Application of NEDD8 activator enzyme inhibitors 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 enzyme inhibitors or their hydrochlorides in the preparation of drugs for treating Japanese encephalitis. This invention utilizes inhibitors from a library of ubiquitinated compounds to target and inhibit key enzymes in the ubiquitination pathway, aiming to identify host factors associated with Japanese encephalitis virus (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, and also provides new targets for therapeutic drugs against JEV-induced Japanese encephalitis. This invention discovers that NEDD8 activator enzyme (NAE) inhibitors or their hydrochlorides possess the property of inhibiting JEV infection of SH-SY5Y cells. This invention provides the application of NEDD8 activator enzyme inhibitors or their hydrochlorides in the preparation of drugs for treating Japanese encephalitis, offering new targets and therapeutic strategies for the prevention and treatment of JEV.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of NEDD8 activator inhibitors or their hydrochloride salts 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 and NEDD8 E3 ligase. This process, similar to ubiquitination, in which NEDD8 activating enzyme, NEDD8 conjugating enzyme, and NEDD8 ligase specifically covalently binds 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.). Among them, the most studied Neddylation substrates are members of the Cullin family, which are scaffold components of Cullin-RING ligase (CRL), and Cullin Neddylation is necessary for CRL activation. Neddylation is a post-translational modification that regulates the activity of substrate proteins. It participates in various cellular processes, including degradation of the ubiquitin-proteasome system, cell division, cytoskeleton remodeling, stress response, and neural cell function, by altering the structure, stability, and subcellular localization of the substrate. NAE is a key enzyme in the entire neddylation process. Studies have shown that overexpression and activation of NAE and neddylation have been observed in various solid tumors, hematological malignancies, and neurodegenerative diseases in humans, 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] Pevonedistat (MLN4924) and its hydrochloride, Pevonedistat hydrochloride (MLN4924hydrochloride), are potent and selective small-molecule inhibitors of NAE. MLN4924 is an adenosine monophosphate (AMP) analog that binds to NEDD8 to form the MLN4924-NEDD8 adduct. The MLN4924-NEDD8 adduct competitively binds to the ATP-binding site of NAE, preventing NAE from catalyzing the formation of the NEDD8-NAE adduct, thereby inhibiting NAE activity. Under physiological conditions, MLN4924 hydrochloride can be converted into the biologically active form MLN4924 to exert its effect (Soucy TA, Smith PG, Milhollen MA, et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009; 458(7239):732-6.). CRL is currently the largest family of ubiquitin ligases, mediating the degradation of approximately 20% of intracellular proteins via the ubiquitin-proteasome system. Its substrates are largely related to tumorigenesis and development. Therefore, by targeting and inhibiting NAE activity with MLN4924, the needdylation modification of Cullin protein can be indirectly blocked, inhibiting CRL function and ultimately leading to the accumulation of large amounts of CRL substrates. This accumulation can induce apoptosis, senescence, and autophagy, thereby exerting an anti-tumor effect (El-Mesery M, Anany MA, Hazem SH, et al. The NEDD8-activating enzyme inhibition with MLN4924 sensitizes human cancer cells of different origins to apoptosis and necroptosis. Arch Biochem Biophys. 2020; 691:108513.). MLN4924 is currently in Phase II clinical trials and is the first small-molecule NAE inhibitor to enter clinical trials. Due to its high efficacy and low toxicity, the results of the Phase I clinical trial also showed that it has good clinical therapeutic effects in acute myeloid leukemia, lymphoma, melanoma, and various solid tumors and hematological malignancies, further proving that inhibiting NAE activity as a therapeutic target for clinical diseases is safe and effective.

[0005] However, there are currently no reports on the therapeutic effects of the NEDD8 activator inhibitor Pevonedistat (MLN4924) and its hydrochloride Pevonedistat hydrochloride (MLN4924 hydrochloride) in Japanese encephalitis. Summary of the Invention

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

[0007] In a first aspect, the present invention provides the use of an inhibitor of NEDD8 activating enzyme or its hydrochloride salt in the preparation of a medicament for treating Japanese encephalitis.

[0008] Furthermore, the NEDD8 activator enzyme inhibitor is Pevonedistat (MLN4924), whose chemical name is ((1S,2S,4R)-4-(4-((S)-2,3-dihydro-1H-inden-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methyl sulfamate, and its chemical structure is shown in Formula I;

[0009] The hydrochloride of Pevonedistat (MLN4924) is Pevonedistat hydrochloride (MLN4924 hydrochloride), whose chemical name is ((1S,2S,4R)-4-(4-((S)-2,3-dihydro-1H-inden-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)carbamate hydrochloride (((1S,2S,4R)-4-(4-(((S)-2,3-dihydro-1H-inden-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methyl sulfamate hydrochloride, and its chemical structure is shown in Formula II).

[0010]

[0011] Furthermore, the use of the NEDD8 activator inhibitor or its hydrochloride in the preparation of drugs that inhibit Japanese encephalitis virus infection and cytotoxicity.

[0012] A second aspect of the present invention provides a medicament for treating Japanese encephalitis, comprising:

[0013] (A) An effective amount of an inhibitor of NEDD8 activating enzyme or its hydrochloride; and

[0014] (B) Pharmaceutically or immunologically acceptable carriers or excipients.

[0015] Furthermore, the NEDD8 activator inhibitor is MLN4924, and its hydrochloride is MLN4924hydrochloride, with chemical structural formulas shown in Formula I and Formula II, respectively.

[0016] The advantages of this invention are:

[0017] 1. This invention uses human neuroblastoma cells (SH-SY5Y) as target cells and utilizes inhibitors from a library of ubiquitination compounds to target and inhibit key enzymes in the ubiquitination pathway in order 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. Simultaneously, it provides new targets for therapeutic drugs targeting JEV-induced Japanese encephalitis. This invention experimentally discovered that NEDD8 activator enzyme (NAE) inhibitors or their hydrochlorides have the property of inhibiting JEV infection of SH-SY5Y cells. Furthermore, it was found that inhibiting the expression of NAE1 protein molecules by transfecting siRNA can reduce JEV infection of SH-SY5Y cells, thus exhibiting an antiviral effect.

[0018] 2. This invention provides the application of NEDD8 activator inhibitors or their hydrochloride salts in the preparation of drugs for treating Japanese encephalitis, providing new targets and therapeutic drugs for the prevention and treatment of Japanese encephalitis virus, and has good market value and clinical application prospects. Attached Figure Description

[0019] Figure 1 Figure A shows partial immunofluorescence detection results of screening a library of ubiquitinated compounds using the JEV in vitro cell culture system, while Figure B shows the statistical graph of the corresponding viral infection inhibition rate.

[0020] Control: JEV-infected SH-SY5Y cells without any inhibitors (blank control group);

[0021] Chloroquine: JEV infection of SH-SY5Y cells with 100uM chloroquine (positive control group);

[0022] Inhibitor: JEVs were added at a concentration of 5 μM to target different key enzymes in the ubiquitin-proteasome system and infected SH-SY5Y cells (experimental group).

[0023] Figure 2 shows the inhibitory effect of the NAE inhibitor Pevonedistat (MLN4924) and its hydrochloride Pevonedistat hydrochloride (MLN4924 hydrochloride) on viral infection. In Figure (A), it is a schematic diagram of the inhibition of JEV infection and cytotoxicity after different concentrations of MLN4924 or MLN4924 hydrochloride were applied to target cells. The main vertical axis represents the amount of JEV virus, and the secondary vertical axis represents the effect on cytotoxicity. Figure (B) is an immunofluorescence detection diagram of the effect of different concentrations of MLN4924 or MLN4924 hydrochloride on viral infectivity after applying them to target cells. Detailed Implementation

[0024] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0025] Example 1

[0026] I. Experimental Materials

[0027] Ubiquitination Compound Library (Cat. No.: HY-L050), purchased from MCE.

[0028] The NAE inhibitor Pevonedistat (MLN4924) (Cat. No.: HY-70062) and its hydrochloride Pevonedistat hydrochloride (MLN4924 hydrochloride) (Cat. No.: HY-10484) were purchased from MCE.

[0029] The human neuroblastoma strain SH-SY5Y was purchased from ATCC, accession number: ATCC CRL-2266.

[0030] II. Experimental Methods

[0031] 1. siRNA interference

[0032] 1.1 RNA transfection

[0033] The transfection procedure should be performed according to the Lipofectamine 2000 instruction manual;

[0034] 1) Sow SH-SY5Y cells in 24-well cell culture plates 12-16 hours in advance to achieve a cell density of 80%-90% at the time of transfection.

[0035] 2) Add 2 μL of Lipofectamine 2000 to 50 μL of opti-MEM and mix gently. Incubate at room temperature for 5 min. Separately, mix 5 μL of 5 μM interfering RNA with 50 μL of opti-MEM. After incubation, add the diluted Lipofectamine 2000 transfection reagent to the diluted RNA and mix gently by pipetting. After incubating at room temperature for 20 min, add the RNA to SH-SY5Y cells and add 400 μL of opti-MEM to bring the final RNA concentration to 50 nM.

[0036] 3) Replace with fresh culture medium containing antibiotics 6-8 hours after transfection.

[0037] 2. Real-time quantitative PCR (RT-PCR) for detecting JEV viral load

[0038] 1) Total RNA was extracted from cells in the control and treatment groups using TRIzol. The specific steps are as follows:

[0039] 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.

[0040] 2) Obtain cDNA from control and interference group cells using the Takara reverse transcription kit. The specific steps are as follows:

[0041] Add the following reaction mixture to the PCR tube.

[0042]

[0043] Mix gently and thoroughly, react at 37°C for 15 min, then heat at 85°C for 5 s to inactivate reverse transcriptase.

[0044] 3) Quantitative Real-Time RT-PCR Detection

[0045] The reaction was carried out using Takara's SYBR Premix Ex Taq kit, and the reaction system is as follows.

[0046]

[0047] 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.

[0048] 3. JEV virus infection of SH-SY5Y cells

[0049] 3.1 JEV virus infection experiment in SH-SY5Y cells

[0050] SH-SY5Y cells were transfected with RNA for 48 hours before 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.

[0051] 3.2 Immunofluorescence staining to detect JEV antigen expression

[0052] 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:

[0053] 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.

[0054] 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.

[0055] 3) Seal: Add 100 μl of 3% BSA to each well and incubate at room temperature for 1 h.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 7) Detect and count the number of green AF 488 positive cell clones under a fluorescence microscope.

[0060] 4. Western blot for protein immunoblotting

[0061] 1) Total protein was extracted from SH-SY5Y cells in different treatment groups using protein lysis buffer.

[0062] 2) After protein quantification, 30 μg of protein was added to a 12.5% ​​polyacrylamide gel for electrophoresis, and the corresponding bands were transferred to a PVDF membrane using an electroporator.

[0063] 3) Block the non-specific sites of the protein with 5% skim milk, then block with NAE or NEDD8 antibody, incubate overnight at 4°C, and wash three times with TBST buffer to remove the primary antibody.

[0064] 4) Then incubate with HRP-labeled secondary antibody at room temperature for 2 hours, followed by washing three times with TBST buffer.

[0065] 5) Finally, develop the color using a colorimetric solution and take photos for analysis.

[0066] 5. Screening of ubiquitinated compound libraries

[0067] 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.

[0068] Control: JEV-infected SH-SY5Y cells without any inhibitors (blank control group);

[0069] Chloroquine: JEV infection of SH-SY5Y cells with 100 μM chloroquine (positive control group);

[0070] Inhibitor: JEVs infected with JEVs targeting different key enzymes in the ubiquitin-proteasome system at a concentration of 5 μM (experimental group).

[0071] 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.

[0072] 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 3.2.

[0073] 6. Inhibitor (MLN4924 or MLN4924 hydrochloride) action

[0074] 1) Grouping: The experiment was divided into a blank control group, a positive drug control group, and an inhibitor interference group. Each drug concentration in each group was set up with 3 replicates, and the experiment was independently repeated three times.

[0075] 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.

[0076] 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 3.2.

[0077] 7. Cytotoxicity assay

[0078] The effect of MLN4924 or MLN4924 hydrochloride on cell proliferation was detected using the CCK-8 assay. The specific steps are as follows:

[0079] 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, MLN4924 or MLN4924 hydrochloride was added, and cell proliferation was assessed after 48 hours of incubation. The original culture medium was discarded, and 110 μL of fresh medium containing 10 μL of CCK-8 was added to each well. After 3 hours of incubation, 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.

[0080] III. Experimental Results

[0081] Using the JEV in vitro cell culture system, screening of a library of ubiquitinated compounds revealed that the NEDD8 activator inhibitor Pevonedistat (MLN4924) or its hydrochloride Pevonedistat hydrochloride (MLN4924 hydrochloride) 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 an inhibitor 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. Candidate drugs were included based on 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. Among them, the NEDD8 activator enzyme inhibitor Pevonedistat (MLN4924) or its hydrochloride Pevonedistat hydrochloride (MLN4924hydrochloride) showed a relatively significant inhibitory effect on JEV after treatment. Figure 1 B).

[0082] Furthermore, the effects on viral infection were examined after treatment with different concentration gradients of the NEDD8 activator inhibitor Pevonedistat (MLN4924) or its hydrochloride (MLN4924 hydrochloride). 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 2A The results obtained by indirect immunofluorescence assay for viral antigens were consistent. Figure 2B These results indicate that the NEDD8 activator inhibitor Pevonedistat (MLN4924) or its hydrochloride Pevonedistat hydrochloride (MLN4924 hydrochloride) can inhibit JEV infection and that the antiviral activity is positively correlated with the drug concentration.

[0083] To clarify the effect of the inhibitory target NAE (neodymium ether activator enzyme) of NEDD8, Pevonedistat (MLN4924) or its hydrochloride (MLN4924 hydrochloride), on JEV infection, the expression of NAE1 protein was detected by Western blotting after transfection with NAE1 siRNA. The results showed that transfection with NAE1 siRNA significantly inhibited the expression of NAE1 protein. Immunofluorescence assays of viral antigens indicated that downregulation of NAE1 significantly reduced JEV infection of SH-SY5Y cells. These results suggest that, compared with control cells, downregulation of NAE1 significantly reduced the infectivity of JEV in SH-SY5Y cells and decreased viral load. Therefore, the NEDD8 activator enzyme NAE could serve as a novel drug target for inhibiting JEV infection of SH-SY5Y cells.

[0084] The above experimental results demonstrate that this invention reveals that NEDD8 activator inhibitors or their hydrochloride salts can significantly inhibit JEV infection in SH-SY5Y cells. Furthermore, it was found that inhibiting NAE1 protein expression can reduce JEV infection, thus exerting an antiviral effect. This invention provides new therapeutic drugs and targets for the prevention and treatment of JEV-induced Japanese encephalitis, possessing significant market value and clinical application prospects.

[0085] 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 use of NEDD8 activator inhibitors or their hydrochloride salts in the preparation of drugs for treating Japanese encephalitis, characterized in that, The NEDD8 activator enzyme inhibitor is MLN4924, whose chemical structure is shown in Formula I; the hydrochloride of MLN4924 is MLN4924 hydrochloride, whose chemical structure is shown in Formula II. Formula I Formula II.

2. The use of the NEDD8 activator inhibitor or its hydrochloride salt according to claim 1 in the preparation of a drug for treating Japanese encephalitis, characterized in that, The use of the NEDD8 activator inhibitor or its hydrochloride in the preparation of drugs that inhibit Japanese encephalitis virus infection and cytotoxicity.

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