP tube. Add an equal volume of isopropanol, mix thoroughly, and precipitate at room temperature for 10 min. Centrifuge at 12,000 rpm for 10 min at 4 °C. Discard the supernatant and add 1 ml of pre-chilled 75% ethanol. Centrifuge at 12,000 rpm for 5 min at 4 °C. Discard the supernatant completely, air-dry the RNA precipitate at room temperature, and dissolve the precipitate in DEPC-treated water to obtain total RNA.

[0033] 2) Obtain cDNA from control and interference group cells using the Takara reverse transcription kit. The specific steps are as follows:

[0034] Add the following reaction mixture to the PCR tube.

[0035]

[0036] Mix gently and thoroughly, react at 37°C for 15 min, then heat at 85°C for 5 s to inactivate reverse transcriptase.

[0037] 3) Quantitative Real-Time RT-PCR Detection

[0038] The reaction was carried out using Takara's SYBR Premix Ex Taq kit, and the reaction system is as follows.

[0039]

[0040] Two-step amplification was performed using a Rotor Gene 3000A instrument: pre-denaturation at 95℃ for 2 min, followed by 40 PCR cycles of 95℃ for 5 s and 60℃ for 30 s.

[0041] 2. JEV virus infection of SH-SY5Y cells

[0042] 2.1 JEV virus infection experiment in SH-SY5Y cells

[0043] Forty-eight hours after transfection of SH-SY5Y cells with plasmid, JEV virus infection experiments were performed. The culture supernatant was aspirated, washed twice with pre-warmed PBS, and inoculated with JEV at an MOI of 0.5. After incubation at 37°C for 2 hours, the virus solution was discarded, and the cells were washed three times with pre-warmed PBS. Fresh culture medium was then added for further culture.

[0044] 2.2 Immunofluorescence staining to detect JEV antigen expression

[0045] After SH-SY5Y cells were infected with the virus, they were cultured for another 48 hours. The expression of viral antigens was detected by immunofluorescence. The specific steps are as follows:

[0046] 1) Cell fixation: Remove the culture medium from the 96-well plate, wash the cells twice with PBS, add 100 μl of pre-cooled methanol to each well, fix at -20℃ for 20 min, and wash the cells three times with pre-cooled PBS.

[0047] 2) Permeabilization: Add 100 μl of 0.1% Triton X-100 to each well of the fixed cells, incubate at room temperature for 15 min, and wash 3 times with pre-cooled PBS.

[0048] 3) Sealing: Add 100 μl of 3% BSA to each well and incubate at room temperature for 1 h.

[0049] 4) Primary antibody incubation: Add 100 μl of JEV-specific rabbit monoclonal antibody GTX125868 (1:1000 dilution) to each well, incubate at room temperature for 1 h, and wash 3 times with pre-cooled PBS.

[0050] 5) Secondary antibody incubation: Add 100 μl of AF 488 fluorescently labeled anti-rabbit IgG (1:1000 dilution) to each well, incubate at room temperature in the dark for 1 h, and wash twice with pre-cooled PBS in the dark.

[0051] 6) Labeling cell nuclei: Add the nuclear fluorescent dye DAPI (1:10000, diluted with PBS) to each well, incubate at room temperature in the dark for 15 min, and wash 3 times with pre-cooled PBS in the dark.

[0052] 7) Detect and count the number of green AF 488 positive cell clones under a fluorescence microscope.

[0053] 3. Screening of ubiquitinated compound libraries

[0054] 1) Grouping: The experiment was divided into a blank control group, a positive drug control group and an experimental group. Each drug in each group was set up with 3 replicates. The experiment was independently repeated three times.

[0055] CTRL: JEV-infected SH-SY5Y cells without any inhibitors (blank control group);

[0056] Chloroquine: JEV infection of SH-SY5Y cells with 100 μM chloroquine (positive control group);

[0057] Drug group: SH-SY5Y cells infected with JEVs targeting different key enzymes in the ubiquitin-proteasome system at a drug concentration of 5 μM (experimental group).

[0058] 2) 12-16 hours in advance, seed SH-SY5Y cells onto 96-well cell culture plates to achieve a cell density of 80%-90% at the time of treatment. Aspirate the culture supernatant, wash twice with PBS pre-warmed to 37°C, add 100 μl of different inhibitors to each well, and incubate at 37°C for 6 hours. The positive control group is given 100 μl of 100 μM chloroquine in whole culture medium, and the blank control group is given an equal volume of whole culture medium.

[0059] Discard the inhibitor solution, add an equal amount of JEV (MOI = 0.5) to each well, incubate at 37°C for 48 h, then discard the virus solution and wash three times with PBS. Immunofluorescence detection is performed using the same method as in step 2.2.

[0060] 4. Action of activator (VII-31)

[0061] 1) Grouping: The experiment was divided into a blank control group, a positive drug control group, and a chemical drug group. Each drug concentration in each group was set up with 3 replicates. The experiment was independently repeated three times.

[0062] 2) 12-16 hours in advance, seed SH-SY5Y cells onto 24-well cell culture plates to achieve a cell density of 80%-90% at the time of treatment. Aspirate the culture supernatant, wash twice with PBS pre-warmed to 37°C, add 500 μl of different concentration gradients of the drug to each well, and incubate at 37°C for 6 hours. The positive control group was given 100 μM chloroquine in whole culture medium, and the blank control group was given an equal volume of whole culture medium.

[0063] Discard the chemical reagent solution, add an equal amount of JEV (MOI = 0.5) to each well, incubate at 37°C for 2 hours, then discard the virus solution. Wash three times with PBS, and add proteinase K solution (1 mg / ml) to remove viral particles bound to the surface of target cells. Subsequent culture and detection of JEV-infected target cells are performed in step 2.2.

[0064] 5. Cytotoxicity assay

[0065] The effect of VII-31 on cell proliferation was detected using the CCK-8 assay. The specific steps are as follows:

[0066] Cells in logarithmic growth phase were collected and seeded at a density of 3000 cells per well in 96-well plates. After overnight cell attachment, VII-31 was added, and cell proliferation was assessed after 48 hours of culture. The original culture medium was discarded, and 110 μL of fresh culture medium containing 10 μL of CCK-8 was added to each well. After 3 hours of culture, the absorbance of each well was measured at 450 nm using a multi-mode microplate reader. The experiment was independently repeated three times, and the average value was calculated.

[0067] III. Experimental Results

[0068] Using the JEV in vitro cell culture system, screening of a library of ubiquitinated compounds revealed that NEDD8 activator VII-31 exhibited good anti-JEV activity, inhibiting JEV infection of SH-SY5Y cells. Figure 1 A). We also set up a blank control group (no drug added, Control), a positive drug control group (100 μM Chloroquine), and a drug treatment group (5 μM). After 6 h of drug treatment, JEV was infected for 48 h. Viral antigen was then detected by indirect immunofluorescence, and the infection rate was calculated and normalized to the Control group. A relative inhibition rate of >50% against JEV (i.e., infection rate <50%, the dotted line in the figure represents infection rate = 50%) and a superior inhibitory effect compared to Chloroquine were used as inclusion criteria for candidate drugs. Among them, the NEDD8 activator VII-31 showed a relatively significant inhibitory effect on JEV after treatment. Figure 1 B).

[0069] Furthermore, the effects on viral infection were examined after treatment with NEDD8 activator VII-31 at different concentration gradients. The results showed that as the inhibitor concentration increased, the viral load of JEV gradually decreased, while different concentrations of inhibitor had no significant effect on cell viability. Figure 2 A) The results obtained by indirect immunofluorescence assay for viral antigens were consistent. Figure 2 B) These results indicate that NEDD8 activator VII-31 can inhibit JEV infection and that its antiviral activity is positively correlated with drug concentration.

[0070] The above experimental results demonstrate that the NEDD8 activator VII-31 can significantly inhibit JEV infection in SH-SY5Y cells, thus exerting an antiviral effect. This invention provides a novel therapeutic drug and target for the prevention and treatment of JEV-induced Japanese encephalitis, possessing significant market value and promising clinical application prospects.

[0071] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. The application of NEDD8 activator in the preparation of drugs for treating Japanese encephalitis, characterized in that, The NEDD8 activator is VII-31, and its structural formula is shown in Formula I: Formula I.

2. The application of the NEDD8 activator according to claim 1 in the preparation of drugs for treating Japanese encephalitis, characterized in that, The application of the NEDD8 activator in the preparation of drugs that inhibit Japanese encephalitis virus infection and cytotoxicity.

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